Conduction System Function Research Articles

Neonatal Deletion of Hand1 and Hand2 within Murine Cardiac Conduction System Reveals a Novel Role for HAND2 in Rhythm Homeostasis.

The cardiac conduction system, a network of specialized cells, is required for the functioning of the heart. The basic helix loop helix factors Hand1 and Hand2 are required for cardiac morphogenesis and have been implicated in cardiac conduction system development and maintenance. Here we use embryonic and post-natal specific Cre lines to interrogate the role of Hand1 and Hand2 in the function of the murine cardiac conduction system. Results demonstrate that loss of HAND1 in the post-natal conduction system does not result in any change in electrocardiogram parameters or within the ventricular conduction system as determined by optical voltage mapping. Deletion of Hand2 within the post-natal conduction system results in sex-dependent reduction in PR interval duration in these mice, suggesting a novel role for HAND2 in regulating the atrioventricular conduction. Surprisingly, results show that loss of both HAND factors within the post-natal conduction system does not cause any consistent changes in cardiac conduction system function. Deletion of Hand2 in the embryonic left ventricle results in inconsistent prolongation of PR interval and susceptibility to atrial arrhythmias. Thus, these results suggest a novel role for HAND2 in homeostasis of the murine cardiac conduction system and that HAND1 loss potentially rescues the shortened HAND2 PR phenotype.

Partial and complete loss of myosin binding protein H-like cause cardiac conduction defects

A premature truncation of MYBPHL in humans and a loss of Mybphl in mice is associated with dilated cardiomyopathy, atrial and ventricular arrhythmias, and atrial enlargement. MYBPHL encodes myosin binding protein H-like (MyBP-HL). Prior work in mice indirectly identified Mybphl expression in the atria and in small puncta throughout the ventricle. Because of its genetic association with human and mouse cardiac conduction system disease, we evaluated the anatomical localization of MyBP-HL and the consequences of loss of MyBP-HL on conduction system function. Immunofluorescence microscopy of normal adult mouse ventricles identified MyBP-HL-positive ventricular cardiomyocytes that co-localized with the ventricular conduction system marker contactin-2 near the atrioventricular node and in a subset of Purkinje fibers. Mybphl heterozygous ventricles had a marked reduction of MyBP-HL-positive cells compared to controls. Lightsheet microscopy of normal perinatal day 5 mouse hearts showed enrichment of MyBP-HL-positive cells within and immediately adjacent to the contactin-2-positive ventricular conduction system, but this association was not apparent in Mybphl heterozygous hearts. Surface telemetry of Mybphl-null mice revealed atrioventricular block and atrial bigeminy, while intracardiac pacing revealed a shorter atrial relative refractory period and atrial tachycardia. Calcium transient analysis of isolated Mybphl-null atrial cardiomyocytes demonstrated an increased heterogeneity of calcium release and faster rates of calcium release compared to wild type controls. Super-resolution microscopy of Mybphl heterozygous and homozygous null atrial cardiomyocytes showed ryanodine receptor disorganization compared to wild type controls. Abnormal calcium release, shorter atrial refractory period, and atrial dilation seen in Mybphl null, but not wild type control hearts, agree with the observed atrial arrhythmias, bigeminy, and atrial tachycardia, whereas the proximity of MyBP-HL-positive cells with the ventricular conduction system provides insight into how a predominantly atrial expressed gene contributes to ventricular arrhythmias and ventricular dysfunction.

Ddx20, DEAD box helicase 20, is essential for the differentiation of oligodendrocyte and maintenance of myelin gene expression.

