Premature MicroRNA-1 Expression Causes Hypoplasia of the Cardiac Ventricular Conduction System.

Mammalian cardiac Purkinje fibers (PFs) are specified from ventricular trabecular myocardium during mid-gestation and undergo limited proliferation before assuming their final form. MicroRNA-1 (miR-1), a negative regulator of proliferation, is normally expressed in the heart at low levels during the period of PF specification and outgrowth, but expression rises steeply after birth, when myocardial proliferation slows and postnatal cardiac maturation and growth commence. Here, we test whether premature up-regulation and overexpression of miR-1 during the period of PF morphogenesis influences PF development and function. Using a mouse model in which miR-1 is expressed under the control of the Myh6 promoter, we demonstrate that premature miR-1 expression leads to PF hypoplasia that persists into adulthood, and miR-1 TG mice exhibit delayed conduction through the ventricular myocardium beginning at neonatal stages. In addition, miR-1 transgenic embryos showed reduced proliferation within the trabecular myocardium and embryonic ventricular conduction system (VCS), a source of progenitor cells for the PF. This repression of proliferation may be mediated by direct translational inhibition by miR-1 of the cyclin dependent kinase Cdk6, a key regulator of embryonic myocardial proliferation. Our results suggest that altering the timing of miR-1 expression can regulate PF development, findings which have implications for our understanding of conduction system development and disease in humans

Premature MicroRNA-1 Expression Causes Hypoplasia of the Cardiac Ventricular Conduction System

Mammalian cardiac Purkinje fibers (PFs) are specified from ventricular trabecular myocardium during mid-gestation and undergo limited proliferation before assuming their final form. MicroRNA-1 (miR-1), a negative regulator of proliferation, is normally expressed in the heart at low levels during the period of PF specification and outgrowth, but expression rises steeply after birth, when myocardial proliferation slows and postnatal cardiac maturation and growth commence. Here, we test whether premature up-regulation and overexpression of miR-1 during the period of PF morphogenesis influences PF development and function. Using a mouse model in which miR-1 is expressed under the control of the Myh6 promoter, we demonstrate that premature miR-1 expression leads to PF hypoplasia that persists into adulthood, and miR-1 TG mice exhibit delayed conduction through the ventricular myocardium beginning at neonatal stages. In addition, miR-1 transgenic embryos showed reduced proliferation within the trabecular myocardium and embryonic ventricular conduction system (VCS), a source of progenitor cells for the PF. This repression of proliferation may be mediated by direct translational inhibition by miR-1 of the cyclin dependent kinase Cdk6, a key regulator of embryonic myocardial proliferation. Our results suggest that altering the timing of miR-1 expression can regulate PF development, findings which have implications for our understanding of conduction system development and disease in humans

Dedifferentiation and Proliferation of Mammalian Cardiomyocytes

It has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle.Here we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1) cardiomyocyte purification from rat hearts, and 2) genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs), while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP) continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit(+).Contradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness, including the expression of c-kit and the capacity for multipotency

Transcriptional Suppression of Connexin43 by TBX18 Undermines Cell-Cell Electrical Coupling in Postnatal Cardiomyocytes

T-box transcription factors figure prominently in embryonic cardiac cell lineage specifications. Mesenchymal precursor cells expressing Tbx18 give rise to the heart's pacemaker, the sinoatrial node (SAN). We sought to identify targets of TBX18 transcriptional regulation in the heart by forced adenoviral overexpression in postnatal cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) transduced with GFP showed sarcolemmal, punctate Cx43 expression. In contrast, TBX18-transduced NRCMs exhibited sparse Cx43 expression. Both the transcript and protein levels of Cx43 were greatly down-regulated within 2 days of TBX18 transduction. Direct injection of TBX18 in the guinea pig heart in vivo inhibited Cx43 expression. The repressor activity of TBX18 on Cx43 was highly specific; protein levels of Cx45 and Cx40, which comprise the main gap junctions in the SAN and conduction system, were unchanged by TBX18. A reporter-based promoter assay demonstrated that TBX18 directly represses the Cx43 promoter. Phenotypically, TBX18-NRCMs exhibited slowed intercellular calcein dye transfer kinetics (421 ± 54 versus control 127 ± 43 ms). Intracellular Ca2+ oscillations in control NRCM monolayers were highly synchronized. In contrast, TBX18 overexpression led to asynchronous Ca2+ oscillations, demonstrating reduced cell-cell coupling. Decreased coupling led to slow electrical propagation; conduction velocity in TBX18 NRCMs slowed by more than 50% relative to control (2.9 ± 0.5 versus 14.3 ± 0.9 cm/s). Taken together, TBX18 specifically and directly represses Cx43 transcript and protein levels. Cx43 suppression leads to significant electrical uncoupling, but the preservation of other gap junction proteins supports slow action potential propagation, recapitulating a key phenotypic hallmark of the SAN

RNA sequencing of mouse sinoatrial node reveals an upstream regulatory role for Islet-1 in cardiac pacemaker cells.

RationaleTreatment of sinus node disease with regenerative or cell-based therapies will require a detailed understanding of gene regulatory networks in cardiac pacemaker cells (PCs).ObjectiveTo characterize the transcriptome of PCs using RNA sequencing and to identify transcriptional networks responsible for PC gene expression.Methods and resultsWe used laser capture microdissection on a sinus node reporter mouse line to isolate RNA from PCs for RNA sequencing. Differential expression and network analysis identified novel sinoatrial node-enriched genes and predicted that the transcription factor Islet-1 is active in developing PCs. RNA sequencing on sinoatrial node tissue lacking Islet-1 established that Islet-1 is an important transcriptional regulator within the developing sinoatrial node.Conclusions(1) The PC transcriptome diverges sharply from other cardiomyocytes; (2) Islet-1 is a positive transcriptional regulator of the PC gene expression program

RNA Sequencing of Mouse Sinoatrial Node Reveals an Upstream Regulatory Role for Islet-1 in Cardiac Pacemaker Cells

RATIONALE: Treatment of sinus node disease with regenerative or cell-based therapies will require a detailed understanding of gene regulatory networks in cardiac pacemaker cells (PCs). OBJECTIVE: To characterize the transcriptome of PCs using RNA sequencing, and to identify transcriptional networks responsible for PC gene expression. METHODS AND RESULTS: We used laser capture micro-dissection (LCM) on a sinus node reporter mouse line to isolate RNA from PCs for RNA sequencing (RNA-Seq). Differential expression and network analysis identified novel SAN-enriched genes, and predicted that the transcription factor Islet-1 (Isl1) is active in developing pacemaker cells. RNA-Seq on SAN tissue lacking Isl1 established that Isl1 is an important transcriptional regulator within the developing SAN. CONCLUSIONS: (1) The PC transcriptome diverges sharply from other cardiomyocytes; (2) Isl1 is a positive transcriptional regulator of the PC gene expression program