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Background. In vivo experiments in Goto-Kakizaki (GK) type 2 diabetic rats have demonstrated reductions in heart rate from a young age. The expression of genes encoding more than 70 proteins that are associated with the generation and conduction of electrical activity in the GK sinoatrial node (SAN) have been evaluated to further clarify the molecular basis of the low heart rate. Materials and Methods. Heart rate and expression of genes were evaluated with an extracellular electrode and real-time RT-PCR, respectively. Rats aged 12-13 months were employed in these experiments. Results. Isolated spontaneous heart rate was reduced in GK heart (161 ± 12 bpm) compared to controls (229 ± 11 bpm). There were many differences in expression of mRNA, and some of these differences were of particular interest. Compared to control SAN, expression of some genes were downregulated in GK-SAN: gap junction, Gja1 (Cx43), Gja5 (Cx40), Gjc1 (Cx45), and Gjd3 (Cx31.9); cell membrane transport, Trpc1 (TRPC1) and Trpc6 (TRPC6); hyperpolarization-activated cyclic nucleotide-gated channels, Hcn1 (HCN1) and Hcn4 (HCN4); calcium channels, Cacna1d (Cav1.3), Cacna1g (Cav3.1), Cacna1h (Cav3.2), Cacna2d1 (Cavα2δ1), Cacna2d3 (Cavα2δ3), and Cacng4 (Cavγ4); and potassium channels, Kcna2 (Kv1.2), Kcna4 (Kv1.4), Kcna5 (Kv1.5), Kcnb1 (Kv2.1), Kcnd3 (Kv4.3), Kcnj2 (Kir2.1), Kcnk1 (TWIK1), Kcnk5 (K2P5.1), Kcnk6 (TWIK2), and Kcnn2 (SK2) whilst others were upregulated in GK-SAN: Ryr2 (RYR2) and Nppb (BNP). Conclusions. This study provides new insight into the changing expression of genes in the sinoatrial node of diabetic heart.

Abstract  Abnormal QT prolongation with the associated arrhythmias is a significant predictor of mortality in diabetic patients. Gap junctional intercellular communication allows electrical coupling between heart muscle cells. The effects of streptozotocin (STZ)-induced diabetes mellitus on the expression and distribution of connexin 43 (Cx43) in ventricular muscle have been investigated. Cx43 mRNA expression was measured in ventricular muscle by quantitative PCR. The distribution of total Cx43, phosphorylated Cx43 (at serine 368) and non-phosphorylated Cx43 was measured in ventricular myocytes and ventricular muscle by immunocytochemistry and confocal microscopy. There was no significant difference in Cx43 mRNA between diabetic rat ventricle and controls. Total and phosphorylated Cx43 were significantly increased in ventricular myocytes and ventricular muscle and dephosphorylated Cx43 was not significantly altered in ventricular muscle from diabetic rat hearts compared to controls. Disturbances in gap junctional intercellular communication, which in turn may be attributed to alterations in balance between total, phosphorylated and dephosporylated Cx43, might partly underlie prolongation of QRS and QT intervals in diabetic heart. [ABSTRACT FROM AUTHOR]

Background: The sinoatrial node (SN) generates the heart rate (HR). Its spontaneous activity is regulated by a complex interplay between the modulation by the autonomic nervous system (ANS) and intrinsic factors including ion channels in SN cells. However, the systemic and intrinsic regulatory mechanisms are still poorly understood. This study aimed to elucidate the sex-specific differences in heart morphology and SN function, particularly focusing on basal HR, expression and function of hyperpolarization-activated HCN4 and HCN1 channels and mRNA abundance of ion channels and mRNA abundance of ion channels contributing to diastolic depolarization (DD) and spontaneous action potentials (APs)., Methods: Body weight, heart weight and tibia length of 2- to 3-month-old male and female mice were measured. Conscious in-vivo HR of male and female mice was recorded via electrocardiography (ECG). Unconscious ex-vivo HR, stroke volume (SV) and ejection fraction (EF) were recorded via echocardiography. Ex-vivo HR was measured via Langendorff apparatus. Volume of atria, ventricles and whole hearts were measured from the ex-vivo hearts by microcomputed tomography (micro-CT). Immunohistochemistry targeting HCN4 and HCN1 was conducted in the SN and RA tissues from both male and female hearts. The funny current ( I f ) of SN cells in 1 nM and following wash-on of 1 μM isoproterenol (ISO) were recorded via whole cell patch clamp. The APs of SN tissue were recorded via sharp microelectrode and optical mapping of membrane voltage. The relative abundance of mRNAs was measured in male and female mice by qPCR., Results: Heart weight to tibia length ratio and heart volume of females were significantly smaller than males. Unconscious in-vivo HR in male mice was higher than that in females. Conscious in-vivo HR, ex-vivo HR, SV, and EF showed no notable difference between male and female mice. Immunohistochemistry revealed HCN4, HCN1, and the sum of HCN4 and HCN1, expression in the SN was notably elevated compared with the RA in both male and females, but there was no sex difference in these channels expression. There were also no significant sex differences in the V 0.5 of I f in SN cells in the presence of 1 nM ISO, however wash-on 1 μM ISO in the same cells induced a significantly increased shift of V 0.5 to more positive voltages in males than in females. The expression of mRNA coding for adrenergic receptor beta-1 (Adrb1) and cholinergic receptors muscarinic 2 (chrm2) in male mice was higher compared with that in female mice. Early diastolic depolarization (EDD) rate in APs from peripheral SN (pSN) from male mice were higher than these in female mice. Mice of both sexes showed equivalent frequency of SN APs and spatial localization of the leading site in control, and similar significant response to ISO 100 nM superfusion., Conclusion: Males display faster in-vivo HR, but not ex-vivo HR, than females associated with increased expression of Adrb1 in male versus female. This suggests a possible difference in the β-adrenergic modulation in males and females, possibly related to the greater ISO response of I f observed in cells from males. The role of hormonal influences or differential expression of other ion channels may explain these sex-specific variations in HR dynamics. Further investigations are necessary to pinpoint the precise molecular substrates responsible for these differences., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Yin, Torre, Marrot, Peters, Feather, Nichols, Logantha, Arshad, Martis, Ozturk, Chen, Liu, Qu, Zi, Cartwright, Proenza, Torrente, Mangoni, Dobrzynski and Atkinson.)

