Halina Dobrzynski

An unexpected requirement for brain-type sodium channels for control of heart rate in the mouse sinoatrial node

Voltage-gated Na+ channels are composed of pore-forming α and auxiliary β subunits. The majority of Na+ channels in the heart contain tetrodotoxin (TTX)-insensitive Nav1.5 α subunits, but TTX-sensitive brain-type Na+ channel α subunits are present and functionally important in the transverse tubules of ventricular myocytes. Sinoatrial (SA) nodal cells were identified in cardiac tissue sections by staining for connexin 43 (which is expressed in atrial tissue but not in SA node), and Na+ channel localization was analyzed by immunocytochemical staining with subtype-specific antibodies and confocal microscopy. Brain-type TTX-sensitive Nav1.1 and Nav1.3 α subunits and all four β subunits were present in mouse SA node, but Nav1.5 α subunits were not. Nav1.1 α subunits were also present in rat SA node. Isolated mouse hearts were retrogradely perfused in a Langendorff preparation, and electrocardiograms were recorded. Spontaneous heart rate and cycle length were constant, and heart rate variability was small under control conditions. In contrast, in the presence of 100 nM TTX to block TTX-sensitive Na+ channels specifically, we observed a significant reduction in spontaneous heart rate and markedly greater heart rate variability, similar to sick-sinus syndrome in man. We hypothesize that brain-type Na+ channels are required because their more positive voltage dependence of inactivation allows them to function at the depolarized membrane potential of SA nodal cells. Our results demonstrate an important contribution of TTX-sensitive brain-type Na+ channels to SA nodal automaticity in mouse heart and suggest that they may also contribute to SA nodal function and dysfunction in human heart.

Requirement of neuronal‐ and cardiac‐type sodium channels for murine sinoatrial node pacemaking

The majority of Na+ channels in the heart are composed of the tetrodotoxin (TTX)‐resistant (KD, 2–6 μm) Nav1.5 isoform; however, recently it has been shown that TTX‐sensitive (KD, 1–10 nm) neuronal Na+ channel isoforms (Nav1.1, Nav1.3 and Nav1.6) are also present and functionally important in the myocytes of the ventricles and the sinoatrial (SA) node. In the present study, in mouse SA node pacemaker cells, we investigated Na+ currents under physiological conditions and the expression of cardiac and neuronal Na+ channel isoforms. We identified two distinct Na+ current components, TTX resistant and TTX sensitive. At 37°C, TTX‐resistant iNa and TTX‐sensitive iNa started to activate at ∼−70 and ∼−60 mV, and peaked at −30 and −10 mV, with a current density of 22 ± 3 and 18 ± 1 pA pF−1, respectively. TTX‐sensitive iNa inactivated at more positive potentials as compared to TTX‐resistant iNa. Using action potential clamp, TTX‐sensitive iNa was observed to activate late during the pacemaker potential. Using immunocytochemistry and confocal microscopy, different distributions of the TTX‐resistant cardiac isoform, Nav1.5, and the TTX‐sensitive neuronal isoform, Nav1.1, were observed: Nav1.5 was absent from the centre of the SA node, but present in the periphery of the SA node, whereas Nav1.1 was present throughout the SA node. Nanomolar concentrations (10 or 100 nm) of TTX, which block TTX‐sensitive iNa, slowed pacemaking in both intact SA node preparations and isolated SA node cells without a significant effect on SA node conduction. In contrast, micromolar concentrations (1–30 μm) of TTX, which block TTX‐resistant iNa as well as TTX‐sensitive iNa, slowed both pacemaking and SA node conduction. It is concluded that two Na+ channel isoforms are important for the functioning of the SA node: neuronal (putative Nav1.1) and cardiac Nav1.5 isoforms are involved in pacemaking, although the cardiac Nav1.5 isoform alone is involved in the propagation of the action potential from the SA node to the surrounding atrial muscle.

