Ventricular Conduction System Research Articles

Visualization of Left Ventricular Endocardium Activation Time during Cardiac Conduction System Pacing

Introduction. Right ventricular myocardial pacing leads to asynchronous contraction of the left ventricle (LV) and can impair its contractility over time. Stimulation of the conduction system of the heart ensures physiological activation of the ventricles.
 The aim. To visualize the pattern of propagation of the electrical front on the activation map of the LV endocardium during conduction system pacing and during right ventricular myocardial pacing.
 Materials and methods. In two patients with the conduction system pacing, who were operated at the National Amosov Institute of Cardiovascular Surgery, invasive LV activation mapping was performed on various options of cardiac pacing using the EnSite Precision TM navigation system. One patient with complete atrioventricular block had nonselective His bundle pacing. The second patient had dilated cardiomyopathy and indications for cardiac resynchronization therapy and ablation of the atrioventricular junction due to chronic tachyform of atrial fibrillation. After an unsuccessful attempt to place LV lead in coronary sinus, a lead for left bundle branch pacing was implanted. Different activation maps of the LV endocardium were built in two patients with two types of cardiac pacing: right ventricular myocardial pacing and cardiac conduction system pacing.
 Results. The total LV endocardial activation time was significantly different in the two studied patients with different pacing options. In a patient with non-selective stimulation of His bundle, the total LV endocardial activation time was 43 ms (QRS width 126 ms, the interval from the stimulus to the peak of the R wave in lead V6 78 ms). After switching to right ventricular myocardial pacing with a decrease in the pacing amplitude, the total LV endocardial activation time prolonged to 90 ms (QRS width 165 ms, the interval from the stimulus to the peak of the R wave in lead V6 117 ms). In a patient with left bundle branch pacing, total LV endocardial activation time was 51 ms (QRS width 160 ms, the interval from the stimulus to the peak of the R wave in lead V6 66 ms). With switching exclusively to right ventricular pacing the total LV endocardial activation time prolonged to 93 ms (stimulated QRS width 180 ms). At the same time, the activation pattern of the LV endocardium was significantly different, since the right ventricular lead was located closer to the apex.
 Conclusions. Conduction system pacing provides rapid physiological LV activation and potentially avoids the negative consequences of asynchronous slow ventricular activation during right ventricular pacing.

Age-related structural and functional changes of the intracardiac nervous system

BackgroundAlthough aging is known to be associated with an increased incidence of both atrial and ventricular arrhythmias, there is limited knowledge about how Schwann cells (SC) and the intracardiac nervous system (iCNS) remodel with age. Here we investigate the differences in cardiac SC, parasympathetic nerve fibers, and muscarinic acetylcholine receptor M2 (M2R) expression in young and old mice. Additionally, we examine age-related changes in cardiac responses to sympathomimetic and parasympathomimetic drugs. Methods and resultsLower SC density, lower SC proliferation and fewer parasympathetic nerve fibers were observed in cardiac and, as a control sciatic nerves from old (20–24 months) compared to young mice (2–3 months). In old mice, CSPG4 was increased in sciatic but not cardiac nerves. Expression of M2R was lower in ventricular myocardium and ventricular conduction system from old mice compared to young mice, while no significant difference was seen in M2R expression in sino-atrial or atrio-ventricular node pacemaker tissue. Heart rate was slower and PQ intervals were longer in Langendorff-perfused hearts from old mice. Ventricular tachycardia and fibrillation were more frequently observed in response to carbachol administration in hearts from old mice versus those from young mice. ConclusionsOn the background of reduced presence of SC and parasympathetic nerve fibers, and of lower M2R expression in ventricular cardiomyocytes and conduction system of aged hearts, the propensity of ventricular arrhythmogenesis upon parasympathomimetic drug application is increased. Whether this is caused by an increase in heterogeneity of iCNS structure and function remains to be elucidated.