(1) Background: The sinus node (SN) is the main pacemaker of the heart. It is characterized by pacemaker cells that lack mitochondria and contractile elements. We investigated the possibility that transcription factors (TFs) and microRNAs (miRs) present in the SN can regulate gene expression that affects SN morphology and function. (2) Methods: From human next-generation sequencing data, a list of mRNAs that are expressed at lower levels in the SN compared with the right atrium (RA) was compiled. The mRNAs were then classified into contractile, mitochondrial or glycogen mRNAs using bioinformatic software, RStudio and Ingenuity Pathway Analysis. The mRNAs were combined with TFs and miRs to predict their interactions. (3) Results: From a compilation of the 1357 mRNAs, 280 contractile mRNAs and 198 mitochondrial mRNAs were identified to be expressed at lower levels in the SN compared with RA. TFs and miRs were shown to interact with contractile and mitochondrial function-related mRNAs. (4) Conclusions: In human SN, TFs (MYCN, SOX2, NUPR1 and PRDM16) mainly regulate mitochondrial mRNAs (COX5A, SLC25A11 and NDUFA8), while miRs (miR-153-3p, miR-654-5p, miR-10a-5p and miR-215-5p) mainly regulate contractile mRNAs (RYR2, CAMK2A and PRKAR1A). TF and miR-mRNA interactions provide a further understanding of the complex molecular makeup of the SN and potential therapeutic targets for cardiovascular treatments.

Background: Although radiofrequency ablation of the cavotricuspid isthmus (CTI), responsible for sustaining atrial flutter, is a highly effective procedure, in extended patients' observations following this procedure, more than every tenth becomes unsuccessful. Therefore, this study aimed to provide helpful information about the anatomy of the CTI in transthoracic echocardiography, which can aid in better planning of the CTI radiofrequency ablation in patients with typical atrial flutter., Materials and Methods: 56 patients with typical atrial flutter after radiofrequency ablation were evaluated at the end of the 24-month observation period. With substernal modified transthoracic echocardiographic (mTTE) evaluation, we identified four main anatomical obstacles impeding radiofrequency ablation. These obstacles were tricuspid annular plane systolic excursion, cavotricuspid isthmus length, cavotricuspid isthmus morphology, and the presence of a prominent Eustachian ridge/Eustachian valve. All intraprocedural radiofrequency ablation data were collected for analysis and correlated with anatomical data., Results: In the 24-month observation period, freedom from atrial flutter was 67.86%. The mean length of the isthmus was 30.34 ± 6.67 mm. The isthmus morphology in 56 patients was categorized as flat (n = 27; 48.2%), concave (n = 10; 17.85%), and pouch (n = 19, 33.9%). A prominent Eustachian ridge was observed in 23 patients (41.1%). Lack of anatomical obstacles in mTTE evaluation resulted in 100% efficacy, while the presence of at least two obstacles significantly increased the risk of unsuccessful ablation with more than two (OR 12.31 p = 0.01). Generally, 8 mm electrodes were the most effective for non-difficult CTI, while 3.5 mm electrodes used with a 3D system had highest performance for complex CTI. Notably, aging was the only factor that worsened the long-term outcome (OR 1.07 p = 0.044)., Conclusions: Preoperative usage of mTTE evaluation helps predict difficulty in cavotricuspid isthmus radiofrequency ablation, thus allowing better planning of the radiofrequency ablation strategy using the most accurate radiofrequency ablation electrode.

The most commonly applied way of teaching students to convey the foundations of human anatomy and physiology involves textbooks and lectures. This way of transmitting knowledge causes difficulties for students, especially in the context of three-dimensional imaging of organ structures, and as a consequence translates into difficulties with imagining them. Even despite the rapid uptake of knowledge dissemination provided by online materials, including courses and webinars, there is a clear need for learning programs featuring first-hand immersive experiences tailored to suit individual study paces. In this paper, we present an approach to enhance a classical study program by combining multi-modality data and representing them in a Mixed Reality (MR)-based environment. The advantages of the proposed approach have been proven by the conducted investigation of the relationship between atrial anatomy, its electrophysiological characteristics, and resulting P wave morphology on the electrocardiogram (ECG). Another part of the paper focuses on the role of the sinoatrial node in ECG formation, while the MR-based visualization of combined micro-computed tomography (micro-CT) data with non-invasive CineECG imaging demonstrates the educational application of these advanced technologies for teaching cardiac anatomy and ECG correlations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Potyagaylo, van Dam, Kuniewicz, Dolega-Dolegowski, Pregowska, Atkinson, Dobrzynski and Proniewska.)

Introduction: The sinus node (SN) is the main pacemaker site of the heart, located in the upper right atrium at the junction of the superior vena cava and right atrium. The precise morphology of the SN in the human heart remains relatively unclear especially the SN microscopical anatomy in the hearts of aged and obese individuals. In this study, the histology of the SN with surrounding right atrial (RA) muscle was analyzed from young non-obese, aged non-obese, aged obese and young obese individuals. The impacts of aging and obesity on fibrosis, apoptosis and cellular hypertrophy were investigated in the SN and RA. Moreover, the impact of obesity on P wave morphology in ECG was also analyzed to determine the speed and conduction of the impulse generated by the SN., Methods: Human SN/RA specimens were dissected from 23 post-mortem hearts (preserved in 4% formaldehyde solution), under Polish local ethical rules. The SN/RA tissue blocks were embedded in paraffin and histologically stained with Masson's Trichrome. High and low-magnification images were taken, and analysis was done for appropriate statistical tests on Prism (GraphPad, USA). 12-lead ECGs from 14 patients under Polish local ethical rules were obtained. The P wave morphologies from lead II, lead III and lead aVF were analyzed., Results: Compared to the surrounding RA, the SN in all four groups has significantly more connective tissue ( P ≤ 0.05) (young non-obese individuals, aged non-obese individuals, aged obese individuals and young obese individuals) and significantly smaller nodal cells ( P ≤ 0.05) (young non-obese individuals, aged non-obese individuals, aged obese individuals, young obese individuals). In aging, overall, there was a significant increase in fibrosis, apoptosis, and cellular hypertrophy in the SN ( P ≤ 0.05) and RA ( P ≤ 0.05). Obesity did not further exacerbate fibrosis but caused a further increase in cellular hypertrophy (SN P ≤ 0.05, RA P ≤ 0.05), especially in young obese individuals. However, there was more infiltrating fat within the SN and RA bundles in obesity. Compared to the young non-obese individuals, the young obese individuals showed decreased P wave amplitude and P wave slope in aVF lead., Discussion: Aging and obesity are two risk factors for extensive fibrosis and cellular hypertrophy in SN and RA. Obesity exacerbates the morphological alterations, especially hypertrophy of nodal and atrial myocytes. These morphological alterations might lead to functional alterations and eventually cause cardiovascular diseases, such as SN dysfunction, atrial fibrillation, bradycardia, and heart failure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Chen, Rams, Zając, Albalawi, Atkinson, Aminu, Mazur, Hołda, Walocha, Gil, Kuniewicz and Dobrzynski.)

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).)

