Circulating Markers for Ischemic Heart Disease

Hypotheses, Objectives and Aims:

Hypotheses:Caspase-3, cleaved and activated, and dystrophin can be detected in human
circulation. The levels of these two markers are elevated during acute myocardial infarction.
Furthermore, the levels of these two proteins are greater in those who develop heart failure
than those who do not.

Objectives:

- To determine whether cleaved caspase-3 and dystrophin can be detected in human
circulation after an acute myocardial infarction

- To compare serum levels of these two markers in those who develop heart failure and
those who do not

Scientific Background and Significance: Apoptosis is a regulated biological process resulting
in cell death (4-9). Caspases, a family of cysteine acid proteases regulate the process, and
in fact, lead to apoptosis. Apoptotic trigger or signal results in the activation of proximal
or initiator caspases (such caspase-8, -9, 10). These initiator caspases then cleave and in
turn activate downstream effector caspases such as caspases-3, -6 and -7. These effector
caspases then cleave various proteins such as those present in cytoskeletons and nucleus like
lamin A, alpha-fodrin and poly (ADP-ribose) polymerase, leading to apoptosis. Caspase-3 is
the key executioner in this apoptotic pathway, responsible totally or critically in the
proteolytic cleavage of cellular and nuclear proteins. Activation of caspase-3 requires
proteolytic processing of its inactive zymogen into active p17 and p12 fragments. The cleaved
caspase-3 can be detected by antibodies specific for this cleaved enzyme (p17 fragment) in
cell lysates by immunoblotting or by an ELISA assay utilizing spectrophotometric
determination with a microplate reader at OD450 nm. Ischemia and reperfusion are known to
cause apoptosis. Therefore, acute MI may be associated with release of the final executioner
of apoptosis that is caspase-3, into the circulation.Another potential marker for acute
deterioration is dystrophin. Dystrophin was originally identified as the x-linked gene whose
mutations in its N-terminus cause cardiomyopathy. Dystrophin provides important structural
support for the cardiac myocyte and its sarcolemmal membrane (10-11). It links actin at its
N-terminus with the dystrophin-associated protein complex and sarcolemma at the C-terminus
and the extracellular matrix of muscle. Mutations cause loss of support and sarcolemmal
instability and myopathy. Myocardial dystrophin translocation and cleavage are associated
with the progression of heart failure and contractile dysfunction. These changes are reversed
following reduction of mechanical stress from ventricular assistance device (12). Since MI is
associated with sarcolemmal instability, dystrophin may also be released into circulation.

Inclusion Criteria:

- men and women, 18 years of age and over with acute myocardial infarction (determined
by positive cardiac markers -CKMB/ troponin) with or without heart failure (dyspnea,
rales, edema, elevated jugular venous pressure, ascites).

- Heart failure can be diagnosed using imaging evidence such as dilated heart, poor
contractile function or echocardiographic Doppler evidence of diastolic dysfunction or
elevated right- or left-sided filling pressures

- A control group of male subjects age 60 and older without history of MI or heart
disease

Exclusion Criteria:

- Subjects unable to give consent

- Subjects who have undergone cardiac or non-cardiac surgery in the 3 months prior to
enrollment