The identification of genetic variants that predispose individuals to cardiovascular disease and a better understanding of their targets would be highly advantageous. Genome-wide association studies have identified variants that associate with QT-interval length (a measure of myocardial repolarization). Three of the strongest associating variants (single-nucleotide polymorphisms) are located in the putative promotor region of CNOT1, a gene encoding the central CNOT1 subunit of CCR4-NOT: a multifunctional, conserved complex regulating gene expression and mRNA stability and turnover. We isolated the minimum fragment of the CNOT1 promoter containing all three variants from individuals homozygous for the QT risk alleles and demonstrated that the haplotype associating with longer QT interval caused reduced reporter expression in a cardiac cell line, suggesting that reduced CNOT1 expression might contribute to abnormal QT intervals. Systematic siRNA-mediated knockdown of CCR4-NOT components in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) revealed that silencing CNOT1 and other CCR4-NOT genes reduced their proliferative capacity. Silencing CNOT7 also shortened action potential duration. Furthermore, the cardiac-specific knockdown of Drosophila orthologs of CCR4-NOT genes in vivo (CNOT1/Not1 and CNOT7/8/Pop2) was either lethal or resulted in dilated cardiomyopathy, reduced contractility or a propensity for arrhythmia. Silencing CNOT2/Not2, CNOT4/Not4 and CNOT6/6L/twin also affected cardiac chamber size and contractility. Developmental studies suggested that CNOT1/Not1 and CNOT7/8/Pop2 are required during cardiac remodeling from larval to adult stages. To summarize, we have demonstrated how disease-associated genes identified by GWAS can be investigated by combining human cardiomyocyte cell-based and whole-organism in vivo heart models. Our results also suggest a potential link of CNOT1 and CNOT7/8 to QT alterations and further establish a crucial role of the CCR4-NOT complex in heart development and function.This article has an associated First Person interview with the first author of the paper.

Highlights d Flies in microgravity exhibit cardiac constriction, remodeling, and diminished output d Heart defects correlate with reduced sarcomeric and extracellular matrix gene expression d Proteosome gene or protein expression is upregulated, suggesting proteostasis imbalance

Somatically acquired Hand1A126FS mutation is not causative of HLHS. Hand1A126FS mutation does exhibit embryonic lethal cardiac defects that reflect a dominant negative function supporting the critical role of Hand1 in cardiogenesis.

Understanding the cellular-molecular substrates of heart disease is key to the development of cardiac specific therapies and to the prevention of off-target effects by non-cardiac targeted drugs. One of the primary targets for therapeutic intervention has been the human ether a go-go (hERG) K+ channel that, together with the KCNQ channel, controls the rate and efficiency of repolarization in human myocardial cells. Neither of these channels plays a major role in adult mouse heart function; however, we show here that the hERG homolog seizure (sei), along with KCNQ, both contribute significantly to adult heart function as they do in humans. In Drosophila, mutations in or cardiac knockdown of sei channels cause arrhythmias that become progressively more severe with age. Intracellular recordings of semi-intact heart preparations revealed that these perturbations also cause electrical remodeling that is reminiscent of the early afterdepolarizations seen in human myocardial cells defective in these channels. In contrast to KCNQ, however, mutations in sei also cause extensive structural remodeling of the myofibrillar organization, which suggests that hERG channel function has a novel link to sarcomeric and myofibrillar integrity. We conclude that deficiency of ion channels with similar electrical functions in cardiomyocytes can lead to different types or extents of electrical and/or structural remodeling impacting cardiac output.

Background - KCNMA1 encodes the α-subunit of the large-conductance Ca 2+ -activated K + channel, K Ca 1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of K Ca 1.1 are limited and KCNMA1 has not been investigated as an AF candidate gene. Methods - The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of K Ca 1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. Results - A complex KCNMA1 variant was identified in one kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of K Ca 1.1 in normal hearts using immunostaining and immunogold electron microscopy. K Ca 1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the K Ca 1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the K Ca 1.1 ortholog, kcnma1b , in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila K Ca 1.1 ortholog, slo , systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo -deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the K Ca 1.1 loss-of-function models. Conclusions - Our data point to a highly conserved role of K Ca 1.1 in sinus node function in humans, mice, zebrafish and fly and suggest that K Ca 1.1 loss of function may predispose to AF.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with high mortality. The cellular origins of PDAC are largely unknown, however, ductal cells, especially centroacinar cells (CACs), have several characteristics in common with PDAC, such as expression of SOX9 and components of the Notch-signaling pathway. Mutations in KRAS and alterations to Notch signaling are common in PDAC; and, both these pathways regulate the transcription factor SOX9. To identify genes regulated by SOX9, we performed siRNA knockdown of SOX9 followed by RNA-seq in PANC-1 cells, a human PDAC cell line. We report 93 differentially expressed (DE) genes, with convergence on alterations to Notch-signaling pathways and ciliogenesis. These results point to SOX9 and Notch activity being in a positive feedback loop; and, SOX9 regulating cilia production in PDAC. We additionally performed ChIP-seq in PANC-1 cells to identify direct targets of SOX9 binding and integrated these results with our DE gene list. Nine of the top ten downregulated genes have evidence of direct SOX9 binding at their promoter regions. One of these targets was the cancer stem cell marker EpCAM. Using whole-mount in situ hybridization to detect epcam transcript in zebrafish larvae, we demonstrated that epcam is a CAC marker and that Sox9 regulation of epcam expression is conserved in zebrafish. Additionally, we generated an epcam null mutant and observed pronounced defects in ciliogenesis during development. Our results provide a link between SOX9, EpCAM, and ciliary repression that can be exploited in improving our understanding of the cellular origins and mechanisms of PDAC.