Guidelines for assessment of cardiac electrophysiology and arrhythmias in small animals.

Cardiac arrhythmias are a major cause of morbidity and mortality worldwide. Although recent advances in cell-based models, including human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), are contributing to our understanding of electrophysiology and arrhythmia mechanisms, preclinical animal studies of cardiovascular disease remain a mainstay. Over the past several decades, animal models of cardiovascular disease have advanced our understanding of pathological remodeling, arrhythmia mechanisms, and drug effects and have led to major improvements in pacing and defibrillation therapies. There exist a variety of methodological approaches for the assessment of cardiac electrophysiology and a plethora of parameters may be assessed with each approach. This guidelines article will provide an overview of the strengths and limitations of several common techniques used to assess electrophysiology and arrhythmia mechanisms at the whole animal, whole heart, and tissue level with a focus on small animal models. We also define key electrophysiological parameters that should be assessed, along with their physiological underpinnings, and the best methods with which to assess these parameters.

The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells.

Notch1 is required to generate the earliest embryonic hematopoietic stem cells (HSCs); however since Notch-deficient embryos die early in gestation, additional functions for Notch in embryonic HSC biology have not been described. We used two complementary genetic models to address this important biological question. Unlike Notch1-deficient mice, mice lacking the conserved Notch1 transcriptional activation domain (TAD) show attenuated Notch1 function in vivo and survive until late gestation, succumbing to multiple cardiac abnormalities. Notch1 TAD-deficient HSCs emerge and successfully migrate to the fetal liver but are decreased in frequency by embryonic day 14.5. In addition, TAD-deficient fetal liver HSCs fail to compete with wild-type HSCs in bone marrow transplant experiments. This phenotype is independently recapitulated by conditional knockout of Rbpj, a core Notch pathway component. In vitro analysis of Notch1 TAD-deficient cells shows that the Notch1 TAD is important to properly assemble the Notch1/Rbpj/Maml trimolecular transcription complex. Together, these studies reveal an essential role for the Notch1 TAD in fetal development and identify important cell-autonomous functions for Notch1 signaling in fetal HSC homeostasis.

Transient Notch Activation Induces Long-Term Gene Expression Changes Leading to Sick Sinus Syndrome in Mice.

RATIONALE: Notch signaling programs cardiac conduction during development, and in the adult ventricle, injury-induced Notch reactivation initiates global transcriptional and epigenetic changes. OBJECTIVE: To determine whether Notch reactivation may stably alter atrial ion channel gene expression and arrhythmia inducibility. METHODS AND RESULTS: To model an injury response and determine the effects of Notch signaling on atrial electrophysiology, we transiently activate Notch signaling within adult myocardium using a doxycycline-inducible genetic system (inducible Notch intracellular domain [iNICD]). Significant heart rate slowing and frequent sinus pauses are observed in iNICD mice when compared with controls. iNICD mice have structurally normal atria and preserved sinus node architecture, but expression of key transcriptional regulators of sinus node and atrial conduction, including Nkx2-5 (NK2 homeobox 5), Tbx3, and Tbx5 are dysregulated. To determine whether the induced electrical changes are stable, we transiently activated Notch followed by a prolonged washout period and observed that, in addition to decreased heart rate, atrial conduction velocity is persistently slower than control. Consistent with conduction slowing, genes encoding molecular determinants of atrial conduction velocity, including Scn5a (Nav1.5) and Gja5 (connexin 40), are persistently downregulated long after a transient Notch pulse. Consistent with the reduction in Scn5a transcript, Notch induces global changes in the atrial action potential, including a reduced dVm/dtmax. In addition, programmed electrical stimulation near the murine pulmonary vein demonstrates increased susceptibility to atrial arrhythmias in mice where Notch has been transiently activated. Taken together, these results suggest that transient Notch activation persistently alters ion channel gene expression and atrial electrophysiology and predisposes to an arrhythmogenic substrate. CONCLUSIONS: Our data provide evidence that Notch signaling regulates transcription factor and ion channel gene expression within adult atrial myocardium. Notch reactivation induces electrical changes, resulting in sinus bradycardia, sinus pauses, and a susceptibility to atrial arrhythmias, which contribute to a phenotype resembling sick sinus syndrome.

Transcriptional and Epigenetic Regulation of Cardiac Electrophysiology.

