Since Dec 2019, coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory
syndrome coronavirus 2(SARS-CoV-2) has resulted in considerable morbidity and mortality
throughout the world. On April 21 2020, Italy and US have registered over 187,000 and 855,000
cases of confirmed COVID-19 disease respectively, with one of the highest death rates in the
world, which stands at more than 25,000 individuals in Italy and 47,000 in US. Epidemiologic
modeling from multiple groups indicate the scope of the problem, with a potential for over
200,000 deaths in the U.S. alone, with large percentage of the population infected. Mortality
from COVID-19 is associated with the presence of severe respiratory illness, although recent
studies indicate 20%-30% of patients have evidence of COVID-19 associated cardiac injury
defined as decline in ejection fraction or troponin I elevation (1,2). Anecdotal evidence
suggests the presence of abnormal EKG findings compatible with myocardial ischemia but
whether this is from epicardial coronary thrombosis, myocarditis, or microvascular thrombosis
remains uncertain although all have been postulated (3). Regardless, patients with cardiac
injury have higher mortality than those without, and sudden cardiac death has been described
in some of these patients (1). Two thirds of patients with cardiovascular history were more
likely to develop cardiac complications (4). Isolated reports have described fulminant
myocarditis in the setting of high viral load with autopsy findings consistent with
inflammatory mononuclear infiltrates (5). However, no systematic autopsy studies of patients
dying from COVID-19 have ever been conducted. The Papa Giovanni XXIII Hospital, Bergamo had
treated about 2000 patients with COVID-19 and performed systematic cardiac autopsies from a
series of 60 patients who died or presented with/without a picture of cardiac injury and were
COVID-19 positive. Detailed clinical data on all these patients are available.

Background Much about the pathogenesis of SARS-CoV-2 and the heart remains unknown.
Angiotensin (AT) converting enzyme 2 (ACE2) is known as the cellular receptor for both
SARS-CoV and SARS-CoV-2 but also as an endogenous counter- regulator of the renin-angiotensin
system (RAS). ACE2 is ubiquitously expressed with the highest levels detected in the
cardiovascular system (cardiomyocytes, cardiac fibroblasts, vascular smooth muscle cells and
endothelial cells) as well as gut, kidneys and lungs. In general, loss of ACE2 increases
susceptibility to cardiovascular disease such as myocardial infarction and hypertension while
gain of function ACE2 has shown protective roles in various models of cardiovascular disease
(6). The bifunctional role of ACE2 as a receptor for SARS-COV-2 but also as a protective
factor against cardiovascular disease means careful understanding of the role of ACE2 during
SARS-CoV-2 is needed. In this proposal investigators will examine cardiac tissues from
patients dying of COVID-19 and examine the effect of infection on the expression of ACE2 on
various cardiac cells. Investigators will also evaluate and validate this hypothesized
mechanism of viral entry by ACE2 and trans-membrane serine protease which promotes entry of
SARS-COV-2 into cells through a separate mechanisms.

Study Aims:

1. Describe the cardiac pathological findings from series of 60 patients dying from
COVID-19 using cardiac samples sent to CVPath Institution from Papa Giovanni XXIII
Hospital, Bergamo, Italy to study the correlations between clinical risk factors and
myocardial findings and difference from other viral myocarditis;

2. Understand the relationship between viral load in cardiac tissues and the extent of
damage seen on myocardial histological sections; and

3. Co-localize the SARS-CoV-2 using RNAscope in situ hybridization, with its entry receptor
ACE2 and serine protease TMPRSS2 (type II transmembrane serine protease) in different
cell types found in the heart, such as endothelial, smooth muscle, myocardial,
fibroblastic and inflammatory cells, to better understand the pathogenesis of the
disease.

Methods:

The study was approved by ethical committee at Papa Giovanni XXIII Hospital and the CVPath
Institute IRB (Institutional Review Board).

All specimens are fixed in 10% buffered formalin. Hearts will be shipped to CVPath in
accordance with all international shipping regulations and U.S. CDC (Centers for Disease
Control and Prevention) guidelines (www.cdc.gov/coronavirus/2019-ncov/hcp/guidance-postmortem
specimens.html). The sample is given a unique identifier and the hearts are weighed after
blood clots have been removed from the cavities, and the heart ventricles are sliced parallel
to the posterior atrioventricular junction to determine absence of any necrosis or fibrosis.
Pulmonary emboli and any right ventricular abnormalities are assessed to rule out any
attributable cause of death. The heart is weighed and radiographed and if any calcification
is observed the epicardial coronary arteries are removed intact away from the heart and
segments decalcified according to the extent of calcification. The rest of the arteries are
submitted for paraffin embedding at 3-4 mm intervals to rule out any significant
atherosclerosis (75 % cross-sectional area stenosis) or any thrombosis.

In total six sections of myocardium (anterior, posterior, and lateral LV (left ventricle),
ventricular septum, anterior and posterior wall of the right ventricle) are routinely taken
transversely, embedded in paraffin, and stained with hematoxylin and eosin (H&E) for
histologic evaluation. If specific pathology is observed, additional sections will be
submitted to determine the etiology of the findings. Histologic examination is performed to
rule out any infiltrative or inflammatory process or any myofiber disarray of the myocardium,
intramyocardial small vessel disease of interstitial or focal fibrosis. Presence of any
cardiomyopathic process will be ruled out by gross and histologic examination. If the
post-mortem interval is short, transmission electron microscopic (TEM) examination will be
performed to determine the presence of virus and the cell type harboring the virus.

Correlations with autopsy findings will be made with available anonymized clinical data
including EKG, echocardiography and cardiac catheterization (where available). The cardiac
pathological characteristics of COVID-19 will be compared to 60 viral myocarditis cases that
have previously been collected in CVPath Registry. A RT-PCR (reverse transcription polymerase
chain reaction) specifically designed for SARS-CoV-2 will be conducted on RNA extracted from
myocardial samples to quantitate the amount of virus in myocardium for each sample. RNA
samples will also be taken from coronary arteries as well. Calculated viral load will be
correlated with the myocardial injury scores including myocardial necrosis, myocardial
infarction, myocarditis, inflammatory cells numbers, type, etc.

RNAscope In-situ hybridization. In situ detection of SARS-CoV-2 with ACE2 and TMPRSS2 in
endothelial (VE-cadherin), myocardial (Cx43 and Myh6), smooth muscle cell (SM22 alpha) and
lymphocytes (CD3, CD4, CD8) will be performed using an RNAscope assay with RNAscope
Probe-V-nCoV2019-S-sense and Probe-V-nCoV2019-S (Advanced Cell Diagnostics) following the
manufacturer's protocols. Correlation will be made with specific cell types mentioned by dual
immunofluorescence as previously described (7) .

This study will result in a greater understanding of the pathology of cardiac injury in
patients with COVID-19. Doing so will open the door to develop new therapeutic options to
treat these patients during an epidemic of unparalleled size and scope.