Empowering artificial intelligence in characterizing the human primary pacemaker of the heart at single cell resolution.

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.

miR-1 as a Key Epigenetic Regulator in Early Differentiation of Cardiac Sinoatrial Region.

A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. In this study, miR-1 potential role in early cardiac chamber differentiation was analyzed through specific signaling pathways. For this, we performed in chick embryos functional experiments by means of miR-1 microinjections into the posterior cardiac precursors-of both primitive endocardial tubes-committed to sinoatrial region fates. Subsequently, embryos were subjected to whole mount in situ hybridization, immunohistochemistry and RT-qPCR analysis. As a relevant novelty, our results revealed that miR-1 increased Amhc1, Tbx5 and Gata4, while this microRNA diminished Mef2c and Cripto expressions during early differentiation of the cardiac sinoatrial region. Furthermore, we observed in this developmental context that miR-1 upregulated CrabpII and Rarß and downregulated CrabpI, which are three crucial factors in the retinoic acid signaling pathway. Interestingly, we also noticed that miR-1 directly interacted with Hdac4 and Calm1/Calmodulin, as well as with Erk2/Mapk1, which are three key factors actively involved in Mef2c regulation. Our study shows, for the first time, a key role of miR-1 as an epigenetic regulator in the early differentiation of the cardiac sinoatrial region through orchestrating opposite actions between retinoic acid and Mef2c, fundamental to properly assign cardiac cells to their respective heart chambers. A better understanding of those molecular mechanisms modulated by miR-1 will definitely help in fields applied to therapy and cardiac regeneration and repair.

Pacemaker Channels and the Chronotropic Response in Health and Disease.

Loss or dysregulation of the normally precise control of heart rate via the autonomic nervous system plays a critical role during the development and progression of cardiovascular disease-including ischemic heart disease, heart failure, and arrhythmias. While the clinical significance of regulating changes in heart rate, known as the chronotropic effect, is undeniable, the mechanisms controlling these changes remain not fully understood. Heart rate acceleration and deceleration are mediated by increasing or decreasing the spontaneous firing rate of pacemaker cells in the sinoatrial node. During the transition from rest to activity, sympathetic neurons stimulate these cells by activating ß-adrenergic receptors and increasing intracellular cyclic adenosine monophosphate. The same signal transduction pathway is targeted by positive chronotropic drugs such as norepinephrine and dobutamine, which are used in the treatment of cardiogenic shock and severe heart failure. The cyclic adenosine monophosphate-sensitive hyperpolarization-activated current (If) in pacemaker cells is passed by hyperpolarization-activated cyclic nucleotide-gated cation channels and is critical for generating the autonomous heartbeat. In addition, this current has been suggested to play a central role in the chronotropic effect. Recent studies demonstrate that cyclic adenosine monophosphate-dependent regulation of HCN4 (hyperpolarization-activated cyclic nucleotide-gated cation channel isoform 4) acts to stabilize the heart rate, particularly during rapid rate transitions induced by the autonomic nervous system. The mechanism is based on creating a balance between firing and recently discovered nonfiring pacemaker cells in the sinoatrial node. In this way, hyperpolarization-activated cyclic nucleotide-gated cation channels may protect the heart from sinoatrial node dysfunction, secondary arrhythmia of the atria, and potentially fatal tachyarrhythmia of the ventricles. Here, we review the latest findings on sinoatrial node automaticity and discuss the physiological and pathophysiological role of HCN pacemaker channels in the chronotropic response and beyond.

Proteomics couples electrical remodelling to inflammation in a murine model of heart failure with sinus node dysfunction.

