key: cord-316018-zrui9i5z
authors: Bristow, Michael R.; Zisman, Lawrence S.; Altman, Natasha L.; Gilbert, Edward M.; Lowes, Brian D.; Minobe, Wayne A.; Slavov, Dobromir; Schwisow, Jessica A.; Rodriguez, Erin M.; Carroll, Ian A.; Keuer, Thomas A.; Buttrick, Peter M.; Kao, David P.
title: Dynamic Regulation of SARS-CoV-2 Binding and Cell Entry Mechanisms in Remodeled Human Ventricular Myocardium
date: 2020-06-24
journal: JACC Basic Transl Sci
DOI: 10.1016/j.jacbts.2020.06.007
sha: 
doc_id: 316018
cord_uid: zrui9i5z

SUMMARY Using serial analysis of myocardial gene expression employing endomyocardial biopsy starting material in a dilated cardiomyopathy cohort, we show that the SARS-CoV-2 cardiac myocyte receptor ACE2 is upregulated with remodeling and with reverse remodeling down-regulates into the normal range. The proteases responsible for virus-cell membrane fusion were expressed but not regulated with remodeling. In addition, a new candidate for CoV-2 cell binding and entry was identified, the integrin ITGA5. The upregulation in ACE2 in remodeled LVs may explain worse outcomes in COVID-19 patients with underlying myocardial disorders, and counteracting ACE2 upregulation is a possible therapeutic approach to minimizing cardiac damage.

Using serial analysis of myocardial gene expression employing endomyocardial biopsy starting material in a dilated cardiomyopathy cohort, we show that the SARS-CoV-2 cardiac myocyte receptor ACE2 is upregulated with remodeling and with reverse remodeling downregulates into the normal range. The proteases responsible for virus-cell membrane fusion were expressed but not regulated with remodeling. In addition, a new candidate for CoV-2 cell binding and entry was identified, the integrin ITGA5. The upregulation in ACE2 in remodeled LVs may explain worse outcomes in COVID-19 patients with underlying myocardial disorders, and counteracting ACE2 upregulation is a possible therapeutic approach to minimizing cardiac damage.

Infection with the SARS-CoV-2 virus, or the virus itself 

The 2020 COVID-19 pandemic has many unique clinical features, including high infectivity, a protean clinical presentation and course, and life-threatening potential (1) . Myocardial involvement is an important pathophysiologic component of some critically ill patients with SARS-CoV-2 (CoV-2) infections (2, 6) , either due to myocarditis (5, 6) or myocardial dysfunction without evidence of inflammation (2) (3) (4) . The prevalence of cardiac complications in patients without underlying heart disease ranges from 20-30% (7) (8) (9) , which when present worsens prognosis. In the initial report of patients receiving intensive care, evidence of cardiac injury was associated with a 50% mortality compared to <10% without such evidence (3) . The prognosis worsens further when evidence of myocardial injury is superimposed on pre-existing cardiovascular disease, with mortality rising to above 60% (3).

A well-established pathway by which the CoV-2 virus gains entry into cells includes membrane attachment by binding to angiotensin converting enzyme 2 (ACE2) (10, 11) , fusion of viral and cell surface membranes through the recruitment of host proteases (12) (13) (14) , and virus internalization followed by assembly of cytoplasmic membranous structures into replication vesicles (15) . In addition, there are other possible mechanisms of virus internalization such as binding to integrins (16, 17) , some of which also bind to ACE2 (18, 19) . Although CoV-2-cell internalization has been investigated in model systems (10) (11) (12) (13) (14) (15) and is beginning to be evaluated in the human heart (20) it is unclear if the virus enters human cardiac myocytes, and if the necessary biologic constituents are expressed in the heart. Of particular importance is the role of ACE2; in human ventricular myocardium ACE2 is a highly functional enzyme present in cardiac myocytes (20) that breaks down angiotensin II (ANG II) to the counter-regulatory peptide angiotensin-(1-7) (21,22). In explanted human heart preparations from patients with end stage reduced ejection fraction heart failure (HFrEF), ACE2 enzyme activity (22) as well as gene expression at the mRNA (20,23) and protein (20,22) levels are upregulated compared to organ donor controls. This is potentially important because upregulated ACE2 might be a mechanism by which CoV-2 myocardial involvement is more prominent in patients with underlying heart muscle disease (20).

