The rapidly evolving coronavirus disease 2019 (COVID-19, caused by severe acute respiratory syndrome coronavirus 2- SARS-CoV-2), has greatly burdened the global healthcare system and led it into crisis in several countries. Lack of targeted therapeutics led to the idea of repurposing broad-spectrum drugs for viral intervention. In vitro analyses of hydroxychloroquine (HCQ)’s anecdotal benefits prompted its widespread clinical repurposing globally. Reports of emerging cardiovascular complications due to its clinical prescription are revealing the crucial role of angiotensin-converting enzyme 2 (ACE2), which serves as a target receptor for SARS-CoV-2. In the present settings, a clear understanding of these targets, their functional aspects and physiological impact on cardiovascular function are critical. In an up-to-date format, we shed light on HCQ’s anecdotal function in stalling SARS-CoV-2 replication and immunomodulatory activities. While starting with the crucial role of ACE2, we here discuss the impact of HCQ on systemic cardiovascular function, its associated risks, and the scope of HCQ-based regimes in current clinical settings. Citing the extent of HCQ efficacy, the key considerations and recommendations for the use of HCQ in clinics are further discussed. Taken together, this review provides crucial insights into the role of ACE2 in SARS-CoV-2-led cardiovascular activity, and concurrently assesses the efficacy of HCQ in contemporary clinical settings.
Keywords: SARS-CoV-2; COVID-19; ACE2; hydroxychloroquine; cardiovascular system; cardiovascular disease (CVD); therapeutics
A new type of pneumonia outbreak surfaced in December 2019 in Wuhan, Hubei province, China, which was caused by a novel coronavirus, viz., severe acute respiratory syndrome coronavirus (SARS-CoV)-2 ]. The pandemic disease, named coronavirus disease 2019 (COVID-19), had 5,867,727 confirmed cases by 29th May 2020 and resulted in 362,238 deaths globally, as sourced by the Coronavirus Resource Center, John Hopkins University (JHU) (https://coronavirus.jhu.edu/). SARS-CoV-2 shares 82% genomic similarity with the other SARS-CoVs, while two other bat-SARS-CoV-like viruses (retrieved from Rhinolophus sinicus, Zhoushan, China), viz., bat-SL-CoVZC45 and bat-SL-CoVZXC21 were found to have >89% similarity [ To date, SARS-CoV-2 has crossed all continental boundaries, and presently Europe and North America have been its major epicenters. The COVID-19 symptoms are comparable to those produced by SARS-CoV and Middle East respiratory syndrome (MERS). However, the earliest estimate showed its lower (2%) fatality rate, while about ~ 20% of COVID-19 patients had developed severe conditions . SARS-CoV-2 tropism to the lungs/respiratory system is prominent, in which it infects the lung cells and causes interstitial pneumonitis that may lead to developing acute respiratory distress syndrome (ARDS) and manifestations related to the cardiovascular (CV) system causing multiple organ failure [. Amongst severe COVID-19 patients, 23% of cases had cardiac injuries [ and, therefore, highlighted this as a common feature that promotes disease severity. Of note, elevated levels of creatinine kinase (CK; >200U/L) in 13% of COVID-19 patients in the general cohort, where most of these lacked any cytokine storm-induced systemic inflammatory response, further affirmed the association of COVID-19 with cardiovascular complications [. The common CV complications reported in COVID-19 patients include arrhythmia, myocardial injury (marked by higher troponin I (hs-cTnI) and CK levels) and myocarditis, acute myocardial infarction, acute heart failure and cardiomyopathy, and disseminated intravascular coagulation (DIC) . Although the association of SARS-CoV-2 infection with these manifestations is now known, preexisting CV comorbidities could further contribute to COVID-19 severity and mortality ]. The earliest report describing a meta-analysis of the COVID-19 clinical cohort revealed a strikingly high existing prevalence of hypertension and cardiovascular disease (CVD) in hospitalized patients, that made them prone to require critical care ]. COVID-19 patients with CVDs were found to have a relatively five-fold higher mortality risk as compared to the patients with no CVD background .
