Endogenous sulfur dioxide deficiency as a driver of cardiomyocyte senescence through abolishing sulphenylation of STAT3 at cysteine 259.

OBJECTIVE: Cardiomyocyte senescence is an important contributor to cardiovascular diseases and can be induced by stressors including DNA damage, oxidative stress, mitochondrial dysfunction, epigenetic regulation, etc. However, the underlying mechanisms for the development of cardiomyocyte senescence remain largely unknown. Sulfur dioxide (SO2) is produced endogenously by aspartate aminotransferase 2 (AAT2) catalysis and plays an important regulatory role in the development of cardiovascular diseases. The present study aimed to explore the effect of endogenous SO2 on cardiomyocyte senescence and the underlying molecular mechanisms. APPROACH AND RESULTS: We interestingly found a substantial reduction in the expression of AAT2 in the heart of aged mice in comparison to young mice. AAT2-knockdowned cardiomyocytes exhibited reduced SO2 content, elevated expression levels of Tp53, p21Cip/Waf, and p16INk4a, enhanced SA-ß-Gal activity, and elevated level of γ-H2AX foci. Notably, supplementation with a SO2 donor ameliorated the spontaneous senescence phenotype and DNA damage caused by AAT2 deficiency in cardiomyocytes. Mechanistically, AAT2 deficiency suppressed the sulphenylation of signal transducer and activator of transcription 3 (STAT3) facilitated its nuclear translocation and DNA-binding capacity. Conversely, a mutation in the cysteine (Cys) 259 residue of STAT3 blocked SO2-induced STAT3 sulphenylation and subsequently prevented the inhibitory effect of SO2 on STAT3-DNA-binding capacity, DNA damage, and cardiomyocyte senescence. Additionally, cardiomyocyte (cm)-specific AAT2 knockout (AAT2cmKO) mice exhibited a deterioration in cardiac function, cardiomegaly, and cardiac aging, whereas supplementation with SO2 donors mitigated the cardiac aging and remodeling phenotypes in AAT2cmKO mice. CONCLUSION: Downregulation of the endogenous SO2/AAT2 pathway is a crucial pathogenic mechanism underlying cardiomyocyte senescence. Endogenous SO2 modifies STAT3 by sulphenylating Cys259, leading to the inhibition of DNA damage and the protection against cardiomyocyte senescence.

Endogenous Hydrogen Sulfide Persulfidates Caspase-3 at Cysteine 163 to Inhibit Doxorubicin-Induced Cardiomyocyte Apoptosis.

Doxorubicin (DOX) is an efficient antitumor anthracycline drug, but its cardiotoxicity adversely affects the prognosis of the patients. In this study, we explored whether endogenous gasotransmitter hydrogen sulfide (H2S) could protect against DOX-induced cardiomyocyte apoptosis and its mechanisms. The results indicated that DOX significantly downregulated endogenous H2S production and endogenous synthetase cystathionine γ-lyase (CSE) expression and obviously stimulated the apoptosis in H9C2 cells. The supplement of H2S donor sodium hydrosulfide (NaHS) or overexpression of CSE inhibited DOX-induced H9C2 cell apoptosis. DOX enhanced the activities of caspase family members in cardiomyocytes, while NaHS attenuated DOX-enhanced caspase-3, caspase-2, and caspase-9 activities by 223.1%, 73.94%, and 52.29%, respectively. Therefore, taking caspase-3 as a main target, we demonstrated that NaHS or CSE overexpression alleviated the cleavage of caspase-3, suppressed caspase-3 activity, and inhibited the cleavage of poly ADP-ribose polymerase (PARP). Mechanistically, we found that H2S persulfidated caspase-3 in H9C2 cells and human recombinant caspase-3 protein, while the thiol-reducing agent dithiothreitol (DTT) abolished H2S-induced persulfidation of caspase-3 and thereby prevented the antiapoptotic effect of H2S on caspase-3 in H9C2 cells. The mutation of caspase-3 C148S and C170S failed to block caspase-3 persulfidation by H2S in H9C2 cells. However, caspase-3 C163S mutation successfully abolished the effect of H2S on caspase-3 persulfidation and the corresponding protection of H9C2 cells. Collectively, these findings indicate that endogenous H2S persulfidates caspase-3 at cysteine 163, inhibiting its activity and cardiomyocyte apoptosis. Sufficient endogenous H2S might be necessary for the protection against myocardial cell apoptosis induced by DOX. The results of the study might open new avenues with respect to the therapy of DOX-stimulated cardiomyopathy.

