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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2019 Apr 29;177(4):836–856. doi: 10.1111/bph.14601

Hydrogen sulfide regulates muscle RING finger‐1 protein S‐sulfhydration at Cys44 to prevent cardiac structural damage in diabetic cardiomyopathy

Xiaojiao Sun 1,*, Dechao Zhao 2,*, Fangping Lu 1, Shuo Peng 1, Miao Yu 1, Ning Liu 1, Yu Sun 1, Haining Du 1, Bingzhu Wang 1, Jian Chen 1, Shiyun Dong 1, Fanghao Lu 1,✉, Weihua Zhang 1,3,✉
PMCID: PMC7024715  PMID: 30734268

Abstract

Background and Purpose

Hydrogen sulfide (H2S) plays important roles as a gasotransmitter in pathologies. Increased expression of the E3 ubiquitin ligase, muscle RING finger‐1 (MuRF1), may be involved in diabetic cardiomyopathy. Here we have investigated whether and how exogenous H2S alleviates cardiac muscle degradation through modifications of MuRF1 S‐sulfhydration in db/db mice.

Experimental Approach

Neonatal rat cardiomyocytes were treated with high glucose (40 mM), oleate (100 μM), palmitate (400 μM), and NaHS (100 μM) for 72 hr. MuRF1 was silenced with siRNA technology and mutation at Cys44. Endoplasmic reticulum stress markers, MuRF1 expression, and ubiquitination level were measured. db/db mice were injected with NaHS (39 μmol·kg−1) for 20 weeks. Echocardiography, cardiac ultrastructure, cystathionine‐γ‐lyase, cardiac structure proteins expression, and S‐sulfhydration production were measured.

Key Results

H2S levels and cystathionine‐γ‐lyase protein expression in myocardium were decreased in db/db mice. Exogenous H2S reversed endoplasmic reticulum stress, including impairment of the function of cardiomyocytes and structural damage in db/db mice. Exogenous H2S could suppress the levels of myosin heavy chain 6 and myosin light chain 2 ubiquitination in cardiac tissues of db/db mice, and MuRF1 was modified by S‐sulfhydration, following treatment with exogenous H2S, to reduce the interaction between MuRF1 and myosin heavy chain 6 and myosin light chain 2.

Conclusions and Implications

Our findings suggest that H2S regulates MuRF1 S‐sulfhydration at Cys44 to prevent myocardial degradation in the cardiac tissues of db/db mice.

Linked Articles

This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc

What is already known

  • The E3 ubiquitin ligase MuRF1 plays a critical role in the regulation of cardiomyopathy.

What this study adds

  • H2S regulates MuRF1 S‐sulfhydration at Cys44 to prevent myocardial degradation in db/db mice.

What is the clinical significance

  • Increased S‐sulfhydration of MuRF1 by H2S may be a useful therapeutic strategy in diabetic cardiomyopathy.

Abbreviations

4‐PBA

sodium phenylbutyrate

C‐7Az

7‐azido‐4‐methylcoumarin

CSE

cystathionine‐γ‐lyase

DCM

diabetic cardiomyopathy

EF

ejection fraction

ER

endoplasmic reticulum

FS

fractional shortening

HG

high glucose

KEGG

Kyoto Encyclopedia of Genes and Genomes

MuRF1

muscle RING finger‐1

MYH6

myosin heavy chain 6

MyL2

myosin light chain 2

NRCMs

neonatal rat cardiomyocytes

Ole

oleate

Pal

palmitate

PPG

propylene glycol

PYR41

4[4‐(5‐nitro‐furan‐2‐ylmethylene)‐3,5‐dioxo‐pyrazolidin‐1‐yl]‐benzoic acid ethyl ester

SSP

SSP4 (3′, 6′‐di(O‐thiosalicyl)fluorescein)

UPR

unfolded protein response

1. INTRODUCTION

The prevalence of diabetes mellitus in the world has increased in the past 20 years and is currently estimated at 9% in the adult population. Patients with diabetes have a twofold to fourfold increased risk of suffering heart disease (Danaei et al., 2011). Increased free fatty acids and hyperglycaemia enhance malignant effects on the heart, causing cardiac dysfunction and structural damage that together constitute a distinct disease state, called diabetic cardiomyopathy (DCM; From et al., 2006; Sorrentino et al., 2017). Diabetic hearts have to increase fatty acid oxidation to maintain ATP production, which is accompanied by enhanced ROS generation and the ROS have adverse effects on cardiac functions (Ni et al., 2016). The main pathophysiological feature of DCM includes a shift in metabolic substrates at the early stage followed by increased ROS generation and induced structural damage, leading to end‐stage cardiac diastolic and systolic dysfunction (Zhao & Yang, 2017).

The protein muscle RING finger‐1 (MuRF1) is a ubiquitin ligase initially identified for its role in the regulation of skeletal muscle atrophy and cardiomyopathy (Bodine et al., 2001). MuRF1 is expressed in striated muscle specifically and is limited to the M‐line and cytoplasm of cardiomyocytes, which have been found to degrade sarcomere proteins, including troponin I, troponin T, and titin (Centner et al., 2001). Several studies have demonstrated that increased ROS production increases MuRF1 expression, which is associated with the degradation of skeletal muscle (Maejima et al., 2014; Sato, Asano, Isono, Ito, & Asano, 2014; Sha et al., 2014).

http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=9532 has been widely recognized in recent years as having physiological importance as an endogenous gas and is accepted as the third gasotransmitter after NO and carbon monoxide (Calvert, Coetzee, & Lefer, 2010; Fu et al., 2012). Increasing evidence suggests that H2S is involved in a wide range of physiological and pathological processes in the cardiovascular system, such as regulation of BP and reduced production of myocardial ROS (Stubbert et al., 2014; Untereiner et al., 2016). Our previous study showed that exogenous H2S could facilitate the clearance of ubiquitin aggregates by promoting autophagy, which contributed to its antioxidative effects in DCM (J. Wu et al., 2017). Nevertheless, the pathogenesis of DCM is complex, and the precise mechanism by which H2S may protect against DCM has not been thoroughly elucidated (Popov, 2013). To date, no information exists on the possible role of H2S in protecting the intact cardiac structure in DCM. The aim of the present study was to investigate whether H2S can modulate MuRF1 S‐sulfhydration to attenuate the cardiac structural damage occurring in DCM.

