Search

Background Hydrogen sulfide (H2S) is increasingly recognised as a key cardiovascular mediator with effects on the heart and vasculature in health and disease [1] , [2] . H2S is generated endogenously via cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS), whilst bioavailability can also be increased by H2S donors. The mitochondrial enzyme, thiosulfate sulfurtransferase (TST) participates in metabolism of H2S to thiosulfate and targeting of this pathway represents an alternative means to increase H2S bioavailability. The present study aimed to investigate the potential role of TST in the cardiovascular system by characterising (i) expression of TST in the murine heart and vasculature and (ii) the cardiovascular phenotype of Tst null (Tst−/−) mice generated using homologous recombination to delete the entire TST gene. Methods To investigate the expression of TST in the cardiovascular system, RNA and protein were prepared from heart and aorta collected from 12 week old male C57BL/6 mice for qRT-PCR and Western blotting. Tissue was formalin fixed, wax embedded and sectioned before immunohistochemical staining. TST activity was assessed in tissue homogenates as previously described [3] . Hepatic tissue expresses TST highly and was used as a positive control for TST expression. Adult male Tst−/− and C57BL/6 N controls underwent high frequency ultrasound scanning (Visualsonics Vevo770) to determine cardiac dimensions and functional performance under 1% isofluorane. Blood pressure (BP) was measured in 12–15wk old male Tst−/− and wild type mice using a Millar catheter inserted into the right carotid artery under ketamine (50 mg/kg)/ medatomidate (0.5 mg/kg) i.p. anaesthesia. Results qRT-PCR showed there was high relative gene expression for TST in the heart compared to liver (n = 4). Western blots confirmed the presence of TST in the heart and aorta, although at significantly lower levels than that found in the liver (n = 3, p = 0.003). Immunohistochemistry localised TST to vascular smooth muscle and cardiomyocytes. All Tst−/− mice survived to adulthood. Deletion of Tst was confirmed with qRT-PCR, Western blotting and enzyme activity assays. Western blotting revealed down-regulation of the H2S synthesising enzymes CSE and CBS in Tst−/− mice compared to WT controls (n = 5/ group). Ultrasound scanning at 6 & 10 weeks of age showed no difference between Tst−/− vs. WT groups for LV ejection fraction, myocardial performance index, LV end systolic or diastolic areas (n = 6/ group, p > 0.05). Systolic BP (Tst−/− 100.5 ± 5.1 mmHg vs. WT 103.5 ± 3.8 mmHg, n = 5/group, p = 0.67) was unchanged in Tst−/− mice relative to WT mice. Conclusion TST is present in the myocardium and in vascular smooth muscle of healthy mice. Although expression is absent in Tst−/− mice, their baseline cardiovascular function appears normal, at least in young healthy males. Down-regulated expression of the synthetic enzymes CSE and CBS reveals a potential feedback system that may serve to reduce H2S synthesis and prevent accumulation when removal through TST-mediated metabolism is reduced, resulting in maintenance of vascular tone and cardiac function.

Background New interventions are required to prevent the loss of cardiomyocytes associated with myocardial ischemia and with the reperfusion injury that follows revascularisation. Recent studies have shown that compounds releasing hydrogen sulfide (H2S) can have cardioprotective effects [1] . However, these compounds are generally effective only at high (μM) concentrations and release is not well controlled, increasing the potential for cytotoxicity. H2S is produced endogenously and targeting the pathways that regulate availability in heart may be a more promising therapeutic approach. The present study aimed to characterise the distribution of the H2S synthesising enzymes, cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE) and mercaptopyruvate sulfurtransferase (MPST), and of H2S degrading enzyme, thiosulfate sulfurtransferase (TST) in the murine heart and in liver, an important site of H2S metabolism. Methods RNA and protein were prepared from heart and liver collected from 10 week old male C57BL/6J mice for quantitative RT-PCR and Western blotting. Further tissue was formalin fixed, processed and wax embedded before sectioning for immunohistochemistry. Results qRT-PCR showed high relative gene expression for all H2S metabolising enzymes in the liver. Cardiac tissue displayed high relative gene expression of TST & CSE, moderate expression of CBS and low expression of MPST. Western blots confirmed the presence of protein for TST & all H2S synthesising enzymes within heart & liver homogenates. Immunohistochemical staining of fixed tissue sections located CSE to vascular smooth muscle in the heart, while CBS & MPST were found in the vascular endothelium. Cardiomyocytes were immunoreactive for all enzymes. In hepatocytes, immunoreactive TST, CBS & MPST are found in hepatocytes throughout the liver, while CSE immunoreactivity is notably intensified around the central vein, responsible for hepatic venous return. Conclusion The murine myocardium and liver have the capacity to regulate local H2S availability via three separate synthetic pathways at least one degradative pathway which have distinctive cellular localisations. Future studies will investigate whether metabolic & synthetic enzyme expression and H2S availability is differentially regulated following ischaemia and reperfusion.

Background Increased H2S availability reduces injury associated with myocardial ischemia (MI) and ischemia–reperfusion. The mitochondrial enzyme thiosulfate sulfurtransferase (TST) has a putative role in removal of H2S and is a potential target to increase H2S bioavailability. This study investigated the hypothesis that TST deletion (Tst−/−) would improve outcome following MI. Methods TST expression was assessed by qRT-PCR and Western blotting. Tst−/− and WT mice were examined in vivo by ultrasound. MI was induced by coronary artery ligation in vivo, or ex vivo inperfused hearts, when 30 min ischemia was followed by 120 min reperfusion. Results qRT-PCR and Western blots confirmed the presence of TST in the murine heart and deletion in Tst−/−. Tst−/− mice survived to adulthood and had normal cardiac function. Expression of the H2S synthesising enzyme, cystathionine gamma-lyase (CSE), was reduced in Tst−/− relative to WT hearts (n = 5, p Discussion TST is present in the heart and deletion does not influence function. Tst−/− mice have reduced cardiac CSE expression, suggesting reduced H2S synthetic capacity. This may underlie the increased susceptibility of Tst−/− mice to myocardial injury and increased mortality. Conclusion TST may act with CSE to regulate H2S availability. Alteration in the balance of these enzymes has no overt physiological effect, but is associated with reduced capacity of the heart to resist ischemic stress.