Expression of the hyperpolarization-activated current, I_f, in cultured adult rat ventricular cardiomyocytes and its modulation by hypertrophic factors

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) current, I_f, is typically overexpressed in myocytes from hypertrophied and failing hearts, where it may play an arrhythmogenic role. Signaling pathways activated by agonists such as angiotensin-II, endothelin-1 and phenylephrine, via G protein-coupled receptors (GPCR), promote myocardial hypertrophy, but their effect on cellular electrophysiological remodeling, particularly I_f expression is largely unknown. Thus, I_f expression was measured by patch-clamp and quantitative RT-PCR measurement in cultured adult rat ventricular cardiomyocytes (VCM) exposed to different culture conditions, that is, in the absence or presence of: fetal bovine serum (FBS, 5%), 0.1 μM angiotensin-II, 0.1 μM endothelin-1 or 20 μM phenylephrine. Membrane capacitance (C_m) was used to estimate cell size and current density in patch-clamped VCM. At 8 days of culture, about 60% of VCM showed I_f. In serum-free medium, I_f density was increased by phenylephrine (2.28 ± 0.51 vs. 0.84 ± 0.30 pA/pF in CTR, p < 0.05) and endothelin-1 (2.20 ± 0.38 vs. 1.03 ± 0.34 pA/pF in control, p < 0.05), not by angiotensin-II (1.60 ± 0.50 pA/pF, not significant vs. control). Similarly, in cells cultured with 5% FBS, phenylephrine and endothelin-1 significantly increased I_f density by 159.3% and 59.5% (p < 0.05 vs. untreated cells), while angiotensin-II did not modify it. The effect of endothelin-1 was abolished by using the selective endothelin receptor type A (ET_1A) antagonist BQ-123 (1 μM). mRNA levels for HCN2 and HCN4 were significantly increased during in vitro culture; exposure to endothelin-1 increased HCN2 mRNA. A similar pattern of I_f overexpression was detected in hypertrophied left ventricular cardiomyocytes from old hypertensive rats. Thus, adult VCM in primary culture undergo changes in I_f expression reminiscent of in vivo hypertrophy; endothelin-1 (but no angiotensin-II) seems to play a role in ionic remodeling.