Hydrogen sulfide generation and signalling in the heart and coronary artery of the pig

Cardiovascular diseases (CVD), such as hypertension, angina, myocardial infarction, arrhythmia, heart failure, and stroke, are some of the most common causes of death around the world. Hydrogen sulfide (H2S) is considered as a bio-modulator gasotransmitter molecule. In recent years, evidence that H2S may have an influential role in several important biological processes, especially in the cardiovascular system (CVS) has increased. Many studies have reported the production of H2S within the body. H2S appears to play a role in the regulation of vascular tone. Therefore, there is accumulated evidence regarding the role of H2S in the vasculature, while there is little information about the role of H2S in the heart. Many cardiovascular pathologies have been associated with low H2S production, such as hypertension, ischaemic heart diseases (IHD), and atherosclerosis. The present study measured H2S generation in the heart by investigating the expression level of H2S synthesising enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and mercaptopyruvate sulfurtransferase (MST) using immunoblotting and investigating the activity of these enzymes via the development of assays using the methylene blue and SF7-AM (sulfidefluor-7 acetoxymethyl ester) methods. We found that CBS and MST were expressed in the heart, but CSE was not detected. CBS detected in the heart was consistently present at a higher MW band, suggesting the less active form, in comparison with the liver that had higher expression of the lower MW form which is thought to be the active form. This may reflect the difference in biological effect of H2S and post-translational modifications (PTMs) in the heart compared to the liver. We found that there was a low amount of H2S produced in the heart compared to the liver using both the methylene blue and SF7-AM methods. Myocardium added to isolated porcine coronary arteries (PCA) in tissue baths produced a relaxation response, which was inhibited by H2S synthesising enzyme/s inhibitors, suggesting that H2S synthesised in the myocardium might regulate coronary artery tone in a paracrine manner. There were no significant effects of SAM (S-adenosyl methionine, a positive CBS modulator) and gender on synthesis of H2S, and degassing on detection of H2S. In contrast, alkaline pH led to an increase of H2S measurement and dialysis of the heart cytosol led to an increase of detection of H2S. Briefly, there was detected enzyme activity in the heart, but it was lower than the liver by both the methylene blue and SF7-AM methods. The detected H2S production in the heart causes relaxation of the coronary artery, which suggests that H2S synthesised in the myocardium might regulate coronary artery tone in a paracrine manner. Measurement of enzyme activity only indicates the potential to produce H2S, not the amount that the tissue actually produces. Measurement of sulfhydration levels may be a way of determining the level of H2S produced in the tissue. In the biotin switch assay (BSA), there was an increase of sulfhydration level of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and MEK1 (mitogen-activated extracellular signal-regulated kinase 1), but the results were variable between different tissues. In the results of the red maleimide assay, there was a decrease in the fluorescence detected in the presence of Na2S and the presence of DTT (dithiothreitol) of GAPDH and MEK1 proteins. There was a difference in the fluorescence of bands which ran at the same place as MEK1 and GAPDH. As a result of running the samples on polyacrylamide gels in non-reducing conditions to detect proteins in the red maleimide assay, we observed the shifting of GAPDH and MEK1 bands to a lower MW in the presence of Na2S and absence of DTT. The same observation was also detected after immunoprecipitation of the heart homogenate and Western blotting. This shift to the lower MW band could be a simpler method for detecting changes in sulfhydration. Therefore, depending on our results of sulfhydration, there is detected sulfhydration in the heart by our assays and sulfhydration is a potential signalling pathway of H2S in the heart and could be used as an index for H2S production in the heart. Next, the present study investigated the effects of three different types of H2S donors (sources, drugs), Na2S fast-releasing H2S salt, GYY4137 slow-releasing H2S donor, and AP39 mitochondria-targeted H2S donor on vascular tone in PCA using organ-bath. Then, this study compared the effects of different types of H2S sources on vascular tone in PCA under different conditions in order to determine whether there is a difference in the signalling pathways activated by these three different H2S sources. The results of this study demonstrated there was no difference in the relaxation responses and mechanisms of relaxation responses between three different H2S donors, Na2S, GYY4137 and AP39. Thus, the studies looking at the vascular responses to H2S using the salts might be useful in determining the response to H2S in PCA. The results of this study demonstrated that there was a significant enhancement of the relaxation responses of all three donors in the presence of L-NAME. These results suggest chemical interaction of H2S with NO might lead to formation of inactive nitrosothiol, which may not be able to contract or relax blood vessels. In contrast to previous studies, the results of this study demonstrated that there was a significant enhancement of the relaxation responses of all three donors in the presence of TEA (non-selective K+ channels blocker) and glibenclamide (KATP channels blocker) and these responses might be due to potassium channels blockade increasing sensitivity to other mechanisms of H2S, such as calcium channels blockade. Interestingly, the blocking of potassium channels by TEA and KATP channels by glibenclamide led to an enhancement of the H2S-mediated relaxation, and these results are in contrast with previous studies that have reported that H2S relaxation was mediated via potassium channels. Furthermore, there was significant inhibition of calcium-induced contractions by all three H2S donors, and this inhibition was maintained after incubation of PCA with all the three different H2S sources for 15, 30 and 60 min. Furthermore, there was a significant inhibition of contraction of PCA induced by BayK8644, L-type voltage-gated calcium channel opener by all three different H2S sources, and thus, these results suggest that H2S acts by blocking calcium channels and also inhibiting a calcium-induced contraction pathway. Hypoxia plays critical roles in IHD. Therefore, studying the role of H2S as an oxygen sensor using three different H2S donors would be useful and interesting. Na2S salt and AP39 caused no significant effect in hypoxia response, hypoxia-induced relaxation, but they caused a significant decrease in the recovery response, reoxygenation-induced contraction. In contrast, GYY4137 caused both a significant enhancement of the hypoxia response and a significant decrease in the recovery response to reoxygenation. Therefore, these results of H2S donors, such as GYY4137 in PCA may be beneficial to decrease ischaemia-reperfusion injury (IRI). Thus, H2S could be important for the CVS and many cardiovascular pathologies, which have been associated with low H2S production. Therefore, H2S donors may be used as a cardiovascular protective drug against IRI. In conclusion, the present study demonstrated that H2S is produced in the heart and causes relaxation of the coronary artery. Sulfhydration measured by our assays in the heart could be used as an index of H2S production and potential signalling pathway for H2S. There was no difference in the relaxation responses and mechanisms of relaxations, such as NO, potassium channels, calcium channels among the three different H2S donors, Na2S, GYY4137 and AP39. There was a difference between the effects of Na2S and AP39 compared to GYY4137 in response to hypoxia and reoxygenation. Therefore, measurement of H2S generation and sulfhydration levels could be used as an index for changes in cardiac dysfunction. Thus, H2S sources could be beneficial in IHD, which are associated with low H2S generation and sulfhydration levels