Biomarkers in the blood and skeletal muscle for the improvement of viral myocarditis diagnostics

Myocarditis (inflammation of the heart) is an inflammatory disease of the heart
that can lead to cardiomyocyte (heart muscle cell) death, the formation of
connective tissue and eventually to severe heart function loss, i.e. heart
failure. The most common cause of infectious myocarditis is a viral infection.
At present approximately 20 different viruses have been described that can
cause viral myocarditis. The pathophysiology of viral myocarditis is
characterized by either direct virus-related cardiac damage (the virus infects
cardiomyocytes and kills them) or cardiac damage because the reaction of the
immune system against the virus also attacks the heart (auto-immune damage).
The clinical presentation of viral myocarditis patients is very diverse and
varies from mild shortness of breath and mild flu-like symptoms to chest pain,
specific aberrations on the ECG, acute heart failure and sometimes sudden
death.
This large variety in clinical presentation makes diagnosing viral myocarditis
difficult. There are currently no specific serological markers available for
viral myocarditis. In addition to the anamnesis, the ECG and/or
echocardiography can be used to rule out other causes of heart failure such as,
myocardial infarction, heart valve failure or congenital heart failure.
However, viral myocarditis often mimics myocardial infarction and patients can
present with additional infarct-like symptoms such as ST-segment elevation on
ECG, wall motion abnormalities, and elevated circulating cardiac troponin
levels. In patients with infarct-like complaints coronary angiography (CAG) is
most often used to distinguish between viral myocarditis and myocardial
infarction, whereby epicardial coronary artery occlusion confirms myocardial
infarction. However, in approximately 4-9% of patients with infarct-like
complaints no coronary artery occlusion is found with CAG. In the majority of
these patients myocardial infarction is still the cause of the complaints. This
is called myocardial infarction with non-occluded coronary arteries (MINOCA).
It is estimated that in about one-third of these patients, acute myocarditis is
the underlying cause. Therefore there is a clinical need to distinguish between
acute (viral) myocarditis and myocardial infarction.
However, to confirm the diagnosis viral myocarditis, histological examination
of heart biopsies (endomyocardial biopsies; EMB) is necessary at present. Even
then the usefulness of the EMB is limited. Viral myocarditis namely induces a
very fragmented inflammation in the heart. The chance therefore, that the
anomaly is not visible in the EMB is substantial. Therefore it is necessary to
take multiple EMB (to a maximum of 5). Even then there is a chance of
false-negative EMB. The collection of EMB is no sinecure for the patient. Such
an invasive method is always accompanied by a chance of complications. In
addition, since the sampling area for EMB in the heart is small, taking EMB at
multiple time-points is generally not done. This makes monitoring disease
progression or therapy effects difficult. Therefore, for a better clinical
management of viral myocarditis, extension of the diagnostic repertoire is
essential: 1) for a more accurate and less demanding diagnosis, 2) to guide
application of therapy and to monitor its effects.
We have recently found increased infiltration of lymphocytes in quadriceps
skeletal muscle, obtained at autopsy, in patients with viral myocarditis and in
a mouse model of viral myocarditis. This suggests that skeletal muscle may be a
*peripheral mirror* that reflects inflammation in the heart in viral
myocarditis. Also, a lot of viruses that infect the heart can also infect
skeletal muscle cells, which suggest that analysis of skeletal muscle may also
reveal viral infection in the heart.
Taken together, these results suggest that skeletal muscle reflects the
pathology of the heart in viral myocarditis and that skeletal muscle biopsies
are a selective diagnostic tool for viral myocarditis.
In addition, at present the viral diagnostics in patients with viral
myocarditis is limited. Whereas over 20 different viruses have been known to
cause myocarditis, currently viral myocarditis patients in general are tested
for only a few virus types. Presently, knowledge regarding the putative
influence(s) of virus type on the pathophysiology of viral myocarditis is
lacking. For this, improvement of the viral diagnostics in viral myocarditis is
a clinical demand.

The primary objectives of this project are the identification of biomarkers in
the blood and skeletal muscle that can improve the diagnosis of viral
myocarditis and the expansion and improvement of the viral diagnostics in these
patients. However, the analysis of the skeletal muscle biopsy of the first 20
patients revealed that the expected increase in intramuscular inflammation was
not observed. Therefore, we will as of 27-2-2019 stop with obtaining skeletal
muscle biopsy from the patients until further notice.

