Abstract
Cardiac injury promotes fibroblasts activation and differentiation into myofibroblasts, which are hypersecretory of multiple cytokines. It is unknown whether any of such cytokines are involved in the electrophysiological remodeling of adult cardiomyocytes. We cultured adult cardiomyocytes for 3 days in cardiac fibroblast conditioned medium (FCM) from adult rats. In whole-cell voltage-clamp experiments, FCM-treated myocytes had 41% more peak inward sodium current (INa) density at −40 mV than myocytes in control medium (p<0.01). In contrast, peak transient outward current (Ito) was decreased by ∼55% at 60 mV (p<0.001). Protein analysis of FCM demonstrated that the concentration of TGF-β1 was >3 fold greater in FCM than control, which suggested that FCM effects could be mediated by TGF-β1. This was confirmed by pre-treatment with TGF-β1 neutralizing antibody, which abolished the FCM-induced changes in both INa and Ito. In current-clamp experiments TGF-β1 (10 ng/ml) prolonged the action potential duration at 30, 50, and 90 repolarization (p<0.05); at 50 ng/ml it gave rise to early afterdepolarizations. In voltage-clamp experiments, TGF-β1 increased INa density in a dose-dependent manner without affecting voltage dependence of activation or inactivation. INa density was −36.25±2.8 pA/pF in control, −59.17±6.2 pA/pF at 0.1 ng/ml (p<0.01), and −58.22±6.6 pA/pF at 1 ng/ml (p<0.01). In sharp contrast, Ito density decreased from 22.2±1.2 pA/pF to 12.7±0.98 pA/pF (p<0.001) at 10 ng/ml. At 1 ng/ml TGF-β1 significantly increased SCN5A (NaV1.5) (+73%; p<0.01), while reducing KCNIP2 (Kchip2; −77%; p<0.01) and KCND2 (KV4.2; −50% p<0.05) mRNA levels. Further, the TGF-β1-induced increase in INa was mediated through activation of the PI3K-AKT pathway via phosphorylation of FOXO1 (a negative regulator of SCN5A). TGF-β1 released by myofibroblasts differentially regulates transcription and function of the main cardiac sodium channel and of the channel responsible for the transient outward current. The results provide new mechanistic insight into the electrical remodeling associated with myocardial injury.
Highlights
[3] Protein analysis of the neonatal rat fibroblast conditioned medium (FCM) has shown that these cells release proinflammatory cytokines, such as transforming growth factor b-1 (TGF-b1), fibroblast growth factor (FGF), interferon-c (IFN-c), cytokine-induced neutrophil chemoattractant (CINC-1), macrophage migration inhibitory factor (MIF), vascular endothelial growth factor (VEGF) and tumor necrosis factor, [2] many of which are significantly upregulated after myocardial injury. [4,5] Neonatal rat fibroblast release significant amounts of IGF-1, endothelin A and leukemia inhibitory factor proteins, which are some of the proteins responsible for hypertrophy in cardiac myocytes and increased collagen synthesis in fibroblasts due to FCM. [6,7]
We have begun to investigate in adult ventricular myocytes the effects of specific cytokines that are known to be released by myofibroblasts and upregulated in the infarcted ventricle. [11,17,18] Here we have focused on the effects of TGF-b1 because our results provide strong evidence that this cytokine may be primarily responsible for the effects produced by FCM on the functional expression of two major ion channels of cultured adult ventricular myocytes
In control experiments the mean peak sodium current measured in freshly dissociated rat ventricular myocytes (Day 0) was similar to that measured after 72 hours in control medium (Day 3)
Summary
Cardiac fibroblasts represent the most abundant cell type in the heart. [1] Myocardial disease promotes fibroblast proliferation and differentiation into myofibroblasts, which are hypersecretory of both ECM components and multiple cytokines and growth factors that are known to affect myocyte morphology and function. [2] Cardiac fibroblast conditioned medium (FCM) from neonatal rats has been shown to protect cardiac myocytes against hypoxia induced injury in isolated cells and the growth factors in such medium had a protective effect against ischemia-reperfusion injury. [3] Protein analysis of the neonatal rat FCM has shown that these cells release proinflammatory cytokines, such as transforming growth factor b-1 (TGF-b1), fibroblast growth factor (FGF), interferon-c (IFN-c), cytokine-induced neutrophil chemoattractant (CINC-1), macrophage migration inhibitory factor (MIF), vascular endothelial growth factor (VEGF) and tumor necrosis factor, [2] many of which are significantly upregulated after myocardial injury. [4,5] Neonatal rat fibroblast release significant amounts of IGF-1, endothelin A and leukemia inhibitory factor proteins, which are some of the proteins responsible for hypertrophy in cardiac myocytes and increased collagen synthesis in fibroblasts due to FCM. [6,7].A number of cytokines may affect the electrical properties of cells. [2] Cardiac fibroblast conditioned medium (FCM) from neonatal rats has been shown to protect cardiac myocytes against hypoxia induced injury in isolated cells and the growth factors in such medium had a protective effect against ischemia-reperfusion injury. Reports on the consequences of culturing neonatal myocytes in cardiac FCM showed increased protein expression, [12] action potential duration (APD) prolongation and KV4.2 downregulation. [13] Pedrotty et al, [14] have shown that exposure of neonatal rat ventricular myocyte (NRVM) monolayers to FCM produced dosedependent reduction in conduction velocity, prolongation of APD, depolarization of the resting membrane potential and reduction of the AP upstroke velocity. The optical APD70 was slightly shorter in the former than in the latter. [15]
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