Oligodendrocytes form myelin sheaths that surround axons, contributing to saltatory conduction and proper central nervous system (CNS) function. Oligodendrocyte progenitor cells (OPCs) are generated during the embryonic stage and differentiate into myelinating oligodendrocytes postnatally. Ddx20 is a multifunctional, DEAD-box helicase involved in multiple cellular processes, including transcription, splicing, microRNA biogenesis, and translation. Although defects in each of these processes result in abnormal oligodendrocyte differentiation and myelination, the involvement of Ddx20 in oligodendrocyte terminal differentiation remains unknown. To address this question, we used Mbp-Cre mice to generate Ddx20 conditional knockout (cKO) mice to allow for the deletion of Ddx20 from mature oligodendrocytes. Mbp-Cre;Ddx20 cKO mice demonstrated small body sizes, behavioral abnormalities, muscle weakness, and short lifespans, with mortality by the age of 2months old. Histological analyses demonstrated significant reductions in the number of mature oligodendrocytes and drastic reductions in the expression levels of myelin-associated mRNAs, such as Mbp and Plp at postnatal day 42. The number of OPCs did not change. A thin myelin layer was observed for large-diameter axons in Ddx20 cKO mice, based on electron microscopic analysis. A bromodeoxyuridine (BrdU) labeling experiment demonstrated that terminal differentiation was perturbed from ages 2 weeks to 7 weeks in the CNS of Mbp-Cre;Ddx20 cKO mice. The activation of mitogen-activated protein (MAP) kinase, which promotes myelination, was downregulated in the Ddx20 cKO mice based on immunohistochemical detection. These results indicate that Ddx20 is an essential factor for terminal differentiation of oligodendrocytes and maintenance of myelin gene expression.

Magnesium: The recent research and developments.

Magnesium is the fourth most abundant mineral in the human body, which facilitates more than 300 enzymatic reactions. Magnesium is essential for nucleic material and protein synthesis, neuromuscular conduction, cardiac contractility, energy metabolism, and immune system function. Gastrointestinal system and kidneys closely regulate magnesium absorption and elimination to maintain adequate storage of magnesium. Magnesium deficiency has been linked to many diseases and poor health outcomes. Magnesium has also been proven to be an effective therapeutic agent in many diseases, such as bronchial asthma, cardiac arrhythmia, and pre-eclampsia.

T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system

Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3+/-) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3+/- mutants. Notably, Tbx3+/- atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus.

Abstract 331: Loss of Myosin Binding Protein H-Like Causes Cardiac Conduction Abnormalities

A premature truncation (R255X) in MYBPHL associates with human dilated cardiomyopathy (DCM) and arrhythmias. Loss of Mybphl in mice causes DCM and arrhythmia. MYBPHL encodes myosin binding protein H-like (MyBP-HL) and is expressed highly in the atria. We hypothesize that MyBP-HL is required for proper conduction system function. Immunofluorescence microscopy on normal human atria showed MyBP-HL staining in all atrial cardiomyocytes with a sarcomere A-band pattern. Atria from the heterozygous (het) MYBPHL R255X mutant carrier lacked MyBP-HL staining. Human induced pluripotent stem cell-derived cardiomyocytes from the het MYBPHL R255X carrier and control cell lines were also examined. MyBP-HL was found in a subset of control cardiomyocytes, whereas R255X cells showed no MyBP-HL, suggesting that the R255X allele exerts a dominant-negative effect on the normal MYBPHL allele. Immunofluorescence microscopy in wild-type (WT) mouse ventricles identified MyBP-HL-positive ventricular cardiomyocytes that co-localized with the ventricular conduction system marker contactin-2 near the atrioventricular node and in a subset of Purkinje fibers. Mybphl het ventricles have 10% as many MyBP-HL-positive cells compared to WT. Surface telemetry revealed atrioventricular block and atrial bigeminy and intracardiac pacing revealed a shorter atrial relative refractory period and inducible atrial tachycardia in Mybphl -null mice. Ca 2+ transients measured with confocal microscopy revealed that isolated Mybphl -null atrial cardiomyocytes had an increased occurrence of triggered Ca 2+ waves and more heterogenous Ca 2+ release than WT controls. Super-resolution microscopy revealed ryanodine receptor disorganization in Mybphl het and null atrial cardiomyocytes compared to WT controls. Abnormal Ca 2+ release, shorter atrial refractory period, and dilated atria could account for the observed atrial arrhythmias, bigeminy, and atrial tachycardia.

Tbx3-Mediated Regulation of Cardiac Conduction System Development and Function: Potential Contributions of Alternative RNA Processing.

In this article, we provide a brief summary of work by us and others to discover the molecular underpinnings of early conduction system development and function. We focus on how the multifunctional protein Tbx3 contributes to acquisition and homeostasis of the tissue-specific properties of the sinoatrial and atrioventricular nodes. We also provide unpublished, preliminary findings supporting the role of Tbx3-regulated alternative RNA processing in the developing conduction system.

