cardiomyocyte isolation Search Results


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  • 99
    Worthington Biochemical neonatal cardiomyocyte isolation system
    Immunocytochemical co-localization of CLP-1, cyclin T and cdk9 in isolated 2 day post natal rat <t>cardiomyocytes.</t> A–C: (A) CLP-1 is in the nucleus of MF20-positive cardiomyocytes; (B) nuclear co-localization of CLP-1 and cyclin T; (C) control (no
    Neonatal Cardiomyocyte Isolation System, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 838 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher primary cardiomyocyte isolation kit
    Transgenic mouse model for Pdzrn3 depletion in <t>cardiomyocytes:</t> a. Schematic of Pdzrn3 deletion in cardiac cells. Control mice were Pdzrn3 f/f . MCM- Pdzrn3 KO mice were MHC-MerCreMer; Pdzrn3 f/f . b. qRT-PCR demonstrates deletion o f Pdzrn3 in adult hearts after 1 month of tamoxifen treatment (n=5). Data are reported as average ± s.e.m. *P
    Primary Cardiomyocyte Isolation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher neonatal rat mouse cardiomyocyte isolation kit
    Molecular events of ROS-dependent PKCδ activation involved in the AGE-BSA-induced <t>cardiomyocyte</t> apoptosis. PKCδ activation is involved in the regulation of AGE-BSA-induced cell apoptosis via ROS production and may play a key role in the development of cardiac mitochondrial dysfunction in rats with diabetes or obesity.
    Neonatal Rat Mouse Cardiomyocyte Isolation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Miltenyi Biotec neonatal cardiomyocyte isolation kit mouse
    ( a , b ) (left) The long-term culture of hIPSC-derived cardiac myocytes yields cell assemblies similar to the culture of mouse primary cardiac myocytes, giving rise to the assumption that such stem cell populations can be used to substitute for primary animal-derived specimens; ( c , d ) Different from the well-ordered  α -actinin apparatus, the connexin-43 distribution does not recapitulate the cell interface accumulation as seen in neonatal cardiac myocyte culture; ( e ) Functional (left) and defect (right) cell ensembles imaged by SEM. The obvious finding that the well-spread cell ensemble is a functional one while the defect one releases surface contacts and reaches a bulk appearance does not absolutely correlate with a loss of contractility. A loss of surface-mediated organization can be regarded inside the bulk assembly. Consequently, spatially-random contracting bulks can be observed; ( f , b ) (right) Aging effects of the contractile apparatus after long-term cultivation. The regular  α -actinin organization with a ladder appearance with pronounced orientation along cells’ longitudes is substituted by a random orientation; ( g ) Ensembles of cardiac myocytes beat synchronously, indicating electrical excitation coupling. While the direction of contraction is random on a plain surface (right), the line pattern-induced cell ensemble organization results in temporally synchronized and spatially-organized contractions. This fulfills a fundamental prerequisite of cardiac tissue function.
    Neonatal Cardiomyocyte Isolation Kit Mouse, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 98/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Miltenyi Biotec psc derived cardiomyocyte isolation kit
    ( a , b ) (left) The long-term culture of hIPSC-derived cardiac myocytes yields cell assemblies similar to the culture of mouse primary cardiac myocytes, giving rise to the assumption that such stem cell populations can be used to substitute for primary animal-derived specimens; ( c , d ) Different from the well-ordered  α -actinin apparatus, the connexin-43 distribution does not recapitulate the cell interface accumulation as seen in neonatal cardiac myocyte culture; ( e ) Functional (left) and defect (right) cell ensembles imaged by SEM. The obvious finding that the well-spread cell ensemble is a functional one while the defect one releases surface contacts and reaches a bulk appearance does not absolutely correlate with a loss of contractility. A loss of surface-mediated organization can be regarded inside the bulk assembly. Consequently, spatially-random contracting bulks can be observed; ( f , b ) (right) Aging effects of the contractile apparatus after long-term cultivation. The regular  α -actinin organization with a ladder appearance with pronounced orientation along cells’ longitudes is substituted by a random orientation; ( g ) Ensembles of cardiac myocytes beat synchronously, indicating electrical excitation coupling. While the direction of contraction is random on a plain surface (right), the line pattern-induced cell ensemble organization results in temporally synchronized and spatially-organized contractions. This fulfills a fundamental prerequisite of cardiac tissue function.
    Psc Derived Cardiomyocyte Isolation Kit, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Charles River Laboratories cardiomyocyte isolation
    AL-LC causes mitochondrial dysfunction and autophagy dysregulation in vitro Using the fluorescent indicator DCFDA, ROS was measured in isolated <t>cardiomyocytes</t> treated with vehicle, Con-LC, AL-LC or AL-LC + Mito-TEMPO. Representative fluorescent and bright field images are shown in the top panels, and quantitative analysis is summarized in the graph below. AL-LC but not Con-LC increased ROS and this was blocked by Mito-TEMPO, indicating that ROS is derived from mitochondria. Scale bar = 100 μm. N = 3. * P = 0.020. Mitochondrial membrane potential was measured using TMRE fluorescent dye in cardiomyocytes following 24-h treatment with vehicle, Con-LC or AL-LC. Representative TMRE fluorescent and bright field microscopy images are shown in the top panels, and TMRE fluorescence signal was quantified and summarized below. AL-LC exposure resulted in loss of mitochondrial membrane potential compared to the other groups. Scale bar = 50 μm. N = 3. * P = 0.031. Quantitative summary of cellular ATP levels in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. N = 3. * P = 0.020. Immunoblot analysis of LC3-II expression in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. GAPDH was used as a loading control. Quantitative results are shown in the graph below for comparison. LC3-II expression was significantly increased in the AL-LC group compared to vehicle and Con-LC. N = 3. * P = 1.9 × 10 −6 . Immunoblot analysis of AL-LC-induced LC3-II expression in the presence of lysosomal inhibitors E64d and Pepstatin A. GAPDH is used as a loading control. Quantitative results in the graph below show that LC3-II levels in the AL-LC group do not exceed vehicle-treated LC3-II levels following lysosomal inhibition, indicating minimal perturbation to initiation. N = 6. * P = 1.4 × 10 −6 , # P = 0.041. Immunoblot and quantitative analysis of p62 expression in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. Results show a decrease in autophagic flux seen by increased p62 accumulation in the AL-LC group. N = 3. * P = 0.033. Confocal imaging of immunofluorescence staining of p62 (red) in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. Mitochondrial co-localization was visualized using mitochondrial protein COX4 (green) co-staining, and nuclear staining with DAPI (blue). Scale bar = 10 μm. Source data are available online for this figure.
    Cardiomyocyte Isolation, supplied by Charles River Laboratories, used in various techniques. Bioz Stars score: 89/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Miltenyi Biotec neonatal cardiomyocyte isolation kit
    ( a , b ) (left) The long-term culture of hIPSC-derived cardiac myocytes yields cell assemblies similar to the culture of mouse primary cardiac myocytes, giving rise to the assumption that such stem cell populations can be used to substitute for primary animal-derived specimens; ( c , d ) Different from the well-ordered  α -actinin apparatus, the connexin-43 distribution does not recapitulate the cell interface accumulation as seen in neonatal cardiac myocyte culture; ( e ) Functional (left) and defect (right) cell ensembles imaged by SEM. The obvious finding that the well-spread cell ensemble is a functional one while the defect one releases surface contacts and reaches a bulk appearance does not absolutely correlate with a loss of contractility. A loss of surface-mediated organization can be regarded inside the bulk assembly. Consequently, spatially-random contracting bulks can be observed; ( f , b ) (right) Aging effects of the contractile apparatus after long-term cultivation. The regular  α -actinin organization with a ladder appearance with pronounced orientation along cells’ longitudes is substituted by a random orientation; ( g ) Ensembles of cardiac myocytes beat synchronously, indicating electrical excitation coupling. While the direction of contraction is random on a plain surface (right), the line pattern-induced cell ensemble organization results in temporally synchronized and spatially-organized contractions. This fulfills a fundamental prerequisite of cardiac tissue function.
    Neonatal Cardiomyocyte Isolation Kit, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 94/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Harvard Bioscience cardiomyocyte isolation
    Angiotensin-forming enzyme activity in <t>cardiomyocyte</t> (CM) and non-cardiomyocyte (NCM) from SHR and WKY female rats. Data indicate differences in (A) chymase activity in CMs (left) and NCMs (right) with respect to estrogen status (intact versus OVX); in NCMs, WKY chymase activity exceeds that of SHR (strain effect). (B) ACE activity is not different between strains nor is it affected by estrogen status. (C) ACE2 activity in CMs is higher in WKY compared to SHR, irrespective of estrogen status; NCMs showed no estrogen or strain effects (2-way ANOVA). Values are reported as mean ± SEM; SHR, N =6 per group. WKY rats, N =6 per group. Black bars represent gonadal intact rats; hatched bars represent OVX rats. ## P
    Cardiomyocyte Isolation, supplied by Harvard Bioscience, used in various techniques. Bioz Stars score: 90/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher neonatal cardiomyocyte isolation system kit
    Inhibition of the cardioprotective effects of E 2 , ERα and BSA-E 2 on LPS-treated ventricular <t>cardiomyocytes</t> by PI3K siRNA treatment. (A and B) Thirty-six hours after being cotransfected with plasmids (3.5 μg ERα or pcDNA3) and siRNA (100 nM siGLO siRNA or PI3K siRNA), ventricular cardiomyocytes were incubated with vehicle, E 2 (10 −8 M) or BSA-E 2 (equivalent to 10 ng/ml free E 2 ) in the presence of LPS for 24 hrs. Cells were harvested and analyzed by RT-PCR to detect TNF-α expression and by immunoblotting using antibodies against TNF-α, ERα and active caspase 3. (C) TUNEL staining of the ventricular cardiomyocytes was performed according to the manufacturer’s protocol PI3K siRNA abolished the reduction of apoptosis induced by E 2 , ERα and BSA-E 2 in LPS-treated ventricular cardiomyocytes. ** P
    Neonatal Cardiomyocyte Isolation System Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher cardiomyocyte isolation enzyme 2 with thermolysin 20x for pierce primary cell isolation kits
    Inhibition of the cardioprotective effects of E 2 , ERα and BSA-E 2 on LPS-treated ventricular <t>cardiomyocytes</t> by PI3K siRNA treatment. (A and B) Thirty-six hours after being cotransfected with plasmids (3.5 μg ERα or pcDNA3) and siRNA (100 nM siGLO siRNA or PI3K siRNA), ventricular cardiomyocytes were incubated with vehicle, E 2 (10 −8 M) or BSA-E 2 (equivalent to 10 ng/ml free E 2 ) in the presence of LPS for 24 hrs. Cells were harvested and analyzed by RT-PCR to detect TNF-α expression and by immunoblotting using antibodies against TNF-α, ERα and active caspase 3. (C) TUNEL staining of the ventricular cardiomyocytes was performed according to the manufacturer’s protocol PI3K siRNA abolished the reduction of apoptosis induced by E 2 , ERα and BSA-E 2 in LPS-treated ventricular cardiomyocytes. ** P
    Cardiomyocyte Isolation Enzyme 2 With Thermolysin 20x For Pierce Primary Cell Isolation Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Immunocytochemical co-localization of CLP-1, cyclin T and cdk9 in isolated 2 day post natal rat cardiomyocytes. A–C: (A) CLP-1 is in the nucleus of MF20-positive cardiomyocytes; (B) nuclear co-localization of CLP-1 and cyclin T; (C) control (no

    Journal:

    Article Title: Pivotal role of cardiac lineage protein-1 (CLP-1) in transcriptional elongation factor P-TEFb complex formation in cardiac hypertrophy

    doi: 10.1016/j.cardiores.2007.03.019

    Figure Lengend Snippet: Immunocytochemical co-localization of CLP-1, cyclin T and cdk9 in isolated 2 day post natal rat cardiomyocytes. A–C: (A) CLP-1 is in the nucleus of MF20-positive cardiomyocytes; (B) nuclear co-localization of CLP-1 and cyclin T; (C) control (no

    Article Snippet: Cardiac cells of 2–4-day-old WKY rats were isolated using a neonatal cardiomyocyte isolation system (Worthington Biochemical Corp, Lakewood, New Jersey, USA).

    Techniques: Isolation

    A. Stretched cardiomyocytes express markers of hypertrophy. Northern blot showing increased ANF (atrial natriuretic factor) mRNA levels in stretched cardiomyocytes. GAPDH is loading control. B. Phosphorylation of STAT 3 increases with continued stretch

    Journal:

    Article Title: Pivotal role of cardiac lineage protein-1 (CLP-1) in transcriptional elongation factor P-TEFb complex formation in cardiac hypertrophy

    doi: 10.1016/j.cardiores.2007.03.019

    Figure Lengend Snippet: A. Stretched cardiomyocytes express markers of hypertrophy. Northern blot showing increased ANF (atrial natriuretic factor) mRNA levels in stretched cardiomyocytes. GAPDH is loading control. B. Phosphorylation of STAT 3 increases with continued stretch

    Article Snippet: Cardiac cells of 2–4-day-old WKY rats were isolated using a neonatal cardiomyocyte isolation system (Worthington Biochemical Corp, Lakewood, New Jersey, USA).

    Techniques: Northern Blot

    CLP-1 dissociates from P-TEFb in cardiomyocytes treated with hypertrophic agents. A. Immunoprecipitated cyclin T1 shows less associated CLP-1 in endothelin-1-treated cardiomyocytes than untreated cells (control). B. Immunoprecipitated cyclin T1 shows

    Journal:

    Article Title: Pivotal role of cardiac lineage protein-1 (CLP-1) in transcriptional elongation factor P-TEFb complex formation in cardiac hypertrophy

    doi: 10.1016/j.cardiores.2007.03.019

    Figure Lengend Snippet: CLP-1 dissociates from P-TEFb in cardiomyocytes treated with hypertrophic agents. A. Immunoprecipitated cyclin T1 shows less associated CLP-1 in endothelin-1-treated cardiomyocytes than untreated cells (control). B. Immunoprecipitated cyclin T1 shows

    Article Snippet: Cardiac cells of 2–4-day-old WKY rats were isolated using a neonatal cardiomyocyte isolation system (Worthington Biochemical Corp, Lakewood, New Jersey, USA).

    Techniques: Immunoprecipitation

    MURF1 colocalizes with RACK1 in cultured cardiac myocytes.  Rat cardiac myocytes were infected with recombinant adenovirus Ad.GFP (A and B) or Ad.MURF1 (C and D) for 24 h in serum free medium followed by induction with PE (B and D) for 48 h. Immunostaining was done with anti-Myc and anti-RACK1 antibodies. This was followed by secondary antibody incubation with anti–rabbit Alexa 568 (red) and anti–mouse AMCA (blue). The green color represents GFP expression.