Spatiotemporal gene expression during cardiac development is a highly regulated process. Activation of key signaling pathways involved in electrophysiological programming, such as Notch and Wnt signaling, occurs in early cardiovascular development and these pathways are reactivated during pathologic remodeling. Direct targets of these signaling pathways have also been associated with inherited arrhythmias such as Brugada syndrome and arrhythmogenic cardiomyopathy. In addition, evidence is emerging from animal models that reactivation of Notch and Wnt signaling during cardiac pathology may predispose to acquired arrhythmias, underscoring the importance of elucidating the transcriptional and epigenetic effects on cardiac gene regulation. Here, we highlight specific examples where gene expression dictates electrophysiological properties in both normal and diseased hearts.

Differential Wnt-mediated programming and arrhythmogenesis in right versus left ventricles.

Several inherited arrhythmias, including Brugada syndrome and arrhythmogenic cardiomyopathy, primarily affect the right ventricle and can lead to sudden cardiac death. Among many differences, right and left ventricular cardiomyocytes derive from distinct progenitors, prompting us to investigate how embryonic programming may contribute to chamber-specific conduction and arrhythmia susceptibility. Here, we show that developmental perturbation of Wnt signaling leads to chamber-specific transcriptional regulation of genes important in cardiac conduction that persists into adulthood. Transcriptional profiling of right versus left ventricles in mice deficient in Wnt transcriptional activity reveals global chamber differences, including genes regulating cardiac electrophysiology such as Gja1 and Scn5a. In addition, the transcriptional repressor Hey2, a gene associated with Brugada syndrome, is a direct target of Wnt signaling in the right ventricle only. These transcriptional changes lead to perturbed right ventricular cardiac conduction and cellular excitability. Ex vivo and in vivo stimulation of the right ventricle is sufficient to induce ventricular tachycardia in Wnt transcriptionally inactive hearts, while left ventricular stimulation has no effect. These data show that embryonic perturbation of Wnt signaling in cardiomyocytes leads to right ventricular arrhythmia susceptibility in the adult heart through chamber-specific regulation of genes regulating cellular electrophysiology.

Notch-Mediated Epigenetic Regulation of Voltage-Gated Potassium Currents.

RATIONALE: Ventricular arrhythmias often arise from the Purkinje-myocyte junction and are a leading cause of sudden cardiac death. Notch activation reprograms cardiac myocytes to an induced Purkinje-like state characterized by prolonged action potential duration and expression of Purkinje-enriched genes. OBJECTIVE: To understand the mechanism by which canonical Notch signaling causes action potential prolongation. METHODS AND RESULTS: We find that endogenous Purkinje cells have reduced peak K+ current, Ito, and IK,slow when compared with ventricular myocytes. Consistent with partial reprogramming toward a Purkinje-like phenotype, Notch activation decreases peak outward K+ current density, as well as the outward K+ current components Ito,f and IK,slow. Gene expression studies in Notch-activated ventricles demonstrate upregulation of Purkinje-enriched genes Contactin-2 and Scn5a and downregulation of K+ channel subunit genes that contribute to Ito,f and IK,slow. In contrast, inactivation of Notch signaling results in increased cell size commensurate with increased K+ current amplitudes and mimics physiological hypertrophy. Notch-induced changes in K+ current density are regulated at least in part via transcriptional changes. Chromatin immunoprecipitation demonstrates dynamic RBP-J (recombination signal binding protein for immunoglobulin kappa J region) binding and loss of active histone marks on K+ channel subunit promoters with Notch activation, and similar transcriptional and epigenetic changes occur in a heart failure model. Interestingly, there is a differential response in Notch target gene expression and cellular electrophysiology in left versus right ventricular cardiac myocytes. CONCLUSIONS: In summary, these findings demonstrate a novel mechanism for regulation of voltage-gated potassium currents in the setting of cardiac pathology and may provide a novel target for arrhythmia drug design.

Canonical wnt signaling regulates atrioventricular junction programming and electrophysiological properties.

RATIONALE: Proper patterning of the atrioventricular canal (AVC) is essential for delay of electrical impulses between atria and ventricles, and defects in AVC maturation can result in congenital heart disease. OBJECTIVE: To determine the role of canonical Wnt signaling in the myocardium during AVC development. METHODS AND RESULTS: We used a novel allele of ß-catenin that preserves ß-catenin's cell adhesive functions but disrupts canonical Wnt signaling, allowing us to probe the effects of Wnt loss of function independently. We show that the loss of canonical Wnt signaling in the myocardium results in tricuspid atresia with hypoplastic right ventricle associated with the loss of AVC myocardium. In contrast, ectopic activation of Wnt signaling was sufficient to induce formation of ectopic AV junction-like tissue as assessed by morphology, gene expression, and electrophysiological criteria. Aberrant AVC development can lead to ventricular pre-excitation, a characteristic feature of Wolff-Parkinson-White syndrome. We demonstrate that postnatal activation of Notch signaling downregulates canonical Wnt targets within the AV junction. Stabilization of ß-catenin protein levels can rescue Notch-mediated ventricular pre-excitation and dysregulated ion channel gene expression. CONCLUSIONS: Our data demonstrate that myocardial canonical Wnt signaling is an important regulator of AVC maturation and electric programming upstream of Tbx3. Our data further suggest that ventricular pre-excitation may require both morphological patterning defects, as well as myocardial lineage reprogramming, to allow robust conduction across accessory pathway tissue.

