Transcriptional Dysregulation Underlies Both Monogenic Arrhythmia Syndrome and Common Modifiers of Cardiac Repolarization.

BACKGROUND: Brugada syndrome (BrS) is an inherited arrhythmia syndrome caused by loss-of-function variants in the cardiac sodium channel gene SCN5A (sodium voltage-gated channel alpha subunit 5) in ≈20% of subjects. We identified a family with 4 individuals diagnosed with BrS harboring the rare G145R missense variant in the cardiac transcription factor TBX5 (T-box transcription factor 5) and no SCN5A variant. METHODS: We generated induced pluripotent stem cells (iPSCs) from 2 members of a family carrying TBX5-G145R and diagnosed with Brugada syndrome. After differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), electrophysiologic characteristics were assessed by voltage- and current-clamp experiments (n=9 to 21 cells per group) and transcriptional differences by RNA sequencing (n=3 samples per group), and compared with iPSC-CMs in which G145R was corrected by CRISPR/Cas9 approaches. The role of platelet-derived growth factor (PDGF)/phosphoinositide 3-kinase (PI3K) pathway was elucidated by small molecule perturbation. The rate-corrected QT (QTc) interval association with serum PDGF was tested in the Framingham Heart Study cohort (n=1893 individuals). RESULTS: TBX5-G145R reduced transcriptional activity and caused multiple electrophysiologic abnormalities, including decreased peak and enhanced "late" cardiac sodium current (INa), which were entirely corrected by editing G145R to wild-type. Transcriptional profiling and functional assays in genome-unedited and -edited iPSC-CMs showed direct SCN5A down-regulation caused decreased peak INa, and that reduced PDGF receptor (PDGFRA [platelet-derived growth factor receptor α]) expression and blunted signal transduction to PI3K was implicated in enhanced late INa. Tbx5 regulation of the PDGF axis increased arrhythmia risk due to disruption of PDGF signaling and was conserved in murine model systems. PDGF receptor blockade markedly prolonged normal iPSC-CM action potentials and plasma levels of PDGF in the Framingham Heart Study were inversely correlated with the QTc interval (P<0.001). CONCLUSIONS: These results not only establish decreased SCN5A transcription by the TBX5 variant as a cause of BrS, but also reveal a new general transcriptional mechanism of arrhythmogenesis of enhanced late sodium current caused by reduced PDGF receptor-mediated PI3K signaling.

Isolation and Characterization of Intact Cardiomyocytes from Frozen and Fresh Human Myocardium and Mouse Hearts.

Procurement and characterization of intact human cells are essential for studies in regenerative medicine and translational medical research. The selection of the currently available approaches to isolate intact cells depends on the age of the hearts. To isolate cardiomyocytes from the fetal or neonatal myocardium, the myocardium can be minced into small tissue blocks followed by enzyme incubation. However, the fetal and neonatal cardiomyocytes are very soft and the morphology changes from long rod or spindle shape to spheres after isolation. Because of the dense packing of the cardiomyocytes and the strong cell-cell connection in adult myocardium, it is difficult to isolate the cardiomyocytes from adult myocardium by enzyme incubation only. A perfusion method is necessary to deliver the enzyme solution to the deep layers of the myocardium. However, intact hearts, which are very rare, are required for the perfusion method. Therefore, lacking methods to efficiently isolate cardiomyocytes from myocardium of various ages builds a barrier between basic research and clinical studies. Here, we describe a method for the isolation of intact cardiomyocytes from fresh or frozen human myocardium or fresh mouse hearts and the quantification of multinucleation, cardiomyocyte size, cell cycle activity, and total cardiomyocyte count per heart. We generalize this fixation-digestion method by isolating cells from a variety of mouse organs, including the liver, lung, and thymus.

Real-time visualization of titin dynamics reveals extensive reversible photobleaching in human induced pluripotent stem cell-derived cardiomyocytes.

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

Anticancer drug-induced cardiac rhythm disorders: Current knowledge and basic underlying mechanisms.

