Amelioration of mitochondrial dysfunction in heart failure through S-sulfhydration of Ca2+/calmodulin-dependent protein kinase II

Abstract

Aims: Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) plays a critical role in the development of heart failure and in the induction of myocardial mitochondrial injury. Recent evidence has shown that hydrogen sulfide (H₂S), produced by the enzyme cystathionine γ-lyase (CSE), improves the cardiac function in heart failure. However, the cellular mechanisms for this remain largely unknown. The present study was conducted to determine the functional role of H₂S in protecting against mitochondrial dysfunction in heart failure through the inhibition of CaMKII using wild type and CSE knockout mouse models.

Results: Treatment with S-propyl-L-cysteine (SPRC) or sodium hydrosulfide (NaHS), modulators of blood H₂S levels, attenuated the development of heart failure in animals, reduced lipid peroxidation, and preserved mitochondrial function. The inhibition CaMKII phosphorylation by SPRC and NaHS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds. Interestingly, CaMKII activity was found to be elevated in CSE knockout (CSE^-/-) mice as compared to wild type animals and the phosphorylation status of CaMKII appeared to relate to the severity of heart failure. Importantly, in wild type mice SPRC was found to promote S-sulfhydration of CaMKII leading to reduced activity of this protein, however, in CSE^-/- mice S-sulfhydration was abolished following SPRC treatment.

Innovation and conclusions: A novel mechanism depicting a role of S-sulfhydration in the regulation of CaMKII is presented. SPRC mediated S-sulfhydration of CaMKII was found to inhibit CAMKII activity and to preserve cardiovascular homeostasis.

Keywords: Ca(2+)/calmodulin-dependent protein kinase II; Heart Failure; Hydrogen Sulfide; Mitochondria; S-sulfhydration.

Fig. 1

H₂S donor had protective effect against heart failure. (A) Treatment with ISO resulted in ventricular dilation and thinning of ventricular wall compared to the vehicle group, which could be improved by SPRC. (B) Masson trichrome stain showed fibrosis of the left ventricular endocardium in each group. The collagen area in each group was quantified by IPP software. (C) Expression levels of Bcl-2, Bax, cleaved caspase 9 (c-CASP 9) and ATPase in ISO-induced heart failure mice treated with SPRC-L (10 mg/kg/d) or SPRC-H (25 mg/kg/d) with or without PAG for 4 weeks. (D) TUNEL-stain showed cardiomyocytes apoptosis in each group. Green: TUNEL staining representing apoptotic cells. Red: Cell stained by PI. Data represent means ± SEM, n = 8–12 for each group. ^**p < 0.01 compared with isoprenaline group, ^##p < 0.01.

Fig. 2

ISO caused more severe HF in CSE^-/-mice. (A) Expression levels of CSE in the heart of wild type (WT) and CSE^-/- mice. (B-C) The echocardiography data showed the changes in ejection fraction, fractional shortening, left ventricular anterior wall systole, left ventricular posterior wall systole, left ventricular internal dimension diastole, left ventricular internal dimension systole, left ventricular end-diastolic volume and left ventricular end-systolic volume in CSE^-/- mice and WT mice treated with or without ISO for 4 weeks. Data represent means ± SEM, n = 8–12 for each group. ^#p < 0.05, ^##p < 0.01, NS = no significance.

Fig. 3

H₂S donor improved cardiac function through CaMKII pathway. (A) Expression levels of CSE, CaMKII, p-CaMKII PLN and p-PLN in ISO-induced HF mice treated with indicated treatments for 4 weeks. (B) Expression levels of CaMKII and ox-CaMKII in ISO-induced HF mice treated with indicated treatments for 4 weeks. (C) The concentration of plasma H₂S in ISO-induced HF mice with indicated treatments for 4 weeks. (D) Quantitative real-time PCR for BNP and ANP. (E) The echocardiography data showed the changes in ejection fraction, fractional shortening, left ventricular internal dimension diastole. SPRC-L = 10 mg/kg/d and SPRC-H = 25 mg/kg/d. PAG: D, L-propargylglycine. KN-93 = 10 μM/kg/d. Data represent means ± SEM, n = 8–12 for each group. ^**p < 0.01 compared with ISO group, ^#p < 0.05, ^##p < 0.01.

Fig. 4

The cardioprotective effect of H₂S donor SPRC through CaMKII pathway relayed on CSE expression. (A) H₂S concentrations of hearts in ISO-induced heart failure with indicated treatments for 4 weeks in WT and CSE^-/- mice. (B) The echocardiographic data in CSE^-/- mice with indicated treatments for 4 weeks. n = 7–9. (C) Expression levels of CaMKII and p-CaMKII in WT and CSE^-/- mice treated with SPRC or NaHS for 4 weeks. Data represent means ± SEM, n = 8–12 for each group. ^*p < 0.05 and ^**p < 0.01 compared with ISO group, ^#p < 0.05, ^##p < 0.01, NS = no significance.