Misinterpretation of the mouse ECG: 'musing the waves of Mus musculus'.

The ECG is a primary diagnostic tool in patients suffering from heart disease, underscoring the importance of understanding factors contributing to normal and abnormal electrical patterns. Over the past few decades, transgenic mouse models have been increasingly used to study pathophysiological mechanisms of human heart diseases. In order to allow extrapolation of insights gained from murine models to the human condition, knowledge of the similarities and differences between the mouse and human ECG is of crucial importance. In this review, we briefly discuss the physiological mechanisms underlying differences between the baseline ECG of humans and mice, and provide a framework for understanding how these inherent differences are relevant to the interpretation of the mouse ECG during pathology and to the translation of the results from the mouse to man.

Engineered tissue geometry and Plakophilin-2 regulate electrophysiology of human iPSC-derived cardiomyocytes.

Engineered heart tissues have been created to study cardiac biology and disease in a setting that more closely mimics in vivo heart muscle than 2D monolayer culture. Previously published studies suggest that geometrically anisotropic micro-environments are crucial for inducing "in vivo like" physiology from immature cardiomyocytes. We hypothesized that the degree of cardiomyocyte alignment and prestress within engineered tissues is regulated by tissue geometry and, subsequently, drives electrophysiological development. Thus, we studied the effects of tissue geometry on electrophysiology of micro-heart muscle arrays (µHM) engineered from human induced pluripotent stem cells (iPSCs). Elongated tissue geometries elicited cardiomyocyte shape and electrophysiology changes led to adaptations that yielded increased calcium intake during each contraction cycle. Strikingly, pharmacologic studies revealed that a threshold of prestress and/or cellular alignment is required for sodium channel function, whereas L-type calcium and rapidly rectifying potassium channels were largely insensitive to these changes. Concurrently, tissue elongation upregulated sodium channel (NaV1.5) and gap junction (Connexin 43, Cx43) protein expression. Based on these observations, we leveraged elongated µHM to study the impact of loss-of-function mutation in Plakophilin 2 (PKP2), a desmosome protein implicated in arrhythmogenic disease. Within µHM, PKP2 knockout cardiomyocytes had cellular morphology similar to what was observed in isogenic controls. However, PKP2-/- tissues exhibited lower conduction velocity and no functional sodium current. PKP2 knockout µHM exhibited geometrically linked upregulation of sodium channel but not Cx43, suggesting that post-translational mechanisms, including a lack of ion channel-gap junction communication, may underlie the lower conduction velocity observed in tissues harboring this genetic defect. Altogether, these observations demonstrate that simple, scalable micro-tissue systems can provide the physiologic stresses necessary to induce electrical remodeling of iPS-CM to enable studies on the electrophysiologic consequences of disease-associated genomic variants.

Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success.

Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persist for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods.

Notch activation of Jagged1 contributes to the assembly of the arterial wall.

BACKGROUND: Notch signaling in vascular smooth muscle precursors is required for smooth muscle differentiation. Jagged1 expression on endothelium activates Notch in vascular smooth muscle precursors including those of neural crest origin to initiate the formation of a smooth muscle layer in a maturing blood vessel. METHODS AND RESULTS: Here, we show that Jagged1 is a direct Notch target in smooth muscle, resulting in a positive feedback loop and lateral induction that propagates a wave of smooth muscle differentiation during aortic arch artery development. In vivo, we show that Notch inhibition in cardiac neural crest impairs Jagged1 messenger RNA expression and results in deficient smooth muscle differentiation and resultant aortic arch artery defects. Ex vivo, Jagged1 ligand activates Notch in neural crest explants and results in activation of Jagged1 messenger RNA, a response that is blocked by Notch inhibition. We examine 15 evolutionary conserved regions within the Jagged1 genomic locus and identify a single Notch response element within the second intron. This element contains a functional Rbp-J binding site demonstrated by luciferase reporter and chromatin immunoprecipitation assays and is sufficient to recapitulate aortic arch artery expression of Jagged1 in transgenic mice. Loss of Jagged1 in neural crest impairs vascular smooth muscle differentiation and results in aortic arch artery defects. CONCLUSIONS: Taken together, these results provide a mechanism for lateral induction that allows for a multilayered smooth muscle wall to form around a nascent arterial endothelial tube and identify Jagged1 as a direct Notch target.

