Nikhil V. Munshi

Acetylation of HMG I(Y) by CBP Turns off IFNβ Expression by Disrupting the Enhanceosome

The transcriptional coactivators CBP and P/CAF are required for activation of transcription from the IFN beta enhanceosome. We show that CBP and P/CAF acetylate HMG I(Y), the essential architectural component required for enhanceosome assembly, at distinct lysine residues, causing distinct effects on transcription. Thus, in the context of the enhanceosome, acetylation of HMG I by CBP, but not by P/CAF, leads to enhanceosome destabilization and disassembly. We demonstrate that acetylation of HMG I(Y) by CBP is essential for turning off IFN beta gene expression. Finally, we show that the acetyltransferase activities of CBP and P/CAF modulate both the strength of the transcriptional response and the kinetics of virus-dependent activation of the IFN beta gene.

The role of HMG I(Y) in the assembly and function of the IFN-beta enhanceosome.

Transcriptional activation of the virus inducible enhancer of the human interferon‐β (IFN‐β) gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF‐κB, ATF‐2/c‐Jun, IRFs and the architectural protein of the mammalian high mobility group I(Y) [HMG I(Y)]. Here, we demonstrate that the first step in enhanceosome assembly, i.e. HMG I(Y)‐dependent recruitment of NF‐κB and ATF‐2/c‐Jun to the enhancer, is facilitated by discrete regions of HMG I and is mediated by allosteric changes induced in the DNA by HMG I(Y) and not by protein–protein interactions between HMG I(Y) and these proteins. However, we show that completion of the enhanceosome assembly process requires protein–protein interactions between HMG I(Y) and the activators. Finally, we demonstrate that once assembled, the IFN‐β enhanceosome is an unusually stable nucleoprotein structure that can activate transcription at high levels by promoting multiple rounds of reinitiation of transcription.

Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors

Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtype-specific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5+ progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols.

Cx30.2 enhancer analysis identifies Gata4 as a novel regulator of atrioventricular delay

The cardiac conduction system comprises a specialized tract of electrically coupled cardiomyocytes responsible for impulse propagation through the heart. Abnormalities in cardiac conduction are responsible for numerous forms of cardiac arrhythmias, but relatively little is known about the gene regulatory mechanisms that control the formation of the conduction system. We demonstrate that a distal enhancer for the connexin 30.2 (Cx30.2, also known as Gjd3) gene, which encodes a gap junction protein required for normal atrioventricular (AV) delay in mice, is necessary and sufficient to direct expression to the developing AV conduction system (AVCS). Moreover, we show that this enhancer requires Tbx5 and Gata4 for proper expression in the conduction system, and Gata4+/- mice have short PR intervals indicative of accelerated AV conduction. Thus, our results implicate Gata4 in conduction system function and provide a clearer understanding of the transcriptional pathways that impact normal AV delay.

Gene Regulatory Networks in Cardiac Conduction System Development

The cardiac conduction system is a specialized tract of myocardial cells responsible for maintaining normal cardiac rhythm. Given its critical role in coordinating cardiac performance, a detailed analysis of the molecular mechanisms underlying conduction system formation should inform our understanding of arrhythmia pathophysiology and affect the development of novel therapeutic strategies. Historically, the ability to distinguish cells of the conduction system from neighboring working myocytes presented a major technical challenge for performing comprehensive mechanistic studies. Early lineage tracing experiments suggested that conduction cells derive from cardiomyocyte precursors, and these claims have been substantiated by using more contemporary approaches. However, regional specialization of conduction cells adds an additional layer of complexity to this system, and it appears that different components of the conduction system utilize unique modes of developmental formation. The identification of numerous transcription factors and their downstream target genes involved in regional differentiation of the conduction system has provided insight into how lineage commitment is achieved. Furthermore, by adopting cutting-edge genetic techniques in combination with sophisticated phenotyping capabilities, investigators have made substantial progress in delineating the regulatory networks that orchestrate conduction system formation and their role in cardiac rhythm and physiology. This review describes the connectivity of these gene regulatory networks in cardiac conduction system development and discusses how they provide a foundation for understanding normal and pathological human cardiac rhythms.

Improving cardiac rhythm with a biological pacemaker

Heart muscle cells in a large animal model are reprogrammed to restore heart rate and function Electronic device therapies, including implantable pacemakers and defibrillators, have revolutionized the management of cardiovascular disease (1). For example, patients with certain slow cardiac rhythms (bradycardia) can experience exercise intolerance, easy fatigability, or circulatory collapse. Given that currently available drugs cannot safely and sustainably elevate heart rate, the only proven treatment for symptomatic bradycardia is permanent pacemaker implantation. Contemporary electronic pacemakers have an extended battery life, contain leads that minimize inflammation and scarring, and possess advanced algorithms to contend with heart rate elevations during exercise. These features allow pacemakers to improve longevity and quality of life in patients who require them. But electronic pacemakers cannot recapitulate all aspects of the endogenous sinoatrial node, the dominant pacemaker in the uninjured heart. In this regard, a recent study by Hu et al. (2) demonstrates the feasibility of a somatic cell reprogramming strategy for creating a biological pacemaker in a large animal preclinical model, raising prospects for clinical translation.