The study design is as follows:
Patient selection: Patients at the Cardiology department at the VUmc that
present with suspected viral myocarditis (show flu-like symptoms combined with
acute loss of heart function) and comply to the ESC/ACC position statement for
EMB will be asked to participate in this study (viral myocarditis group).
Because we require distinguishing biomarkers, we want to obtain blood from
patients that developed cardiac inflammation through another cause also, i.e.
patients with acute myocardial infarction (AMI). In AMI cardiac damage and
cardiac inflammation are also induced, although not as a result of viral
infection but as a result of impaired perfusion of the heart. Therefore,
patients that present at the Cardiology department with AMI will be asked to
participate in this study also (AMI group). These are patients with CAG-proven
epicardial coronary occlusion reflecting AMI.
Because viral myocarditis can mimic AMI, we will also include patients who
present with infarct-like complaints, but show non-occluded epicardial coronary
arteries in CAG. Based on subsequent MRI analyses, these patients will later be
subdivided into the AMI group (in case of MINOCA) or the viral myocarditis
group.


Study-related proceedings: From all patients that are enrolled in this study
three extra tubes of blood will be drawn. This will be done within 0-2 days
after admission to the hospital. We plan to let this coincide (as much as
possible) with a time point when blood is drawn for diagnostic purposes. From
patients who are scheduled for CAG we aim to take the blood shortly after the
CAG procedure. Participants in the viral myocarditis group are also asked to
collect a faeces sample.
After approximately 6 month follow-up, from participants in the viral
myocarditis group again three extra tubes of blood will be drawn.

One blood sample will be collected in a coagulation tube to obtain serum. Two
blood samples will be collected in EDTA coated tubes to isolate plasma and
peripheral blood mononuclear cells (PBMCs). Serum and plasma will be aliquoted
and stored at -80*C. Also the PBMCs will be stored in liquid nitrogen.
Following consent from the patient, one third of the isolated PBMCs will be
used for direct monocyte isolation to study their characteristics post AMI.
Faeces samples will be collected in stool containers and stored at -20*C. One
skeletal muscle biopsy will be frozen immediately (-80*C) for viral PCR and one
will be fixed immediately in formalin for (immuno)histochemical biomarker
research. All samples will receive an experimental code before storage. Left
over material from EMB that were taken from these patients on medical
indication for diagnostic purposes will be used for diagnostic biomarker
validation and viral diagnosis. In theory this will create two groups within
the viral myocarditis group: 1) patients with viral myocarditis confirmed by
analysis of EMB, 2) patients with symptoms of viral myocarditis but with
negative EMB (these patients may have viral myocarditis but a false-negative
biopsy analysis or despite the symptoms they may not have viral myocarditis.

Promising biomarkers, identified in different mouse models of myocarditis that
we are now studying will be analyzed in patients with (suspected or confirmed)
viral myocarditis. In addition our findings from the quadriceps muscle and
heart tissue obtained at autopsy of viral myocarditis patients will be analyzed
in the living patient. In mice we induce different manifestations of viral
myocarditis i.e. acute and chronic (auto-immune) myocarditis. We will use
Coxsackievirus B3 and Parvo B19 virus; two viruses that most frequently appear
to be responsible for inducing viral myocarditis in humans. In these models we
will analyze the blood and quadriceps skeletal muscle for diagnostic biomarkers
that are able to discern viral myocarditis in different stages of the disease.
Biomarkers in the blood/skeletal muscle will be compared with heart function
measurements, histological anomalies in the heart, viral titers and the
incidence and severity of auto-immunity. Promising biomarkers, identified in
the animal models will be analyzed in the blood, quadriceps skeletal muscle
biopsies and EMB of patients in the viral myocarditis group. Hereby, biomarkers
in the blood/skeletal muscle will be compared with heart function measurements,
ECG, (immuno)histological analysis of EBM and to data obtained from routine
diagnostic blood measurements (hemoglobin, blood cell counts, electrolytes,
blood gasses, cardiac troponins, CK-MB, CRP). Furthermore, these biomarkers
will be compared among the two (possible) different groups within the viral
myocarditis group and the AMI group.

As for the viral diagnostics we will determine virus type and titer in the
blood, quadriceps skeletal muscle biopsies, faeces and the EBM. In the heart
and skeletal muscle biopsies we will analyze for the presence and titer of a
wide range of cardiotropic viruses using PCR. Also here, the viral data
obtained will be compared to heart function measurements, ECG,
(immuno)histological analysis of EBM and to data obtained from routine
diagnostic blood measurements. Furthermore, this data regarding virus
type/titer will be compared among the different patient groups and to the
different analyzed biomarkers.

Promising biomarkers, identified in the viral myocarditis patients will then be
tested in the blood of patients with acute myocardial infarction also. In this
way we hope to identify biomarkers that we can use to diagnose viral
myocarditis with high incidence and to improve the viral diagnostics in these
patients.

Burden and risk for patients are minimal: only taking blood.
With taking blood, local phlebitis and thrombophlebitis are possible adverse
events, but they are very rare. With taking skeletal muscle biopsies, local
muscle pain, haematoma and a small chance of bleeding, infection and a local
temporary numb feeling are possible side-effects. Patients that develop any of
these side-effects will be given adequate treatment.