Variation in a Left Ventricle-Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function.

The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. GWAS (Genome-wide association studies) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r2>0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5' to the gene encoding the basic helix-loop-helix transcription factor HAND1 (heart- and neural crest derivatives-expressed protein 1). Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis-regulatory activity. Two evolutionarily conserved GATA transcription factor cis-binding elements within this enhancer are bound by GATA4 and are necessary for cis-regulatory activity, as shown by in vitro DNA binding assays. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify 3 additional single nucleotide polymorphisms (SNPs), located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis-regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays, disrupts GATA4 DNA-binding. Modeling 2 of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in electrophoretic mobility shift assay, and this enhancer in total, is required for VCS development and function in mice and perhaps humans.

Impact of aortic root morphology on the implantation depth of aortic valve prosthesis during trans-catheter aortic valve replacement in patients with native bicuspid aortic valve stenosis

Objective: To investigate the impact of aortic root morphology on the implantation depth of aortic valve prosthesis during trans-catheter aortic valve replacement (TAVR) in bicuspid aortic valve patients. Methods: Clinical data of 40 patients with native bicuspid aortic valve stenosis who underwent TAVR using the self-expandable prosthesis (the Venus A-valve) from 2014 to 2017 in Fuwai Hospital was retrospectively analyzed. The patients were divided into non-deep implantation group (implant depth ≤10 mm by instant angiogram after implantation,29 cases) and deep implantation group (implant depth> 10 mm by instant angiogram after implantation,11 cases).Pre-procedural aortic root characteristics (e.g. calcification, angle and dimensions) were assessed by CT. The impact of aortic root morphology on the implantation depth and clinical outcomes were also evaluated. Results: The age was (75.1±5.9) years with equal representation from the raphe-type and non-raphe type (52.5%(21/40) and 47.5%(19/40)).The bigger aorta angle ((56.5±4.5)° vs. (47.4±9.4)°, P=0.004),more frequent mild-calcification (HU850, <200 mm(3)) or severe-calcification(HU850, >1 000 mm(3)) of aortic leaflets (7/11 vs. 4/29, P=0.006), as well as higher ratio of left ventricular outflow tract perimeter to annulus perimeter ((109.2±7.5)% vs. (101.5±6.5)%, P=0.004) were found in the deep implantation group compared to the non-deep implantation group. The new in-hospital onset of bundle-branchheart-block or atrioventricular block conduction disturbance rate was higher in the deep implantation group than in the non-deep implantation group (6/11 vs. 2/29, P=0.030).Left ventricular ejection fraction was similar between deep implantation group and non-deep implantation group at baseline((49.9±8.9)% vs. (55.8±10.4)%, P=0.117), and was significantly lower in the deep implantation group than in the non-deep implantation group at 30 days after implantation ((51.6±12.8)% vs. (60.9±8.1)%, P=0.020). Conclusion: Aortic root morphology of bicuspid aortic valve patients is associated with implantation depth of the prosthesis during TAVR, which affects the conduction system and left ventricular function during and post TAVR.

Relation of Left Ventricular Fractional Shortening to Needfor Permanent Pacemaker After Transcatheter AorticValve Implantation

Transcatheter aortic valve implantation (TAVI) can potentially alter conduction system function due to the mechanical force applied to the conduction system by the proximal edge of the valve, particularly the CoreValve. Some reasons for post-TAVI advanced atrioventricular block have been identified. We investigated whether the degree of the motion of the basal left ventricular (LV) walls impacted the development of advanced atrioventricular block post-TAVI. A total of 407 patients (82.1 ± 6.2 years) without prior permanent pacemakers (PPMs) underwent TAVI using CoreValve (70%) or Edwards-SAPIEN (30%) prosthetic devices. The LV fractional shortening (FS) of the basal segments was measured in each patient, and the association between FS and PPM requirement, or new-onset left bundle branch block (LBBB) was evaluated. During hospitalization, 64 patients (15.7%) required PPM implantation, and 128 patients (31.4%) required PPM implantation or developed new LBBB. Independent predictors of PPM implantation included preprocedural right bundle branch block, CoreValve prosthetic device, valve implantation depth, and FS. Patients with high FS (≥40%, upper tertile) had a 2.5-fold increased risk of PPM implantation (p = 0.004) and a 1.8-fold increased risk of PPM or new LBBB (p = 0.020). Every 10% increment in FS was consistently associated with an adjusted 42% increased risk of PPM implantation (p = 0.015) and with an adjusted 43% increased risk of PPM implantation or new LBBB (p = 0.005). Thus, in our cohort, LV FS was independently associated with the need for PPM implantation during hospitalization. Hence, this simple echocardiographic measure can be used to identify patients who are at risk after TAVI.

