Phospholamban S-sulfydration mediates H2S –dependent lusitropic effect in rat heart

H2S has recently emerged as an important mediator of mammalian cardiovascular homeostasis under both physiological and pathological conditions. H2S is active on the vessels in which it regulates the vascular tone, as well as on the heart, in which it inhibits contractility, also playing cardioprotection. The purpose of the present study was to evaluate in the Langendorff perfused rat hearts the effect of H2S also in relation to the diastolic function, an aspect which so far has been neglected. Besides the classic negative inotropic effect, NaHS (10-12÷10-7M) induced a dose-dependent inhibition of cardiac relaxation, as indicated by the reduction of –(LVdP/dt)max, significant at all concentrations tested, with a decrease of about 50% at higher concentrations. In addition, NaHS reduced HTR, an index of contraction–relaxation coupling, used to simultaneously evaluate variations in contraction and relaxation and to quantify drug-induced changes in myocardial lusitropy. This is of note since the shorter time needed to reach the 50% of LVP decline, suggests, for the first time, a negative lusitropic control elicited by H2S. In addition, NaHS (10-9 M) treatment induced Akt and eNOS phosphorylation consistent with an Akt-dependent eNOS activation and an increase of S-Sulfhydration of both cytosolic and membrane proteins. The range of S-Sulfhydrated proteins appeared to include the band corresponding to the regulatory protein phospholamban (PLN). Western Blotting of membrane fraction revealed two bands corresponding to the apparent molecular weights of PLN monomer (6kDa) and dimer (12 kDa) as putative targets for S-Sulfhydration. This was confirmed by immunoprecipitation of the membrane protein fraction with anti-PLN antibody on both control and NaHS treated hearts. Densitometric analysis of the bands corresponding to 6 and 12 kDa revealed an increased PLN S-Sulfhydration in NaHS-treated hearts, which was particularly evident in the case of the 12kDa band. Our data extend the role of H2S also to cardiac relaxation and suggest PLN S-sulfhydration as an alternative mechanism for SERCA2a regulation, with putative consequences on the H2S-dependent negative inotropic and lusitropic actions.