AIMS: In patients with heart failure (HF), concomitant sinus node dysfunction (SND) is an important predictor of mortality, yet its molecular underpinnings are poorly understood. Using proteomics, this study aimed to dissect the protein and phosphorylation remodelling within the sinus node in an animal model of HF with concurrent SND. METHODS AND RESULTS: We acquired deep sinus node proteomes and phosphoproteomes in mice with heart failure and SND and report extensive remodelling. Intersecting the measured (phospho)proteome changes with human genomics pharmacovigilance data, highlighted downregulated proteins involved in electrical activity such as the pacemaker ion channel, Hcn4. We confirmed the importance of ion channel downregulation for sinus node physiology using computer modelling. Guided by the proteomics data, we hypothesized that an inflammatory response may drive the electrophysiological remodeling underlying SND in heart failure. In support of this, experimentally induced inflammation downregulated Hcn4 and slowed pacemaking in the isolated sinus node. From the proteomics data we identified proinflammatory cytokine-like protein galectin-3 as a potential target to mitigate the effect. Indeed, in vivo suppression of galectin-3 in the animal model of heart failure prevented SND. CONCLUSION: Collectively, we outline the protein and phosphorylation remodeling of SND in heart failure, we highlight a role for inflammation in electrophysiological remodelling of the sinus node, and we present galectin-3 signalling as a target to ameliorate SND in heart failure.

Decreased connexin 40 expression of the sinoatrial node mediates ischemic stroke-induced arrhythmia in mice.

BACKGROUND: Arrhythmia is the most common cardiac complication after ischemic stroke. Connexin 40 is the staple component of gap junctions, which influences the propagation of cardiac electrical signals in the sinoatrial node. However, the role of connexin 40 in post-stroke arrhythmia remains unclear. METHODS: In this study, a permanent middle cerebral artery occlusion model was used to simulate the occurrence of an ischemic stroke. Subsequently, an electrocardiogram was utilized to record and assess variations in electrocardiogram measures. In addition, optical tissue clearing and whole-mount immunofluorescence staining were used to confirm the anatomical localization of the sinoatrial node, and the sinoatrial node tissue was collected for RNA sequencing to screen for potential pathological mechanisms. Lastly, the rAAV9-Gja5 virus was injected with ultrasound guidance into the heart to increase Cx40 expression in the sinoatrial node. RESULTS: We demonstrated that the mice suffering from a permanent middle cerebral artery occlusion displayed significant arrhythmia, including atrial fibrillation, premature ventricular contractions, atrioventricular block, and abnormal electrocardiogram parameters. Of note, we observed a decrease in connexin 40 expression within the sinoatrial node after the ischemic stroke via RNA sequencing and western blot. Furthermore, rAAV9-Gja5 treatment ameliorated the occurrence of arrhythmia following stroke. CONCLUSIONS: In conclusion, decreased connexin 40 expression in the sinoatrial node contributed to the ischemic stroke-induced cardiac arrhythmia. Therefore, enhancing connexin 40 expression holds promise as a potential therapeutic approach for ischemic stroke-induced arrhythmia.

Segregation of morphogenetic regulatory function of Shox2 from its cell fate guardian role in sinoatrial node development.

Shox2 plays a vital role in the morphogenesis and physiological function of the sinoatrial node (SAN), the primary cardiac pacemaker, manifested by the formation of a hypoplastic SAN and failed differentiation of pacemaker cells in Shox2 mutants. Shox2 and Nkx2-5 are co-expressed in the developing SAN and regulate the fate of the pacemaker cells through a Shox2-Nkx2-5 antagonistic mechanism. Here we show that simultaneous inactivation of Nkx2-5 in the SAN of Shox2 mutants (dKO) rescued the pacemaking cell fate but not the hypoplastic defects, indicating uncoupling of SAN cell fate determination and morphogenesis. Single-cell RNA-seq revealed that the presumptive SAN cells of Shox2-/- mutants failed to activate pacemaking program but remained in a progenitor state preceding working myocardium, while both wildtype and dKO SAN cells displayed normal pacemaking cell fate with similar cellular state. Shox2 thus acts as a safeguard but not a determinant to ensure the pacemaking cell fate through the Shox2-Nkx2-5 antagonistic mechanism, which is segregated from its morphogenetic regulatory function in SAN development.

D-galactose causes sinoatrial node dysfunction: from phenotype to mechanism.

