The cardiac conduction system is a network of cells responsible for the rhythmic and coordinated excitation of the heart. Components of the murine conduction system, including the peripheral Purkinje fibers, are morphologically indistinguishable from surrounding cardiomyocytes, and a paucity of molecular markers exists to identify these cells. The murine conduction system develops in close association with the endocardium. Using the recently identified CCS-lacZ line of reporter mice, in which lacZ expression delineates the embryonic and fully mature conduction system, we tested the ability of several endocardial-derived paracrine factors to convert contractile cardiomyocytes into conduction-system cells as measured by ectopic reporter gene expression in the heart. In this report we show that neuregulin-1, a growth and differentiation factor essential for ventricular trabeculation, is sufficient to induce ectopic expression of the lacZ conduction marker. This inductive effect of neuregulin-1 was restricted to a window of sensitivity between 8.5 and 10.5 days postcoitum. Using the whole mouse embryo culture system, neuregulin-1 was shown to regulate lacZ expression within the embryonic heart, whereas its expression in other tissues remained unaffected. We describe the electrical activation pattern of the 9.5-days postcoitum embryonic mouse heart and show that treatment with neuregulin-1 results in electrophysiological changes in the activation pattern consistent with a recruitment of cells to the conduction system. This study supports the hypothesis that endocardial-derived neuregulins may be the major endogenous ligands responsible for inducing murine embryonic cardiomyocytes to differentiate into cells of the conduction system.

Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than previously published methods.

Background Notch signaling in vascular smooth muscle precursors is required for smooth muscle differentiation. Jagged1 expression on endothelium activates Notch in vascular smooth muscle precursors including those of neural crest origin to initiate the formation of a smooth muscle layer in a maturing blood vessel. Methods and Results Here, we show that Jagged1 is a direct Notch target in smooth muscle, resulting in a positive feedback loop and lateral induction that propagates a wave of smooth muscle differentiation during aortic arch artery development. In vivo, we show that Notch inhibition in cardiac neural crest impairs Jagged1 mRNA expression and results in deficient smooth muscle differentiation and resultant aortic arch artery defects. Ex vivo, Jagged1 ligand activates Notch in neural crest explants and results in activation of Jagged1 mRNA, a response that is blocked by Notch inhibition. We examine 15 evolutionary conserved regions within the Jagged1 genomic locus and identify a single Notch response element within the second intron. This element contains a functional Rbp-J binding site demonstrated by luciferase reporter and chromatin immunoprecipitation assays and is sufficient to recapitulate aortic arch artery expression of Jagged1 in transgenic mice. Loss of Jagged1 in neural crest impairs vascular smooth muscle differentiation and results in aortic arch artery defects. Conclusions Taken together, these results provide a mechanism for lateral induction that allows for a multilayered smooth muscle wall to form around a nascent arterial endothelial tube and identify Jagged1 as a direct Notch target.

Heart failure represents a major cause of morbidity and mortality worldwide. Single-cell transcriptomics have revolutionized our understanding of cell composition and associated gene expression. Through integrated analysis of single-cell and single-nucleus RNA-sequencing data generated from 27 healthy donors and 18 individuals with dilated cardiomyopathy, here we define the cell composition of the healthy and failing human heart. We identify cell-specific transcriptional signatures associated with age and heart failure and reveal the emergence of disease-associated cell states. Notably, cardiomyocytes converge toward common disease-associated cell states, whereas fibroblasts and myeloid cells undergo dramatic diversification. Endothelial cells and pericytes display global transcriptional shifts without changes in cell complexity. Collectively, our findings provide a comprehensive analysis of the cellular and transcriptomic landscape of human heart failure, identify cell type-specific transcriptional programs and disease-associated cell states and establish a valuable resource for the investigation of human heart failure.