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The sinus node (SN) serves as the primary pacemaker of the heart and is the first component of the cardiac conduction system. Due to its anatomical properties and sample scarcity, the cellular composition of the human SN has been historically challenging to study. Here, we employed a novel deep learning deconvolution method, namely Bulk2space, to characterise the cellular heterogeneity of the human SN using existing single-cell datasets of non-human species. As a proof of principle, we used Bulk2Space to profile the cells of the bulk human right atrium using publicly available mouse scRNA-Seq data as a reference. 18 human cell populations were identified, with cardiac myocytes being the most abundant. Each identified cell population correlated to its published experimental counterpart. Subsequently, we applied the deconvolution to the bulk transcriptome of the human SN and identified 11 cell populations, including a population of pacemaker cardiomyocytes expressing pacemaking ion channels (HCN1, HCN4, CACNA1D) and transcription factors (SHOX2 and TBX3). The connective tissue of the SN was characterised by adipocyte and fibroblast populations, as well as key immune cells. Our work unravelled the unique single cell composition of the human SN by leveraging the power of a novel machine learning method., (© 2024. The Author(s).)

Mir-133a-3p is the most abundant myocardial microRNA. The impact of mir-133a-3p on cardiac electrophysiology is poorly explored. In this study, we investigated the effects of mir-133a-3p on the main ionic currents critical for action potential (AP) generation and electrical activity of the heart. We used conventional ECG, sharp microelectrodes and patch-clamp to clarify a role of mir-133a-3p in normal cardiac electrophysiology in rats after in vivo and in vitro transfection. Mir-133a-3p caused no changes to pacemaker APs and automaticity in the sinoatrial node. No significant changes in heart rate (HR) were observed in vivo; however, miR transfection facilitated HR increase in response to β-adrenergic stimulation. Mir-133a-3p induced repolarization abnormalities in the atrial working myocardium and the L-type calcium current (I Ca,L ) was significantly increased. The main repolarization currents, including the transient outward (I to ), ultra-rapid (I K,ur ), and inward rectifier (I K1 ) remained unaffected in atrial cardiomyocytes. Mir-133a-3p affected both I Ca,L and I to in ventricular cardiomyocytes. Systemic administration of mir-133a-3p induced QT-interval prolongation. Bioinformatic analysis revealed protein phosphatase 2 (PPP2CA/B) and Kcnd3 (encoding K v 4.3 channels generating I to ) as the main miR-133a-3p targets in the heart. No changes in mRNA expression of Cacna1c (encoding Ca v 1.2 channels generating I Ca,L ) and Kcnd3 were seen in mir-133a-3p treated rats. However, the expression of Ppp2cA, encoding PPP2CA, and Kcnip2 encoding KChIP2, a K v 4.3 regulatory protein, were significantly decreased. The accumulation of mir-133a-3p in cardiac myocytes causes chamber-specific electrophysiological changes. The suppression of PPP2CA, involved in adrenergic signal transduction, and Kchip2 may indirectly mediate mir-133a-3p-induced augmentation of I Ca,L and attenuation of I to ., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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Background: The anatomical substrate for the mid-mural ventricular hyperechogenic zone remains uncertain, but it may represent no more than ultrasound reflected from cardiomyocytes orientated orthogonally to the ultrasonic beam. We sought to ascertain the relationship between the echogenic zone and the orientation of the cardiomyocytes., Methods: We used 3D echocardiography, diffusion tensor imaging, and microcomputed tomography to analyze the location and orientation of cardiomyocytes within the echogenic zone., Results: We demonstrated that visualization of the echogenic zone is dependent on the position of the transducer and is most clearly seen from the apical window. Diffusion tensor imaging and microcomputed tomography show that the echogenic zone seen from the apical window corresponds to the position of the circumferentially orientated cardiomyocytes. An oblique band seen in the parasternal view relates to cardiomyocytes orientated orthogonally to the ultrasonic beam., Conclusions: The mid-mural ventricular hyperechogenic zone represents reflected ultrasound from cardiomyocytes aligned orthogonal to the ultrasonic beam. The echogenic zone does not represent a space, a connective tissue sheet, a boundary between ascending and descending limbs of a hypothetical helical ventricular myocardial band, nor an abrupt change in cardiomyocyte orientation., (© 2016, Wiley Periodicals, Inc.)