Significant advances in cancer treatment have resulted in decreased cancer related mortality for many malignancies with some cancer types now considered chronic diseases. Despite these improvements, there is increasing recognition that many cancer patients or cancer survivors can develop cardiovascular diseases, either due to the cancer itself or as a result of anticancer therapy. Much attention has focused on heart failure; however, other cardiotoxicities, notably cardiac rhythm disorders, can occur without underlying cardiomyopathy. Supraventricular tachycardias occur in cancer patients treated with cytotoxic chemotherapy (anthracyclines, gemcitabine, cisplatin and alkylating-agents) or kinase-inhibitors (KIs) such as ibrutinib. Ventricular arrhythmias, with a subset of them being torsades-de-pointes (TdP) favored by QTc prolongation have been reported: this may be the result of direct hERG-channel inhibition or a more recently-described mechanism of phosphoinositide-3-kinase inhibition. The major anticancer drugs responsible for QTc prolongation in this context are KIs, arsenic trioxide, anthracyclines, histone deacetylase inhibitors, and selective estrogen receptor modulators. Anticancer drug-induced cardiac rhythm disorders remain an underappreciated complication even by experienced clinicians. Moreover, the causal relationship of a particular anticancer drug with cardiac arrhythmia occurrence remains challenging due in part to patient comorbidities and complex treatment regimens. For example, any cancer patient may also be diagnosed with common diseases such as hypertension, diabetes or heart failure which increase an individual's arrhythmia susceptibility. Further, anticancer drugs are generally usually used in combination, increasing the challenge around establishing causation. Thus, arrhythmias appear to be an underappreciated adverse effect of anticancer agents and the incidence, significance and underlying mechanisms are now being investigated.

Hypertrophic cardiomyopathy-linked mutation in troponin T causes myofibrillar disarray and pro-arrhythmic action potential changes in human iPSC cardiomyocytes.

BACKGROUND: Mutations in cardiac troponin T (TnT) are linked to increased risk of ventricular arrhythmia and sudden death despite causing little to no cardiac hypertrophy. Studies in mice suggest that the hypertrophic cardiomyopathy (HCM)-associated TnT-I79N mutation increases myofilament Ca sensitivity and is arrhythmogenic, but whether findings from mice translate to human cardiomyocyte electrophysiology is not known. OBJECTIVES: To study the effects of the TnT-I79N mutation in human cardiomyocytes. METHODS: Using CRISPR/Cas9, the TnT-I79N mutation was introduced into human induced pluripotent stem cells (hiPSCs). We then used the matrigel mattress method to generate single rod-shaped cardiomyocytes (CMs) and studied contractility, Ca handling and electrophysiology. RESULTS: Compared to isogenic control hiPSC-CMs, TnT-I79N hiPSC-CMs exhibited sarcomere disorganization, increased systolic function and impaired relaxation. The Ca-dependence of contractility was leftward shifted in mutation containing cardiomyocytes, demonstrating increased myofilament Ca sensitivity. In voltage-clamped hiPSC-CMs, TnT-I79N reduced intracellular Ca transients by enhancing cytosolic Ca buffering. These changes in Ca handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. The myofilament Ca sensitizer EMD57033 produced similar action potential triangulation in control hiPSC-CMs. CONCLUSIONS: The TnT-I79N hiPSC-CM model not only reproduces key cellular features of TnT-linked HCM such as myofilament disarray, hypercontractility and diastolic dysfunction, but also suggests that this TnT mutation causes pro-arrhythmic changes of the human ventricular action potential.

Genome-wide association and pathway analysis of left ventricular function after anthracycline exposure in adults.

BACKGROUND: Anthracyclines are important chemotherapeutic agents, but their use is limited by cardiotoxicity. Candidate gene and genome-wide studies have identified putative risk loci for overt cardiotoxicity and heart failure, but there has been no comprehensive assessment of genomic variation influencing the intermediate phenotype of anthracycline-related changes in left ventricular (LV) function. The purpose of this study was to identify genetic factors influencing changes in LV function after anthracycline chemotherapy. METHODS: We conducted a genome-wide association study (GWAS) of change in LV function after anthracycline exposure in 385 patients identified from BioVU, a resource linking DNA samples to de-identified electronic medical record data. Variants with P values less than 1×10 were independently tested for replication in a cohort of 181 anthracycline-exposed patients from a prospective clinical trial. Pathway analysis was performed to assess combined effects of multiple genetic variants. RESULTS: Both cohorts were middle-aged adults of predominantly European descent. Among 11 candidate loci identified in discovery GWAS, one single nucleotide polymorphism near PR domain containing 2, with ZNF domain (PRDM2), rs7542939, had a combined P value of 6.5×10 in meta-analysis. Eighteen Kyoto Encyclopedia of Gene and Genomes pathways showed strong enrichment for variants associated with the primary outcome. Identified pathways related to DNA repair, cellular metabolism, and cardiac remodeling. CONCLUSION: Using genome-wide association we identified a novel candidate susceptibility locus near PRDM2. Variation in genes belonging to pathways related to DNA repair, metabolism, and cardiac remodeling may influence changes in LV function after anthracycline exposure.

Azithromycin Causes a Novel Proarrhythmic Syndrome.

