Genetics of sinoatrial node function and heart rate disorders.

The sinoatrial node (SAN) is the primary pacemaker of the mammalian heart, initiating its electrical activation and ensuring that the heart's functional cardiac output meets physiological demand. SAN dysfunction (SND) can cause complex cardiac arrhythmias that can manifest as severe sinus bradycardia, sinus arrest, chronotropic incompetence and increased susceptibility to atrial fibrillation, among other cardiac conditions. SND has a complex aetiology, with both pre-existing disease and heritable genetic variation predisposing individuals to this pathology. In this Review, we summarize the current understanding of the genetic contributions to SND and the insights that they provide into this disorder's underlying molecular mechanisms. With an improved understanding of these molecular mechanisms, we can improve treatment options for SND patients and develop new therapeutics.

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.

Development of a new histological identification method of human sinoatrial node suitable for immunohistochemical study.

Histological identification of the human sinoatrial node (SAN) remains a challenge. Conventional identification methods, such as Lev's method, have certain limitations. The aim of our study was to develop a new histological identification method that could properly identify the sinoatrial node, applicable to the immunohistochemical study of intra-nodal structures. Thirty-nine human autopsied hearts were included in this study. The cases included 23 men and 16 women ranging in age from 20 to 99 years. The sinoatrial area from eight control samples was cut in the vertical section using the conventional Lev's method. In our new method, called the "En face one-block method," the sinoatrial node was cut in "En face" at the junction of the right border of the right appendage and superior vena cava, placed in one long cassette, and serially cut using a microtome. Immunostaining was performed using primary antibodies against CD31, podoplanin (D2-40), S-100, and other proteins. The average area of the SAN on the slide glass in our new method was 32.2 mm2, which was significantly larger than that (3.59 mm2) of the control samples by Lev's method. The SAN area was positively correlated with age (r = 0.357; p = 0.026), especially in women (r = 0.626; p = 0.0095). The SAN group had significantly lower percentage of CD31-positive blood capillaries, higher percentage of podoplanin-positive lymphatic channels, and S-100-positive peripheral nerves. We successfully developed a novel cutting method applicable to immunohistochemical studies, with which we could provide a bird's-eye view of the sinoatrial nodes.

Eosinophilic Infiltration of the Sino-Atrial Node in Sudden Cardiac Death Caused by Long QT Syndrome.

Sudden death is defined as the unexpected death of a healthy person that occurs within the first hour of the onset of symptoms or within 24 h of the victim being last seen alive. In some of these cases, rare deleterious variants of genes associated with inherited cardiac disorders can provide a highly probable explanation for the fatal event. We report the case of a 21-year-old obese woman who lost consciousness suddenly in a public place and was pronounced dead after hospital admission. Clinical autopsy showed an inconclusive gross examination, while in the histopathological analysis an eosinophilic inflammatory focus and interstitial fibrosis in the sino-atrial node were found. Molecular autopsy revealed an intronic variant in the KCNQ1 gene (c.683 + 5G > A), classified as likely pathogenic for long QT syndrome according to the guidelines provided by the American College of Medical Genetics and Genomics. Therefore, there were many anomalies that could have played a role in the causation of the sudden death, such as the extreme obesity, the cardiac anomalies and the KNCQ1 variant. This case depicts the difficult interpretation of rare cardiac structural abnormalities in subjects carrying rare variants responsible for inherited arrhythmic disorders and the challenge for the forensic pathologist to make causal inferences in the determinism of the unexpected decease.

Whole genome sequencing identifies a deletion mutation in the unknown-functional KCNG2 from familial sick sinus syndrome.

