HAND1 loss-of-function within the embryonic myocardium reveals survivable congenital cardiac defects and adult heart failure

Abstract Aims To examine the role of the basic Helix-loop-Helix (bHLH) transcription factor HAND1 in embryonic and adult myocardium. Methods and results Hand1 is expressed within the cardiomyocytes of the left ventricle (LV) and myocardial cuff between embryonic days (E) 9.5–13.5. Hand gene dosage plays an important role in ventricular morphology and the contribution of Hand1 to congenital heart defects requires further interrogation. Conditional ablation of Hand1 was carried out using either Nkx2.5 knockin Cre (Nkx2.5Cre) or α-myosin heavy chain Cre (αMhc-Cre) driver. Interrogation of transcriptome data via ingenuity pathway analysis reveals several gene regulatory pathways disrupted including translation and cardiac hypertrophy-related pathways. Embryo and adult hearts were subjected to histological, functional, and molecular analyses. Myocardial deletion of Hand1 results in morphological defects that include cardiac conduction system defects, survivable interventricular septal defects, and abnormal LV papillary muscles (PMs). Resulting Hand1 conditional mutants are born at Mendelian frequencies; but the morphological alterations acquired during cardiac development result in, the mice developing diastolic heart failure. Conclusion Collectively, these data reveal that HAND1 contributes to the morphogenic patterning and maturation of cardiomyocytes during embryogenesis and although survivable, indicates a role for Hand1 within the developing conduction system and PM development.

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Regulation of Cardiac Conduction and Arrhythmias by Ankyrin/Spectrin-Based Macromolecular Complexes

Journal of Cardiovascular Development and Disease ◽

10.3390/jcdd8050048 ◽

2021 ◽

Vol 8 (5) ◽

pp. 48

Author(s):

Drew Nassal ◽

Jane Yu ◽

Dennison Min ◽

Cemantha Lane ◽

Rebecca Shaheen ◽

...

Keyword(s):

Ion Channels ◽

Cardiac Disease ◽

Conduction System ◽

Cytoskeletal Proteins ◽

Cardiac Conduction ◽

Proper Function ◽

Cardiac Conduction System ◽

Loss Of Function ◽

Macromolecular Complexes ◽

Conduction Disturbances

The cardiac conduction system is an extended network of excitable tissue tasked with generation and propagation of electrical impulses to signal coordinated contraction of the heart. The fidelity of this system depends on the proper spatio-temporal regulation of ion channels in myocytes throughout the conduction system. Importantly, inherited or acquired defects in a wide class of ion channels has been linked to dysfunction at various stages of the conduction system resulting in life-threatening cardiac arrhythmia. There is growing appreciation of the role that adapter and cytoskeletal proteins play in organizing ion channel macromolecular complexes critical for proper function of the cardiac conduction system. In particular, members of the ankyrin and spectrin families have emerged as important nodes for normal expression and regulation of ion channels in myocytes throughout the conduction system. Human variants impacting ankyrin/spectrin function give rise to a broad constellation of cardiac arrhythmias. Furthermore, chronic neurohumoral and biomechanical stress promotes ankyrin/spectrin loss of function that likely contributes to conduction disturbances in the setting of acquired cardiac disease. Collectively, this review seeks to bring attention to the significance of these cytoskeletal players and emphasize the potential therapeutic role they represent in a myriad of cardiac disease states.

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P3826Gene therapy for cardiac conduction system dysfunction in heart failure

European Heart Journal ◽

10.1093/eurheartj/ehz745.0668 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

L Stuart ◽

I Y Oh ◽

Y Wang ◽

S Nakao ◽

T Starborg ◽

...

