Mirko Baruscotti

Molecular Architecture of the Human Sinus Node Insights Into the Function of the Cardiac Pacemaker

Background— Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity. Methods and Results— Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Nav1.5, Kv4.3, Kv1.5, ERG, Kir2.1, Kir6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Cav1.3, Cav3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of Kv4.2, Kir6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking. Conclusions— Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.

Current-dependent Block of Rabbit Sino-Atrial Node I f Channels by Ivabradine

“Funny” (f-) channels have a key role in generation of spontaneous activity of pacemaker cells and mediate autonomic control of cardiac rate; f-channels and the related neuronal h-channels are composed of hyperpolarization-activated, cyclic nucleotide–gated (HCN) channel subunits. We have investigated the block of f-channels of rabbit cardiac sino-atrial node cells by ivabradine, a novel heart rate-reducing agent. Ivabradine is an open-channel blocker; however, block is exerted preferentially when channels deactivate on depolarization, and is relieved by long hyperpolarizing steps. These features give rise to use-dependent behavior. In this, the action of ivabradine on f-channels is similar to that reported of other rate-reducing agents such as UL-FS49 and ZD7288. However, other features of ivabradine-induced block are peculiar and do not comply with the hypothesis that the voltage-dependence of block is entirely attributable to either the sensitivity of ivabradine-charged molecules to the electrical field in the channel pore, or to differential affinity to different channel states, as has been proposed for UL-FS49 (DiFrancesco, D. 1994. Pflugers Arch. 427:64–70) and ZD7288 (Shin, S.K., B.S. Rotheberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91–101), respectively. Experiments where current flows through channels is modified without changing membrane voltage reveal that the ivabradine block depends on the current driving force, rather than voltage alone, a feature typical of block induced in inwardly rectifying K+ channels by intracellular cations. Bound drug molecules do not detach from the binding site in the absence of inward current through channels, even if channels are open and the drug is therefore not “trapped” by closed gates. Our data suggest that permeation through f-channel pores occurs according to a multiion, single-file mechanism, and that block/unblock by ivabradine is coupled to ionic flow. The use-dependence resulting from specific features of If block by ivabradine amplifies its rate-reducing ability at high spontaneous rates and may be useful to clinical applications.

Heteromeric HCN1–HCN4 channels: a comparison with native pacemaker channels from the rabbit sinoatrial node

‘Funny‐’ (f‐) channels of cardiac sino‐atrial node (SAN) cells are key players in the process of pacemaker generation and mediate the modulatory action of autonomic transmitters on heart rate. The molecular components of f‐channels are the hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels. Of the four HCN isoforms known, two (HCN4 and HCN1) are expressed in the rabbit SAN at significant levels. However, the properties of f‐channels of SAN cells do not conform to specific features of the two isoforms expressed locally. For example, activation kinetics and cAMP sensitivity of native pacemaker channels are intermediate between those reported for HCN1 and HCN4. Here we have explored the possibility that both HCN4 and HCN1 isoforms contribute to the native If in SAN cells by co‐assembling into heteromeric channels. To this end, we used heterologous expression in human embryonic kidney (HEK) 293 cells to investigate the kinetics and cAMP response of the current generated by co‐transfected (HCN4 + HCN1) and concatenated (HCN4‐HCN1 (4–1) tandem or HCN1‐HCN4 (1–4) tandem) rabbit constructs and compared them with those of the native f‐current from rabbit SAN. 4–1 tandem, but not co‐transfected, currents had activation kinetics approaching those of If; however, the activation range of 4–1 tandem channels was more negative than that of the f‐channel and their cAMP sensitivity were poorer (although that of 1–4 tandem channels was normal). Co‐transfection of 4–1 tandem channels with minK‐related protein 1(MiRP1) did not alter their properties. HCN1 and HCN4 may contribute to native f‐channels, but a ‘context’‐dependent mechanism is also likely to modulate the channel properties in native tissues.

Deep bradycardia and heart block caused by inducible cardiac-specific knockout of the pacemaker channel gene Hcn4

Cardiac pacemaking generation and modulation rely on the coordinated activity of several processes. Although a wealth of evidence indicates a relevant role of the If (“funny,” or pacemaker) current, whose molecular constituents are the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and particularly HCN4, work with mice where Hcn genes were knocked out, or functionally modified, has challenged this view. However, no previous studies used a cardiac-specific promoter to induce HCN4 ablation in adult mice. We report here that, in an inducible and cardiac-specific HCN4 knockout (ciHCN4-KO) mouse model, ablation of HCN4 consistently leads to progressive development of severe bradycardia (∼50% reduction of original rate) and AV block, eventually leading to heart arrest and death in about 5 d. In vitro analysis of sinoatrial node (SAN) myocytes isolated from ciHCN4-KO mice at the mean time of death revealed a strong reduction of both the If current (by ∼70%) and of the spontaneous rate (by ∼60%). In agreement with functional results, immunofluorescence and Western blot analysis showed reduced expression of HCN4 protein in SAN tissue and cells. In ciHCN4-KO animals, the residual If was normally sensitive to β-adrenergic receptor (β-AR) modulation, and the permanence of rate response to β-AR stimulation was observed both in vivo and in vitro. Our data show that cardiac HCN4 channels are essential for normal heart impulse generation and conduction in adult mice and support the notion that dysfunctional HCN4 channels can be a direct cause of rhythm disorders. This work contributes to identifying the molecular mechanism responsible for cardiac pacemaking.