Sick sinus syndrome (SSS) is a term used for a variety of disorders defined by abnormal cardiac impulse formation and by abnormal propagation from the heart's sinoatrial node. In this study, we present a case from a Chinese family in which two closely related individuals had the symptoms and electrocardiographic evidence of SSS. We hypothesized that multiple individuals affected by the disease in the family was an indication of its genetic predisposition, and thus performed high-throughput sequencing for the participants from the family to detect potential disease-associated variants. One of the potential variants that was identified was a KCNG2 gene variant (NC_000018.9: g.77624068_77624079del). Further bioinformatic analysis showed that the observed variant may be a pathogenic mutation. The results of protein-protein docking and whole cell patch-clamp measurements implied that the deletion variant in KCNG2 could affect its binding the KV2.1 protein, and finally affect the function of Kv channel, which is an important determinant in regulation of heartbeat. Therefore, we inferred that the variable KCNG2 gene may affect the function of Kv channel by changing the binding conformation of KCNG2 and KV2.1 proteins and then adversely affect propagation from the sinoatrial node and cardiac impulse formation by changing the action potential repolarization of heart cells. In summary, our findings suggested that the dominant KCNG2 deletion variant in the examined Chinese family with SSS may be a potential disease-associated variant.

Tongyang Huoxue Decoction (TYHX) Ameliorating Hypoxia/Reoxygenation-Induced Disequilibrium of Calcium Homeostasis and Redox Imbalance via Regulating Mitochondrial Quality Control in Sinoatrial Node Cells.

Sick sinus syndrome (SSS) is a disease with bradycardia or arrhythmia. The pathological mechanism of SSS is mainly due to the abnormal conduction function of the sinoatrial node (SAN) caused by interstitial lesions or fibrosis of the SAN or surrounding tissues, SAN pacing dysfunction, and SAN impulse conduction accompanied by SAN fibrosis. Tongyang Huoxue Decoction (TYHX) is widely used in SSS treatment and amelioration of SAN fibrosis. It has a variety of active ingredients to regulate the redox balance and mitochondrial quality control. This study mainly discusses the mechanism of TYHX in ameliorating calcium homeostasis disorder and redox imbalance of sinoatrial node cells (SANCs) and clarifies the protective mechanism of TYHX on the activity of SANCs. The activity of SANCs was determined by CCK-8 and the TUNEL method. The levels of apoptosis, ROS, and calcium release were analyzed by flow cytometry and immunofluorescence. The mRNA and protein levels of calcium channel regulatory molecules and mitochondrial quality control-related molecules were detected by real-time quantitative PCR and Western Blot. The level of calcium release was detected by laser confocal. It was found that after H/R treatment, the viability of SANCs decreased significantly, the levels of apoptosis and ROS increased, and the cells showed calcium overload, redox imbalance, and mitochondrial dysfunction. After treatment with TYHX, the cell survival level was improved, calcium overload and oxidative stress were inhibited, and mitochondrial energy metabolism and mitochondrial function were restored. However, after the SANCs were treated with siRNA (si-ß-tubulin), the regulation of TYHX on calcium homeostasis and redox balance was counteracted. These results suggest that ß-tubulin interacts with the regulation of mitochondrial function and calcium release. TYHX may regulate mitochondrial quality control, maintain calcium homeostasis and redox balance, and protect SANCs through ß-tubulin. The regulation mechanism of TYHX on mitochondrial quality control may also become a new target for SSS treatment.

Age-related histologic features of the sinoatrial node from normal human hearts during the first 10 decades of life: a study of 200 cases.

Knowledge of the histologic constituency of the sinoatrial (SA) node is based on small studies with unevenly distributed ages and subjective assessments of nodal composition, leading to difficulties in interpreting what constitutes true pathology of the SA node. SA nodes from two-hundred normal hearts (10 male and 10 female from each of the first 10 decades of life) were digitally analyzed to assess their histologic composition. Both nodal area and nodal fat content (≥5%) showed a quadratic relationship with age, peaking in the fifth to eighth decades of life. Increased fat content was also more prevalent with increased BMI (≥25 kg/m2). No differences between sexes were observed. Mean nodal collagen ranged from 7.1% to 50.3%, without a statistically significant differences by age or body mass index (BMI). The data suggests that the designation of pathologic fibrosis should be reserved for SA nodes with >50% collagen content. These findings expand and refine our understanding of the anatomy of the SA node.

cAMP-dependent regulation of HCN4 controls the tonic entrainment process in sinoatrial node pacemaker cells.