GATA-Binding Factor 6 Contributes to Atrioventricular Node Development and Function.

Several transcription factors regulate cardiac conduction system (CCS) development and function but the role of each in specifying distinct CCS components remains unclear. GATA-binding factor 6 (GATA6) is a zinc-finger transcription factor that is critical for patterning the cardiovascular system. However, the role of GATA6 in the embryonic heart and CCS has never been shown. We report that Gata6 is expressed abundantly in the proximal CCS during midgestation in mice. Myocardial-specific deletion of the carboxyl zinc-finger of Gata6 induces loss of hyperpolarizing cyclic nucleotide-gated channel, subtype 4 staining in the compact atrioventricular node with some retention of hyperpolarizing cyclic nucleotide-gated channel, subtype 4 staining in the atrioventricular bundle, but has no significant effect on the connexin-40-positive bundle branches. Furthermore, myocardial-specific deletion of the carboxyl zinc-finger of Gata6 alters atrioventricular conduction in postnatal life as assessed by surface and invasive electrophysiological evaluation, as well as decreasing the number of ventricular myocytes and inducing compensatory myocyte hypertrophy. Myocardial-specific deletion of the carboxyl zinc-finger of Gata6 is also associated with downregulation of the transcriptional repressor ID2 and the cardiac sodium-calcium exchanger NCX1 in the proximal CCS, where GATA6 transactivates both of these factors. Finally, carboxyl zinc-finger deletion of Gata6 reduces cell-cycle exit of TBX3+ myocytes in the developing atrioventricular bundle during the period of atrioventricular node specification, which results in fewer TBX3+ cells in the proximal CCS of mature mutant mice. GATA6 contributes to the development and postnatal function of the murine atrioventricular node by promoting cell-cycle exit of specified cardiomyocytes toward a conduction system lineage.

Myocardial deletion of transcription factor CHF1/Hey2 results in altered myocyte action potential and mild conduction system expansion but does not alter conduction system function or promote spontaneous arrhythmias

CHF1/Hey2 is a Notch-responsive basic helix-loop-helix transcription factor involved in cardiac development. Common variants in Hey2 are associated with Brugada syndrome. We hypothesized that absence of CHF1/Hey2 would result in abnormal cellular electrical activity, altered cardiac conduction system (CCS) development, and increased arrhythmogenesis. We isolated neonatal CHF/Hey2-knockout (KO) cardiac myocytes and measured action potentials and ion channel subunit gene expression. We also crossed myocardial-specific CHF1/Hey2-KO mice with cardiac conduction system LacZ reporter mice and stained for conduction system tissue. We also performed ambulatory ECG monitoring for arrhythmias and heart rate variability. Neonatal cardiomyocytes from CHF1/Hey2-KO mice demonstrate a 50% reduction in action potential dV/dT, a 50-75% reduction in SCN5A, KCNJ2, and CACNA1C ion channel subunit gene expression, and an increase in delayed afterdepolarizations from 0/min to 12/min. CHF1/Hey2 cKO CCS-lacZ mice have a ∼3-fold increase in amount of CCS tissue. Ambulatory ECG monitoring showed no difference in cardiac conduction, arrhythmias, or heart rate variability. Wild-type cells or animals were used in all experiments. CHF1/Hey2 may contribute to Brugada syndrome by influencing the expression of SCN5A and formation of the cardiac conduction system, but its absence does not cause baseline conduction defects or arrhythmias in the adult mouse.-Hartman, M. E., Liu, Y., Zhu, W.-Z., Chien, W.-M., Weldy, C. S., Fishman, G. I., Laflamme, M. A., Chin, M. T. Myocardial deletion of transcription factor CHF1/Hey2 results in altered myocyte action potential and mild conduction system expansion but does not alter conduction system function or promote spontaneous arrhythmias.

