Molecular composition and functional properties of f-channels in murine embryonic stem cell-derived pacemaker cells

Abstract

Mouse embryonic stem cells (mESCs) differentiate into all cardiac phenotypes, and thus represent an important potential source for cardiac regenerative therapies. Here we characterize the molecular composition and functional properties of “funny” (f-) channels in mESC-derived pacemaker cells. Following differentiation, a fraction of mESC-derived myocytes exhibited action potentials characterized by a slow diastolic depolarization and expressed the I_f current. I_f plays an important role in the pacemaking mechanism of these cells since ivabradine (3 μM), a specific f-channel inhibitor, inhibited I_f by about 50% and slowed rate by about 25%. Analysis of I_f kinetics revealed the presence of two populations of cells, one expressing a fast- and one a slow-activating I_f; the two components are present both at early and late stages of differentiation and had also distinct activation curves. Immunofluorescence analysis revealed that HCN1 and HCN4 are the only isoforms of the pacemaker channel expressed in these cells. Rhythmic cells responded to β-adrenergic and muscarinic agonists: isoproterenol (1 μM) accelerated and acetylcholine (0.1 μM) slowed spontaneous rate by about 50 and 12%, respectively. The same agonists caused quantitatively different effects on I_f: isoproterenol shifted activation curves by about 5.9 and 2.7 mV and acetylcholine by − 4.0 and − 2.0 mV in slow and fast I_f-activating cells, respectively. Accordingly, β1- and β2-adrenergic, and M2-muscarinic receptors were detected in mESC-derived myocytes. Our data show that mESC-derived pacemaker cells functionally express proteins which underlie generation and modulation of heart rhythm, and can therefore represent a potential cell substrate for the generation of biological pacemakers.

Introduction

The pacemaker of the heart is located in the sinoatrial node (SAN), a region structurally and functionally different from the rest of the myocardium. Its function is to generate spontaneous action potentials and drive the heartbeat with a mechanism allowing fine tuning of rate by the autonomic nervous system [1], [2], [3]. Sinoatrial myocytes and autorhythmic cells in general (like embryonic ventricular myocytes) exhibit action potentials with a slow diastolic depolarization, which at the termination of an action potential drives the membrane potential to the threshold for the next one. It is established that an important role in initiating the diastolic depolarization and modulating its rate, hence the rate of spontaneous activity, is played by the pacemaker I_f current [4], [5]. I_f flows through Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, the molecular correlates of f-channels; four HCN isoforms have been identified so far which are all expressed with different densities in different regions of the heart [6], [7], [8].

Evidence confirming the basic function of HCN channels in the generation, development and modulation of pacemaker activity has recently been provided in mice and humans [9], [10], [11], [12], [13], [14].

The specific role of f/HCN channels in pacemaking makes their properties essential in the development of new tools aiming to heart rate control, such as the biological pacemakers [15]. Overexpression of HCN channels can indeed provide a depolarizing stimulus sufficient to induce, in vitro and in vivo, quiescent myocytes to beat spontaneously [16], [17], [18], [19], [20]. An alternative approach proven to be effective in pacing the heart has employed human embryonic stem cell-derived autorhythmic agglomerates [21], [22]. Although the mechanism by which ES cells can pace silent cardiac tissue has not been fully elucidated, it is long known that ES cells can differentiate toward a cardiac pacemaker phenotype [23], [24], [25], [26].

While it is well established that ESC-derived cardiomyocytes express the I_f current [23], [26], [27] and that HCN channels, whether native or overexpressed, underlie generation of spontaneous activity [28], [29], a detailed analysis of the HCN isoforms expressed in ESC-derived cells is still lacking. In this work, we have analyzed the HCN composition of both spontaneously beating embryoid bodies (EBs) and isolated autorhythmic cells. Further, we have analyzed the basic properties of I_f, its involvement in pacemaking and its modulation by neurotransmitters and which type of G-protein-coupled receptors underlie the I_f-mediated autonomic response.