2. METHODS

2.1. Experimental animals

All animal care and experimental procedures complied with the guidelines of China National Institute of Health (http://www.most.gov.cn/fggw/zfwj/zfwj2006/200609/t20060930_54389.htm) and were approved by the Animal Ethical Committee of Harbin Medical University. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology. Female, leptin receptor‐deficient (db/db) mice (RRID:IMSR_JAX:000697) on a C57BL/6 (RRID:MGI:5657888) background (n = 90, 8–10 weeks old) and their corresponding wild‐type littermates (n = 30) were purchased from the Animal Model Institute of Nanjing (Nanjing, China). The animals were housed under diurnal lighting conditions (12h light and 12h dark) and fed standard mouse chow and water throughout the study period. Half of the db/db mice and their wild‐type littermates were treated with http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6278 (39 μmol·kg−1; i.p.) every 2 days for 20 weeks (Yang et al., 2008).

2.2. Echocardiography analysis

Cardiac functions of mice were assessed using an echocardiography system (GE VIVID7 10S, St. CT., Fairfield, USA) after 6, 12, and 20 weeks of treatment by NaHS. Mice were lightly anaesthetized with tribromoethanol (Avertin; 240 mg·kg−1, i.p.) and the mouse body temperature was maintained as close to 37°C as possible during the entire process (Papaioannou & Fox, 1993). Left ventricular parameters were measured including ejection fraction (EF, %) and fractional shortening (FS, %).

2.3. Transmission electron microscopy

The mice were killed with an overdose of anaesthetic and left ventricular cardiac tissues were collected and fixed in 2.5% glutaraldehyde in 0.1‐M sodium phosphate buffer (pH 7.3) at 4°C overnight and then treated with 1% osmium tetroxide for 2 hr. The left ventricular cardiac tissues were stained en bloc with saturated uranyl acetate for 1 hr and dehydrated in ethanol, embedded in epoxy resin. Ultrathin sections were cut on a Reichert Ultracut (Leica, Nussloch, Germany), and the ultrathin sections were stained with uranyl acetate and lead citrate by standard methods. All ultrastructural analyses were performed in a blinded and non‐biased manner from photomicrographs captured using the Philips CM120 electron microscope.

2.4. Staining of F‐actin

To assess the integrity of myocardial fibres, cardiac tissues were stained with phalloidin (Solarbio, China; Watkins, Borthwick, & Arthur, 2011). Briefly, hearts were washed with cold PBS to remove blood and then frozen in cold isopentane and stored at ‐80°C for less than a week. Frozen sections (5‐10 μm) were incubated with 20‐μM phalloidin in PBS for 30 min and were then washed three times with PBS. The nuclei were labelled with DAPI (1:1,000, Solarbio, China). Visualization of filamentous actin (F‐actin) in the frozen sections of mouse hearts was carried out using a fluorescence microscope (Olympus, XSZ‐D2, Tokyo, Japan) with excitation by a 495 nm laser.

2.5. Measurement of H2S level in cardiac tissues

The levels of H2S in the frozen cardiac tissues were determined by fluorescence, using 7‐azido‐4‐methylcoumarin (C‐7Az; Sigma, St Louis, MO), which is known to respond selectively to H2S (B. Chen et al., 2013). Cardiac tissues were incubated with 50‐μmol·L−1 C‐7Az PBS for 30 min followed by washing with PBS three times. Visualization of the turn‐on fluorescence response of C‐7Az to H2S in cardiac tissues was implemented using fluorescence microscopy with the excitation of a 720 nm laser. These results confirmed that excitation fluorescence imaging could be used to detect H2S through the triggered fluorescence response of C‐7Az.

2.6. Immunohistochemistry

The antibody‐based procedures used here comply with the recommendations made by the British Journal of Pharmacology. Isolated cardiac tissues from db/db mice and wild‐type mice were washed with PBS and fixed in 4% paraformaldehyde overnight before being embedded in paraffin. Embedded left ventricles were cut into 4 μm sections to demonstrate the structure of the cardiac muscle. Sections were stained with haematoxylin/eosin for the detection of morphology.

2.7. Culture of neonatal rat cardiomyocytes and treatment

Neonatal rat cardiomyocytes (NRCMs) were isolated from 1‐ to 3‐day‐old Sprague Dawley rats (RRID:RGD_70508) by enzymic digestion with 0.25% trypsin (Sigma). Briefly, neonatal Sprague Dawley rats was anaesthetized (isoflurane) and fixed on a platform, the skin disinfected with a medical cotton ball, soaked in 75% medical alcohol. The whole heart was excised and placed in cold PBS, washing to remove the blood. The left heart was isolated, cut into pieces and the pieces of cardiac tissue were digested with 0.25% trypsin in a water bath at 37°C for 5–10 min. This step of digestion was repeated eight times. Finally, the digestion was terminated by adding the same volume of DMEM (HyClone, USA) containing 10% FBS (NQBB, Australia). The mixture contained NRCMs was centrifuged in 1,000× g at 4°C for 10 min, the supernatant removed and the cell pellet resuspended in medium. The cell suspension was then allowed to attach for 1.5 hr and the non‐attached cells (NRCMs) were washed off and collected. The NRCMs were plated onto 35‐mm dishes at a density of 1 × 106 cells·ml−1 for a monolayer and cultured in low glucose (5.56 mM) DMEM (HyClone) containing 10% FBS (NQBB) and 100‐units·ml−1 penicillin/streptomycin for 72 hr before use. The cultured NRCMs were randomly divided into several groups as follows: control (low glucose, 5.5 mM), high glucose (HG; 40 mM) + http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1055 (Pal, 400 μM) + http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1054 (Ole, 100 μM), HG + Pal + Ole + NaHS (100 μM), HG + Pal + Ole + PPG (10 nM, an irreversible competitive http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=279#1444 inhibitor), HG + Pal + Ole + N‐acetyl cysteine (NAC; 100 μM, an inhibitor of ROS), HG + Pal + Ole + http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=8616 (20 μM, an inhibitor of proteasome activity), HG + Pal + Ole + NaHS+ DTT (20 μM, an inhibitor of disulfide bonds), HG + Pal + Ole + PYR41 (3 μM, an inhibitor of ubiquitin‐activating enzyme [E1]), HG + Pal + Ole + 4‐PBA (5 mM, an inhibitor of endoplasmic reticulum [ER] stress), and control + http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5351 (100 μM, an inducer of ER stress). Drugs were added directly into the culture for 48 hr. NRCMs were treated with HG, palmitate and oleate to model the myocardiocytes under conditions of hyperglycaemia and hyperlipidaemia, as in diabetes.