    Journal: The Journal of Cell Biology

    Article Title: Muscle ring finger protein-1 inhibits PKC? activation and prevents cardiomyocyte hypertrophy

    doi: 10.1083/jcb.200402033

    Figure Lengend Snippet: MURF1 colocalizes with RACK1 in cultured cardiac myocytes. Rat cardiac myocytes were infected with recombinant adenovirus Ad.GFP (A and B) or Ad.MURF1 (C and D) for 24 h in serum free medium followed by induction with PE (B and D) for 48 h. Immunostaining was done with anti-Myc and anti-RACK1 antibodies. This was followed by secondary antibody incubation with anti–rabbit Alexa 568 (red) and anti–mouse AMCA (blue). The green color represents GFP expression.

    Article Snippet: NRVM were isolated using the neonatal cardiomyocyte isolation kit (Worthington) and were plated on laminin.

    Techniques: Cell Culture, Infection, Recombinant, Immunostaining, Incubation, Expressing

    Ca 2+ imaging of cardiomyocytes from WT and mutant hearts.

    Journal:

    Article Title: Early cardiac hypertrophy in mice with impaired calmodulin regulation of cardiac muscle Ca2+ release channel

    doi: 10.1172/JCI29515

    Figure Lengend Snippet: Ca 2+ imaging of cardiomyocytes from WT and mutant hearts.

    Article Snippet: Neonatal cardiomyocytes were prepared from the ventricular muscle of mice approximately 1 day old using Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp.) according to the manufacturer’s protocol.

    Techniques: Imaging, Mutagenesis

    Effect of PE and DHA on α-actinin and ANF expression. Cardiomyocytes were treated with PE and DHA as described in the legend of Figure 1 . Solid arrows indicate the expression of sarcomeric α-actinin in z-bands (green fluorescence) whereas dotted arrows indicate expression of ANF (red fluorescence). Results are a typical representation of five experiments.

    Journal: Journal of molecular and genetic medicine : an international journal of biomedical research

    Article Title: Docosahexaenoic acid inhibits protein kinase C translocation/activation and cardiac hypertrophy in rat cardiomyocytes

    doi:

    Figure Lengend Snippet: Effect of PE and DHA on α-actinin and ANF expression. Cardiomyocytes were treated with PE and DHA as described in the legend of Figure 1 . Solid arrows indicate the expression of sarcomeric α-actinin in z-bands (green fluorescence) whereas dotted arrows indicate expression of ANF (red fluorescence). Results are a typical representation of five experiments.

    Article Snippet: Materials The cardiomyocyte isolation kit was purchased from Worthington Biochemical Corporation (Lakewood, NJ).

    Techniques: Expressing, Fluorescence

    Effect of PE and DHA on PKCα translocation in cardiomyocytes. Cardiomyocytes were treated with PE and DHA as described in the legend of Figure 1 . Proteins were detected using anti-PKCα specific antibody and visualized with Alexa 546- (red fluorescence) labelled anti-mouse as described in the Material and Methods section. Cells were examined under a fluorescence microscope and images were captured using a MagnaFire digital camera (Optronics) for analysis. Results are a typical representation of three experiments.

    Journal: Journal of molecular and genetic medicine : an international journal of biomedical research

    Article Title: Docosahexaenoic acid inhibits protein kinase C translocation/activation and cardiac hypertrophy in rat cardiomyocytes

    doi:

    Figure Lengend Snippet: Effect of PE and DHA on PKCα translocation in cardiomyocytes. Cardiomyocytes were treated with PE and DHA as described in the legend of Figure 1 . Proteins were detected using anti-PKCα specific antibody and visualized with Alexa 546- (red fluorescence) labelled anti-mouse as described in the Material and Methods section. Cells were examined under a fluorescence microscope and images were captured using a MagnaFire digital camera (Optronics) for analysis. Results are a typical representation of three experiments.

    Article Snippet: Materials The cardiomyocyte isolation kit was purchased from Worthington Biochemical Corporation (Lakewood, NJ).

    Techniques: Translocation Assay, Fluorescence, Microscopy

    Effect of PE and DHA on cell surface area. Cardiomyocytes were treated with either ethanol (control), serum free media or DHA (5 μM in serum-free conditions) for 24 hr and then incubated with/without PE (100 μM) for 48 hr. Data are expressed as mean ± SEM for examination of 10 cells in each group from three separate experiments and were analyzed by ANOVA and Tukey's multiple comparison tests. Significant differences within groups are reported. ***P

    Journal: Journal of molecular and genetic medicine : an international journal of biomedical research

    Article Title: Docosahexaenoic acid inhibits protein kinase C translocation/activation and cardiac hypertrophy in rat cardiomyocytes

    doi:

    Figure Lengend Snippet: Effect of PE and DHA on cell surface area. Cardiomyocytes were treated with either ethanol (control), serum free media or DHA (5 μM in serum-free conditions) for 24 hr and then incubated with/without PE (100 μM) for 48 hr. Data are expressed as mean ± SEM for examination of 10 cells in each group from three separate experiments and were analyzed by ANOVA and Tukey's multiple comparison tests. Significant differences within groups are reported. ***P

    Article Snippet: Materials The cardiomyocyte isolation kit was purchased from Worthington Biochemical Corporation (Lakewood, NJ).

    Techniques: Incubation

    Effect of PE and DHA on PKC activity. Cardiomyocytes were treated with PE and DHA as described in the legend of Figure 1 . The total PKC activity in membrane fractions of cardiomyocytes was assayed as described in the Material and Methods section. Results are expressed as the mean±SE for three experiments and analyzed by ANOVA and Tukey's multiple comparison tests. Significant differences within groups are reported. ***P

    Journal: Journal of molecular and genetic medicine : an international journal of biomedical research

    Article Title: Docosahexaenoic acid inhibits protein kinase C translocation/activation and cardiac hypertrophy in rat cardiomyocytes

    doi:

    Figure Lengend Snippet: Effect of PE and DHA on PKC activity. Cardiomyocytes were treated with PE and DHA as described in the legend of Figure 1 . The total PKC activity in membrane fractions of cardiomyocytes was assayed as described in the Material and Methods section. Results are expressed as the mean±SE for three experiments and analyzed by ANOVA and Tukey's multiple comparison tests. Significant differences within groups are reported. ***P

    Article Snippet: Materials The cardiomyocyte isolation kit was purchased from Worthington Biochemical Corporation (Lakewood, NJ).

    Techniques: Activity Assay

    Effect of PE and DHA on PKCα distribution in membranes. Cardiomyocytes were treated with PE and DHA as described in the legend of Figure 1 . Membranes were isolated and separated on 8% SDS-PAGE as described in the Material and Methods section. PKCα was detected by Western blotting using anti-PKCα antibodies and the relative distribution of PKCα was determined by densitometric analysis. Results are a typical representation of three experiments.

    Journal: Journal of molecular and genetic medicine : an international journal of biomedical research

    Article Title: Docosahexaenoic acid inhibits protein kinase C translocation/activation and cardiac hypertrophy in rat cardiomyocytes

    doi:

    Figure Lengend Snippet: Effect of PE and DHA on PKCα distribution in membranes. Cardiomyocytes were treated with PE and DHA as described in the legend of Figure 1 . Membranes were isolated and separated on 8% SDS-PAGE as described in the Material and Methods section. PKCα was detected by Western blotting using anti-PKCα antibodies and the relative distribution of PKCα was determined by densitometric analysis. Results are a typical representation of three experiments.

    Article Snippet: Materials The cardiomyocyte isolation kit was purchased from Worthington Biochemical Corporation (Lakewood, NJ).

    Techniques: Isolation, SDS Page, Western Blot

    Inhibition of FGF signaling in cardiomyocytes in vivo results in Cx43 lateralization. (a) Immunostaining of left ventricle at 40 weeks. Sections were stained for Cx43, pan-cadherin, or desmoplakin (red), HA (FGFR1DN, white), actin (green) and DAPI (blue).

    Journal: Experimental cell research

    Article Title: Cardiomyocyte FGF signaling is required for Cx43 phosphorylation and cardiac gap junction maintenance

    doi: 10.1016/j.yexcr.2013.05.022

    Figure Lengend Snippet: Inhibition of FGF signaling in cardiomyocytes in vivo results in Cx43 lateralization. (a) Immunostaining of left ventricle at 40 weeks. Sections were stained for Cx43, pan-cadherin, or desmoplakin (red), HA (FGFR1DN, white), actin (green) and DAPI (blue).

    Article Snippet: Primary neonatal rat cardiomyocytes were isolated from hearts of 1–2 day-old Sprague-Dawley rats using the cardiomyocyte isolation system (Worthington biochemical corporation, Lakewood, New Jersey, USA) according to instructions.

    Techniques: Inhibition, In Vivo, Immunostaining, Staining

    Inhibition of FGF signaling decreases Cx43 localization at cell–cell contacts, but does not significantly affect ZO-1 and N-cadherin. (a) Cx43 mislocalization in primary rat cardiomyocytes lacking FGF signaling. Primary rat cardiomyocytes, infected

    Journal: Experimental cell research

    Article Title: Cardiomyocyte FGF signaling is required for Cx43 phosphorylation and cardiac gap junction maintenance

    doi: 10.1016/j.yexcr.2013.05.022

    Figure Lengend Snippet: Inhibition of FGF signaling decreases Cx43 localization at cell–cell contacts, but does not significantly affect ZO-1 and N-cadherin. (a) Cx43 mislocalization in primary rat cardiomyocytes lacking FGF signaling. Primary rat cardiomyocytes, infected

    Article Snippet: Primary neonatal rat cardiomyocytes were isolated from hearts of 1–2 day-old Sprague-Dawley rats using the cardiomyocyte isolation system (Worthington biochemical corporation, Lakewood, New Jersey, USA) according to instructions.

    Techniques: Inhibition, Infection

    FGF signaling in cardiomyocytes affects Cx43 phosphorylation. Western analysis of total protein extracted from primary rat cardiomyocytes infected with either Ad-GFP (MOI 5) or Ad-FGFR1DN (MOI 0.5-10) with incremental doses of MOI, or left untreated (NT,

    Journal: Experimental cell research

    Article Title: Cardiomyocyte FGF signaling is required for Cx43 phosphorylation and cardiac gap junction maintenance

    doi: 10.1016/j.yexcr.2013.05.022

    Figure Lengend Snippet: FGF signaling in cardiomyocytes affects Cx43 phosphorylation. Western analysis of total protein extracted from primary rat cardiomyocytes infected with either Ad-GFP (MOI 5) or Ad-FGFR1DN (MOI 0.5-10) with incremental doses of MOI, or left untreated (NT,

    Article Snippet: Primary neonatal rat cardiomyocytes were isolated from hearts of 1–2 day-old Sprague-Dawley rats using the cardiomyocyte isolation system (Worthington biochemical corporation, Lakewood, New Jersey, USA) according to instructions.

    Techniques: Western Blot, Infection

    Disruption of cardiomyocyte FGF signaling in adult mice leads to heart failure and premature death. (a) Survival curves of control (A) and aMHC-FGFR1DN (AF) mice ( n =20 in each group). Transgene expression was induced at the age of 10 weeks (Doxycycline

    Journal: Experimental cell research

    Article Title: Cardiomyocyte FGF signaling is required for Cx43 phosphorylation and cardiac gap junction maintenance

    doi: 10.1016/j.yexcr.2013.05.022

    Figure Lengend Snippet: Disruption of cardiomyocyte FGF signaling in adult mice leads to heart failure and premature death. (a) Survival curves of control (A) and aMHC-FGFR1DN (AF) mice ( n =20 in each group). Transgene expression was induced at the age of 10 weeks (Doxycycline

    Article Snippet: Primary neonatal rat cardiomyocytes were isolated from hearts of 1–2 day-old Sprague-Dawley rats using the cardiomyocyte isolation system (Worthington biochemical corporation, Lakewood, New Jersey, USA) according to instructions.

    Techniques: Mouse Assay, Expressing

    Phosphorylation of MnSOD by p38β kinase. ( A ) In-vitro kinase assay is performed with purified MnSOD in the indicated amount as a substrate in the presence of activated, GST-tagged purified p38β kinase in the presence or absence of the specific kinase inhibitor, SB203580. As a positive control, the kinase activity is also assayed on a well known substrate, ATF2. ( B ) In-vitro kinase assay is performed with endogenous p38β kinase immunoprecipitated from cultured rat cardiomyocytes and 1 µg of MnSOD or ATF2 as substrate in the presence or absence of the kinase inhibitor. A western blot of the isolated kinase is shown as a control.

    Journal: PLoS ONE

    Article Title: Mitochondrial p38? and Manganese Superoxide Dismutase Interaction Mediated by Estrogen in Cardiomyocytes

    doi: 10.1371/journal.pone.0085272

    Figure Lengend Snippet: Phosphorylation of MnSOD by p38β kinase. ( A ) In-vitro kinase assay is performed with purified MnSOD in the indicated amount as a substrate in the presence of activated, GST-tagged purified p38β kinase in the presence or absence of the specific kinase inhibitor, SB203580. As a positive control, the kinase activity is also assayed on a well known substrate, ATF2. ( B ) In-vitro kinase assay is performed with endogenous p38β kinase immunoprecipitated from cultured rat cardiomyocytes and 1 µg of MnSOD or ATF2 as substrate in the presence or absence of the kinase inhibitor. A western blot of the isolated kinase is shown as a control.

    Article Snippet: Then, cardiomyocytes were isolated using a neonatal cardiomyocyte isolation system kit (Worthington), as reported previously , .

    Techniques: In Vitro, Kinase Assay, Purification, Positive Control, Activity Assay, Immunoprecipitation, Cell Culture, Western Blot, Isolation

    Physical association between p38β and MnSOD. ( A ) Endogenous p38β is immunoprecipitated (IP: p38β) from cardiomyocytes after H/R with or without E2 at 10 nM, and the p38β-containing immunocomplex blotted for MnSOD. A western blot of p38β protein is also shown as a loading control. Nonspecific IgG is used as a specificity control in place of antibody specific to p38β in immunoprecipitation (IP: NS IgG). ( B ) Endogenous MnSOD is immunoprecipitated (IP: MnSOD) from treated cardiomyocytes as indicated, then the MnSOD-containing immunocomplex is blotted for p38β. Nonspecific IgG is used as a specificity control in place of antibody specific to MnSOD in immunoprecipitation (IP: NS IgG).

    Journal: PLoS ONE

    Article Title: Mitochondrial p38? and Manganese Superoxide Dismutase Interaction Mediated by Estrogen in Cardiomyocytes

    doi: 10.1371/journal.pone.0085272

    Figure Lengend Snippet: Physical association between p38β and MnSOD. ( A ) Endogenous p38β is immunoprecipitated (IP: p38β) from cardiomyocytes after H/R with or without E2 at 10 nM, and the p38β-containing immunocomplex blotted for MnSOD. A western blot of p38β protein is also shown as a loading control. Nonspecific IgG is used as a specificity control in place of antibody specific to p38β in immunoprecipitation (IP: NS IgG). ( B ) Endogenous MnSOD is immunoprecipitated (IP: MnSOD) from treated cardiomyocytes as indicated, then the MnSOD-containing immunocomplex is blotted for p38β. Nonspecific IgG is used as a specificity control in place of antibody specific to MnSOD in immunoprecipitation (IP: NS IgG).