However, it is unclear if ACE2 is upregulated in intact hearts with less severe pathologic remodeling/dysfunction than in explanted hearts from cardiac transplant recipients, who in contrast to most organ donor controls have been treated with renin-angiotensin (RAS) system inhibitors (24,25). In addition, it is not clear if the proteases that prime and facilitate membrane fusion are expressed or regulated in the remodeled, failing human heart. Finally, there is limited information on the status of integrins, particularly those that can bind to ACE2 or to the CoV-2 virus itself, to potentially effect virus-cell internalization (16) . In order to obtain information on these mechanisms in nonfailing and remodeled intact human heart we analyzed data from a serial analysis of myocardial gene expression cohort study (26), where reverse remodeled LVs were compared to unchanged ventricles exposed to the same pharmacologic regimen, with each subject serving as their own control.

Control subjects were four individuals with LVEFs ≥50% (mean ± SD of 59±7%) who had endomyocardial biopsies to rule out myocarditis or other infiltrative processes, and had no histopathologic abnormalities. Heart failure patients with reduced LVEF (HFrEF) and pathologic 

We used both baseline comparisons between NF controls and F/NDC plus changes from baseline in F/NDC patients to construct an ordered classification of four degrees of remodeling association (Supplement , Table S2 ). For baseline studies, where only microarray data were available, alpha was set at <0.05. As described in the Supplement (Table S2 ), in serially evaluated N/NDC patients these results were then linked to the reverse remodeling mRNA results measured by both the microarray and RNA-Seq platforms. The estimated alpha levels based on achieving P <0.05 in more than one condition including directionality requirements are given in the Supplement (Table S2) : unequivocal evidence, 0.0001; evidence, 0.002; and possible evidence, 0.08. The statistical significance of all gene expression data was analyzed in the same way, by non-parametric methods using Wilcoxon rank sum or signed rank tests.

Correlation analysis was by Spearman's Rho LVEF data for which evidence of non-normal distribution was absent (26), and baseline characteristics were analyzed by t-tests or contingency table analysis.

Because several analyzed gene expression groups had small Ns (NF, 4; RNA-Seq 6 R and 6 NR; LOCF 3 month F/NDC, 5 Rs and 3NRs) we used means and standard deviations (SD) or

for change values standard errors of the mean (SEM) as estimates of central tendency and dispersion, in both the small N groups and larger groups to which these data were compared.

For microarray data (30 R and 16 NR) group size was sufficient for data to be also presented as medians and 25%, 75% interquartile ranges. Thus in gene expression analyses conducted exclusively in groups where sample sizes were >6 nonparametric statistics and median, IQR data are presented, but when smaller gene expression group sizes were analyzed or compared nonparametric significance tests plus means and SDs or SEMs are used.

R, GraphPad Prism and XLSTAT/Excel were the statistical software packages used.

Baseline characteristics for the 46 F/NDC subjects and 4 NF controls are given in Table 1 .

At baseline the F/NDC and NF characteristics were similar, except for measurements and biomarkers of ventricular dysfunction/remodeling. In F/NDC patients LVEF was more reduced than RVEF (Table 1 , Figure 1A ), but both were P <0.05 vs. NF. Right heart catheterization data trended abnormal in F/NDC but no measurement was P <0.05 vs. NF. NPPB was elevated in F/NDC compared to NF (P = 0.035), but norepinephrine was not significantly different (P = 0.44). These data describe a relatively young (mean age 45.6±13.2 years), well compensated

HFrEF population with moderate LV dysfunction/remodeling (LVEF 27.2±9.0 %, LV end diastolic volume 232±93 ml). The baseline characteristics of the Responder and Nonresponder groups are also given in Table S3 . A markedly greater duration of heart failure in Nonresponders (55.7±67.0 months vs. 7.0±10 months in Responders, P = 0.011) was the only difference.

The protocol mandated ACE inhibitor background therapy, and diuretics as needed.

