Circulating Markers That Underlie the Transition From Compensated Hypertrophy to Heart Failure

Heart failure (HF) is a complex clinical syndrome and major public health problem claiming
the lives of >500,000 per year. It is the leading Diagnosis Related Group (DRG) for hospital
discharges in the US. There are approximately 1-3 million admissions annually for acute
decompensated heart failure. The mortality rate in Classes III and IV heart failure is 14-18%
each year (1). At present, nearly 5 million patients have HF in the U.S. (2). Early detection
and prevention remain key measures in treating subjects with this form of heart disease.
Discovering and defining circulating markers that underlie the transition between compensated
hypertrophy and acute heart failure represent an area of important need in the detection and
prevention effort. The existing marker BNP is useful in helping differentiate those with
heart failure from those who have other conditions. However, the large variation of abnormal
BNP levels in those who carry a diagnosis of heart failure makes BNP level unreliable as a
predictor for the transition between compensated and decompensated heart failure.

Apoptosis contributes to, and perhaps, is the cause of myocyte death that underlies the
progression of heart dysfunction and the transition between stable compensated heart failure
and acute deterioration (3). 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.

The primary goals of this pilot study are to determine whether 1) activated caspase-3 can be
detected in human circulation and if so are there diurnal rhythm variations and serial
changes in the levels over time, 2) whether its level is increased during acute decompensated
heart failure, and 3) whether transition between acute and stable heart failure is correlated
with a decrease in its level.

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). In the present pilot study, we will test the
hypothesis that dystrophin can be released and detected in human circulation during acute
deterioration of heart failure. We will further test whether 1) its level is increased during
acute decompensated heart failure, and 2) whether transition between acute and stable heart
failure is correlated with a decrease in its level, and 3) whether there are serial changes
of these levels over time.

BNP is a known marker for stressed myocardium and has been used to detect myocardial
stretching and stress in heart disease. IL-6 and TNF alpha are both inflammatory markers and
have been shown to be elevated in inflammatory state such as heart failure. CRP is another
inflammatory marker. Knowing their levels is helpful in correlating the serum caspase-3 p17
level with those known serum factors in CHF.

By checking levels of these markers every 3 months (+/- one month) in stable HF patients for
two years, our goal is to see if those with a "spike" in level predict adverse outcome in
CHF. Ultimately, if such is the case, we can identify patients with a "spike" or cumulative
higher caspase-3 fragment as at high risk for morbidity/mortality. Identification of such
patients may cause us to treat them more proactively to attempt to alter outcome.

We will also obtain blood samples from control subjects to measure baseline levels and
determine if there are diurnal rhythm variations.

Inclusion Criteria:

- Individuals aged >18yrs

- stable or decompensated heart failure, irrespective of LVEF

- decompensated heart failure clinical symptoms such as dyspnea, rales, edema, elevated
jugular venous pressure, or ascites

- Imaging evidence of heart failure (cardiomegaly, poor contractile function or
echocardiographic Doppler evidence of diastolic dysfunction or elevated right- or
left-sided filling pressures)

- Healthy individuals with no prior history of heart attack or heart failure will be
recruited to use as controls.

Exclusion Criteria:

- Subjects who are unable to give informed consent

- Subjects who had undergone cardiac or non-cardiac surgery in the 3 months before
enrollment

- Pregnant subjects are not excluded