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. 2020 Feb;177(4):836-856.
doi: 10.1111/bph.14601. Epub 2019 Apr 29.

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
Affiliations

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

Xiaojiao Sun et al. Br J Pharmacol. 2020 Feb.

Abstract

Background and purpose: Hydrogen sulfide (H2 S) 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 H2 S 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: H2 S levels and cystathionine-γ-lyase protein expression in myocardium were decreased in db/db mice. Exogenous H2 S reversed endoplasmic reticulum stress, including impairment of the function of cardiomyocytes and structural damage in db/db mice. Exogenous H2 S 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 H2 S, to reduce the interaction between MuRF1 and myosin heavy chain 6 and myosin light chain 2.

Conclusions and implications: Our findings suggest that H2 S 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.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
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
Figure 2
Figure 2
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
Figure 3
Figure 3
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
Figure 4
Figure 4
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
Figure 5
Figure 5
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
Figure 6
Figure 6
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
Figure 7
Figure 7
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
Figure 8
Figure 8
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
Figure 9
Figure 9
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
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