Translational medicine. Improving cardiac rhythm with a biological pacemaker.

Heart muscle cells in a large animal model are reprogrammed to restore heart rate and function Electronic device therapies, including implantable pacemakers and defibrillators, have revolutionized the management of cardiovascular disease (1). For example, patients with certain slow cardiac rhythms (bradycardia) can experience exercise intolerance, easy fatigability, or circulatory collapse. Given that currently available drugs cannot safely and sustainably elevate heart rate, the only proven treatment for symptomatic bradycardia is permanent pacemaker implantation. Contemporary electronic pacemakers have an extended battery life, contain leads that minimize inflammation and scarring, and possess advanced algorithms to contend with heart rate elevations during exercise. These features allow pacemakers to improve longevity and quality of life in patients who require them. But electronic pacemakers cannot recapitulate all aspects of the endogenous sinoatrial node, the dominant pacemaker in the uninjured heart. In this regard, a recent study by Hu et al. (2) demonstrates the feasibility of a somatic cell reprogramming strategy for creating a biological pacemaker in a large animal preclinical model, raising prospects for clinical translation.

MyoR Modulates Cardiac Conduction by Repressing Gata4

ABSTRACT The cardiac conduction system coordinates electrical activation through a series of interconnected structures, including the atrioventricular node (AVN), the central connection point that delays impulse propagation to optimize cardiac performance. Although recent studies have uncovered important molecular details of AVN formation, relatively little is known about the transcriptional mechanisms that regulate AV delay, the primary function of the mature AVN. We identify here MyoR as a novel transcription factor expressed in Cx30.2+ cells of the AVN. We show that MyoR specifically inhibits a Cx30.2 enhancer required for AVN-specific gene expression. Furthermore, we demonstrate that MyoR interacts directly with Gata4 to mediate transcriptional repression. Our studies reveal that MyoR contains two nonequivalent repression domains. While the MyoR C-terminal repression domain inhibits transcription in a context-dependent manner, the N-terminal repression domain can function in a heterologous context to convert the Hand2 activator into a repressor. In addition, we show that genetic deletion of MyoR in mice increases Cx30.2 expression by 50% and prolongs AV delay by 13%. Taken together, we conclude that MyoR modulates a Gata4-dependent regulatory circuit that establishes proper AV delay, and these findings may have wider implications for the variability of cardiac rhythm observed in the general population.

Phenotypically silent Cre recombination within the postnatal ventricular conduction system

The cardiac conduction system (CCS) is composed of specialized cardiomyocytes that initiate and maintain cardiac rhythm. Any perturbation to the normal sequence of electrical events within the heart can result in cardiac arrhythmias. To understand how cardiac rhythm is established at the molecular level, several genetically modified mouse lines expressing Cre recombinase within specific CCS compartments have been created. In general, Cre driver lines have been generated either by homologous recombination of Cre into an endogenous locus or Cre expression driven by a randomly inserted transgene. However, haploinsufficiency of the endogenous gene compromises the former approach, while position effects negatively impact the latter. To address these limitations, we generated a Cre driver line for the ventricular conduction system (VCS) that preserves endogenous gene expression by targeting the Contactin2 (Cntn2) 3’ untranslated region (3’UTR). Here we show that Cntn23’UTR-IRES-Cre-EGFP/+ mice recombine floxed alleles within the VCS and that Cre expression faithfully recapitulates the spatial distribution of Cntn2 within the heart. We further demonstrate that Cre expression initiates after birth with preservation of native Cntn2 protein. Finally, we show that Cntn23’UTR-IRES-Cre-EGFP/+ mice maintain normal cardiac mechanical and electrical function. Taken together, our results establish a novel VCS-specific Cre driver line without the adverse consequences of haploinsufficiency or position effects. We expect that our new mouse line will add to the accumulating toolkit of CCS-specific mouse reagents and aid characterization of the cell-autonomous molecular circuitry that drives VCS maintenance and function.

CRISPR (Clustered Regularly Interspaced Palindromic Repeat)/Cas9 System: A Revolutionary Disease-Modifying Technology

As clinicians, we are equipped with an ever-expanding armamentarium of drug and device therapies that have extended the lifespan of countless patients with cardiovascular disease. Nevertheless, it is likely that future therapies aimed at the root cause of disease, rather than secondary effects, will improve clinical outcomes in our patients yet further. Imagine if we could restore expression of a critical protein that is reduced as a consequence of remodeling associated with heart failure? Alternatively, what if a mutated gene in one of your patients with familial hypertrophic cardiomyopathy could actually be corrected such that they were essentially cured of their disease? Although these scenarios would have been inconceivable just a few years ago, molecular genetic tools now exist in principle to modify genomes and essentially fix a variety of diseases that affect the heart and blood vessels. To achieve this seemingly impossible objective, several research groups have swiftly repurposed a bacterial system for adaptive immunity, called the CRISPR (Clustered Regularly Interspaced Palindromic Repeat)/Cas9 System, as a revolutionary genome editing tool in mammalian tissues.1 For a genome modification technology to be translatable to human disease, it must be able to navigate the complex milieu of the human genome and precisely cut the DNA only at the …