SARS-CoV-2 interacts with an ACE (Angiotensin-converting enzyme) homolog, viz., transmembrane angiotensin-converting enzyme 2 (ACE2) to enter border-line host cells including type II pneumocytes, perivascular pericytes, macrophages, and cardiac cardiomyocytes . ACE2 is a carboxy-monopeptidase and an essential component of the renin-angiotensin system (RAS), where it critically participates in maintaining normal CV functions while its dysregulation, observed in multiple CVDs, includes hypertension, myocarditis, and heart failure [. Expression of ACE2 on pericytes and cardiomyocytes brought heart and CV tissues to potential risk for SARS-CoV-2 infection, and therefore explained a higher prevalence of CV complications in COVID-19 patients. With the evolving COVID-19 pandemic situation, tremendous pressure and a lack of targeted anti-viral or vaccine prompted researchers and clinicians to consider all available therapeutic options. In this context, the two aminoquinolines, viz., Chloroquine (CQ) and Hydroxychloroquine (HCQ, a less-toxic derivative of CQ) were repurposed widely as therapeutic options for COVID-19. In multiple reports earlier, CQ was shown to be effective in inhibiting SARS-CoV viral replication in vitro . This evidence prompted an early assessment of CQ and HCQ efficacies against SARS-CoV-2 [, where in post-SARS-CoV-2 infection HCQ was found to impair viral replication more effectively than CQ [1. These preliminary in vitro findings pave the way to assess the therapeutic application of HCQ in clinical stud]. As of May 29, 2020, searching with “COVID” and “Hydroxychloroquine” terms, 206 clinical trials including that of the National Institutes of Health (NIH) are in progress to assess the therapeutic utility of HCQ globally (details available at https://clinicaltrials.gov/ct2/home). HCQ’s anecdotal repurposing is now being extensively exercised in clinics worldwide. However, in the light of SARS-CoV-2 infection, ACE2 function, and emerging CV challenges, we lacked a clear understanding of HCQ’s pharmacology, mode of action, benefits, and inevitable risks for COVID-19 patients. In this review, we provide insights into the crucial part ACE2 plays in SARS-CoV-2 infection and its significance in systemic cardiovascular function and reviewed the impact of HCQ on SARS-CoV-2 replication and immunomodulatory activities. Taking readouts from clinical COVID-19 studies so far, we reviewed cardiovascular risk and the benefits of HCQ in current clinical settings. We further brief on key considerations in HCQ repurposing and its future perspectives.
2. SARS-CoV-2, ACE2, and Cardiovascular Challenges
SARS-CoV-2 is a non-segmented, single-stranded (ss), positive (+) sense RNA virus . It belongs to the family of enveloped RNA beta-coronavirus. Out of seven known species of beta-coronavirus, only three (SARS, MERS, and COVID-19) cause potentially fatal human disease. SARS-CoV-2 produces a 50–200 nanometers virion that is constituted by four structural proteins, viz., the S (spike), E (envelope), M (membrane), and N (nucleocapsid), wherein the N protein is aligned with its RNA genome, while the S, M, and E proteins collectively constitute the viral envelope . The S protein at the SARS-CoV-2 envelop resembles a spike projection that serves as a tool for it to enter the host cell . Phylogenetic analysis revealed 99% similarity of S protein comparing SARS-CoV-2 and SARS-CoV  and therefore reaffirmed the evidence that SARS-CoV-2 exploits the same ACE2 receptor  that originally served as a functional receptor for SARS-CoV [.