Fig. 5

H₂S donor had protective effect against oxidative through CaMKII pathway. (A) The levels of p-CaMKII and p-PLN in H9c2 cardiomyocytes with indicated treatments. (B) The levels of CaMKII activity were decreased by SPRC in a time-dependent manner. (C) SPRC inhibited CaMKII activity depended on CSE. (D) SPRC and CaMKII inhibitor KN-93 decreased CaMKII activity. (E) The activity of caspase 9 was decreased after treatment with SPRC or KN-93. (F) SPRC and KN-93 increased the mitochondrial membrane potential. Data represent means ± SEM, n = 6. ^*p < 0.05, ^**p < 0.01 compared with H₂O₂ group, ^##p < 0.01.

Fig. 6

H₂S donor regulated CaMKII activity to protect against oxidative stress-induced mitochondrial dysfunctionin vitro. (A) The maximum intracellular Ca²⁺ elevation was decreased by SPRC treatment in H9c2 cardiomyocytes. (B) The change of mitochondria membrane potential in H9c2 cardiomyocytes treated with SPRC. (C) The concentration of ATP in H9c2 cardiomyocytes. (D-F) The activity of SOD and the content of MDA and GSH were measured in the mitochondria isolated from H9c2 cells. (G and H) The activity of caspase 9 and caspase 3 in H9c2 cardiomyocytes. Data represent means ± SEM, n = 6. ^*p < 0.05 and ^**p < 0.01, ^##p < 0.01.

Fig. 7

H₂S donor decreased the CaMKII activity throughS-sulfhydration of CaMKII. (A) H₂S stimulated CaMKII S-sulfhydration in H9c2 cardiomyocytes in a time-dependent manner. (B) SPRC stimulated CaMKII S-sulfhydration in H9c2 cardiomyocytes in a dose-dependent manner. (C) SPRC stimulated CaMKII S-sulfhydration under oxidative stress. (D) Levels of CaMKII S-sulfhydration of hearts in ISO-induced heart failure mice with indicated treatments. DTT: dithiothreitol, SHY-CaMKII: S-sulfhydrated CaMKII. Data represent means ± SEM, n = 6 in vitro, and n = 8–12 for each group in vivo.^##p < 0.01, NS: no significance.

Fig. 8

CaMKII Cys⁶mutant abolished the regulatory effect of H₂S donor on CaMKII activity. (A) The mutant CaMKII (C6S) reduced H₂S donor-induced CaMKII S-sulfhydration in H9c2 cardiomyocytes. (B) The levels of p-CaMKII and p-PLN in H9c2 cardiomyocytes with indicated treatments after transfected with mutation or WT plasmid. (C) The change of mitochondria membrane potential in H9c2 cardiomyocytes treated with mutation or WT plasmid. (D-F) The cell viability, LDH leakage rate and the activity of caspase 9 were measured after transfected with mutation or wild type plasmid. ^##p < 0.01, NS: no significance.

Fig. 9

Signaling pathway underlying the effects of H₂S donor on CaMKII. Opening of mPTP induced by activated CaMKII results in mitochondrial dysfunction and apoptosis of myocardium in heart failure. H₂S donor could release H₂S in myocardium. The S-sulfhydration of CaMKII at cysteine-6 by H₂S could restore the CaMKII activity and mitochondria dysfunction, to decrease apoptosis and oxidative stress in heart failure.

Fig. S1

SPRC could release H₂S depending on CSE. CSE is a H₂S generating enzyme relies on pyridoxal 5′-phosphate (PLP). In cell free buffer, SPRC could release H₂S only in the presence of CSE and PLP. Data represent means ± SEM, n = 6 for each group.

Fig. S2

SPRC had protective effect against heart failure. (A) Administration of H₂S donor SPRC (25 mg/kg/d) increased survival rate of mice compared with isoprenaline (ISO)-treated. n = 30. (B) H&E staining of sections from the left ventricular myocardium of ISO-induced heart failure mice subjected to SPRC, enalapril or PAG for 4 weeks.

Fig. S3

H₂S donor protected against oxidative stress and mitochondrial dysfunction in heart failure. (A-B) Activities of SOD and contents of MDA in left ventricular tissues of different groups treated with SPRC-L (10 mg/kg/d) or SPRC-H (25 mg/kg/d) with or without PAG in ISO-induced heart failure mice for 4 weeks. (C) The concentration of ATP in ISO-induced heart failure mice treated with SPRC or PAG for 4 weeks.

Fig. S4

SPRC could not improve heart tissue injury induced by ISO in CSE^-/- mice. (A) H&E staining of sections from the left ventricular myocardium with indicated treatments for 4 weeks in CSE^-/- mice. (B) Masson trichrome stain showed fibrosis of the left ventricular endocardium in each group.

Fig. S5

H₂S donor regulated CaMKII activity to protect against oxidative stress-induced cell death. (A and B) After H₂O₂ treatment with or without H₂S donor, the cell viability and the number of apoptotic cells were measured.

Fig. S6

CaMKII (C6S) mutation did not change the activity of CaMKII. After transfected with mutation or wild type plasmid for 48 h, the protein were extracted and subjected to Western Blot.