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a role in post-transcriptional gene regulation. It is generally accepted that their main mechanism of action is the negative regulation of gene expression, through binding to specific regions in messenger RNA (mRNA) and repressing protein translation. By interrupting protein synthesis, miRNAs can effectively turn genes off and influence many basic processes in the body, such as developmental and apoptotic behaviours of cells and cardiac organogenesis. Their importance is highlighted by inhibiting or overexpressing certain miRNAs, which will be discussed in the context of coronary artery disease, atrial fibrillation, bradycardia, and heart failure. Dysregulated levels of miRNAs in the body can exacerbate or alleviate existing disease, and their omnipresence in the body makes them reliable as quantifiable markers of disease. This review aims to provide a summary of miRNAs as biomarkers and their interactions with targets that affect cardiac health, and intersperse it with current therapeutic knowledge. It intends to succinctly inform on these topics and guide readers toward more comprehensive works if they wish to explore further through a wide-ranging citation list.

Patients with pulmonary arterial hypertension (PAH) have a high burden of arrhythmias, including arrhythmias arising from sinus node dysfunction, and the aim of this study was to investigate the effects of PAH on the sinus node. In the rat, PAH was induced by an injection of monocrotaline. Three weeks after injection, there was a decrease of the intrinsic heart rate (heart rate in the absence of autonomic tone) as well as the normal heart rate, evidence of sinus node dysfunction. In the sinus node of PAH rats, there was a significant downregulation of many ion channels and Ca 2+ -handling genes that could explain the dysfunction: HCN1 and HCN4 (responsible for pacemaker current, I f ), Cav1.2, Cav1.3 and Cav3.1 (responsible for L- and T-type Ca 2+ currents, I Ca,L and I Ca,T ), NCX1 (responsible for Na + -Ca 2+ exchanger) and SERCA2 and RYR2 (Ca 2+ -handling molecules). In the sinus node of PAH rats, there was also a significant upregulation of many fibrosis genes that could also help explain the dysfunction: vimentin, collagen type 1, elastin, fibronectin and transforming growth factor β1. In summary, in PAH, there is a remodelling of ion channel, Ca 2+ -handling and fibrosis genes in the sinus node that is likely to be responsible for the sinus node dysfunction. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Heart failure is associated with atrioventricular (AV) node dysfunction, and AV node dysfunction in the setting of heart failure is associated with an increased risk of mortality and heart failure hospitalisation. This study aims to understand the causes of AV node dysfunction in heart failure by studying changes in the whole nodal transcriptome. The mouse transverse aortic constriction model of pressure overload-induced heart failure was studied; functional changes were assessed using electrocardiography and echocardiography and the transcriptome of the AV node was quantified using RNAseq. Heart failure was associated with a significant increase in the PR interval, indicating a slowing of AV node conduction and AV node dysfunction, and significant changes in 3,077 transcripts (5.6% of the transcriptome). Many systems were affected: transcripts supporting AV node conduction were downregulated and there were changes in transcripts identified by GWAS as determinants of the PR interval. In addition, there was evidence of remodelling of the sarcomere, a shift from fatty acid to glucose metabolism, remodelling of the extracellular matrix, and remodelling of the transcription and translation machinery. There was evidence of the causes of this widespread remodelling of the AV node: evidence of dysregulation of multiple intracellular signalling pathways, dysregulation of 109 protein kinases and 148 transcription factors, and an immune response with a proliferation of neutrophils, monocytes, macrophages and B lymphocytes and a dysregulation of 40 cytokines. In conclusion, inflammation and a widespread transcriptional remodelling of the AV node underlies AV node dysfunction in heart failure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wilson, Zi, Smith, Hussain, D’Souza, Dobrzynski and Boyett.)

Purpose: The left ventricular summit (LVS) is a source of difficult-to-treat arrhythmias because of anatomical limitations. The aim of this study was to perform detailed research of the left atrial appendage (LAA) anatomy of cadaveric hearts to analyze their complex anatomy and coverage of the LVS., Methods and Results: Eighty human formalin fixed hearts (mean age 44.4 ± 15.5, 27.5% females) were investigated. Each LAA size, type, and its relationship to the LVS were analyzed, as well as possible access sites for mapping/ablating electrode. Four types of LAA were observed over two LVS sites that are either accessible or not. The highest coverage over an inaccessible LVS area was observed in the Broccoli type, followed by the Windsock then the Chicken Wing and finally the Cactus types; over the accessible area of the LVS was observed in the Windsock, then in the Chicken Wing, then in the Cactus, and finally in the Broccoli types. The attainable coverage for electrode access is diminished from 25 to 65% because of the complex pectinate muscles and sharp angles. The highest density of the LAA floor made by pectinate muscles can be found in the Broccoli type (p < 0.005), while the Chicken Wing had the highest number of paper-thin-like pouches., Conclusions: The LAA appears to be a promising entry for ablation-qualified patients with the LV summit originate arrhythmias. The complex internal structure of the LAA may complicate ablation procedures. More prominent appendages are promising in more extensive mapping areas over the LVS., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Recent clinical reports have emphasized the clinical significance of the left ventricular summit (LVS), a specific triangular epicardial area, as the source of ventricular arrhythmias where radiofrequency ablation is of great difficulty., Materials and Methods: The macroscopic morphology of the LVS has been assessed in 80 autopsied and 48 angio-computed tomography (CT) human hearts. According to Yamada's equation, the size was calculated based on the distance to the first, most prominent septal perforator., Results: The size of the LVS varies from 33.69 to 792.2 mm2, is highly variable, and does not correlate with body mass index, sex, or age in general. The mean size of the LVS was 287.38 ± 144.95 mm2 in autopsied and angio-CT (p = 0.44). LVS is mostly disproportionately bisected by cardiac coronary veins to superior-inaccessible and inferior-accessible areas. The superior aspect dominates over the inferior in both groups (p = 0.04). The relation between superior and inferior groups determines three possible arrangements: the most common type is superior domination (50.2%), then inferior domination (26.6%), and finally, equal distribution (17.2%). In 10.9%, the inferior aspect is absent. Only 16.4% of the LVS were empty, without additional trespassing coronary arteries., Conclusions: The difference in size and content of the LVS is significant, with no correlation to any variable. The size depends on the anatomy of the most prominent septal perforator artery. The superior, inaccessible aspect dominates, and the LVS is seldom free from additional coronary vessels, thus making this region hazardous for electrophysiological procedures.