Spironolactone was administered as tolerated, and an angiotensin blocking agent (ARB) could be substituted in ACEI intolerant patients. Table 1 gives the ACEI and ARB doses in enalapril and losartan equivalents (30) , at both baseline and the average dose during the 12 month study. All 4 of the NF controls were on ACEIs for suspected and ultimately unproved heart muscle disease, and 44 of the 46 patients were on an ACEI at baseline. There is no difference in ACEI mg dose between F/NDC and NF patients, and no difference between reverse remodeling Responders and Nonresponders in average dose of ACEIs or ARBs. (Table S1) , and follow the same pattern, except that the LVEF change from baseline was larger than in the entire cohort , Table S3 ) exhibited any change in the F/NDC group.

At baseline NPPB, PLN and ATP2A2 exhibited changes similar to those in previously reported RT-PCR data (27) . NPPB was upregulated by 8.8 fold in F/NDC, and its expression vs. Integrins Table 3 contains NF vs. F/NDC baseline data for 10 integrins previously reported to bind to ACE2 (18,19), facilitate viral internalization (17) or be associated with LV remodeling (36) or cardiac myocyte injury protection (37) . Only one, the laminin binding integrin ITGA7, has not been reported to be involved in pathogenic virus cell internalization. Five integrins in Table 3 bind to the RGD motif, recently identified in the CoV-2 spike protein binding domain (16) and

used by multiple viruses for binding to integrins. Of the ACE2 binding integrins, ITGA5 expression was 1.28 fold higher in F/NDC compared to NF (P = 0.039) while ITGB1 and ITGA2

were not different. ITGA5 has also been associated with virus internalization (17) and LV remodeling where it decreases with LVAD treatment (36) . Of the six non-ACE2 binding integrins listed in Table 3 

Changes in Responders or Nonresponder fold changes based on mean values are given in Table 2 , while median and IQR values for microarray data are presented in Tables S3 and S4 .

In microarray measurements, the near twofold upregulated ACE2 at baseline was downregulated as LV remodeling improved, declining to 0.675 fold (an approximate 1.5 fold decrease, P <0.0001). Three other RAS genes (ACE, AGT, AGTR2) and only one of the proteases shown in Table 2 exhibited changed expression on reverse remodeling. The protease that was downregulated in F/NDC (Table S3 , CTSLL3) was not changed with reverse remodeling (fold change from baseline 1.03, P = 0.62 (Table S3) ). Although in the R vs NR comparison serine protease 1 (PRSS1) was downregulated on reverse remodeling (fold change 0.90, P = 0.042 (Table S3) (Tables 2 and S4 ) were similar to microarray measurements.

As expected, these genes generally changed their expression in directions opposite to their baseline levels ( Table 2, Table S4 ). NPPB, a counter-regulatory peptide considered the premier biomarker of pathologically remodeled ventricular myocardium in tissue (27, 35) 

We also evaluated the expression of five proteases that participate in fusion of viral and cell membranes (12-14,31-33) when triggered by SARS coronavirus binding to ACE2 (42, 43) .

Expression of CTSL1 (13), TMPRSS11D (13, 14) , ADAM17 (31) and FURIN (32, 33) were detected by both microarray and RNA-Seq platforms. In contrast, TMPRSS2 (12) (13) (14) , recently implicated in CoV-2-membrane fusion in model systems (12) , was low abundance as measured by microarray and could not be detected by RNA-Seq. None of these proteases and only one (CTSLL3) of an additional panel of 11 others exhibited differences between NF and F/NDC, and none changed expression in Responders with reverse remodeling. However, this does not exclude the possibility that protease protein abundance or enzyme activity may have changed with remodeling.