If and 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.

Properties of ivabradine-induced block of HCN1 and HCN4 pacemaker channels

Ivabradine is a ‘heart rate‐reducing’ agent able to slow heart rate, without complicating side‐effects. Its action results from a selective and specific block of pacemaker f‐channels of the cardiac sinoatrial node (SAN). Investigation has shown that block by ivabradine requires open f‐channels, is use dependent, and is affected by the direction of current flow. The constitutive elements of native pacemaker channels are the hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels, of which four isoforms (HCN1–4) are known; in rabbit SAN tissue HCN4 is expressed strongly, and HCN1 weakly. In this study we have investigated the blocking action of ivabradine on mouse (m) HCN1 and human (h) HCN4 channels heterologously expressed in HEK 293 cells. Ivabradine blocked both channels in a dose‐dependent way with half‐block concentrations of 0.94 μm for mHCN1 and 2.0 μm for hHCN4. Properties of block changed substantially for the two channels. Block of hHCN4 required open channels, was strengthened by depolarization and was relieved by hyperpolarization. Block of mHCN1 did not occur, nor was it relieved, when channels were in the open state during hyperpolarization; block required channels to be either closed, or in a transitional state between open and closed configurations. The dependence of block upon current flow was limited for hHCN4, and not significant for mHCN1 channels. In summary our results indicate that ivabradine is an ‘open‐channel’ blocker of hHCN4, and a ‘closed‐channel’ blocker of mHCN1 channels. The mode of action of ivabradine on the two channels is discussed by implementing a simplified version of a previously developed model of f‐channel kinetics.

The cardiac pacemaker current.

In mammals cardiac rate is determined by the duration of the diastolic depolarization of sinoatrial node (SAN) cells which is mainly determined by the pacemaker I(f) current. f-channels are encoded by four members of the hyperpolarization-activated cyclic nucleotide-gated gene (HCN1-4) family. HCN4 is the most abundant isoform in the SAN, and its relevance to pacemaking has been further supported by the discovery of four loss-of-function mutations in patients with mild or severe forms of cardiac rate disturbances. Due to its selective contribution to pacemaking, the I(f) current is also the pharmacological target of a selective heart rate-reducing agent (ivabradine) currently used in the clinical practice. Albeit to a minor extent, the I(f) current is also present in other spontaneously active myocytes of the cardiac conduction system (atrioventricular node and Purkinje fibres). In working atrial and ventricular myocytes f-channels are expressed at a very low level and do not play any physiological role; however in certain pathological conditions over-expression of HCN proteins may represent an arrhythmogenic mechanism. In this review some of the most recent findings on f/HCN channels contribution to pacemaking are described.

The pacemaker current: from basics to the clinics.

Activation of the pacemaker (“funny,” If) current during diastole is the main process underlying generation of the diastolic depolarization and spontaneous activity of cardiac pacemaker cells. If modulation by autonomic transmitters is responsible for the chronotropic regulation of heart rate. Given its role in pacemaking, If has been a major target of investigation aimed to exploit its rate‐controlling function in a clinical perspective. In this short review, we describe some of the most recent clinically relevant applications of the concept of If‐based pacemaking.

If-dependent modulation of pacemaker rate mediated by cAMP in the presence of ryanodine in rabbit sino-atrial node cells

I(f) contributes to generation and autonomic control of spontaneous activity of cardiac pacemaker cells through a cAMP-dependent, Ca(2+)-independent mechanism of rate regulation. However, disruption of Ca(2+) release from sarcoplasmic reticulum (SR) by ryanodine (Ry) has been recently shown to slow spontaneous rate and inhibit beta-adrenergic receptor (betaAR)-induced rate acceleration, leading to the suggestion that the target of betaAR modulation of pacemaking is the intracellular Ca(2+)-regulatory process. We have investigated whether the Ry-induced decrease of betaAR rate modulation alternatively involves disruption of the betaAR-adenylate-cyclase-cAMP-I(f) mechanism. Prolonged exposure to Ry (3 microM, >2 min) slowed spontaneous rate of pacemaker cells by 29.8% via a depolarizing shift of take-off potential (TOP) without significantly changing early diastolic depolarization rate. Ry depressed rate acceleration caused by isoproterenol (Iso) (1 microM, 23.6% in control vs. 8.0%), but did not modify that caused by two membrane-permeable cAMP analogs, CPT-cAMP (300 microM, 17.7% vs. 17.3%) and Rp-cAMPs (50 microM, 18.0% vs. 20.6%). Consistent with the rate effect, exposure to Ry decreased the shift induced by Iso, but not that induced by either cAMP analog on the I(f)-activation curve. We conclude that disruption of Ry receptor function and SR Ca(2+) release depresses betaAR-induced modulation of heart rate, but does not impair cAMP-dependent rate acceleration mediated by I(f). However, abolishment of normal Ca(2+) homeostasis may result in the failure of betaAR agonists to sufficiently elevate cAMP near f-channels. The molecular basis for Ca(2+)-dependent interference in beta-adrenergic signaling remains to be determined.

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

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.