Myocardial Notch signaling reprograms cardiomyocytes to a conduction-like phenotype.

BACKGROUND: Notch signaling has previously been shown to play an essential role in regulating cell fate decisions and differentiation during cardiogenesis in many systems including Drosophila, Xenopus, and mammals. We hypothesized that Notch may also be involved in directing the progressive lineage restriction of cardiomyocytes into specialized conduction cells. METHODS AND RESULTS: In hearts where Notch signaling is activated within the myocardium from early development onward, Notch promotes a conduction-like phenotype based on ectopic expression of conduction system-specific genes and cell autonomous changes in electrophysiology. With the use of an in vitro assay to activate Notch in newborn cardiomyocytes, we observed global changes in the transcriptome, and in action potential characteristics, consistent with reprogramming to a conduction-like phenotype. CONCLUSIONS: Notch can instruct the differentiation of chamber cardiac progenitors into specialized conduction-like cells. Plasticity remains in late-stage cardiomyocytes, which has potential implications for engineering of specialized cardiovascular tissues.

Human Cardiac Pericytes Are Susceptible to SARS-CoV-2 Infection.

COVID-19 is associated with serious cardiovascular complications, with incompletely understood mechanism(s). Pericytes have key functions in supporting endothelial cells and maintaining vascular integrity. We demonstrate that human cardiac pericytes are permissive to SARS-CoV-2 infection in organotypic slice and primary cell cultures. Viral entry into pericytes is mediated by endosomal proteases, and infection leads to up-regulation of inflammatory markers, vasoactive mediators, and nuclear factor kappa-B-dependent cell death. Furthermore, we present evidence of cardiac pericyte infection in COVID-19 myocarditis patients. These data demonstrate that human cardiac pericytes are susceptible to SARS-CoV-2 infection and suggest a role for pericyte infection in COVID-19.

Cardiac radiation improves ventricular function in mice and humans with cardiomyopathy.

BACKGROUND: Rapidly dividing cells are more sensitive to radiation therapy (RT) than quiescent cells. In the failing myocardium, macrophages and fibroblasts mediate collateral tissue injury, leading to progressive myocardial remodeling, fibrosis, and pump failure. Because these cells divide more rapidly than cardiomyocytes, we hypothesized that macrophages and fibroblasts would be more susceptible to lower doses of radiation and that cardiac radiation could therefore attenuate myocardial remodeling. METHODS: In three independent murine heart failure models, including models of metabolic stress, ischemia, and pressure overload, mice underwent 5 Gy cardiac radiation or sham treatment followed by echocardiography. Immunofluorescence, flow cytometry, and non-invasive PET imaging were employed to evaluate cardiac macrophages and fibroblasts. Serial cardiac magnetic resonance imaging (cMRI) from patients with cardiomyopathy treated with 25 Gy cardiac RT for ventricular tachycardia (VT) was evaluated to determine changes in cardiac function. FINDINGS: In murine heart failure models, cardiac radiation significantly increased LV ejection fraction and reduced end-diastolic volume vs. sham. Radiation resulted in reduced mRNA abundance of B-type natriuretic peptide and fibrotic genes, and histological assessment of the LV showed reduced fibrosis. PET and flow cytometry demonstrated reductions in pro-inflammatory macrophages, and immunofluorescence demonstrated reduced proliferation of macrophages and fibroblasts with RT. In patients who were treated with RT for VT, cMRI demonstrated decreases in LV end-diastolic volume and improvements in LV ejection fraction early after treatment. CONCLUSIONS: These results suggest that 5 Gy cardiac radiation attenuates cardiac remodeling in mice and humans with heart failure. FUNDING: NIH, ASTRO, AHA, Longer Life Foundation.

Acute Glycogen Synthase Kinase-3 Inhibition Modulates Human Cardiac Conduction.