    Article Snippet: Then, cardiomyocytes were isolated using a neonatal cardiomyocyte isolation system kit (Worthington), as reported previously , .

    Techniques: Immunoprecipitation, Western Blot

    Identification of mitochondrial p38β. ( A ) Upper , Immnoblots of total p38β and phosphorylated p38β (p-p38β) from mitochondria (Mito) and cytosol (Cyto) of rat cardiomyocytes are shown. Final [E2] = 10 nM. Immunoblots of CoxIV and actin are shown also as loading controls for mitochondrial and cytosolic fractions, respectively. Lower Left , CoxIV activity. Mitochondria were isolated and assayed for Cox IV activity and citrate synthase activity. CoxIV activity was then normalized to that of citrate synthase, and is presented with quantitative analysis. Lower Right , NAO fluorescence. Cells were stained with NAO, a marker of mitochondrial biogenesis, and analyzed by flow cytometry. A representative graph from triplicate experiments is presented ( B ) Mitochondrial p-p38β. Upper , the ratio of dual phosphorylated p38β (p-p38β) over total p38β in mitochondria after indicated treatments is expressed in graph. Lower , the percentage of mitochondrial p38β localized to mitochondria is shown in graph as the total mitochondrial p38β over total cellular p38β pool. ( C ) Immunocytochemistry of p38β in cardiomyocytes. Primary antibody specific to p38β is used to stain for intracellular p38β ( left ), and the cells are co-stained with MitoTracker, a fluorescent probe for mitochondria ( middle ). The two images are then merged to demonstrate co-localization of p38β with mitochondria ( right ). The white scale bar represents 25 µm. ( D ) Mitochondrial membrane potential by JC-1 fluorescence. Upper , Flow cytometry analysis of JC-1 stained cells after the indicated treatments shows mitochondrial membrane potential changes reflected by the ratio of the red (FL2 district) and the green (FL1) fluorescent signals. Lower , Red/green JC-1 fluorescence ratio is represented in graph with quantitative analysis. * p

    Journal: PLoS ONE

    Article Title: Mitochondrial p38? and Manganese Superoxide Dismutase Interaction Mediated by Estrogen in Cardiomyocytes

    doi: 10.1371/journal.pone.0085272

    Figure Lengend Snippet: Identification of mitochondrial p38β. ( A ) Upper , Immnoblots of total p38β and phosphorylated p38β (p-p38β) from mitochondria (Mito) and cytosol (Cyto) of rat cardiomyocytes are shown. Final [E2] = 10 nM. Immunoblots of CoxIV and actin are shown also as loading controls for mitochondrial and cytosolic fractions, respectively. Lower Left , CoxIV activity. Mitochondria were isolated and assayed for Cox IV activity and citrate synthase activity. CoxIV activity was then normalized to that of citrate synthase, and is presented with quantitative analysis. Lower Right , NAO fluorescence. Cells were stained with NAO, a marker of mitochondrial biogenesis, and analyzed by flow cytometry. A representative graph from triplicate experiments is presented ( B ) Mitochondrial p-p38β. Upper , the ratio of dual phosphorylated p38β (p-p38β) over total p38β in mitochondria after indicated treatments is expressed in graph. Lower , the percentage of mitochondrial p38β localized to mitochondria is shown in graph as the total mitochondrial p38β over total cellular p38β pool. ( C ) Immunocytochemistry of p38β in cardiomyocytes. Primary antibody specific to p38β is used to stain for intracellular p38β ( left ), and the cells are co-stained with MitoTracker, a fluorescent probe for mitochondria ( middle ). The two images are then merged to demonstrate co-localization of p38β with mitochondria ( right ). The white scale bar represents 25 µm. ( D ) Mitochondrial membrane potential by JC-1 fluorescence. Upper , Flow cytometry analysis of JC-1 stained cells after the indicated treatments shows mitochondrial membrane potential changes reflected by the ratio of the red (FL2 district) and the green (FL1) fluorescent signals. Lower , Red/green JC-1 fluorescence ratio is represented in graph with quantitative analysis. * p

    Article Snippet: Then, cardiomyocytes were isolated using a neonatal cardiomyocyte isolation system kit (Worthington), as reported previously , .

    Techniques: Western Blot, Activity Assay, Isolation, Fluorescence, Staining, Marker, Flow Cytometry, Cytometry, Immunocytochemistry

    Mitochondrial p38β activity and viability. ( A ) p38β is immunoprecipitated from mitochondria isolated from cultured rat cardiomyocytes and subjected to kinase assays using 32 P-labeled ATP and ATF2 as its substrate after indicated treatments. Thus radiolabeled ATF2 is shown with quantitative analysis of the band intensity. Representative western blots of ATF2 protein and immunoprecipitated mitochondrial p38β are shown at the bottom as a control. [E2] = 10 nM. * p

    Journal: PLoS ONE

    Article Title: Mitochondrial p38? and Manganese Superoxide Dismutase Interaction Mediated by Estrogen in Cardiomyocytes

    doi: 10.1371/journal.pone.0085272

    Figure Lengend Snippet: Mitochondrial p38β activity and viability. ( A ) p38β is immunoprecipitated from mitochondria isolated from cultured rat cardiomyocytes and subjected to kinase assays using 32 P-labeled ATP and ATF2 as its substrate after indicated treatments. Thus radiolabeled ATF2 is shown with quantitative analysis of the band intensity. Representative western blots of ATF2 protein and immunoprecipitated mitochondrial p38β are shown at the bottom as a control. [E2] = 10 nM. * p

    Article Snippet: Then, cardiomyocytes were isolated using a neonatal cardiomyocyte isolation system kit (Worthington), as reported previously , .

    Techniques: Activity Assay, Immunoprecipitation, Isolation, Cell Culture, Labeling, Western Blot

    MnSOD activity and anion superoxide. ( A ) After indicated treatments, mitochondria isolated from cardiomyocytes are assayed for MnSOD activity. The final dismutase activity is expressed relative to that under normoxia (control), with quantitative analysis. * p

    Journal: PLoS ONE

    Article Title: Mitochondrial p38? and Manganese Superoxide Dismutase Interaction Mediated by Estrogen in Cardiomyocytes

    doi: 10.1371/journal.pone.0085272

    Figure Lengend Snippet: MnSOD activity and anion superoxide. ( A ) After indicated treatments, mitochondria isolated from cardiomyocytes are assayed for MnSOD activity. The final dismutase activity is expressed relative to that under normoxia (control), with quantitative analysis. * p

    Article Snippet: Then, cardiomyocytes were isolated using a neonatal cardiomyocyte isolation system kit (Worthington), as reported previously , .

    Techniques: Activity Assay, Isolation

    A working model of E2-dependent protection of a cardiomyocyte under H/R stress. Following H/R or simulated I/R, there is a burst of mitochondrial ROS. The oxygen radicals stimulate p38α MAPK, which phosphorylates and activates p53. p53 in turn

    Journal: Cardiovascular Research

    Article Title: Oestrogen prevents cardiomyocyte apoptosis by suppressing p38?-mediated activation of p53 and by down-regulating p53 inhibition on p38?

    doi: 10.1093/cvr/cvq265

    Figure Lengend Snippet: A working model of E2-dependent protection of a cardiomyocyte under H/R stress. Following H/R or simulated I/R, there is a burst of mitochondrial ROS. The oxygen radicals stimulate p38α MAPK, which phosphorylates and activates p53. p53 in turn

    Article Snippet: Myocytes were isolated from the hearts of 1- to 3-day-old rats (Sprague–Dawley) using a neonatal cardiomyocyte isolation system kit (Worthington) as reported previously.

    Techniques:

    ( A ) Apoptosis assay. After H/R with or without PFT (1 µM) or siRNA p53, apoptotic cardiomyocytes were identified by annexin V-FITC. The apoptotic cells appeared green, and the nuclei co-stained with Hoechst 33342 blue. N, normoxia. The white scale

    Journal: Cardiovascular Research

    Article Title: Oestrogen prevents cardiomyocyte apoptosis by suppressing p38?-mediated activation of p53 and by down-regulating p53 inhibition on p38?

    doi: 10.1093/cvr/cvq265

    Figure Lengend Snippet: ( A ) Apoptosis assay. After H/R with or without PFT (1 µM) or siRNA p53, apoptotic cardiomyocytes were identified by annexin V-FITC. The apoptotic cells appeared green, and the nuclei co-stained with Hoechst 33342 blue. N, normoxia. The white scale

    Article Snippet: Myocytes were isolated from the hearts of 1- to 3-day-old rats (Sprague–Dawley) using a neonatal cardiomyocyte isolation system kit (Worthington) as reported previously.

    Techniques: Apoptosis Assay, Staining

    Mitochondrial internalization in cardiomyocytes. (A) Representative florescent micrographs of isolated rat liver mitochondria labeled with pHrodo co-incubated with cardiomyocytes for 4 hour (middle panel) or 24 hours (right panel). Control cardiomyocytes (no mitochondria) are shown in the left panel. In all images the blue stain is DAPI; red, pHrodo labeled mitochondria, green is 488 phalloidin (f-actin). Scale bars are 25 µm. Results show internalization of transplanted mitochondria at 4 and 24 hours. (B) ATP content in cardiomyocytes: ATP content (nmol/10 3 cells) in control, no mitochondria (white bars) and cardiomyocytes co-incubated with mitochondria (black bars). (C) Mitochondrial uptake at 1, 4 and 24 hour co-incubation. The mean±sd for n = 5 experiments is shown. Results indicate that ATP content is significantly increased (p

    Journal: Biology Open

    Article Title: Actin-dependent mitochondrial internalization in cardiomyocytes: evidence for rescue of mitochondrial function

    doi: 10.1242/bio.201511478

    Figure Lengend Snippet: Mitochondrial internalization in cardiomyocytes. (A) Representative florescent micrographs of isolated rat liver mitochondria labeled with pHrodo co-incubated with cardiomyocytes for 4 hour (middle panel) or 24 hours (right panel). Control cardiomyocytes (no mitochondria) are shown in the left panel. In all images the blue stain is DAPI; red, pHrodo labeled mitochondria, green is 488 phalloidin (f-actin). Scale bars are 25 µm. Results show internalization of transplanted mitochondria at 4 and 24 hours. (B) ATP content in cardiomyocytes: ATP content (nmol/10 3 cells) in control, no mitochondria (white bars) and cardiomyocytes co-incubated with mitochondria (black bars). (C) Mitochondrial uptake at 1, 4 and 24 hour co-incubation. The mean±sd for n = 5 experiments is shown. Results indicate that ATP content is significantly increased (p

    Article Snippet: Cardiomyocytes isolation and culture Neonatal rat cardiomyocytes were isolated from 1-day-old Lewis rat pups the Neonatal Cardiomyocyte Isolation System (Worthington Biochemicals, Lakewood, NJ, USA ( ).

    Techniques: Isolation, Labeling, Incubation, Staining

    Inhibition of mitochondrial internalization. (A) Representative florescent micrographs of 2-day cardiomyocytes co-incubated with 1×10 7 rat liver mitochondria labeled with pHrodo red following 4 hours co-incubation. Actin-dependent endocytosis and phagocytosis was inhibited with cytochalasin D (CytoD), caveola-dependent-clathrin dependent endocytosis was inhibited with methyl-β–cyclodextrin (MβCD), tunneling nano tube formation was inhibited with nocodazole (Noco) and macro pinocytosis was blocked with 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) at 10 µM, 50 µM or 100 µM . Control cardiomyocytes were co-incubated with mitochondria without inhibitor (no inhibitor), As separate control, cardiomyocytes without mitochondria (no mitochondria) is also shown. Internalized mitochondria are red (pHrodo labeled mitochondria). Nuclei are shown in blue (DAPI). Scale bars are 25 µm. (B) Internalization of mitochondria per nucleus as determined by florescent microscopy following 4 hours of co-incubation. (C) ATP content (nmol/10 3 cells). Results are shown as the mean and standard deviation for n = 5 in each group. Statistical differences at p

    Journal: Biology Open

    Article Title: Actin-dependent mitochondrial internalization in cardiomyocytes: evidence for rescue of mitochondrial function

    doi: 10.1242/bio.201511478

    Figure Lengend Snippet: Inhibition of mitochondrial internalization. (A) Representative florescent micrographs of 2-day cardiomyocytes co-incubated with 1×10 7 rat liver mitochondria labeled with pHrodo red following 4 hours co-incubation. Actin-dependent endocytosis and phagocytosis was inhibited with cytochalasin D (CytoD), caveola-dependent-clathrin dependent endocytosis was inhibited with methyl-β–cyclodextrin (MβCD), tunneling nano tube formation was inhibited with nocodazole (Noco) and macro pinocytosis was blocked with 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) at 10 µM, 50 µM or 100 µM . Control cardiomyocytes were co-incubated with mitochondria without inhibitor (no inhibitor), As separate control, cardiomyocytes without mitochondria (no mitochondria) is also shown. Internalized mitochondria are red (pHrodo labeled mitochondria). Nuclei are shown in blue (DAPI). Scale bars are 25 µm. (B) Internalization of mitochondria per nucleus as determined by florescent microscopy following 4 hours of co-incubation. (C) ATP content (nmol/10 3 cells). Results are shown as the mean and standard deviation for n = 5 in each group. Statistical differences at p

    Article Snippet: Cardiomyocytes isolation and culture Neonatal rat cardiomyocytes were isolated from 1-day-old Lewis rat pups the Neonatal Cardiomyocyte Isolation System (Worthington Biochemicals, Lakewood, NJ, USA ( ).