Nitric oxide inhibits endothelial cell apoptosis by inhibiting cysteine-dependent SOD1 monomerization.

Endothelial cell apoptosis is an important pathophysiology in many cardiovascular diseases. The gasotransmitter nitric oxide (NO) is known to regulate cell survival and apoptosis. However, the mechanism underlying the effect of NO remains unclear. In this research, by targeting cytosolic copper/zinc superoxide dismutase (SOD1) monomerization, we aimed to explore how NO inhibited endothelial cell apoptosis. We showed that treatment with the NO synthase (NOS) inhibitor nomega-nitro-l-arginine methyl ester hydrochloride (L-NAME) significantly decreased the endogenous NO content of endothelial cells, facilitated the formation of SOD1 monomers, inhibited dismutase activity, and promoted reactive oxygen species (ROS) accumulation in human umbilical vein endothelial cells (HUVECs); by contrast, supplementation with the NO donor sodium nitroprusside (SNP) upregulated NO content, prevented the formation of SOD1 monomers, enhanced dismutase activity, and reduced ROS accumulation in L-NAME-treated HUVECs. Mechanistically, tris(2-carboxyethyl) phosphine hydrochloride (TCEP), a specific reducer of cysteine thiol, increased SOD1 monomer formation, thus preventing the NO-induced increase in dismutase activity and the decrease in ROS. Furthermore, SNP inhibited HUVEC apoptosis caused by the decrease in endogenous NO, whereas TCEP abolished this protective effect of SNP. In summary, our data reveal that NO protects endothelial cells against apoptosis by inhibiting cysteine-dependent SOD1 monomerization to enhance SOD1 activity and inhibit oxidative stress.

Endogenous Taurine Downregulation Is Required for Renal Injury in Salt-Sensitive Hypertensive Rats via CBS/H2S Inhibition.

Although taurine is known to exert an antihypertensive effect, it is unclear whether it is involved in the mechanism for hypertension-related target organ injury. To reveal the role of endogenous taurine in renal injury formation during salt-sensitive hypertension and clarify its mechanisms, both salt-sensitive Dahl rats and salt-resistant SS-13BN rats were fed a high-salt diet (8% NaCl) and given 2% taurine for 6 weeks. Rat systolic blood pressure (SBP) was measured by the tail-cuff method and artery catheterization. Kidney ultrastructure was observed under an electron microscope. Taurine content and mRNA and protein levels of taurine synthases, cysteine dioxygenase type 1 (CDO1) and cysteine sulfinic acid decarboxylase (CSAD), were decreased in Dahl rats fed a high-salt diet. However, taurine supplementation and the resulting increase in renal taurine content reduced the increased SBP and improved renal function and structural damage in high-salt diet-fed Dahl rats. In contrast, taurine did not affect SS-13BN SBP and renal function and structure. Taurine intervention increased the renal H2S content and enhanced cystathionine-ß-synthase (CBS) expression and activity in Dahl rats fed a high-salt diet. Taurine reduced the renin, angiotensin II, and aldosterone contents and the levels of oxidative stress indices in Dahl rat renal tissues but increased antioxidant capacity, antioxidant enzyme activity, and protein expression. However, taurine failed to achieve this effect in the renal tissue of SS-13BN rats fed a high-salt diet. Pretreatment with the CBS inhibitor HA or renal CBS knockdown inhibited H2S generation and subsequently blocked the effect of taurine on renin, superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2) levels in high-salt-stimulated Dahl renal slices. In conclusion, the downregulation of endogenous taurine production resulted in a decrease in the renal CBS/H2S pathway. This decrease subsequently promoted renin-angiotensin-aldosterone system (RAAS) activation and oxidative stress in the kidney, ultimately contributing to renal injury in salt-sensitive Dahl rats.