Research Purpose: The sinus node (SN) is the heart's primary pacemaker. Key ion channels (mainly the funny channel, HCN4) and Ca 2+ -handling proteins in the SN are responsible for its function. Transcription factors (TFs) regulate gene expression through inhibition or activation and microRNAs (miRs) do this through inhibition. There is high expression of macrophages and mast cells within the SN connective tissue. 'Novel'/unexplored TFs and miRs in the regulation of ion channels and immune cells in the SN are not well understood. Using RNAseq and bioinformatics, the expression profile and predicted interaction of key TFs and cell markers with key miRs in the adult human SN vs. right atrial tissue (RA) were determined., Principal Results: 68 and 60 TFs significantly more or less expressed in the SN vs. RA respectively. Among those more expressed were ISL1 and TBX3 (involved in embryonic development of the SN) and 'novel' RUNX1-2, CEBPA, GLI1-2 and SOX2. These TFs were predicted to regulate HCN4 expression in the SN. Markers for different cells: fibroblasts (COL1A1), fat (FABP4), macrophages (CSF1R and CD209), natural killer (GZMA) and mast (TPSAB1) were significantly more expressed in the SN vs. RA. Interestingly, RUNX1-3, CEBPA and GLI1 also regulate expression of these cells. MiR-486-3p inhibits HCN4 and markers involved in immune response., Major Conclusions: In conclusion, RUNX1-2, CSF1R, TPSAB1, COL1A1 and HCN4 are highly expressed in the SN but not miR-486-3p. Their complex interactions can be used to treat SN dysfunction such as bradycardia. Interestingly, another research group recently reported miR-486-3p is upregulated in blood samples from severe COVID-19 patients who suffer from bradycardia., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.)

Heart failure (HF) is frequently accompanied with the sinoatrial node (SAN) dysfunction, which causes tachy-brady arrhythmias and increased mortality. MicroRNA (miR) alterations are associated with HF progression. However, the transcriptome of HF human SAN, and its role in HF-associated remodeling of ion channels, transporters, and receptors responsible for SAN automaticity and conduction impairments is unknown. We conducted comprehensive high-throughput transcriptomic analysis of pure human SAN primary pacemaker tissue and neighboring right atrial tissue from human transplanted HF hearts (n = 10) and non-failing (nHF) donor hearts (n = 9), using next-generation sequencing. Overall, 47 miRs and 832 mRNAs related to multiple signaling pathways, including cardiac diseases, tachy-brady arrhythmias and fibrosis, were significantly altered in HF SAN. Of the altered miRs, 27 are predicted to regulate mRNAs of major ion channels and neurotransmitter receptors which are involved in SAN automaticity (e.g. HCN1, HCN4, SLC8A1) and intranodal conduction (e.g. SCN5A, SCN8A) or both (e.g. KCNJ3, KCNJ5). Luciferase reporter assays were used to validate interactions of miRs with predicted mRNA targets. In conclusion, our study provides a profile of altered miRs in HF human SAN, and a novel transcriptome blueprint to identify molecular targets for SAN dysfunction and arrhythmia treatments in HF., (© 2021. The Author(s).)

Background: Purkinje fibers (PFs) control timing of ventricular conduction and play a key role in arrhythmogenesis in heart failure (HF) patients. We investigated the effects of HF on PFs., Methods: Echocardiography, electrocardiography, micro-computed tomography, quantitative polymerase chain reaction, immunohistochemistry, volume electron microscopy, and sharp microelectrode electrophysiology were used., Results: Congestive HF was induced in rabbits by left ventricular volume- and pressure-overload producing left ventricular hypertrophy, diminished fractional shortening and ejection fraction, and increased left ventricular dimensions. HF baseline QRS and corrected QT interval were prolonged by 17% and 21% (mean±SEMs: 303±6 ms HF, 249±11 ms control; n=8/7; P =0.0002), suggesting PF dysfunction and impaired ventricular repolarization. Micro-computed tomography imaging showed increased free-running left PF network volume and length in HF. mRNA levels for 40 ion channels, Ca 2+ -handling proteins, connexins, and proinflammatory and fibrosis markers were assessed: 50% and 35% were dysregulated in left and right PFs respectively, whereas only 12.5% and 7.5% changed in left and right ventricular muscle. Funny channels, Ca 2+ -channels, and K + -channels were significantly reduced in left PFs. Microelectrode recordings from left PFs revealed more negative resting membrane potential, reduced action potential upstroke velocity, prolonged duration (action potential duration at 90% repolarization: 378±24 ms HF, 249±5 ms control; n=23/38; P <0.0001), and arrhythmic events in HF. Similar electrical remodeling was seen at the left PF-ventricular junction. In the failing left ventricle, upstroke velocity and amplitude were increased, but action potential duration at 90% repolarization was unaffected., Conclusions: Severe volume- followed by pressure-overload causes rapidly progressing HF with extensive remodeling of PFs. The PF network is central to both arrhythmogenesis and contractile dysfunction and the pathological remodeling may increase the risk of fatal arrhythmias in HF patients.

[Figure: see text].