BACKGROUND: The widely used macrolide antibiotic azithromycin increases risk of cardiovascular and sudden cardiac death, although the underlying mechanisms are unclear. Case reports, including the one we document here, demonstrate that azithromycin can cause rapid, polymorphic ventricular tachycardia in the absence of QT prolongation, indicating a novel proarrhythmic syndrome. We investigated the electrophysiological effects of azithromycin in vivo and in vitro using mice, cardiomyocytes, and human ion channels heterologously expressed in human embryonic kidney (HEK 293) and Chinese hamster ovary (CHO) cells. METHODS AND RESULTS: In conscious telemetered mice, acute intraperitoneal and oral administration of azithromycin caused effects consistent with multi-ion channel block, with significant sinus slowing and increased PR, QRS, QT, and QTc intervals, as seen with azithromycin overdose. Similarly, in HL-1 cardiomyocytes, the drug slowed sinus automaticity, reduced phase 0 upstroke slope, and prolonged action potential duration. Acute exposure to azithromycin reduced peak SCN5A currents in HEK cells (IC50=110±3 µmol/L) and Na+ current in mouse ventricular myocytes. However, with chronic (24 hour) exposure, azithromycin caused a ≈2-fold increase in both peak and late SCN5A currents, with findings confirmed for INa in cardiomyocytes. Mild block occurred for K+ currents representing IKr (CHO cells expressing hERG; IC50=219±21 µmol/L) and IKs (CHO cells expressing KCNQ1+KCNE1; IC50=184±12 µmol/L), whereas azithromycin suppressed L-type Ca++ currents (rabbit ventricular myocytes, IC50=66.5±4 µmol/L) and IK1 (HEK cells expressing Kir2.1, IC50=44±3 µmol/L). CONCLUSIONS: Chronic exposure to azithromycin increases cardiac Na+ current to promote intracellular Na+ loading, providing a potential mechanistic basis for the novel form of proarrhythmia seen with this macrolide antibiotic.

Contrasting Nav1.8 Activity in Scn10a-/- Ventricular Myocytes and the Intact Heart.

BACKGROUND: Genome-wide association studies have implicated variants in SCN10A, which encodes Nav1.8, as modulators of cardiac conduction. Follow-up work has indicated the SCN10A sequence includes an intronic enhancer for SCN5A. Yet the role of the Nav1.8 protein in the myocardium itself is still unclear. To investigate this, we use homozygous knockout mice (Scn10a-/-) generated by disruption of exons 4 and 5, leaving the Scn5a enhancer intact. METHODS AND RESULTS: We previously reported that pharmacologic blockade of Nav1.8 in wild-type animals blunts action potential prolongation by ATX-II at slow drive rates (≤1 Hz). Here we present evidence of the same blunting in Scn10a-/- compared to wild-type ventricular myocytes, supporting the conclusion that Nav1.8 contributes to late sodium current at slow rates. In contrast to earlier studies, we found no differences in electrocardiographic parameters between genotypes. Low-dose ATX-II exposure in lightly anesthetized animals and Langendorff-perfused hearts prolonged QTc and generated arrhythmias to the same extent in wild-type and Scn10a-/-. RNA sequencing failed to identify full-length Scn10a transcripts in wild-type or knockout isolated ventricular myocytes. However, loss of late current in Scn10a-/- myocytes was replicated independently in a blinded set of experiments. CONCLUSIONS: While Scn10a transcripts are not detectible in ventricular cardiomyocytes, gene deletion results in reproducible loss of late sodium current under extreme experimental conditions. However, there are no identifiable consequences of this Scn10a deletion in the intact mouse heart at usual rates. These findings argue that common variants in SCN10A that affect ventricular conduction do so by modulating SCN5A.

De novo KCNB1 mutations in epileptic encephalopathy.

OBJECTIVE: Numerous studies have demonstrated increased load of de novo copy number variants or single nucleotide variants in individuals with neurodevelopmental disorders, including epileptic encephalopathies, intellectual disability, and autism. METHODS: We searched for de novo mutations in a family quartet with a sporadic case of epileptic encephalopathy with no known etiology to determine the underlying cause using high-coverage whole exome sequencing (WES) and lower-coverage whole genome sequencing. Mutations in additional patients were identified by WES. The effect of mutations on protein function was assessed in a heterologous expression system. RESULTS: We identified a de novo missense mutation in KCNB1 that encodes the KV 2.1 voltage-gated potassium channel. Functional studies demonstrated a deleterious effect of the mutation on KV 2.1 function leading to a loss of ion selectivity and gain of a depolarizing inward cation conductance. Subsequently, we identified 2 additional patients with epileptic encephalopathy and de novo KCNB1 missense mutations that cause a similar pattern of KV 2.1 dysfunction. INTERPRETATION: Our genetic and functional evidence demonstrate that KCNB1 mutation can result in early onset epileptic encephalopathy. This expands the locus heterogeneity associated with epileptic encephalopathies and suggests that clinical WES may be useful for diagnosis of epileptic encephalopathies of unknown etiology.