It is highly debated how cyclic adenosine monophosphate-dependent regulation (CDR) of the major pacemaker channel HCN4 in the sinoatrial node (SAN) is involved in heart rate regulation by the autonomic nervous system. We addressed this question using a knockin mouse line expressing cyclic adenosine monophosphate-insensitive HCN4 channels. This mouse line displayed a complex cardiac phenotype characterized by sinus dysrhythmia, severe sinus bradycardia, sinus pauses and chronotropic incompetence. Furthermore, the absence of CDR leads to inappropriately enhanced heart rate responses of the SAN to vagal nerve activity in vivo. The mechanism underlying these symptoms can be explained by the presence of nonfiring pacemaker cells. We provide evidence that a tonic and mutual interaction process (tonic entrainment) between firing and nonfiring cells slows down the overall rhythm of the SAN. Most importantly, we show that the proportion of firing cells can be increased by CDR of HCN4 to efficiently oppose enhanced responses to vagal activity. In conclusion, we provide evidence for a novel role of CDR of HCN4 for the central pacemaker process in the sinoatrial node.

Identification of Key Small Non-Coding MicroRNAs Controlling Pacemaker Mechanisms in the Human Sinus Node.

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 Ca2+-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 (Cav)1.3, and Cav3.1. 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.

Channelopathies of voltage-gated L-type Cav1.3/α1D and T-type Cav3.1/α1G Ca2+ channels in dysfunction of heart automaticity.

The heart automaticity is a fundamental physiological function in vertebrates. The cardiac impulse is generated in the sinus node by a specialized population of spontaneously active myocytes known as "pacemaker cells." Failure in generating or conducting spontaneous activity induces dysfunction in cardiac automaticity. Several families of ion channels are involved in the generation and regulation of the heart automaticity. Among those, voltage-gated L-type Cav1.3 (α1D) and T-type Cav3.1 (α1G) Ca2+ channels play important roles in the spontaneous activity of pacemaker cells. Ca2+ channel channelopathies specifically affecting cardiac automaticity are considered rare. Recent research on familial disease has identified mutations in the Cav1.3-encoding CACNA1D gene that underlie congenital sinus node dysfunction and deafness (OMIM # 614896). In addition, both Cav1.3 and Cav3.1 channels have been identified as pathophysiological targets of sinus node dysfunction and heart block, caused by congenital autoimmune disease of the cardiac conduction system. The discovery of channelopathies linked to Cav1.3 and Cav3.1 channels underscores the importance of Ca2+ channels in the generation and regulation of heart's automaticity.

Protective mechanism of SIRT1 on Hcy-induced atrial fibrosis mediated by TRPC3.

High plasma levels of homocysteine (Hcy) are regarded as a risk factor for atrial fibrillation (AF), which is closely associated with the pathological consequence of atrial fibrosis and can lead to heart failure with a high mortality rate; here, we show that atrial fibrosis is mediated by the relationship between canonical transient receptor potential 3 (TRPC3) channels and sirtuin type 1 (SIRT1) under the stimulation of Hcy. The left atrial appendage was obtained from patients with either sinus rhythm (SR) or AF and used to evaluate the relationship between the concentration of Hcy and a potential mechanism of cardiac fibrosis mediated by TRPC3 and SIRT1. We next performed transverse aortic constriction (TAC) in mouse to investigate the relationship. The mechanisms underlying atrial fibrosis involving TRPC3 and SIRT1 proteins were explored by co-IP, BLI and lentivirus transfection experiments. qPCR and WB were performed to analyse gene and protein expression, respectively. The higher level of atrial fibrosis was observed in the HH mouse group with a high Hcy diet. Such results suggest that AF patients may be more susceptible to atrial fibrosis and possess a high probability of progressing to hyperhomocysteinemia. Moreover, our findings are consistent with the hypothesis that TRPC3 channel up-regulation leads to abnormal accumulation of collagen, with the down-regulation of SIRT1 as an aetiological factor of high Hcy, which in turn predisposes to atrial fibrosis and strongly enhances the possibility of AF.

Mitochondrial thioredoxin-2 maintains HCN4 expression and prevents oxidative stress-mediated sick sinus syndrome.