    Techniques: Inhibition, Incubation, Labeling, Microscopy, Standard Deviation

    14–3–3 recruitment to chromatin is important for transcriptional elongation of GATA-4 and of fetal cardiac genes. (A) ChIP–qPCR performed from mouse heart tissues showing 14–3–3 binding at the ANP and β -MHC promoters in CaMKII δ -WT or CaMKII δ -KO mice 21 days after sham operation or TAC. qPCR reactions were also performed at GAPDH and α-actin promoters that are not responsive to pressure overload hypertrophy: results are expressed as percentage input over signals with IgG antibody and represent average ± SD ( n = 3); p values from Student's t test are indicated. (B) Immunoblotting of cardiomyocytes expressing reduced 14–3–3 (si14–3–3) or normal 14–3–3 (siControl). (C) ChIP–qPCR, showing p-RNAPII S2 at the promoter and internal exon of ANP , β-MHC , GATA-4 , GAPDH and actin in primary neonatal rat cardiomyocytes with siCt or si14–3–3 and stimulated with phenylephrine (PE) for 24 h. (D) ChIP–qPCR assay from mouse heart chromatin, showing p-RNAPII S2 at the ANP , β-MHC , actin and GAPDH exonic region in CaMKII δ -WT or CaMKII δ -KO animals, 21 days after sham operation or TAC. (E) ChIP–qPCR assay, showing p-RNAPII S2 at the ANP , β-MHC , actin and GAPDH promoter regions in CaMKII δ -WT or CaMKII δ -KO animals, 21 days after sham operation or TAC: results are expressed as percentage input over signals with IgG antibody, and represent average ± SD ( n = 3); p values from Student's t -test are indicated

    Journal: The Journal of Pathology

    Article Title: Control of histone H3 phosphorylation by CaMKIIδ in response to haemodynamic cardiac stress

    doi: 10.1002/path.4489

    Figure Lengend Snippet: 14–3–3 recruitment to chromatin is important for transcriptional elongation of GATA-4 and of fetal cardiac genes. (A) ChIP–qPCR performed from mouse heart tissues showing 14–3–3 binding at the ANP and β -MHC promoters in CaMKII δ -WT or CaMKII δ -KO mice 21 days after sham operation or TAC. qPCR reactions were also performed at GAPDH and α-actin promoters that are not responsive to pressure overload hypertrophy: results are expressed as percentage input over signals with IgG antibody and represent average ± SD ( n = 3); p values from Student's t test are indicated. (B) Immunoblotting of cardiomyocytes expressing reduced 14–3–3 (si14–3–3) or normal 14–3–3 (siControl). (C) ChIP–qPCR, showing p-RNAPII S2 at the promoter and internal exon of ANP , β-MHC , GATA-4 , GAPDH and actin in primary neonatal rat cardiomyocytes with siCt or si14–3–3 and stimulated with phenylephrine (PE) for 24 h. (D) ChIP–qPCR assay from mouse heart chromatin, showing p-RNAPII S2 at the ANP , β-MHC , actin and GAPDH exonic region in CaMKII δ -WT or CaMKII δ -KO animals, 21 days after sham operation or TAC. (E) ChIP–qPCR assay, showing p-RNAPII S2 at the ANP , β-MHC , actin and GAPDH promoter regions in CaMKII δ -WT or CaMKII δ -KO animals, 21 days after sham operation or TAC: results are expressed as percentage input over signals with IgG antibody, and represent average ± SD ( n = 3); p values from Student's t -test are indicated

    Article Snippet: Primary neonatal rat cardiomyocytes isolated using the Worthington Neonatal CardioMyocytes System (Worthington, USA) were co-transfected with a GFP-tagged plasmid and siControl (siCt) or siRNA against 14–3–3 (si14–3–3) from Dharmacon (Thermo Scientific, USA) using lipofectamine (Invitrogen).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Aqueous Normal-phase Chromatography, Mouse Assay, Expressing

    Development and optimization of high throughput optical recording of VF2.1.Cl in cardiomyocytes. (A) Schematic of workflow for HTS transmembrane potential assay. VF2.1.Cl, VoltageFluor2.1.Cl voltage sensitive dye. (B) Schematic of workflow to isolate

    Journal: Frontiers in Physiology

    Article Title: An Automated Platform for Assessment of Congenital and Drug-Induced Arrhythmia with hiPSC-Derived Cardiomyocytes

    doi: 10.3389/fphys.2017.00766

    Figure Lengend Snippet: Development and optimization of high throughput optical recording of VF2.1.Cl in cardiomyocytes. (A) Schematic of workflow for HTS transmembrane potential assay. VF2.1.Cl, VoltageFluor2.1.Cl voltage sensitive dye. (B) Schematic of workflow to isolate

    Article Snippet: Neonatal atrial and ventricular rat cardiomyocytes were isolated with the neonatal rat cardiomyocyte isolation kit (Worthington, New Jersey, USA) (Toraason et al., ; Macgregor et al., ) and cultured at 37°C with 5% CO2 .

    Techniques: High Throughput Screening Assay

    Transgenic mouse model for Pdzrn3 depletion in cardiomyocytes: a. Schematic of Pdzrn3 deletion in cardiac cells. Control mice were Pdzrn3 f/f . MCM- Pdzrn3 KO mice were MHC-MerCreMer; Pdzrn3 f/f . b. qRT-PCR demonstrates deletion o f Pdzrn3 in adult hearts after 1 month of tamoxifen treatment (n=5). Data are reported as average ± s.e.m. *P

    Journal: bioRxiv

    Article Title: Repression of Pdzrn3 is required for heart maturation and protects against heart failure

    doi: 10.1101/2020.07.29.226597

    Figure Lengend Snippet: Transgenic mouse model for Pdzrn3 depletion in cardiomyocytes: a. Schematic of Pdzrn3 deletion in cardiac cells. Control mice were Pdzrn3 f/f . MCM- Pdzrn3 KO mice were MHC-MerCreMer; Pdzrn3 f/f . b. qRT-PCR demonstrates deletion o f Pdzrn3 in adult hearts after 1 month of tamoxifen treatment (n=5). Data are reported as average ± s.e.m. *P

    Article Snippet: Ventricular neonatal cardiomyocytes were then isolated and prepared as recommended (Pierce™ primary cardiomyocyte isolation kit).

    Techniques: Transgenic Assay, Mouse Assay, Quantitative RT-PCR

    Myocardial reactivation of Pdzrn3 alters post natal cardiomyocyte maturation a . Electron micrographs of part of an intercalated disc between two cardiac of control and Pdzrn3 OE heart at 8 weeks of age (scale bars represent 2 μm) and in right part, schema of ID distribution. b . Immunolabeling of Cx43, ZO1, β catenin and α actinin in hearts from control and Pdzrn3 OE mice at 2 weeks. Scale bars represent 10 μm. c . Western blot analysis of indicated proteins in heart tissues retrieved from control and Pdzrn3 OE mice at 1 and 2 weeks (W) of age. Relative expression of Cx43, from Western blots. Significance vs control *, P

    Journal: bioRxiv

    Article Title: Repression of Pdzrn3 is required for heart maturation and protects against heart failure

    doi: 10.1101/2020.07.29.226597

    Figure Lengend Snippet: Myocardial reactivation of Pdzrn3 alters post natal cardiomyocyte maturation a . Electron micrographs of part of an intercalated disc between two cardiac of control and Pdzrn3 OE heart at 8 weeks of age (scale bars represent 2 μm) and in right part, schema of ID distribution. b . Immunolabeling of Cx43, ZO1, β catenin and α actinin in hearts from control and Pdzrn3 OE mice at 2 weeks. Scale bars represent 10 μm. c . Western blot analysis of indicated proteins in heart tissues retrieved from control and Pdzrn3 OE mice at 1 and 2 weeks (W) of age. Relative expression of Cx43, from Western blots. Significance vs control *, P

    Article Snippet: Ventricular neonatal cardiomyocytes were then isolated and prepared as recommended (Pierce™ primary cardiomyocyte isolation kit).

    Techniques: Immunolabeling, Mouse Assay, Western Blot, Expressing

    Cardiac specific overexpression of Pdzrn3 impairs cardiomyocyte elongation a. The sphericity index was determined by measuring the area (A) and the circumference (P) of myocytes. The ratio of an “ideal” round cell is close to 1, whereas that of an elongated cell is closer to 0. b. Quantification of the cardiomyocyte sphericity index in heart sections from patients with non-decompensated (control HCM), decompensated HCM (HF-HCM), and from decompensated primitive or ischemic dilated cardiomyopathy (HF-DCM). (n=3 in control-HCM, n=4 in HF-HCM, n=6 in HF-DCM). Mean ± s.e.m. *P

    Journal: bioRxiv

    Article Title: Repression of Pdzrn3 is required for heart maturation and protects against heart failure

    doi: 10.1101/2020.07.29.226597

    Figure Lengend Snippet: Cardiac specific overexpression of Pdzrn3 impairs cardiomyocyte elongation a. The sphericity index was determined by measuring the area (A) and the circumference (P) of myocytes. The ratio of an “ideal” round cell is close to 1, whereas that of an elongated cell is closer to 0. b. Quantification of the cardiomyocyte sphericity index in heart sections from patients with non-decompensated (control HCM), decompensated HCM (HF-HCM), and from decompensated primitive or ischemic dilated cardiomyopathy (HF-DCM). (n=3 in control-HCM, n=4 in HF-HCM, n=6 in HF-DCM). Mean ± s.e.m. *P

    Article Snippet: Ventricular neonatal cardiomyocytes were then isolated and prepared as recommended (Pierce™ primary cardiomyocyte isolation kit).

    Techniques: Over Expression

    Characterization of cardiac damage after MI by TTC and TUNEL staining. (A) Experiment design for short term cardiac damage assessment. (B) representative images of WT and KO hearts stained with TTC 24 h after MI. (C) Quantification of infarct size (IS), area at risk (AAR) of left ventricle (LV), (D) LDH and Troponin release in the serum at 24 h after MI as measured by ELISA. (E) representative images of TUNEL staining on rat neonatal cardiomyocytes, transfected with indicated microRNA mimics or scramble controls and treated for 24 h of ischemia and the quantification of TUNEL positive cells. (F,G) Representative images of TUNEL staining on the left ventricle 24 h after MI and their quantifications.

    Journal: Frontiers in Physiology

    Article Title: Loss of miR-132/212 Has No Long-Term Beneficial Effect on Cardiac Function After Permanent Coronary Occlusion in Mice

    doi: 10.3389/fphys.2020.00590

    Figure Lengend Snippet: Characterization of cardiac damage after MI by TTC and TUNEL staining. (A) Experiment design for short term cardiac damage assessment. (B) representative images of WT and KO hearts stained with TTC 24 h after MI. (C) Quantification of infarct size (IS), area at risk (AAR) of left ventricle (LV), (D) LDH and Troponin release in the serum at 24 h after MI as measured by ELISA. (E) representative images of TUNEL staining on rat neonatal cardiomyocytes, transfected with indicated microRNA mimics or scramble controls and treated for 24 h of ischemia and the quantification of TUNEL positive cells. (F,G) Representative images of TUNEL staining on the left ventricle 24 h after MI and their quantifications.

    Article Snippet: Neonatal Rat Cardiomyocytes Isolation and Hypoxia Treatment Neonatal rat cardiomyocytes isolation was performed with Pierce Primary Cardiomyocyte Isolation Kit (Life Technologies, Cat. 88281) following manufacture’s instruction.

    Techniques: TUNEL Assay, Staining, Enzyme-linked Immunosorbent Assay, Transfection

    (A,B,C) Effects of unmodified MSC exosomes on cardiomyocyte apoptosis. (B,C) Nuclei were labeled with Hoechst (blue), TUNEL-positive nuclei were labeled with Alexa 488 (green), and cardiomyocytes were stained with Troponin T (red). Scale bar: 50 µm. ( D ) Effect of unmodified MSC exosomes on cardiomyocyte proliferation. Data are mean ± sd, by one-way ANOVA with Tukey post-test (*p

    Journal: Scientific Reports

    Article Title: The microRNA regulatory landscape of MSC-derived exosomes: a systems view

    doi: 10.1038/s41598-018-19581-x

    Figure Lengend Snippet: (A,B,C) Effects of unmodified MSC exosomes on cardiomyocyte apoptosis. (B,C) Nuclei were labeled with Hoechst (blue), TUNEL-positive nuclei were labeled with Alexa 488 (green), and cardiomyocytes were stained with Troponin T (red). Scale bar: 50 µm. ( D ) Effect of unmodified MSC exosomes on cardiomyocyte proliferation. Data are mean ± sd, by one-way ANOVA with Tukey post-test (*p

    Article Snippet: Cardiomyocyte proliferation and apoptosis Cardiomyocytes were isolated from murine hearts derived from 18 day old ICR mice using the Pierce cardiomyocyte isolation kit (Thermofisher) and cultured according to the manufacturer’s protocol .

    Techniques: Labeling, TUNEL Assay, Staining

    (A) Loading of miR-199a-3p and miR-130 into MSC exosomes. ( B,D,E ) Effects of miR-199a-3p loaded exosomes on cardiomyocyte apoptosis. (C,F,G) Effects of miR-199a-3p loaded exosomes on cardiomyocyte proliferation. (D,E) Nuclei were labeled with Hoechst (blue), TUNEL-positive nuclei were labeled with Alexa Fluor 488 (green), cardiomyocytes were stained with Troponin T (red). Scale bar = 50 µm. (F,G) Nuclei were labeled with Hoechst (blue), newly proliferated nuclei were labeled with EDU (green), and cardiomyocytes were stained with Troponin T (red). Scale bar = 50 µm. ( H,I ) miR-199a-3p promotes cardiomyocyte proliferation through Crim1 and Caspase 9 downregulation. (J,K,L,M) Expression of apoptosis markers in hydrogen peroxide treated cardiomyocytes following pretreatment with 2 µg/ml of MSC exosomes or 2 µg/ml of miR-199a-loaded MSC exosomes. n = 3 for A,B,C,H,I ; n = 4 for J,K,L,M . Data are presented as mean ± SD, with *p

    Journal: Scientific Reports

    Article Title: The microRNA regulatory landscape of MSC-derived exosomes: a systems view

    doi: 10.1038/s41598-018-19581-x

    Figure Lengend Snippet: (A) Loading of miR-199a-3p and miR-130 into MSC exosomes. ( B,D,E ) Effects of miR-199a-3p loaded exosomes on cardiomyocyte apoptosis. (C,F,G) Effects of miR-199a-3p loaded exosomes on cardiomyocyte proliferation. (D,E) Nuclei were labeled with Hoechst (blue), TUNEL-positive nuclei were labeled with Alexa Fluor 488 (green), cardiomyocytes were stained with Troponin T (red). Scale bar = 50 µm. (F,G) Nuclei were labeled with Hoechst (blue), newly proliferated nuclei were labeled with EDU (green), and cardiomyocytes were stained with Troponin T (red). Scale bar = 50 µm. ( H,I ) miR-199a-3p promotes cardiomyocyte proliferation through Crim1 and Caspase 9 downregulation. (J,K,L,M) Expression of apoptosis markers in hydrogen peroxide treated cardiomyocytes following pretreatment with 2 µg/ml of MSC exosomes or 2 µg/ml of miR-199a-loaded MSC exosomes. n = 3 for A,B,C,H,I ; n = 4 for J,K,L,M . Data are presented as mean ± SD, with *p

    Article Snippet: Cardiomyocyte proliferation and apoptosis Cardiomyocytes were isolated from murine hearts derived from 18 day old ICR mice using the Pierce cardiomyocyte isolation kit (Thermofisher) and cultured according to the manufacturer’s protocol .