With the population aging, age-related sinoatrial node dysfunction (SND) has been on the rise. Sinoatrial node (SAN) degeneration is an important factor for the age-related SND development. However, there is no suitable animal modeling method in this field. Here, we investigated whether D-galactose could induce SAN degeneration and explored the associated mechanism. In vivo, twelve C57BL/6 mice were divided into Control and D-galactose group to receive corresponding treatments. Senescence was confirmed by analyzing the hair and weight; cardiac function was evaluated through echocardiography, cerebral blood flux and serum-BNP; the SAN function was evaluated by electrocardiogram; fibrotic change was evaluated by Masson's trichrome staining and oxidative stress was assessed through DHE staining and serum indicators. Mechanism was verified through immunofluorescence-staining and Western blotting. In vitro, mouse-atrial-myocytes were treated with D-galactose, and edaravone was utilized as the ROS scavenger. Senescence, oxidative stress, proliferation ability and mechanism were verified through various methods, and intuitive evidence was obtained through electrophysiological assay. Finally, we concluded that D-galactose can be used to induce age-related SND, in which oxidative stress plays a key role, causing PITX2 ectopic expression and downregulates SHOX2 expression, then through the downstream GATA4/NKX2-5 axis, results in pacing-related ion channels dysfunction, and hence SND development.

Computational insight into energy control balance by Ca2+ and cAMP-PKA signaling in pacemaker cells.

Cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling controls sinoatrial node cell (SANC) function by affecting the degree of coupling between Ca2+ and membrane clocks. PKA is known to phosphorylate ionic channels, Ca2+ pump and release from the sarcoplasmic reticulum, and enzymes controlling ATP production in the mitochondria. While the PKA cytosolic targets in SANC have been extensively explored, its mitochondrial targets and its ability to maintain SANC energetic balance remain to be elucidated. To investigate the role of PKA in SANC energetics, we tested three hypotheses: (i) PKA is an important regulator of the ATP supply-to-demand balance, (ii) Ca2+ regulation of energetics is important for maintenance of NADH level and (iii) abrupt reduction in ATP demand first reduces the AP firing rate and, after dropping below a certain threshold, leads to a reduction in ATP. To gain mechanistic insights into the ATP supply-to-demand matching regulators, a modified model of mitochondrial energy metabolism was integrated into our coupled-clock model that describes ATP demand. Experimentally, increased ATP demand was accompanied by maintained ATP and NADH levels. Ca2+ regulation of energetics was found by the model to be important in the maintenance of NADH and PKA regulation was found to be important in the maintenance of intracellular ATP and the increase in oxygen consumption. PKA inhibition led to a biphasic reduction in AP firing rate, with the first phase being rapid and ATP-independent, while the second phase was slow and ATP-dependent. Thus, SANC energy balance is maintained by both Ca2+ and PKA signaling.

Tbx5 overexpression in embryoid bodies increases TAK1 expression but does not enhance the differentiation of sinoatrial node cardiomyocytes.

Genetic studies place Tbx5 at the apex of the sinoatrial node (SAN) transcriptional program. To understand its role in SAN differentiation, clonal embryonic stem (ES) cell lines were made that conditionally overexpress Tbx5, Tbx3, Tbx18, Shox2, Islet-1, and MAP3k7/TAK1. Cardiac cells differentiated using embryoid bodies (EBs). EBs overexpressing Tbx5, Islet1, and TAK1 beat faster than cardiac cells differentiated from control ES cell lines, suggesting possible roles in SAN differentiation. Tbx5 overexpressing EBs showed increased expression of TAK1, but cardiomyocytes did not differentiate as SAN cells. EBs showed no change in the expression of the SAN transcription factors Shox2 and Islet1 and decreased expression of the SAN channel protein HCN4. EBs constitutively overexpressing TAK1 direct cardiac differentiation to the SAN fate but have reduced phosphorylation of its targets, p38 and Jnk. This opens the possibility that blocking the phosphorylation of TAK1 targets may have the same impact as forced overexpression. To test this, we treated EBs with 5z-7-Oxozeanol (OXO), an inhibitor of TAK1 phosphorylation. Like TAK1 overexpressing cardiac cells, cardiomyocytes differentiated in the presence of OXO beat faster and showed increased expression of SAN genes (Shox2, HCN4, and Islet1). This suggests that activation of the SAN transcriptional network can be accomplished by blocking the phosphorylation of TAK1.