Birds developed endothermy and four-chambered high-performance heart independently from mammals. Though avian embryos are extensively studied and widely used as various models for heart research, little is known about cardiac physiology of adult birds. Meanwhile, cardiac electrophysiology is in search for easily accessible and relevant model objects which resemble human myocardium in the pattern of repolarizing currents (I Kr , I Ks , I Kur and I to ). This study focuses on the configuration of electrical activity and electrophysiological phenotype of working myocardium in adult Japanese quails (Coturnix japonica). The resting membrane potential and action potential (AP) waveform in quail atrial myocardium were similar to that in working myocardium of rodents. Using whole-cell patch clamp and sharp glass microelectrodes, we demonstrated that the repolarization of quail atrial and ventricular myocardium is determined by voltage-dependent potassium currents I Kr , I Ks and I to - the latter was previously considered as an exclusive evolutionary feature of mammals. The specific blockers of these currents, dofetilide (3 μmol l -1 ), HMR 1556 (30 μmol l -1 ) and 4-aminopyridine (3 mmol l -1 ), prolonged AP in both ventricular and atrial myocardial preparations. The expression of the corresponding channels responsible for these currents in quail myocardium was investigated with quantitative RT-PCR and western blotting. In conclusion, the described pattern of repolarizing ionic currents and channels in quail myocardium makes this species a novel and suitable experimental model for translational cardiac research and reveals new information related to the evolution of cardiac electrophysiology in vertebrates., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Heart rate follows a diurnal variation, and slow heart rhythms occur primarily at night., Objective: The lower heart rate during sleep is assumed to be neural in origin, but here we tested whether a day-night difference in intrinsic pacemaking is involved., Methods: In vivo and in vitro electrocardiographic recordings, vagotomy, transgenics, quantitative polymerase chain reaction, Western blotting, immunohistochemistry, patch clamp, reporter bioluminescence recordings, and chromatin immunoprecipitation were used., Results: The day-night difference in the average heart rate of mice was independent of fluctuations in average locomotor activity and persisted under pharmacological, surgical, and transgenic interruption of autonomic input to the heart. Spontaneous beating rate of isolated (ie, denervated) sinus node (SN) preparations exhibited a day-night rhythm concomitant with rhythmic messenger RNA expression of ion channels including hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4). In vitro studies demonstrated 24-hour rhythms in the human HCN4 promoter and the corresponding funny current. The day-night heart rate difference in mice was abolished by HCN block, both in vivo and in the isolated SN. Rhythmic expression of canonical circadian clock transcription factors, for example, Brain and muscle ARNT-Like 1 (BMAL1) and Cryptochrome (CRY) was identified in the SN and disruption of the local clock (by cardiomyocyte-specific knockout of Bmal1) abolished the day-night difference in Hcn4 and intrinsic heart rate. Chromatin immunoprecipitation revealed specific BMAL1 binding sites on Hcn4, linking the local clock with intrinsic rate control., Conclusion: The circadian variation in heart rate involves SN local clock-dependent Hcn4 rhythmicity. Data reveal a novel regulator of heart rate and mechanistic insight into bradycardia during sleep., (Copyright © 2020 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Aim: This study is aimed at investigation of electrophysiological effects of α1-adrenoreceptor (α1-AR) stimulation in the rat superior vena cava (SVC) myocardium, which is one of the sources of proarrhythmic activity., Methods: α1-ARs agonists (phenylephrine-PHE or norepinephrine in presence of atenolol-NE + ATL) were applied to SVC and atrial tissue preparations or isolated cardiomyocytes, which were examined using optical mapping, glass microelectrodes or whole-cell patch clamp. α1-ARs distribution was evaluated using immunofluorescence. Kir2.X mRNA and protein level were estimated using RT-PCR and Western blotting., Results: PHE or NE + ATL application caused a significant suppression of the conduction velocity (CV) of excitation and inexcitability in SVC, an increase in the duration of electrically evoked action potentials (APs), a decrease in the maximum upstroke velocity (dV/dt max ) and depolarization of the resting membrane potential (RMP) in SVC to a greater extent than in atria. The effects induced by α1-ARs activation in SVC were attenuated by protein kinase C inhibition (PKC). The whole-cell patch clamp revealed PHE-induced suppression of outward component of I K1 inward rectifier current in isolated SVC, but not atrial myocytes. These effects can be mediated by α1A subtype of α-ARs found in abundance in rat SVC. The basal I K1 level in SVC was much lower than in atria as a result of the weaker expression of Kir2.2 channels., Conclusion: Therefore, the reduced density of I K1 in rat SVC cardiomyocytes and sensitivity of this current to α1A-AR stimulation via PKC-dependent pathways might lead to proarrhythmic conduction in SVC myocardium by inducing RMP depolarization, AP prolongation, CV and dV/dt max decrease., (© 2020 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background: KCNMA1 encodes the α-subunit of the large-conductance Ca 2+ -activated K + channel, K Ca 1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of K Ca 1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene., Methods: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of K Ca 1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized., Results: A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of K Ca 1.1 in normal hearts using immunostaining and immunogold electron microscopy. K Ca 1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the K Ca 1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the K Ca 1.1 ortholog, kcnma1b , in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila K Ca 1.1 ortholog, slo , systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo -deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the K Ca 1.1 loss-of-function models., Conclusions: Our data point to a highly conserved role of K Ca 1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that K Ca 1.1 loss of function may predispose to AF.

Background: The sinoatrial/sinus node (SAN) is the primary pacemaker of the heart. In humans, SAN is surrounded by the paranodal area (PNA). Although the PNA function remains debated, it is thought to act as a subsidiary atrial pacemaker (SAP) tissue and become the dominant pacemaker in the setting of sinus node disease (SND). Large animal models of SND allow characterization of SAP, which might be a target for novel treatment strategies for SAN diseases., Methods: A goat model of SND was developed ( n = 10) by epicardially ablating the SAN and validated by mapping of emergent SAP locations through an ablation catheter and surface electrocardiogram (ECG). Structural characterization of the goat SAN and SAP was assessed by histology and immunofluorescence techniques., Results: When the SAN was ablated, SAPs featured a shortened atrioventricular conduction, consistent with the location in proximity of atrioventricular junction. SAP recovery time showed significant prolongation compared to the SAN recovery time, followed by a decrease over a follow-up of 4 weeks. Like the SAN tissue, the SAP expressed the main isoform of pacemaker hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and Na + /Ca 2+ exchanger 1 (NCX1) and no high conductance connexin 43 (Cx43). Structural characterization of the right atrium (RA) revealed that the SAN was located at the earliest activation [i.e., at the junction of the superior vena cava (SVC) with the RA] and was surrounded by the paranodal-like tissue, extending down to the inferior vena cava (IVC). Emerged SAPs were localized close to the IVC and within the thick band of the atrial muscle known as the crista terminalis (CT)., Conclusions: SAN ablation resulted in the generation of chronic SAP activity in 60% of treated animals. SAP displayed development over time and was located within the previously discovered PNA in humans, suggesting its role as dominant pacemaker in SND. Therefore, SAP in goat constitutes a promising stable target for electrophysiological modification to construct a fully functioning pacemaker., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Soattin, Borbas, Caldwell, Prendergast, Vohra, Saeed, Hoschtitzky, Yanni, Atkinson, Logantha, Borbas, Garratt, Morris and Dobrzynski.)

Physiological systems vary in a day-night manner anticipating increased demand at a particular time. Heart is no exception. Cardiac output is primarily determined by heart rate and unsurprisingly this varies in a day-night manner and is higher during the day in the human (anticipating increased day-time demand). Although this is attributed to a day-night rhythm in post-translational ion channel regulation in the heart's pacemaker, the sinus node, by the autonomic nervous system, we investigated whether there is a day-night rhythm in transcription. RNAseq revealed that ~ 44% of the sinus node transcriptome (7134 of 16,387 transcripts) has a significant day-night rhythm. The data revealed the oscillating components of an intrinsic circadian clock. Presumably this clock (or perhaps the master circadian clock in the suprachiasmatic nucleus) is responsible for the rhythm observed in the transcriptional machinery, which in turn is responsible for the rhythm observed in the transcriptome. For example, there is a rhythm in transcripts responsible for the two principal pacemaker mechanisms (membrane and Ca 2+ clocks), transcripts responsible for receptors and signalling pathways known to control pacemaking, transcripts from genes identified by GWAS as determinants of resting heart rate, and transcripts from genes responsible for familial and acquired sick sinus syndrome.