Abstract 203: Measurement of Cardiac Function in a Porcine Model of Cardiac Arrest Due to Massive Exsanguination

Background: While extremity hemorrhage can be stymied with a well placed tourniquet, vascular injury within the thorax and abdomen is not amenable to direct compression. Uncontrolled, noncompressible torso hemorrhage (NCTH) and the resultant cardiac arrest (CA) may account for up to 80% of preventable deaths in combat. We developed a porcine model of CA to test novel surgical interventions and are reporting measures of cardiovascular function during a simulated NCTH. Methods: Ten anesthetized Yorkshire pigs underwent tracheostomy, arterial and central line placement. Animals were awakened and sedated with midazolam titrated to a bispectral index of 75 ± 5. Spontaneously breathing, the animals were subjected to a computerized hemorrhage of 80% total blood volume (TBVH) over 20 minutes or until death, defined as mean arterial pressure (MAP)=0 mmHg and etCO2&lt;10. During hemorrhage, if minute ventilation decreased 25% from baseline (BL) for &gt;20 seconds, volume control ventilation was initiated. The Millar MPVS Ultra system continuously collected left ventricular (LV) and descending aortic pressures, as well as LV volume data via indwelling catheters. A repeated measures one-way ANOVA with Tukey’s procedure was performed on data collected at BL, 25%, 44%, 62% TBV hemorrhage, and CA, defined as a MAP&lt;20 mmHg. Data are presented as means±SEM. Results: Weight was 63.8±2.8kg. TBVH at CA was 2941±130ml (71.0±1.5%). Time to CA was 18:22±00:40min. In Table 1, * denotes a significant change (p&lt;0.05) in a variable from previous hemorrhage phase as marked by #. Conclusions: We developed a porcine model of CA secondary to exsanguination. We found that despite decreasing left ventricular volumes and pressures, cardiac contractility and conduction system function were maintained throughout hemorrhage. This suggests that lifesaving interventions (such as endovascular aortic balloon occlusion and extracorporeal life support) may be possible despite profound hypovolemia.

Gene regulatory elements of the cardiac conduction system

The coordinated contraction of the heart relies on the generation and conduction of the electrical impulse. Aberrations of the function of the cardiac conduction system have been associated with various arrhythmogenic disorders and increased risk of sudden cardiac death. The genetics underlying conduction system function have been investigated using functional studies and genome-wide association studies. Both methods point towards the involvement of ion channel genes and the transcription factors that govern their activity. A large fraction of disease- and trait-associated sequence variants lie within non-coding sequences, enriched with epigenetic marks indicative of regulatory DNA. Although sequence conservation as a result of functional constraint has been a useful property to identify transcriptional enhancers, this identification process has been advanced through the development of techniques such as ChIP-seq and chromatin conformation capture technologies. The role of variation in gene regulatory elements in the cardiac conduction system has recently been demonstrated by studies on enhancers of SCN5A/SCN10A and TBX5. In both studies, a region harbouring a functionally implicated single-nucleotide polymorphism was shown to drive reproducible cardiac expression in a reporter gene assay. Furthermore, the risk variant of the allele abrogated enhancer function in both cases. Functional studies on regulatory DNA will likely receive a boost through recent developments in genome modification technologies.

Changes in spatial ventricular gradient and QRS-T angle induced by strict 5-days immobilization, and effects of exercise countermeasure