ACE2 is present in alveolar epithelial cells and frequently localized at the cell membrane of enterocytes (intestine), pericytes, cardiomyocytes, and macrophages . ACE2 at the surface of pericytes and cardiomyocytes serves a vital activity of the RAS by maintaining normal CV functions by catalyzing the Ang (angiotensin) I and II . SARS-CoV-2′s S protein primarily binds to the ACE2 of alveolar epithelial cells in the respiratory tissues that enable its further access to the systemic circulation, reaching cardiomyocytes in the heart and pericytes and endothelial cells in the macro-vessels (Figure 1A). Endocytosis-driven internalization of ACE2 on the membrane of cardiomyocytes, pericytes, and endothelial cells by SARS-CoV-2 results in omitting ACE2 from the cell surface and potentially raises the risk of CV complications in COVID-19 patients . The loss of ACE2 carboxypeptidase function was earlier shown to compromise cardiac function . A higher ACE2 level in patients with existing CVD and/or hypertension was also suggested to increase the susceptibility to SARS-CoV-2 infection . In light of this information, clinical readouts from six studies, including 1527 COVID-19 patients, revealed 17.1%, 16.4%, and 9.7% prevalence of hypertension, cardiac & cerebrovascular disease, and diabetes, respectively . Prevalence of these CVD comorbidities was found to be higher in patients requiring ICU than the non-ICU patient groups. Analyses of mortalities in a cohort of 44,672 COVID-19 patients from Wuhan, China also showed 10.5%, 7.3%, and 6% mortalities in patients having CVD, diabetes, and hypertension, respectively, significantly greater than the overall mortality rate (2.3%) for COVID-19 patients . To date, nine clinical studies from China  have comprehensively assessed CV comorbidities in COVID-19 patient cohorts and yielded similar clinical results (Figure 1B). However, disparities in testing, standardization and options for standard procedure in clinical studies from China and elsewhere  have impacted the quantitative clinical outcomes. To assess the cardiovascular outcomes of SARS-CoV-2 infection in a recent report, Liu et al. reported a significantly higher level of circulating Ang II in COVID-19 patients than the controls; circulating Ang II in levels COVID-19 patients also correlated well with viral load . Of note, these results were consistent with reduced ACE2 activity. They again underlined the crucial role of RAS in COVID-19 disease and reaffirmed the focus on the cardio-protective function of ACE2, where an alteration in its activity may substantially impact the cardiovascular outcomes [. Therefore, in light of these reports, ACE2 has gained recognition as a key and central target in COVID-19 pathology and associated CV complications. Taking note of SARS-CoV-2 infection severity, here we review the frequent clinical cardiovascular complications observed in COVID-19 patients and further shed light on the potential involvement of ACE2 activity.
Figure 1. SARS-CoV-2, angiotensin converting enzyme 2 (ACE2), and cardiovascular complications. (A) Transmembrane ACE2 receptor facilitates SARS-CoV-2 entry to host cell primarily in the lungs, and then the vascular system, postulating cardiovascular complications by causing inflammation and myocardial dysfunction. SARS-CoV-2 access to the systemic circulation via the lungs potentiates heart infection, while its direct infection of associated pericytes and endothelial cells may cause vascular endothelial dysfunction. Cardiac SARS-CoV-2 infection causes micro-vessel dysfunction, and elevated immunoreactivity disrupts atherosclerotic plaques leading to the progression of the acute coronary syndromes. SARS-CoV-2 infection of alveolar pneumocytes (type II) cells progressively develops the systemic inflammation and elevated immunoreactivity that eventually produces the ‘cytokine storm’, marked by elevated IL-6, IL-7, IL-22, and CXCL10 cytokine levels. It potentiates T-cell and macrophage activation infiltrating infected myocardial tissues and may produce severe cardiac damage and myocarditis, leading to heart failure. Cytokine storm may further increase damage of cardiac monocytes causing myocardial dysfunction and subsequent development of arrhythmia. These events cumulatively produce cardiac dysfunction. (B) Manifestation (%) of cardiovascular complications in hospitalized COVID-19 patients reported in key clinical studies exhibiting comorbidities including hypertension, cardiovascular disease (CVD), cerebrovascular disease, coronary artery disease and rate of cardiac injury, shock, heart failure, and arrhythmia in low (LS), and high severity (HS) patient groups. p values indicate *** (<0.001), ** (<0.01), and * (<0.05) statistical significance.