Atrial natriuretic peptide signaling co-regulates lipid metabolism and ventricular conduction system gene expression in the embryonic heart

It has been shown that atrial natriuretic peptide (ANP) and its high affinity receptor (NPRA) are involved in the formation of ventricular conduction system (VCS). Inherited genetic variants in fatty acid oxidation (FAO) genes are known to cause conduction abnormalities in newborn children. Although the effect of ANP on energy metabolism in noncardiac cell types is well documented, the role of lipid metabolism in VCS cell differentiation via ANP/NPRA signaling is not known. In this study, histological sections and primary cultures obtained from E11.5 mouse ventricles were analyzed to determine the role of metabolic adaptations in VCS cell fate determination and maturation. Exogenous treatment of E11.5 ventricular cells with ANP revealed a significant increase in lipid droplet accumulation, FAO and higher expression of VCS marker Cx40. Using specific inhibitors, we further identified PPARγ and FAO as critical downstream regulators of ANP-mediated regulation of metabolism and VCS formation.

Personalized accelerated physiologic pacing.

Heart failure with preserved ejection fraction (HFpEF) is increasingly prevalent with a high socioeconomic burden. Pharmacological heart rate lowering was recommended to improve ventricular filling in HFpEF. This article discusses the misperceptions that have resulted in an overprescription of beta-blockers, which in all likelihood have untoward effects on patients with HFpEF, even if they have atrial fibrillation or coronary artery disease as a comorbidity. Directly contradicting the lower heart rate paradigm, faster heart rates provide haemodynamic and structural benefits, amongst which lower cardiac filling pressures and improved ventricular capacitance may be most important. Safe delivery of this therapeutic approach is feasible with atrial and ventricular conduction system pacing that aims to emulate or enhance cardiac excitation to maximize the haemodynamic benefits of accelerated pacing. This conceptual framework was first tested in the myPACE randomized controlled trial of patients with pre-existing pacemakers and preclinical or overt HFpEF. This article provides the background and path towards this treatment approach.

The transcription factor EBF1 non-cell-autonomously regulates cardiac growth and differentiation.

Reciprocal interactions between non-myocytes and cardiomyocytes regulate cardiac growth and differentiation. Here, we report that the transcription factor Ebf1 is highly expressed in non-myocytes and potently regulates heart development. Ebf1-deficient hearts display myocardial hypercellularity and reduced cardiomyocyte size, ventricular conduction system hypoplasia, and conduction system disease. Growth abnormalities in Ebf1 knockout hearts are observed as early as embryonic day 13.5. Transcriptional profiling of Ebf1-deficient embryonic cardiac non-myocytes demonstrates dysregulation of Polycomb repressive complex 2 targets, and ATAC-Seq reveals altered chromatin accessibility near many of these same genes. Gene set enrichment analysis of differentially expressed genes in cardiomyocytes isolated from E13.5 hearts of wild-type and mutant mice reveals significant enrichment of MYC targets and, consistent with this finding, we observe increased abundance of MYC in mutant hearts. EBF1-deficient non-myocytes, but not wild-type non-myocytes, are sufficient to induce excessive accumulation of MYC in co-cultured wild-type cardiomyocytes. Finally, we demonstrate that BMP signaling induces Ebf1 expression in embryonic heart cultures and controls a gene program enriched in EBF1 targets. These data reveal a previously unreported non-cell-autonomous pathway controlling cardiac growth and differentiation.

Computational Modelling Enabling In Silico Trials for Cardiac Physiologic Pacing.

Conduction system pacing (CSP) has the potential to achieve physiological-paced activation by pacing the ventricular conduction system. Before CSP is adopted in standard clinical practice, large, randomised, and multi-centre trials are required to investigate CSP safety and efficacy compared to standard biventricular pacing (BVP). Furthermore, there are unanswered questions about pacing thresholds required to achieve optimal pacing delivery while preventing device battery draining, and about which patient groups are more likely to benefit from CSP rather than BVP. In silico studies have been increasingly used to investigate mechanisms underlying changes in cardiac function in response to pathologies and treatment. In the context of CSP, they have been used to improve our understanding of conduction system capture to optimise CSP delivery and battery life, and noninvasively compare different pacing methods on different patient groups. In this review, we discuss the in silico studies published to date investigating different aspects of CSP delivery.

