key: cord-0868001-xy8sm6ma
authors: Bos, J. Martijn; Hebl, Virginia B.; Oberg, Ann L.; Sun, Zhifu; Herman, Daniel S.; Teekakirikul, Polakit; Seidman, J. G.; Seidman, Christine E.; dos Remedios, Cristobal G.; Maleszewski, Joseph J.; Schaff, Hartzell V.; Dearani, Joseph A.; Noseworthy, Peter A.; Friedman, Paul A.; Ommen, Steve R.; Brozovich, Frank V.; Ackerman, Michael J.
title: Marked Up-Regulation of ACE2 in Hearts of Patients with Obstructive Hypertrophic Cardiomyopathy: Implications for SARS-CoV-2-Mediated COVID-19
date: 2020-04-28
journal: Mayo Clin Proc
DOI: 10.1016/j.mayocp.2020.04.028
sha: 155105d7f2ae165512a1466dd5e6c0fd6eb59c29
doc_id: 868001
cord_uid: xy8sm6ma

Abstract Objective To explore the transcriptomic differences between patients with hypertrophic cardiomyopathy (HCM) and controls. Patients and Methods RNA was extracted from cardiac tissue flash frozen at therapeutic surgical septal myectomy for 106 patients with HCM and from 39 healthy donor hearts. Expression profiling of 37,846 genes was performed using the Illumina Human HT-12v3 Expression BeadChip. All HCM patients were genotyped for pathogenic variants causing HCM. Technical validation was performed using quantitative real-time PCR (qRT-PCR) and Western blot. This study was started on January 1, 1999 and final analysis was completed on April 20, 2020. Results Overall, 22% of the transcriptome (8443 genes) was expressed differentially between HCM and control tissues. Analysis by genotype revealed that gene expression changes were similar among genotypic subgroups of HCM, with only 4-6% of the transcriptome exhibiting differential expression between genotypic subgroups. qRT-PCR confirmed differential expression in 92% of tested transcripts. Notably, in the context of COVID-19, the transcript for ACE2, a negative regulator of the angiotensin system, was the single most up-regulated gene in HCM (fold-change 3.53, q-value=1.30x10-23), which was confirmed with qRT-PCR in triplicate (fold-change 3.78; p=5.22x10-4), and Western blot confirmed a >5-fold over-expression of ACE2 protein (fold-change 5.34, p=1.66x10-6). Conclusions Over 20% of the transcriptome is expressed differentially between HCM and control tissues. Importantly, ACE2 was the most up-regulated gene in HCM indicating perhaps the heart’s compensatory effort to mount an anti-hypertrophic, anti-fibrotic response. However, given that the SARS-CoV-2 uses ACE2 for viral entry, this 5-fold increase in ACE2 protein may confer increased risk for COVID-19 manifestations and outcomes in patients with increased ACE2 transcript expression and protein levels in the heart.

Hypertrophic cardiomyopathy (HCM) affects approximately 1 in 500 individuals 1 and is among the leading causes of identifiable sudden cardiac death (SCD) in the young. 2 HCM is often a genetic disease, typically with autosomal dominant inheritance, that is defined clinically as cardiac hypertrophy without physiologic explanation. Hundreds of pathogenic variants in many HCM-susceptibility genes have been identified, most of which encode components of the sarcomere. [3] [4] [5] [6] [7] [8] [9] [10] [11] However, genetic tests are negative in approximately 50% of all unrelated patients with HCM that is diagnosed by clinical studies. 4 Additionally, the transcriptional changes that cause and result from HCM, with and without pathogenic variants, remains largely unknown as prior studies analyzed data from small numbers of patients. 5, 6 To better identify common transcriptional changes that represent fundamental, and heretofore unrecognized, pathogenic responses of human HCM, we performed transcriptome analysis of human HCM tissues.

We designed a case-control study to identify the mRNAs differentially expressed in HCMaffected myocardium versus control myocardium. All patients signed informed consent, and protocols were approved by Mayo Clinic's Institutional Review Board or the Human Research Ethics Committee of the University of Sydney. This study was started on January 1, 1999 and final analysis was completed on April 20, 2020.

All patients undergoing therapeutic surgical septal myectomy for symptomatic relief of obstructive HCM between January 1, 1999 and December 31, 2010 were eligible for inclusion in this study. The diagnosis of HCM was made by an experienced cardiologist from Mayo Clinic's HCM Clinic based on physical exam, ECG, and echocardiographic/cardiac MRI findings.