Background: The hyperpolarization-activated current, If, plays an important role in sinoatrial node (SAN) pacemaking. Surprisingly, the distribution of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in human SAN has only been investigated at the mRNA level. Our aim was to define the expression pattern of HCN proteins in human SAN and different atrial regions., Methods and Results: Entire SAN complexes were isolated from failing (n=5) and nonfailing (n=9) human hearts cardioplegically arrested in the operating room. Three-dimensional intramural SAN structure was identified as the fibrotic compact region around the SAN artery with Connexin 43-negative pacemaker cardiomyocytes visualized in Masson's trichrome and immunostained cryosections. SAN protein was precisely isolated from the adjacent frozen SAN tissue blocks using a 16G biopsy needle. The purity of the SAN protein was confirmed by Connexin 43 immunoblot. All 3 HCN isoform proteins were detected in SAN. HCN1 was predominantly distributed in the human SAN with a 125.1±40.2 (n=12) expression ratio of SAN to right atrium. HCN2 and HCN4 expression levels were higher in SAN than in atria, with SAN to right atrium ratios of 6.1±0.9 and 4.6±0.6 (n=12), respectively., Conclusions: This is the first study to conduct precise 3D molecular mapping of the human SAN by isolating pure pacemaker SAN tissue. All 3 cardiac HCN isoforms had higher expression in the SAN than in the atria. HCN1 was almost exclusively expressed in SAN, emphasizing its utility as a new specific molecular marker of the human SAN and as a potential target of specific treatments intended to modify sinus rhythm., (© 2015 American Heart Association, Inc.)

Understanding the mechanisms of fatal ventricular arrhythmias is of great importance. In view of the many electrophysiological differences that exist between animal species and humans, the acquisition of basic electrophysiological data in the intact human heart is essential to drive and complement experimental work in animal and in-silico models. Over the years techniques have been developed to obtain basic electrophysiological signals directly from the patients by incorporating these measurements into routine clinical procedures which access the heart such as cardiac catheterisation and cardiac surgery. Early recordings with monophasic action potentials provided valuable information including normal values for the in vivo human heart, cycle length dependent properties, the effect of ischaemia, autonomic nervous system activity, and mechano-electric interaction. Transmural recordings addressed the controversial issue of the mid myocardial "M" cell. More recently, the technique of multielectrode mapping (256 electrodes) developed in animal models has been extended to humans, enabling mapping of activation and repolarisation on the entire left and right ventricular epicardium in patients during cardiac surgery. Studies have examined the issue of whether ventricular fibrillation was driven by a "mother" rotor with inhomogeneous and fragmented conduction as in some animal models, or by multiple wavelets as in other animal studies; results showed that both mechanisms are operative in humans. The simpler spatial organisation of human VF has important implications for treatment and prevention. To link in-vivo human electrophysiological mapping with cellular biophysics, multielectrode mapping is now being combined with myocardial biopsies. This technique enables region-specific electrophysiology changes to be related to underlying cellular biology, for example: APD alternans, which is a precursor of VF and sudden death. The mechanism is incompletely understood but related to calcium cycling and APD restitution. Multielectrode sock mapping during incremental pacing enables epicardial sites to be identified which exhibit marked APD alternans and sites where APD alternans is absent. Whole heart electrophysiology is assessed by activation repolarisation mapping and analysis is performed immediately on-site in order to guide biopsies to specific myocardial sites. Samples are analysed for ion channel expression, Ca(2+)-handling proteins, gap junctions and extracellular matrix. This new comprehensive approach to bridge cellular and whole heart electrophysiology allowed to identify 20 significant changes in mRNA for ion channels Ca(2+)-handling proteins, a gap junction channel, a Na(+)-K(+) pump subunit and receptors (particularly Kir 2.1) between the positive and negative alternans sites., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Mutations in the cardiac Na(+) channel gene (SCN5A) can adversely affect electric function in the heart, but effects can be age dependent. We explored the interacting effects of Scn5a disruption and aging on the pathogenesis of sinus node dysfunction in a heterozygous Scn5a knockout (Scn5a(+/-)) mouse model., Methods and Results: We compared functional, histological, and molecular features in young (3 to 4 month) and old (1 year) wild type and Scn5a(+/-) mice. Both Scn5a disruption and aging were associated with decreased heart rate variability, reduced sinoatrial node automaticity, and slowed sinoatrial conduction. They also led to increased collagen and fibroblast levels and upregulated transforming growth factor-β(1) (TGF-β(1)) and vimentin transcripts, providing measures of fibrosis and reduced Nav1.5 expression. All these effects were most noticeable in old Scn5a(+/-) mice. Na(+) channel inhibition by Nav1.5-E3 antibody directly increased TGF-β(1) production in both cultured human cardiac myocytes and fibroblasts. Finally, aging was associated with downregulation of a wide range of ion channel and related transcripts and, again, was greatest in old Scn5a(+/-) mice. The quantitative results from these studies permitted computer simulations that successfully replicated the observed sinoatrial node phenotypes shown by the different experimental groups., Conclusions: These results implicate a tissue degeneration triggered by Nav1.5 deficiency manifesting as a TGF-β(1)-mediated fibrosis accompanied by electric remodeling in the sinus node dysfunction associated with Scn5a disruption or aging. The latter effects interact to produce the most severe phenotype in old Scn5a(+/-) mice. In demonstrating this, our findings suggest a novel regulatory role for Nav1.5 in cellular biological processes in addition to its electrophysiologic function.