Purpose: The spatial QRS-T angle (SA) and ventricular gradient (SVG) are classical ECG parameters that provide information on cardiac conduction system function and on ventricular action potential duration heterogeneity. Wide SA and reduced SVG are related to pathological conditions. Our aims were: 1) to assess the effects of 5 days immobilization (-6 degrees bed-rest, BR) on SA and SVG; 2) to evaluate the effectiveness of 25 min/day standing exercise (unilateral and bilateral heel raise, bilateral squatting (60°, 90° and 120°), bilateral and cross-hopping) as possible countermeasure. Methods: 10 healthy men (mean age 30±6) were enrolled in a cross-over design (wash out period: 2 months; control, CTRL, and exercise, EXE, groups) at DLR (Cologne, Germany) as part of the European Space Agency BR studies. High fidelity (1000 Hz) Holter ECG (12-leads, Mortara Instrument) was acquired before (PRE), the last day of BR (HDT5), and four days after the BR conclusion (POST). The night period (23:00-06:30) was selected for the analysis. X,Y,Z leads were derived (inverse Dower matrix) and vectorcardiogram computed. Selective beat averaging was used to obtain averages of P-QRS-T complexes preceded by the same stable heart rate (10 msec bin amplitude, in the range 900-1200 msec). For each averaged waveform (i.e., one for each bin), Twave maximum amplitude (Tmax), VG and SVG were computed. Results: Compared to PRE, a reduction in SVG (11%±3% and 9%±1%) and corresponding widening in SA (27%±9% and 21%±5%) were found at HDT5 in both CTRL and EXE, respectively. The EXE countermeasure significantly reduced both SVG and SA changes compared to CTRL. Tmax presented the same changes, being reduced of 8%±3% in CTRL and only of 4%±2% in EXE. At POST, in both groups SA was restored to PRE values; conversely, while in EXE the SVG returned to PRE values, in CTRL it was found still reduced by 3%. Also, while Tmax was back to PRE values in EXE, it was increased by 5% in CTRL. Conclusions: Despite the short-term -6° BR immobilization, cardiac adaptation to this deconditioning condition affected ventricular repolarization during the night period, thus widening SA and reducing SVG as result of increased heterogeneity, thus increasing the risk of arrhythmia development. Selective beat averaging allowed quantifying these changes. Application of 25 min/day standing exercise reduced, but not canceled, these effects. This should be taken into account in patients with cardiovascular diseases, when immobilized in bed, to proper adjust the pharmacological therapy or set the rehabilitation training, in order to avoid further complications.

The effect of connexin40 deficiency on ventricular conduction system function during development

The aim of this study was to characterize ventricular activation patterns in normal and connexin40-deficient mice in order to dissect the role of connexin40 in developing the conduction system. We performed optical mapping of epicardial activation between ED9.5-18.5 and analysed ventricular activation patterns and times of left ventricular activation. Mouse embryos deficient for connexin40 were compared with normal and heterozygous littermates. Morphology of the primary interventricular ring (PIR) was delineated with the help of T3-LacZ transgene. Four major types of ventricular activation patterns characterized by primary breakthrough in different parts of the heart were detected during development: PIR, left ventricular apex, right ventricular apex, and dual right and left ventricular apices. Activation through PIR was frequently present at the early stages until ED12.5. From ED14.5, the majority of hearts showed dual left and right apical breakthrough, suggesting functionality of both bundle branches. Connexin40-deficient embryos showed initially a delay in left bundle branch function, but the right bundle branch block, previously described in the adults, was not detected in ED14.5 embryos and appeared only gradually with 80% penetrance at ED18.5. The switch of function from the early PIR conduction pathway to the mature apex to base activation is dependent upon upregulation of connexin40 expression in the ventricular trabeculae. The early function of right bundle branch does not depend on connexin40. Quantitative analysis of normal mouse embryonic ventricular conduction patterns will be useful for interpretation of effects of mutations affecting the function of the cardiac conduction system.

TBX5 drives Scn5a expression to regulate cardiac conduction system function

Cardiac conduction system (CCS) disease, which results in disrupted conduction and impaired cardiac rhythm, is common with significant morbidity and mortality. Current treatment options are limited, and rational efforts to develop cell-based and regenerative therapies require knowledge of the molecular networks that establish and maintain CCS function. Recent genome-wide association studies (GWAS) have identified numerous loci associated with adult human CCS function, including TBX5 and SCN5A. We hypothesized that TBX5, a critical developmental transcription factor, regulates transcriptional networks required for mature CCS function. We found that deletion of Tbx5 from the mature murine ventricular conduction system (VCS), including the AV bundle and bundle branches, resulted in severe VCS functional consequences, including loss of fast conduction, arrhythmias, and sudden death. Ventricular contractile function and the VCS fate map remained unchanged in VCS-specific Tbx5 knockouts. However, key mediators of fast conduction, including Nav1.5, which is encoded by Scn5a, and connexin 40 (Cx40), demonstrated Tbx5-dependent expression in the VCS. We identified a TBX5-responsive enhancer downstream of Scn5a sufficient to drive VCS expression in vivo, dependent on canonical T-box binding sites. Our results establish a direct molecular link between Tbx5 and Scn5a and elucidate a hierarchy between human GWAS loci that affects function of the mature VCS, establishing a paradigm for understanding the molecular pathology of CCS disease.

