Journal: PLoS ONE

Article Title: Cardiac-Oxidized Antigens Are Targets of Immune Recognition by Antibodies and Potential Molecular Determinants in Chagas Disease Pathogenesis

doi: 10.1371/journal.pone.0028449

Figure Lengend Snippet: Identification of proteins recognized by immune sera from chagasic patients.

Article Snippet: Human recombinant titin (0.1 mg/ml, Novus Biologicals) was sonicated three times for 30 sec each at 4°C, and then in vitro oxidized with 4-HNE (30 μM) or H 2 O 2 (100 μM) for 1 h at 37°C.

Techniques: Variant Assay, Binding Assay

Journal: PLoS ONE

Article Title: Cardiac-Oxidized Antigens Are Targets of Immune Recognition by Antibodies and Potential Molecular Determinants in Chagas Disease Pathogenesis

doi: 10.1371/journal.pone.0028449

Figure Lengend Snippet: Recombinant titin was in vitro oxidized with 4-HNE (30 µM) or H 2 O 2 (100 µM). An enzyme-linked immune-sorbent assay (ELISA) was performed to titer the antibody level against recombinant and oxidized titin in sera samples of normal healthy controls, seropositive chagasic patients, and seronegative subjects with cardiomyopathy of other etiologies (OCM). n = 25/group (* p <0.05).

Article Snippet: Human recombinant titin (0.1 mg/ml, Novus Biologicals) was sonicated three times for 30 sec each at 4°C, and then in vitro oxidized with 4-HNE (30 μM) or H 2 O 2 (100 μM) for 1 h at 37°C.

Techniques: Recombinant, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Cardiac-Oxidized Antigens Are Targets of Immune Recognition by Antibodies and Potential Molecular Determinants in Chagas Disease Pathogenesis

doi: 10.1371/journal.pone.0028449

Figure Lengend Snippet: Sprague Dawley rats were infected with T. cruzi and treated with phenyl-α-tert-butyl nitrone (PBN, antioxidant) as detailed in . ( A ) Recombinant titin was in vitro oxidized with 4-HNE (30 µM) or H 2 O 2 (100 µM). An ELISA was performed to titer the antibody levels against recombinant and oxidized titin in sera of normal rats, and rats that were chronically infected and treated with PBN. ( B ) Rat heart homogenates (normal and in vitro oxidized with 4-HNE or H 2 O 2 ) were resolved by 1D-GE and Western blotting was performed with sera from normal (panel a) , chronically infected (panel b) and infected/PBN-treated (panel c) rats. Coomassie blue staining of membranes (panel d) confirmed the equal loading of samples. n = 6/group (* p<0.05 ). ( C ) Densitometry analysis of protein bands (marked by asterisks in B ).

Article Snippet: Human recombinant titin (0.1 mg/ml, Novus Biologicals) was sonicated three times for 30 sec each at 4°C, and then in vitro oxidized with 4-HNE (30 μM) or H 2 O 2 (100 μM) for 1 h at 37°C.

Techniques: Infection, Recombinant, In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot, Staining

Journal: International journal of molecular sciences

Article Title: Disruption of Z-Disc Function Promotes Mechanical Dysfunction in Human Myocardium: Evidence for a Dual Myofilament Modulatory Role by Alpha-Actinin 2.

doi: 10.3390/ijms241914572

Figure Lengend Snippet: Figure 2. Ultrastructural analysis of the A868T variant patient heart. (A,B) Representative electron microscopy of (A) normal and (B) A868T variant myocardium. Scale bars are 800 nm. (C–F) Repre- sentative immunofluorescence images with α-actinin 2 (green) and titin (red) antibodies. Scale bars are 10 µm. Additional immunofluorescence images are shown in Supplementary Figures S2 and S3. (G) Sarcomere length measured from EM images (5 EM images of the same patient’s heart and 5 EM images of the same donor heart were analyzed). (H) Myofibrillar width measured from EM images (5 EM images of the same patient’s heart and 5 EM images of the same donor heart were analyzed). (I) Z-disc width measured from EM images (5 EM images of the same patient’s heart and 5 EM images of the same donor heart were analyzed). (J,K) Antibody fluorescence intensity quantification. (L) Sarcomere length measured from immunofluorescence images. (M) Z-bodies quantification measured from immunofluorescence images. (N) Titin spots measured from immunofluorescence images. Data are shown as mean ± S.E; the data points are technical replicates. * p < 0.05. ** p < 0.01.

