Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node

Abstract

Several studies of the pacemaker mechanisms in mammalian cells, most of which were carried out in cells isolated from the rabbit sinoatrial node (SAN), have highlighted the role of the I_f current. While the distribution of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, the molecular correlates of f-channels, is known at the mRNA level, the identification of f-channel proteins in this tissue is still undetermined. Here we investigate HCN protein expression in the rabbit pacemaker region. We found that HCN4 is the main isoform, and set therefore to analyze its distribution within the SAN and surrounding areas with the aim of correlating protein expression and pacemaking function. The analysis was carried out in tissue slices and single cells of the intercaval area, which includes the crista terminalis (CT), the SAN, and the septum interatrialis (SI). Immunolabeling, in situ hybridization, qRT-PCR analysis, and electrophysiological recordings identified the SAN as a region characterized by high HCN4 signal and current levels, while the expression in the CT and in the SI was either negligible or absent. Detailed analysis of the central SAN area showed that cells are predominantly distributed in islets interconnected by cell prolongations, and single-cell HCN4 labeling suggested sites of channel clustering. Our data indicate that in the rabbit SAN, HCN4 proteins are major constituents of native f-channels, and their distribution matches closely the SAN as defined morphologically and electrophysiologically. Until recently, the SAN was identified as the region where Cx43 and atrial natriuretic peptide are not expressed; we propose here that expression of HCN4 is an appropriate tool to map and identify the cardiac SAN pacemaker region.

Introduction

In mammals, the pacemaker region of the heart (SAN) is located in the posterior wall of the right atrium; a coarse anatomical definition identifies this structure as the intercaval region adjacent to the atrial muscle of the CT extending from the superior to near the inferior vena cava (SVC, IVC). The histological investigation reveals a heterogeneous architecture with myocytes sparsely distributed in a dense connective matrix [1], [2]. The identification of the SAN as the leading pacemaker zone of the rabbit heart was originally accomplished by electrophysiological mapping studies and later confirmed in rabbit and other species. Since the repertoire of transcribed mRNAs and expressed proteins changes in different types of myocytes, in more recent years criteria based on molecular properties have also been extensively employed to identify SAN cells. For example SAN cells, unlike adjacent atrial working myocytes, are rich in connexin 45 (Cx45) and (in the rabbit) neurofilament M (NF-M), but lack connexin 43 (Cx43) and atrial natriuretic peptide (ANP). The specific pattern of mRNA/protein expression can be used therefore as a cellular “footprint” of the SAN area [3].

The electrical oscillatory property of SAN cells reflects the lack of a stable resting potential and is associated with the slow diastolic depolarization of the action potential. Among the various ion channels and transporters contributing to spontaneous activity, the “funny” (f-) channels play a key role in the generation of the early fraction of the diastolic depolarization and in the autonomic modulation of cardiac rhythm, achieved through the control of the diastolic depolarization rate [4]. Importantly, it has recently been confirmed that the pacemaker current is present in the human SAN [5].

Crystallographic and electrophysiological investigation of the molecular structure of HCN channels indicates that they assemble as tetramers [6]. Extensive work has addressed the issue of the isoform composition of native f-channels, and both homomeric and heteromeric assemblies of the four isoforms known have been demonstrated in vitro and/or in vivo[7], [8], [9], [10], but conclusive evidence on the identification of HCN isoforms contributing to f-channels are still missing.

mRNA signals and the corresponding protein expression are well correlated in the mouse SAN [11], [12]; however, while electrophysiological, morphological, structural, and histochemical data on SAN tissue are largely based on experiments in the rabbit heart, no direct measurements of HCN protein in the rabbit SAN are yet available.

Several data indicate that HCN4 is the major HCN subunit composing f-channels, including evidence for the high expression of the mRNA of HCN4 relative to other HCN isoforms in nodal tissue [11], [13], [14], [15], [16] and for the contribution of HCN4 to kinetic and ionic properties of native f-channels in the SAN [8], as well as recent evidence correlating mutations of HCN4 protein in humans to sinus bradycardia or more complex cardiac pathologies involving disturbances of rhythm [17].

We present here a set of experiments aimed to identify the distribution of the HCN4 protein in the rabbit SAN region. Our results indicate that the HCN4 isoform is abundantly expressed in nodal cells, but is virtually absent in surrounding atrial myocytes. Thus, HCN4 can be considered as a specific molecular marker of the nodal pacemaker cells.