Data_Sheet_1_Long-Term Regulation of Excitation–Contraction Coupling and Oxidative Stress in Cardiac Myocytes by Pirfenidone.pdf

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.

吡非尼酮（Pirfenidone, PFD）可用于治疗人类肺纤维化。将其给予患有不同类型心血管疾病的动物后，可显著改善其心脏功能。本研究针对其对心肌细胞的功能影响展开了探究。实验中将细胞分为两组：一组在正常培养条件下培养1~2天，另一组则在添加1 mM吡非尼酮的培养基中培养相同时长。随后对两组细胞施加电刺激，以评估其收缩能力与细胞内钙离子（Ca²+）水平。吡非尼酮处理可同时提升心肌细胞的收缩峰值，以及收缩与舒张的动力学特性。此外，钙离子瞬变的幅度与动力学特性也得到了增强。本研究同时在全细胞膜片钳条件下探究了兴奋-收缩耦联（Excitation–contraction coupling, ECC）。与此前的研究报道一致，吡非尼酮可使钙离子电流（Ca²+ current, ICa）的密度提升一倍。值得注意的是，研究同时观察到钙离子瞬变的幅度出现了类似的提升。因此，兴奋-收缩耦联的增益并未发生改变。同样地，吡非尼酮并未改变咖啡因诱导的钙离子释放峰值幅度，提示在肌浆网钙离子负载维持恒定的条件下，吡非尼酮可促进钙触发钙释放（Ca2+-induced–Ca2+-release, CICR）。相平面分析结果显示，吡非尼酮可促进肌丝的钙离子脱敏现象，而这一效应可通过升高钙离子水平来代偿，以维持收缩功能。有趣的是，尽管钠钙交换体（Na+/Ca2+ exchanger, NCX）的表达水平未受影响，但在咖啡因存在的情况下，经吡非尼酮处理的细胞内钙离子信号衰减速度较对照组慢50%，这提示吡非尼酮可下调该交换体的活性。吡非尼酮还可抑制活性氧的产生，无论是在基础培养条件下，还是在氧化应激刺激（乙醛与过氧化氢）存在的情况下。与之相反，一氧化氮的产生则呈现出两种情况：在心房肌细胞中一氧化氮产生增加，而在心室肌细胞中则无明显变化。研究同时检测了内皮型一氧化氮合酶（endothelial nitric oxide synthase, eNOS）与神经元型一氧化氮合酶（neuronal nitric oxide synthase, nNOS）的蛋白表达水平。内皮型一氧化氮合酶的表达水平未出现显著变化。与之形成对比的是，神经元型一氧化氮合酶则呈现出双向调控：在心室肌细胞中其活性受到抑制，而在心房肌细胞中则被激活。因此，在心房肌细胞中，神经元型一氧化氮合酶的上调足以促进一氧化氮的合成。上述研究结果加深了我们对吡非尼酮作用分子机制的认识，同时也有助于阐释其对应的心脏保护效应。