TGF-β1, Released by Myofibroblasts, Differentially Regulates Transcription and Function of Sodium and Potassium Channels in Adult Rat Ventricular Myocytes

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 (I-Na) density at -40 mV than myocytes in control medium (p<0.01). In contrast, peak transient outward current (I-to) was decreased by similar to 55% at 60 mV (p<0.001). Protein analysis of FCM demonstrated that the concentration of TGF-beta 1 was >3 fold greater in FCM than control, which suggested that FCM effects could be mediated by TGF-beta 1. This was confirmed by pre-treatment with TGF-beta 1 neutralizing antibody, which abolished the FCM-induced changes in both I-Na and I-to. In current-clamp experiments TGF-beta 1 (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-beta 1 increased I-Na density in a dose-dependent manner without affecting voltage dependence of activation or inactivation. I-Na 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, I-to 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-beta 1 significantly increased SCN5A (Na(V)1.5) (+73%; p<0.01), while reducing KCNIP2 (Kchip2; -77%; p<0.01) and KCND2 (K(V)4.2; -50% p<0.05) mRNA levels. Further, the TGF-beta 1-induced increase in I-Na was mediated through activation of the PI3K-AKT pathway via phosphorylation of FOXO1 (a negative regulator of SCN5A). TGF-beta 1 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.