[Effect of blood-letting puncture of "Well-points" on hippocampal mitophagy-related protein expression in rats with acute brain injury due to hypobaric hypoxia].

OBJECTIVE: To study the effect of blood-letting puncture at "Well-points" of the twelve meridians on hippocampal mitophagy of hypobaric hypoxia-induced brain injury (HHIBI) rats, so as to explore its biological mechanisms underlying improvement of high altitude hypoxia-induced brain injury. METHODS: Male SD rats were randomly divided into normal control group （n=9）， and model and blood-letting groups which were further divided into 6, 12, 24, 48 and 72 h subgroups (n=9 in each subgroup). The HHIBI model was established by putting the rats into a hypobaric hypoxia chamber (equivalent to 5 000 m above sea level).The blood-letting groups were given blood-letting therapy at "Shaoshang"(LU11), "Shangyang"(LI1), "Zhongchong"(PC9), "Guanchong"(SJ1), "Shaochong"(HT9), "Shaoze"(SI9), once a day for 7 days. H.E. staining was used to observe the histopatholo-gical changes of hippocampus tissue. Serum hypoxia inducible factor(HIF)-1α and vascular endothelial growth factor(VEGF) contents were assayed using ELISA, and the expression levels of hippocampal Beclin-1 and LC3-Ⅱ proteins detected using Western blot. RESULTS: Compared with the normal control group, the levels of serum HIF-1α and VEGF at each time point, and the expressions of hippocampal Beclin-1 at 12 and 24 h, LC3-Ⅱat each time point were significantly increased in the model group (P<0.05, P<0.01); while in comparison with the model group, the levels of serum HIF-1α and VEGF contents, and the expressions of Beclin-1 at 12 h, LC3-Ⅱ at 24, 48 and 72 h were further significantly up-regulated in the blood-letting group (P<0.01, P<0.05). H.E. staining revealed that the pyramidal cells in the hippocampal CA1 region had a disordered arrangement, and some of them presented swelling with loose and pale cytoplasm or vacuolation at 6, 12 and 24 h, and showed indistinct nucleolus, irregular shape, pyknosis and deep staining and an obvious edema at 48 and 72 h, which was relatively milder in the blood-letting group. CONCLUSION: Blood-letting of "Well-points" can up-regulate serum HIF-1α and VEGF contents and hippocampal Beclin-1 and LC3-Ⅱ (mitophagy related proteins) expressions in HHIBI rats, which may contribute to its effect in reducing hypoxic brain injury.

Shensu IV prevents glomerular podocyte injury in nephrotic rats via promoting lncRNA H19/DIRAS3-mediated autophagy.

Shensu IV is a Chinese prescription well-known for its function in treating chronic kidney diseases. However, the potential mechanisms underlying how Shensu IV exerts its effects remain unclear. In the present study, we investigated the effects of Shensu IV on glomerular podocyte injury in nephrotic rats and puromycin-induced injury in cultured podocytes, and assessed the associated molecular mechanisms. Liquid chromatography-mass spectrometry (LC-MS) results showed that the main components of Shensu IV were l-Carnitine, P-lysoPC (LPC) 16:0, Coumaroyl tyramine, Tetramethylpyrazine, LPC 18:1, Choline, (S,S)-Butane-2,3-diol, and Scopoletin. We further found that nephrotic rats displayed pathological alterations in kidney tissues and ultrastructural changes in glomerular podocytes; however, these effects were reversed with Shensu IV treatment. Compared with the control, the numbers of autophagosomes were markedly reduced in the model group, but not in the Shensu IV treatment group. Furthermore, the expression of p62 was significantly higher in the model group than in the controls, whereas the LC3-II/I ratio was significantly lower; however, these changes were not observed when Shensu IV was administered. The protective effects of Shensu IV were further confirmed in podocytes displaying puromycin-induced injury. Compared with control group, the expression of long non-coding RNA (lncRNA) H19, mTOR, p-mTOR, and p62 was significantly increased in the puromycin group, whereas that of distinct subgroup of the RAS family member 3 (DIRAS3) was significantly decreased, as was the LC3-II/I ratio. The opposite results were obtained for both shH19- and Shensu IV-treated cells. Collectively, our data demonstrated that Shensu IV can prevent glomerular podocyte injury in nephrotic rats and puromycin-treated podocytes, likely via promoting lncRNA H19/DIRAS3-regulated autophagy.

