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Background: The human heart requires a complex ensemble of specialized cell types to perform its essential function. A greater knowledge of the intricate cellular milieu of the heart is critical to increase our understanding of cardiac homeostasis and pathology. As recent advances in low-input RNA sequencing have allowed definitions of cellular transcriptomes at single-cell resolution at scale, we have applied these approaches to assess the cellular and transcriptional diversity of the nonfailing human heart. Methods: Microfluidic encapsulation and barcoding was used to perform single nuclear RNA sequencing with samples from 7 human donors, selected for their absence of overt cardiac disease. Individual nuclear transcriptomes were then clustered based on transcriptional profiles of highly variable genes. These clusters were used as the basis for between-chamber and between-sex differential gene expression analyses and intersection with genetic and pharmacologic data. Results: We sequenced the transcriptomes of 287 269 single cardiac nuclei, revealing 9 major cell types and 20 subclusters of cell types within the human heart. Cellular subclasses include 2 distinct groups of resident macrophages, 4 endothelial subtypes, and 2 fibroblast subsets. Comparisons of cellular transcriptomes by cardiac chamber or sex reveal diversity not only in cardiomyocyte transcriptional programs but also in subtypes involved in extracellular matrix remodeling and vascularization. Using genetic association data, we identified strong enrichment for the role of cell subtypes in cardiac traits and diseases. Intersection of our data set with genes on cardiac clinical testing panels and the druggable genome reveals striking patterns of cellular specificity. Conclusions: Using large-scale single nuclei RNA sequencing, we defined the transcriptional and cellular diversity in the normal human heart. Our identification of discrete cell subtypes and differentially expressed genes within the heart will ultimately facilitate the development of new therapeutics for cardiovascular diseases.

Gene therapy carries the potential to treat more than 10,000 human monogenic diseases and benefit an even greater number of complex polygenic conditions. The repurposing of CRISPR/Cas9, an ancient bacterial immune defense system, into a gene-editing technology has armed researchers with a revolutionary tool for gene therapy. However, as the breadth of research and clinical applications of this technology continues to expand, outstanding technical challenges and ethical considerations will need to be addressed before clinical applications become commonplace. Here, we review CRISPR/Cas9 technology and discuss its benefits and limitations in research and the clinical context, as well as ethical considerations surrounding the use of CRISPR gene editing.

Background: Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. Methods: We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH. Results: Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses. Conclusions: Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.

Background— Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary arterioles, characterized by increased pulmonary arterial pressure and right ventricular failure. The cause of PAH is complex, but aberrant proliferation of the pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells is thought to play an important role in its pathogenesis. Understanding the mechanisms of transcriptional gene regulation involved in pulmonary vascular homeostasis can provide key insights into potential therapeutic strategies. Methods and Results— We demonstrate that the activity of the transcription factor myocyte enhancer factor 2 (MEF2) is significantly impaired in the PAECs derived from subjects with PAH. We identified MEF2 as the key cis-acting factor that regulates expression of a number of transcriptional targets involved in pulmonary vascular homeostasis, including microRNAs 424 and 503, connexins 37, and 40, and Krűppel Like Factors 2 and 4, which were found to be significantly decreased in PAH PAECs. The impaired MEF2 activity in PAH PAECs was mediated by excess nuclear accumulation of 2 class IIa histone deacetylases (HDACs) that inhibit its function, namely HDAC4 and HDAC5. Selective, pharmacological inhibition of class IIa HDACs led to restoration of MEF2 activity in PAECs, as demonstrated by increased expression of its transcriptional targets, decreased cell migration and proliferation, and rescue of experimental pulmonary hypertension models. Conclusions— Our results demonstrate that strategies to augment MEF2 activity hold potential therapeutic value in PAH. Moreover, we identify selective HDAC IIa inhibition as a viable alternative approach to avoid the potential adverse effects of broad spectrum HDAC inhibition in PAH.