    Techniques: Labeling, TUNEL Assay, Staining, Expressing

    No change in cardiac hypertrophy in miR22-KO mice in response to HF feeding ( A ) miR-22 expression in the heart of C57Bl/6 mice fed control diet and HF diet and db/db mice evaluated by qPCR ( n = 4–5). ( B ) Analysis of cardiac hypertrophy in miR22-WT and miR22-KO mice fed control diet and HF diet, evaluated by heart weight normalized by tibia length (HW/TL). ( C ) Wheat germ agglutinin (WGA) staining of heart transverse sections. Quanti3cation of relative cardiomyocyte area is displayed at the right panel; bars, 40 μm ( n =3). ( D ) Fibrosis area of heart transverse sections evaluated by Sirius Red/Fast Green collagen staining; bars, 1 mm ( n = 3–4). ( E ) Higher magni3cations of 3brosis area of heart transverse sections evaluated by Sirius Red/Fast Green collagen staining. ( F ) Quanti3cation of relative 3brosis area of heart transverse sections. ( G ) Measurement of hydroxyproline content in the heart ( n =3). * vs miR22WT-control ( P

    Journal: Clinical science (London, England : 1979)

    Article Title: Loss of microRNA-22 prevents high-fat diet induced dyslipidemia and increases energy expenditure without affecting cardiac hypertrophy

    doi: 10.1042/CS20171368

    Figure Lengend Snippet: No change in cardiac hypertrophy in miR22-KO mice in response to HF feeding ( A ) miR-22 expression in the heart of C57Bl/6 mice fed control diet and HF diet and db/db mice evaluated by qPCR ( n = 4–5). ( B ) Analysis of cardiac hypertrophy in miR22-WT and miR22-KO mice fed control diet and HF diet, evaluated by heart weight normalized by tibia length (HW/TL). ( C ) Wheat germ agglutinin (WGA) staining of heart transverse sections. Quanti3cation of relative cardiomyocyte area is displayed at the right panel; bars, 40 μm ( n =3). ( D ) Fibrosis area of heart transverse sections evaluated by Sirius Red/Fast Green collagen staining; bars, 1 mm ( n = 3–4). ( E ) Higher magni3cations of 3brosis area of heart transverse sections evaluated by Sirius Red/Fast Green collagen staining. ( F ) Quanti3cation of relative 3brosis area of heart transverse sections. ( G ) Measurement of hydroxyproline content in the heart ( n =3). * vs miR22WT-control ( P

    Article Snippet: Briefly, cardiomyocytes were isolated by enzymatic dissociation of one day old neonatal rat hearts using the Neonatal Cardiomyocyte Isolation kit (Cellutron, Life Technologies).

    Techniques: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Whole Genome Amplification, Staining

    Molecular events of ROS-dependent PKCδ activation involved in the AGE-BSA-induced cardiomyocyte apoptosis. PKCδ activation is involved in the regulation of AGE-BSA-induced cell apoptosis via ROS production and may play a key role in the development of cardiac mitochondrial dysfunction in rats with diabetes or obesity.

    Journal: Aging and Disease

    Article Title: Pkcδ Activation is Involved in ROS-Mediated Mitochondrial Dysfunction and Apoptosis in Cardiomyocytes Exposed to Advanced Glycation End Products (Ages)

    doi: 10.14336/AD.2017.0924

    Figure Lengend Snippet: Molecular events of ROS-dependent PKCδ activation involved in the AGE-BSA-induced cardiomyocyte apoptosis. PKCδ activation is involved in the regulation of AGE-BSA-induced cell apoptosis via ROS production and may play a key role in the development of cardiac mitochondrial dysfunction in rats with diabetes or obesity.

    Article Snippet: Neonatal rat ventricular myocytes primary culture NRVMs were prepared and cultured using a Neonatal Rat/Mouse Cardiomyocyte Isolation Kit (nc-6031; Cellutron Life Technology, Baltimore, MD, USA) which was previously described [ ].

    Techniques: Activation Assay

    AGE-BSA-induced cardiomyocyte apoptosis is mediated through ROS-dependent PKCδ activation NRVM and H9c2 cells were exposed to AGE-BSA (300 μg/ml) for 24 h. Cells were co-treated with bryostatin 1, a PKCδ activator (100 nM), rottlerin, a PKCδ inhibitor (3 μM), NAC (500 μM), Rote (0.1 μM), Apo (10 μM) or siPKCδ (1 μg). ( A ) Expression and phosphorylation of PKCδ and ( B, C, D ) apoptosis-related proteins were examined by western blot analyses. These are cropped blots, full-length blots of PKCδ and pPKCδ are presented in Suppl. Figure S5 . β-Actin was used as a loading control. N-acetylcysteine, NAC; Rote, rotenone; APO, apocynin; SC, scramble.

    Journal: Aging and Disease

    Article Title: Pkcδ Activation is Involved in ROS-Mediated Mitochondrial Dysfunction and Apoptosis in Cardiomyocytes Exposed to Advanced Glycation End Products (Ages)

    doi: 10.14336/AD.2017.0924

    Figure Lengend Snippet: AGE-BSA-induced cardiomyocyte apoptosis is mediated through ROS-dependent PKCδ activation NRVM and H9c2 cells were exposed to AGE-BSA (300 μg/ml) for 24 h. Cells were co-treated with bryostatin 1, a PKCδ activator (100 nM), rottlerin, a PKCδ inhibitor (3 μM), NAC (500 μM), Rote (0.1 μM), Apo (10 μM) or siPKCδ (1 μg). ( A ) Expression and phosphorylation of PKCδ and ( B, C, D ) apoptosis-related proteins were examined by western blot analyses. These are cropped blots, full-length blots of PKCδ and pPKCδ are presented in Suppl. Figure S5 . β-Actin was used as a loading control. N-acetylcysteine, NAC; Rote, rotenone; APO, apocynin; SC, scramble.

    Article Snippet: Neonatal rat ventricular myocytes primary culture NRVMs were prepared and cultured using a Neonatal Rat/Mouse Cardiomyocyte Isolation Kit (nc-6031; Cellutron Life Technology, Baltimore, MD, USA) which was previously described [ ].

    Techniques: Activation Assay, Expressing, Western Blot

    AGE-BSA-induced cardiomyocyte apoptosis is mediated through PKCδ activation. ( A ) The diagram depicts the domain organization of GFP-PKCδ. The GFP-PKCδ derivatives, including the wild-type (WT) and the kinase-deficient mutant (KD; K376R). Cells were treated with AGE-BSA (300 μg/ml) and ( B ) rottlerin (1-5 μM) or ( C ) PKCδ silencing. ( D ) Cells were transfected with GFP-fused PKCδ (GFP PKCδ-WT) at different doses as indicated and with ( E ) 1 μg rottlerin (1-5 μM). ( F G ) H9c2 cells or ( H ) neonatal rat ventricular myocytes (NRVM) were exposed to AGEs (300 μg/ml) with or without (GFP PKCδ-KD) transfection or transfected with GFP PKCδ-WT in the presence of rottlerin (3 μM) or not. These are cropped blots, full-length blots of PKCδ and pPKCδ are presented in Suppl. Figure S4 . SC, scramble; WT, wild type; KD, kinase-deficient; All the proteins were analyzed by western blotting using β-actin as a loading control.

    Journal: Aging and Disease

    Article Title: Pkcδ Activation is Involved in ROS-Mediated Mitochondrial Dysfunction and Apoptosis in Cardiomyocytes Exposed to Advanced Glycation End Products (Ages)

    doi: 10.14336/AD.2017.0924

    Figure Lengend Snippet: AGE-BSA-induced cardiomyocyte apoptosis is mediated through PKCδ activation. ( A ) The diagram depicts the domain organization of GFP-PKCδ. The GFP-PKCδ derivatives, including the wild-type (WT) and the kinase-deficient mutant (KD; K376R). Cells were treated with AGE-BSA (300 μg/ml) and ( B ) rottlerin (1-5 μM) or ( C ) PKCδ silencing. ( D ) Cells were transfected with GFP-fused PKCδ (GFP PKCδ-WT) at different doses as indicated and with ( E ) 1 μg rottlerin (1-5 μM). ( F G ) H9c2 cells or ( H ) neonatal rat ventricular myocytes (NRVM) were exposed to AGEs (300 μg/ml) with or without (GFP PKCδ-KD) transfection or transfected with GFP PKCδ-WT in the presence of rottlerin (3 μM) or not. These are cropped blots, full-length blots of PKCδ and pPKCδ are presented in Suppl. Figure S4 . SC, scramble; WT, wild type; KD, kinase-deficient; All the proteins were analyzed by western blotting using β-actin as a loading control.

    Article Snippet: Neonatal rat ventricular myocytes primary culture NRVMs were prepared and cultured using a Neonatal Rat/Mouse Cardiomyocyte Isolation Kit (nc-6031; Cellutron Life Technology, Baltimore, MD, USA) which was previously described [ ].

    Techniques: Activation Assay, Mutagenesis, Transfection, Western Blot

    ( a , b ) (left) The long-term culture of hIPSC-derived cardiac myocytes yields cell assemblies similar to the culture of mouse primary cardiac myocytes, giving rise to the assumption that such stem cell populations can be used to substitute for primary animal-derived specimens; ( c , d ) Different from the well-ordered  α -actinin apparatus, the connexin-43 distribution does not recapitulate the cell interface accumulation as seen in neonatal cardiac myocyte culture; ( e ) Functional (left) and defect (right) cell ensembles imaged by SEM. The obvious finding that the well-spread cell ensemble is a functional one while the defect one releases surface contacts and reaches a bulk appearance does not absolutely correlate with a loss of contractility. A loss of surface-mediated organization can be regarded inside the bulk assembly. Consequently, spatially-random contracting bulks can be observed; ( f , b ) (right) Aging effects of the contractile apparatus after long-term cultivation. The regular  α -actinin organization with a ladder appearance with pronounced orientation along cells’ longitudes is substituted by a random orientation; ( g ) Ensembles of cardiac myocytes beat synchronously, indicating electrical excitation coupling. While the direction of contraction is random on a plain surface (right), the line pattern-induced cell ensemble organization results in temporally synchronized and spatially-organized contractions. This fulfills a fundamental prerequisite of cardiac tissue function.

    Journal: Journal of Functional Biomaterials

    Article Title: Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

    doi: 10.3390/jfb7010001

    Figure Lengend Snippet: ( a , b ) (left) The long-term culture of hIPSC-derived cardiac myocytes yields cell assemblies similar to the culture of mouse primary cardiac myocytes, giving rise to the assumption that such stem cell populations can be used to substitute for primary animal-derived specimens; ( c , d ) Different from the well-ordered α -actinin apparatus, the connexin-43 distribution does not recapitulate the cell interface accumulation as seen in neonatal cardiac myocyte culture; ( e ) Functional (left) and defect (right) cell ensembles imaged by SEM. The obvious finding that the well-spread cell ensemble is a functional one while the defect one releases surface contacts and reaches a bulk appearance does not absolutely correlate with a loss of contractility. A loss of surface-mediated organization can be regarded inside the bulk assembly. Consequently, spatially-random contracting bulks can be observed; ( f , b ) (right) Aging effects of the contractile apparatus after long-term cultivation. The regular α -actinin organization with a ladder appearance with pronounced orientation along cells’ longitudes is substituted by a random orientation; ( g ) Ensembles of cardiac myocytes beat synchronously, indicating electrical excitation coupling. While the direction of contraction is random on a plain surface (right), the line pattern-induced cell ensemble organization results in temporally synchronized and spatially-organized contractions. This fulfills a fundamental prerequisite of cardiac tissue function.

    Article Snippet: Cell Culture For isolation of primary neonatal mouse cardiac myocytes, the Neonatal Heart Dissociation Kit and the Neonatal Cardiomyocyte Isolation Kit for mice (No. 130-098-373, No. 130-100-825, Miltenyi Biotec, Bergisch Gladbach, Germany) are used according to the manufacturer recommendations.

    Techniques: Derivative Assay, Functional Assay

    Fluorescence ( a ) and bright-field ( b ) light microscopy recordings of a typical murine cardiomyocyte specimen situation. A multicellular ensemble in near-confluent cell density is orientated along the polymer line pattern series; ( a , c ) (red and green inserts) Dual color overlay of the intracellular α -actinin (red) distribution and the intercellular connexin-43 distribution (green). The red signal closely follows the cell shapes, indicating that all cells are densely filled with α -actinin. Image (c) represents a higher magnification of the green rectangle with the α -actinin recording; ( d , e ) (blue insert) Distribution of the gap junction protein connexin-43. The pearl chain-like accumulations are located at the cell interfaces inside the cell contacts. A more homogeneous distribution of small signals all over the cytosol indicates non-recruited protein; ( f , g ) SEM recording of murine cardiac myocytes cultivated on line-patterned substrate. Vital cells are orientated along the fillets, while a randomly-distributed residual of rounded cells indicates the loss of cells during preparation from heart tissue; ( f ) Near confluent inoculation does not impede the organizing influence of line patterns, but it reduces the ratio of long cell protrusions. Such protrusions are more apparent at low cell densities (g). The golden overlay in (g) hints at the chamfer component of the surface pattern. An abstract sketch of the alternating chamfer to line pattern is given in Figure 1 .

    Journal: Journal of Functional Biomaterials

    Article Title: Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

    doi: 10.3390/jfb7010001

    Figure Lengend Snippet: Fluorescence ( a ) and bright-field ( b ) light microscopy recordings of a typical murine cardiomyocyte specimen situation. A multicellular ensemble in near-confluent cell density is orientated along the polymer line pattern series; ( a , c ) (red and green inserts) Dual color overlay of the intracellular α -actinin (red) distribution and the intercellular connexin-43 distribution (green). The red signal closely follows the cell shapes, indicating that all cells are densely filled with α -actinin. Image (c) represents a higher magnification of the green rectangle with the α -actinin recording; ( d , e ) (blue insert) Distribution of the gap junction protein connexin-43. The pearl chain-like accumulations are located at the cell interfaces inside the cell contacts. A more homogeneous distribution of small signals all over the cytosol indicates non-recruited protein; ( f , g ) SEM recording of murine cardiac myocytes cultivated on line-patterned substrate. Vital cells are orientated along the fillets, while a randomly-distributed residual of rounded cells indicates the loss of cells during preparation from heart tissue; ( f ) Near confluent inoculation does not impede the organizing influence of line patterns, but it reduces the ratio of long cell protrusions. Such protrusions are more apparent at low cell densities (g). The golden overlay in (g) hints at the chamfer component of the surface pattern. An abstract sketch of the alternating chamfer to line pattern is given in Figure 1 .

    Article Snippet: Cell Culture For isolation of primary neonatal mouse cardiac myocytes, the Neonatal Heart Dissociation Kit and the Neonatal Cardiomyocyte Isolation Kit for mice (No. 130-098-373, No. 130-100-825, Miltenyi Biotec, Bergisch Gladbach, Germany) are used according to the manufacturer recommendations.