We considered that integrins could be a potential participant in CoV-2 cell entry, and found evidence or unequivocal evidence of remodeling-associated up-regulation in five of the ten investigated. ITGA5, which rated evidence of an association with remodeling, can bind to ACE2

(19) and an RBD domain in CoV-2 (16), and thus is a candidate for involvement in CoV-2 cell binding and internalization. The only integrin that achieved unequivocal evidence of remodeling association was the laminin binding cardiac and skeletal muscle integrin ITGA7, which has not been reported to bind to ACE2 or pathogenic viruses and which, like ACE2 and NPPB, can be viewed in a counter-regulatory context (37) . Five of the ten integrins evaluated contained motifs for binding to an RGD domain, common in pathogenic viruses as a means of cell surface binding and virus internalization, and recently identified in CoV-2 (16) . Of these, only ITGA5 and ITGB3 were up-regulated, meaning if Cov-2 did utilize RGD-integrin binding for internalization these two monomers might predispose to greater cell entry. However, these integrins do not dimerize with each other (44) , and the ITGA5-ITGB1 or ITGAV-ITGB3 protein product heterodimers, both commonly used by pathogenetic viruses for cell entry, would need to be formed. If RGD binding is disregarded (17) , the other up-regulated integrin (rated as possible evidence) was ITGA4, which also doesn't dimerize with ITGB3 (44) . Alternatively, upregulated integrins could affect virus replication in cardiac myocytes via interaction with integrin linked kinase (45), a pseudokinase adaptor molecule known to bind to ITGB1 and ITGB3 (46) .

Despite substantial evidence that myocardial involvement is common and potentially devastating in COVID-19 disease, identification of CoV-2 virus in cardiac myocytes has not been reported. Cardiac findings on autopsy of COVID-19 patients are limited to a single case of severe pulmonary involvement where on tissue examination no myocardial pathologic findings were observed (47) , and two pulmonary death cases where post-mortem myocardial needle biopsy findings were deemed likely to be secondary to pre-existing underlying conditions (48) .

One of the two needle biopsy subjects had a negative tissue block CoV-2 PCR (48) . There is thus far only a single case report of an EmBx in a COVID-19 PCR-proven case, in a patient in cardiogenic shock (49) . This patient had electron microscopy imaged coronavirus in ventricular myocardial interstitial cells but not in cardiac myocytes, despite evidence of myofibril lysis (49) .

However, based on remodeling associated upregulation in ACE2 and the demonstration that all other myocardial cell entry constituents exist in human ventricular myocardium, it would be surprising if CoV-2 can't bind to, enter, replicate and damage human cardiac myocytes.

Myocytes contribute approximately 70% of tissue volume in the human left ventricle (50) , and ACE2 is definitely cardiac myocyte-expressed according to cell marker findings (20), single cell RNA data (51) , and the degree of enzyme activity in vivo (21) and in explanted hearts (22).Thus cardiac myocytes appear to be a vulnerable target for CoV-2, and this needs to be addressed by further histopathologic investigation in hearts exhibiting CoV-2 associated myocardial dysfunction.

As has been noted by others (52), ACE2 and its receptor for the spike protein binding domain are an attractive therapeutic target for treating or preventing CoV-2 infections. ACE2 small molecule inhibitors have been developed (53) and dramatically lower ACE2 activity in failing human LV preparations, at nanomolar concentrations (22). However, ACE2 is an important counter-regulatory enzyme in the heart, responsible for converting angiotensin II to angiotensin-(1-7) that is antiproliferative, antifibrotic and a vasorelaxant. Based on gene ablation ACE2 is considered an "essential regulator of heart function" (54) . It follows that any ACE2

inhibitor would need to block CoV-2 binding without decreasing enzyme activity, which may be possible through Ab inhibition (11) or the use of decoy receptors (55) . Another possibility would be to deploy an ACE2 activator such as diminazene (54) if an ACE2-CoV-2 receptor inhibitor diminishes ACE2 activity. However, if any of these approaches are taken it would be important to rule out other mechanisms of CoV-2 cell entry that would be uninhibited, such as virusintegrin binding.