Lethal arrhythmias in Tbx3 -deficient mice reveal extreme dosage sensitivity of cardiac conduction system function and homeostasis

TBX3 is critical for human development: mutations in TBX3 cause congenital anomalies in patients with ulnar-mammary syndrome. Data from mice and humans suggest multiple roles for Tbx3 in development and function of the cardiac conduction system. The mechanisms underlying the functional development, maturation, and maintenance of the conduction system are not well understood. We tested the requirements for Tbx3 in these processes. We generated a unique series of Tbx3 hypomorphic and conditional mouse mutants with varying levels and locations of Tbx3 activity within the heart, and developed techniques for evaluating in vivo embryonic conduction system function. Disruption of Tbx3 function in different regions of the developing heart causes discrete phenotypes and lethal arrhythmias: sinus pauses and bradycardia indicate sinoatrial node dysfunction, whereas preexcitation and atrioventricular block reveal abnormalities in the atrioventricular junction. Surviving Tbx3 mutants are at increased risk for sudden death. Arrhythmias induced by knockdown of Tbx3 in adults reveal its requirement for conduction system homeostasis. Arrhythmias in Tbx3-deficient embryos are accompanied by disrupted expression of multiple ion channels despite preserved expression of previously described conduction system markers. These findings indicate that Tbx3 is required for the conduction system to establish and maintain its correct molecular identity and functional properties. In conclusion, Tbx3 is required for the functional development, maturation, and homeostasis of the conduction system in a highly dosage-sensitive manner. TBX3 and its regulatory targets merit investigation as candidates for human arrhythmias.

The emerging genetic landscape underlying cardiac conduction system function

Proper function of an organized Cardiac Conduction System (CCS) is vital to the survival of metazoans ranging from fly to man. The routine use of non-invasive electrocardiogram measures in the diagnosis and monitoring of cardiovascular health has established a trove of reliable CCS functional data in both normal and diseased cardiac states. Recent combination of echocardiogram (ECG) data with genome-wide association studies has identified genomic regions implicated in ECG variability which impact CCS function. In this study, we review the substantial recent progress in this area, highlighting the identification of novel loci, confirming the importance of previously implicated loci in CCS function, and exploring potential links between genes with important roles in developmental processes and variation in function of the CCS.

Developmental Origin, Growth, and Three-Dimensional Architecture of the Atrioventricular Conduction Axis of the Mouse Heart

The clinically important atrioventricular conduction axis is structurally complex and heterogeneous, and its molecular composition and developmental origin are uncertain. To assess the molecular composition and 3D architecture of the atrioventricular conduction axis in the postnatal mouse heart and to define the developmental origin of its component parts. We generated an interactive 3D model of the atrioventricular junctions in the mouse heart using the patterns of expression of Tbx3, Hcn4, Cx40, Cx43, Cx45, and Nav1.5, which are important for conduction system function. We found extensive figure-of-eight rings of nodal and transitional cells around the mitral and tricuspid junctions and in the base of the atrial septum. The rings included the compact node and nodal extensions. We then used genetic lineage labeling tools (Tbx2(+/Cre), Mef2c-AHF-Cre, Tbx18(+/Cre)), along with morphometric analyses, to assess the developmental origin of the specific components of the axis. The majority of the atrial components, including the atrioventricular rings and compact node, are derived from the embryonic atrioventricular canal. The atrioventricular bundle, including the lower cells of the atrioventricular node, in contrast, is derived from the ventricular myocardium. No contributions to the conduction system myocardium were identified from the sinus venosus, the epicardium, or the dorsal mesenchymal protrusion. The atrioventricular conduction axis comprises multiple domains with distinctive molecular signatures. The atrial part proliferates from the embryonic atrioventricular canal, along with myocytes derived from the developing atrial septum. The atrioventricular bundle and lower nodal cells are derived from ventricular myocardium.