Keyword(s):

Heart Failure ◽

Ion Channel ◽

Transgene Expression ◽

Ex Vivo ◽

Sinus Node ◽

Conduction System ◽

Cardiac Conduction ◽

Cardiac Conduction System ◽

Gfp Expression ◽

Free Running

Abstract Background and purpose Heart failure (HF) is characterised by generalised dysfunction of the cardiac conduction system (CCS). Ion channel and structural remodelling in the CCS have been widely demonstrated in animal models of cardiovascular disease. As Purkinje fibres (PFs) are minute strands of tissue, little is known about their ultrastructure and remodelling in disease. Furthermore, given the role for microRNAs (miRs) in CCS molecular remodelling, we aimed to develop a tissue specific method for delivering therapeutic transgenes, such as miR sponges. Methods New Zealand rabbits were used for PF ultrastructural studies. HF was induced via pressure and volume overload. Free running PFs were processed for serial block face scanning electron microscopy (SBF-SEM). Manual contrast-based segmentation techniques were used on IMOD software to determine the 3D cellular ultrastructure. To target transgene expression to the CCS, adenoviral plasmids were cloned expressing a GFP reporter gene. GFP transcription was placed under control of the KCNE1 promoter, a K+ channel subunit expressed throughout the CCS, or the HCN4 promoter, a key pacemaker ion channel, to target the sinus node. The strong ubiquitous cytomegalovirus (CMV) promoter was used as a positive control. Adenovirus was produced using via transfection into the 293A cell line for viral packaging and amplification. Results Purkinje cells (PCs) formed a central core within PFs, encapsulated by an extensive collagen matrix. PCs were uninucleated and spindle shaped with an irregular membrane. Gap junctions were abundant and distributed along the lateral surface of cells, and there was a trend towards decreased expression in HF (p=0.0526, n=3 cells analysed per group). Hypertrophy and nuclear membrane breakdown were evident in HF PCs, the latter facilitating mitochondrial entry. Using the CMV-GFP adenoviral construct, abundant GFP expression was conferred in ex vivo sinus node tissue, isolated sinus node myocytes, and neonatal ventricular rat cardiomyocytes (NRCMs). The KCNE1 promoter conferred relatively high GFP expression in NRCMs, greater than that from the HCN4 promoter. In isolated sinus node myocytes, the HCN4 promoter conferred greater transgene expression than in NRCMs. In ex vivo sinus node tissue, only the CMV construct was capable of driving significant GFP expression. Notably, expression was largely confined to the sinus node, with only sparse expression detected in the surrounding atrial muscle. Conclusions SBF-SEM revealed ultrastructure of free running PFs in situ, and uncovered novel structural changes in HF that are likely to be pro-arrhythmic. Preliminary data suggest that 1.2 kb and 0.8 kb fragments of the HCN4 promoter are capable of driving sinus node specific transgene expression. Further tests are warranted to confirm the utility of these promoters to express therapeutic transgenes, such as miR sponges to competitively inhibit miR activity in vitro and in vivo. Acknowledgement/Funding The British Heart Foundation

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Nkx2.5 is essential to establish normal heart rate variability in the zebrafish embryo

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00223.2016 ◽

2017 ◽

Vol 313 (3) ◽

pp. R265-R271 ◽

Cited By ~ 4

Author(s):

Jamie K. Harrington ◽

Robert Sorabella ◽

Abigail Tercek ◽

Joseph R. Isler ◽

Kimara L. Targoff

Keyword(s):

