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

Journal: Skeletal muscle

Article Title: N-terminal titin fragment: a non-invasive, pharmacodynamic biomarker for microdystrophin efficacy.

doi: 10.1186/s13395-023-00334-y

Figure Lengend Snippet: Fig. 2 The SOMAmer detects the previously characterized N-terminal fragment of titin in DMD patient urine. A SOMAmer detects the N-terminal fragment (amino acids 1–194) of titin. B Western blot using anti-TTN mouse monoclonal antibody [clone: 7D3] against amino acids 1–110 shows the presence of the fragment in DMD (lanes 7–14) urine, as well its absence in age-matched healthy controls (lanes 1–6). C Human urine ELISA confirms increases in the titin fragment seen in the SOMAscan panel and Western blot. D Normalization of the ELISA results using creatinine, E specific gravity, and F cystatin C show significant increases in the urinary titin fragment in DMD urine. For individual plots of normalization values, see Supplementary Fig. 1

Article Snippet: The N-terminal fragment was detected with titin (7D3) antibody (Novus Biologicals).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Skeletal muscle

Article Title: N-terminal titin fragment: a non-invasive, pharmacodynamic biomarker for microdystrophin efficacy.

doi: 10.1186/s13395-023-00334-y

Figure Lengend Snippet: Fig. 4 Circulating titin shows changes at lower levels of expressed microdystrophin when compared to CK-MM in preclinical DMD models. A The Somalogic aptamer detects the N terminal portion (AA 1–194) of the titin protein. This region was predicted to be highly conserved across mice, dogs, and humans. B Titin showed a response in vastus lateralis at > 50% microdystrophin levels, while an increase or no change was observed in CK-MM in the GRMD dogs 90 days post-treatment. C Circulating titin was also decreased in the plasma of mdx mice at lower levels of expressed microdystrophin in quadriceps when compared to CK-MM

Article Snippet: The N-terminal fragment was detected with titin (7D3) antibody (Novus Biologicals).

Techniques: Clinical Proteomics

Journal: Skeletal muscle

Article Title: N-terminal titin fragment: a non-invasive, pharmacodynamic biomarker for microdystrophin efficacy.

doi: 10.1186/s13395-023-00334-y

Figure Lengend Snippet: Fig. 3 CK8-μDys5 reduces urinary titin in the presence of 40–50% microdystrophin-positive fibers. A Group 1 (microdystrophin < 10% positive fibers in the vastus lateralis) shows no changes in urinary titin or serum CK activity 360 days post-treatment. B Group 2 (microdystrophin > 10% positive fibers in the vastus lateralis) shows decreases in urinary titin at day 180 and 360, while serum CK activity was trending at day 180, but significantly changed at day 360 only

Article Snippet: The N-terminal fragment was detected with titin (7D3) antibody (Novus Biologicals).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Mechanobiological Specialization of Choroid Plexus Macrophages Defined by Titin Expression

doi: 10.64898/2026.01.20.700716

Figure Lengend Snippet: Titin-expressing macrophages are present in the choroid plexus. (A) Heatmap of the top eight differentially expressed genes in each of the three ChP macrophage sub-clusters. Gene expression levels are represented on a normalized gradient scale. (B) DAB immunohistochemical stain of a 5 µm FFPE section of control ChP tissue using a CD163 antibody. CD163-positive macrophages are marked by brown staining, while cell nuclei are counterstained blue with hematoxylin, indicating their distribution within the vascularized ChP tissue. Scale bar = 40 µm. (C) Violin plots illustrating TTN expression levels in ChP samples from Alzheimer’s disease and control conditions. Each plot shows the distribution, density, and variability of TTN transcript counts in ChP macrophages within each condition. (D) Schematic representation of the experimental workflow for evaluation of TTN RNA enrichment in macrophages, involving tissue dissociation, CD163 positive cell enrichment, RNA isolation, cDNA synthesis, PCR amplification using isoform-specific TTN primers, and ΔΔCq calculation with GAPDH used for normalization. (E) Fold changes, computed from ΔΔCq, in TTN isoform expression relative to GAPDH in CD163-bead enriched macrophages relative to whole tissue. The x-axis represents different primer sets (1, 3, 7, and 12), and the y-axis shows fold changes (log scale). Each point represents a single donor sample/primer set pair. (F) Schematic representation of the TTN gene with highlighted primer binding sites, with genomic coordinates on chromosome 2, indicating selected splicing sites and isoforms of TTN. (G) Immunofluorescence images of ChP with anti-CD68 (red) and anti-titin (green) primary antibodies, co-stained with DAPI (blue), showing cytoplasmic titin protein in ChP macrophage cytoplasm. Merged images are in right column. Scale bars = 20 µm.

