Faculty Bibliography

SCN5A encodes the alpha subunit of the major cardiac sodium channel NaV1.5. Mutations in SCN5A are associated with conduction disease and ventricular fibrillation (VF); however, the mechanisms that link loss of sodium channel function to arrhythmic instability remain unresolved. Here, we generated a large-animal model of a human cardiac sodium channelopathy in pigs, which have cardiac structure and function similar to humans, to better define the arrhythmic substrate. We introduced a nonsense mutation originally identified in a child with Brugada syndrome into the orthologous position (E558X) in the pig SCN5A gene. SCN5AE558X/+ pigs exhibited conduction abnormalities in the absence of cardiac structural defects. Sudden cardiac death was not observed in young pigs; however, Langendorff-perfused SCN5AE558X/+ hearts had an increased propensity for pacing-induced or spontaneous VF initiated by short-coupled ventricular premature beats. Optical mapping during VF showed that activity often began as an organized focal source or broad wavefront on the right ventricular (RV) free wall. Together, the results from this study demonstrate that the SCN5AE558X/+ pig model accurately phenocopies many aspects of human cardiac sodium channelopathy, including conduction slowing and increased susceptibility to ventricular arrhythmias.

Cardiac Purkinje cells are important triggers of ventricular arrhythmias associated with heritable and acquired syndromes; however, the mechanisms responsible for this proarrhythmic behavior are incompletely understood. Here, through transcriptional profiling of genetically labeled cardiomyocytes, we identified expression of Purkinje cell protein-4 (Pcp4), a putative regulator of calmodulin and Ca2+/calmodulin-dependent kinase II (CaMKII) signaling, exclusively within the His-Purkinje network. Using Pcp4-null mice and acquired cardiomyopathy models, we determined that reduced expression of PCP4 is associated with CaMKII activation, abnormal electrophysiology, dysregulated intracellular calcium handling, and proarrhythmic behavior in isolated Purkinje cells. Pcp4-null mice also displayed profound autonomic dysregulation and arrhythmic behavior in vivo. Together, these results demonstrate that PCP4 regulates cardiac excitability through both Purkinje cell-autonomous and central mechanisms and identify this modulator of CaMKII signaling as a potential arrhythmia-susceptibility candidate.

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 approximately 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.

Genome-wide association studies (GWAS) have implicated SCN10A, which encodes a nociceptor-associated voltage-gated sodium channel subunit, as a modulator of cardiac conduction; however, this role has traditionally been ascribed to SCN5A, which is highly expressed in cardiac muscle. SCN10A is believed to affect cardiac conduction either directly through cardiomyocytes or indirectly via intracardiac neurons. In this issue of the JCI, van den Boogaard and colleagues introduce a third possibility: that the SCN10A locus acts as an enhancer of SCN5A gene expression. The authors demonstrate that SCN10A expression is negligible within human and murine hearts, and that a T-box enhancer within the SCN10A locus drives SCN5A expression within cardiomyocytes. This work reasserts SCN5A as the key determinant of cardiac conduction and highlights the importance of deciphering the functionality of coding versus noncoding regions when interpreting GWAS data.

To date, over 65 mutations in the gene encoding Cx43 (connexin43) have been linked to the autosomal-dominant disease ODDD (oculodentodigital dysplasia). A subset of these patients experience bladder incontinence which could be due to underlying neurogenic deterioration or aberrant myogenic regulation. BSMCs (bladder smooth muscle cells) from wild-type and two Cx43 mutant lines (Cx43G60S and Cx43I130T) that mimic ODDD exhibit a significant reduction in total Cx43. Dye transfer studies revealed that the G60S mutant was a potent dominant-negative inhibitor of co-expressed Cx43, a property not equally shared by the I130T mutant. BSMCs from both mutant mouse strains were defective in their ability to contract, which is indicative of phenotype changes due to harbouring the Cx43 mutants. Upon stretching, Cx43 levels were significantly elevated in controls and mutants containing BSMCs, but the non-muscle myosin heavy chain A levels were only reduced in cells from control mice. Although the Cx43G60S mutant mice showed no difference in voided urine volume or frequency, the Cx43I130T mice voided less frequently. Thus, similar to the diversity of morbidities seen in ODDD patients, genetically modified mice also display mutation-specific changes in bladder function. Furthermore, although mutant mice have compromised smooth muscle contraction and response to stretch, overriding bladder defects in Cx43I130T mice are likely to be complemented by neurogenic changes.

The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC) reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN). When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.