OBJECTIVE: Sick sinus syndrome (SSS) is associated with loss of HCN4 (hyperpolarization-activated cyclic nucleotide-gated potassium channel 4) function in the cardiac conduction system. The underlying mechanism for SSS remains elusive. This study is to investigate how mitochondrial oxidative stress induces HCN4 downregulation associated with in sick sinus syndrome. METHODS AND RESULTS: Trx2lox/lox mice were crossed with α-myosin heavy chain (α-Mhc)-Cre and Hcn4-CreERT2 deleter mice to generate Trx2 deletion mice in the whole heart (Trx2cKO) and in the conduction system (Trx2ccsKO), respectively. Echocardiography was applied to measure hemodynamics and heart rhythm. Histological analyses, gene profiling and chromatin immunoprecipitation were performed to define the mechanism by which thioredoxin-2 (Trx2) regulates HCN4 expression and cardiac function. Trx2cKO mice displayed dilated cardiomyopathy, low heart rate, and atrial ventricular block (AVB) phenotypes. Immunofluorescence revealed that HCN4 expression was specifically reduced within the sinoatrial node in Trx2cKO mice. Interestingly, Trx2ccsKO mice displayed low heart rate and AVB without dilated cardiomyopathy. Both mRNA and protein levels of HCN4 were reduced in the sinoatrial node, suggesting transcriptional HCN4 regulation upon Trx2 deletion. ChIP indicated that the binding of MEF2 to the HCN4 enhancer was not altered by Trx2 deletion; however, histone 3 acetylation at the MEF2 binding site was decreased, and expression of histone deacetylase 4 (HDAC4) was elevated following Trx2 deletion. Moreover, HDAC4 binding to the HCN4 enhancer was mediated by MEF2. Mitochondrial ROS were increased by Trx2 deletion and importantly, mitochondria-specific ROS scavenger MitoTEMPO suppressed HDAC4 elevation, HCN4 reduction, and sinus bradycardia in Trx2ccsKO mice. CONCLUSION: In the conduction system, Trx2 is critical for maintaining HCN4-mediated normal heart rate. Loss of Trx2 reduces HCN4 expression via a mitochondrial ROS-HDAC4-MEF2C pathway and subsequently induces sick sinus syndrome in mice.

Fibrosis independent atrial fibrillation in older patients is driven by substrate leukocyte infiltration: diagnostic and prognostic implications to patients undergoing cardiac surgery.

BACKGROUND: The objectives of the study were to characterize and quantify cellular inflammation and structural remodeling of human atria and correlate findings with molecular markers of inflammation and patient surrogate outcome. METHODS: Voluntary participants undergoing heart surgery were enrolled in the study and blood samples were collected prior to surgery, and right atrium samples were harvested intraoperatively. Blood samples were analyzed by flow cytometry and complete blood counts. Atrial samples were divided for fixed fibrosis analysis, homogenized for cytokine analysis and digested for single cell suspension flow cytometry. RESULTS: A total of 18 patients were enrolled and samples assessed. Isolated cells from the atria revealed a CD45+ population of ~ 20%, confirming a large number of leukocytes. Further characterization revealed this population as 57% lymphocytes and 26% monocyte/macrophages (MoΦ), with the majority of the latter cells being classical (CD14++/CD16-). Interstitial fibrosis was present in 87% of samples and correlated significantly with patient age. Older patients (> 65) had significantly more atrial fibrosis and cellular inflammation. AFib patients had no distinguishing feature of atrial fibrosis and had significantly greater CD45+ MoΦ, increased expression of MMP9 and presented with a significant correlation in length of stay to CCL-2/MCP-1 and NLR (neutrophil-to-lymphocyte ratio). CONCLUSION: Atrial fibrosis is correlated with age and not determinate to AFib. However, severity of atrial leukocyte infiltration and markers of matrix degradation are determinant to AFib. This also correlated with CCL2 (or MCP-1) and NLR-indicative of marked inflammation. These data show the potential importance of diagnostic and prognostic assessments that could inform clinical decision making in regard to the intensity of AFib patient management.