2.8. ROS level analysis

The NRCMs were treated with HG (40 mM) + Pal (400 μM) + Ole (100 μM), HG + Pal + Ole + NaHS (100 μM), HG + Pal + Ole + PPG (10 nM), or HG + Pal + Ole + NAC (100 μM) for 2, 4, 8, and 12 hr, respectively. The production of ROS was measured by DHE or DCFH. Briefly, the NRCMs were inoculated into 24‐well plate and treated under different conditions. The NRCMs were washed with PBS three times and then incubated (45 min, 37°C, in the dark) in serum‐free media containing DHE (25 μM) or DCFH (10 μM). After incubation, NRCMs were washed and the DHE or DCFH retained was detected with a fluorescence microscope (Olympus, XSZ‐D2).

2.9. Western blotting

NRCMs or heart tissues were lysed in cell lysis buffer (Beyotime, Shanghai, China) or RIPA (Beyotime) separately, containing 1% PMSF. The protein lysates were centrifuged at 4°C, 12,000× g for 30 min. The supernatant was collected, and the protein concentration of each sample was evaluated by the BCA Protein Assay kit (Beyotime). Proteins were separated by SDS‐PAGE and then transferred onto PVDF membranes (Millipore). After blocking with 5% BSA at room temperature, the membranes were blotted with primary antibodies overnight at 4°C as follows: rabbit‐anti‐CSE (ProteinTech group, 12217‐1‐AP, 1:500, RRID:AB_2087497), rabbit‐anti‐ATF4 (ProteinTech group, 10835‐1‐AP, 1:1000, RRID:AB_2058600), rabbit‐anti‐Bip (ProteinTech group, 11587‐1‐AP, 1:1000, RRID:AB_2119855), rabbit‐anti‐CHOP (ProteinTech group, 15204‐1‐AP, 1:1000, RRID:AB_2292610), rabbit‐anti‐p‐PERK (Santa Cruz Biotechnology, sc32577, 1:500, RRID:AB_2293243), rabbit‐anti‐PERK (ProteinTech group, 20582‐1‐AP, 1:1000, RRID:AB_10695760), rabbit‐anti‐p‐eIF2α (Cell Signaling Technology, 3398S, 1:1000, RRID:AB_2096481), rabbit‐anti‐eIF2α (ProteinTech group, 11233‐1‐AP, 1:1000, RRID:AB_2246321), rabbit‐anti‐myosin heavy chain 6 (MYH6; ProteinTech group, 22281‐1‐AP, 1:1000, RRID:AB_2736822), rabbit‐anti‐myosin light chain 2 (MyL2; ProteinTech group, 10906‐1‐AP, 1:1000, RRID:AB_2147453), rabbit‐anti‐MuRF1 (ProteinTech group, 55456‐1‐AP, RRID:AB_11232209, 1:1000), and rabbit‐anti‐ubiquitin (ProteinTech group, 10201‐2‐AP, 1:1000, RRID:AB_671515). β‐Tubulin (ProteinTech group, 10068‐1‐AP, 1:1000, RRID:AB_2303998) was used as control. Membranes were washed with PBS three times and then incubated with the corresponding secondary antibodies at a 1:5,000 dilution for 1.5 hr at room temperature. The specific complex was visualized using ECL plus luminescent image analyser (ProteinSample).

2.10. S‐sulfhydration assay

S‐sulfhydration was performed as described previously (Meng, Zhao, Xie, Han, & Ji, 2018). Briefly, NRCMs or heart tissues were homogenized in HEN buffer (composition; 250‐mM HEPES‐NaOH, pH 7.7: 1‐mM EDTA; 0.1‐mM neocuproine), supplemented with 100‐mM deferoxamine and centrifuged at 13,000× g for 30 min at 4°C. NRCMs or heart tissue lysates were added to blocking buffer (HEN buffer with 2.5% SDS and 20‐mM methyl methanethiosulfonate) at 50°C for 60 min with frequent vortexing. Methyl methanethiosulfonate was later removed by acetone, and the proteins were precipitated at −20°C for 60 min. After acetone removal, proteins were resuspended in HENS buffer (HEN buffer with 1% SDS). To the suspension was added 4‐mM N‐[6‐(biotinamido)hexyl]‐3‐(2‐pyridyldithio) propionamide (biotin‐HPDP) in DMSO without ascorbic acid. After incubation for 3 hr at 25°C, biotinylated proteins were precipitated by streptavidin‐agarose beads, which were later washed with HENS buffer. The biotinylated proteins were eluted by SDS‐PAGE and subjected to Western blotting analysis using antibodies against MuRF1.

2.11. Measurement of intracellular levels of polysulfide

Intracellular production of polysulfide was monitored using a newly developed fluorescent probe, SSP4, with slight modifications (Kimura et al., 2015). Briefly, NRCMs were loaded with 50‐μM SSP4 in a serum‐free DMEM containing 0.003% Cremophor EL for 15 min at 37°C in the dark. After being washed, SSP4 was detected using the fluorescence microscope (Olympus, XSZ‐D2).

2.12. Immunoprecipitation

Briefly, NRCMs or heart tissue lysates were diluted to a concentration of 2 mg·ml−1. About 500 μg per sample of protein was used for immunoprecipitation. Protein A/G magnetic beads for immunoprecipitation were conjugated with rabbit‐anti‐MuRF1 antibody (10‐μg antibody per 500‐μg protein) and incubated with NRCMs or heart tissue lysates overnight at 4°C with gentle rotation. Beads were collected using centrifugation at 4°C, 10,000× g for 5 min, and beads were washed with cell lysis buffer containing 1% PMSF, three times. The precipitates were diluted with loading buffer and boiled for 10 min at 100°C and later used for Western blotting analyses to detect potential interacting proteins.

2.13. siRNA transfection

NRCMs were treated according to the manufacturer's instructions with MuRF1 small‐interfering RNA (siRNA; mouse; Sangon Biotech, China) for 48 hr to inhibit MuRF1 expression. NRCMs (70% confluent) were seeded in six‐well plates, incubated with DMEM containing 10% FBS without penicillin/streptomycin. Transfection of NRCMs by siRNA was achieved using Lipofectamine RNAi MAX (Invitrogen). In brief, MuRF1 siRNA with the transfection reagent was incubated for 15 min to form complexes, which were subsequently added to plates containing cells and medium. The cells were incubated at 37°C in a CO2 incubator for further analysis.