Background The sinus node (SN) is the primary pacemaker of the heart. SN myocytes possess distinctive action potential morphology with spontaneous diastolic depolarization because of a unique expression of ion channels and Ca 2+ -handling proteins. MicroRNAs (miRs) inhibit gene expression. The role of miRs in controlling the expression of genes responsible for human SN pacemaking and conduction has not been explored. The aim of this study was to determine miR expression profile of the human SN as compared with that of non-pacemaker atrial muscle. Methods and Results SN and atrial muscle biopsies were obtained from donor or post-mortem hearts (n=10), histology/immunolabeling were used to characterize the tissues, TaqMan Human MicroRNA Arrays were used to measure 754 miRs, Ingenuity Pathway Analysis was used to identify miRs controlling SN pacemaker gene expression. Eighteen miRs were significantly more and 48 significantly less abundant in the SN than atrial muscle. The most interesting miR was miR-486-3p predicted to inhibit expression of pacemaking channels: HCN1 (hyperpolarization-activated cyclic nucleotide-gated 1), HCN4, voltage-gated calcium channel (Ca v )1.3, and Ca v 3.1. A luciferase reporter gene assay confirmed that miR-486-3p can control HCN4 expression via its 3' untranslated region. In ex vivo SN preparations, transfection with miR-486-3p reduced the beating rate by ≈35±5% ( P <0.05) and HCN4 expression ( P <0.05). Conclusions The human SN possesses a unique pattern of expression of miRs predicted to target functionally important genes. miR-486-3p has an important role in SN pacemaker activity by targeting HCN4, making it a potential target for therapeutic treatment of SN disease such as sinus tachycardia.

Bradyarrhythmias are an important cause of mortality in heart failure and previous studies indicate a mechanistic role for electrical remodelling of the key pacemaking ion channel HCN4 in this process. Here we show that, in a mouse model of heart failure in which there is sinus bradycardia, there is upregulation of a microRNA (miR-370-3p), downregulation of the pacemaker ion channel, HCN4, and downregulation of the corresponding ionic current, I f , in the sinus node. In vitro, exogenous miR-370-3p inhibits HCN4 mRNA and causes downregulation of HCN4 protein, downregulation of I f , and bradycardia in the isolated sinus node. In vivo, intraperitoneal injection of an antimiR to miR-370-3p into heart failure mice silences miR-370-3p and restores HCN4 mRNA and protein and I f in the sinus node and blunts the sinus bradycardia. In addition, it partially restores ventricular function and reduces mortality. This represents a novel approach to heart failure treatment.

Mechanisms for human sinoatrial node (SAN) dysfunction are poorly understood and whether human SAN excitability requires voltage-gated sodium channels (Nav) remains controversial. Here, we report that neuronal (n)Nav blockade and selective nNav1.6 blockade during high-resolution optical mapping in explanted human hearts depress intranodal SAN conduction, which worsens during autonomic stimulation and overdrive suppression to conduction failure. Partial cardiac (c)Nav blockade further impairs automaticity and intranodal conduction, leading to beat-to-beat variability and reentry. Multiple nNav transcripts are higher in SAN vs atria; heterogeneous alterations of several isoforms, specifically nNav1.6, are associated with heart failure and chronic alcohol consumption. In silico simulations of Nav distributions suggest that I Na is essential for SAN conduction, especially in fibrotic failing hearts. Our results reveal that not only cNav but nNav are also integral for preventing disease-induced failure in human SAN intranodal conduction. Disease-impaired nNav may underlie patient-specific SAN dysfunctions and should be considered to treat arrhythmias.

Background: Functional properties of the sinoatrial node (SAN) are known to differ between sexes. Women have higher resting and intrinsic heart rates. Sex determines the risk of developing certain arrhythmias such as sick sinus syndrome, which occur more often in women. We believe that a major contributor to these differences is in gender specific ion channel expression., Methods: qPCR was used to compare ion channel gene expression in the SAN and right atrium (RA) between male and female rats. Histology, immunohistochemistry and signal intensity analysis were used to locate the SAN and determine abundance of ion channels. The effect of nifedipine on extracellular potential recording was used to determine differences in beating rate between sexes., Results: mRNAs for Cav1.3, Kir3.1, and Nkx2-5, as well as expression of the L-Type Ca²⁺ channel protein, were higher in the female SAN. Females had significantly higher intrinsic heart rates and the effect of nifedipine on isolated SAN preparations was significantly greater in male SAN. Computer modelling using a SAN cell model demonstrated a higher propensity of pacemaker-related arrhythmias in females., Conclusion: This study has identified key differences in the expression of Cav1.3, Kir3.1 and Nkx2-5 at mRNA and/or protein levels between male and female SAN. Cav1.3 plays an important role in the pacemaker function of the SAN, therefore the higher intrinsic heart rate of the female SAN could be caused by the higher expression of Cav1.3. The differences identified in this study advance our understanding of sex differences in cardiac electrophysiology and arrhythmias.

Athletes are prone to supraventricular rhythm disturbances including sinus bradycardia, heart block, and atrial fibrillation. Mechanistically, this is attributed to high vagal tone and cardiac electrical and structural remodeling. Here, we consider the supporting evidence for these three pro-arrhythmic mechanisms in athletic human cohorts and animal models, featuring current controversies, emerging data, and future directions of relevance to the translational research agenda.

In adult mammalian hearts, atrioventricular rings (AVRs) surround the atrial orifices of atrioventricular valves and are hotbed of ectopic activity in patients with focal atrial tachycardia. Experimental data offering mechanistic insights into initiation and maintenance of ectopic foci is lacking. We aimed to characterise AVRs in structurally normal rat hearts, identify arrhythmia predisposition and investigate mechanisms underlying arrhythmogenicity. Extracellular potential mapping and intracellular action potential recording techniques were used for electrophysiology, qPCR for gene and, Western blot and immunohistochemistry for protein expression. Conditions favouring ectopic foci were assessed by simulations. In right atrial preparations, sinus node (SN) was dominant and AVRs displayed 1:1 impulse conduction. Detaching SN unmasked ectopic pacemaking in AVRs and pacemaker action potentials were SN-like. Blocking pacemaker current I f , and disrupting intracellular Ca 2+ release, prolonged spontaneous cycle length in AVRs, indicating a role for SN-like pacemaker mechanisms. AVRs labelled positive for HCN4, and SERCA2a was comparable to SN. Pacemaking was potentiated by isoproterenol and abolished with carbachol and AVRs had abundant sympathetic nerve endings. β 2 -adrenergic and M 2 -muscarinic receptor mRNA and β 2 -receptor protein were comparable to SN. In computer simulations of a sick SN, ectopic foci in AVR were unmasked, causing transient suppression of SN pacemaking.

Cardiovascular complications are common in type 1 diabetes mellitus (TIDM) and there is an increased risk of arrhythmias as a result of dysfunction of the cardiac conduction system (CCS). We have previously shown that, in vivo , there is a decrease in the heart rate and prolongation of the QRS complex in streptozotocin-induced type 1 diabetic rats indicating dysfunction of the CCS. The aim of this study was to investigate the function of the ex vivo CCS and key proteins that are involved in pacemaker mechanisms in TIDM. RR interval, PR interval and QRS complex duration were significantly increased in diabetic rats. The beating rate of the isolated sinoatrial node (SAN) preparation was significantly decreased in diabetic rats. The funny current density and cell capacitance were significantly decreased in diabetic nodal cells. Western blot showed that proteins involved in the function of the CCS were significantly decreased in diabetic rats, namely: HCN4, Ca v 1.3, Ca v 3.1, Cx45, and NCX1 in the SAN; RyR2 and NCX1 in the atrioventricular junction and Cx40, Cx43, Cx45, and RyR2 in the Purkinje network. We conclude that there are complex functional and cellular changes in the CCS in TIDM. The changes in the proteins involved in the function of this electrical system are expected to adversely affect action potential generation and propagation, and these changes are likely to be arrhythmogenic.