Abstract EC.10: Transcriptional Heterogeneity Of The Postnatal Cardiac Conduction System

The cardiac conduction system (CCS) is a network of specialized cardiomyocytes responsible for the coordinated spreading of electrical impulses throughout the heart for synchronized contractions. The CCS is largely made up of the sinoatrial node (SAN), atrioventricular node (AVN) and the fast-conducting ventricular conduction system (VCS), which is further divided into the His bundle, Bundle branches and Purkinje fibers. Although these CCS structures were anatomically discovered >100 years ago, their molecular constituents are not fully known. In this study, CCS cells, making up ~0.05% of myocytes (~500 cells/heart), were isolated from early postnatal hearts (postnatal day 1 to 4) of fluorescent reporter mice (Cntn2-Cre;Rosa26TdTomato), purified using fluorescence-activated cell sorting, and subjected to single-cell RNA-sequencing. Unbiased cluster analysis on ~7000 CCS cells demonstrated distinct transcriptomic profiles in the SAN, AVN, proximal VCS ( His bundle, bundle branches) and distal VCS ( Purkinje fibers). Conforming to previous studies, global conduction markers, such as Cntn2 and Id2, were present throughout the entire CCS, while a VCS-specific transcription factor, Irx3 , was expressed only in the VCS. A comparison between proximal and distal VCS components revealed enrichments of Igfbp7 , Lyz2 and Sfrp5 in the proximal VCS, and Scn10a , Psd3 and Wisp1 in the distal VCS. Sub-clustering analysis followed by validation with immunostaining further identified unique molecular profiles of the SAN ( Shox2 , Vsnl1 ), AVN ( Rspo3 ), His bundle ( S100a6 , Pcp4 ) and bundle branches ( Nppa , Mest ). Data integrating publicly available ATAC-seq in SAN, AV junction, and atrial and ventricular cardiomyocytes as well as Tbx3-ChIP-seq, suggested that these regional markers are possibly regulated by two CCS-specific transcription factors, Irx3 and Tbx3 . More importantly, overexpressing Irx3 and/or Tbx3 in cultured neonatal mouse atrial and ventricular myocytes recapitulated these region-specific expression patterns in a dish. Together, our study provides a comprehensive view of the molecular profiles within the postnatal CCS at a high-resolution and show Irx3 and Tbx3 as genetic regulators establishing heterogeneities of the CCS.

Loss of the Tbx5 gene in the ventricular conduction system induces spontaneous ventricular arrhythmias

The ventricular conduction system (VCS) and the working myocardium (WM) derive from the same myogenic progenitors during the embryonic development. The VCS development is regulated by numerous genes including the T-box transcription factor 5 (Tbx5). In human, Tbx5 mutations cause Holt-Oram syndrome, an autosomal dominant disease associated with heart abnormalities such as arrhythmias. In adult mice, the deletion of the Tbx5 gene provoked a slowing of ventricular conduction due to a loss of the gap junction Connexin 40 and dysfunction of cardiac channels. However, it is still unknown how Tbx5 deletion affects the development and the function of the VCS. The aim is to study the consequences of the Tbx5 deletion in embryonic progenitors of the ventricular conduction system on cardiac function in adult mice. Conditional deletion of Tbx5 alleles in mice was induced by expressing the Cre recombinase under the control of the Connexin 40 (Cx40) gene. We studied two groups of mutants: Tbx5 deletion at embryonic days E13-14 targeting both the ventricular conduction system and the working myocardium (“VCS + WM” group; n = 40) and mice deleted at E18-P2 targeting only the ventricular conduction system (“VCS” group; n = 17). Surface ECGs were achieved monthly until 3 months of age, when immunostaining was performed on the left VCS. Immunostaining showed a reduced expression of the two major connexines in the ventricle (Cx40 and Cx43) and a reduced number of conductive cells in both mutants, suggesting a defective maturation of the VCS (Fig. 1). Furthermore, the loss of Cx43 was correlated with that of Tbx5, reflecting a direct transcriptional effect. Surface ECG revealed a significant increase of the ventricular activation duration in both mutants (P < 0.01). Moreover, premature ventricular contractions (PVCs) and a left QRS axis deviation were observed in both mutants. Finally, these PVCs disappeared with age in both mutants under resting conditions, and are unmasked when the heart is challenged with a β-1 adrenergic agonist. Our results provide new insights into Tbx5 targets in the VCS. Tbx5 deletion impacts the maturation and the anatomy of the VCS as proved by the deviation of the ventricular electrical axis, which even occurs when the VCS is already formed. Tbx5 appears to have a direct impact on the Gja1 gene, encoding the gap junction Cx43, which is reduced at the junction between the VCS and the WM in both mutants and may be responsible for PVCs.