BACKGROUND: We previously demonstrated that the cell adhesion protein contactin2 (Cntn2) is enriched in Purkinje cells of the cardiac conduction system (CCS). The objective of this study was generation of a mouse embryonic stem cell (mESC) reporter line that allows identification of Purkinje-like cardiomyocytes in vitro. METHODS AND RESULTS: mESC were generated from transgenic mice carrying a BAC Cntn2-eGFP reporter gene and were subsequently transduced with lentivirus coding for a MHCalpha-mCherry cardiomyocyte reporter gene. Immunostaining analysis confirmed that mESC expressed markers of pluripotency (Oct3/4; Klf4) and spontaneously differentiated into cells of all three germ layers in the absence of LIF (alpha-smooth muscle actin, beta-tubulin, alpha-fetoprotein). Spontaneous or serum-free directed cardiac differentiation resulted in generation of spontaneously beating cardiomyocytes, of which single positive MHCalpha-mCherry cells comprised 57.76% +/- 3.7% and MHCalpha-mCherry/Cntn2-eGFP cells comprised 1.9% +/- 0.9% of the total population as determined by fluorescence-activated cell sorting (FACS) (n = 5 differentiation cultures). Quantitative real-time PCR analysis of FACS isolated double-positive cells verified cardiomyocyte-specific transcript expression (Mlc2v: 33-fold, P <.01; Nkx2.5: 178-fold, P <.001) compared to MHCalpha-mCherry/Cntn2-eGFP-negative noncardiomyocytes. Moreover, double-positive cells expressed significantly elevated levels of CCS-specific transcripts compared to mCherry single-positive cardiomyocytes (Cntn2: 31-fold, P <.001; Cx40: 8-fold, P <.01). Action potential recordings of double-positive cells demonstrated distinct plateau phase and elongated action potential duration (APD50 = 79.9 +/- 10.4 ms, APD90 = 170.2 +/- 17.5 ms, n = 11) compared with eGFP-negative cardiomyocytes (APD50 = 53.4 +/- 9.4 ms, APD90 = 120.6 +/- 17.3 ms, n = 15), consistent with their assignment as Purkinje-like derivatives. CONCLUSIONS: We established an mESC reporter line that allows for the identification and enrichment of ventricular CCS derivatives. This model should be useful for downstream studies of CCS development and pathology, including the role of Purkinje cells as arrhythmogenic triggers. Cntn2 may also be a useful marker of CCS-like cells derived from human embryonic stem and/or induced pluripotent stem cells.

BACKGROUND: Triggered activity in cardiac Purkinje cells (PCs) has been implicated in the genesis of inherited and acquired ventricular arrhythmias. PC arrhythmogenicity is attributed, at least in part, to dysregulation of intracellular calcium homeostasis; however, the molecular basis for differential calcium handling in PCs vs ventricular myocytes (VMs) is incompletely characterized. The goal of this study was to identify and characterize novel molecular mechanisms responsible for altered calcium handling in PCs. METHODS AND RESULTS: Compound transgenic mice harboring both a Cntn2-eGFP BAC reporter and an alpha-MHC-Cre/floxed tdTomato reporter were used to isolate cardiac PCs (eGFP(+)/tomato(+)) from VMs (eGFP(-)/tomato(+)) by FACS. Gene profiling was performed on total RNA extracted from each cell population. The transcript encoding Purkinje cell protein 4 (PCP4), a modulator of calmodulin-dependent signaling, was significantly enriched in PCs. Restricted expression of PCP4 protein in PCs was confirmed by immunohistochemistry. PCP4 knockout mice were obtained and crossed with the Cntn2-eGFP reporter mouse, and isolated VMs and PCs from wild-type (WT) and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) dynamics recorded by microfluorometry. PCP4(-/-) PCs displayed significantly slower kinetics of relaxation [tau(decay)] than that observed in wild-type PCs (328.9 +/- 24.9 ms vs 235.8 +/- 20.7 ms, P <.01). PCP4(-/-)) PCs were also more likely to develop rate dependent early aftertransients [63.6% (21/33) vs 8.3% (3/36) at 0.5 Hz, P <.0001] and delayed aftertransients [36.4% (12/33) vs 8.3% (3/36) at 0.5 Hz, P <.01). Intraperitoneal injection of caffeine and epinephrine in adult WT mice (n = 5) elicited no arrhythmias, whereas injection into PCP4(-/-)) mice elicited PVCs (4/5, P <.5) and bidirectional VT (1/5). CONCLUSIONS: PCP4 is preferentially expressed in the murine ventricular conduction system, where it modulates intracellular calcium homeostasis. Loss of function of PCP4 results in an increased propensity for the development of early and delayed aftertransients and triggered arrhythmias.

MicroRNA cluster miR-17-92 has been implicated in cardiovascular development and function, yet its precise mechanisms of action in these contexts are uncertain. This study aimed to investigate the role of miR-17-92 in morphogenesis and function of cardiac and smooth muscle tissues. To do so, a mouse model of conditional overexpression of miR-17-92 in cardiac and smooth muscle tissues was generated. Extensive cardiac functional studies identified a dose-dependent induction of dilated, hypertrophic cardiomyopathy, and arrhythmia inducibility in transgenic animals, which correlated with premature mortality (98.3+/-42.5 d, P<0.0001). Expression analyses revealed the abundance of Pten transcript, a known miR-17-92 target, to be inversely correlated with miR-17-92 expression levels and heart size. In addition, we demonstrated through 3'-UTR luciferase assays and expression analyses that Connexin43 (Cx43) is a novel direct target of miR-19a/b and its expression is suppressed in transgenic hearts. Taken together, these data demonstrate that dysregulated expression of miR-17-92 during cardiovascular morphogenesis results in a lethal cardiomyopathy, possibly in part through direct repression of Pten and Cx43. This study highlights the importance of miR-17-92 in both normal and pathological functions of the heart, and provides a model that may serve as a useful platform to test novel antiarrhythmic therapeutics.-Danielson, L. S., Park, D. S., Rotllan, N., Chamorro-Jorganes, A., Guijarro, M. V., Fernandez-Hernando, C., Fishman, G. I., Phoon, C. K. L., Hernando, E. Cardiovascular dysregulation of miR-17-92 causes a lethal hypertrophic cardiomyopathy and arrhythmogenesis.