The coronavirus disease 2019 caused by SARS-CoV-2 infections imposes a major threat for the world's healthcare systems and is leading to thousands of deaths. Angiotensin-converting enzyme 2 (ACE2) has been identified as a potential receptor for SARS coronavirus 1 and is also considered the main receptor for SARS-CoV-2. 2 SARS-CoV-2 binds to ACE2 via its glycosylated outer membrane spike proteins. ACE2 is highly expressed in the lung and heart, and is known for its vital role in the cardiovascular system. 3-5 Although SARS-CoV-2 mainly invades alveolar epithelial cells, it can also cause myocardial injury, as assessed by increased troponin T and NT-proBNP levels accompanying increased cardiovascular symptoms in COVID-19-infected patients. 6,7 It is unclear whether elevated biomarkers of cardiac injury (or long-term effects on the cardiovascular system) are directly caused by viral infection of cardiac tissue or are secondary to hypoxia and systemic inflammation. However, patients with underlying cardiovascular disease represent a significant proportion of the patients who may suffer from a severe course after COVID-19 infection. 8 This situation may be aggravated by findings showing that ACE inhibitors, which are often used to treat cardiovascular diseases, augment the expression of the SARS-CoV-2 receptor ACE2 in lung cells. 9 This is probably mediated by an effect on angiotensin II, which is known to reduce ACE2 expression. 9 Thus, ACE inhibition decreases angiotensin II, leading to an indirect up-regulation of ACE2. 9 The effect of angiotensin II receptor blockers (ARBs), which primarily target the angiotensin receptor 1, is unclear. One may speculate that ARBs indirectly reduce ACE2 levels by augmenting free angiotensin II levels, which in turn is expected to downregulate ACE2 via activating the angiotensin receptor 2. However, the effect of the two different treatments on the expression of ACE2 in the heart requires further investigation.Therefore, we used single nuclei RNA sequencing to determine the expression of ACE and ACE2 in the different cell types of the human heart. Gene expression signatures were detected in cardiac tissues of five patients with aortic stenosis (AS) and two patients with heart failure with reduced ejection fraction (HFrEF) ( Figure 1A) and compared with with samples of one healthy donor heart (age: 63 years, male) that was not used for transplantation. After single nuclei RNA sequencing, data were pooled, and unsupervised clustering was performed with a total of 57 601 nuclei. We found 18 distinct clusters. Using cell type-specific gene markers, major cell types were annotated, including cardiomyocytes (six clusters), fibroblasts (one cluster), endothelial cells (three clusters), leucocytes (two clusters), pericytes (one cluster), and smooth muscle cells (one cluster) ( Figure 1B-D). ACE2 was expressed in cardiomyocytes (Cluster 0 and 1) and mural cells, particularly pericytes (Cluster 4), and was detected at a lower expression level in fibroblasts, endothelial cell, and leucocytes...
Background: Dilated cardiomyopathy (DCM) is a leading cause of death in children with heart failure. The outcome of pediatric heart failure treatment is inconsistent and large cohort studies are lacking. Progress may be achieved through personalized therapy that takes age- and disease-related pathophysiology, pathology and molecular fingerprints into account. We present snRNA-seq from pediatric DCM patients as the next step in identifying cellular signatures. Methods: We performed single nuclei RNA sequencing with heart tissues from six children with DCM with an age of 0.5, 0.75, 5, 6, 12 and 13 years. Unsupervised clustering of 18,211 nuclei led to the identification of 14 distinct clusters with 6 major cell types. Results: The number of nuclei in fibroblast clusters increased with age in DCM patients, a finding that was confirmed by histological analysis and was consistent with an age-related increase in cardiac fibrosis quantified by cardiac magnetic resonance imaging. Fibroblasts of DCM patients over 6 years of age showed a profoundly altered gene expression pattern with enrichment of genes encoding fibrillary collagens, modulation of proteoglycans, switch in thrombospondin isoforms and signatures of fibroblast activation. Additionally, a population of cardiomyocytes with a high pro-regenerative profile was identified in infant DCM patients, but was absent in > 6-year-old children. This cluster showed high expression of cell cycle activators such as cyclin D family members, increased glycolytic metabolism and antioxidative genes and alterations in ß-adrenergic signaling genes. Conclusions: Novel insights into the cellular transcriptomes of hearts from pediatric DCM patients provide remarkable age-dependent changes in the expression patterns of fibroblast and cardiomyocyte genes with less fibrotic but enriched pro-regenerative signatures in infants.
The mechanism underlying dilated cardiomyopathy (DCM) in children without a known genetic disorder are unclear. In contrast to adult DCM patients, there is an unmet need for therapeutic options that improve survival in pediatric DCM. Therefore, we performed single nuclei RNA sequencing (snRNA-seq) from heart tissue obtained from children undergoing heart transplantation due to severe heart failure. We processed heart tissue from 6 children with DCM (EF: 18.67±2.11%) of an age of 0.5, 0.75, 5, 6, 12 and 13 years (y). After snRNA-seq, unsupervised clustering was performed identifying 8 major cell types, including cardiomyocytes (CM), fibroblasts (FB), endothelial cells, leukocytes, pericytes, smooth muscle cells, neuronal-like cells and an endothelial-fibroblast-like cluster. Relative numbers of FB clusters correlated with increasing age of the children which was clinically validated by measuring late enhancement (LE) with cardiac magnetic resonance imaging in 68 pediatric DCM patients. The mean age of patients with LE was 5.86±0.53y vs. 2.36±0.53y in patients without LE (p<0.05). Further analysis of unique highly expressed genes (DEGs) between the 3 age groups identified a profound alteration of gene expression in FB clusters. FBs of explants of <1y old patients showed high expression of anti-fibrotic, development and remodeling associated genes. In contrast, FBs of 12–13y old children highly expressed pro-fibrotic and FB activation associated genes as transforming growth factor beta binding protein (6.63 fold), cytochrome P450 1B1 (3.79 fold), and periostin (7.67 fold) (all p<0.05). Moreover, we observed a switch in collagen expression patterns and in thrombospondin isoforms (from THBS1 to THBS4). Furthermore, our analysis revealed most profound transcriptional changes in CMs. We identified a cluster of CMs with a pro-regenerative profile in <1y old patients, which could not be detected during adolescence. This CM cluster showed high expression of genes associated with proliferation (e.g. cyclin D2), glycolytic metabolism and anti-oxidant markers. Increased cyclin D2 was confirmed by immunostaining (1.43 fold higher in <1y vs. 12–13y). Since all of these gene expression patterns might be affected by the underlying disease of the pediatric heart recipients, we explored their expression in FBs and CMs of postnatal vs. adult mice. Importantly, we could recapitulate the vast majority of the findings from humans in the mice experiments. Together, these data demonstrate an age-dependent decrease in CM numbers concomitant with increased FBs in pediatric DCM. FBs of <1y old pediatric patients revealed a distinct collagen expression profile and showed lower levels of pro-fibrotic genes. CMs of <1y old donors where characterized with a regeneration enabling gene expression profile, which include pro-proliferative genes. The expression patterns of the CMs indicates, that regeneration might also occur in humans during the firs year of life. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): Dr. Rolf M. Schwiete Stiftung, DZHK
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