Nav1-8, a new marker of Purkinje fibers, provides insight into defective conduction system maturation in a mouse model of congenital cardiomyopathy

The transcription factor Nkx2-5 is known to be a key regulator of cardiac development and NKX2-5 mutations have been found in numerous cardiac diseases associated with conduction defects. This factor is required for the differentiation of Purkinje fibers (PF), which have been implicated in triggering ventricular arrhythmias. A better understanding of the development of the Purkinje system in normal and Nkx2-5 mutant hearts could provide insight into their involvement in conduction defects in cardiac diseases. However, analysis of the Purkinje network development is hampered by the lack of specific lineage tracers. Recently, the voltage-gated sodium channel Nav1-8 short transcript has been shown to be expressed in the ventricular conduction system (VCS). This study aims to characterize the expression domain of Nav1-8 in the murine VCS to determine whether it is a suitable tool for tracing conductive cells. This would also help to identify defects in conductive recruitment in congenital disease. Nav1-8-Flp mice were crossed with a GFP reporter line for genetic tracing analyses in WT and Nkx2-5 heterozygous mice. The specificity and efficiency of Nav1-8 labelling was measured from P1 to P21 based on the number of GFP-positive (Nav1-8 derivatives), Cntn2-positive (VCS) and double positive cells (Nav1-8 derivatives in the VCS). GFP reporter expression starts at birth and is restricted to the PF at the distal part of the VCS. The specificity of Nav1-8 as a marker of PF increased over time between P1 and P21, suggesting that Nav1.8 is expressed in PF progenitor cells prior to their recruitment to the conduction system. However, its efficiency is limited to 20% of PF being marked at a mature stage of development (P21). In Nkx2-5 mutant hearts, GFP labelling within the left ventricle delimits the PF scaffold and is almost absent in the complex ramifications of the PF network. This anatomical distribution is consistent with the hypoplastic Purkinje system of these mice. However, unlike in control hearts, additional GFP+ cells are found in the bundle branches at the proximal part of the VCS in Nkx2-5 mutants. Our study shows that the sodium channel Nav1-8 represents a unique and highly specific lineage tracer of the peripheral PF in mature hearts. In addition, we show an enlargement of the Nav1-8 expression domain to the bundle branches in Nkx2-5 mutants. This indicates that Nkx2-5 may influence cell fate choice within the VCS.

Nkx2-5 Loss of Function in the His-Purkinje System Hampers Its Maturation and Leads to Mechanical Dysfunction.

The ventricular conduction or His-Purkinje system (VCS) mediates the rapid propagation and precise delivery of electrical activity essential for the synchronization of heartbeats. Mutations in the transcription factor Nkx2-5 have been implicated in a high prevalence of developing ventricular conduction defects or arrhythmias with age. Nkx2-5 heterozygous mutant mice reproduce human phenotypes associated with a hypoplastic His-Purkinje system resulting from defective patterning of the Purkinje fiber network during development. Here, we investigated the role of Nkx2-5 in the mature VCS and the consequences of its loss on cardiac function. Neonatal deletion of Nkx2-5 in the VCS using a Cx40-CreERT2 mouse line provoked apical hypoplasia and maturation defects of the Purkinje fiber network. Genetic tracing analysis demonstrated that neonatal Cx40-positive cells fail to maintain a conductive phenotype after Nkx2-5 deletion. Moreover, we observed a progressive loss of expression of fast-conduction markers in persistent Purkinje fibers. Consequently, Nkx2-5-deleted mice developed conduction defects with progressively reduced QRS amplitude and RSR' complex associated with higher duration. Cardiac function recorded by MRI revealed a reduction in the ejection fraction in the absence of morphological changes. With age, these mice develop a ventricular diastolic dysfunction associated with dyssynchrony and wall-motion abnormalities without indication of fibrosis. These results highlight the requirement of postnatal expression of Nkx2-5 in the maturation and maintenance of a functional Purkinje fiber network to preserve contraction synchrony and cardiac function.

