key: cord-0786760-sj603x7m
authors: Lu, Dongchao; Chatterjee, Shambhabi; Xiao, Ke; Riedel, Isabelle; Wang, Yibin; Foo, Roger; Bär, Christian; Thum, Thomas
title: MicroRNAs targeting the SARS-CoV-2 entry receptor ACE2 in cardiomyocytes
date: 2020-09-03
journal: J Mol Cell Cardiol
DOI: 10.1016/j.yjmcc.2020.08.017
sha: 26819e78c8edab040b9eda40549c57614eb49fca
doc_id: 786760
cord_uid: sj603x7m

The World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19) as a public health emergency of international concern as more than 15 million cases were reported by 24th July 2020. Angiotensin-converting enzyme 2 (ACE2) is a COVID-19 entry receptor regulating host cell infection. A recent study reported that ACE2 is expressed in cardiomyocytes. In this study, we aimed to explore if there are microRNA (miRNA) molecules which target ACE2 and which may be exploited to regulate the SARS-CoV-2 receptor. Our data reveal that both Ace2 mRNA and Ace2 protein levels are inhibited by miR-200c in rat primary cardiomyocytes and importantly, in human iPSC-derived cardiomyocytes. We report the first miRNA candidate that can target ACE2 in cardiomyocytes and thus may be exploited as a preventive strategy to treat cardiovascular complications of COVID-19.

The novel coronavirus disease 2019 (COVID-19) outbreak occurred in December 2019 and in the following months this epidemic was declared as a global health emergency by the World Health Organization (WHO) with more than 600 thousand deaths reported till date [1] . Similar to SARS-CoV and the Middle East respiratory syndrome (MERS), severe symptoms such as acute respiratory distress syndrome (ARDS) and multiple organ failure were observed in nearly 20% patients suffering from COVID-19 infection [2] .

Angiotensin-converting enzyme 2 (ACE2) helps to maintain the balance of blood pressure and electrolyte in the human body. It also reduces the Angiotensin II levels in the circulation by suppressing the renin-angiotensin-aldosterone system conferring J o u r n a l P r e -p r o o f Journal Pre-proof anti-hypertensive effects [3] . Recently, it has also been reported as a receptor for the spike protein of SARS-CoV-2 and plays pivotal roles during the COVID-19 infection [4] . Notably, COVID-19 patients with severe symptoms also seem to suffer from various other health conditions including cardiovascular disease (CVD), hypertension and diabetes [5] . Importantly, ACE2 is expressed in cardiomyocytes and elevated in patients with heart diseases [6] . Thus, it is likely that the extent of COVID-19 infection gets more pronounced by ACE2 in patients with comorbidities, which in turn may cause additional myocardial damage. MiRNAs are highly conserved small non-coding RNAs which are ~20-22 nucleotides in length and can negatively regulate gene expression. Several studies have reported that miRNAs could modulate ACE2 expression in a variety of cell types and diseases [7] , but whether miRNA could be potential preventive target for SARS-CoV-2 is still unknown.

In our pervious review, we summarized the state of art of investigation and potential therapeutic strategies for COVID-19 patients with cardiac disease [8] . In this study, we investigate several in-silico identified miRNAs which could regulate ACE2 in vitro and therefore provide a potential strategy to develop novel therapeutic candidates for COVID-19.

As one of the main receptors of SARS-CoV-2 [4], ACE2 is considered as a potential target for COVID-19 prevention. We first examined the expression of Ace2 in different mouse organs. Ace2 showed higher expression in kidney, lung and heart when compared to liver or spleen ( Figure 1A ). To further investigate ACE2 expression pattern in different cell types (cardiomyocyte, fibroblast and endothelial cell) from human and rat were tested. Interestingly, Ace2 was highly expressed in neonatal rat cardiomyocytes (NRCMs) compared to neonatal rat cardiac fibroblasts (NRCFs) ( Figure 1B) . Similarly, ACE2 demonstrated higher expression in human induced J o u r n a l P r e -p r o o f Journal Pre-proof pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) compared to human cardiac fibroblasts and endothelial cells ( Figure 1C ). Our next aim was to identify miRNAs which could potentially regulate ACE2 expression. A bioinformatics search using TargetScan was applied to predict miRNAs binding to the three prime untranslated region (3'UTR) of Ace2. Amongst the in silico candidates, miR-429, -200b, -200c were selected for further validation based on the highest conservation for the 3'UTR binding site ( Figure 1D ). Collectively, these data suggest that cardiomyocytes are probably the major cell type which expresses ACE2 in the heart and miR-429, -200b and -200c may present as promising targets to regulate ACE2 expression.

