Faculty Bibliography

The generation and propagation of the cardiac impulse is the central function of the cardiac conduction system (CCS). Impulse initiation occurs in nodal tissues that have high levels of automaticity, but slow conduction properties. Rapid impulse propagation is a feature of the ventricular conduction system, which is essential for synchronized contraction of the ventricular chambers. When functioning properly, the CCS produces ~2.4 billion heartbeats during a human lifetime and orchestrates the flow of cardiac impulses, designed to maximize cardiac output. Abnormal impulse initiation or propagation can result in brady- and tachy-arrhythmias, producing an array of symptoms, including syncope, heart failure or sudden cardiac death. Underlying the functional diversity of the CCS are gene regulatory networks that direct cell fate towards a nodal or a fast conduction gene program. In this review, we will discuss our current understanding of the transcriptional networks that dictate the components of the CCS, the growth factor-dependent signaling pathways that orchestrate some of these transcriptional hierarchies and the effect of aberrant transcription factor expression on mammalian conduction disease.

Rapid impulse propagation in the heart is a defining property of pectinated atrial myocardium (PAM) and the ventricular conduction system (VCS) and is essential for maintaining normal cardiac rhythm and optimal cardiac output. Conduction defects in these tissues produce a disproportionate burden of arrhythmic disease and are major predictors of mortality in heart failure patients. Despite the clinical importance, little is known about the gene regulatory network that dictates the fast conduction phenotype. Here, we have used signal transduction and transcriptional profiling screens to identify a genetic pathway that converges on the NRG1-responsive transcription factor ETV1 as a critical regulator of fast conduction physiology for PAM and VCS cardiomyocytes. Etv1 was highly expressed in murine PAM and VCS cardiomyocytes, where it regulates expression of Nkx2-5, Gja5, and Scn5a, key cardiac genes required for rapid conduction. Mice deficient in Etv1 exhibited marked cardiac conduction defects coupled with developmental abnormalities of the VCS. Loss of Etv1 resulted in a complete disruption of the normal sodium current heterogeneity that exists between atrial, VCS, and ventricular myocytes. Lastly, a phenome-wide association study identified a link between ETV1 and bundle branch block and heart block in humans. Together, these results identify ETV1 as a critical factor in determining fast conduction physiology in the heart.

Fever is a highly conserved systemic response to infection dating back over 600 million years. Although conferring a survival benefit, fever can negatively impact the function of excitable tissues, such as the heart, producing cardiac arrhythmias. Here we show that mice lacking fibroblast growth factor homologous factor 2 (FHF2) have normal cardiac rhythm at baseline, but increasing core body temperature by as little as 3 degrees C causes coved-type ST elevations and progressive conduction failure that is fully reversible upon return to normothermia. FHF2-deficient cardiomyocytes generate action potentials upon current injection at 25 degrees C but are unexcitable at 40 degrees C. The absence of FHF2 accelerates the rate of closed-state and open-state sodium channel inactivation, which synergizes with temperature-dependent enhancement of inactivation rate to severely suppress cardiac sodium currents at elevated temperatures. Our experimental and computational results identify an essential role for FHF2 in dictating myocardial excitability and conduction that safeguards against temperature-sensitive conduction failure.

Long QT syndrome (LQTS) is characterized by ventricular arrhythmias and sudden cardiac death. Purkinje cells (PC) within the specialized cardiac conduction system have unique electrophysiological properties that we hypothesize may produce the primary sources of arrhythmia in heritable LQTS. LQTS type 3 (LQT3) transgenic mice harboring the DeltaKPQ(+/-) mutation were crossed with Contactin2-EGFP BAC transgenic mice, which express a fluorescent reporter gene within the Purkinje fiber network. Isolated ventricular myocytes (VMs) (EGFP(-)) and PCs (EGFP(+)) from wild type and DeltaKPQ mutant hearts were compared using the whole-cell patch clamp technique and microfluorimetry of calcium transients. Increased late sodium current was seen in DeltaKPQ-PCs and DeltaKPQ-VMs, with larger density in DeltaKPQ-PCs. Marked prolongation of action potential duration of DeltaKPQ-PCs was seen compared to DeltaKPQ-VMs. DeltaKPQ-PCs, but not DeltaKPQ-VMs, exhibited frequent early afterdepolarizations, which corresponded to repetitive oscillations of intracellular calcium. Abnormalities in cell repolarization were reversed with exposure to mexiletine. We present the first direct experimental evidence that PCs are uniquely sensitive to LQT3 mutations, displaying electrophysiological behavior that is highly pro-arrhythmic.

