N-acetylcysteine reverses diastolic dysfunction and hypertrophy in familial hypertrophic cardiomyopathy

Abstract

S-glutathionylation of cardiac myosin-binding protein C (cMyBP-C) induces Ca(2+) sensitization and a slowing of cross-bridge kinetics as a result of increased oxidative signaling. Although there is evidence for a role of oxidative stress in disorders associated with hypertrophic cardiomyopathy (HCM), this mechanism is not well understood. We investigated whether oxidative myofilament modifications may be in part responsible for diastolic dysfunction in HCM. We administered N-acetylcysteine (NAC) for 30 days to 1-mo-old wild-type mice and to transgenic mice expressing a mutant tropomyosin (Tm-E180G) and nontransgenic littermates. Tm-E180G hearts demonstrate a phenotype similar to human HCM. After NAC administration, the morphology and diastolic function of Tm-E180G mice was not significantly different from controls, indicating that NAC had reversed baseline diastolic dysfunction and hypertrophy in our model. NAC administration also increased sarco(endo)plasmic reticulum Ca(2+) ATPase protein expression, reduced extracellular signal-related kinase 1/2 phosphorylation, and normalized phosphorylation of phospholamban, as assessed by Western blot. Detergent-extracted fiber bundles from NAC-administered Tm-E180G mice showed nearly nontransgenic (NTG) myofilament Ca(2+) sensitivity. Additionally, we found that NAC increased tension cost and rate of cross-bridge reattachment. Tm-E180G myofilaments were found to have a significant increase in S-glutathionylation of cMyBP-C, which was returned to NTG levels upon NAC administration. Taken together, our results indicate that oxidative myofilament modifications are an important mediator in diastolic function, and by relieving this modification we were able to reverse established diastolic dysfunction and hypertrophy in HCM.

Keywords: S-glutathionylation; cardiac myosin-binding protein C; diastolic dysfunction; oxidative stress; sarcomeres.

Fig. 1.

N-acetylcysteine (NAC) reduces ERK1/2 phosphorylation but not β-myosin heavy chain expression. A: tropomyosin (Tm)-E180G mice display increased ERK1/2 phosphorylation, which is reduced to nontransgenic (NTg) levels with NAC administration. B: representative Western blot images of phosphorylated (p)-ERK1/2 and total ERK1/2. C: Tm-E180G mice have increased β-myosin heavy chain expression, which is not reduced with NAC administration. D: representative 6% SDS-PAGE gel image showing β-myosin heavy chain expression. Data were analyzed by 2-way ANOVA, followed by Tukey's post hoc test for multiple comparisons and represented as means ± SE; n = 3–6 hearts/group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 2.

NAC alters expression and posttranslational modification of Ca²⁺ regulatory proteins. A: NAC-administered mice have significantly increased sarco(endo)plasmic reticulum Ca²⁺ ATPase 2 (SERCA2) expression over controls. B: phosphorylation of phospholamban (PLN) at Ser¹⁶ is unchanged in all groups. C: p-PLN at Thr¹⁷ is significantly increased in Tm-E180G mice and is returned to NTg levels following NAC administration. D: representative Western blot images of SERCA2, p-PLN Ser¹⁶, p-PLN Thr¹⁷, and total PLN. Data were analyzed by 2-way ANOVA followed by Tukey's post hoc test for multiple comparisons and represented as means ± SE; n = 3–6 hearts/group. *P ≤ 0.05; **P ≤ 0.01.

Fig. 3.

NAC desensitizes the myofilament to Ca²⁺ in NTg and Tm-E180G myofilaments. A: comparison of pCa-tension relationships of NTg, NTg NAC, Tm-E180G, and Tm-E180G NAC mice. B: comparison of Ca²⁺ sensitivity of ATPase rates among NTg, NTg NAC, Tm-E180G, and Tm-E180G NAC mice. Data are represented as means ± SE; n = 6–7 fibers, 3–5 hearts, per group.

Fig. 4.

NAC increases cross-bridge kinetics in NTg and Tm-E180G myofilaments. A: in untreated NTg and Tm-E180G mice, there is no difference in tension cost. Upon NAC administration, there was a significant increase in tension cost of both NTg and Tm-E180G mice. B: NAC increases the rate of tension redevelopment (k_tr) in both NTg and Tm-E180G mice. Data are represented as means ± SE; n = 6–7 fibers, 3–5 hearts, per group.

Fig. 5.

NAC alters posttranslational modification of myofilament proteins. A: phosphorylation of cTnI in Tm-E180G mice was decreased compared with NTg NAC mice. B: phosphorylation of cMyBP-C was increased in Tm-E180G mice. C: representative Western blot images of p-cMyBP-C Ser²⁸², total cMyBP-C, p-cTnI Ser^23/24, and total cTnI. Data were analyzed by 2-way ANOVA followed by Tukey's post hoc test for multiple comparisons and represented as means ± SE; n = 4–6 hearts/group. *P ≤ 0.05.

Fig. 6.

NAC decreased S-glutathionylation of cMyBP-C and tropomyosin. cMyBP-C glutathionylation was significantly increased in Tm-E180G mice, which was reduced to NTg control and NTg NAC-levels after NAC administration. Tropomyosin S-glutathionylation was significantly reduced in NTg NAC- and Tm-E180G NAC-administered mice compared with NTg and Tm-E180G controls. Data were analyzed by 2-way ANOVA followed by Tukey's post hoc test for multiple comparisons and represented as means ± SE; n = 4 hearts/group. *P ≤ 0.05; **P ≤ 0.01.

Fig. 7.

NAC decreases myosin heavy chain carbonylation. Myosin heavy chain carbonylation was increased significantly in Tm-E180G mice, which was reduced to NTg control and NTg NAC levels after NAC administration. Actin, troponin T, and Tm carbonylation were unchanged. Data were analyzed by 2-way ANOVA followed by Tukey's post hoc test for multiple comparisons and represented as means ± SE; n = 3 hearts/group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.