Diagnosis was corroborated by histologic examination of the patient's surgical septal myectomy specimen. A representative portion of myectomy specimen was flash frozen at the time of excision and subsequently stored at -80 o C. Data on patient age, sex, age at diagnosis, New York

Heart Association (NYHA) classification, blood pressure, heart rate, family history of HCM, and family history of SCD were extracted from each patient's electronic medical record.

Echocardiographic parameters were extracted from each patient's pre-operative echocardiography study. Degree of endocardial and interstitial fibrosis was assessed semiquantitatively, at the time of resection, by a cardiovascular pathologist (JJM).

A cohort of control tissue was procured from the University of Sydney consisting of donor hearts for which there was not a suitable transplant recipient. A normal phenotype had been confirmed by cardiac examination, ECG, and echocardiogram obtained within 24 hours prior to explantation.

DNA was extracted from the HCM myectomy and control donor heart tissues using the Qiagen PureGene DNA Purification Kit (Qiagen, Inc.) according to the manufacturer's protocol.

Briefly, cells were lysed with detergent, RNA was removed using an RNase enzyme, proteins were removed by salt precipitation, and DNA was recovered with alcohol precipitation.

Damaging variants in 10 genes implicated in sarcomeric HCM (ACTC1, MYBPC3, MYH6,   MYH7, MYL2, MYL3, TNNC1, TNNI3, TNNT2, TPM1 ) and 3 genes known to mimic HCM (GLA, LAMP2, PRKAG2) were studied using filter-based hybridization capture, as described previously. 7 

The tissue sections were de-paraffinized in xylene, dipped in decreasing concentrations of ethyl alcohol, and then rehydrated in distilled water. Antigen retrieval for ACE2 was performed by placing slides in preheated Citrate as the retrieval solution in a steamer at 98°C for 40 minutes. and xylene were performed prior to permanent coverslipping. All slides were graded by a cardiac pathologist (JJM), and graded for intensity (0 = no staining, 1+ = dot-like sarcoplasmic staining, 2+=diffuse sarcoplasmic staining) and distribution (scoring 0 = 0% of cells, 1 = < 25%, 2 = 26-75% and 3 > 75% of cells) of ACE2 staining.

Specimens were allocated randomly to arrays using randomized block methods in order to avoid confounding of biological and experimental effects. Illumina BeadStudio Version 3.1.3 with gene expression module 3.4 was used to process raw data without background correction and normalization. The gene level expression data was exported and analyzed in R (http://www.rproject.org/). Briefly, the un-normalized raw data was first log2 transformed and evaluated for potential outlier samples and bead chip effects by graphic and dimension reduction approaches (density plot, M-A plot, and principal components analysis).

Outlier samples were excluded for further analyses and the remaining good samples were normalized together using fastlo 10 , a model-based, intensity-dependent, normalization method that produces results essentially the same as those from cyclic loess 10 , but in a fraction of the time. Gene level expression was compared between HCM and normal tissue or between genotype subgroups overall followed by pair-wise contrasts via ANOVA linear models together with false discovery rates (FDR) 11 . Genes with a FDR q-value < 0.05 were considered statistically significant for HCM and normal tissue comparison.

For the pair-wise comparisons between different genotype subgroups of HCM, a p-value < 0.05 was considered statistically significant. A less stringent cut-off was used here in an effort to elucidate differences between two similar disease conditions, acknowledging the fact that the chance of a false positive result is higher. A cut-off for biological significance was set as an absolute fold change > 1.5 between genotype subsets of HCM.

qRT-PCR data was analyzed after calculating 2 -∆Ct for the average ∆Ct value (transcript of interest minus GAPDH control) for the triplicate replicates of each sample. A one-tailed t-test was used. A fold-change was calculated by the 2 -∆∆Ct method, 12 taking 2 -∆Ct for the overall average of all cases divided by 2 -∆Ct for the overall average of all controls.

All HCM participants (n=121) provided written informed consent to participate in this Mayo 

Notably, the ACE2-encoded angiotensin I/angiotensin II converting enzyme subtype 2, an important counter-regulator of the renin-angiotensin-aldosterone system (RAAS) involved in hypertrophy, fibrosis, and vasoconstriction, was the most up-regulated gene in HCM tissues (3.5fold increase vs. controls, confirmed by qRT-PCR; Figures 1 and 2) . Western blot analyses indicated 5.3-fold increased ACE2 protein expression compared to control (p<0.001; Figure   3A ). Additionally, immunohistochemistry cardiomyocyte staining of cardiac myectomy tissue from 14 HCM patients showed significantly increased ACE2-antibody staining intensity (p=0.002) and distribution (p<0.001) of ACE2 protein compared to 8 control samples ( Figure   3B ).

