Endothelial cell dysfunction is one of the main reasons for type II diabetes vascular complications. Hydrogen sulphide (H₂S) has antioxidative effect, but its regulation on mitochondrial dynamics and mitophagy in aortic endothelial cells under hyperglycaemia and hyperlipidaemia is unclear. Rat aortic endothelial cells (RAECs) were treated with 40 mM glucose and 200 μM palmitate to imitate endothelium under hyperglycaemia and hyperlipidaemia, and 100 μM NaHS was used as an exogenous H₂S donor. Firstly, we demonstrated that high glucose and palmitate decreased H₂S production and CSE expression in RAECs. Then, the antioxidative effect of H₂S was proved in RAECs under high glucose and palmitate to reduce mitochondrial ROS level. We also showed that exogenous H₂S inhibited mitochondrial apoptosis in RAECs under high glucose and palmitate. Using Mito Tracker and transmission electron microscopy assay, we revealed that exogenous H₂S decreased mitochondrial fragments and significantly reduced the expression of p‐Drp‐1/Drp‐1 and Fis1 compared to high‐glucose and high‐palmitate group, whereas it increased mitophagy by transmission electron microscopy assay. We demonstrated that exogenous H₂S facilitated Parkin recruited by PINK1 by immunoprecipitation and immunostaining assays and then ubiquitylated mitofusin 2 (Mfn2), which illuminated the mechanism of exogenous H₂S on mitophagy. Parkin siRNA suppressed the expression of Mfn2, Nix and LC3B, which revealed that it eliminated mitophagy. In summary, exogenous H₂S could protect RAECs against apoptosis under high glucose and palmitate by suppressing oxidative stress, decreasing mitochondrial fragments and promoting mitophagy. Based on these results, we proposed a new mechanism of H₂S on protecting endothelium, which might provide a new strategy for type II diabetes vascular complication.

Excessive autophagy induced by extravagant oxidative stress is the main reason for diabetes-induced vascular endothelial cells dysfunction. Hydrogen sulfide (H2S) has anti-oxidative effects but its regulation on excessive autophagy of vascular endothelial cells is unclear. In this study, aorta of db/db mice (28 weeks old) and rat aortic endothelial cells (RAECs) treated with 40 mM glucose and 500 μM palmitate acted as type II diabetic animal and cellular models, respectively, and 100 μMNaHS was used as an exogenous H2S donor. The apoptosis level was measured by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) staining and Hoechst 33342/PI staining. The activities of SOD, CAT and respiratory complexes were also measured. The mRNA levels of SOD and CAT were detected by real-time PCR. AMPK-siRNA was used to detect the effect of AMPK on autophagy. Western blotting was used to detected the protein level. H2S production was decreased (p < 0.05, p < 0.01) both in vitro and in vivo; NaHS treatment rescued this impairment (p < 0.05, p < 0.01). The expression of adhesive proteins was increased (p < 0.05, p < 0.01) both in vitro and in vivo; NaHS attenuated (p < 0.05, p < 0.01) these alterations. NaHS could protect endothelial cells against apoptosis induced by type II diabetes (p < 0.05, p < 0.01). Furthermore, the expressions and activities of SOD and CAT were impaired (p < 0.05, p < 0.01) in endothelial cells of diabetes II; NaHS treatment attenuated (p < 0.05) this impairment. NaHS also increased ATP production (p < 0.05) and activities of respiratory complexes (p < 0.05), and the ratio of p-AMPK to AMPK was also decreased by NaHS (p < 0.01). The level of autophagy in endothelial cells was also decreased (p < 0.05, p < 0.01) by NaHS treatment and AMPK-siRNA treatment. The expression of Nrf2 in the nuclei was increased (p < 0.05) by NaHS treatment. Exogenous H2S might protect arterial endothelial cells by suppressing excessive autophagy induced by oxidative stress through the Nrf2-ROS-AMPK signaling pathway.

Excessive ROS accumulation contributes to cardiac injury in type 2 diabetes mellitus. Hydrogen sulfide (H₂S) is a vital endogenous gasotransmitter to alleviate cardiac damage in diabetic cardiomyopathy (DCM). However, the underlying mechanisms remain unclear. In this study, we investigated the effects of NaHS administration in db/db mice via intraperitoneal injection for 20 weeks and the treatment of high glucose (HG), palmitate (PA) and NaHS in HL-1 cardiomyocytes for 48 h, respectively. H₂S levels were decreased in hearts of db/db mice and HL-1 cardiomyocytes exposed to HG and PA, which were restored by NaHS. Exogenous H₂S activated the nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPx4)/glutathione (GSH) pathway, suppressed ferroptosis and mitigated mitochondrial apoptosis in db/db mice. However, these effects were abrogated after Nrf2 knockdown. NaHS treatment elevated the ubiquitination level of Kelch-like ECH-associated protein (Keap1) by preserving its E3 ligase synoviolin (Syvn1), resulting in Nrf2 nuclear translocation. H₂S facilitated the sulfhydration of Syvn1-cys115 site, a post-translational modification. Transfecting Syvn1 C115A in cardiomyocytes exposed to HG and PA partially attenuated the effects of NaHS on Nrf2 and cell death. Our findings suggest that exogenous H₂S regulates Nrf2/GPx4/GSH pathway by promoting the Syvn1-Keap1 interaction to reduce ferroptosis and mitochondrial apoptosis in DCM.

