33Background: COVID-19 patients with comorbidities such as hypertension or heart failure (HF) are 34 associated with poor clinical outcomes. Angiotensin-converting enzyme 2 (ACE2), the critical enzyme 35 for SARS-CoV-2 infection, is broadly expressed in many organs including heart. However, the cellular 36 distribution of ACE2 in the human heart, particularly the failing heart is unknown. 37Methods: We analyzed single-cell RNA sequencing (scRNA-seq) data in both normal and failing hearts, 38 and characterized the ACE2 gene expression profile in various cell subsets, especially in cardiomyocyte : medRxiv preprint 3 subsets, as well as its interaction with gene networks relating to various defense and immune responses 40 at the single cell level. 41Results: The results demonstrated that ACE2 is present in cardiomyocytes (CMs), endothelial cells, 42 fibroblasts and smooth muscle cells in the heart, while the number of ACE2-postive (ACE2+) CMs and 43 ACE2 gene expression in these CMs are significantly increased in the failing hearts. Interestingly, both 44 brain natriuretic peptides (BNP) and atrial natriuretic peptide (ANP) are significantly up-regulated in 45 the ACE2+ CMs. Further analysis shows that ANP, BNP and ACE2 may form a negative feedback loop 46 with a group of genes associated with the development of heart failure. To our surprise, we found that 47

RATIONALE: Myocardial infarction (MI) elicits cardiac fibroblast activation and ECM (extracellular matrix) deposition to maintain the structural integrity of the heart. Recent studies demonstrate that Fap (fibroblast activation protein)—a prolyl-specific serine protease—is an important marker of activated cardiac fibroblasts after MI. OBJECTIVE: This study aims to test whether Fap is a critical regulator of cardiac repair after MI and to uncover the underlying cellular and molecular mechanisms for better treatment of MI. METHODS AND RESULTS: We found that Fap is upregulated in patient cardiac fibroblasts after cardiac injuries, while plasma Fap is downregulated and functions as a prognostic marker for cardiac repair. Genetic or pharmacological inhibition of Fap in mice significantly improved cardiac function after MI. Histological and transcriptomic analyses showed that Fap inhibition leads to increased angiogenesis in the peri-infarct zone, which promotes ECM deposition and alignment by cardiac fibroblasts and prevents their overactivation, thereby limiting scar expansion. Mechanistically, we found that BNP (brain natriuretic peptide) is a novel substrate of Fap that mediates postischemic angiogenesis. Fap degrades BNP to inhibit vascular endothelial cell migration and tube formation. Pharmacological inhibition of Fap in Nppb (encoding pre-proBNP) or Npr1 (encoding the BNP receptor)-deficient mice showed no cardioprotective effects, suggesting that BNP is a physiological substrate of Fap. CONCLUSIONS: This study identifies Fap as a negative regulator of cardiac repair and a potential drug target to treat MI. Inhibition of Fap stabilizes BNP to promote angiogenesis and cardiac repair.

Aims: COVID-19 patients with comorbidities such as hypertension or heart failure (HF) are associated with poor clinical outcomes. The cellular distribution of Angiotensin-converting enzyme 2 (ACE2), the critical enzyme for SARS-CoV-2 infection, in the human heart is unknown. We explore the underlying mechanism that leads to increased susceptibility to SARS-CoV-2 in patients with cardiovascular diseases and patients of cardiac dysfunction have increased risk of multi-organ injury compared with patients of normal cardiac function.Methods and Results: We analyzed single-cell RNA sequencing (scRNA-seq) data in both normal and failing hearts. The results demonstrated that ACE2 is present in cardiomyocytes (CMs) and non-CMs, while the number of ACE2-postive (ACE2+) CMs and ACE2 gene expression in these CMs are significantly increased in the failing hearts. Interestingly, both brain natriuretic peptides (BNP) and atrial natriuretic peptide (ANP) are significantly up-regulated in the ACE2+ CMs, which is consistent with other studies that ACE2, ANP, and BNP increased in HF patients. We found that genes related to virus entry, virus replication and suppression of interferon-gamma signaling are all up-regulated in failing CMs, and the increase was significantly higher in ACE2+ CMs, suggesting that these CMs may be more vulnerable to virus infection. As the level of expression of both ACE2 and BNP in CMs were up-regulated, we further performed retrospective analysis of the plasma BNP levels and clinical outcomes of 91 COVID-19 patients from a single-center. Patients with higher plasma BNP were associated with significantly higher mortality and expression levels of inflammatory and infective markers.Conclusion: In the failing heart, the upregulation of ACE2 and virus infection associated genes could potentially facilitate SARS-CoV-2 virus entry and replication in these vulnerable cardiomyocyte subsets. COVID-19 patients with higher plasma BNP levels had poorer clinical outcomes. These observations may allude to a potential regulatory association between ACE2 and BNP in mediating myocarditis associated with COVID-19.