2.14. LC–MS/MS analysis

To identify the lysine ubiquitylome of three cardiac tissues of db/db mice after the treatment of NaHS, the above extracted proteins were digested with proteases and later subjected to Kub peptide enrichment. To enrich Kub peptide, tryptic peptides dissolved in NETN buffer (100‐mM NaCl, 1‐mM EDTA, 50‐mM Tris–HCl, 0.5% NP‐40, pH 8.0) were incubated with pre‐washed antibody beads (PTM Biolabs) at 4°C overnight with gentle shaking. The beads were washed four times with NETN buffer and twice with double‐distilled H2O. The bound peptides were eluted from the beads with 0.1% TFA. The eluted fractions were combined and vacuum‐dried. The resulting peptides were cleaned with C18 ZipTip (Millipore) according to the manufacturer's instructions followed by LC–MS/MS analysis (Pang et al., 2015; Yu et al., 2014). The resulting peptides were then analysed by nano‐HPLC–MS/MS on a Q Exactive mass spectrometer (Thermo Fisher Scientific). Minimum peptide length was set to 7. All of the other parameters in MaxQuant were set to default values. The site localization probability was set as >0.75.

2.15. Bioinformatic analysis

Pathway and functional annotation enrichment was performed using DAVID bioinformatics resources (RRID:SCR_001881). Cellular localization of identified proteins was further analysed on the basis of information available from Gene Ontology (http://www.geneontology.org/). Biological function classifications and signalling pathway analysis were performed with the tools on DAVID Bio‐informatics Resources 2011 (http://david.abcc.ncifcrf.gov/). All statistics reported are Benjamini–Hochberg corrected P values. Molecular function categorization was performed using PANTHER (RRID:SCR_004869). Statistical analysis of enriched motifs was performed using motif‐x. Network analysis was performed using Ingenuity Pathway Analysis (RRID:SCR_008653).

2.16. Computational method

To date, the three‐dimensional structure of MuRF1 has not been resolved; therefore, homology modelling, a promising tool to predict protein structure based on a template containing similar sequence, was applied in this study using Phrye2. The sequence of MuRF1 was obtained from Uniprot (RRID:SCR_002380). According to a sequence alignment by BLAST (RRID:SCR_004870), the crystal structure and active site of MuRF1 were calculated by the website of protein plus (RRID:SCR_005375).

2.17. Point mutation of MuRF1

Adenoviruses expressing GFP and MuRF1‐GFP were purchased from Cyagen Biosciences Inc. (Guangzhou, China). The full‐length mouse MuRF1 with a single mutation of Cys44 to alanine, and GFP cDNA was inserted into pM vector (Cyagen Biosciences) between the Kozak and T2A sites. The adenovirus was added directly to NRCMs, and after 4–6 hr for transfection, new fresh medium was added. The NRCMs were treated in different conditions 24 hr after transfection, and the related proteins were detected by Western blotting.

2.18. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology. All data are presented as the mean ± SD. Data were analysed using one‐way ANOVA test followed by Bonferroni post hoc tests, as appropriate. The threshold of P < 0.05 was designated statistically significant for all tests. All statistical analyses were performed using Prism 5 (GraphPad, La Jolla, CA, USA; RRID:SCR_002798).

2.19. Materials

Compounds used in these experiments were supplied as follows: 4‐PBA, biotin‐HPDP, deferoxamine, NAC, NaHS, MG‐132, oleate, palmitate, PPG, PYR41, thapsigargin and tribromoethanol were from Sigma (St Louis, MO). SSP was supplied by Dojindo Molecular Technologies Inc. (Rockville, MD). DHE and DHCF were supplied by Boster (Wuhan, China).

2.20. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017).

3. RESULTS

3.1. Exogenous H2S improves DCM in db/db mice

To investigate the effects of H2S on DCM, the ratio of heart weight/body weight and blood glucose level were measured. We found that the ratio of heart weight/body weight and blood glucose level both significantly increased in db/db mice compared with those in db/db + NaHS after 20 weeks (Tables S1–S3). To further characterise the effects of H2S on cardiac function, we used echocardiography and found that EF and FS decreased at 12 and 20 weeks in db/db mice, whereas they were improved after treatment with NaHS (Figure 1a). The transmission electron microscopy assay indicated that exogenous H2S reduced the degradation of cardiac sarcomeres compared with those in db/db mice at 12 and 20 weeks (Figures 1b and S1). To further confirm the effect of H2S on cardiac structure, we assayed F‐actin through phalloidin staining (green). The results showed that F‐actin was degraded in db/db mice and exogenous H2S prevented this degradation (Figure 1c). These results indicate that exogenous H2S exerted protective effects against DCM.

Figure 1.

Figure 1

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Exogenous H2S protected cardiac function and cardiac ultrastructure in db/db mice. Female db/db mice and their wild‐type littermates were injected with NaHS (39 μmol·kg−1) or saline every 2 days for 20 weeks. (a) Representative M‐mode echocardiograms of hearts (left) and quantitative analysis of EF and FS by echocardiography (right) at 6, 12, and 20 weeks treated with NaHS are shown. (b) Cardiac ultrastructure of mice was examined with transmission electron microscope at 6, 12, and 20 weeks treated with NaHS,. Bar = 2 μm. (c) Cardiac structure was detected by phalloidin staining (green) of F‐actin at 6, 12, and 20 weeks treated with NaHS,. Data shown are means ±SD; n = 8, *P < 0.05, significantly different as indicated; one‐way ANOVA

3.2. H2S level and CSE expression in db/db mice

The levels of H2S and CSE expression declined in db/db mice (Sun et al., 2018; J. Wu et al., 2017). The results showed that H2S level and expression of CSE were reduced at 12 and 20 weeks in db/db mice but there was no significant change at 6 weeks, as shown C‐7Az probe staining and Western blotting, respectively (Figure 2). Exogenous H2S enhanced the H2S level and CSE expression in the hearts of db/db mice, demonstrating that the change in H2S is relevant to the maintenance of cardiac structure.

Figure 2.