Aims: Action potential duration (APD) alternans is an established precursor or arrhythmia and sudden cardiac death. Important differences in fundamental electrophysiological properties relevant to arrhythmia exist between experimental models and the diseased in vivo human heart. To investigate mechanisms of APD alternans using a novel approach combining intact heart and cellular cardiac electrophysiology in human in vivo., Methods and Results: We developed a novel approach combining intact heart electrophysiological mapping during cardiac surgery with rapid on-site data analysis to guide myocardial biopsies for laboratory analysis, thereby linking repolarization dynamics observed at the organ level with underlying ion channel expression. Alternans-susceptible and alternans-resistant regions were identified by an incremental pacing protocol. Biopsies from these sites (n = 13) demonstrated greater RNA expression in Calsequestrin (CSQN) and Ryanodine (RyR) and ion channels underlying IK1 and Ito at alternans-susceptible sites. Electrical restitution properties (n = 7) showed no difference between alternans-susceptible and resistant sites, whereas spatial gradients of repolarization were greater in alternans-susceptible than in alternans-resistant sites (P = 0.001). The degree of histological fibrosis between alternans-susceptible and resistant sites was equivalent. Mathematical modelling of these changes indicated that both CSQN and RyR up-regulation are key determinants of APD alternans., Conclusion: Combined intact heart and cellular electrophysiology show that regions of myocardium in the in vivo human heart exhibiting APD alternans are associated with greater expression of CSQN and RyR and show no difference in restitution properties compared to non-alternans regions. In silico modelling identifies up-regulation and interaction of CSQN with RyR as a major mechanism underlying APD alternans., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Cardiac arrhythmias are a leading cause of cardiovascular death. It has long been accepted that life-threatening cardiac arrhythmias (ventricular tachycardia, ventricular fibrillation, and sudden cardiac death) are more likely to occur in the morning after waking. It is perhaps less well recognized that there is a circadian rhythm in cardiac pacemaking and other electrophysiological properties of the heart. In addition, there is a circadian rhythm in other arrhythmias, for example, bradyarrhythmias and supraventricular arrhythmias. Two mechanisms may underlie this finding: (1) a central circadian clock in the suprachiasmatic nucleus in the hypothalamus may directly affect the electrophysiology of the heart and arrhythmogenesis via various neurohumoral factors, particularly the autonomic nervous system; or (2) a local circadian clock in the heart itself (albeit under the control of the central clock) may drive a circadian rhythm in the expression of ion channels in the heart, which in turn varies arrhythmic substrate. This review summarizes the current understanding of the circadian rhythm in cardiac electrophysiology, arrhythmogenesis, and the underlying molecular mechanisms., (Copyright © 2018. Published by Elsevier Inc.)

Key Points: The sinoatrial node (SAN) is the primary pacemaker of the heart. SAN dysfunction, or 'sick sinus syndrome', can cause excessively slow heart rates and pauses, leading to exercise limitation and syncope, currently treated by implantation of an electronic pacemaker. 'Biopacemaking' utilises gene therapy to restore pacemaker activity by manipulating gene expression. Overexpressing the HCN pacemaker ion channel has been widely used with limited success. We utilised bradycardic rat subsidiary atrial pacemaker tissue to evaluate alternative gene targets: the Na + /Ca 2+ exchanger NCX1, and the transcription factors TBX3 and TBX18 known to be involved in SAN embryonic development. TBX18 overexpression restored normal SAN function, as assessed by increased rate, improved heart rate stability and restoration of isoprenaline response. TBX3 and NCX1 were not effective in accelerating the rate of subsidiary atrial pacemaker tissue. Gene therapy targeting TBX18 could therefore have the potential to restore pacemaker function in human sick sinus syndrome obviating electronic pacemakers., Abstract: The sinoatrial node (SAN) is the primary pacemaker of the heart. Disease of the SAN, sick sinus syndrome, causes heart rate instability in the form of bradycardia and pauses, leading to exercise limitation and syncope. Biopacemaking aims to restore pacemaker activity by manipulating gene expression, and approaches utilising HCN channel overexpression have been widely used. We evaluated alternative gene targets for biopacemaking to restore normal SAN pacemaker physiology within bradycardic subsidiary atrial pacemaker (SAP) tissue, using the Na + /Ca 2+ exchanger NCX1, and the transcription factors TBX3 and TBX18. TBX18 expression in SAP tissue restored normal SAN function, as assessed by increased rate (SAN 267.5 ± 13.6 bpm, SAP 144.1 ± 8.6 bpm, SAP-TBX18 214.4 ± 14.4 bpm; P < 0.001), improved heart rate stability (standard deviation of RR intervals fell from 39.3 ± 7.2 ms to 6.9 ± 0.8 ms, P < 0.01; root mean square of successive differences of RR intervals fell from 41.7 ± 8.2 ms to 6.1 ± 1.2 ms, P < 0.01; standard deviation of points perpendicular to the line of identity of Poincaré plots (SD1) fell from 29.5 ± 5.8 ms to 7.9 ± 2.0 ms, P < 0.05) and restoration of isoprenaline response (increases in rates of SAN 65.5 ± 1.3%, SAP 28.4 ± 3.4% and SAP-TBX18 103.3 ± 10.2%; P < 0.001). These changes were driven by a TBX18-induced switch in the dominant HCN isoform in SAP tissue, with a significant upregulation of HCN2 (from 1.01 × 10 -5  ± 2.2 × 10 -6 to 2.8 × 10 -5  ± 4.3 × 10 -6 arbitrary units, P < 0.001). Biophysically detailed computer modelling incorporating isoform-specific HCN channel electrophysiology confirmed that the measured changes in HCN abundance could account for the observed changes in beating rates. TBX3 and NCX1 were not effective in accelerating the rate of SAP tissue., (© 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Background: In vivo experiments in Goto-Kakizaki (GK) type 2 diabetic rats have demonstrated reductions in heart rate from a young age. The expression of genes encoding more than 70 proteins that are associated with the generation and conduction of electrical activity in the GK sinoatrial node (SAN) have been evaluated to further clarify the molecular basis of the low heart rate., Materials and Methods: Heart rate and expression of genes were evaluated with an extracellular electrode and real-time RT-PCR, respectively. Rats aged 12-13 months were employed in these experiments., Results: Isolated spontaneous heart rate was reduced in GK heart (161 ± 12 bpm) compared to controls (229 ± 11 bpm). There were many differences in expression of mRNA, and some of these differences were of particular interest. Compared to control SAN, expression of some genes were downregulated in GK-SAN: gap junction, Gja1 (Cx43), Gja5 (Cx40), Gjc1 (Cx45), and Gjd3 (Cx31.9); cell membrane transport, Trpc1 (TRPC1) and Trpc6 (TRPC6); hyperpolarization-activated cyclic nucleotide-gated channels, Hcn1 (HCN1) and Hcn4 (HCN4); calcium channels, Cacna1d (Ca v 1.3), Cacna1g (Ca v 3.1), Cacna1h (Ca v 3.2), Cacna2d1 (Ca v α 2 δ 1), Cacna2d3 (Ca v α 2 δ 3), and Cacng4 (Cav γ 4); and potassium channels, Kcna2 (K v 1.2), Kcna4 (K v 1.4), Kcna5 (K v 1.5), Kcnb1 ( K v 2.1), Kcnd3 (K v 4.3), Kcnj2 (K ir 2.1), Kcnk1 (TWIK1), Kcnk5 (K 2P 5.1), Kcnk6 (TWIK2), and Kcnn2 (SK2) whilst others were upregulated in GK-SAN: Ryr2 (RYR2) and Nppb (BNP)., Conclusions: This study provides new insight into the changing expression of genes in the sinoatrial node of diabetic heart.

