Effects And Mechanisms Of Hydrogen Sulfide On Myocardial Hypertrophy

Myocardial hypertrophy, as one of the most prevalent damages on target organs in hypertension, constitutes an independent risk factor for myocardial ischaemia, myocardial infarction and heart failure. Hydrogen sulfide (H2S) is now accepted as the third "gasotransmitter" after nitric oxide and carbon monoxide. Accumulating evidence suggests that H2S influences a wide range of physiological and pathological processes especially in the cardiovascular system synthesized from L-Cysteine by cystathionine γ-lyase (CSE), including modulation of blood vessel tone, cardioprotection, anti-atherosclerosis, inhibition vascular smooth muscle proliferation and myocardial apoptosis. In the present work, we used a slow-releasing H2S donor drug, GYY4137, to examine the effect of H2S on myocardial hypertrophy in spontaneously hypertensive rats (SHR) and angiotension Ⅱ (Ang Ⅱ) induced neonatal rat cardiomyocytes.Male spontaneously hypertensive rats (SHR) at 12 weeks of age were randomly divided into 4 groups assigned different dosages of GYY4137:0 (SHR group),10 (GYY10 group),25 (GYY25 group) or 50 (GYY50 group) mg/kg/day. GYY4137 was given by intraperitoneal injection once daily over a 4-week period. Age-matched normotensive Wistar-Kyoto (WKY) rats served as controls (WKY group). SHR and WKY control groups received the same volume of physiological saline instead of drug once daily over the same time period. During treatment, systolic blood pressure (SBP) was measured by the tail-cuff method. After 4-week’s treatment, cardiac geometry and function were evaluated with echocardiography. Invasive hemodynamic parameters were measured from carotid artery. Cardiac index, area of cardiomyocyte and mRNA expression of atrial natriuretic peptide (ANP) were measured as indices of cardiac hypertrophy. Superoxide production in myocardium was detected with DHE staining. Expression of mRNA and protein were detected by real time PCR and Western Blot respectively. Another male SHRs and WKY rats with 16 week age of old were given GYY4137 for 8 weeks, and blood pressure, cardiac geometry and function, degree of myocardial hypertrophy were also detected.Neonatal rat cardiomyocytes were pretreated with GYY4137 (12.5μM,25 μM and 5μM) for 4 h followed by Ang Ⅱ (100 nM) stimulation for a further 24 h. Then area of cardiomyocyte and mRNA expression of ANP were measured. Levels of ROS in cardiomyocytes were measured with DCFH-DA staining. Expressions of proteins were down-regulated with Si RNA technology. Activity of promoter was measured with Luciferase Reporter Assay. Binding Activity between proteins and promoter were measure with ChIP. Expression of mRNA and protein were detected by real time PCR and Western Blot respectively. Nuclear translocation of proteins was visualized with Immunofluorescence staining.Human myocardium samples of left ventricle were collected during procedure of cardiac valve replacement and classified into those with no myocardial hypertrophy group (control group) and those with hypertrophy (hypertrophy group). Samples were subjected to histological or molecular biological analysis.After 4-week’s treatment for SHR of 12-week age old, SHR in groups of GYY25 and GYY50, but not GYY10, displayed decreased blood pressure. Cardiac functions were not different between 5 groups. However, three doses of GYY4137 improved myocardial structure, decreased cardiac index and area of cardiomyocyte, suppressed the mRNA expression of ANP, down-regulated oxidative stress levels in myocardium, increased the expression of anti-oxidative related proteins including NF-E2-related factor 2 (Nrf2), thioredoxin (Trx) and heme oxygenase-1 (HO-1), inhibited phosphorylation of extracellular signal regulated kinase (ERK), P38 and c-Jun NH2-terminal kinase (JNK). After 8-week’s treatment for SHR of 16-week age old, SHR in groups of GYY25 and GYY50, but not GYY10, displayed decreased blood pressure. However, three doses of GYY4137 improved myocardial hypertrophy and cardiac function.GYY4137 also decreased areas of cardiomyocyte surface, suppressed mRNA expressions of ANP, attenuated levels of oxidative stress, and inhibited phosphorylations of ERK, P38 and JNK. However, combination of GYY4137(50 μM) and excess ROS scavenger tempol (500 μM) had a synergetic inhibitive effect on Ang Ⅱ-induced ANP gene expression, which suggested that there might be other mechanisms involved in attenuating effect of H2S on myocardial hypertrophy. Neonatal rat cardiomyocytes were transfected with ANP promoter-luciferase fusion plasmid. We found that Ang Ⅱ-enhanced ANP promoter activity was inhibited by H2S. Quantitative PCR for Kruppel-like transcription factors (KLF) mRNA expression were performed in neonatal rat cardiomyocytes. We found that mRNA expression of KLF5, but not other KLFs, was decreased by H2S treatment followed by Ang Ⅱ stimulation. H2S also inhibited KLF5 expression in myocardium of SHR. ChIP assay showed that Ang Ⅱ increased the recruitment of KLF5 to the ANP promoter, and this was decreased by H2S. KLF5 siRNA attenuated the effect of H2S on decreasing ANP promoter activity and ANP mRNA expression, suggesting that H2S suppresses ANP transcript activity in a KLF5-dependent manner. Neonatal rat cardiomyocytes were transiently transfected with luciferase reporter plasmids containing the KLF5 promoter (-2300/+236). These reporter assays revealed a higher KLF5 promoter activity with Ang Ⅱ stimulation, and H2S reversed this effect. In a series of deletion constructs, the attenuating effects of H2S on KLF5 promoter activity were observed in-2300 Luc,-363 Luc and -67 Luc, of which the 5’-ends correspond to 2300bp,363bp and 67bp from the transcription start site respectively. However, H2S-induced inhibition of KLF5 promoter activity was abolished in-32 Luc. It appeared that SP-1, an important site between 67bp and 32 bp on the upstream of KLF5 promoter sequence, might be responsible for the attenuating effect of H2S on KLF5 transcription. EMS A assay suggested H2S decreased binding activity between SP-1 and DNA. And ChIP assay confirmed that H2S decreased SP-1 binding activity to the KLF5 promoter which was enhanced after Ang II stimulation. Moreover, the attenuating effect of H2S on KLF5 promoter activity and KLF5 mRNA expression was weakened after SP-1 protein knockdown with siRNA. Western blot and immunofluorescence staining also confirmed that H2S decreased KLF5 protein level in the nucleus of Ang II-stimulated neonatal rat cardiomyocytes.We found that plasma H2S concentration was lower in patients exhibiting myocardial hypertrophy that in those without hypertrophy. We also found lower CSE expression but higher ANP and KLF5 expression in myocardium from patients with hypertrophy.All of the data suggests that decrease of KLF5 expression by H2S might a comprehensive consequence which is related to slighter phosphorylation of MAPKs, lower SP-1 binding activity with KLF5 promoter and weaker promoter activity of KLF5. H2S also reduced the amount of KLF5 in nuclei, and then inhibited the binding between KLF5 and ANP promoter, down-regulated ANP expression and attenuated myocardial hypertrophy. These results highlight a novel role for KLF5 in the protective effect of H2S on myocardial hypertrophy.