Sulphenylation of CypD at Cysteine 104: A Novel Mechanism by Which SO2 Inhibits Cardiomyocyte Apoptosis.

Objectives: The study was designed to explore the role of endogenous gaseous signaling molecule sulfur dioxide (SO2) in the control of cardiomyocyte apoptosis and its molecular mechanisms. Methods: Neonatal mouse cardiac myocytes (NMCMs) and H9c2 cells were used in the cell experiments. The endogenous SO2 pathway including SO2 level and the expression of SO2-generating enzyme aspartate aminotransferase 1/2 (AAT1/2) were detected in NMCMs. The apoptosis of cardiomyocytes was examined by a TUNEL assay. The cleavage and the activity of apoptotic proteins caspase9 and caspase3 were measured. The content of ATP, the opening of mitochondrial permeability transition pore (mPTP), and the cytochrome c (cytc) leakage were detected by immunofluorescence. The sulphenylation of cyclophilin-D (CypD) was detected by biotin switch analysis. The four CypD mutant plasmids in which cysteine sites were mutated to serine were constructed to identify the SO2-affected site in vitro. Results: ISO down-regulated the endogenous SO2/AAT pathway of cardiomyocytes in association with a significant increase in cardiomyocyte apoptosis, demonstrated by the increases in apoptosis, cleaved-caspase3/caspase3 ratio, and caspase3 activity. Furthermore, ISO significantly reduced ATP production in H9c2 cells, but the supplement of SO2 significantly restored the content of ATP. ISO stimulated mPTP opening, resulting in an increase in the release of cytc, which further increased the ratio of cleaved caspase9/caspase9 and enhanced the protein activity of caspase9. While, the supplementation of SO2 reversed the above effects. Mechanistically, SO2 did not affect CypD protein expression, but sulphenylated CypD and inhibited mPTP opening, resulting in an inhibition of cardiomyocyte apoptosis. The C104S mutation in CypD abolished SO2-induced sulphenylation of CypD, and thereby blocked the inhibitory effect of SO2 on the mPTP opening and cardiomyocyte apoptosis. Conclusion: Endogenous SO2 sulphenylated CypD at Cys104 to inhibit mPTP opening, and thus protected against cardiomyocyte apoptosis.

Postural Tachycardia Syndrome in Children and Adolescents: Pathophysiology and Clinical Management.

Postural tachycardia syndrome (POTS), characterized by chronic (≥6 months) orthostatic intolerance symptoms with a sustained and excessive heart rate increase while standing without postural hypotension, is common in children and adolescents. Despite the unclear pathogenesis of POTS, the present opinion is that POTS is a heterogeneous and multifactorial disorder that includes altered central blood volume, abnormal autonomic reflexes, "hyperadrenergic" status, damaged skeletal muscle pump activity, abnormal local vascular tension and vasoactive factor release, mast cell activation, iron insufficiency, and autoimmune dysfunction. A number of pediatric POTS patients are affected by more than one of these pathophysiological mechanisms. Therefore, individualized treatment strategies are initiated in the management of POTS, including basal non-pharmacological approaches (e.g., health education, the avoidance of triggers, exercise, or supplementation with water and salt) and special pharmacological therapies (e.g., oral rehydration salts, midodrine hydrochloride, and metoprolol). As such, the recent progress in the pathogenesis, management strategies, and therapeutic response predictors of pediatric POTS are reviewed here.