Article Snippet: The fixed tissue was washed 3× for 10 min in Permeabilization Buffer (10% FBS, 0.2% Triton X-100 in PBS) and then incubated with Collagenase Permeabilization Buffer (Collagenase II and IV in permeabilization buffer) for 30 min. Immunostaining was followed by incubating the primary antibody for α-Actinin 2 (GeneTex #GTX103219) or Titin (Novus #NBP1-88071) overnight at 4 ◦C.

Techniques: Variant Assay, Electron Microscopy

Journal: International journal of molecular sciences

Article Title: Disruption of Z-Disc Function Promotes Mechanical Dysfunction in Human Myocardium: Evidence for a Dual Myofilament Modulatory Role by Alpha-Actinin 2.

doi: 10.3390/ijms241914572

Figure Lengend Snippet: Figure 6. Summary of proposed pathophysiological pathways. (A) Communication pathways. Communication Pathway 1 (CP1) proposes a relationship between α-actinin 2 and actin that regulates thin filament activation. Communication Pathway 2 (CP2) proposes an interaction between α-actinin 2/titin/myosin that controls LS, thick filament activation, and cross-bridges kinetics. (B) Model for α-actinin 2 variant A868T influences cardiac function, leading to pathology. Summary of the ultrastructural and mechanical changes caused by the A868T variant on α-actinin 2 and the predicted role in the Z-disc of the sarcomere.

Article Snippet: The fixed tissue was washed 3× for 10 min in Permeabilization Buffer (10% FBS, 0.2% Triton X-100 in PBS) and then incubated with Collagenase Permeabilization Buffer (Collagenase II and IV in permeabilization buffer) for 30 min. Immunostaining was followed by incubating the primary antibody for α-Actinin 2 (GeneTex #GTX103219) or Titin (Novus #NBP1-88071) overnight at 4 ◦C.

Techniques: Activation Assay, Variant Assay

Journal: Nature Communications

Article Title: SMYD2 glutathionylation contributes to degradation of sarcomeric proteins

doi: 10.1038/s41467-018-06786-x

Figure Lengend Snippet: SMYD2 Cys13 glutathionylation or oxidation reduces myofibril integrity. a , b Monitoring the myofibril alignment in rat neonatal cardiomyocytes upon incubation of AMA (2 µg/mL) for 12 h: no expression ( a ) and ectopic expression of SMYD2 WT or C13S ( b ). Immunostainings were done by using antibodies to SMYD2, HA (green), or titin (α-titin-NT, red). About 30 cells were photographed and examined for myofibril alignment or directionality by FiberFit software. Images represent the major myofibril structure in individual conditions. Scale bars, 10 µm. c Analysis of myofibril alignment in cardiomyocytes. Individual cell images were analyzed by the FiberFit software to determine the fiber dispersion parameter ( k ) values that represent the degree of fiber alignment. High k values represent the aligned networks, whereas low k values represent the disordered networks. The median values with 95% CI are shown, n = 3 independent experiments. Difference is significant by one-way ANOVA, followed by Tukey’s post-hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Primary antibodies include titin rabbit antibody (Novus biologicals, Cat# NBP 1-88071, α-titin-NT) (1:100 dilution), SMYD2 mouse antibody (Sigma, Cat# SAB1407760) (1:100 dilution), SMYD2 rabbit antibody (Cell Signaling, Cat# 9734) (1:100 dilution), HA-tag (6E2) mouse antibody conjugated with Alexa Fluor 488 (Cell Signaling, Cat# 2350 S) and α-actinin mouse antibody (Abcam, Cat# ab9465) (1:100 dilution).