Glycogen synthase kinase 3 (GSK-3) inhibition has emerged as a potential therapeutic target for several diseases, including cancer. However, the role for GSK-3 regulation of human cardiac electrophysiology remains ill-defined. We demonstrate that SB216763, a GSK-3 inhibitor, can acutely reduce conduction velocity in human cardiac slices. Combined computational modeling and experimental approaches provided mechanistic insight into GSK-3 inhibition-mediated changes, revealing that decreased sodium-channel conductance and tissue conductivity may underlie the observed phenotypes. Our study demonstrates that GSK-3 inhibition in human myocardium alters electrophysiology and may predispose to an arrhythmogenic substrate; therefore, monitoring for adverse arrhythmogenic events could be considered.

Single-cell transcriptomics reveals cell-type-specific diversification in human heart failure.

Heart failure represents a major cause of morbidity and mortality worldwide. Single-cell transcriptomics have revolutionized our understanding of cell composition and associated gene expression. Through integrated analysis of single-cell and single-nucleus RNA-sequencing data generated from 27 healthy donors and 18 individuals with dilated cardiomyopathy, here we define the cell composition of the healthy and failing human heart. We identify cell-specific transcriptional signatures associated with age and heart failure and reveal the emergence of disease-associated cell states. Notably, cardiomyocytes converge toward common disease-associated cell states, whereas fibroblasts and myeloid cells undergo dramatic diversification. Endothelial cells and pericytes display global transcriptional shifts without changes in cell complexity. Collectively, our findings provide a comprehensive analysis of the cellular and transcriptomic landscape of human heart failure, identify cell type-specific transcriptional programs and disease-associated cell states and establish a valuable resource for the investigation of human heart failure.

An iPS-derived in vitro model of human atrial conduction.

Atrial fibrillation (AF) is the most common arrhythmia in the United States, affecting approximately 1 in 10 adults, and its prevalence is expected to rise as the population ages. Treatment options for AF are limited; moreover, the development of new treatments is hindered by limited (1) knowledge regarding human atrial electrophysiological endpoints (e.g., conduction velocity [CV]) and (2) accurate experimental models. Here, we measured the CV and refractory period, and subsequently calculated the conduction wavelength, in vivo (four subjects with AF and four controls), and ex vivo (atrial slices from human hearts). Then, we created an in vitro model of human atrial conduction using induced pluripotent stem (iPS) cells. This model consisted of iPS-derived human atrial cardiomyocytes plated onto a micropatterned linear 1D spiral design of Matrigel. The CV (34-41 cm/s) of the in vitro model was nearly five times faster than 2D controls (7-9 cm/s) and similar to in vivo (40-64 cm/s) and ex vivo (28-51 cm/s) measurements. Our iPS-derived in vitro model recapitulates key features of in vivo atrial conduction and may be a useful methodology to enhance our understanding of AF and model patient-specific disease.

Cardiac radiotherapy induces electrical conduction reprogramming in the absence of transmural fibrosis.

Cardiac radiotherapy (RT) may be effective in treating heart failure (HF) patients with refractory ventricular tachycardia (VT). The previously proposed mechanism of radiation-induced fibrosis does not explain the rapidity and magnitude with which VT reduction occurs clinically. Here, we demonstrate in hearts from RT patients that radiation does not achieve transmural fibrosis within the timeframe of VT reduction. Electrophysiologic assessment of irradiated murine hearts reveals a persistent supraphysiologic electrical phenotype, mediated by increases in NaV1.5 and Cx43. By sequencing and transgenic approaches, we identify Notch signaling as a mechanistic contributor to NaV1.5 upregulation after RT. Clinically, RT was associated with increased NaV1.5 expression in 1 of 1 explanted heart. On electrocardiogram (ECG), post-RT QRS durations were shortened in 13 of 19 patients and lengthened in 5 patients. Collectively, this study provides evidence for radiation-induced reprogramming of cardiac conduction as a potential treatment strategy for arrhythmia management in VT patients.

Tissue-tissue interactions during morphogenesis of the outflow tract.

The heart forms as a linear heart tube that loops and septates to produce a mature four-chambered structure. The single vessel emerging from the embryonic heart, the truncus arteriosus, divides into the aorta and the pulmonary artery as part of this septation process, and a series of additional morphogenetic events result in the proper alignment and orientation of the cardiac outflow tract. Recent evidence indicates that this process involves the complex interactions of multiple cell types including primary and secondary heart fields, neural crest, pharyngeal mesenchyme, endoderm, and endothelium. Among the many signals that mediate tissue-tissue interactions during the formation of the outflow tract, we have focused on the role of the Notch signaling pathway. Here, we focus on recent advances in our understanding of Notch-mediated regulation of cardiac development with specific attention to the formation of the cardiac outflow tract.