Figure 2

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H2S level and CSE expression was decreased in db/db mice. (a) 7‐Azido‐4‐methylcoumarin staining was used to measure H2S levels in cardiac tissues at 20 weeks, with and without NaHS. (b–d) Quantification of CSE expression in cardiac tissues was carried out by Western blotting at 6, 12, and 20 weeks treated with NaHS, respectively. Data shown are means ±SD; n = 6, *P < 0.05, significantly different as indicated; one‐way ANOVA

3.3. Exogenous H2S ameliorated ER stress in vivo and in vitro

It is well known that H2S has antioxidant effects. NRCMs were cultured with HG, palmitate and oleate to model the hyperglycaemia and hyperlipidaemia of Type 2 diabetes (S. Li et al., 2016). Under these conditions, we assayed ROS production in NRCMs, using DHE and DCFH‐DA staining. Production of ROS increased in the HG + Pal + Ole group compared with the control group. Exogenous H2S and NAC (another antioxidant) reduced ROS production, while the CSE inhibitor, PPG, up‐regulated ROS production (Figure S2). To investigate whether H2S could suppress ER stress in db/db mice, we measured the expression of ER stress‐associated proteins, p‐PERK (Thr981)/PERK, Bip, ATF4, p‐eIF2α(Ser51)/eIF2α, and CHOP, These marker proteins were up‐regulated in 12‐ and 20‐week‐old db/db mice but exogenous H2S reversed these alterations (Figure 3a,b). To further study whether H2S could suppress ER stress in vitro, ER stress‐associated proteins were detected in NRCMs. We found that exogenous H2S also attenuated ER stress in vitro (Figure 3c). NAC (an ROS scavenger) treatment also decreased ER stress effect, like NaHS, indicating the suppressioin of ER stress by H2S might be related to its antioxidative effect.

Figure 3.

Figure 3

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Effect of exogenous H2S on levels of ER stress in vivo and in vitro. (a, b) The expression of protein markers of ER stress, in cardiac tissues, was assessed by Western blotting, in db/db mice at 12 and 20 weeks, treated with NaHS. (c) Western blotting analysis the expression of protein markers of ER stress in NRCMs, incubated with HG (40 mM) + palmitate (Pal, 400 μM) + oleate (Ole, 100 μM), HG + Pal + Ole + NaHS (100 μM), HG + Pal + Ole + PPG (10 nM, an irreversible competitive CSE inhibitor), and HG + Pal + Ole + NAC (100 μM, an inhibitor of ROS) for 72 hr. Data shown are means ±SD; n = 6, *P < 0.05, significantly different as indicated; one‐way ANOVA

3.4. Enrichment and clustering analysis of quantitative cardiac structural protein data sets, based on gene ontology annotations

To confirm that the ubiquitination levels of cardiac structural proteins were regulated by NaHS treatment of db/db mice, we analysed the cardiac structural protein proteome data set for four enrichment gene ontology categories: cellular component, molecular function, biological process, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The cellular component category (Figure 4a) showed that NaHS treatment had a marked effect on the expression of cardiac structural proteins. Moreover, the enrichment analysis of molecular functions (Figure 4b) showed that proteins involved in the structural constituent of cytoskeleton, muscle, and motor activity were enriched after NaHS treatment. In the biological process category (Figure 4c), proteins related to regulation of muscle system processes, muscle contraction, regulation of heart contraction, tissue morphogenesis, and tissue regeneration were also enriched with NaHS treatment. To identify cellular pathways regulated by NaHS treatment, we performed a pathway clustering analysis of the H2S‐responsive proteome for pathways from the KEGG (Figure 4d). The results showed that hypertrophic cardiomyopathy, dilated cardiomyopathy, and cardiac muscle contraction were the most prominent pathways enriched in quantiles with increased protein level in db/db mice treated by NaHS, suggesting a role of exogenous H2S in these pathways. In summary, gene ontology enrichment and KEGG pathway‐based cluster analysis results shiowed that the ubiquitination levels of cardiac structural proteins showed many differences in db/db mice from those treated by NaHS based on enrichment.

Figure 4.

Figure 4

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Enrichment and clustering analysis, based on gene ontology annotations, of quantitative data sets of cardiac structural proteins. Using KEGG standard peptides, normalized affinity enrichment followed by label‐free quantitative proteomics strategy, quantitative lysine ubiquitylation analysis was performed in a pair of mouse myocardial tissues. Proteins were classified by GO annotation into three categories: biological process, cellular compartment, and molecular function. For each category, we used the Functional Annotation Tool of DAVID Bioinformatics Resources 6.7 to identify enriched GO against the background of Homo sapiens. A two‐tailed Fisher's exact test was employed to test the enrichment of the protein‐containing IPI entries against all IPI proteins. Correction for multiple hypothesis testing was carried out using standard false discovery rate control methods. The GO with a corrected P‐value <0.05 is considered significant. The quantified Kub proteins in this study were divided, according to the quantification ratio, to generate four quantiles: Q1 (0–15%), Q2 (15–50%), Q3 (50–85%), and Q4 (85–100%). The relative fold change was db/db versus db/db + NaHS. The green colour means Z score <0, and this ratio is less than the average. The red colour means Z score >0, and this ratio is more than the average. (a) Cellular component, (b) molecular function, (c) biological process, and (d) KEGG pathway analysis were identified by bioinformatic analysis

3.5. Exogenous H2S attenuated the degradation of MYH6 and MyL2 in vivo

Our earlier experiments h had demonstrated that there was structural damage in the hearts of db/db mice (Figure 1b). Further analysis showed that the expression of two structural proteins, MYH6 and MyL2, were lower in the cardiac tissues of db/db mice, compared with those of db/db mice treated by NaHS (Figure 5a). The level of ubiquitination induced by ER stress can lead to degradation of cardiac structural proteins (Baskin & Taegtmeyer, 2011; Wiersma et al., 2017; Willis, Schisler, Li, et al., 2009; Willis, Schisler, Portbury, & Patterson, 2009). In our study, the ubiquitination level in the hearts of db/db mice was up‐regulated in untreated mice, compared with that in db/db mice treated by NaHS (Figure 5b). MuRF1, an E3 ubiquitin ligases, is associated with cardiac structure and skeletal muscle degradation (S. N. Chen et al., 2012; Gielen et al., 2012; Kedar et al., 2004; Maejima et al., 2014). Our results revealed that the expression of MuRF1 was increased in the heart of db/db mice and that MuRF1 was decreased after treatment with NaHS (Figure 5c). Further experiments using immunoprecipitation showed that exogenous H2S weakened the interaction between MuRF1 and MYH6 and MyL2 (Figure 5d). Taken together, the present findings indicate that exogenous H2S could attenuate the degradation of cardiac structural proteins by MuRF1 in the hearts of db/db mice.

Figure 5.

