Abstract
Pirfenidone (PFD) is used to treat human pulmonary fibrosis. Its administration to animals with distinct forms of cardiovascular disease results in striking improvement in cardiac performance. Here, its functional impact on cardiac myocytes was investigated. Cells were kept 1–2 days under either control culture conditions or the presence of PFD (1 mM). Subsequently, they were subjected to electrical stimulation to assess the levels of contractility and intracellular Ca2+. The PFD treatment promoted an increase in both peak contraction and kinetics of shortening and relaxation. Moreover, the amplitude and kinetics of Ca2+ transients were enhanced as well. Excitation–contraction coupling (ECC) was also investigated, under whole-cell patch-clamp conditions. In keeping with a previous report, PFD increased twofold the density of Ca2+ current (ICa). Notably, 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 peak amplitude of caffeine-induced Ca2+ release, indicating stimulation of Ca2+-induced–Ca2+-release (CICR) at constant sarcoplasmic reticulum Ca2+ load. A phase-plane analysis indicated that PFD promotes myofilament Ca2+ desensitization, which is being compensated by higher levels of Ca2+ to promote contraction. Interestingly, although the expression of the Na+/Ca2+ exchanger (NCX) was unaffected, the decay of Ca2+ signal in the presence of caffeine was 50% slower in PFD-treated cells (compared with controls), suggesting that PFD downregulates the activity of the exchanger. PFD also inhibited the production of reactive oxygen species, under both, basal conditions and the presence of oxidative insults (acetaldehyde and peroxide hydrogen). Conversely, the production of nitric oxide was either increased (in atrial myocytes) or remained unchanged (in ventricular myocytes). Protein levels of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) were also investigated. eNOS values did not exhibit significant changes. By contrast, a dual regulation was observed for nNOS, which consisted of inhibition and stimulation, in ventricular and atrial myocytes, respectively. In the latter cells, therefore, an up-regulation of nNOS was sufficient to stimulate the synthesis of NO. These findings improve our knowledge of molecular mechanisms of PFD action and may also help in explaining the corresponding cardioprotective effects.
Highlights
Excitation–contraction coupling (ECC) is of pivotal relevance for cardiac contractility
The procedure was approved by the Institutional Animal Care and Use Committee (IACUC— CINVESTAV, 0199-16), complies with the Mexican Official Norm NOM-062-ZOO-1999 and is in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011)
Our previous work has shown that the density of ICa is larger in PFD-treated myocytes than in controls (Ramos-Mondragón et al, 2012)
Summary
Excitation–contraction coupling (ECC) is of pivotal relevance for cardiac contractility. An action potential (AP) activates voltage-gated Ca2+ channels of the plasma membrane (CaV1.2), allowing entry of extracellular Ca2+ (ICa) that activates ryanodine receptors (RyR2) and a consequent release of Ca2+ from the sarcoplasmic reticulum (SR). The resulting increase in cytosolic Ca2+ (Ca2+ transient) activates the contractile machinery, and relaxation occurs as the levels of Ca2+ decay thanks to the activity of both the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) and the Na+/Ca2+ exchanger (NCX; for reviews see Bers, 2002; Eisner et al, 2017; Landstrom et al, 2017). Many studies have reported that PFD improves cardiac performance in animal models of: atrial fibrillation (AF, Lee et al, 2006), myocardial infarction (Nguyen et al, 2010; Li et al, 2017; Adamo et al, 2018), Duchenne muscular dystrophy (Van Erp et al, 2006), pressure overload (Mirkovic et al, 2002; Wang et al, 2013; Yamagami et al, 2015), hypertrophy (Yamazaki et al, 2012), diabetic cardiomyopathy (Miric et al, 2001), and diphtheritic myocarditis (Adamo et al, 2018)
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