Background: Aging is associated with an increased incidence of atrioventricular nodal (AVN) dysfunction., Objective: The aim of this study was to investigate the structural and functional remodeling in the atrioventricular junction (AVJ) with aging., Methods: Electrophysiology, histology, and immunohistochemistry experiments on male Wistar Hannover rats aged 3 months (n = 24) and 2 years (n = 15) were performed. Atrio-His (AH) interval, Wenkebach cycle length (WBCL), and AVN effective refractory period (AVNERP) were measured. Cesium (2 mM) was used to block hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, while ryanodine (2 μM) was used to block ryanodine 2 (RyR2) channels. Protein expression from different regions of the AVJ was studied using immunofluorescence. The expression of connexins (connexin 43 and connexin 40), ion channels (Hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), voltage sensitive sodium channel (Na v 1.5), and L-Type calcium channel (Ca v 1.3)), and calcium handling proteins (RyR2 and sarco/endoplasmic reticulum calcium ATPaset type 2a (SERCA2a)) were measured. Morphological characteristics were studied with histology., Results: Without drugs to block HCN and RyR2 channels, there was prolongation of the AH interval, WBCL, and AVNERP (P < .05) with aging. In young rats only, cesium prolonged the AH interval, WBCL, and AVNERP (P < .01). Ryanodine prolonged the AH interval and WBCL (P < .01) in both young and old rats. Immunofluorescence revealed that with aging, connexin 43, HCN4, Na v 1.5, and RyR2 downregulate in the regions of the AVJ and connexin 40, SERCA2a, and Ca v 1.3 upregulate (P < .05). Aging results in cellular hypertrophy, loosely packed cells, a decrease in the number of nuclei, and an increase in collagen content., Conclusion: Heterogeneous ion channel expression changes were observed in the AVJ with aging. For the first time, we have shown that HCN and RyR2 play an important role in AVN dysfunction with aging., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Downregulation of the pacemaking ion channel, HCN4 (hyperpolarization-activated cyclic nucleotide gated channel 4), and the corresponding ionic current, I f , underlies exercise training-induced sinus bradycardia in rodents. If this occurs in humans, it could explain the increased incidence of bradyarrhythmias in veteran athletes, and it will be important to understand the underlying processes., Objective: To test the role of HCN4 in the training-induced bradycardia in human athletes and investigate the role of microRNAs (miRs) in the repression of HCN4., Methods and Results: As in rodents, the intrinsic heart rate was significantly lower in human athletes than in nonathletes, and in all subjects, the rate-lowering effect of the HCN selective blocker, ivabradine, was significantly correlated with the intrinsic heart rate, consistent with HCN repression in athletes. Next-generation sequencing and quantitative real-time reverse transcription polymerase chain reaction showed remodeling of miRs in the sinus node of swim-trained mice. Computational predictions highlighted a prominent role for miR-423-5p. Interaction between miR-423-5p and HCN4 was confirmed by a dose-dependent reduction in HCN4 3'-untranslated region luciferase reporter activity on cotransfection with precursor miR-423-5p (abolished by mutation of predicted recognition elements). Knockdown of miR-423-5p with anti-miR-423-5p reversed training-induced bradycardia via rescue of HCN4 and I f . Further experiments showed that in the sinus node of swim-trained mice, upregulation of miR-423-5p (intronic miR) and its host gene, NSRP1, is driven by an upregulation of the transcription factor Nkx2.5., Conclusions: HCN remodeling likely occurs in human athletes, as well as in rodent models. miR-423-5p contributes to training-induced bradycardia by targeting HCN4. This work presents the first evidence of miR control of HCN4 and heart rate. miR-423-5p could be a therapeutic target for pathological sinus node dysfunction in veteran athletes., (© 2017 The Authors.)

Prolonged action potential duration, reduced action potential firing rate, upstroke velocity and rate of diastolic depolarization have been demonstrated in atrioventricular node (AVN) cells from streptozotocin (STZ)‑induced diabetic rats. To further clarify the molecular basis of these electrical disturbances, the mRNA profiles encoding a variety of proteins associated with the generation and conduction of electrical activity in the AVN, were evaluated in the STZ‑induced diabetic rat heart. Expression of mRNA was measured in AVN biopsies using reverse transcription‑quantitative polymerase chain reaction techniques. Notable differences in mRNA expression included upregulation of genes encoding membrane and intracellular Ca2+ transport, including solute carrier family 8 member A1, transient receptor potential channel 1, ryanodine receptor 2/3, hyperpolarization‑activated cyclic‑nucleotide 2 and 3, calcium channel voltage‑dependent, β2 subunit and sodium channels 3a, 4a, 7a and 3b. In addition to this, potassium channels potassium voltage‑gated channel subfamily A member 4, potassium channel calcium activated intermediate/small conductance subfamily N α member 2, potassium voltage‑gated channel subfamily J members 3, 5, and 11, potassium channel subfamily K members 1, 2, 3 and natriuretic peptide B (BNP) were upregulated in AVN of STZ heart, compared with controls. Alterations in gene expression were associated with upregulation of various proteins including the inwardly rectifying, potassium channel Kir3.4, NCX1 and BNP. The present study demonstrated notable differences in the profile of mRNA encoding proteins associated with the generation, conduction and regulation of electrical signals in the AVN of the STZ‑induced diabetic rat heart. These data will provide a basis for a substantial range of future studies to investigate whether variations in mRNA translate into alterations in electrophysiological function.