Interestingly, ACE2 is located on the X-chromosome and therefore sex-differences could be expected. Thus, we performed a sex-corrected analysis of our expression data for ACE2 and observed that there was still a 3. 

Subgroup analyses were performed comparing the three largest genotypic subsets; MYBPC3+, MYH7+, and genotype negative-HCM. Pair-wise comparisons of MYH7+ and MYBPC3+, MYH7+ and genotype negative-HCM, and MYBC3+ and genotype negative-HCM were performed. There were no gene expression changes that met a false discovery rate q-value < 0.1, suggesting a high probability of false positive findings.

However, given that the disease states under comparison are, as previously documented, phenotypically indistinguishable (at clinical, gross anatomic, and microscopic levels), we hypothesized that gene expression changes due to genotype subgroup might be subtle.

Therefore, we accepted a higher false positive rate in order to reveal potentially important differences in gene expression. After adjusting to meet a p-value < 0.05, there were ~1000 -2000 gene expression changes in each subgroup comparison totaling approximately 4-6% of genes tested. Most differentially expressed genes had low, absolute fold changes and high false discovery rate q-value. The overall 3-way comparison of gene expression among these 3 genotypic subgroups of HCM revealed that 94% of gene expression changes were shared. Since additional differences between genotypic subgroups were subtle, we have summarized them in the Supplemental data.

Our analysis of 106 HCM myectomy tissues and 39 control tissues identified 8443 differentially expressed genes, 22% of all genes analyzed. These genes participate in 1075 Molecular

Functions and 4272 Processes as defined by the Gene Ontology Consortium. Remarkably, the most differentially expressed genes were not previously identified in hypertrophic pathways.

Whether this reflects the relatively small sizes of study groups, variability in age and treatments, background genotypes, or other factors is unknown. Previous genotype-phenotype studies have not identified a gene-specific profile. [16] [17] [18] Nevertheless, we suggest that the newly identified differentially expressed genes warrant further investigation.

Given the current and devastating COVID-19 pandemic (> 2.5 million confirmed cases) that has claimed over 172,000 lives worldwide in less than 4 months (04/21/2020), it was noteworthy that the most up-regulated gene in HCM samples was ACE2 (3.5-fold; q-value = 1.30x10 -23 ). ACE2

protein was also increased > 5-fold by Western blot analyses (p< 0.001). ACE2 encodes angiotensin converting enzyme subtype 2, which has important compensatory roles in modulating excessive activation of the RAAS as occurs in hypertension (HTN), congestive heart failure (CHF), and atherosclerosis. In its soluble form, ACE2 acts as a carboxypeptidase cleaving the pro-hypertrophic polypeptides angiotensin I and angiotensin II to angiotensin 1-9 and angiotensin 1-7 respectively, thereby producing counter-regulating, vasodilating, and potentially anti-hypertrophic/anti-fibrotic polypeptides ( Figure 3C) . 19 Accordingly, we speculate that up-regulation of both ACE2 transcript and ACE2 protein levels might be a compensatory, counter-regulatory signaling response ('patho-responsive') in patients with obstructive HCM. This was echoed in a recent paper by Liu et al. in which the investigators studied the transcriptome of HCM mouse models and found that pro-fibrotic pathways initiated by increase of endothelin-1(ET1) were the main drivers of HCM pathogenesis in mice through miRNA-29 and TGFβ signaling. However, using our preliminary microarray data that was derived from the patients in our study and made publicly available (GSE36961), this differential expression of the TGFβ-signaling genes was not observed. Instead, the increased transcript levels of ACE2 were noted prompting the speculation that ACE2 overexpression might be a compensatory response. 20 Lastly, ACE2, located in the X-chromosome, was upregulated significantly in female patients with HCM (1.33 fold compared to males: p<0.01), and while only one gene, findings like these could start to shed light and form the basis to understanding some of the underlying (epigenetic) contributions to the significantly different outcomes that are observed for women with HCM. [21] [22] [23] 