Figure 5

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Exogenous H2S protected cardiac structure in db/db mice. (a) The expression of cardiac structural proteins MYH6 and MyL2 was measured with Western blotting at 20 weeks treated with NaHS. (b) The ubiquitination level of cardiac tissues was tested by Western blotting. (c) Quantification of MuRF1 expression in cardiac tissues was carried out by Western blotting. (d) Cardiac tissues lysate were immunoprecipitated with anti‐MuRF1 antibody and then immunoblotted with antibodies specific for MYH6 and MyL2. Data shown are means ±SD; n = 6, *P < 0.05, significantly different as indicated; one‐way ANOVA

3.6. Exogenous H2S down‐regulated the ubiquitination level of cardiac structural proteins by reducing the expression of MuRF1 in vitro

We further investigated whether exogenous H2S protected cardiac structural proteins through down‐regulation of MuRF1. Expression of MuRF1 in NRCMs was increased under the HG + Pal + Ole conditions, and this increased reversed with exogenous H2S. The protein levels of MYH6 and MyL2 declined under HG + Pal + Ole conditions and were restored by exogenous H2S. Two different enzyme inhibitors, MG132 (26S proteasome inhibitor, 25 nM) and PYR41 (E1 ubiquitin‐activating enzyme inhibitor, 3 μM) inhibited the degradation of MYH6 and MyL2 in NRCMs (Figure 6a,b). Moreover, the ubiquitination level declined when E1 ubiquitin‐activating enzyme was inhibited in NRCMs (Figure 6c). There is evidence that ER stress initiates ubiquitinylated degradation (Schlossarek, Frey, & Carrier, 2014; Swatek & Komander, 2016; P. Xu et al., 2009) and we therefore treated NRCMs with thapsigargin (100 μM, an inducer of ER stress) and 4‐PBA (an inhibitor of ER stress) to assess their effects on ubiquitination levels in our model. As expected, ubiquitination levels in NRCMs were raised by thapsigargin, but decreased by 4‐PBA (Figure 6d). Our results also confirmed that the ubiquitination level was decreased in both NaHS and NAC groups. PPG (an irreversible competitive CSE inhibitor) elevated the ubiquitination level (Figure 6e). To analyse how H2S suppressed ubiquitination, immunoprecipitation was used to measure the interaction between MuRF1 and MYH6 and MyL2 in vitro. The results showed that exogenous H2S weakened the interaction between MuRF1 and MYH6 and MyL2 whereas PPG improved the interaction between MuRF1 and MYH6 and MyL2. The interaction between MuRF1 and MYH6 and MyL2 was also enhanced by NAC (Figure 6f). Taken together, those findings demonstrate that exogenous H2S could decrease the expression of MuRF1 and the ubiquitination levels in vitro.

Figure 6.

Figure 6

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Effect of exogenous H2S on ubiquitination level of cardiac structural proteins in neonatal rat cardiomyocytes. (a, b) NRCMs exposed to HG + Pal + Ole conditions for 72 hr and then treated with MG132 (20 μM, an inhibitor of proteasome) for 30 min, or PYR41 (3 μM, an inhibitor of ubiquitin‐activating enzyme [E1]), for 2 hr, Expression of MYH6, MyL2, and MuRF1 were measured with Western blotting. (c) The ubiquitination level in NRCMs treated with PYR41, was assessed by Western blotting. (d) The ubiquitination level was assessed by Western blotting in NRCMs under HG + Pal + Ole conditions, with 4‐PBA (5 mM, an inhibitor of endoplasmic reticulum stress) and control + thapsigargin (Tg, 100 μM, an inducer of endoplasmic reticulum stress). Drugs were added directly into the culture for 48 hr. (e) The ubiquitination level in NRCMs was assessed by Western blotting, under conditions of HG (40 mM) + palmitate (Pal, 400 μM) + oleate (Ole, 100 μM), HG + Pal + Ole + NaHS (100 μM), HG + Pal + Ole + PPG (10 nM, an irreversible competitive CSE inhibitor), and HG + Pal + Ole + NAC (100 μM, an inhibitor of ROS) for 72 hr. (f) NRCMs were immunoprecipitated with anti‐MuRF1 antibody and then immunoblotted with antibodies specific for MYH6 and MyL2. Data shown are means ±SD; n = 6, *P < 0.05, significantly different as indicated; one‐way ANOVA

3.7. MuRF1 siRNA and mutation attenuated the degradation of cardiac structural proteins

To further study the role of MuRF1 in the degradation of cardiac structural proteins, we used MuRF1 siRNA to knock down the expression of MuRF1 in NRCMs,, using scramble siRNA as a negative control (Figure 7a). When the expression of MuRF1 was blocked, the protein level of MYH6 and MyL2 was not reduced (Figure 7b). MuRF1 contains a RING‐type zinc finger region, containing 20‐80 amino acids. We used bioinformatics methods to analyse and predict the structure of the active site of MuRF1. MuRF1 consists of 350 amino acids and contains 17 cysteine residues, and is involved in ubiquitination through to its RING‐type zinc finger domain. The bioinformatics analysis of MuRF1 predicted six active centres. However, only two of active centres had the greatest correlation with cysteine residues. They were the sequences from residues 23 to 79 and from residue 117 to 159 amino acid, and they were exactly RING‐type zinc finger domain and B‐box‐type zinc finger domain. Based on this analysis, we mutated the Cys44 in the RING‐type zinc finger domain to alanine. The Cys44 plays a crucial role in the activity of MuRF1 (Figure 7c). Mutants of MuRF1 at Cys44 were transfected into NRCMs. The expression of MYH6 and MyL2 increased after transfection with the mutant MuRF1, compared with the vector group (Figure 7d). The ubiquitination level declined, as mutant MuRF1 abolished its ubiquitin ligase function (Figure 7e). Immunoprecipitation was used to detect the interaction between MuRF1 and MYH6 and MyL2. The mutant MuRF1 could not interact with MYH6 and MyL2 (Figure 7f). The present studies suggest that MuRF1 plays a pivotal role in the degradation of cardiac structural proteins.

Figure 7.

Figure 7

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Deletion of MuRF1 could attenuate the degradation of cardiac sarcomere. (a, b) After MuRF1 siRNA or scramble siRNA was transfected into NRCMs for 24 hr, the cells were exposed to HG + Pal + Ole conditions for 72 hr. MuRF1, MYH6, and MyL2 protein expressions were measured with Western blotting. (c) The active centre of MuRF1 was predicted by computational method. (d) A mutant MuRF1 (Cys44 to alanine) and the vector control were transfected into NRCMs for 12 hr and then exposed to HG + Pal + Ole conditions in the presence or absence of NaHS (100 μM) for 72 hr. Expression of MYH6, MyL2, and MuRF1 was detected by Western blotting. (e) The ubiquitination level. (f) Extracts of NRCMs were immunoprecipitated with anti‐MuRF1 antibody and then immunoblotted with antibodies specific for MYH6 and MyL2. Data shown are means ±SD; n = 6, *P < 0.05, significantly different as indicated; one‐way ANOVA