To test this hypothesis, NRCMs were first transfected with miRNA mimics pre-miR- Figure 2E ). Collectively, these data provide direct evidence that miR-200c can regulate the expression of ACE2 in both rat and human cardiomyocytes.

In this study, high expression of Ace2 was observed in mouse kidney, lung and heart.

A recent study employed single cell sequencing analysis of heart tissue showing that ACE2 expression is enriched in the cardiomyocyte fraction and is further elevated specifically in cardiomyocytes of patients with heart disease [6]. Our findings reaffirm these observations that ACE2 is abundant in cardiomyocytes compared to other cardiac cell types. Strikingly, viral particles were observed in the endo-myocardial biopsy from patients infected with SARS-CoV-2 [10]. Another multi-organ autopsy study of COVID-19 patients revealed the presence of SARS-CoV-2 viral RNA in the heart tissue [11] . Surprisingly, the SARS-Cov-2 mRNA was found in the heart tissue biopsy of patients who had already recovered from SARS-CoV-2 infection [12] .

Moreover, several recent studies described that hiPSC-CMs can be efficiently infected with SARS-CoV-2 particles and such a platform can be further utilized for screening of potential anti-viral medicines [9, 13] 

Neonatal rat cardiomyocytes (NRCMs) and Neonatal rat cardiac fibroblasts (NRCFs) were isolated from one to three-day old rat pups by using the Neonatal Heart Dissociation Kit (Miltenyi). NRCMs were cultured in MEM (BioConcept) medium with 

RNA isolation from in vitro and mouse organ tissue was performed by using Trifast (Peqlab) as described in manufacturer's instructions. Isolated RNA (500ng) was reversed transcribed with random primer using iScript Select cDNA synthesis kit (Bio-Rad). Real-Time quantitative PCR was done with iQ SYBR Green mix (Bio-Rad) on C1000 Touch Thermocycler (Bio-Rad) using specific primer pairs. The specific primers for target genes are as follows (5' Cell pellets were lysed in 1X Cell lysis buffer (Cell Signaling) and isolated protein was measured by Bradford (Bio-Rad) method. 30 μg of protein was loaded for each sample on SDS-polyacrylamide gel to resolve the proteins. Proteins were transferred to polyvinylidene fluoride membrane in Mini PROTEAN Tetra cell (Bio-Rad). Specific proteins were identified by following antibodies: ACE2 (Proteintech), and Actb (Cell Signaling). HRP conjugated secondary antibody (Cell Signaling) was used for detection of bands. Band intensity was calculated by Image J software.

Luciferase reporter vector were constructed by using pMIR-Report plasmid (Ambion# Am5795) containing the binding site (or mutated) with miR-200c in 3'UTR of ACE2

(179-185). HEK 293T cells were transfected with wild type (WT) or mutated (MUT) luciferase reporter plasmid with pre-miR-negative control#2 (Life Technologies), pre-miR-200c (Life Technologies, PM11714) and beta-Gal control plasmid (Promega).

Luciferase activity was performed according to manufacturer's instructions to study the direct binding of miR-200c in 3'UTR of ACE2 normalizing with beta-Gal values via utilizing beta-Gal kit (Promega) and Luciferase Assay System (Promega).

All data were analyzed using GraphPad Prism software. Data are presented as mean±SEM, and an unpaired 2-tailed t-test was performed to calculate the significance between groups

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