Heart Rate ◽

Heart Rate Variability ◽

Heart Development ◽

Conduction System ◽

Cardiac Conduction ◽

Loss Of Function ◽

Normal Heart ◽

Wild Type ◽

Cardiac Health ◽

Normal Heart Rate

Heart rate variability (HRV) has become an important clinical marker of cardiovascular health and a research measure for the study of the cardiac conduction system and its autonomic controls. While the zebrafish ( Danio rerio) is an ideal vertebrate model for understanding heart development, HRV has only recently been investigated in this system. We have previously demonstrated that nkx2.5 and nkx2.7, two homologues of Nkx2–5 expressed in zebrafish cardiomyocytes, play vital roles in maintaining cardiac chamber-specific characteristics. Given observed defects in ventricular and atrial chamber identities in nkx2.5−/− embryos coupled with conduction system abnormalities in murine models of Nkx2.5 insufficiency, we postulated that reduced HRV would serve as a marker of poor cardiac health in nkx2.5 mutants and in other zebrafish models of human congenital heart disease. Using live video image acquisition, we derived beat-to-beat intervals to compare HRV in wild-type and nkx2.5−/− embryos. Our data illustrate that the nkx2.5 loss-of-function model exhibits increased heart rate and decreased HRV when compared with wild type during embryogenesis. These findings validate HRV analysis as a useful quantitative tool for assessment of cardiac health in zebrafish and underscore the importance of nkx2.5 in maintaining normal heart rate and HRV during early conduction system development.

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Conduction System Pacing for Cardiac Resynchronisation

Arrhythmia & Electrophysiology Review ◽

10.15420/aer.2020.45 ◽

2021 ◽

Vol 10 (1) ◽

pp. 51-58

Author(s):

Parikshit S Sharma ◽

Pugazhendhi Vijayaraman

Keyword(s):

Heart Failure ◽

Ejection Fraction ◽

Conduction System ◽

Cardiac Conduction ◽

Cardiac Conduction System ◽

Reduced Ejection Fraction ◽

Ventricular Dyssynchrony ◽

Permanent Pacing ◽

Patients With Heart Failure ◽

Cardiac Resynchronisation

Conduction system pacing (CSP) is a technique of pacing that involves implantation of permanent pacing leads along different sites of the cardiac conduction system and includes His bundle pacing and left bundle branch pacing. There is an emerging role for CSP to achieve cardiac resynchronisation in patients with heart failure with reduced ejection fraction and inter-ventricular dyssynchrony. In this article, the authors review these strategies for resynchronisation and the available data on the use of CSP in overcoming dyssynchrony.

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Characterization of embryonic cardiac pacemaker and atrioventricular conduction physiology in Xenopus laevis using noninvasive imaging

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00807.2003 ◽

2004 ◽

Vol 286 (6) ◽

pp. H2035-H2041 ◽

Cited By ~ 9

Author(s):

Heather L. Bartlett ◽

Thomas D. Scholz ◽

Fred S. Lamb ◽

Daniel L. Weeks

Keyword(s):

Xenopus Laevis ◽

Congenital Heart ◽

Morphological Changes ◽

Heart Defects ◽

Cardiac Pacemaker ◽

Conduction System ◽

Cardiac Conduction ◽

Model Organisms ◽

Morphological Evidence ◽

Video Images

Congenital heart defects often include altered conduction as well as morphological changes. Model organisms, like the frog Xenopus laevis, offer practical advantages for the study of congenital heart disease. X. laevis embryos are easily obtained free living, and the developing heart is readily visualized. Functional and morphological evidence for a conduction system is available for adult frog hearts, but information on the normal properties of embryonic heart contraction is lacking, especially in intact animals. With the use of fine glass microelectrodes, we were able to obtain cardiac recordings and make standard electrophysiological measurements in 1-wk-old embryos ( stage 46). In addition, a system using digital analysis of video images was adapted for measurement of the standard cardiac intervals and compared with invasive measurements. Video images were obtained of the heart in live, pharmacologically paralyzed, stage 46 X. laevis embryos. Normal values for the timing of the cardiac cycle were established. Intervals determined by video analysis ( n = 53), including the atrial and ventricular cycle lengths (473 ± 10 ms and 464 ± 19 ms, respectively) and the atrioventricular interval (169 ± 5 ms) were not statistically different from those determined by intrathoracic cardiac recordings. We also present the data obtained from embryos treated with standard medications that affect the human conduction system. We conclude that the physiology of embryonic X. laevis cardiac conduction can be noninvasively studied by using digital video imaging. Additionally, we show the response of X. laevis embryonic hearts to chronotropic agents is similar but not identical to the response of the human heart.