Biological Pacemakers: Still a Dream?

A biological pacemaker is one or more types of cellular components that, when implanted into certain regions of the heart, produce electrical stimuli that mimic that of the body's natural pacemaker cells. Somatic gene transfer, cell fusion, or cell transplantation provide a way to realize it as somatic reprogramming strategies, which involve transfer of genes encoding transcription factors to transform working myocardium into a surrogate sinoatrial node, are furthest along in the possibilities. The idea, no doubt, is bright and appealing. The objective herein intends to dig into the subject trying to find out how realizable it really is.

Induced cardiac pacemaker cells survive metabolic stress owing to their low metabolic demand.

Cardiac pacemaker cells of the sinoatrial node initiate each and every heartbeat. Compared with our understanding of the constituents of their electrical excitation, little is known about the metabolic underpinnings that drive the automaticity of pacemaker myocytes. This lack is largely owing to the scarcity of native cardiac pacemaker myocytes. Here, we take advantage of induced pacemaker myocytes generated by TBX18-mediated reprogramming (TBX18-iPMs) to investigate comparative differences in the metabolic program between pacemaker myocytes and working cardiomyocytes. TBX18-iPMs were more resistant to metabolic stresses, exhibiting higher cell viability upon oxidative stress. TBX18-induced pacemaker myocytes (iPMs) expensed a lower degree of oxidative phosphorylation and displayed a smaller capacity for glycolysis compared with control ventricular myocytes. Furthermore, the mitochondria were smaller in TBX18-iPMs than in the control. We reasoned that a shift in the balance between mitochondrial fusion and fission was responsible for the smaller mitochondria observed in TBX18-iPMs. We identified a mitochondrial inner membrane fusion protein, Opa1, as one of the key mediators of this process and demonstrated that the suppression of Opa1 expression increases the rate of synchronous automaticity in TBX18-iPMs. Taken together, our data demonstrate that TBX18-iPMs exhibit a low metabolic demand that matches their mitochondrial morphology and ability to withstand metabolic insult.

A newly identified missense mutation in CLCA2 is associated with autosomal dominant cardiac conduction block.

BACKGROUND: Progressive cardiac conduction defect (PCCD), also known as Lenegre-Lev disease, is one of the most common heart conduction abnormalities. Previous studies have screened for known mutation sites that cause heart block in a 68-person family with a history of PCCD, revealed no mutations. OBJECTIVE: To screen pathogenic genes of the PCCD family and to study the function of the gene mutations related to heart block diseases. METHODS: Whole exome sequencing (WES) was performed on two PCCD patients and one non-PCCD family member to find the related pathogenic gene. After family co-segregation and preliminary functional analysis, we identified the mutant gene CLCA2. To study the function of this gene, we constructed mutant-gene mice using CRISPR-Cas9 technology, and electrocardiogram monitoring was performed after genotype verification. RESULTS: The CLCA2 c.G1725T mutation was identified and co-segregated with the phenotype. The analysis showed that the CLCA2 c.G1725T mutation is harmful and mainly affects protein glycosylation. Immunofluorescence staining revealed that CLCA2 was highly expressed in the sinoatrial node (SAN) tissues. Electrocardiogram monitoring of the mice revealed that CLCA2 point mutations induced mild conduction block and ectopic pacemakers. CONCLUSION: Our findings indicate that a novel heterozygous missense mutation c.G1725T of the CLCA2 gene may be associated with heart block disease and the mutation in this gene may lead to sinus node lesions and conduction blocking.

Sinoatrial node remodels in chronic sleep-restricted rats.