Article Snippet: For titin immunofluorescence, primary antibody incubation with rabbit anti-titin (Proteintech Catalog #27867-1-AP) at 1:250 dilution and mouse anti-CD68 antibody (Millipore Cat.#168M-95) at 1:250 was conducted overnight at 4°C, followed by detection with secondary donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, Cat. #A21206) (1:500) and goat anti-mouse Alexa Fluor 647 (Thermo Fisher Scientific, Cat. #A32728), with nuclei counterstaining with DAPI.

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Staining, Control, Isolation, cDNA Synthesis, Amplification, Binding Assay, Immunofluorescence

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: bioRxiv

Article Title: PITX2C Deficiency Promotes Arrhythmogenic Remodeling via Oxidative Stress in Atrial Myocytes

doi: 10.64898/2026.03.27.714813

Figure Lengend Snippet: Sarcomere structure was assessed in neonatal rat atrial myocytes (NRAMs) transfected with non-targeting (Ctl) or Pitx2c (KD) siRNA. [A] Confocal microscopy of immunofluorescently labelled Myomesin (M-band), F-actin (Z-disk), and DAPI (nuclei). Scale bars depict 50 microns. Quantification of [B] sarcomere length [C] sarcomere organization and [D] M-band density (as a % of total cell area) [A,B] Ctl: n=105 cells, KD: n=106 cells. [D] Ctl: n=124 cells, KD: n=126 cells [E] Confocal microscopy of immunofluorescently labelled Titin, F-actin, and DAPI. Scale bars depict 50 microns and [E’] representation of co-localization of Titin and F-actin signal along one myofibril. [F] Quantification of Titin and F-actin co-localization. Ctl: n=154 cells, KD: 140 cells. Statistical analyses were performed using Student’s t-test [B-D, F] to study differences between groups. Data are expressed as mean ± SEM.

Article Snippet: Primary antibodies used were alpha-Actinin (1:100, Sigma A7732), Myomesin (1:20, DSHB mMac) and Titin (1:100, DHSB 9 D10).

Techniques: Transfection, Confocal Microscopy

Journal: bioRxiv

Article Title: PITX2C Deficiency Promotes Arrhythmogenic Remodeling via Oxidative Stress in Atrial Myocytes

doi: 10.64898/2026.03.27.714813

Figure Lengend Snippet: Neonatal rat atrial myocytes (NRAMs) transfected with non-targeting (Ctl) or Pitx2c (KD) siRNA were treated with 1mM N-acetylcysteine (NAC) for 24 hours. [A] Quantification of mitochondrial specific reactive oxygen species using MitoSOX relative to Ctl cells in Ctl or KD NRAMs. Ctl: 76 cells, KD: 79 cells, KD + NAC: 96 cells. Intracellular calcium rate kinetics were quantified including [B] beat rate, [C] time to peak, [D] time to 90% decay, and [E] time to 50% decay. Early [F] and delayed [G] afterdepolarizations via calcium traces and quantified. Ctl: n=68 cells, KD: n=47, KD + NAC: 72 cells. [H] Quantification of Titin and F-actin co-localization. Ctl: 60 cells, KD: 91 cells, KD + NAC: 76 cells. Statistical analyses were performed using Student’s t-test [A-E, H] or Fisher’s exact test [F,G] to study differences between groups. Data are expressed as mean ± SEM.

Article Snippet: Primary antibodies used were alpha-Actinin (1:100, Sigma A7732), Myomesin (1:20, DSHB mMac) and Titin (1:100, DHSB 9 D10).

Techniques: Transfection