Does Protein Sulfuration Take Place in the Heart?

Abstract

Endogenous hydrogen sulfide (H2S) has been suggested to play a physiological role through protein sulfuration (also referred as sulfhydration) in the regulation of cardiac contractility. The sulfuration is a process by which H2S combines to free cysteine residue on proteins to create persulfide (protein‐S‐SH), changing protein conformation, such as in calcium channels, and functions. Since any new molecule of free H2S produced will immediately combine with the iron present on myoglobin or will be oxidized, our objective was to determine whether increasing endogenous H2S production can actually increase the pool of H2S combined with cysteine residues in the heart.To achieve this goal, we infused exogenous H2S up to a level that will alter cardiac contractility in order to create conditions wherein soluble H2S concentrations increase to levels higher than those reached by endogenous sulfide in the heart. The presence of sulfurated protein in the heart was identified by measuring gaseous H2S released after reducing disulfide bonds. TCEP was chosen as a potent reducing agent instead of dithiothreitol (DTT), previously used for this purpose in the literature, as DTT possesses two thiol groups (−SH) a significant source of H2S. Hearts were removed from control animals and animals exposed to 0.8 mg/min NaHS until cardiac depression‐induced hypotension was produced. The hearts were immediately homogenized in glycine‐NaOH buffer (pH 9), to prevent any evaporation of gaseous H2S. The homogenate was transferred into the 50‐ml chamber filled with sodium phosphate buffer (final pH ~6). At this pH, HS− will easily convert into gaseous H2S, in keeping with H2S partial pressure, and measured in the chamber headspace. The headspace was continuously flowed with N2, and H2S was directly measured by a voltammetry H2S analyzer before and after TCEP (5 mM).We found that the level of H2S produced by TCEP was similar and very low (below 0.5 nmol/g of protein) in both the control and intoxicated hearts, despite levels of free H2S in the blood above 10 microM in the latter. In contrast, the pool of H2S in albumin solution (10% in saline) incubated with H2S for 10 min, increased as a function of H2S concentrations using 0, 2, and 20 microM NaHS and reaching 10.6 ± 1.1, 11.8 ± 0.7 and 13.3 ± 0.4 nmol/g respectively. The change in H2S remained however extremely modest.Toxic levels of soluble H2S were unable to increase the pool of H2S liberated from the heart in a strong reducing environment. This suggests that free H2S in the heart is immediately trapped by other compounds such metallo‐proteins, e.g. myoglobin, present in the heart or is oxidized in the mitochondria before reacting with proteins, in contrast to a solution of albumin unable to produce these effects. Even in the latter, the pool of H2S produced by an increase in free H2S appears to be extremely small and to require toxic levels of sulfide to be affected.