Purkinje Cardiomyocytes of the Adult Ventricular Conduction System Are Highly Diploid but Not Uniquely Regenerative

Adult hearts are characterized by inefficient regeneration after injury, thus, the features that support or prevent cardiomyocyte (CM) proliferation are important to clarify. Diploid CMs are a candidate cell type that may have unique proliferative and regenerative competence, but no molecular markers are yet known that selectively identify all or subpopulations of diploid CMs. Here, using the conduction system expression marker Cntn2-GFP and the conduction system lineage marker Etv1CreERT2, we demonstrate that Purkinje CMs that comprise the adult ventricular conduction system are disproportionately diploid (33%, vs. 4% of bulk ventricular CMs). These, however, represent only a small proportion (3%) of the total diploid CM population. Using EdU incorporation during the first postnatal week, we demonstrate that bulk diploid CMs found in the later heart enter and complete the cell cycle during the neonatal period. In contrast, a significant fraction of conduction CMs persist as diploid cells from fetal life and avoid neonatal cell cycle activity. Despite their high degree of diploidy, the Purkinje lineage had no enhanced competence to support regeneration after adult heart infarction.

Ventricular Parasystole in Cardiomyopathy Patients: A Link Between His-Purkinje System Damage and Ventricular Fibrillation

BackgroundThe clinical relevance and prognostic implications of ventricular parasystole are unknown. ObjectivesThis study sought to assess the prevalence of ventricular parasystole in patients with implantable cardioverter-defibrillators (ICDs) and ventricular parasystole’s association with ventricular arrhythmias and conduction system abnormalities. MethodsThis study retrospectively evaluated patients who underwent ICD interrogation at a single center between June 1, 2019, and August 31, 2020, and reviewed all available ICD and electrocardiogram data. This study identified patients with ventricular parasystole and compared the prevalence of ventricular fibrillation (VF), ventricular tachycardia (VT), and new conduction system abnormalities in those with ≥5 years of intrinsic QRS-complex electrocardiograms to those without parasystole. ResultsThis study included 374 patients (age 57 ± 21 years, 72% male, 45% nonischemic, 32% ischemic cardiomyopathy), of which, 104 (28%) had VT only, 39 (10%) VF only, and 10 (3%) both VT/VF. Ventricular parasystole was identified in 33 patients (9%); parasystolic foci were predominantly from the His-Purkinje system. Compared with those without parasystole, patients with parasystole had a significantly higher rate of VF (36% vs 11%; P < 0.01), but not VT (42% vs 29%; P = 0.12). Patients with parasystole, compared with those without parasystole, had a higher prevalence of new conduction abnormalities, particularly progressive intraventricular conduction delay (11 of 18 [61%] vs 12 of 83 [14%]; P < 0.01) and new right bundle branch block (4 of 18 [22%] vs 1 of 83 [1%]; P < 0.01). ConclusionsVentricular parasystole was strongly associated with new conduction system abnormalities and VF in patients who have cardiomyopathy with ICDs, suggesting a potential link between VF and His-Purkinje damage in this patient population.

Pacing interventions in non-responders to cardiac resynchronization therapy.

Non-responders to Cardiac Resynchronization Therapy (CRT) represent a high-risk, and difficult to treat population of heart failure patients. Studies have shown that these patients have a lower quality of life and reduced life expectancy compared to those who respond to CRT. Whilst the first-line treatment for dyssynchronous heart failure is "conventional" biventricular epicardial CRT, a range of novel pacing interventions have emerged as potential alternatives. This has raised the question whether these new treatments may be useful as a second-line pacing intervention for treating non-responders, or indeed, whether some patients may benefit from these as a first-line option. In this review, we will examine the current evidence for four pacing interventions in the context of treatment of conventional CRT non-responders: CRT optimization; multisite left ventricular pacing; left ventricular endocardial pacing and conduction system pacing.