Emerging Signaling Regulation of Sinoatrial Node Dysfunction.

PURPOSE OF REVIEW: The sinoatrial node (SAN), the natural pacemaker of the heart, is responsible for generating electrical impulses and initiating each heartbeat. Sinoatrial node dysfunction (SND) causes various arrhythmias such as sinus arrest, SAN block, and tachycardia/bradycardia syndrome. Unraveling the underlying mechanisms of SND is of paramount importance in the pursuit of developing effective therapeutic strategies for patients with SND. This review provides a concise summary of the most recent progress in the signaling regulation of SND. RECENT FINDINGS: Recent studies indicate that SND can be caused by abnormal intercellular and intracellular signaling, various forms of heart failure (HF), and diabetes. These discoveries provide novel insights into the underlying mechanisms SND, advancing our understanding of its pathogenesis. SND can cause severe cardiac arrhythmias associated with syncope and an increased risk of sudden death. In addition to ion channels, the SAN is susceptible to the influence of various signalings including Hippo, AMP-activated protein kinase (AMPK), mechanical force, and natriuretic peptide receptors. New cellular and molecular mechanisms related to SND are also deciphered in systemic diseases such as HF and diabetes. Progress in these studies contributes to the development of potential therapeutics for SND.

Remodelling and dysfunction of the sinus node in pulmonary arterial hypertension.

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 Ca2+-handling genes that could explain the dysfunction: HCN1 and HCN4 (responsible for pacemaker current, If), Cav1.2, Cav1.3 and Cav3.1 (responsible for L- and T-type Ca2+ currents, ICa,L and ICa,T), NCX1 (responsible for Na+-Ca2+ exchanger) and SERCA2 and RYR2 (Ca2+-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 ß1. In summary, in PAH, there is a remodelling of ion channel, Ca2+-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'.

What makes the sinoatrial node tick? A question not for the faint of heart.

Even before the sinoatrial node (SAN) was discovered, cardiovascular science was engaged in an active investigation of when and why the heart would beat. After the electrochemical theory of bioelectric membrane potentials was formulated and the first action potentials were measured in contracting muscle cells, the field became divided: some investigators studied electrophysiology and ion channels, others studied muscle contraction. It later became known that changes in intracellular Ca2+ cause contraction. The pacemaking field was reunited by the coupled-clock theory of pacemaker cell function, which integrated intracellular Ca2+ cycling and transmembrane voltage into one rhythmogenic system. In this review, we will discuss recent discoveries that contextualize the coupled-clock system, first described in isolated SAN cells, into the complex world of SAN tissue: heterogeneous local Ca2+ releases, generated within SAN pacemaker cells and regulated by the other cell types within the SAN cytoarchitecture, variably co-localize and synchronize to give rise to relatively rhythmic impulses that emanate from the SAN to excite the heart. We will ultimately conceptualize the SAN as a brain-like structure, composed of intercommunicating meshworks of multiple types of pacemaker cells and interstitial cells, intertwined networks of nerves and glial cells and more. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Human Sinoatrial Node Pacemaker Activity: Role of the Slow Component of the Delayed Rectifier K+ Current, IKs.

