Search

Background: Lamin A/C (LMNA) mutations are the second most common cause of familial dilated cardiomyopathy (DCM). LMNA encodes nuclear lamins which are intermediate filaments of the inner nuclear membrane. Patients with LMNA DCM present with early signs of arrhythmias and often display a rapid deterioration in cardiac function. Previous induced pluripotent derived stem cell (iPSC) modeling of cardiac laminopathy did not demonstrate defects in cardiomyocyte (CM) contractility nor provide evidence of arrhythmogenesis. Developing a robust human model that recapitulates disease manifestations of LMNA cardiomyopathy is a critical step for understanding CM-intrinsic defects contributing to disease pathogenesis. Purpose: As such, our lab hypothesized that iPSC-CMs derived from a laminopathy patient would recapitulate the contractile dysfunction of the disease if studied in monolayer using flexible matrigel substrate. Methods: Patients with symptomology indicative of LMNA DCM were recruited for clinical testing. Peripheral blood mononuclear cells were isolated and reprogrammed into iPSCs. CMs were generated using a chemical differentiation method and then subjected to molecular and functional assessment. Cardioexcyte96 based detection of impedance was utilized for analysis of contractility of iPSC-CMs in a monolayer. Results/Conclusions: We identified a large pedigree with DCM in which affected individuals had a putatively pathogenic mutation within LMNA. Western blot analyses demonstrated that lamin A/C can be detected in iPSC-CMs with reduced expression in mutant cells. Mutant iPSC-CMs in a monolayer demonstrate both increased time for contraction (+55.1 msec P

Introduction: Current screening for mutations in human disease is turning increasingly to next-generation methods that map short reads to a reference sequence. We report here an unusual variant that was undetected by next generation sequencing in a patient diagnosed with Jervell Lange-Nielsen syndrome (JLNS) and initial results in an induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model. Methods and Results: A diagnosis of JLNS was made in a middle-aged woman with congenital deafness and QT intervals as long as 800 msec. However, next-generation sequencing found only a heterozygous KCNQ1 mutation, R518X. Convinced by the clinical phenotype that a second causative variant was highly likely, we used Sanger sequencing of PCR KCNQ1 amplicons to identify a 36-basepair poly-adenine tract, encoding 12 lysines, inserted within the coding sequence at the 5’ end of exon 15. Electrophysiological studies in patient-specific IPSC-CMs revealed marked prolongation of ventricular-like action potentials (Figure). Conclusion: Long inserts of the type we identified here have not been previously reported in the long QT syndromes. We speculate that next generation-based short reads containing this variant could not be mapped to a reference sequence and thus this type of variant will be missed by next-generation analysis unless bioinformatics filters are specifically modified to include this possibility. Validation of this long QT syndrome iPSC-CM model provides a human cell based platform for drug discovery and mechanistic studies to further our understanding of disease pathogenesis.

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.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.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.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.

Introduction: Cardiomyocytes carry the pump function of the heart. What molecular and cellular mechanisms control proliferation of cardiomyocytes is an unresolved question with high impact on regenerative medicine. Hypothesis: The growth factor neuregulin1 (NRG1) and its tyrosine kinase receptor ErbB4 control cardiomyocyte proliferation during prenatal development. NRG1 and ErbB4 are expressed in the adult heart. We hypothesized that activating NRG1 signaling stimulates cell cycle reentry and division of a subpopulation of differentiated cardiomyocytes. Methods: We determined cardiomyocyte cell cycle reentry and division in vitro using immunofluorescence microscopy and live cell imaging as read-out assays. We tested the in vivo proliferative effect of controlling NRG1 signaling at the level of its receptor using inducible cardiomyocyte-specific ErbB4 knockout mice and ErbB4 transgenic mice. Results: NRG1 induced cell cycle reentry of large, striated, and rod-shaped cardiomyocytes that express cardiac contractile proteins, consistent with a differentiated phenotype. Live cell imaging demonstrated in real time that NRG1 induces differentiated cardiomyocytes to undergo karyokinesis (nuclear division) and cytokinesis (cytoplasmic division), followed by separation into two differentiated daughter cardiomyocytes. During cell division, cardiomyocytes disassembled their contractile fibrils in the regions of the mitotic spindle and the cleavage furrow. NRG1 induced karyokinesis in 33% of mononucleated and in 1% of binucleated cardiomyocytes. Mononucleated cardiomyocytes completed cytokinesis, while binucleated cardiomyocytes did not. In vivo, postnatal genetic inactivation of the NRG1 receptor ErbB4 reduced cycling of differentiated mononucleated cardiomyocytes (Ctr. 5% vs. ko 0%, P < 0.01). In contrast, transgenic expression of ErbB4 increased cycling of differentiated mononucleated cardiomyocytes (Ctr. 7.2% vs. tg 22.6%, P < 0.05). Conclusions: ErbB4 is required and NRG1 and ErbB4 are sufficient to induce proliferation of a subpopulation of differentiated cardiomyocytes. NRG1 and the pathway that it regulates may provide new therapeutic targets to enhance mammalian cardiac regeneration. This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).