Computer Three-Dimensional Reconstruction of the Sinoatrial Node

Background—There is an effort to build an anatomically and biophysically detailed virtual heart, and, although there are models for the atria and ventricles, there is no model for the sinoatrial node (SAN). For the SAN to show pacemaking and drive atrial muscle, theoretically, there should be a gradient in electrical coupling from the center to the periphery of the SAN and an interdigitation of SAN and atrial cells at the periphery. Any model should include such features. Methods and Results—Staining of rabbit SAN preparations for histology, middle neurofilament, atrial natriuretic peptide, and connexin (Cx) 43 revealed multiple cell types within and around the SAN (SAN and atrial cells, fibroblasts, and adipocytes). In contrast to atrial cells, all SAN cells expressed middle neurofilament (but not atrial natriuretic peptide) mRNA and protein. However, 2 distinct SAN cell types were observed: cells in the center (leading pacemaker site) were small, were organized in a mesh, and did not express Cx43. In contrast, cells in the periphery (exit pathway from the SAN) were large, were arranged predominantly in parallel, often expressed Cx43, and were mixed with atrial cells. An ≈2.5-million-element array model of the SAN and surrounding atrium, incorporating all cell types, was constructed. Conclusions—For the first time, a 3D anatomically detailed mathematical model of the SAN has been constructed, and this shows the presence of a specialized interface between the SAN and atrial muscle.

Sophisticated Architecture is Required for the Sinoatrial Node to Perform Its Normal Pacemaker Function

Structure‐Function Relationships of the SA Node. The hearts pacemaker, the sinoatrial node, does not consist of a group of uniform sinoatrial node cells embedded in atrial muscle. Instead, it is a heterogeneous tissue with multiple cell types and a complex structure. Evidence suggests that from the periphery to the center of the sinoatrial node, there is a gradient in action potential shape, pacemaking, ionic current densities, connexin expression, Ca2+ handling, myofilament density, and cell size. This complexity may be necessary for the sinoatrial node to pacemake under diverse conditions, drive the more hyperpolarized atrial muscle, and resist proarrhythmic perturbations.

Cellular Mechanisms of Sinoatrial Activity

The rhythmic beating of the heart is the result of action potentials initiated in a small specialized tissue, the sinoatrial (SA) node. With a wealth of recent information on the cellular electrophysiology, molecular biology, and microscopic morphology of the SA node, the machinery underlying its robust pacemaking during a variety of physiologic and pathologic conditions is beginning to be fully understood. This chapter discusses (1) the ionic events (and their molecular basis) responsible for the spontaneous activity in SA node cells, and (2) the sophisticated organization of different types of SA node cells within the SA node as a whole to make a robust and dependable pacemaker for the heart.

Heterogeneous sinoatrial node of rabbit heart-molecular and electrical mapping and biophysical reconstruction

Electrophysiology and immunocytochemistry have been used to map the detailed properties of cells cross the pacemaker of the heart, the sinoatrial node. The electrical activities and expressions of various proteins across the sinoatrial node change smoothly with distance and so can be modelled by a gradient in parameter values. Experimental data have been integrated into computational models of the SA node at cellular and tissue levels and mechanisms underlying initiation and propagation of pacemaker activity of the heart identified.

Molecular Investigation into the Human Atrioventricular Node in Heart Failure

The atrioventricular node (AVN), the molecular basis has been studied in animal models; however, the human AVN remains poorly explored at the molecular level in heart failure patients. We studied ex vivo donor human hearts rejected for transplantation (n=6) and end-stage failing hearts with cardiomyopathy of various etiologies (n=6). Microdissection and quantitative PCR (qPCR) were used to anatomically map mRNA expression in both failing and non-failing hearts from tissue sections through the AVN, atrial and ventricular muscle. In the failing ventricle significant (P<0.05) downregulation is apparent for vimentin, hERG and Kir3.4 and trend to downregulation for Nav1.5, Cav1.2, Tbx3, Kir2.1, NCX1, Cx43 and Cx45. In the failing atrium, there is non-significant trend in upregulation for Nav1.5, HCN2, Cav3.1, Kv1.5, Kir3.1, RYR2 and significant upregulation for Cx40 and trend in downregulation for hERG, Kir2.1 and NCX1, and significant downregulation for HCN4 and Kir3.4. In the failing AVN there is significant (P<0.05) downregulation for vimentin, collagen, Tbx3, Kir3.1, Kir3.4, Cx45 and HCN4; there is also trend toward downregulation for HCN2, Kv1.5, Kir2.1 and RYR2. In the failing AVN there is significant (P<0.05) upregulation for Cx40, HCN1 and Cav3.1; and trend in upregulation for Nav1.5 and Cx43. For several transcripts, we also analysed corresponding protein expression via immunofluorescence. The protein expression data on the AVN for HCN1, Cav3.1 and Cx40 support qPCR data. Remodelling of AVN in heart failure might contribute to prolonged AV conduction time and could explain the prolonged PR interval that occurs in heart failure patients.