Dysfunction of the specialized cardiac conduction system (CCS) is associated with life-threatening arrhythmias. Strategies to derive CCS cells, including rare Purkinje cells (PCs), would facilitate models for mechanistic studies and drug discovery and also provide new cellular materials for regenerative therapies. A high-throughput chemical screen using CCS:lacz and Contactin2:egfp (Cntn2:egfp) reporter embryonic stem cell (ESC) lines was used to discover a small molecule, sodium nitroprusside (SN), that efficiently promotes the generation of cardiac cells that express gene profiles and generate action potentials of PC-like cells. Imaging and mechanistic studies suggest that SN promotes the generation of PCs from cardiac progenitors initially expressing cardiac myosin heavy chain and that it does so by activating cyclic AMP signaling. These findings provide a strategy to derive scalable PCs, along with insight into the ontogeny of CCS development.

The cardiac Purkinje fiber network is comprised of highly specialized cardiomyocytes responsible for the synchronous excitation and contraction of the ventricles. Computational modeling, experimental animal studies and intracardiac electrical recordings from patients with heritable and acquired forms of heart disease suggest that Purkinje cells (PC) may also serve as critical triggers of life-threatening arrhythmias. Nonetheless, owing to the difficulty in isolating and studying this rare population of cells, the precise role of PC in arrhythmogenesis and the underlying molecular mechanisms responsible for their pro-arrhythmic behavior are not fully characterized. Conceptually, a stem cell-based model system might facilitate studies of PC-dependent arrhythmia mechanisms and serve as a platform to test novel therapeutics. Here, we describe the generation of murine embryonic stem cells (ESC) harboring pan-cardiomyocyte and PC-specific reporter genes. We demonstrate that the dual reporter gene strategy may be used to identify and isolate the rare ESC-derived PC (ESC-PC) from a mixed population of cardiogenic cells. ESC-PC display transcriptional signatures and functional properties, including action potentials, intracellular calcium cycling and chronotropic behavior comparable to endogenous PC. Our results suggest that stem-cell derived PC are a feasible new platform for studies of developmental biology, disease pathogenesis and screening for novel anti-arrhythmic therapies

KEY POINTS: Na(+) current (INa ) results from the integrated function of a molecular aggregate (the voltage-gated Na(+) channel complex) that includes the beta subunit family. Mutations or rare variants in Scn1b (encoding the beta1 and beta1B subunits) have been associated with various inherited arrhythmogenic syndromes, including Brugada syndrome and sudden unexpected death in patients with epilepsy. We used Scn1b null mice to understand better the relation between Scn1b expression, and cardiac electrical function. Loss of Scn1b caused, among other effects, increased amplitude of tetrodotoxin-sensitive INa , delayed after-depolarizations, triggered beats, delayed Ca(2+) transients, frequent spontaneous calcium release events and increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) homeostasis were prevented by 100 nm tetrodotoxin. We propose that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na(+) channel alpha subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis. ABSTRACT: Na(+) current (INa ) is determined not only by the properties of the pore-forming voltage-gated Na(+) channel (VGSC) alpha subunit, but also by the integrated function of a molecular aggregate (the VGSC complex) that includes the VGSC beta subunit family. Mutations or rare variants in Scn1b (encoding the beta1 and beta1B subunits) have been associated with various inherited arrhythmogenic syndromes, including cases of Brugada syndrome and sudden unexpected death in patients with epilepsy. Here, we have used Scn1b null mouse models to understand better the relation between Scn1b expression, and cardiac electrical function. Using a combination of macropatch and scanning ion conductance microscopy we show that loss of Scn1b in juvenile null animals resulted in increased tetrodotoxin-sensitive INa but only in the cell midsection, even before full T-tubule formation; the latter occurred concurrent with increased message abundance for the neuronal Scn3a mRNA, suggesting increased abundance of tetrodotoxin-sensitive NaV 1.3 protein and yet its exclusion from the region of the intercalated disc. Ventricular myocytes from cardiac-specific adult Scn1b null animals showed increased Scn3a message, prolonged action potential repolarization, presence of delayed after-depolarizations and triggered beats, delayed Ca(2+) transients and frequent spontaneous Ca(2+) release events and at the whole heart level, increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) homeostasis were prevented by 100 nm tetrodotoxin. Our results suggest that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na(+) channel alpha subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis in ventricular myocytes.