Myocardial hypertrophy is an adaptive response to hemodynamic demands. Although angiogenesis is critical to support the increase in heart mass with matching blood supply, it may also promote a hypertrophic response. Previously, we showed that cardiac angiogenesis induced by placental growth factor (PlGF), promotes myocardial hypertrophy through the paracrine action of endothelium-derived NO, which triggers the degradation of regulator of G protein signaling 4 (RGS4) to activate the Akt/mTORC1 pathways in cardiomyocytes. Here, we investigated whether miRNAs contribute to the development of hypertrophic response associated with myocardial angiogenesis. We show that miR-182 is upregulated concurrently with the development of hypertrophy in PlGF mice, but not when hypertrophy was blocked by concomitant expression of PlGF and RGS4, or by PlGF expression in eNOS−/− mice. Anti-miR-182 treatment inhibits the hypertrophic response and prevents the Akt/mTORC1 activation in PlGF mice and NO-treated cardiomyocytes. miR-182 reduces the expression of Bcat2, Foxo3 and Adcy6 to regulate the hypertrophic response in PlGF mice. Particularly, depletion of Bcat2, identified as a new miR-182 target, promotes AktSer473/p70-S6KThr389 phosphorylation and cardiomyocyte hypertrophy. LV pressure overload did not upregulate miR-182. Thus, miR-182 is a novel target of endothelial-cardiomyocyte crosstalk and plays an important role in the angiogenesis induced-hypertrophic response.

Rationale: Sprouting angiogenesis is governed by the concept of tip/stalk cells that guides our understanding of the transition from vascular sprouting to maturation and ultimately quiescence. The VEGF and Notch signaling pathways have been extensively described in regulating the discrimination between these two cell populations. However, several additional tip and stalk cell specific genes have been identified. To date, unresolved questions remain, and our understanding of the mechanisms by which these signaling processes are integrated is incomplete. Objective: We set out to investigate novel mechanisms by which signaling pathways involving two G protein coupled receptors (GPCRs), expressed in a mutually exclusive fashion in the tip/stalk cell populations, are intricately linked in vascular development. Methods/Results: Using a combination of in vivo and in vitro techniques, we demonstrate the critical role of crosstalk between APLNR and CXCR4 in vascular maturation. We show robust flow induced expression of the stalk cell specific APLNR, that leads to marked suppression of CXCR4 expression, a mechanism to achieve tip cell restricted expression of the latter. Retinas from Apln (ligand), Aplnr (receptor) and endothelial specific Aplnr deleted mice show retarded vascular expansion, reduced vascularized area and fewer vascular branch points. These phenotypes are in part due to increased expression of Cxcr4 in Apln-/- and Aplnr-/- retinal vessels as Cxcr4 inhibition through a selective inhibitor can ameliorate the Aplnr phenotype. The crosstalk between the two GPCRs was found to involve a key shear responsive microRNA, miR-139-5p, which is upregulated by APLN/APLNR signaling and directly targets CXCR4 in endothelial cells. In accordance, Apln-/- and Aplnr-/- retinal endothelial cells showed depleted levels of miR-139-5p. Lastly, we demonstrate that atorvastatin, an HMG-CoA reductase inhibitor shown to enhance APLNR signaling, can induce miR-139-5p expression and rescue the vascular phenotypes associated with APLN/APLNR deficiency. Conclusions: These findings provide key mechanistic insights into a critical microRNA based crosstalk between two GPCR signaling cascades, which regulates important steps in vascular maturation.

We have read with great interest the article by Lathen and colleagues titled “The ERG–APLNR Axis Controls Pulmonary Venule Endothelial Proliferation in Pulmonary Veno-Occlusive Disease,” and congratulate the authors for their thorough approach using human samples and modern genetic techniques.1 However, there are 2 key points that, in our opinion, deserve attention. First, following a literature review, the authors conclude that “These studies point to the concept that Aplnr signaling has direct and unique effects on the venous circulation.” However, the majority of the cited studies do not describe venous-specific mechanisms. Cheng et al2 demonstrated that apelin administration decreased both the mean arterial …