Abstract 12222: The Significance of Concealed Fasciculo-Ventricular Connections in His Bundle Pacing

Introduction: It is almost 100 years ago since Mahaim described so-called paraspecific connections between the ventricular conduction axis and the crest of the muscular ventricular septum, believing such pathways to be ubiquitous. Hypothesis: These pathways have yet to be considered as potential pathways for septal activation during His bundle pacing Methods: So as to explore the hypothesis that specialized septal pathways provide the substrate for septal activation during His bundle pacing, we compared the findings from 22 serially sectioned histological datasets and 96 different individuals undergoing pacing of the His bundle. Results: We found extensive histologically specialized pathways connecting the branching bundle with the crest of the muscular ventricular septum in almost four-fifths of the histological datasets. A delta wave was observed in nine-tenths of the individuals undergoing His bundle pacing. Capture of the delta wave varied according to voltage, a finding which correlated with the distance of the septal pathways from the site of penetration of the ventricular conduction axis. Ventricular activation times remained normal in the presence of the delta wave. A significant decrease in ventricular activation times was observed in individuals with disease of the ventricular conduction system. Conclusions: Analysis of the effect of delta wave on ventricular activation times permits us to conclude that His bundle pacing captures the fasciculo-ventricular connections initially described by Mahaim as being paraspecific and ubiquitous.

Abstract P3092: Transcriptomic Landscape Of The Postnatal Atrioventricular Conduction System

The atrioventricular conduction system (AVCS) is a network of specialized cardiomyocytes responsible for coordinated spreading of electrical impulses throughout the heart to allow synchronized contractions. Specifically, it is composed of the AV node (AVN), which delays the electrical signal, and the ventricular conduction system (VCS) (i.e. His bundle, bundle branches and Purkinje fibers), which rapidly propagates the signal throughout the ventricles. Although these AVCS structures were discovered &gt;100 years ago, its molecular constituents are not fully known. In this study, AVCS cells, making up ~0.05% of myocytes (~500 cells/heart), were isolated from early postnatal hearts (Postnatal day 1 to 4) of fluorescent reporter mice (Cntn2- Cre;Rosa26TdTomato), purified using fluorescence-activated cell sorting, and were subjected to single-cell RNA-sequencing. Unbiased cluster analysis on ~7000 AVCS cells demonstrated distinct transcriptomic profiles in the AVN, proximal-VCS (i.e. His bundle and bundle branches) and distal-VCS (i.e. Purkinje fibers). Conforming to previous studies, global conduction markers, such as Etv1 and Kcne1, were present throughout the entire AVCS, whereas a VCS-specific TF, Irx3 , was expressed only in the VCS. Additionally, Tbx3, Pcp4, Nppa and Lyz2 were enriched in the proximal, not distal, VCS. Sub-clustering analysis of AVN and proximal-VCS further identified unique molecular profiles, such as Shox2 (in compact AVN) and Rspo3 (likely in lower nodal region connecting AVN and proximal-VCS). Moreover, we uncovered a potential regulatory relationship between Irx3 and Tbx3 in the AVCS, mediating their downstream targets, Gja5 and Gja1 . In Irx3 and Tbx3 -positive proximal-VCS, Gja5 and Gja1 expressions were lower compared to only Irx3 -positive distal-VCS, whereas in only Tbx3 -positive AVN, both Gja5 and Gja1 expressions were undetected. Importantly, this regional difference was recapitulated in cultured neonatal mouse atrial and ventricular myocytes overexpressing Irx3 and/or Tbx3 . Overall, our study provides a comprehensive view of the cellular and molecular heterogeneity within the postnatal AVCS at a high-resolution and reveals novel gene regulatory mechanisms that may govern the development of the AVCS.

