Prevention of Hypertrophy by Overexpression of Kv4.2 in Cultured Neonatal Cardiomyocytes

Abstract

Background-Prolonged action potentials (APs) and decreased transient outward K ϩ currents (I to ) are consistent findings in hypertrophic myocardium. However, the connection of these changes with cardiac hypertrophy is unknown. The present study investigated the effects of changes in I to and the associated alterations in AP on myocyte hypertrophy induced by phenylephrine. Methods and Results-Chronic incubation of cultured neonatal ventricular rat myocytes (NVRMs) with phenylephrine (PE) reduced I to density and prolonged AP duration, leading to a 2-fold increase in the net Ca 2ϩ influx per beat and a 1.4-fold increase in Ca 2ϩ -transient amplitude. PE treatment of chronically paced (2-Hz) NVRM also induced increases in cell size, protein/DNA ratio, atrial natriuretic factor mRNA expression, as well as ␤/␣ myosin mRNA ratio. These hypertrophic changes were associated with a 2.4-fold increase in activation of nuclear factor of activated T-cells (NFAT), indicating increased activity of the Ca 2ϩ -dependent phosphatase calcineurin. Overexpression of Kv4.2 channels using adenovirus prevented the AP duration prolongation as well as the increases in Ca 2ϩ influx and Ca 2ϩ -transient amplitude induced by PE. Kv4.2 overexpression also prohibited the PE-induced increases in cell size, protein/DNA ratio, atrial natriuretic factor expression, ␤/␣ myosin mRNA ratio, and NFAT activation.Conclusions-Our results demonstrate that PE-mediated hypertrophy in NRVMs seems to require I to reductions and AP prolongation associated with increased Ca 2ϩ influx and Ca 2ϩ transients as well as calcineurin activation. The clinical implications of these studies and the possible involvement of other signaling pathways are discussed. (Circulation.

Calcineurin Increases Cardiac Transient Outward K+ Currents via Transcriptional Up-regulation of Kv4.2 Channel Subunits

“…In addition, overexpression of CAIN (21), a specific inhibitory peptide of Cn, inhibited NFAT activation and prevented hypertrophy following treatment with PE, confirming the essential role of Cn signaling in ␣ 1 AR-induced hypertrophy. Consistent with previous studies (8,34), Fig. 4 (C and D) and Table 1 show that I to,f densities along with G slope and G max were decreased in PEtreated myocytes compared with control myocytes without PE.…”

“…Each of the lysed samples was added to 7 ml of ScintiSafe TM 30% liquid scintillation mixture (Fisher) in a 20-ml polyethylene scintillation vial (Fisher) and subjected to liquid scintillation counting. Electrophysiological Recordings in NRVMs-Whole-cell voltage-clamp recordings were done as described previously (8,25) at room temperature. Whole-cell currents were filtered at 2 kHz (Axon 200A amplifier).…”

“…To achieve this, we overexpressed either CAIN or constitutively active Cn in NRVMs in the presence of 100 mol/ liter PE for 48 h. As expected from a previous study (8), PE treatment induced marked myocyte hypertrophy based on cell microscopy, cell capacitance data, and […”

“…Cn Minimizes I to,f Reductions following Chronic ␣ 1 AR Stimulation-Because several previous studies have suggested that Cn activation in cardiac hypertrophy is responsible for reductions in I to,f (8,21), we investigated the regulation of I to,f by Cn in an in vitro hypertrophic model induced by ␣ 1 AR stimulation. To achieve this, we overexpressed either CAIN or constitutively active Cn in NRVMs in the presence of 100 mol/ liter PE for 48 h. As expected from a previous study (8), PE treatment induced marked myocyte hypertrophy based on cell microscopy, cell capacitance data, and […”

Ca2+ Controls Functional Expression of the Cardiac K+ Transient Outward Current via the Calcineurin Pathway

“…As reported, the Ca 2ϩ /calmodulin-activated phosphatase calcineurin and its downstream transcriptional effector calcineurin-nuclear factor of activated T-cells (NFAT) have been implicated as transducers of the pathological hypertrophic response (43). Importantly, decreased I to and downregulated expression of ventricular Kv4 mRNAs are consistent findings in the hypertrophic myocardium, which can lead to increases in Ca 2ϩ influx through L-type Ca 2ϩ channels during longer action potential (44). On the other hand, we suggest that, in this pathological condition, increased diastolic [Ca 2ϩ ] i might induce down-regulation of I to .…”