ACE2 comprises an 805-amino acid (aa; Mr 110,000 glycoprotein) long endothelium-bound carboxy-mono-peptidase that consists of a 17-aa N-terminal peptide (catalytic domain-oriented extracellularly) and a C-terminal anchor integrated into the membrane. ACE2 is catalytically a zinc metalloprotease and the only homolog of ACE known in humans . ACE2 is part of the RAS that plays a crucial function in maintaining normal cardiovascular functions, while dysfunction in RAS contributes to CVDs, including hypertension, myocarditis, coronary heart disease, and heart failure . RAS is constituted by a set of catalytic enzymes that includes angiotensinogen, renin, Ang II, Ang II receptors (AT1R and AT2R), and ACE . Among these, ACE2 has a crucial role to play by catalyzing Ang II to Ang (1–7) or Ang I to Ang (1–9) . ACE2 can access substrate/peptide in the circulation, and it is known for its circulatory presence and catalytic function in the blood and body fluid. Given its carboxy-monopeptidase activity, ACE2 primarily trims the COOH-terminal phenylalanine residue from Ang II . ACE2-led trimming of Ang II to Ang (1–7) is a significant event in the RAS, since the role of Ang II is critically implicated in producing hypertension by promoting vasoconstriction, fibrosis, Na+ retention, and pro-inflammation and pro-oxidant activities. At the same time, elevated levels of Ang (1–7) peptide inhibits the Ang II/AT1R axis and induces anti-inflammatory, anti-oxidant, anti-fibrotic, and vasodilatory activities (Figure 2A) [56,58]. Therefore, ACE2 activity switches on the processing of Ang II in the classical RAS system and loss of ACE2 or its function could put the RAS system to an overall higher Ang II level .These cardioprotective activities of ACE2 are regulated through the Ang I (1–9)/AT2R and Ang I (1–7)/MasR axes .On the contrary, ACE degrades Ang (1–7) and forms ANG II that results in promoting inflammation, fibrosis, and high blood pressure (Figure 2). The role of ACE2 was also implicated in the hydrolysis of apelin and des-arginine bradykinin (des-Arg1-BK) apelin peptides, wherein des-Arg1-BK was shown to have a pro-inflammatory function via stimulating the B1 receptor  (Figure 2). Besides its critical role in the CV system, ACE2 was earlier discovered to be a key binding receptor for SARS-CoV and NL63 (HCoVNL63) coronaviruses [30,60], while recently it was identified to be a SARS-CoV-2 receptor . ACE2 is also shown to play a key role in acute respiratory/lung injury caused by influenza viruses viz., H1N1, H5N1, and H7N9 [62,63,64].
The steps of viral entry, replication, and protein synthesis/processing are key druggable targets for antiviral drugs (Figure 3A). In the context of the utility of quinines, Savarino et al. were first to suggest the benefits of HCQ and CQ for the treatment of SARS-CoV . They postulated the involvement of endocytosis in viral entry and associated immune response, where the latter could be a result of the activation inflammatory cytokines contributing further to the severity of viral infection, and therefore hinted at the potential benefits of HCQ and CQ to intervene in the underlying mechanism . An in vitro study by Kayaerts et al. in the subsequent year confirmed the potency of CQ in inhibiting SARS-CoV replication in Vero E6 cells , whereas, Vincent et al. showed a dose-dependent inhibition of viral replication in Vero E6 cells, in both cases, either immediate or 3–5 h post-viral infection . Of note, they showed that CQ treated cells had a lesser viral infection, and CQ could impair the terminal glycosylation of the ACE2 receptor, reducing SARS-CoV–ACE2 affinity and eventually diminishing the infection rate. These results emphasized the utility of HCQ for coronavirus prophylaxis . Multiple recent in vitro reports as described in the earlier section [18,19,85,100] further implicated the role of HCQ in the inhibition of SARS-CoV-2 replication. However, we presently lack molecular insights into the mode of action of HCQ/CQ against SARS-CoV-2. Learning from available evidence of its function primarily involves three aspects of its antiviral functions including: (i) inhibition of viral entry by affecting receptor glycosylation, (ii) control of virus replication by abolishing the pH-dependent endosome-mediated viral entry, and (iii) restriction of viral protein’s post-translational modification.
The data used for the analyses described in this manuscript (Figure 2B,D) were obtained from the GTEx Portal on 05/13/20 and/or dbGaP accession number phs000424.vN.pN on 05/13/2020.
Conflicts of Interest
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.