Alterations in calcium homeostasis in the intracellular endo/sarcoplasmic reticulum (ER/SR) and mitochondria of cardiomyocytes cause cell death via the SR and mitochondrial apoptotic pathway, contributing to ventricular dysfunction. However, the role of the calcium-sensing receptor (CaR) in cardiac hypertrophy and heart failure has not been studied. This study examined the possible involvement of CaR in the SR and mitochondrial apoptotic pathway in an experimental model of heart failure. In Wistar rats, cardiac hypertrophy and heart failure were induced by subcutaneous injection of isoproterenol (Iso). Calindol, an activator of CaR, and calhex231, an inhibitor of CaR, were administered by caudal vein injection. Cardiac remodeling and left ventricular function were then analyzed in these rats. After 2, 4, 6 and 8 weeks after the administration of Iso, the rats developed cardiac hypertrophy and failure. The cardiac expression of ER chaperones and related apoptotic proteins was significantly increased in the failing hearts. Furthermore, the expression of ER chaperones and the apoptotic rate were also increased with the administration of calindol, whereas the expression of these proteins was reduced with the treatment of calhex231. We also induced cardiac hypertrophy and failure via thoracic aorta constriction (TAC) in mice. After 2 and 4 weeks of TAC, the expression of ER chaperones and apoptotic proteins were increased in the mouse hearts. Furthermore, Iso induced ER stress and apoptosis in cultured cardiomyocytes, while pretreatment with calhex231 prevented ER stress and protected the myocytes against apoptosis. To further investigate the effect of CaR on the concentration of intracellular calcium, the calcium concentration in the SR and mitochondria was determined with Fluo-5N and x-rhod-1 and the mitochondrial membrane potential was examined with JC-1 using laser confocal microscopy. After treatment with Iso for 48 hours, activation of CaR reduced [Ca(2+)]SR, increased [Ca(2+)]m, decreased the mitochondrial membrane potential, increased the expression of ER stress chaperones and related apoptotic proteins, and induced the release of cytochrome c from the mitochondria. Our results demonstrated that CaR activation caused Ca(2+) release from the SR into the mitochondria and induced cardiomyocyte apoptosis through the SR and mitochondrial apoptotic pathway in failing hearts.

Diabetic cardiomyopathy (DCM) is a serious complication of diabetes. Hydrogen sulphide (H₂S), a newly found gaseous signalling molecule, has an important role in many regulatory functions. The purpose of this study is to investigate the effects of exogenous H₂S on autophagy and its possible mechanism in DCM induced by type II diabetes (T2DCM). In this study, we found that sodium hydrosulphide (NaHS) attenuated the augment in left ventricular (LV) mass and increased LV volume, decreased reactive oxygen species (ROS) production and ameliorated H₂S production in the hearts of db/db mice. NaHS facilitated autophagosome content degradation, reduced the expression of P62 (a known substrate of autophagy) and increased the expression of microtubule-associated protein 1 light chain 3 II. It also increased the expression of autophagy-related protein 7 (ATG7) and Beclin1 in db/db mouse hearts. NaHS increased the expression of Kelch-like ECH-associated protein 1 (Keap-1) and reduced the ubiquitylation level in the hearts of db/db mice. 1,4-Dithiothreitol, an inhibitor of disulphide bonds, increased the ubiquitylation level of Keap-1, suppressed the expression of Keap-1 and abolished the effects of NaHS on ubiquitin aggregate clearance and ROS production in H9C2 cells treated with high glucose and palmitate. Overall, we concluded that exogenous H₂S promoted ubiquitin aggregate clearance via autophagy, which might exert its antioxidative effect in db/db mouse myocardia. Moreover, exogenous H₂S increased Keap-1 expression by suppressing its ubiquitylation, which might have an important role in ubiquitin aggregate clearance via autophagy. Our findings provide new insight into the mechanisms responsible for the antioxidative effects of H₂S in the context of T2DCM.

Aims: Calcium-sensing receptor (CaR) acts as a G protein coupled receptor that mediates the increase of the intracellular Ca²⁺ concentration. The expression of CaR has been confirmed in various cell types, including cardiomyocytes, smooth muscle cells, neurons and vascular endothelial cells. However, whether CaR is expressed and functions in cardiac fibroblasts has remained unknown. The present study investigated whether CaR played a role in cardiac fibroblast proliferation and extracellular matrix (ECM) secretion, both in cultured rat neonatal cardiac fibroblasts and in a model of cardiac hypertrophy induced by isoproterenol (ISO). Methods and Results: Immunofluorescence, immunohistochemistry and Western blot analysis revealed the presence of CaR in cardiac fibroblasts. Calcium and calindol, a specific activator of CaR, elevated the intracellular calcium concentration in cardiac fibroblasts. Pretreatment of cardiac fibroblasts with calhex231, a specific inhibitor of CaR, U73122 and 2-APB attenuated the calindol- and extracellular calcium-induced increase in intracellular calcium ([Ca²⁺]_i). Cardiac fibroblast proliferation and migration were assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), cell count and the cell scratch assay. ECM production was detected by expression of matrix metalloproteinase-3 and -9 (MMP-3 and -9). Activation of CaR promoted cardiac fibroblast proliferation and migration and ECM secretion. More importantly, calhex231, suppressed cardiac fibroblast proliferation and migration and MMP-3 and -9 expression. To further investigate the effect of CaR on cardiac fibrosis, a model of ISO-induced cardiac hypertrophy was established. Pretreatment with calhex231 prevented cardiac fibrosis and decreased the expression of MMP-3 and -9 expression. Conclusions: Our results are the first report that CaR plays an important role in Ca²⁺ signaling involved in cardiac fibrosis through the phospholipase C- inositol 3,4,5 phosphate (PLC-IP₃) pathway.