Abstract 4626: Optimal Dose of Adv-hHCN4 Creates Stable and Long-lasting Biopacing Activity in Cultured Ventricular Cardiomyocytes

Abstract

Listen

Translate article

Purpose: Hyperpolarization-activated cyclic nucleotide-gated (HCN) genes have been successfully used as a strategy for recreating cardiac biological pacemakers in animal models. However, optimal dose of HCN and toxicity from HCN overexpression have not been investigated. Therefore, we assessed the effects of various titers of adenoviral human HCN4-GFP vector (Adv-hHCN4) on cardiomyocytes. Methods: Neonatal rat ventricular myocytes (NRVMs) were isolated, selected and cultured on microelectrode arrays to assess their automaticities. Morphology and apoptosis with and without HCN or Ca 2+ channel inhibitor were also assessed. Results: Beating rates significantly increased in NRVMs after hHCN4 infection (Fig. 1 ). For example, the rates were gradually increased to 235±11 beat/min on day 7 after hHCN4 infection with 1×10 5 PFU/array. In contrast, control cells showed low rates. NRVMs with ≥10 6 PFU/array Adv-hHCN4 reached faster rates early and subsequently stopped beating (Fig. 1 ). In addition, myocytes with ≥10 6 PFU/array Adv-hHCN4 underwent significant apoptosis (>50%) which potentially resulted from hHCN4 overexpression and was blocked by the HCN channel blocker Cs + (1 mM), but not by the Ca 2+ channel inhibitor nifedipine. In addition, myocytes infected with ≥10 6 PFU/array Adv-GFP maintained normal morphology and rate. Our data demonstrate that hHCN4 transfer significantly and dose-dependently increased beating rates of NRVMs. However, overexpression of HCN could cause apoptosis. Therefore, an optimal dose of HCN gene is important for reducing toxicity and creating stable and long-lasting biopacing activity in cardiomyocytes in vitro, and probably also in vivo. Figure 1. Effects of hHCN4 infection on automaticities of neonatal rat ventricular myocytes. Each data point represents an averaged beating rate (mean ± SE) from 8 to 10 arrays. Various titers (1×10 5 to 1×10 7 PFU/500,000 cells per array) of Adv-Hhcn4 (expect control) were added to the arrays after measurements on day 0 (see the arrow)