The pacemaker activity of the sinoatrial node (SAN) has been studied extensively in animal species but is virtually unexplored in humans. Here we assess the role of the slowly activating component of the delayed rectifier K+ current (IKs) in human SAN pacemaker activity and its dependence on heart rate and ß-adrenergic stimulation. HEK-293 cells were transiently transfected with wild-type KCNQ1 and KCNE1 cDNA, encoding the α- and ß-subunits of the IKs channel, respectively. KCNQ1/KCNE1 currents were recorded both during a traditional voltage clamp and during an action potential (AP) clamp with human SAN-like APs. Forskolin (10 µmol/L) was used to increase the intracellular cAMP level, thus mimicking ß-adrenergic stimulation. The experimentally observed effects were evaluated in the Fabbri-Severi computer model of an isolated human SAN cell. Transfected HEK-293 cells displayed large IKs-like outward currents in response to depolarizing voltage clamp steps. Forskolin significantly increased the current density and significantly shifted the half-maximal activation voltage towards more negative potentials. Furthermore, forskolin significantly accelerated activation without affecting the rate of deactivation. During an AP clamp, the KCNQ1/KCNE1 current was substantial during the AP phase, but relatively small during diastolic depolarization. In the presence of forskolin, the KCNQ1/KCNE1 current during both the AP phase and diastolic depolarization increased, resulting in a clearly active KCNQ1/KCNE1 current during diastolic depolarization, particularly at shorter cycle lengths. Computer simulations demonstrated that IKs reduces the intrinsic beating rate through its slowing effect on diastolic depolarization at all levels of autonomic tone and that gain-of-function mutations in KCNQ1 may exert a marked bradycardic effect during vagal tone. In conclusion, IKs is active during human SAN pacemaker activity and has a strong dependence on heart rate and cAMP level, with a prominent role at all levels of autonomic tone.

The role of P21-activated kinase (Pak1) in sinus node function.

Sinoatrial node (SAN) dysfunction (SND) and atrial arrhythmia frequently occur simultaneously with a hazard ratio of 4.2 for new onset atrial fibrillation (AF) in SND patients. In the atrial muscle attenuated activity of p21-activated kinase 1 (Pak1) increases the risk for AF by enhancing NADPH oxidase 2 dependent production of reactive oxygen species (ROS). However, the role of Pak1 dependent ROS regulation in SAN function has not yet been determined. We hypothesize that Pak1 activity maintains SAN activity by regulating the expression of the hyperpolarization activated cyclic nucleotide gated cation channel (HCN). To determine Pak1 dependent changes in heart rate (HR) regulation we quantified the intrinsic sinus rhythm in wild type (WT) and Pak1 deficient (Pak1-/-) mice of both sexes in vivo and in isolated Langendorff perfused hearts. Pak1-/- hearts displayed an attenuated HR in vivo after autonomic blockage and in isolated hearts. The contribution of the Ca2+ clock to pacemaker activity remained unchanged, but Ivabradine (3 µM), a blocker of HCN channels that are a membrane clock component, eliminated the differences in SAN activity between WT and Pak1-/- hearts. Reduced HCN4 expression was confirmed in Pak1-/- right atria. The reduced HCN activity in Pak1-/- could be rescued by class II HDAC inhibition (LMK235), ROS scavenging (TEMPOL) or attenuation of Extracellular Signal-Regulated Kinase (ERK) 1/2 activity (SCH772984). No sex specific differences in Pak1 dependent SAN regulation were determined. Our results establish Pak1 as a class II HDAC regulator and a potential therapeutic target to attenuate SAN bradycardia and AF susceptibility.

Insulin signaling is critical for sinoatrial node maintenance and function.