Endogenous Sulfur Dioxide Inhibits Vascular Calcification in Association with the TGF-ß/Smad Signaling Pathway.

The study was designed to investigate whether endogenous sulfur dioxide (SO2) plays a role in vascular calcification (VC) in rats and its possible mechanisms. In vivo medial vascular calcification was induced in rats by vitamin D3 and nicotine for four weeks. In vitro calcification of cultured A7r5 vascular smooth muscle cells (VSMCs) was induced by calcifying media containing 5 mmol/L CaCl2. Aortic smooth muscle (SM) α-actin, runt-related transcription factor 2 (Runx2), transforming growth factor-ß (TGF-ß) and Smad expression was measured. VC rats showed dispersed calcified nodules among the elastic fibers in calcified aorta with increased aortic calcium content and alkaline phosphatase (ALP) activity. SM α-actin was markedly decreased, but the osteochondrogenic marker Runx2 concomitantly increased and TGF-ß/Smad signaling was activated, in association with the downregulated SO2/aspartate aminotransferase (AAT) pathway. However, SO2 supplementation successfully ameliorated vascular calcification, and increased SM α-actin expression, but inhibited Runx2 and TGF-ß/Smad expression. In calcified A7r5 VSMCs, the endogenous SO2/AAT pathway was significantly downregulated. SO2 treatment reduced the calcium deposits, calcium content, ALP activity and Runx2 expression and downregulated the TGF-ß/Smad pathway in A7r5 cells but increased SM α-actin expression. In brief, SO2 significantly ameliorated vascular calcification in association with downregulation of the TGF-ß/Smad pathway.

Hyperhomocysteinemia exaggerates adventitial inflammation and angiotensin II-induced abdominal aortic aneurysm in mice.

RATIONALE: A number of epidemiological studies have suggested an association of hyperhomocysteinemia (HHcy) and abdominal aortic aneurysm (AAA), but discrepancies exist. In addition, we lack direct evidence supporting a causal role. OBJECTIVE: We determined the association and contribution of HHcy to AAA formation. METHODS AND RESULTS: We first performed a meta-analysis of studies involving 1489 subjects and found a strong association of HHcy and AAA (odds ratio, 7.39). Next, we used angiotensin II-infused male apolipoprotein E-deficient mice and tested whether HHcy contributes to AAA pathogenesis. Homocysteine (Hcy) supplement (1.8 g/L) in drinking water resulted in mild HHcy. Intriguingly, HHcy greatly increased the incidence of angiotensin II-induced AAA and aortic dissection in apolipoprotein E-deficient mice (vehicle versus Hcy: 50% versus 100%; P<0.05). Histology indicated HHcy markedly exaggerated aortic adventitial inflammation. Increased levels of proinflammatory interleukin-6 and monocyte chemoattractant protein-1 were preferentially colocalized within adventitial fibroblasts in HHcy plus angiotensin II mice, which suggested the importance of adventitial fibroblasts activation in Hcy-aggravated AAA. Hcy sequentially stimulated adventitial fibroblasts transformation into myofibroblasts, secretion of interleukin-6 and monocyte chemoattractant protein-1, and consequent recruitment of monocytes/macrophages to adventitial fibroblasts, which was abolished by the NADPH oxidase inhibitor diphenyliodonium. NADPH oxidase 4, but not other homologs of NADPH oxidase, was significantly upregulated by Hcy in adventitial fibroblasts, whereas NADPH oxidase 4 small interfering RNA silencing diminished Hcy-induced adventitial fibroblasts activation. Finally, folic acid supplement (0.071 µg/g per day) markedly reduced HHcy-aggravated angiotensin II-induced AAA formation in apolipoprotein E-deficient mice. CONCLUSIONS: HHcy may aggravate AAA formation at least partially via activating adventitial fibroblast NADPH oxidase 4.