A molecular basis for HCN channel over-expression.

Background: Myocardial hypertrophy and failure is characterized by remodeling of ventricular cardiomyocytes (VCM), leading to electrophysiological abnormalities responsible for severe arrhythmias. A functional over-expression of the f-current occurs in VCM isolated from both hypertrophied rat hearts and failing human hearts, where it might contribute to arrhythmias. Four different genes encoding for the f-channel are known and termed Hyperpolarization-activated Cyclic-Nucleotide-gated Channels (HCN 1-4). No information is available on the influence of cardiac disease on isoform expression, and its relationship with the functional occurrence of the f-current. This study aimed to investigate the effect of hypertrophic factors on HCN expression in primary cultures of adult rat VCM, a well-established model for studying the molecular events involved in myocardial remodeling. Methods and Results: VCM isolated from the left ventricle of adult rat hearts were cultured either in control condition or in the presence of 0.1 µM endothelin-1 (ET-1) for 4-9 days. Total mRNA was converted to cDNA by reverse transcription. Primers and probes were designed to amplify cDNA of HCN1-4 genes and quantify mRNA by Real-Time PCR. Cultured VCM expressed HCN2, 3 and 4 isoforms, the rank of expression being HCN2>>HCN4>HCN3. The specific mRNA for HCN3,4 isoforms significantly increased with time (HCN3: +19.7±0.9% at 6 days and +24.5±1.5% at 7 days, p<0.001 vs. 0-4 days; HCN4: +48.5±4.9% at 6 days and +93.2±7.4% at 7 days, p<0.001 vs. 0-4 days). At 6 day of culture, exposure to ET-1 caused a selective and significant increase of mRNA coding for HCN2 with respect to controls (+141.3±54.8% vs. +38.5±12.2%, p<0.001). These results were in agreement with patch-clamp measurements of f-current in cultured cells. The technique was used to measure (Cm), an index of cell size, and f-current, evoked by hyperpolarization to -120 mV. At 6-9 days of culture, membrane capacitance, an index of cell size (+72%) and f-current density (+171%) markedly augmented as compared to 3-5 days. f-current density was significantly higher in cells exposed to ET-1 (3.7±0.5pA/pF, n=31) than in control cells (2.3±0.4pA/pF, n=40) (p<0.05). Conclusions: Ventricular cardiomyocytes express HCN2,3,4 isoforms with a predominance of HCN2. In cultured cells, chronic exposure to ET-1 increases the mRNA expression of the HCN2 isoform and the functional expression of f-current density, with respect to control conditions. These results suggest that hypertrophic factors such as ET-1 play a major role in cardiac electrophysiological remodeling.