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.

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.heart failure | pediatrics H eart failure, a leading public health problem worldwide (1), is linked to the loss of cardiomyocytes (2-4). The only currently available, definitive therapy-heart transplantation-is limited by donor availability. New approaches, such as cell transplantation, have shown encouraging results in clinical trials (5, 6). However, a third, complementary strategy has emerged, based on stimulating endogenous regenerative mechanisms. One approach for developing such regeneration strategies is to examine the cellular mechanisms of myocardial growth, since mechanisms of regeneration should be similar to the mechanisms of development.Although stem and progenitor cells are important for morphogenesis of the myocardium, developmental growth in a number of nonhuman species is largely driven by cardiomyocyte proliferation (7-9). In biological models that, unlike adult humans, regenerate myocardium, cardiomyocyte proliferation is important for regeneration as well as postnatal heart growth (10, 11). For example, in mice, developmental cardiomyocyte proliferation continues for up to day 7 after birth, which coincides with the loss of regenerative capacity (11,12). The close temporal relationship between cardiomyocyte proliferation and heart regeneration in animals raises the question of whether and to what age and extent cardiomyocyte proliferation plays a role in humans. The answer may help us understand the endogenous regenerative potential of the human heart and possibly indicate strategies for stimulating cardiomyocyte proliferation to reg...

Background Numerous studies have demonstrated increased load of de novo copy number variants (CNVs) or single nucleotide variants (SNVs) 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 (WGS). 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 KV2.1 voltage-gated potassium channel. Functional studies demonstrated a deleterious effect of the mutation on KV2.1 function leading to a loss of ion selectivity and gain of a depolarizing inward cation conductance. Subsequently, we identified two additional patients with epileptic encephalopathy and de novo KCNB1 missense mutations that cause a similar pattern of KV2.1 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.

SUMMARYNumerous 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.

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 electrophysiologic 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±3μM) and Na+ current in mouse ventricular myocytes. However, with chronic (24hour) 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±21μM) and IKs (CHO cells expressing KCNQ1+KCNE1; IC50=184±12μM), while azithromycin suppressed L-type Ca++ currents (rabbit ventricular myocytes; IC50=66.5±4μM) and IK1 (HEK cells expressing Kir2.1; IC50=44±3μM). 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.

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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.

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 subjects identified from BioVU, a resource linking DNA samples to de-identified electronic medical record data. Variants with p-values <1×10−5 were independently tested for replication in a cohort of 181 anthracycline-exposed subjects 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 (SNP) near PR Domain Containing 2, With ZNF Domain (PRDM2), rs7542939, had a combined p-value of 6.5×10−7 in meta-analysis. Eighteen Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways showed strong enrichment for variants associated with the primary outcome. Identified pathways related to DNA repair, cellular metabolism, and cardiac remodeling. Conclusions 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.