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The Emerging Role of Cardiac Conduction System Pacing as a Treatment for Heart Failure

Current Heart Failure Reports ◽

10.1007/s11897-020-00474-y ◽

2020 ◽

Vol 17 (5) ◽

pp. 288-298

Author(s):

Nadine Ali ◽

Mathew Shun Shin ◽

Zachary Whinnett

Keyword(s):

Heart Failure ◽

Cardiac Function ◽

Left Bundle Branch Block ◽

Conduction System ◽

Cardiac Conduction ◽

Bundle Branch Block ◽

His Bundle ◽

His Bundle Pacing ◽

Patients With Heart Failure ◽

Ventricular Activation

Abstract Purpose of Review The aim of cardiac resynchronization therapy (CRT) is to improve cardiac function by delivering more physiological cardiac activation to patients with heart failure and conduction abnormalities. Biventricular pacing (BVP) is the most commonly used method for delivering CRT; it has been shown in large randomized controlled trials to significantly improve morbidity and mortality in patients with heart failure. However, BVP delivers only modest reductions in ventricular activation time and is only beneficial in patients with prolonged QRS duration. In this review, we explore conduction system pacing as a method for delivering more effective ventricular resynchronization and to extend pacing therapy for heart failure to patients without left bundle branch block (LBBB). Recent Findings The aim of conduction system pacing is to provide physiological ventricular activation by directly stimulating the conduction system. Current modalities include His bundle and left conduction system pacing. His bundle pacing is the most established method; it has the potential to correct left bundle branch block and deliver more effective ventricular resynchronization than BVP. This translates into greater acute haemodynamic improvements and observational data suggests that His-CRT results in improvements in cardiac function and symptoms. AV-optimized His bundle pacing is being investigated in patients with heart failure and long PR interval without LBBB, to see if this improves exercise capacity. More recently, a technique for pacing the left bundle branch has been developed. Early studies show potential advantages including low and stable capture thresholds. Summary Conduction system pacing can deliver more effective ventricular resynchronization than BVP, which has the potential to deliver greater improvements in cardiac function. It may also provide the opportunity to extend pacing therapy for heart failure to patients who do not have LBBB. Further data is required from randomized trials to assess these promising pacing techniques.

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PRC1 Stabilizes Cardiac Contraction by Regulating Cardiac Sarcomere Assembly and Cardiac Conduction System Construction

International Journal of Molecular Sciences ◽

10.3390/ijms222111368 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11368

Author(s):

Xixia Peng ◽

Gang Feng ◽

Yanyong Zhang ◽

Yuhua Sun

Keyword(s):

Cardiac Development ◽

Critical Role ◽

Conduction System ◽

Cardiac Contraction ◽

Cardiac Conduction ◽

Cardiac Conduction System ◽

Loss Of Function ◽

Atrioventricular Canal ◽

Cardiac Sarcomere

Cardiac development is a complex process that is strictly controlled by various factors, including PcG protein complexes. Several studies have reported the critical role of PRC2 in cardiogenesis. However, little is known about the regulation mechanism of PRC1 in embryonic heart development. To gain more insight into the mechanistic role of PRC1 in cardiogenesis, we generated a PRC1 loss-of-function zebrafish line by using the CRISPR/Cas9 system targeting rnf2, a gene encoding the core subunit shared by all PRC1 subfamilies. Our results revealed that Rnf2 is not involved in cardiomyocyte differentiation and heart tube formation, but that it is crucial to maintaining regular cardiac contraction. Further analysis suggested that Rnf2 loss-of-function disrupted cardiac sarcomere assembly through the ectopic activation of non-cardiac sarcomere genes in the developing heart. Meanwhile, Rnf2 deficiency disrupts the construction of the atrioventricular canal and the sinoatrial node by modulating the expression of bmp4 and other atrioventricular canal marker genes, leading to an impaired cardiac conduction system. The disorganized cardiac sarcomere and defective cardiac conduction system together contribute to defective cardiac contraction. Our results emphasize the critical role of PRC1 in the cardiac development.

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