Techniques: Incubation, Expressing, Software, Dispersion

Journal: Nature Communications

Article Title: SMYD2 glutathionylation contributes to degradation of sarcomeric proteins

doi: 10.1038/s41467-018-06786-x

Figure Lengend Snippet: SMYD2 Cys13 glutathionylation or oxidation leads to degradation of sarcomeric proteins. a Sarcomeric protein levels in response to AMA in differentiated H9c2 cells that express SMYD2 WT or C13S. b Levels of titin in response to AMA in HL-1 cells expressing SMYD2 WT or C13S. Extracts of left ventricle (LV) and soleus muscle isolated from 6.5-months old rat were used as standards (lane 1 and lane 2) to show the position of N2B-titin or N2A-titin isoforms, respectively. c Sarcomeric protein levels in response to AMA after SMYD2 knockdown. d , e Sarcomeric protein levels in response to AMA after incubation of ARP-100 (MMP-2 inhibitor) ( d ) or MMP-2 knockdown ( e ). f , g The cell viability in response to AMA after incubation of ARP-100 (1 µM), calpastatin (calpain 1 inhibitor, 5 µM) ( f ) or MMP-2 knockdown ( g ). In all conditions, differentiated H9c2 ( a , c – g ) or HL-1 cells ( b ) were treated with AMA (2 µg/mL) for 12 h. Lysates were analyzed by Western blotting ( a , c – e , g ) or Coomassie staining ( b ). Cell viability was analyzed by Trypan blue assay. Data represent the mean ± SD, n = 3 independent experiments. Difference is significant by two-way ANOVA followed by Bonferroni’s post-hoc test ( f ) and one-way ANOVA followed by Tukey’s post-hoc test ( a – e , g ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Primary antibodies include titin rabbit antibody (Novus biologicals, Cat# NBP 1-88071, α-titin-NT) (1:100 dilution), SMYD2 mouse antibody (Sigma, Cat# SAB1407760) (1:100 dilution), SMYD2 rabbit antibody (Cell Signaling, Cat# 9734) (1:100 dilution), HA-tag (6E2) mouse antibody conjugated with Alexa Fluor 488 (Cell Signaling, Cat# 2350 S) and α-actinin mouse antibody (Abcam, Cat# ab9465) (1:100 dilution).

Techniques: Expressing, Isolation, Knockdown, Incubation, Western Blot, Staining

Journal: Nature Communications

Article Title: SMYD2 glutathionylation contributes to degradation of sarcomeric proteins

doi: 10.1038/s41467-018-06786-x

Figure Lengend Snippet: SMYD2 Cys13 glutathionylation induces dissociation of SMYD2 from N2A and Hsp90. a , b SMYD2 glutathionylation disrupts its interaction with Hsp90. Purified SMYD2-SH and SMYD2-SSG were incubated with GST-Hsp90 bound to glutathione beads, and eluted sample was analyzed ( a ). Hsp90 was co-immunoprecipitated with SMYD2 WT or C13S from HEK293 cells in response to AMA with glucose deprivation ( b ). c , d SMYD2 glutathionylation disrupts its interaction with N2A. Purified SMYD2-SH and SMYD2-SSG were incubated with GST-N2A bound to glutathione beads, and eluted sample was analyzed ( c ). FLAG-N2A was co-immunoprecipitated with SMYD2 WT or C13S in HEK293 cells in response to AMA with glucose deprivation ( d ). e SMYD2 subjected to glutathionylation decreases its binding with N2A. SMYD2 WT or C13S was pre-incubated with H 2 O 2 in the absence or presence of glutathione for 15 min, then mixed with GST-N2A bound to glutathione beads for 1 h. Eluted samples were analyzed. f , g Colocalization of titin and SMYD2 decreases upon incubation of AMA in rat neonatal cardiomyocytes expressing SMYD2 WT versus C13S. Immunostainings of cardiomyocytes with antibodies to titin (α-titin-NT, red), HA, or SMYD2 (green) are shown with enlarged areas for details (the red boxes) ( f ). Pearson’s correlation coefficients were calculated to determine colocalization of titin and SMYD2 ( g ). About 30 cells were analyzed in individual conditions. Images represent the major colocalization pattern in individual experiments. Scale bars, 10 µm. Data represent the mean ± SD, n = 3 independent experiments