Chronic Sleep Restriction (CSR) is known as a risk factor for cardiovascular diseases. However, the structural changes of Sinoatrial (SA) node cells have received less attention. This study aimed to evaluate the effects of CSR on SA node in an animal model using stereological methods. Adult male Sprague-Dawley rats were randomly divided into CSR, grid-floor, and control groups. The CSR procedure was designed such a way that the animals had a full cycle of sleep (6 hours) per day, while they were unable to have a Rapid Eye Movement (REM) sleep during the remaining 18 hours. This was induced by a multiplatform box containing water. The grid-floor animals were placed in the same multiplatform box with a grid-floor covering to prevent falling in water. After 21 days, the right atria were dissected out. Then, the location of the SA node was determined and evaluated by stereological techniques. The total volume of the SA node, the total volume of the main node cells, the volume of the connective tissue, and mean volume of the node cells were respectively enlarged by 60%, 47%, 68%, and 51% in the CSR animals compared to the grid-floor rats (p < 0.05). However, no significant changes were detected in these parameters in the control and grid-floor animals. The population of the main node cells remained constant in all animal groups. In addition, the three-dimensional reconstruction of the SA node in the CSR group showed a hypertrophied appearance. In conclusion, CSR induced hypertrophic changes in the rats' SA node structures without alteration in the number of main node cells.

Interleukin-10 treatment attenuates sinus node dysfunction caused by streptozotocin-induced hyperglycaemia in mice.

Aims: Diabetes, characterized by hyperglycaemia, causes sinus node dysfunction (SND) in several rodent models. Interleukin (IL)-10, which is a potent anti-inflammatory cytokine, has been reported to decrease in obese and diabetic patients. We tested the hypothesis that administration of IL-10 inhibits the development of SND caused by hyperglycaemia in streptozotocin (STZ)-induced diabetic mice. Methods and results: Six-week old CL57/B6 (WT) mice were divided into the following groups: control, STZ injection, and STZ injection with systemic administration of IL-10. IL-10 knockout mice were similarly treated. STZ-induced hyperglycaemia for 8 weeks significantly depressed serum levels of IL-10, but increased several proinflammatory cytokines in WT mice. STZ-induced hyperglycaemia-reduced resting heart rate (HR), and attenuated HR response to isoproterenol in WT mice. In isolated perfused heart experiments, corrected-sinus node recovery time was prolonged in WT mice with STZ injection. Sinus node tissue isolated from the WT-STZ group showed fibrosis, abundant infiltration of macrophages, increased production of reactive oxygen species (ROS), and depressed hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4). However, the changes observed in the WT-STZ group were significantly attenuated by IL-10 administration and were further exaggerated in IL-10 knockout mice. In cultured cells, preincubation of IL-10 suppressed hyperglycaemia-induced apoptotic and profibrotic signals, and overproduction of ROS. IL-10 markedly inhibited the high glucose-induced p38 activation, and activated signal transducer and activator of transcription (STAT) 3 phosphorylation. Conclusions: Our results suggest that IL-10 attenuates ROS production, inflammation and fibrosis, and plays an important role in the inhibition of hyperglycaemia-induced SND by suppression of HCN4 downregulation. In addition, IL-10-mediated inhibition of p38 is dependent on STAT3 phosphorylation.

Identification of CACNA1D variants associated with sinoatrial node dysfunction and deafness in additional Pakistani families reveals a clinical significance.

Sinoatrial node dysfunction and deafness (SANDD) syndrome is rare and characterized by a low heart beat and severe-to-profound deafness. Additional features include fatigue, dizziness, and episodic syncope. The sinoatrial node (SAN) drives heart automaticity and continuously regulates heart rate. The CACNA1D gene encoding the Cav1.3 protein expressed in inner hair cells, atria and SAN, induces loss-of-function in channel activity and underlies SANDD. To date, only one variant c.1208_1209insGGG:p.(G403_V404insG) has been reported for SANDD syndrome. We studied five Pakistani families with SANDD and characterized a new missense variant p.(A376V) in CACNA1D in one family, and further characterized the founder variant p.(G403_V404insG) in four additional pedigrees. We show that affected individuals in the four families which segregate p.(G403_V404insG) share a 1.03 MB haplotype on 3p21.1 suggesting they share a common distant ancestor. In conclusion, we identified new and known variants in CACNA1D in five Pakistani families with SANDD. This study is of clinical importance as the CACNA1D founder variant is only observed in families from the Khyber Pakhtunkhwa (KPK) province, in Pakistan. Therefore, screening patients with congenital deafness for SAN dysfunction in this province could ensure adequate follow-up and prevent cardiac failure associated with SAN.