Angiotensin converting enzyme 2 (ACE2), a counter-regulatory enzyme robustly expressed in human left and right ventricles and the major pathway for breaking down angiotensin II into the antihypertrophic, antifibrotic and vasorelaxant peptide angiotensin-(1-7), has been hijacked by SARS coronaviruses as the binding site for initiation of cell entry. We show that ACE2 is upregulated in eccentrically remodeled/failing LVs, and then decreases expression with reverse remodeling. This regulatory behavior was independent of RAS inhibitors as doses of ACEIs or ARBs weren't different in patients that reverse remodeled compared to those that didn't. ACE2's remodeling expression behavior was identical to NPPB, another counter-regulatory gene that leads to generation of the antiproliferative vasodilator peptide BNP. Upregulated ACE2 should be viewed as beneficial to a remodeled, failing heart, although it may increase the risk of CoV-2 cell invasion and cytopathology. It stands that any therapeutic approach involving ACE2 should focus on inhibiting CoV-2 binding, while maintaining enzyme activity.

The data presented are another example of reverse translation, where an enzyme that was originally characterized and shown to be upregulated in failing, pathologically remodeled human hearts was discovered to be the cell transducer for a worldwide pandemic. The precise biologic mechanisms involved in SARS coronavirus cell entry and damage are now being investigated at the basic science level, and therapeutic strategies involving ACE2 inhibition or intervention at downstream events may result in forward translation back to the clinical setting. 

Clinical characteristics of coronavirus disease 2019 in China

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Association of cardiac injury with mortality in hospitalized patients with COVID-19 in Wuhan, China

Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China

Cardiac involvement in a patient with coronavirus disease 2019 (COVID-19)

Coronavirus fulminant myocarditis saved with glucocorticoid and human immunoglobulin

Cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (COVID-19)

COVID-19 and the cardiovascular system

Sanchis-Gomar F. Cardiac troponin I in patients with coronavirus disease 2019 (COVID-19): evidence from a meta-analysis

Cryo-EM structure of the 2019-nCoV spike in the perfusion conformation

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites

Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease

Biogenesis and dynamics of the coronavirus replicative structures

A potential role for integrins in host cell entry by SARS-CoV-2

Beyond RGD: virus interactions with integrins

Therapeutic Molecular Phenotype of beta-Blocker-Associated Reverse-Remodeling in Nonischemic Dilated Cardiomyopathy

Histamine H2 Receptor Polymorphisms, Myocardial Transcripts, and Heart Failure (from the Multi-Ethnic Study of Atherosclerosis and Beta-Blocker Effect on Remodeling and Gene Expression Trial)

Myocardial microRNAs associated with reverse remodeling in human heart failure

Target doses of heart failure medical therapy and blood pressure: Insights from the CHAMP-HF registry

Modulation of TNF-alpha-converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-alpha production and facilitates viral entry

The spike glycoprotein of the new coronavirus-2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites

Changes in gene expression in the intact human heart: down-regulation of alpha-myosin heavy chain in hypertrophied, failing ventricular myocardium

Myocardial gene expression in dilated cardiomyopathy treated with beta-blocking agents

Integrin expression during reverse remodeling in the myocardium of heart failure patients

Integrins protect cardiomyocytes from ischemia/reperfusion injury

B-type natriuretic peptide: beyond a diagnostic

Angiotensin-(1-7) Is an endogenous ligand for the G protein-coupled receptor Mas

Angiotensin-(1-7) inhibits growth of cardiac myocytes through activation of the mas receptor

A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9

Ready, set, fuse! The coronavirus spike protein and acquisition of fusion competence

Mechanisms of coronavirus cell entry mediated by the viral spike protein

The integrins

Novel role for integrin-linked kinase in modulation of coxsackievirus B3 replication and virus-induced cardiomyocyte injury

Mechanisms that regulate adaptor binding to beta-integrin cytoplasmic tails

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Pathological study of the 2019 novel coronavirus disease (COVID-19) through postmortem core biopsies

Myocardial localization of coronavirus in COVID-19 cardiogenic shock

The application of stereological methods for estimating structural parameters in the human heart

Single-cell RNA analysis on ACE2 expression provides insight into SARS-CoV-2 blood entry and heart injury

Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target

Substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ACE2) inhibitors

Angiotensin-converting enzyme 2 is an essential regulator of heart function

Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor

The potential actions of angiotensinconverting enzyme II (ACE2) activator diminazene aceturate (DIZE) in various diseases

mineralocorticoid receptor antagonist, NEP = neutral endopeptidase, ACE = angiotensin converting enzyme, MASR = MAS1 (also known as MAS) receptor