Journal: Communications Biology

Article Title: A maladaptive feedback mechanism between the extracellular matrix and cytoskeleton contributes to hypertrophic cardiomyopathy pathophysiology

doi: 10.1038/s42003-022-04278-9

Figure Lengend Snippet: Immunoblot analysis of L-type calcium channel ( a , b ) and β 1 integrin ( c , d ) protein expression performed on total heart homogenate pooled from groups of 5 pre- (10–15-wk-old) or post-cardiomyopathic (30–50-week-old) cTnI-G203S mice and age-matched wt counterparts. Representative immunoblots probed with L-type calcium channel α 1C subunit (Ca V 1.2, a ) or β 1 integrin ( c ) antibody, then GAPDH monoclonal antibody. Densitometry analysis of Ca V 1.2 ( b ) or β 1 integrin ( d ) protein expression, normalized to associated GAPDH expression. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA ( b ) or Kruskal-Wallis test ( d ) determined statistical significance. Densitometry analysis of relative mTOR expression (calculated as Phospho-mTOR/Total mTOR) performed on cytoplasmic ( e ) and nuclear ( f ) fractions pooled from groups of five pre- or post-cardiomyopathic cTnI-G203S mice and age-matched wt counterparts. β-tubulin and histone H2B antibodies were used as loading controls for cytoplasmic and nuclear fractions respectively. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA ( e ) or Kruskal-Wallis test ( f ) determined statistical significance. g Schematic indicating structural-functional link between the L-type calcium channel, cytoskeletal network, mitochondria, integrin and the extracellular matrix in wt and cTnI-G203S cardiac myocytes. A disrupted cytoskeletal architecture in cTnI-G203S cardiac myocytes may trigger a maladaptive feedback mechanism between increased cytoskeletal and extracellular matrix stiffness, resulting in a hypermetabolic mitochondrial state.

Article Snippet: Blots were probed with the following primary antibodies: rabbit polyclonal anti-Ca V 1.2 (Alomone, ACC-003, 1:200) or rabbit monoclonal anti-β 1 integrin (D6S1W) (Cell Signaling Technology 34971, 1:1000).

Techniques: Western Blot, Expressing, Functional Assay

Journal: iScience

Article Title: Exendin-4 promotes retinal ganglion cell survival and function by inhibiting calcium channels in experimental diabetes

doi: 10.1016/j.isci.2023.107680

Figure Lengend Snippet: Involvement of intracellular Ca 2+ , CaM, calcineurin and PP1 in suppression of I LCa by Ex-4 (A and B) Representative recordings from an RGC at −10 mV, showing that during internal infusion of Ca 2+ -free solution (containing 10 mM BAPTA; control), Ex-4 failed to suppress the I LCa , and summary data are shown in (B) (n = 6). (C and E) Current traces of two RGCs, showing that during internal dialysis of 5 mg/mL heparin (C), but not ryanodine (50 μM, E), the Ex-4-induced suppression of I LCa was seen. (D and F) Bar charts summarizing the effects of Ex-4 on the I LCa amplitudes in the presence of heparin (n = 7) (D) or ryanodine (n = 7) (F). (G, I, and K) Representative recordings obtained from three different RGCs showing that Ex-4 no longer reduced the I LCa amplitudes during the internal infusion of 100 μM W-7 (G), 50 μM FK-506 (I) and 1 μM OA (K) to block CaM, calcineurin and PP1, respectively. (H, J, and L) Bar charts summarizing the results regarding the effects of W-7 (n = 7) (H), FK-506 (n = 8) (J) or OA (n = 8) (L). (M and N) Current traces of an RGC, showing that perfusion of C2 Ceramide (500 nM) suppressed the I LCa (M), and summary data are shown in (N) (n = 6). Data are presented as mean ± SD, n.s., p > 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 by paired t test. (O) Representative immunoblots showed changes in the expression of L-VGCC subunits Cav1.2 and p -Cav1.2 in membrane components in retinas from the control, DM rats treated with or without Ex-4 eye drops. Na + -K + -ATPase served as loading control. (P and Q) Densitometric analysis revealing a significant increase in Cav1.2 (P) and p -Cav1.2 (Q) protein levels in DM retinas. Topical administration of Ex-4 significantly decreased Cav1.2 and p -Cav1.2 levels in DM retinas. (R) Quantitative analysis of p -Cav1.2 relative to Cav1.2 total protein is shown in the bar graph. The level of p -Cav1.2/Cav1.2 did not change in DM retinas, but significantly decreased in the DM + Ex-4 group. n = 3 for each group. Data are presented as mean ± SD. n.s., p > 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 by post hoc Tukey’s multiple comparisons test after one-way ANOVA. See also Figure S3 .

Article Snippet: Rabbit polyclonal anti-Cav1.2 (CACNA1C) , Alomone , Cat#ACC-003; RRID: AB_2039771.

Techniques: Blocking Assay, Western Blot, Expressing, Membrane, Eye Drops

Journal: iScience

Article Title: Exendin-4 promotes retinal ganglion cell survival and function by inhibiting calcium channels in experimental diabetes

doi: 10.1016/j.isci.2023.107680

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Cav1.2 (CACNA1C) , Alomone , Cat#ACC-003; RRID: AB_2039771.