Beyond the potential relevance of ACE2 expression in HCM hearts and its disease pathogenesis, in its membrane-bound state, ACE2 plays an important role as a functional receptor required for viral entry and subsequent viral replication for the SARS-CoV family of viruses 24, 25 , and thereby may in fact contribute to the increased morbidity and mortality from SARS-CoV-2 in adult patients with a variety of heart diseases. 26 The currently endemic SARS-CoV-2 is a member of the SARS-family of coronaviruses that bind to membrane-bound ACE2 via its viral spike protein. 27 ACE2 is expressed in many other tissues including the intestinal and vascular epithelium kidneys, and the heart. However, expression in cardiomyocytes is quite low. In fact, in a study of cardiac cell samples from donor hearts, both ACE2 and TMPRSS2 showed highest expression in the heart tissue's pericyte sub-population rather than in the cardiomyocytes. 28 Nevertheless, widespread expression of ACE2 may contribute to multi-organ dysfunction seen in patients with 30 The marked 5-fold increase in ACE2 protein in HCM may provide a mechanism to explain higher rates of severe outcomes in COVID19 patients who also have cardiovascular comorbidities, as well as the direct cardiac damage caused by SARS-CoV-2 infections. While the incidence of COVID-19 seems highest in the elderly or immunocompromised, a large number of affected patients and those requiring hospitalization suffer from significant co-morbidities, including highly prevalent cardiovascular diseases, such as HTN or CHF. A large meta-analysis of over 46,000 patients in China showed the most common co-morbidities were HTN (17±7%, 95% CI 14 -22%), diabetes mellitus (8±6% 95% CI 6-11%), and cardiovascular disease (5±4%, 95% CI 4-7%) 31 . These percentages were much higher in patients requiring hospitalization or even ICU admission. In two separate inpatient studies, pre-existing HTN was present in 30%

(and up to 60% for ICU or non-surviving patients) as were concomitant cardiovascular disease (15%, increased to 13-25% in ICU or non-surviving patients). 32, 33 There is mounting evidence for cardiotropism with SARS-CoV-2 infection and direct cardiac toxicity. 32 The effect of ACE (ACEi) inhibitors or angiotensin receptor blockers (ARBs) is under active investigation. Although initial reports suggested potentially worse outcomes in patients with COVID-19 who were on ACE inhibitors, 35, 36 a subsequent review article concluded that there was insufficient evidence for this claim prompting all major cardiac societies to advise that heart disease patients treated with these medications should continue them. 37 On the other hand, ARBs could attenuate the impact of SARS-CoV-2 by blocking the damaging effects resulting from a viral-mediated decrease of ACE2 and subsequent increase of damaging angiotensin II. In fact, experimental studies of the related virus, SARS-CoV, showed that down-regulation of ACE2 exacerbated lung injury, and treatment with the ARB losartan mitigated these effects. 24 Furthermore, a large study among 1128 patients with HTN and diagnosed with COVID-19

showed that unadjusted mortality rate was significantly lower in those whose HTN was treated with ACEi/ARBs (3.7% vs. 9.8%; p = 0.001). This risk remained consistently lower when performed as a propensity score-matched analysis with adjustment of imbalanced variables, such as age gender, co-morbidities, and in-hospital medications (adjusted HR, 0.37; 95% CI, 0.15-0.89; p = 0.03). 38 In light of these findings, a clinical trial has been launched testing the effect of losartan in study eligible patients with COVID-19 (NCT04312009, Figure 4 ). In addition, clinical grade human recombinant soluble ACE2 (hrsACE2) can block early stages of SARS-CoV-2 infection significantly by preventing the virus from entering the cell 39 highlighting the crucial and dual role of ACE2 in health and disease (Figure 4) . A clinical trial to test hrsACE2 in patients was commenced recently in Europe (EudraCT2020-001172-15).

We studied only HCM patients undergoing therapeutic surgical septal myectomy, representing one phenotypic subset of HCM. However, this study design had the inherent bias as the procedure provided the only means to ethically obtain heart tissue. It therefore remains to be determined whether ACE2 elevation was a marker of this state of the disease alone (obstructive HCM) and whether it persisted following the relief of LVOTO. Whether ACE2 elevation is present in the hearts of patients with non-obstructive HCM, other cardiomyopathies, HTN, or other forms of acquired heart disease is unknown.

To overcome the logistical and ethical issue of obtaining healthy heart tissue, we used a common source for healthy heart tissue 40-44 as controls, both due to the scarcity of this reagent and the need for these control tissues to be procured and flash-frozen to preserve RNA 

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ACE2-accentuating diseases like obstructive HCM warrants further investigation. None of these entities were involved in this study in any manner.