Abstract EC101: Premature Stop Mutations In Mybphl Share The Common Pathomechanism Of Preventing Protein Incorporation Into The Cardiac Myofilament

Cardiomyopathy and arrhythmia are diseases with significant genetic linkage. Cardiac myosin binding protein-C (cMyBP-C) is a myofilament associated protein with a C-terminal composed of repeating immunoglobulin (Ig) and fibronectin III (FnIII) domains that bind to the myosin thick filaments and titin. Truncating mutations in the C-terminal of MyBP-C result in improper myofilament incorporation and degradation of the truncated protein and is a major cause of hypertrophic cardiomyopathy. Myosin-binding protein-H like ( MyBP-HL ) is a protein that was recently identified that is structurally related to MyBP-C. Both cMyBP-C and MyBP-HL contain repeated C-terminal Ig and FnIII domains that bind to myosin. MyBP-HL is different in that it is primarily expressed in the atria where it interacts with the myofilament. MyBP-HL premature stop mutations are associated with improper myofilament incorporation, and has been linked to ventricular conduction system abnormalities, atria enlargement, and dilated cardiomyopathy (DCM) in humans. The gnomAD database reports 9 stop-gain mutations for MYBPHL in humans including: Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255 and Tyr307. Prior investigation modeled the MyBP-HL Arg255X mutation and showed no MyBP-HL localization into the myofilament. A null mouse model showed that loss of MyBP-HL resulted in arrhythmia and cardiomyopathy, similar to that observed in humans. We hypothesize that all MyBP-HL truncation mutations will share a common mechanism by failing to produce MyBP-HL that integrates into the sarcomere. We test this hypothesis by transfecting neonatal rat cardiomyocytes with MyBP-HL constructs containing these mutations. We tested Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250 and Arg255 MyBP-HL mutations. We measured co-localization events between MyBP-HL mutants and MyBP-C in atrial and ventricular overexpressed cells and observed that stop gained mutations disrupt normal localization as we expected. Moreover, we tested wild-type MyBP-HL overexpression, and both showed to be able to incorporate in a similar pattern to cMyBP-C C-zone doublets. These truncating mutations in MYBP-HL may increase risk for developing arrhythmias, atria enlargement and DCM.

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.

Application of Three-Dimensional Culture Method in the Cardiac Conduction System Research.

Congenital heart defects (CHD) are the most common type of birth defects. Several human case studies and genetically altered animal models have identified abnormalities in the development of ventricular conduction system (VCS) in the heart. While cell-based therapies hold promise for treating CHDs, translational efforts are limited by the lack of suitable in vitro models for feasibility and safety studies. A better understanding of cell differentiation pathways can lead to development of cell-based therapies for individuals living with CHD/VCS disorders. Here, we describe a new and reproducible 3-D cell culture method for studying cardiac cell lineage differentiation in vitro. We used primary ventricular cells isolated from embryonic day 11.5 (E11.5) mouse embryos, which can differentiate into multiple cardiac cell types including VCS cells. We compared 3-D cultures with three types of basement membrane extracts (BME) for their abilities to support E11.5 ventricular cell differentiation. In addition, the effects of atrial natriuretic peptide (ANP) and an inhibitor for its high affinity receptor were tested on cell differentiation in 3-D cultures. Following the cell culture, protocols for immunofluorescence imaging, cell extraction and protein isolation from the 3-D culture matrix and in-cell western methods are described. Further, these approaches can be used to study the effects of various ligands and genetic interventions on VCS cell development. We propose that these methodologies may also be extended for differentiation studies using other sources of stem cells such as induced pluripotent stem cells.

Pathophysiological study of the loss of function of the Tbx5 gene in the ventricular conduction system