Insulin and insulin-like growth factor 1 (IGF-1) signaling regulate cellular growth and glucose metabolism in the myocardium. However, their physiological role in the cells of the cardiac conduction system has never been explored. Therefore, we sought to determine the spatiotemporal function of insulin/IGF-1 receptors in the sinoatrial node (SAN). We generated cardiac conduction cell-specific inducible IGF-1 receptor (IGF-1R) knockout (KO) (CSIGF1RKO), insulin receptor (IR) KO (CSIRKO), and IR/IGF-1R double-KO (CSDIRKO) mice and evaluated their phenotypes. Telemetric electrocardiography revealed regular sinus rhythm in CSIGF1RKO mice, indicating that IGF-1R is dispensable for normal pacemaking. In contrast, CSIRKO and CSDIRKO mice exhibited profound sinus bradycardia. CSDIRKO mice showed typical sinus node dysfunction characterized by junctional rhythm and sinus pauses on electrocardiography. Interestingly, the lack of an insulin receptor in the SAN cells of CSIRKO and CSDIRKO mice caused sinus nodal fibrosis. Mechanistically, hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) protein expression significantly decreased in the CSIRKO and CSDIRKO mice relative to the controls. A patch-clamp study of the SAN cells of CSIRKO mice revealed a significant decrease in the funny current, which is responsible for spontaneous diastolic depolarization in the SAN. This result suggested that insulin receptor loss reduces the heart rate via downregulation of the HCN4 channel. Additionally, HCN1 expression was decreased in CSDIRKO mice, explaining their sinus node dysfunction. Our results reveal a previously unrecognized role of insulin/IGF-1 signaling in sinus node structural maintenance and pacemaker function.

The method of sinus node-like pacemaker cells from human induced pluripotent stem cells by BMP and Wnt signaling.

The embryonic development of sinus nodes (SAN) is co-regulated by multiple signaling pathways. Among these, the bone morphogenetic protein (BMP) and Wnt signaling pathways are involved in the development of SAN. In this study, the effects of BMP and Wnt signaling on the differentiation of SAN-like pacemaker cells (SANLPCs) were investigated. Human induced pluripotent stem cells (hiPSCs) were divided into four groups: control, BMP4, CHIR-3, and BMP4 + CHIR (CHIR: a Wnt signaling activator). The samples were tested at day (D) 15 of differentiation. The final protocol for the activation of BMP signaling at D0-D3 and reactivation of Wnt signaling at D5-D7 in the differentiation of hiPSCs were determined. The results showed that the mRNA levels of pacemaker markers (TBX18, SHOX2, TBX3, HCN4, and HCN1) were higher in the BMP4 + CHIR group than in the control group, and working myocardial genes were downregulated. The immunofluorescence assay revealed that the expression of SHOX2 and HCN4 increased in the BMP4 + CHIR group compared to that in the other groups. In addition, the results of patch clamps revealed that a funny current of higher density and typical SAN action potentials were recorded, except in the control group, in which the L-type calcium current was higher in the BMP4 + CHIR group than in the other groups. Finally, the proportion of SANLPCs (cTnT+ NKX2.5-) was further enhanced by the combination of BMP4 and CHIR treatment. In summary, the combination of BMP and Wnt signaling promotes the differentiation of SANLPCs from hiPSCs.

Depressed HCN4 function in the type 2 diabetic sinoatrial node.

Diabetic patients often have impaired heart rate (HR) control. HR is regulated both intrinsically within the sinoatrial node (SAN) and via neuronal input. Previously, we found lower ex vivo HR in type 2 diabetic rat hearts, suggesting impaired HR generation within the SAN. The major driver of pacemaking within the SAN is the activity of hyperpolarisation-activated cyclic nucleotide-gated 4 (HCN(4)) channels. This study aimed to investigate whether the lower intrinsic HR in the type 2 diabetic heart is due to changes in HCN4 function, protein expression and/ or distribution. The intrinsic HR response to HCN4 blockade was determined in isolated Langendorff-perfused hearts of Zucker type 2 Diabetic Fatty (ZDF) rats (DM) and their non-diabetic ZDF littermates (nDM). HCN4 protein expression and membrane localisation were determined using western blot and immunofluorescence, respectively. We found that the intrinsic HR was lower in DM compared to nDM hearts. The change in intrinsic HR in response to HCN4 blockade with ivabradine was diminished in DM hearts, which normalised the intrinsic HR between the groups. HCN4 protein expression was decreased in the SAN of DM compared to nDM controls with no change in the fraction of HCN4 localised to the membrane of SAN cardiomyocytes. The lower intrinsic HR in DM is likely due to decreased HCN4 expression and depressed HCN4 function. Our study provides a novel understanding into the intrinsic mechanisms underlying altered HR control in type 2 diabetes.