    Techniques: Fluorescence, Light Microscopy

    ( a ) Survey of a group of neonatal murine cardiac myocytes on a smooth glass substrate. The cells are stained for nuclei (blue), gap junctions via connexin-43 immunostaining (green) and the contractile apparatus via  α -actinin immuno-staining (red); ( b ) (insert in (a))Exclusive presentation of the  α -actinin signal component; ( c ) Exclusive presentation of the connexin-43 signal component. Besides the accumulation along pearl chain-like structures located along the intercellular borders, a faint non-specific signal is also visible inside the nuclei due to a cross-over in the acquisition channel. ( d )–( f ) are SEM recordings of neonatal cardiac myocytes cultivated on plain glass surface. The cells are flat and polygonal with an irregular polygonal shape.

    Journal: Journal of Functional Biomaterials

    Article Title: Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

    doi: 10.3390/jfb7010001

    Figure Lengend Snippet: ( a ) Survey of a group of neonatal murine cardiac myocytes on a smooth glass substrate. The cells are stained for nuclei (blue), gap junctions via connexin-43 immunostaining (green) and the contractile apparatus via α -actinin immuno-staining (red); ( b ) (insert in (a))Exclusive presentation of the α -actinin signal component; ( c ) Exclusive presentation of the connexin-43 signal component. Besides the accumulation along pearl chain-like structures located along the intercellular borders, a faint non-specific signal is also visible inside the nuclei due to a cross-over in the acquisition channel. ( d )–( f ) are SEM recordings of neonatal cardiac myocytes cultivated on plain glass surface. The cells are flat and polygonal with an irregular polygonal shape.

    Article Snippet: Cell Culture For isolation of primary neonatal mouse cardiac myocytes, the Neonatal Heart Dissociation Kit and the Neonatal Cardiomyocyte Isolation Kit for mice (No. 130-098-373, No. 130-100-825, Miltenyi Biotec, Bergisch Gladbach, Germany) are used according to the manufacturer recommendations.

    Techniques: Staining, Immunostaining

    AL-LC causes mitochondrial dysfunction and autophagy dysregulation in vitro Using the fluorescent indicator DCFDA, ROS was measured in isolated cardiomyocytes treated with vehicle, Con-LC, AL-LC or AL-LC + Mito-TEMPO. Representative fluorescent and bright field images are shown in the top panels, and quantitative analysis is summarized in the graph below. AL-LC but not Con-LC increased ROS and this was blocked by Mito-TEMPO, indicating that ROS is derived from mitochondria. Scale bar = 100 μm. N = 3. * P = 0.020. Mitochondrial membrane potential was measured using TMRE fluorescent dye in cardiomyocytes following 24-h treatment with vehicle, Con-LC or AL-LC. Representative TMRE fluorescent and bright field microscopy images are shown in the top panels, and TMRE fluorescence signal was quantified and summarized below. AL-LC exposure resulted in loss of mitochondrial membrane potential compared to the other groups. Scale bar = 50 μm. N = 3. * P = 0.031. Quantitative summary of cellular ATP levels in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. N = 3. * P = 0.020. Immunoblot analysis of LC3-II expression in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. GAPDH was used as a loading control. Quantitative results are shown in the graph below for comparison. LC3-II expression was significantly increased in the AL-LC group compared to vehicle and Con-LC. N = 3. * P = 1.9 × 10 −6 . Immunoblot analysis of AL-LC-induced LC3-II expression in the presence of lysosomal inhibitors E64d and Pepstatin A. GAPDH is used as a loading control. Quantitative results in the graph below show that LC3-II levels in the AL-LC group do not exceed vehicle-treated LC3-II levels following lysosomal inhibition, indicating minimal perturbation to initiation. N = 6. * P = 1.4 × 10 −6 , # P = 0.041. Immunoblot and quantitative analysis of p62 expression in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. Results show a decrease in autophagic flux seen by increased p62 accumulation in the AL-LC group. N = 3. * P = 0.033. Confocal imaging of immunofluorescence staining of p62 (red) in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. Mitochondrial co-localization was visualized using mitochondrial protein COX4 (green) co-staining, and nuclear staining with DAPI (blue). Scale bar = 10 μm. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Lysosomal dysfunction and impaired autophagy underlie the pathogenesis of amyloidogenic light chain-mediated cardiotoxicity

    doi: 10.15252/emmm.201404190

    Figure Lengend Snippet: AL-LC causes mitochondrial dysfunction and autophagy dysregulation in vitro Using the fluorescent indicator DCFDA, ROS was measured in isolated cardiomyocytes treated with vehicle, Con-LC, AL-LC or AL-LC + Mito-TEMPO. Representative fluorescent and bright field images are shown in the top panels, and quantitative analysis is summarized in the graph below. AL-LC but not Con-LC increased ROS and this was blocked by Mito-TEMPO, indicating that ROS is derived from mitochondria. Scale bar = 100 μm. N = 3. * P = 0.020. Mitochondrial membrane potential was measured using TMRE fluorescent dye in cardiomyocytes following 24-h treatment with vehicle, Con-LC or AL-LC. Representative TMRE fluorescent and bright field microscopy images are shown in the top panels, and TMRE fluorescence signal was quantified and summarized below. AL-LC exposure resulted in loss of mitochondrial membrane potential compared to the other groups. Scale bar = 50 μm. N = 3. * P = 0.031. Quantitative summary of cellular ATP levels in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. N = 3. * P = 0.020. Immunoblot analysis of LC3-II expression in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. GAPDH was used as a loading control. Quantitative results are shown in the graph below for comparison. LC3-II expression was significantly increased in the AL-LC group compared to vehicle and Con-LC. N = 3. * P = 1.9 × 10 −6 . Immunoblot analysis of AL-LC-induced LC3-II expression in the presence of lysosomal inhibitors E64d and Pepstatin A. GAPDH is used as a loading control. Quantitative results in the graph below show that LC3-II levels in the AL-LC group do not exceed vehicle-treated LC3-II levels following lysosomal inhibition, indicating minimal perturbation to initiation. N = 6. * P = 1.4 × 10 −6 , # P = 0.041. Immunoblot and quantitative analysis of p62 expression in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. Results show a decrease in autophagic flux seen by increased p62 accumulation in the AL-LC group. N = 3. * P = 0.033. Confocal imaging of immunofluorescence staining of p62 (red) in cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC. Mitochondrial co-localization was visualized using mitochondrial protein COX4 (green) co-staining, and nuclear staining with DAPI (blue). Scale bar = 10 μm. Source data are available online for this figure.

    Article Snippet: Adult rats for cardiomyocyte isolation were purchased from Charles River Laboratory (male Wistar rats, 180–220 g, catalog #003).

    Techniques: In Vitro, Isolation, Derivative Assay, Microscopy, Fluorescence, Expressing, Inhibition, Imaging, Immunofluorescence, Staining

    Temporal analysis of AL-LC-induced toxicity and dysregulation of autophagy Lysosomal labeling in cardiomyocytes following exposure to vehicle, Con-LC or AL-LC using LysoTracker red at 3, 6 and 12 h post-AL-LC exposure. Quantitation of fluorescent signal is shown in the graph. N = 3. * P = 0.011, # P = 0.032, ** P = 0.029. Dysregulation of autophagy following AL-LC exposure was monitored using a GFP-LC3 cleavage assay. Degradation of the GFP-LC3 fusion protein was monitored via immunoblotting against GFP antibody at 2, 6, and 12 h post-AL-LC exposure. A significant delay in the degradation of GFP-LC fusion protein was seen by accumulation of GFP-LC fusion protein in cardiomyocytes starting 6 h following exposure to AL-LC. N = 5. * P = 0.003, # P = 0.038. Mitochondrial membrane potential was measured using TMRE fluorescent dye in cardiomyocytes 6, 12 and 24 h following treatment with vehicle or AL-LC. TMRE fluorescence signal was quantified and summarized in the graph. AL-LC exposure resulted in loss of mitochondrial membrane potential compared to the other groups at 12 and 24 h post-AL-LC exposure. N = 3. * P = 0.020, # P = 0.029. Using fluorescent indicator DCFDA, ROS was measured in isolated cardiomyocytes treated with vehicle, Con-LC and AL-LC for 3, 12 and 24 h. Quantitative analysis is summarized in the graph. AL-LC but not Con-LC increased ROS only at 24 h following AL-LC exposure. N = 3. * P = 0.004. Schematic illustration of temporal events involved in AL-LC-induced pathology. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Lysosomal dysfunction and impaired autophagy underlie the pathogenesis of amyloidogenic light chain-mediated cardiotoxicity

    doi: 10.15252/emmm.201404190

    Figure Lengend Snippet: Temporal analysis of AL-LC-induced toxicity and dysregulation of autophagy Lysosomal labeling in cardiomyocytes following exposure to vehicle, Con-LC or AL-LC using LysoTracker red at 3, 6 and 12 h post-AL-LC exposure. Quantitation of fluorescent signal is shown in the graph. N = 3. * P = 0.011, # P = 0.032, ** P = 0.029. Dysregulation of autophagy following AL-LC exposure was monitored using a GFP-LC3 cleavage assay. Degradation of the GFP-LC3 fusion protein was monitored via immunoblotting against GFP antibody at 2, 6, and 12 h post-AL-LC exposure. A significant delay in the degradation of GFP-LC fusion protein was seen by accumulation of GFP-LC fusion protein in cardiomyocytes starting 6 h following exposure to AL-LC. N = 5. * P = 0.003, # P = 0.038. Mitochondrial membrane potential was measured using TMRE fluorescent dye in cardiomyocytes 6, 12 and 24 h following treatment with vehicle or AL-LC. TMRE fluorescence signal was quantified and summarized in the graph. AL-LC exposure resulted in loss of mitochondrial membrane potential compared to the other groups at 12 and 24 h post-AL-LC exposure. N = 3. * P = 0.020, # P = 0.029. Using fluorescent indicator DCFDA, ROS was measured in isolated cardiomyocytes treated with vehicle, Con-LC and AL-LC for 3, 12 and 24 h. Quantitative analysis is summarized in the graph. AL-LC but not Con-LC increased ROS only at 24 h following AL-LC exposure. N = 3. * P = 0.004. Schematic illustration of temporal events involved in AL-LC-induced pathology. Source data are available online for this figure.

    Article Snippet: Adult rats for cardiomyocyte isolation were purchased from Charles River Laboratory (male Wistar rats, 180–220 g, catalog #003).

    Techniques: Labeling, Quantitation Assay, Cleavage Assay, Fluorescence, Isolation

    Lysosomal dysfunction contributes to AL-LC-induced dysregulation of autophagy and consequent cellular dysfunction and death in vitro and in vivo Lysosomal labeling using LysoTracker red in cardiomyocytes following exposure to vehicle, Con-LC or AL-LC is shown (top panels), with corresponding bright field images shown below. Quantitation of fluorescent signal is shown in the graph on the right. Scale bar = 50 μm. N = 6. * P = 0.009. Alterations in lysosomal pH were measured using LysoSensor in cardiomyocytes treated with vehicle, Con-LC or AL-LC. pH changes were measured by calculating the ratio between green (basic) and red (acidic) LysoSensor signal. Quantitation is shown on the right indicating loss of lysosomal acidity in AL-LC treated cardiomyocytes. Scale bar = 10 μm. N = 8. * P = 0.038. Quantitative PCR analysis of cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC reveals changes in mRNA encoding the lysosomal gene products cathepsin D and vacuolar ATPase subunits 1 and 2. All three targets were significantly downregulated in the AL-LC group. N = 3. * P = 0.015, # P = 0.002, ** P = 1.9 × 10 −4 . Quantitative PCR analysis of cardiomyocytes following 24-h exposure to vehicle, Con-LC and AL-LC reveals decreased mRNA level of the lysosomal transcriptional factor TFEB. N = 4. * P = 0.003. Protein expression of TFEB in cardiomyocytes was measured using immunoblot analysis following 24-h exposure to vehicle, Con-LC or AL-LC. AL-LC-induced downregulation of TFEB protein expression was prevented by rapamycin treatment. N = 3. * P = 0.043, # P = 0.032. TFEB was overexpressed in cardiomyocytes using adenovirus, and adenoviral GFP overexpression was used as a control. Contractile function was measured following exposure to vehicle or AL-LC. In cells overexpressing TFEB, AL-LC-induced decreased cell shortening was rescued compared to GFP-expressing myocytes. N = 5. * P = 0.002. TFEB was overexpressed in cardiomyocytes using adenovirus. Adeno-GFP was used as a control. Calcium transient amplitude was measured following exposure to either vehicle or AL-LC. In cells overexpressing TFEB, AL-LC-induced decrease in calcium amplitude was rescued compared to control groups. N = 5. * P = 0.002. Hearts were isolated 2 days post-injection of vehicle, Con-LC or AL-LC from zebrafish overexpressing TFEB, or control mRNA. Hearts were stained for TUNEL-labeled nuclei with a DAPI counterstain. Cell death was calculated as a percent of TUNEL-positive nuclei to total cell number. Scale bar = 20 μm. N = 3–5 per group. * P = 0.003, # P = 0.002. Kaplan–Meier analysis of survival following injection of Con-LC or AL-LC in zebrafish overexpressing control or TFEB mRNA. Survival was monitored daily. During the time course of transient overexpression of TFEB, AL-LC-induced mortality was rescued significantly. N = 25 per group. * P = 0.0003.

    Journal: EMBO Molecular Medicine

    Article Title: Lysosomal dysfunction and impaired autophagy underlie the pathogenesis of amyloidogenic light chain-mediated cardiotoxicity

    doi: 10.15252/emmm.201404190

    Figure Lengend Snippet: Lysosomal dysfunction contributes to AL-LC-induced dysregulation of autophagy and consequent cellular dysfunction and death in vitro and in vivo Lysosomal labeling using LysoTracker red in cardiomyocytes following exposure to vehicle, Con-LC or AL-LC is shown (top panels), with corresponding bright field images shown below. Quantitation of fluorescent signal is shown in the graph on the right. Scale bar = 50 μm. N = 6. * P = 0.009. Alterations in lysosomal pH were measured using LysoSensor in cardiomyocytes treated with vehicle, Con-LC or AL-LC. pH changes were measured by calculating the ratio between green (basic) and red (acidic) LysoSensor signal. Quantitation is shown on the right indicating loss of lysosomal acidity in AL-LC treated cardiomyocytes. Scale bar = 10 μm. N = 8. * P = 0.038. Quantitative PCR analysis of cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC reveals changes in mRNA encoding the lysosomal gene products cathepsin D and vacuolar ATPase subunits 1 and 2. All three targets were significantly downregulated in the AL-LC group. N = 3. * P = 0.015, # P = 0.002, ** P = 1.9 × 10 −4 . Quantitative PCR analysis of cardiomyocytes following 24-h exposure to vehicle, Con-LC and AL-LC reveals decreased mRNA level of the lysosomal transcriptional factor TFEB. N = 4. * P = 0.003. Protein expression of TFEB in cardiomyocytes was measured using immunoblot analysis following 24-h exposure to vehicle, Con-LC or AL-LC. AL-LC-induced downregulation of TFEB protein expression was prevented by rapamycin treatment. N = 3. * P = 0.043, # P = 0.032. TFEB was overexpressed in cardiomyocytes using adenovirus, and adenoviral GFP overexpression was used as a control. Contractile function was measured following exposure to vehicle or AL-LC. In cells overexpressing TFEB, AL-LC-induced decreased cell shortening was rescued compared to GFP-expressing myocytes. N = 5. * P = 0.002. TFEB was overexpressed in cardiomyocytes using adenovirus. Adeno-GFP was used as a control. Calcium transient amplitude was measured following exposure to either vehicle or AL-LC. In cells overexpressing TFEB, AL-LC-induced decrease in calcium amplitude was rescued compared to control groups. N = 5. * P = 0.002. Hearts were isolated 2 days post-injection of vehicle, Con-LC or AL-LC from zebrafish overexpressing TFEB, or control mRNA. Hearts were stained for TUNEL-labeled nuclei with a DAPI counterstain. Cell death was calculated as a percent of TUNEL-positive nuclei to total cell number. Scale bar = 20 μm. N = 3–5 per group. * P = 0.003, # P = 0.002. Kaplan–Meier analysis of survival following injection of Con-LC or AL-LC in zebrafish overexpressing control or TFEB mRNA. Survival was monitored daily. During the time course of transient overexpression of TFEB, AL-LC-induced mortality was rescued significantly. N = 25 per group. * P = 0.0003.

