SMYD2 glutathionylation contributes to degradation of sarcomeric proteins

Abstract

Reactive oxygen species (ROS) contribute to the etiology of multiple muscle-related diseases. There is emerging evidence that cellular stress can lead to destabilization of sarcomeres, the contractile unit of muscle. However, it is incompletely understood how cellular stress induces structural destabilization of sarcomeres. Here we report that glutathionylation of SMYD2 contributes to a loss of myofibril integrity and degradation of sarcomeric proteins mediated by MMP-2 and calpain 1. We used a clickable glutathione approach in a cardiomyocyte cell line and found selective glutathionylation of SMYD2 at Cys13. Biochemical analysis demonstrated that SMYD2 upon oxidation or glutathionylation at Cys13 loses its interaction with Hsp90 and N2A, a domain of titin. Upon dissociation from SMYD2, N2A or titin is degraded by activated MMP-2, suggesting a protective role of SMYD2 in sarcomere stability. Taken together, our results support that SMYD2 glutathionylation is a novel molecular mechanism by which ROS contribute to sarcomere destabilization.

Fig. 1

SMYD2 is glutathionylated in response to ROS. a A scheme for a clickable glutathione approach: a glutathione synthetase mutant (GS M4), which synthesizes azido-glutathione (γGlu-Cys-azido-Ala, N₃-GSH), was expressed in differentiated H9c2 cells. After incubation of azido-Ala, cells were subjected to ROS. Glutathionylated proteins in lysates were identified after click reaction. b In-gel fluorescence detection of glutathionylated proteins. H9c2 cells expressing GS M4 were incubated with azido-Ala (0.6 mM) for 20 h and treated with H₂O₂ or antimycin A (AMA). Collected lysates were then subjected to click reaction with rhodamine-alkyne for fluorescence detection. c Identification of individual glutathionylated proteins. Glutathionylated proteins were subjected to click reaction with biotin-alkyne and pull-downs with streptavidin-agarose, and detected by western blotting with individual antibodies, including SMYD2, Hsp90, actin, and myosin-heavy chain (MHC). d The level of glutathionylation on SMYD2. Lysates were subjected to click reaction with 2-kD PEG-alkyne. The mass shift of SMYD2 was analyzed by Western blotting. The level of glutathionylation on SMYD2 (SMYD2-SSG-N₃) was calculated by dividing the amount of glutathionylated SMYD2 (upper band) by a total amount of SMYD2 (upper and bottom bands) in the blot after click reaction. Data represent the mean ± SD. All data are representative of 3 independent experiments

Fig. 2

SMYD2 is selectively glutathionylated at Cys13. a The structure (PDB: 3RIB) and domains of SMYD2, and an enlarged area around Cys13 in SMYD2. b Sequence alignment around Cys13 of SMYD2 with other members of the SMYD family. c–e In-gel analysis of SMYD2 glutathionylation with azido-glutathione. Purified SMYD2 WT or C13S was mixed with azido-glutathione in vitro and treated with H₂O₂ or diamide for 15 min. SMYD2 glutathionylation was detected by fluorescence (c, d) or a mass shift (e) after click reaction with rhodamine-alkyne or 2-kD PEG-alkyne, respectively. f Quantifying the molar ratio of rhodamine to SMYD2 concentrations after incubation of SMYD2 with oxidized azido-glutathione (N₃-GSSG-N₃) and click reaction with rhodamine-alkyne. Data represent the mean ± SD. g Mass spectrometry analysis of glutathionylated SMYD2. SMYD2 glutathionylated by azido-glutathione was conjugated with biotin-alkyne and digested by trypsin. The digested peptides were enriched by streptavidin-agarose and eluted for MALDI-TOF analysis. h Detection of SMYD2 Cys13 glutathionylation in HEK293 cells stably expressing GS M4 (HEK293/GS M4). After transfection of SMYD2 WT or C13S, cells were incubated with azido-Ala. After inducing glutathionylation, collected lysates were subjected to click reaction with biotin-alkyne before pull-downs with streptavidin-agarose and western blotting. All data are representative of 3 independent experiments

Fig. 3

SMYD2 Cys13 glutathionylation or oxidation decreases cell viability. a Levels of SMYD2 WT and C13S in differentiated H9c2 cells. b Viability of differentiated H9c2 cells expressing SMYD2 WT or C13S after exposure to H₂O₂ (25 µM), AMA, (2 µg/mL), angiotensin II (Ag II, 1 µM), or a nitric oxide (NO) donor (NONOate, 100 µM) for 24 h. c, d Viability of differentiated H9c2 cells with overexpression (c) or knockdown (d) of SMYD2 after treatment of AMA (2 µg/mL) for 24 h. Data represent the mean ± SD, n = 3 independent experiments. Difference is significant by two-way ANOVA followed by Bonferroni’s post-hoc test (b, c) and one-way ANOVA followed by Tukey’s post-hoc test (d), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

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

Fig. 5

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

Fig. 6

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 H₂O₂ 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

Fig. 7

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)