Double Regulation of Cardiac Excitation-Contraction Coupling and Oxidant Stress by Pirfenidone

Abstract

Pirfenidone (PFD) is well-known for improving cardiac performance in models of disease. Thus, here we have investigated its functional effects on cardiomyocytes. Cells were cultured under control culture conditions or the presence of PFD (1 mM, 1-2d). Subsequently, they were subjected to electrical stimulation to examine the levels of intracellular Ca2+ and contractility. Remarkably, PFD improved both peak contraction and kinetics of shortening and relaxation. Moreover, the amplitude and kinetics of Ca2+ transients were also enhanced. Excitation-contraction coupling (ECC) was also investigated, under whole-cell patch-clamp. In keeping with a previous report, PFD elevated twofold the activity of CaV1.2. Besides, a similar increase in the magnitude of Ca2+ transients was also observed. Thus the gain of ECC was unaltered. Likewise, PFD did not alter the caffeine-sensitive Ca2+ store, indicating stimulation of Ca2+-induced Ca2+-release at constant sarcoplasmic reticulum Ca2+ load. Interestingly, although the expression of the Na+/Ca2+ exchanger (NCX) was unaffected, the decay of Ca2+ signal during caffeine applications was 50% slower in PFD-treated cells (compared with controls), indicating that PFD downregulates the NCX activity. PFD also inhibited the production of reactive oxygen species, under both, basal conditions and the presence of oxidant agents (acetaldehyde and peroxide hydrogen). Conversely, the amount of nitric oxide (NO) was either, increased (in atrial myocytes) or unchanged (in ventricular myocytes). Western-blot analysis showed that endothelial nitric oxide synthase expression remains unaffected, while a dual regulation was observed for that of neuronal isoform (nNOS) which consisted of inhibition and stimulation, in ventricular and atrial myocytes, respectively. Thus, in atrial cardiomyocytes, an up-regulation of nNOS was enough to augment the synthesis of NO. Apart from improving our knowledge of the molecular mechanisms of PFD action, these findings may also help in explaining the corresponding cardioprotective effects.