    Article Snippet: Adult rats for cardiomyocyte isolation were purchased from Charles River Laboratory (male Wistar rats, 180–220 g, catalog #003).

    Techniques: In Vitro, In Vivo, Labeling, Quantitation Assay, Real-time Polymerase Chain Reaction, Expressing, Over Expression, Isolation, Injection, Staining, TUNEL Assay

    Restoration of autophagic flux with rapamycin attenuates AL-LC-induced cellular dysfunction and cell death in vitro Immunoblot analysis of p62 on cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC in the absence or presence of 10 nM rapamycin. Quantitative results summarized below show decreased p62 expression following rapamycin treatment. N = 3. * P = 0.003, # P = 0.006. Mitochondrial function is rescued by rapamycin treatment, shown by quantitative analysis of mitochondrial membrane potential using TMRE dye in cardiomyocytes following exposure to vehicle, Con-LC or AL-LC for 24 h in the presence or absence of rapamycin. N = 3. * P = 0.046, # P = 0.005 between indicated groups. ROS levels are reduced by treatment with rapamycin, shown using DCFDA in cardiomyocytes exposed to vehicle, Con-LC or AL-LC. N = 3. * P = 2.3 × 10 −4 , # P = 0.002 between indicated groups. Contractile function was measured in cardiomyocytes exposed to AL-LC for 24 h in the absence or presence of rapamycin with or without chloroquine (2.5 μM). Quantitative analysis was performed by calculating percent cell shortening. N = 3. * P = 3.3 × 10 −4 , # P = 0.007. Calcium transient amplitude, measured in isolated cardiomyocytes, was quantified following exposure to vehicle or AL-LC in the presence or absence of rapamycin with or without chloroquine. Representative tracings are shown, and quantitative analysis in the graph shows a rescue of AL-LC-induced decrease in calcium transient amplitude following rapamycin treatment. N = 3, * P = 0.002, # P = 0.006. Rapamycin treatment reduces apoptosis in cardiomyocytes exposed to AL-LC. TUNEL staining was performed to quantify cell death in cardiomyocytes following exposure to vehicle, Con-LC or AL-LC with or without rapamycin treatment. Cell death was measured as percent TUNEL-positive nuclei relative to total cell number. N = 3. * P = 0.015, # P = 0.035 between indicated groups. Verification that rapamycin rescue was autophagy dependent was demonstrated using chloroquine (2.5 μM) administered to AL-LC + rapamycin-treated cardiomyocytes. p62 accumulation was measured using immunoblot analysis, with GAPDH as a loading control. N = 5. * P = 0.023. Cardiomyocytes were treated with chloroquine in the presence of rapamycin, and immunoblot analysis was performed to probe for active caspase 3 levels, normalized to GAPDH expression. N = 3. * P = 0.007. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Lysosomal dysfunction and impaired autophagy underlie the pathogenesis of amyloidogenic light chain-mediated cardiotoxicity

    doi: 10.15252/emmm.201404190

    Figure Lengend Snippet: Restoration of autophagic flux with rapamycin attenuates AL-LC-induced cellular dysfunction and cell death in vitro Immunoblot analysis of p62 on cardiomyocytes following 24-h exposure to vehicle, Con-LC or AL-LC in the absence or presence of 10 nM rapamycin. Quantitative results summarized below show decreased p62 expression following rapamycin treatment. N = 3. * P = 0.003, # P = 0.006. Mitochondrial function is rescued by rapamycin treatment, shown by quantitative analysis of mitochondrial membrane potential using TMRE dye in cardiomyocytes following exposure to vehicle, Con-LC or AL-LC for 24 h in the presence or absence of rapamycin. N = 3. * P = 0.046, # P = 0.005 between indicated groups. ROS levels are reduced by treatment with rapamycin, shown using DCFDA in cardiomyocytes exposed to vehicle, Con-LC or AL-LC. N = 3. * P = 2.3 × 10 −4 , # P = 0.002 between indicated groups. Contractile function was measured in cardiomyocytes exposed to AL-LC for 24 h in the absence or presence of rapamycin with or without chloroquine (2.5 μM). Quantitative analysis was performed by calculating percent cell shortening. N = 3. * P = 3.3 × 10 −4 , # P = 0.007. Calcium transient amplitude, measured in isolated cardiomyocytes, was quantified following exposure to vehicle or AL-LC in the presence or absence of rapamycin with or without chloroquine. Representative tracings are shown, and quantitative analysis in the graph shows a rescue of AL-LC-induced decrease in calcium transient amplitude following rapamycin treatment. N = 3, * P = 0.002, # P = 0.006. Rapamycin treatment reduces apoptosis in cardiomyocytes exposed to AL-LC. TUNEL staining was performed to quantify cell death in cardiomyocytes following exposure to vehicle, Con-LC or AL-LC with or without rapamycin treatment. Cell death was measured as percent TUNEL-positive nuclei relative to total cell number. N = 3. * P = 0.015, # P = 0.035 between indicated groups. Verification that rapamycin rescue was autophagy dependent was demonstrated using chloroquine (2.5 μM) administered to AL-LC + rapamycin-treated cardiomyocytes. p62 accumulation was measured using immunoblot analysis, with GAPDH as a loading control. N = 5. * P = 0.023. Cardiomyocytes were treated with chloroquine in the presence of rapamycin, and immunoblot analysis was performed to probe for active caspase 3 levels, normalized to GAPDH expression. N = 3. * P = 0.007. Source data are available online for this figure.

    Article Snippet: Adult rats for cardiomyocyte isolation were purchased from Charles River Laboratory (male Wistar rats, 180–220 g, catalog #003).

    Techniques: In Vitro, Expressing, Isolation, TUNEL Assay, Staining

    ( a , b ) (left) The long-term culture of hIPSC-derived cardiac myocytes yields cell assemblies similar to the culture of mouse primary cardiac myocytes, giving rise to the assumption that such stem cell populations can be used to substitute for primary animal-derived specimens; ( c , d ) Different from the well-ordered  α -actinin apparatus, the connexin-43 distribution does not recapitulate the cell interface accumulation as seen in neonatal cardiac myocyte culture; ( e ) Functional (left) and defect (right) cell ensembles imaged by SEM. The obvious finding that the well-spread cell ensemble is a functional one while the defect one releases surface contacts and reaches a bulk appearance does not absolutely correlate with a loss of contractility. A loss of surface-mediated organization can be regarded inside the bulk assembly. Consequently, spatially-random contracting bulks can be observed; ( f , b ) (right) Aging effects of the contractile apparatus after long-term cultivation. The regular  α -actinin organization with a ladder appearance with pronounced orientation along cells’ longitudes is substituted by a random orientation; ( g ) Ensembles of cardiac myocytes beat synchronously, indicating electrical excitation coupling. While the direction of contraction is random on a plain surface (right), the line pattern-induced cell ensemble organization results in temporally synchronized and spatially-organized contractions. This fulfills a fundamental prerequisite of cardiac tissue function.

    Journal: Journal of Functional Biomaterials

    Article Title: Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

    doi: 10.3390/jfb7010001

    Figure Lengend Snippet: ( a , b ) (left) The long-term culture of hIPSC-derived cardiac myocytes yields cell assemblies similar to the culture of mouse primary cardiac myocytes, giving rise to the assumption that such stem cell populations can be used to substitute for primary animal-derived specimens; ( c , d ) Different from the well-ordered α -actinin apparatus, the connexin-43 distribution does not recapitulate the cell interface accumulation as seen in neonatal cardiac myocyte culture; ( e ) Functional (left) and defect (right) cell ensembles imaged by SEM. The obvious finding that the well-spread cell ensemble is a functional one while the defect one releases surface contacts and reaches a bulk appearance does not absolutely correlate with a loss of contractility. A loss of surface-mediated organization can be regarded inside the bulk assembly. Consequently, spatially-random contracting bulks can be observed; ( f , b ) (right) Aging effects of the contractile apparatus after long-term cultivation. The regular α -actinin organization with a ladder appearance with pronounced orientation along cells’ longitudes is substituted by a random orientation; ( g ) Ensembles of cardiac myocytes beat synchronously, indicating electrical excitation coupling. While the direction of contraction is random on a plain surface (right), the line pattern-induced cell ensemble organization results in temporally synchronized and spatially-organized contractions. This fulfills a fundamental prerequisite of cardiac tissue function.

    Article Snippet: Cell Culture For isolation of primary neonatal mouse cardiac myocytes, the Neonatal Heart Dissociation Kit and the Neonatal Cardiomyocyte Isolation Kit for mice (No. 130-098-373, No. 130-100-825, Miltenyi Biotec, Bergisch Gladbach, Germany) are used according to the manufacturer recommendations.

    Techniques: Derivative Assay, Functional Assay

    Fluorescence ( a ) and bright-field ( b ) light microscopy recordings of a typical murine cardiomyocyte specimen situation. A multicellular ensemble in near-confluent cell density is orientated along the polymer line pattern series; ( a , c ) (red and green inserts) Dual color overlay of the intracellular α -actinin (red) distribution and the intercellular connexin-43 distribution (green). The red signal closely follows the cell shapes, indicating that all cells are densely filled with α -actinin. Image (c) represents a higher magnification of the green rectangle with the α -actinin recording; ( d , e ) (blue insert) Distribution of the gap junction protein connexin-43. The pearl chain-like accumulations are located at the cell interfaces inside the cell contacts. A more homogeneous distribution of small signals all over the cytosol indicates non-recruited protein; ( f , g ) SEM recording of murine cardiac myocytes cultivated on line-patterned substrate. Vital cells are orientated along the fillets, while a randomly-distributed residual of rounded cells indicates the loss of cells during preparation from heart tissue; ( f ) Near confluent inoculation does not impede the organizing influence of line patterns, but it reduces the ratio of long cell protrusions. Such protrusions are more apparent at low cell densities (g). The golden overlay in (g) hints at the chamfer component of the surface pattern. An abstract sketch of the alternating chamfer to line pattern is given in Figure 1 .

    Journal: Journal of Functional Biomaterials

    Article Title: Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

    doi: 10.3390/jfb7010001

    Figure Lengend Snippet: Fluorescence ( a ) and bright-field ( b ) light microscopy recordings of a typical murine cardiomyocyte specimen situation. A multicellular ensemble in near-confluent cell density is orientated along the polymer line pattern series; ( a , c ) (red and green inserts) Dual color overlay of the intracellular α -actinin (red) distribution and the intercellular connexin-43 distribution (green). The red signal closely follows the cell shapes, indicating that all cells are densely filled with α -actinin. Image (c) represents a higher magnification of the green rectangle with the α -actinin recording; ( d , e ) (blue insert) Distribution of the gap junction protein connexin-43. The pearl chain-like accumulations are located at the cell interfaces inside the cell contacts. A more homogeneous distribution of small signals all over the cytosol indicates non-recruited protein; ( f , g ) SEM recording of murine cardiac myocytes cultivated on line-patterned substrate. Vital cells are orientated along the fillets, while a randomly-distributed residual of rounded cells indicates the loss of cells during preparation from heart tissue; ( f ) Near confluent inoculation does not impede the organizing influence of line patterns, but it reduces the ratio of long cell protrusions. Such protrusions are more apparent at low cell densities (g). The golden overlay in (g) hints at the chamfer component of the surface pattern. An abstract sketch of the alternating chamfer to line pattern is given in Figure 1 .

    Article Snippet: Cell Culture For isolation of primary neonatal mouse cardiac myocytes, the Neonatal Heart Dissociation Kit and the Neonatal Cardiomyocyte Isolation Kit for mice (No. 130-098-373, No. 130-100-825, Miltenyi Biotec, Bergisch Gladbach, Germany) are used according to the manufacturer recommendations.

    Techniques: Fluorescence, Light Microscopy

    ( a ) Survey of a group of neonatal murine cardiac myocytes on a smooth glass substrate. The cells are stained for nuclei (blue), gap junctions via connexin-43 immunostaining (green) and the contractile apparatus via  α -actinin immuno-staining (red); ( b ) (insert in (a))Exclusive presentation of the  α -actinin signal component; ( c ) Exclusive presentation of the connexin-43 signal component. Besides the accumulation along pearl chain-like structures located along the intercellular borders, a faint non-specific signal is also visible inside the nuclei due to a cross-over in the acquisition channel. ( d )–( f ) are SEM recordings of neonatal cardiac myocytes cultivated on plain glass surface. The cells are flat and polygonal with an irregular polygonal shape.

    Journal: Journal of Functional Biomaterials

    Article Title: Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

    doi: 10.3390/jfb7010001

    Figure Lengend Snippet: ( a ) Survey of a group of neonatal murine cardiac myocytes on a smooth glass substrate. The cells are stained for nuclei (blue), gap junctions via connexin-43 immunostaining (green) and the contractile apparatus via α -actinin immuno-staining (red); ( b ) (insert in (a))Exclusive presentation of the α -actinin signal component; ( c ) Exclusive presentation of the connexin-43 signal component. Besides the accumulation along pearl chain-like structures located along the intercellular borders, a faint non-specific signal is also visible inside the nuclei due to a cross-over in the acquisition channel. ( d )–( f ) are SEM recordings of neonatal cardiac myocytes cultivated on plain glass surface. The cells are flat and polygonal with an irregular polygonal shape.

    Article Snippet: Cell Culture For isolation of primary neonatal mouse cardiac myocytes, the Neonatal Heart Dissociation Kit and the Neonatal Cardiomyocyte Isolation Kit for mice (No. 130-098-373, No. 130-100-825, Miltenyi Biotec, Bergisch Gladbach, Germany) are used according to the manufacturer recommendations.