Sinus node dysfunction: current understanding and future directions.

The sinoatrial node (SAN) is the primary pacemaker of the heart. Normal SAN function is crucial in maintaining proper cardiac rhythm and contraction. Sinus node dysfunction (SND) is due to abnormalities within the SAN, which can affect the heartbeat frequency, regularity, and the propagation of electrical pulses through the cardiac conduction system. As a result, SND often increases the risk of cardiac arrhythmias. SND is most commonly seen as a disease of the elderly given the role of degenerative fibrosis as well as other age-dependent changes in its pathogenesis. Despite the prevalence of SND, current treatment is limited to pacemaker implantation, which is associated with substantial medical costs and complications. Emerging evidence has identified various genetic abnormalities that can cause SND, shedding light on the molecular underpinnings of SND. Identification of these molecular mechanisms and pathways implicated in the pathogenesis of SND is hoped to identify novel therapeutic targets for the development of more effective therapies for this disease. In this review article, we examine the anatomy of the SAN and the pathophysiology and epidemiology of SND. We then discuss in detail the most common genetic mutations correlated with SND and provide our perspectives on future research and therapeutic opportunities in this field.

ß-tubulin contributes to Tongyang Huoxue decoction-induced protection against hypoxia/reoxygenation-induced injury of sinoatrial node cells through SIRT1-mediated regulation of mitochondrial quality surveillance.

BACKGROUND: TYHX-Tongyang Huoxue decoction has been used clinically for nearly 40 years. The ingredients of TYHX are Radix Astragali (Huangqi), Red Ginseng (Hongshen), Rehmannia Glutinosa (Dihuang), Common Yam Rhizome (Shanyao) and Cassia-bark-tree Bark (Rougui). Our previous experiments confirmed that TYHX can protect sinoatrial node cells. However, its mechanism of action is not completely understood yet. PURPOSE: The present study aimed to determine the protective effects of TYHX against Sinus node cell injury under hypoxic stress and elucidate the underlying mechanisms of protection. METHODS: Through RNA sequencing analysis and network pharmacology analysis, we found significant differences in mitochondrial-related genes before and after hypoxia-mimicking SNC, resolved the main regulatory mechanism of TYHX. Through the intervention of TYHX on SNC, a series of detection methods such as laser confocal, fluorescence co-localization, mitochondrial membrane potential and RT-PCR. The regulatory effect of TYHX on ß-tubulin in sinoatrial node cells was verified by in vitro experiments. The mechanism of action of TYHX and its active ingredient quercetin to maintain mitochondrial homeostasis and protect sinoatrial node cells through mitophagy, mitochondrial fusion/fission and mitochondrial biosynthesis was confirmed. RESULTS: Through RNA sequencing analysis, we found that there were significant differences in mitochondrial related genes before and after SNC was modeled by hypoxia. Through pharmacological experiments, we showed that TYHX could inhibit the migration of Drp1 to mitochondria, inhibit excessive mitochondrial fission, activate mitophagy and increase the mitochondrial membrane potential. These protective effects were mainly mediated by ß-tubulin. Furthermore, the active component quercetin in TYHX could inhibit excessive mitochondrial fission through SIRT1, maintain mitochondrial energy metabolism and protect SNCs. Our results showed that protection of mitochondrial function through the maintenance of ß-tubulin and activation of SIRT1 is the main mechanism by which TYHX alleviates hypoxic stress injury in SNCs. The regulatory effects of TYHX and quercetin on mitochondrial quality surveillance are also necessary. Our findings provide empirical evidence supporting the use of TYHX as a targeted treatment for sick sinus syndrome. CONCLUSION: Our data indicate that TYHX exerts protective effects against sinus node cell injury under hypoxic stress, which may be associated with the regulation of mitochondrial quality surveillance (MQS) and inhibition of mitochondrial homeostasis-mediated apoptosis.