Aims: Plasmalemmal Ca(2+)-ATPase (PMCA) is involved in Ca(2+) handling and the regulation of intracellular signalling pathways in the heart. However, there is no information on its functioning in heart hypertrophy and failure. We aimed to investigate the Ca(2+)-transporting ability of PMCA, Na(+)/Ca(2+) exchanger (NCX), and sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a), as well as the amplitude of Ca(2+) transients and cell shortening in myocytes isolated from rat hearts at various time intervals after myocardial infarction (MI)., Methods and Results: The rate of Ca(2+) transport by PMCA, NCX, and SERCA2a was estimated from the rate constants of decay of electrically and caffeine-evoked Ca(2+) transients in left ventricular myocytes isolated 1 week, 1 month, and 3 months after MI. One week, 1 month, and 3 months after MI, the transporting function of PMCA decreased by 27, 41, and 67%, respectively, compared with that in time-matched sham animals. This was accompanied by increased amplitude of Ca(2+) transients, cell shortening, and SR Ca(2+) content. Carboxyeosin, a blocker of PMCA, increased the amplitude of shortening in cells extracted from control hearts. This effect was absent 1 and 3 months after MI. PMCA1, 2, and 4 mRNAs were unchanged in the ventricular muscle 3 months after MI when compared with time-matched sham animals. The transporting function of NCX was increased by 65% only 3 months after MI, whereas that of SERCA2a was decreased by approximately 18% at all three time points after MI., Conclusion: The ability of PMCA to transport Ca(2+) progressively decreases over 3 months after MI. This decrease may contribute to the increase in amplitude of Ca(2+) transients and myocyte shortening.

All the myocytes within the heart have the capacity to conduct the cardiac impulse. A population of myocytes is specialized so as to generate the cardiac impulse and then to conduct it from the atrial to the ventricular chambers. This population has become known as the conduction system. Anatomists who seek to demonstrate the location of the components of this system must contend with the fact that the components of the system cannot be distinguished from the working myocardial elements by gross dissection. In important presentations to the German Pathological Society in 1910, rules were suggested for the histological distinction of these conducting cells. These rules proposed that the myocytes, to be considered as part of the conduction system, should be histologically discrete, traceable from section to section in serially prepared material, and if to be considered as tracts, should be insulated by fibrous tissue from the adjacent myocytes. Immunohistochemical techniques have now been developed that better demonstrate the distinction between the cells specialized to conduct from working myocytes. These new techniques, for the most part, confirm the accuracy of the initial descriptions. They also reveal additional areas with the characteristics of conduction tissues. These additional areas are located in a paranodal area adjacent to the sinus node, in the vestibules of both atrioventricular valvar orifices, and in a partial ring around the aortic root. In this review, we describe all these features, emphasizing the relationship of the newly recognized components to the established parts of the cardiac conduction system, and how the new findings need to be assessed in the light of the old criteria., ((c) 2008 Wiley-Liss, Inc.)

The majority of Na+ channels in the heart are composed of the tetrodotoxin (TTX)-resistant (KD, 2-6 microm) 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 degrees C, TTX-resistant iNa and TTX-sensitive iNa started to activate at approximately -70 and approximately -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 microm) 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.