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.