Moderate and high amounts of tamoxifen in αMHC-MerCreMer mice induce a DNA damage response, leading to heart failure and death.

Numerous mouse models have utilized Cre-loxP technology to modify gene expression. Adverse effects of Cre recombinase activity have been reported, including in the heart. However, the mechanisms associated with cardiac Cre toxicity are largely unknown. Here, we show that expression of Cre in cardiomyocytes induces a DNA damage response, resulting in cardiomyocyte apoptosis, cardiac fibrosis and cardiac dysfunction. In an effort to increase the recombination efficiency of a widely used tamoxifen-sensitive Cre transgene under control of the α-myosin-heavy-chain promoter (αMHC-MerCreMer), we observed myocardial dysfunction and decreased survival, which were dependent on the dose of tamoxifen injected. After excluding a Cre-independent contribution by tamoxifen, we found that Cre induced myocardial fibrosis, activation of pro-fibrotic genes and cardiomyocyte apoptosis. Examination of the molecular mechanisms showed activation of DNA damage response signaling and p53 stabilization in the absence of loxP sites, suggesting that Cre induced illegitimate DNA breaks. Cardiomyocyte apoptosis was also induced by expressing Cre using adenoviral transduction, indicating that the effect was not dependent on genomic integration of the transgene. Cre-mediated homologous recombination at loxP sites was dose-dependent and had a ceiling effect at ∼80% of cardiomyocytes showing recombination. By titrating the amount of tamoxifen to maximize recombination while minimizing animal lethality, we determined that 30 µg tamoxifen/g body weight/day injected on three consecutive days is the optimal condition for the αMHC-MerCreMer system to induce recombination in the Rosa26-lacZ strain. Our results further highlight the importance of experimental design, including the use of appropriate genetic controls for Cre expression.

Cardiomyocyte proliferation contributes to heart growth in young humans.

The human heart is believed to grow by enlargement but not proliferation of cardiomyocytes (heart muscle cells) during postnatal development. However, recent studies have shown that cardiomyocyte proliferation is a mechanism of cardiac growth and regeneration in animals. Combined with evidence for cardiomyocyte turnover in adult humans, this suggests that cardiomyocyte proliferation may play an unrecognized role during the period of developmental heart growth between birth and adolescence. We tested this hypothesis by examining the cellular growth mechanisms of the left ventricle on a set of healthy hearts from humans aged 0-59 y (n = 36). The percentages of cardiomyocytes in mitosis and cytokinesis were highest in infants, decreasing to low levels by 20 y. Although cardiomyocyte mitosis was detectable throughout life, cardiomyocyte cytokinesis was not evident after 20 y. Between the first year and 20 y of life, the number of cardiomyocytes in the left ventricle increased 3.4-fold, which was consistent with our predictions based on measured cardiomyocyte cell cycle activity. Our findings show that cardiomyocyte proliferation contributes to developmental heart growth in young humans. This suggests that children and adolescents may be able to regenerate myocardium, that abnormal cardiomyocyte proliferation may be involved in myocardial diseases that affect this population, and that these diseases might be treatable through stimulation of cardiomyocyte proliferation.

Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury.

Many organs rely on undifferentiated stem and progenitor cells for tissue regeneration. Whether differentiated cells themselves can contribute to cell replacement and tissue regeneration is a controversial question. Here, we show that differentiated heart muscle cells, cardiomyocytes, can be induced to proliferate and regenerate. We identify an underlying molecular mechanism for controlling this process that involves the growth factor neuregulin1 (NRG1) and its tyrosine kinase receptor, ErbB4. NRG1 induces mononucleated, but not binucleated, cardiomyocytes to divide. In vivo, genetic inactivation of ErbB4 reduces cardiomyocyte proliferation, whereas increasing ErbB4 expression enhances it. Injecting NRG1 in adult mice induces cardiomyocyte cell-cycle activity and promotes myocardial regeneration, leading to improved function after myocardial infarction. Undifferentiated progenitor cells did not contribute to NRG1-induced cardiomyocyte proliferation. Thus, increasing the activity of the NRG1/ErbB4 signaling pathway may provide a molecular strategy to promote myocardial regeneration.