Techniques: Recombinant, Protease Inhibitor, Software

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Cardiac Fibroblasts Promote Ferroptosis in Atrial Fibrillation by Secreting Exo-miR-23a-3p Targeting SLC7A11

doi: 10.1155/2022/3961495

Figure Lengend Snippet: Canines electrophysiological examination by programmed stimulation and the expression of ion channel. (a) Canines electrocardiogram during programmed stimulation. In Sham group, the state at which atrial fibrillation (AF) was not reached. In Pacing group, the state at which AF was reached and the AF sustained more than 5 seconds. Compared with Pacing group, AF susceptibility attenuated by GW4869 injection because most irregular rhythm shortens than 5 seconds. (b) Effective refractory period of different parts of the atrium. GW4869 increased the ERP of RSPV and LIPV in Pacing group. ( n =6). (c) Difference in AF inducibility, shown by the number of episodes. ( n =6). (d) Difference in mean AF durations. ( n =6). (e, f) Representative gel bands depicting KCa3.1 and Cav1.2 protein expression using specific antibodies. GAPDH was used as the loading control. ( n =5). (g) Total iron level in atrial tissue. ( n =3). (h) MDA level in atrial tissue. ( n =3). Data are presented as the mean ± SD. Statistical significance was determined using one-way ANOVA with a post hoc Dunnett test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. Sham group; # P < 0.05, ## P < 0.01, and ### P < 0.001 vs. Pacing group. Abbreviations: RA: right atrium; RSPV: right superior pulmonary vein; RIPV: right inferior pulmonary vein; LA: left atrium; LSPV: left superior pulmonary vein; LIPV: left inferior pulmonary vein; dERP: dispersion effective refractory period.

Article Snippet: Subsequently, the membranes were incubated with primary antibodies against GAPDH (Abcam, ab181602; 1 : 10000), Cav1.2 (Alomone, acc-003; 1 : 500), KCa3.1 (Bioss, bs-6675r; 1 : 500), CD63 (Biorbyt, orb11597; 1 : 1000), CD81 (Abcam, ab109201; 1 : 1000), TSG101 (Sigma, av38773; 1 : 500), FTH1 (Abcam, ab65080; 1 : 1000), GPX4 (Abcam, ab125066; 1 : 1000), and SLC7A11 (Biorbyt, orb325112; 1 : 500) overnight at 4°C.

Techniques: Expressing, Injection

Journal: Mediators of Inflammation

Article Title: Tumor Necrosis Factor Alpha Inhibits L-Type Ca 2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

doi: 10.1155/2016/5972302

Figure Lengend Snippet: Sequence of primers and PCR conditions for the different subunits of L-VDCC.

Article Snippet: To block nonspecific binding to proteins, 10% horse serum was applied on the slices for two h. The slices were incubated with the primary antibodies to Ca V 1.2 and Ca V 1.3 (subunits of L-VDCC, Alomone Labs., Cat. numbers ACC-003 and ACC-311, resp., Jerusalem, Israel), both antibodies at a dilution 1 : 50, overnight at 4°C.

Techniques: Sequencing, Amplification

Journal: Mediators of Inflammation

Article Title: Tumor Necrosis Factor Alpha Inhibits L-Type Ca 2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

doi: 10.1155/2016/5972302

Figure Lengend Snippet: Detection of mRNA for L-VDCC subunits in guinea pig tracheal smooth muscle, as revealed by RT-PCR. (a) In airway smooth muscle, the PCR products at 470 and 459 bp length correspond to Ca V 1.2 and Ca V 1.3 cDNA, respectively. In this tissue, Ca V 1.1 and Ca V 1.4 were not found. Positive controls for these subunits were skeletal muscle (SKM, ~500 bp) and retina (~200 bp). Lane at the left corresponds to 1 Kb Plus DNA Ladder. (b) Representative PCR blots for Ca V 1.2 and Ca V 1.3 from nonsensitized (NS, n = 3) and sensitized (S, n = 4) smooth muscles. The first column in each blot corresponds to a negative control without template. The lower panel displays constitutive cDNA of GAPDH. (c) Densitometry data analysis for mRNA from Ca V 1.2 and Ca V 1.3 subunits showing no statistical significance between NS and S. Bars correspond to mean ± SEM.

Article Snippet: To block nonspecific binding to proteins, 10% horse serum was applied on the slices for two h. The slices were incubated with the primary antibodies to Ca V 1.2 and Ca V 1.3 (subunits of L-VDCC, Alomone Labs., Cat. numbers ACC-003 and ACC-311, resp., Jerusalem, Israel), both antibodies at a dilution 1 : 50, overnight at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: Mediators of Inflammation

Article Title: Tumor Necrosis Factor Alpha Inhibits L-Type Ca 2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

doi: 10.1155/2016/5972302

Figure Lengend Snippet: Immunofluorescence for Ca V 1.3 in nonsensitized and sensitized guinea pig tracheal smooth muscle. The first column shows immunoreactivity for Ca V 1.3 (stained green) in nonsensitized (a) and sensitized tissues (e); notice that Ca V 1.3 is located in the airway smooth muscle (SM) and epithelium (EPI, pointed by arrows); blocking peptide completely eliminated the fluorescence (i). The second and the third columns illustrate smooth muscle α -actin (stained red; (b), (f), (j)) and cell nuclei (DAPI, stained blue; (c), (g), (k)). The last column depicts merged images of the former three columns ((d), (h), (l)). In these merged images, Ca V 1.3 is seen to be colocalized with α -actin (stained yellow) on the smooth muscle.

Article Snippet: To block nonspecific binding to proteins, 10% horse serum was applied on the slices for two h. The slices were incubated with the primary antibodies to Ca V 1.2 and Ca V 1.3 (subunits of L-VDCC, Alomone Labs., Cat. numbers ACC-003 and ACC-311, resp., Jerusalem, Israel), both antibodies at a dilution 1 : 50, overnight at 4°C.

Techniques: Immunofluorescence, Staining, Blocking Assay, Fluorescence