3.8. Polysulfidation protected cardiac structural proteins in DCM

Protein polysulfidation plays critical role in post‐translational modification by targeting specific cysteine residue(s) which could change local protein structure and activity (Akaike et al., 2017). Polysulfidation could activate sulfhydryl groups, which are believed to mediate numerous cellular responses initiated by H2S (Greiner et al., 2013; Shimizu et al., 2017). SSP4, a newly developed fluorescent probe, was used to detect the production of polysulfides in the cardiac tissues of db/db mice. Polysulfides were observed in WT + NaHS and db/db + NaHS groups but not in the WT and db/db groups (Figure 8a). Moreover, biotin‐switch assay was also used to detect protein polysulfidation. The results showed that NaHS enhanced polysulfidation formation both in the hearts of WT and db/db mice (Figure 8b). We also identified the effect of H2S on polysulfide formation in vitro. NaHS elevated polysulfidation levels in NRCMs and DTT (1 mM, an inhibitor of polysulfidate formation, which abolished the effect of NaHS on polysulfidation formation (Figure 8c). The biotin‐switch assay also showed that exogenous H2S promoted polysulfidation formation and DTT abolished the effect of NaHS on polysulfidation formation (Figure 8d). After transfection of NRCMs with the Cys44 mutant MuRF1, the polysulfidation of MuRF1 was decreased compared with the vector with NaHS group (Figure 8e). The results indicate that H2S can promote polysulfidation formation and attenuate the degradation of MYH6 and MyL2.

Figure 8.

Figure 8

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Polysulfidation played a crucial role in improvement of DCM in db/db mice. (a) The polysulfidation level was measured with the fluorescent probe, SSP4, in cardiac tissues. Bar = 100 μm. (b) Quantification of polysulfidation in cardiac tissues was carried out by Western blotting. (c, d) Polysulfidation production in NRCMs was assayed, using the fluorescent probe, SSP4, or by Western blotting. DTT (1 mM, 30 min, an inhibitor of disulfide bonds). Bar = 100 μm. (e) Polysulfidation production in NRCMs, transfected with MuRF1‐Cys44 mutant, was assessed by Western blotting. Data shown are means ±SD; n = 6, *P < 0.05, significantly different as indicated; one‐way ANOVA

4. DISCUSSION

In the present study, we found that (a) H2S level and CSE protein expression level in the myocardium were decreased in db/db mice; (b) exogenous H2S reversed ER stress, including impairment of the function of cardiomyocytes and structural damage in db/db mice; (c) exogenous H2S could suppress MYH6 and MyL2 ubiquitination level in cardiac tissues of db/db mice; and (d) MuRF1 was modified by S‐sulfhydration following treatment with exogenous H2S to reduce the interaction between MuRF1 and MYH6 and MyL2. These data suggested that an impaired H2S/CSE system contributed to cardiac structural damage. Moreover, our results revealed the possibility that H2S supplementation might be a significant therapeutic approach to attenuate cardiac damage in diabetic patients.

In diabetic hearts, cardiomyocytes have to increase fatty acid oxidation to maintain ATP production, which is accompanied by increased ROS production, which has adverse effects on cell functions (Pulinilkunnil & Rodrigues, 2006). ROS may damage DNA, proteins, and membrane lipids, leading to significant apoptosis, fibrosis, and heart failure with preserved EF, similar to restrictive cardiomyopathy (Zhang, Perino, Ghigo, Hirsch, & Shah, 2013). This condition subsequently evolves into heart failure with reduced EF, resembling dilated cardiomyopathy. In our study, we found that EF (%) and FS (%) were decreased, the Z‐line was ruptured with a degraded M‐line, and the sarcoplasmic reticulum (SR) was significantly swollen in 20‐week‐old db/db mice. Therefore, db/db mice were considered to be exhibiting cardiomyopathy at 20 weeks.

Hyperglycaemia and hyperlipidaemia enhance oxidative stress, and ROS accumulation may promote protein misfolding and aggregation in the ER, which is called ER stress, and contributes to the aetiology and pathogenesis of Type 2 diabetes. ER protein misfolding activates the unfolded protein response (UPR), a conserved signalling system that initiates many processes to restore proteostasis (Calamai, Chiti, & Dobson, 2005). Our present study demonstrates that hyperglycaemia and hyperlipidaemia induce ER stress in cardiac muscle, contributing to cardiac dysfunctions. Three markers of ER stress (Bip, p‐eIF2α, and p‐PERK) were markedly increased in db/db mice. When the UPR fails to resolve ER stress, maladaptive features of the UPR, called maladaptive stress, are responsible for the tissue damage and organ dysfunction (C. Xu, Bailly‐Maitre, & Reed, 2005). Bip, as a molecular chaperone, regulates the unfolded proteins. PERK and eIF2α constitute the core stress regulator of UPR and transduce signals from ER to the cytosol and nucleus during ER stress (Ron & Walter, 2007). CHOP, which is a transcription factor, decreases Bcl‐2 transcription and induces apoptosis. As our present results have shown, ER stress was induced in db/db mice and in NRCMs exposed to HG + Pal + Ole conditions. However, levels of ER stress can be attenuated by NaHS and NAC, the antioxidant and ROS scavenger. The misfolded proteins are damaged by ER‐associated degradation (Calamai et al., 2005). ER‐associated degradation is a four‐step quality control process for removing misfolded proteins from the ER by the cytosolic ubiquitin‐proteasome system (Olzmann, Kopito, & Christianson, 2013). Ubiquitin is attached to a substrate lysine, through an enzymic cascade, which includes an ubiquitin‐activating enzyme (E1), an ubiquitin‐conjugating enzyme (E2), and an ubiquitin‐protein ligase (E3; C. Chen et al., 2014). E1 is the first step in ubiquitination. PYR41 is a low MW inhibitor of E1 ubiquitin‐activating enzyme, which suppresses the subsequent ubiquitination reaction. In our study, when NRCMs were treated with PYR41 under HG + Pal + Ole conditions; the ubiquitination of cardiomyocyte proteins was suppressed. E3 ubiquitin ligases target them for degradation by cytosolic proteasomes. The MuRF family consists of RING finger E3 ubiquitin ligases expressed specifically in cardiac and skeletal muscles. MuRF1 levels are decreased in denervation‐induced skeletal muscle atrophy, and MuRF1 is located in the M‐line region and colocalizes with α‐actinin, one of the major components of the Z‐disc in heart tissues (Vidal & Hetz, 2012). In addition, MuRF1 regulates microtubule dynamics, myofilament structure, and muscle metabolism, including carbohydrate metabolism (Bogomolovas, Gasch, & Simkovic, 2014; Hirner et al., 2008; Perera, Holt, Mankoo, & Gautel, 2011). In this study, we found that increased expression and activity of the ubiquitin proteasome proteolytic pathway, that is, a marked up‐regulation of MuRF1, play essential roles in muscle rupture in DCM. Using LC–MS/MS protein analysis, we identified 147 proteins that were hyper‐ubiquitinated in the heart of db/db mice, compared to those after treatment with NaHS, and six proteins belong to cardiac muscle structural proteins (Table S4). The ubiquitination levels of two cardiac muscle structural proteins MYH6 and MyL2, after treatment with NaHS were clearly lower than those in the hearts of untreated db/db mice. The ubiquitin‐26S proteasome system, as a cellular house‐keeping system, degrades the selected target proteins by the 26S proteasome (Marshall, Li, Gemperline, Book, & Vierstra, 2015), which is a 2.5‐MDa, self‐compartmentalized proteolytic machine located in the cytosol (Finley, 2009). It includes two distinct subunits, the 20S core protease and the 19S regulatory particle (Bhattacharyya, Yu, Mim, & Matouschek, 2014) and it plays a central important role in the ubiquitin‐26S proteasome system. In this study, MG132, a proteasome inhibitor, decreased the degradation of MYH6 and MyL2 in NRCMs HG + Pal + Ole conditions.