    Techniques: Staining, Immunostaining

    Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1 flox/flox mice crossed with Myh6-Cre transgenic mice enabled generation of cardiac-specific Etv1 mutant mice. ( A ) Heart and tail RT-PCR analysis of DNA from Etv1 WT ( Etv1 flox/flox ) and Etv1 cKO ( Etv1 flox/flox , Myh6-Cre ) mice demonstrate heart specific deletion of Etv1 exon 11. ( B ) Immunoblot assessment of Etv1 WT and Etv1 cKO tissue extracts. Protein from cerebellum and right atrial lysates were electrophoresed and immunoblotted to detect ETV1 (top) and Vinculin (bottom). ( C ) Immunofluorescence evaluation of ETV1 expression in 10-week-old Etv1 WT and Etv1 cKO cerebellar Purkinje cells, atrial myocytes, and His-Purkinje system (HPS). Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). ( D ) Representative surface ECG traces of 10-week-old Etv1 WT and Etv1 c KO mice. Conduction intervals from 10–12-week-old Etv1 cKO mice compared to Etv1 WT mice showed significantly prolonged P wave and QRS intervals. 20% of Etv1 cKO mice displayed an RsR’ pattern. LA, left atria; LV, left ventricle. Data represent mean ± SEM. Scale bars: 25 um.

    Journal: Scientific Reports

    Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes

    doi: 10.1038/s41598-018-28239-7

    Figure Lengend Snippet: Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1 flox/flox mice crossed with Myh6-Cre transgenic mice enabled generation of cardiac-specific Etv1 mutant mice. ( A ) Heart and tail RT-PCR analysis of DNA from Etv1 WT ( Etv1 flox/flox ) and Etv1 cKO ( Etv1 flox/flox , Myh6-Cre ) mice demonstrate heart specific deletion of Etv1 exon 11. ( B ) Immunoblot assessment of Etv1 WT and Etv1 cKO tissue extracts. Protein from cerebellum and right atrial lysates were electrophoresed and immunoblotted to detect ETV1 (top) and Vinculin (bottom). ( C ) Immunofluorescence evaluation of ETV1 expression in 10-week-old Etv1 WT and Etv1 cKO cerebellar Purkinje cells, atrial myocytes, and His-Purkinje system (HPS). Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). ( D ) Representative surface ECG traces of 10-week-old Etv1 WT and Etv1 c KO mice. Conduction intervals from 10–12-week-old Etv1 cKO mice compared to Etv1 WT mice showed significantly prolonged P wave and QRS intervals. 20% of Etv1 cKO mice displayed an RsR’ pattern. LA, left atria; LV, left ventricle. Data represent mean ± SEM. Scale bars: 25 um.

    Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

    Techniques: Knock-Out, Mouse Assay, Transgenic Assay, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Expressing

    Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. ( A ) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). ( B ) Quantitative RT-PCR analysis of Etv1 , NKX2–5 , GJA5 , SCN5A , and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). ( C ) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. ( D ) hiPSC-CM Na V peak conductance (gNa V -peak). gNa V -peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P

    Journal: Scientific Reports

    Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes

    doi: 10.1038/s41598-018-28239-7

    Figure Lengend Snippet: Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. ( A ) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). ( B ) Quantitative RT-PCR analysis of Etv1 , NKX2–5 , GJA5 , SCN5A , and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). ( C ) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. ( D ) hiPSC-CM Na V peak conductance (gNa V -peak). gNa V -peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P

    Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

    Techniques: Activation Assay, Derivative Assay, Expressing, Transduction, Quantitative RT-PCR, Patch Clamp

    Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. ( A ) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh ) comparing 10–12-week-old Etv1 WT ( Etv1 flox/flox ) and Etv1 cKO ( Etv1 flox/flox , Myh6-Cre ) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5 , Gja5 , and Scn5a expression displayed versus control, Etv1 WT (n = 4). ( B ) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, Na V 1.5, and Vinculin (loading control). ( C ) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). ( D ) Immunofluorescence evaluation of NKX2–5, Cx40, and Na V 1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. ( E ) Immunofluorescence evaluation of NKX2–5, Cx40, and Na V 1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P

    Journal: Scientific Reports

    Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes

    doi: 10.1038/s41598-018-28239-7

    Figure Lengend Snippet: Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. ( A ) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh ) comparing 10–12-week-old Etv1 WT ( Etv1 flox/flox ) and Etv1 cKO ( Etv1 flox/flox , Myh6-Cre ) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5 , Gja5 , and Scn5a expression displayed versus control, Etv1 WT (n = 4). ( B ) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, Na V 1.5, and Vinculin (loading control). ( C ) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). ( D ) Immunofluorescence evaluation of NKX2–5, Cx40, and Na V 1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. ( E ) Immunofluorescence evaluation of NKX2–5, Cx40, and Na V 1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P

    Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

    Techniques: Expressing, Quantitative RT-PCR, FACS, Purification, Immunofluorescence

    ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT ( Etv1 flox/flox ) and Etv1 cKO ( Etv1 flox/flox , Myh6- Cre) mice in a Cntn2 -EGFP background (n = 4). ( A ) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. ( B ) Voltage dependence of steady-state activation. Voltage at half activation (V 0.5 , activation) was calculated to assess significant differences among experimental groups. ( C ) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V 0.5 , inactivation) was calculated to assess significant differences among experimental groups. ( D ) Time course of recovery from inactivation. Tau of recovery (τ recovery ) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent mean ± SEM. *P

    Journal: Scientific Reports

    Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes

    doi: 10.1038/s41598-018-28239-7

    Figure Lengend Snippet: ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT ( Etv1 flox/flox ) and Etv1 cKO ( Etv1 flox/flox , Myh6- Cre) mice in a Cntn2 -EGFP background (n = 4). ( A ) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. ( B ) Voltage dependence of steady-state activation. Voltage at half activation (V 0.5 , activation) was calculated to assess significant differences among experimental groups. ( C ) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V 0.5 , inactivation) was calculated to assess significant differences among experimental groups. ( D ) Time course of recovery from inactivation. Tau of recovery (τ recovery ) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent mean ± SEM. *P

    Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

    Techniques: Patch Clamp, Mouse Assay, Activation Assay

    Angiotensin-forming enzyme activity in cardiomyocyte (CM) and non-cardiomyocyte (NCM) from SHR and WKY female rats. Data indicate differences in (A) chymase activity in CMs (left) and NCMs (right) with respect to estrogen status (intact versus OVX); in NCMs, WKY chymase activity exceeds that of SHR (strain effect). (B) ACE activity is not different between strains nor is it affected by estrogen status. (C) ACE2 activity in CMs is higher in WKY compared to SHR, irrespective of estrogen status; NCMs showed no estrogen or strain effects (2-way ANOVA). Values are reported as mean ± SEM; SHR, N =6 per group. WKY rats, N =6 per group. Black bars represent gonadal intact rats; hatched bars represent OVX rats. ## P

    Journal: Journal of cellular physiology

    Article Title: Blunting of estrogen modulation of cardiac cellular chymase/RAS activity and function in SHR

    doi: 10.1002/jcp.26179

    Figure Lengend Snippet: Angiotensin-forming enzyme activity in cardiomyocyte (CM) and non-cardiomyocyte (NCM) from SHR and WKY female rats. Data indicate differences in (A) chymase activity in CMs (left) and NCMs (right) with respect to estrogen status (intact versus OVX); in NCMs, WKY chymase activity exceeds that of SHR (strain effect). (B) ACE activity is not different between strains nor is it affected by estrogen status. (C) ACE2 activity in CMs is higher in WKY compared to SHR, irrespective of estrogen status; NCMs showed no estrogen or strain effects (2-way ANOVA). Values are reported as mean ± SEM; SHR, N =6 per group. WKY rats, N =6 per group. Black bars represent gonadal intact rats; hatched bars represent OVX rats. ## P

    Article Snippet: The heart was then cannulated through the aorta on an Easycell System for Cardiomyocyte Isolation (Harvard Apparatus, Holliston, MA) and perfused at 37°C with calcium-free perfusion buffer at a flow rate of 4 ml/min for 5 min until the effluent became clear.

    Techniques: Activity Assay

    Relationship between cardiac myocyte chymase activity and diastolic function, defined by E/e′, and left ventricular hypertrophy, defined by LV mass. Linear relationships (solid line) and 95% confidence intervals (dotted line) between (A) CM chymase activity and E/e′ in WKY rats, and (B) CM chymase activity and LV mass in SHRs. Worsening diastolic function, indicated by elevations in E/e′, was related to chymase activity (top) in WKYs, while in SHRs, it was related to LV mass.

    Journal: Journal of cellular physiology

    Article Title: Blunting of estrogen modulation of cardiac cellular chymase/RAS activity and function in SHR

    doi: 10.1002/jcp.26179

    Figure Lengend Snippet: Relationship between cardiac myocyte chymase activity and diastolic function, defined by E/e′, and left ventricular hypertrophy, defined by LV mass. Linear relationships (solid line) and 95% confidence intervals (dotted line) between (A) CM chymase activity and E/e′ in WKY rats, and (B) CM chymase activity and LV mass in SHRs. Worsening diastolic function, indicated by elevations in E/e′, was related to chymase activity (top) in WKYs, while in SHRs, it was related to LV mass.

    Article Snippet: The heart was then cannulated through the aorta on an Easycell System for Cardiomyocyte Isolation (Harvard Apparatus, Holliston, MA) and perfused at 37°C with calcium-free perfusion buffer at a flow rate of 4 ml/min for 5 min until the effluent became clear.

    Techniques: Activity Assay

    Inhibition of the cardioprotective effects of E 2 , ERα and BSA-E 2 on LPS-treated ventricular cardiomyocytes by PI3K siRNA treatment. (A and B) Thirty-six hours after being cotransfected with plasmids (3.5 μg ERα or pcDNA3) and siRNA (100 nM siGLO siRNA or PI3K siRNA), ventricular cardiomyocytes were incubated with vehicle, E 2 (10 −8 M) or BSA-E 2 (equivalent to 10 ng/ml free E 2 ) in the presence of LPS for 24 hrs. Cells were harvested and analyzed by RT-PCR to detect TNF-α expression and by immunoblotting using antibodies against TNF-α, ERα and active caspase 3. (C) TUNEL staining of the ventricular cardiomyocytes was performed according to the manufacturer’s protocol PI3K siRNA abolished the reduction of apoptosis induced by E 2 , ERα and BSA-E 2 in LPS-treated ventricular cardiomyocytes. ** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Akt mediates 17β-estradiol and/or estrogen receptor-α inhibition of LPS-induced tumor necresis factor-α expression and myocardial cell apoptosis by suppressing the JNK1/2-NFκB pathway

    doi: 10.1111/j.1582-4934.2009.00669.x

    Figure Lengend Snippet: Inhibition of the cardioprotective effects of E 2 , ERα and BSA-E 2 on LPS-treated ventricular cardiomyocytes by PI3K siRNA treatment. (A and B) Thirty-six hours after being cotransfected with plasmids (3.5 μg ERα or pcDNA3) and siRNA (100 nM siGLO siRNA or PI3K siRNA), ventricular cardiomyocytes were incubated with vehicle, E 2 (10 −8 M) or BSA-E 2 (equivalent to 10 ng/ml free E 2 ) in the presence of LPS for 24 hrs. Cells were harvested and analyzed by RT-PCR to detect TNF-α expression and by immunoblotting using antibodies against TNF-α, ERα and active caspase 3. (C) TUNEL staining of the ventricular cardiomyocytes was performed according to the manufacturer’s protocol PI3K siRNA abolished the reduction of apoptosis induced by E 2 , ERα and BSA-E 2 in LPS-treated ventricular cardiomyocytes. ** P

    Article Snippet: Cardiomyocyte culture Neonatal cardiomyocytes were isolated and cultured using the commercial Neonatal Cardiomyocyte Isolation System Kit according to manufacture’s directions (Cellutron Life Technology, Highland Park, NJ).

    Techniques: Inhibition, Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing, TUNEL Assay, Staining

    A schematic representation showing how E 2 , BSA-E 2 and ERα expression inhibit JNK1/2-mediated LPS-induced TNF-α expression and cardiomyocyte apoptosis through activation of Akt. Activation of toll-like receptor 4 (TLR4) by specific LPS binding induces JNK1/2 activation, which in turn leads to the activation of transcription factor NFκB. Therefore, phosphated IκB marked with a polyubiquitin chain by β-TrCP/HOS (E3, an ubiquitin ligase) is targeted for proteasomal degradation. After transcription and translation, trimeric TNF-α is inserted into the cell membrane. TNF-α converting enzyme (TACE) cleaves the pro-form of TNF-α off the cell membrane, which yields a soluble form of TNF-α (sTNF-α) that then binds to TNF receptor-I (TNFR-I) through the autocrine loop to activate downstream caspases, thereby executing cardiomyocyte apoptosis. E 2 , acting through cytosolic ERα or membrane ERα, and ERα overexpression alone inside the cells block LPS-induced JNK1/2 activation by activating the PI3K–Akt pathway. This blockade helps to maintain the association between IκB and NFκB and to inhibit nuclear localization of NFκB, thereby inhibiting cardiomyocyte apoptosis.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Akt mediates 17β-estradiol and/or estrogen receptor-α inhibition of LPS-induced tumor necresis factor-α expression and myocardial cell apoptosis by suppressing the JNK1/2-NFκB pathway

    doi: 10.1111/j.1582-4934.2009.00669.x

    Figure Lengend Snippet: A schematic representation showing how E 2 , BSA-E 2 and ERα expression inhibit JNK1/2-mediated LPS-induced TNF-α expression and cardiomyocyte apoptosis through activation of Akt. Activation of toll-like receptor 4 (TLR4) by specific LPS binding induces JNK1/2 activation, which in turn leads to the activation of transcription factor NFκB. Therefore, phosphated IκB marked with a polyubiquitin chain by β-TrCP/HOS (E3, an ubiquitin ligase) is targeted for proteasomal degradation. After transcription and translation, trimeric TNF-α is inserted into the cell membrane. TNF-α converting enzyme (TACE) cleaves the pro-form of TNF-α off the cell membrane, which yields a soluble form of TNF-α (sTNF-α) that then binds to TNF receptor-I (TNFR-I) through the autocrine loop to activate downstream caspases, thereby executing cardiomyocyte apoptosis. E 2 , acting through cytosolic ERα or membrane ERα, and ERα overexpression alone inside the cells block LPS-induced JNK1/2 activation by activating the PI3K–Akt pathway. This blockade helps to maintain the association between IκB and NFκB and to inhibit nuclear localization of NFκB, thereby inhibiting cardiomyocyte apoptosis.

    Article Snippet: Cardiomyocyte culture Neonatal cardiomyocytes were isolated and cultured using the commercial Neonatal Cardiomyocyte Isolation System Kit according to manufacture’s directions (Cellutron Life Technology, Highland Park, NJ).

    Techniques: Expressing, Activation Assay, Binding Assay, Over Expression, Blocking Assay