Article Snippet: Primary antibodies include titin rabbit antibody (Novus biologicals, Cat# NBP 1-88071, α-titin-NT) (1:100 dilution), SMYD2 mouse antibody (Sigma, Cat# SAB1407760) (1:100 dilution), SMYD2 rabbit antibody (Cell Signaling, Cat# 9734) (1:100 dilution), HA-tag (6E2) mouse antibody conjugated with Alexa Fluor 488 (Cell Signaling, Cat# 2350 S) and α-actinin mouse antibody (Abcam, Cat# ab9465) (1:100 dilution).

Techniques: Purification, Incubation, Immunoprecipitation, Binding Assay, Expressing

Journal: Nature Communications

Article Title: SMYD2 glutathionylation contributes to degradation of sarcomeric proteins

doi: 10.1038/s41467-018-06786-x

Figure Lengend Snippet: SMYD2-N2A dissociation contributes to degradation of sarcomeric proteins. a , b N2A is degraded by MMP-2, and SMYD2 protects N2A from degradation. Purified N2A was incubated with active MMP-2 in a time-dependent manner ( a ) or with an increasing amount of SMYD2 ( b ). c , d N2A is degraded by calpain 1, and SMYD2 protects N2A from degradation. Purified N2A was incubated with calpain 1 in a time-dependent manner ( c ) or with an increasing amount of SMYD2 ( d ). Data are representative of 4 independent experiments. e , f Titin in isolated myofibrils is degraded by MMP-2, and SMYD2 protects titin from degradation. Myofibrils isolated from mouse gastrocnemius muscle were incubated with active MMP-2 in the absence and presence of SMYD2 ( e ). Extracts of soleus muscle and left ventricle (LV) isolated from 6.5-months old rat were used as standards (lane 1 and lane 6). Levels of titin degradation by measuring the ratio of T1 or T2 to MHC ( f ). In all conditions, proteins were analyzed by Coomassie stains. Data represent the mean ± SD, n = 3 independent experiments. Difference is significant by one-way ANOVA followed by Tukey’s post-hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. g A proposed mechanism of sarcomere destabilization upon SMYD2 glutathionylation or oxidation. Under unstressed conditions, SMYD2-Hsp90 binds with and protects N2A of titin (top). Under stressed conditions, ROS lead to activation of MMP-2 and calpain 1 while inducing glutathionylation (or other oxidations) of SMYD2, which is then dissociated from N2A or titin, allowing for sarcomeric protein degradation by MMP-2 and calpain 1. It remains to be analyzed how SMYD2 glutathionylation or oxidation contributes to degradation of α-actinin and troponin I (bottom)

Article Snippet: Primary antibodies include titin rabbit antibody (Novus biologicals, Cat# NBP 1-88071, α-titin-NT) (1:100 dilution), SMYD2 mouse antibody (Sigma, Cat# SAB1407760) (1:100 dilution), SMYD2 rabbit antibody (Cell Signaling, Cat# 9734) (1:100 dilution), HA-tag (6E2) mouse antibody conjugated with Alexa Fluor 488 (Cell Signaling, Cat# 2350 S) and α-actinin mouse antibody (Abcam, Cat# ab9465) (1:100 dilution).

Techniques: Purification, Incubation, Isolation, Activation Assay