H2S, as a novel gasotransmitter, exerts a range of physiological and pathophysiological effects, particularly in the cardiovascular system (Nagpure & Bian, 2016; Wallace & Wang, 2015). Accumulating evidence suggests that production of endogenous H2S or the administration of exogenous H2S can attenuate myocardial injury after ischaemia or reperfusion and decrease BP and resist oxidative stress (X. Chen et al., 2017; L. Li et al., 2008; D. Wu et al., 2015). In our study, we noticed that the expression of CSE, a key enzyme to produce H2S in cardiovascular system, was reduced in db/db mice (Figure 2c,d). Immunoprecipitation was carried out to detect the interaction between CSE and ubiquitin, we found that CSE was modified by ubiquitin and degraded by the ubiquitin‐proteasome system. After treatment with NaHS, the ubiquitination of CSE was decreased (Figure S3). H2S acts as a secondary messenger, by directly modulating the function of downstream targets (Mustafa et al., 2009). PPG is a specific inhibitor of CSE and thus inhibited endogenous H2S production and increased ER stress and ubiquitination. H2S also covalently modifies cysteine residues on target proteins in a process known as S‐sulfhydration (Meng et al., 2016; Paul & Snyder, 2012; Paul & Snyder, 2015). In this instance, sulfur derived from H2S binds to the thiol group of a target cysteine residue to form a hydropersulfide moiety (–SSH; Lu, Kavalier, Lukyanov, & Gross, 2013; Pennick, Poole, Dennis, & Smyth, 2012). We found that NaHS increased the S‐sulfhydration in MuRF1 (Figure 8) but that DTT, a blocker of S‐sulfhydration, decreased MuRF1 S‐sulfhydration. In addition, NaHS regulated MuRF1 activity and inhibited the interaction of MuRF1 with MYH6 or MyL2. After MuRF1 was mutated at Cys44, the S‐sulfhydration level of MuRF1 was decreased, and the interaction between MuRF1 and MYH6 and MyL2 was inhibited.

In conclusion, this study has provided evidence that H2S increased MuRF1 S‐sulfhydration at Cys44 to regulate MYH6 and MyL2 ubiquitination, thereby preventing cardiac muscle degradation in our model of DCM in db/db mice (Figure 9). H2S may be a useful therapeutic strategy in DCM in the future.

Figure 9.

Figure 9

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The role of H2S in regulation of the degradation of cardiac structural proteins in a model of Type 2 diabetes. In Type 2 diabetes, increasing amounts of ROS promote ubiquitination and MuRF1 expression, which then lead to cardiac sarcomere degradation. H2S decreases ubiquitination levels and MuRF1 expression. Also, H2S can modulate the S‐sulfhydration on MuRF1 to decrease its activity and thus to protect cardiac structural proteins

AUTHOR CONTRIBUTIONS

X.S., D.Z., F.L., F.L., and W.Z. participated in the research design. X.S., S.P., M.Y., and Y.S. conducted the experiments. X.S., Y.S., H.D., J.C., and B.W. performed the data analysis. X.S., N.L., S.D., and W.Z. wrote the manuscript. All authors reviewed and revised the final version of manuscript and approved manuscript submission.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14207, https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14208, and https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14206, and as recommended by funding agencies, publishers, and other organisations engaged with supporting research.

Supporting information

Table S1. Blood glucose level (mM)

Table S2. Body Weight (g)

Table S3. Heart Weight/Body Weigh

Table S4. List of immunoprecipitated cardiac proteins from db/db mice with significantly different lysine ubiquitination presented as a fold change compared to db/db mice with treatment of NaHS.

Figure S1. HE staining of mice cardiac tissues.

Figure S2. Detection of ROS level by DHE and DCFH staining in NRCMs. NRCMs were treated under different conditions for 2 h, 4 h, 8 h and 12 h and the production of ROS was measured by DHE or DCFH.

Figure S3. Immunoprecipitation of mice heart tissues. **p < 0.01, ***p < 0.001.

Click here for additional data file. (621KB, pdf)

ACKNOWLEDGEMENT

This work was supported by the National Natural Science Foundation of China (81570340 and 81670344).

Sun X, Zhao D, Lu F, et al. Hydrogen sulfide regulates muscle RING finger‐1 protein S‐sulfhydration at Cys44 to prevent cardiac structural damage in diabetic cardiomyopathy. Br J Pharmacol. 2020;177:836–856. 10.1111/bph.14601

Contributor Information

Fanghao Lu, Email: lufanghao1973@126.com.

Weihua Zhang, Email: zhangwh116@126.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1. Blood glucose level (mM)

Table S2. Body Weight (g)

Table S3. Heart Weight/Body Weigh

Table S4. List of immunoprecipitated cardiac proteins from db/db mice with significantly different lysine ubiquitination presented as a fold change compared to db/db mice with treatment of NaHS.

Figure S1. HE staining of mice cardiac tissues.

Figure S2. Detection of ROS level by DHE and DCFH staining in NRCMs. NRCMs were treated under different conditions for 2 h, 4 h, 8 h and 12 h and the production of ROS was measured by DHE or DCFH.

Figure S3. Immunoprecipitation of mice heart tissues. **p < 0.01, ***p < 0.001.

Click here for additional data file. (621KB, pdf)

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