Patients with Holt-Oram syndrome suffer from cardiac conduction disorders and are often associated with mutations in the gene encoding the cardiac transcription factor Tbx5. In mice, it has been shown that Tbx5 is essential for maintaining the structure and function of the atrioventricular node and His bundle. Conditional deletion of Tbx5 in the adult mouse heart is associated with conduction defects, slowing of the ventricular conduction system (VCS), QRS prolongation, bundle branch blocks and atrial fibrillation. However, there were no data on the morphological and functional consequences of a cardiac deletion of Tbx5 during embryonic development. The aim is to analyze the morphological and functional consequences in the adult heart of the deletion of the Tbx5 gene in the progenitor cells of the cardiac VCS at the fetal stage. Tbx5 gene was inactivated in VCS progenitors using Cx40-CreERT2:Tbx5 floxed/floxed transgenic mice. Cre-mediated recombination was induced by oral gavage of pregnant females at E13-14 during trabecular compaction. Surface ECGs were achieved monthly until 3 months of age, when immunofluorescence staining was performed on mouse hearts. In 3-week-old Tbx5Fl/Fl mice ( n = 16), ECG analysis showed a decrease in P- ( P < 0.01) and T-wave amplitudes ( P < 0.01) and an increased QRS interval duration ( P < 0.05) compared to controls ( n = 12). At 2 months, the mice did not show any worsening of the phenotype. In addition, severe functional defects in Tbx5Fl/Fl mice, including atrial susceptibility, extrasystoles, and arrhythmias were observed by 3 weeks of age. Moreover, left bundle branches block and slowing of ventricular depolarization were also detected in Tbx5Fl/Fl mice. A β-adrenergic stimulation revealed a large tendency for atrial fibrillation and extrasystoles in 88% of Tbx5Fl/Fl mice against 50% of Tbx5Fl/+ mice and 43% of control mice. In 2-month-old Tbx5Fl/Fl mice, Purkinje Fiber cells composing the fast-VCS are still present, however, they lose the expression of Contactin-2 and Connexin-40. Furthermore, we observed a drastic loss of Connexin-43 in Tbx5-deleted contractile cardiomyocytes that are close to the VCS, which may have resulted in decreased signal propagation. In this study, we demonstrated that Tbx5 deletion in VCS progenitors prevents their maturation and hence the expression of rapid conduction markers causing disturbances in the propagation of ventricular electrical activity.

P172 AN ACCESSORY PATHWAY WITHOUT REENTRANT TACHYCARDIA

Abstract A 65–year–old patient, hypertensive, dyslipidemic, strong smoker. He has heart disease with hypokinetic evolution EF 47%, mild MI. Negative Dipyridamole–stress echocardiography dated January 2008. Recently atrial fibrillation (AF) is found and anticoagulant therapy was started. Dyspnea and weight loss occurred in past two months. Detected keratinizing squamous pulmonary Ca with negative markers. This injury was judged to be inoperable, and the patient get indications for chemotherapy and radiotherapy. The patient also starts complaining of dyspnea and chest pain on exertion: Not serological modification, not ischemic ECG. Echocardiogram LVEF 47–50% highlights diffuse hypokinesia, regular valves. Ergometric test was not evaluable for ischemia. Coronary angiography shows stenosis of proximal and middle anterior interventricular coronary and non–critical lesions of circumflex and right coronary. Ecodobutamine test was positive for ischemia and arrhythmias (AF and wide QRS tachycardias in recovery phase), but negative for angor. The patient was treated with CT and IVA I – II. angioplasty Monitoring shows AF with high penetrance, wide QRS tachycardia runs and very short TVNS. ECG 1: During atrial tachycardia, show broad QRS conduction to BBSX alternating with narrow QRS with the same RR interval. ECG 2. show fusion between the wavefront conducted with narrow QRS and that with wide QRS. These are supraventricular arrhythmias that run along the atrioventricular nodal pathway (NAV) in the narrow QRS tracts and take an accessory pathway in the wide QRS run. The Mahaim fibers have decremental conductivities like the AV node, therefore high ventricular frequencies does not occur AF appeared during ecodobutamine, but some beats were conducted by an anomalous path in a discontinuous way); they are generally directed from the atrium or the NAV, towards another part of the right ventricular conduction system, with atrium wave or NAV – RB, therefore the early activation of the RB generates a widened QRS, with a LBB morphology. ECG 3 Re–entry on Mahaim pathway, ventricular activation widened through Mahaim fiber, which generate LBB morphology with retrograde atrial depolarization. In this case there is no reentry, but we have an atrial tachycardia conducted alternately on an accessory pathway and across the NAV. Given the patient‘s clinical problems and the total absence of symptoms, the patient remains asymptomatic during therapy with amiodarone and beta–blocker.