Department of Pediatrics, Division of Molecular Cardiovascular
Biology, Children's Hospital Medical Center, 3333 Burnet Ave,
Cincinnati, OH 45229-3039
Correspondence should be addressed to Jeffery D. Molkentin molkj0@chmcc.org
To the editor−Heart failure afflicts an estimated 4−5
million individuals in the United States each year at a cost of approximately
$12 billion1,
2. Of this group, a few hundred thousand have
advanced heart failure, characterized by a 2-year mortality rate of greater
than 90% (3). Many intracellular signaling
pathways and second messenger systems are involved in cardiac hypertrophy
and heart failure. One potential central molecule associated with cardiac
hypertrophy and the progressive nature of heart failure is calcium. Intracellular
calcium regulates myocardial contractility and relaxation by coordinated release
and sequestration of calcium from the sarcolemma and sarcoplasmic reticulum.
Heart failure is associated with increased intracellular basal calcium concentrations,
decreased amplitude of the systolic calcium transient and defective sequestration
of calcium in the sarcoplasmic reticulum during diastole4. These
alterations in calcium handling are thought to play an essential part in the
progressive deterioration of cardiac function in heart failure
Alterations in intracellular calcium handling associated with heart failure
might also be expected to activate calcium-sensitive intracellular signaling
factors, including PKC isoforms, MAPK signaling factors and calcineurin. Calcineurin,
a calcium-regulated phosphatase, is sufficient to mediate cardiac hypertrophy
in transgenic mouse hearts when overexpressed in an activated form5.
Cyclosporin and FK506, both calcineurin inhibitors, can prevent cardiac hypertrophy
and dilated myopathy in rodent models of heart disease6. Therefore,
calcineurin may be important in cardiac reactive responses; however, it is
uncertain whether these findings have relevance to human heart disease.
Activated calcineurin protein levels are increased in failing human hearts.
Human left ventricular protein extracts (400 ug) were immunoprecipitated with
excess calmodulin antibody (4 ug) and protein G agarose in calcium-free buffer.
Precipitated proteins were then analyzed by western blot to quantify calmodulin
and calcineurin protein levels. As activated calcineurin is complexed with
calmodulin, these data indicate levels of activated calcineurin. Calcineurin
levels were normalized to the amount of immunoprecipitated calmodulin in each
reaction (histogram). Western blot analysis of unprecipitated GAPDH levels
demonstrates equal integrity of the extracts. The data are quantified as percent
activation s.e.m. Significance was demonstrated by an unpaired
t-test with software from Instat (GraphPad, San Diego, California).
Calcineurin is activated by prolonged increases in intracellular calcium
concentration7, thus it might be activated in human heart failure.
To determine if calcineurin was associated with human heart failure, we developed
an assay specific for calcineurin activity, to bypass the technical difficulties
associated with human tissue samples. This assay involves immunoprecipitation
of calmodulin from tissue homogenates followed by a calcineurin-specific western
blot. Because activated calcineurin is complexed with calmodulin, this assay
identifies the portion of calcineurin in the activated state.
The normal control individuals were three males and two females, ages 22−74;
the heart failure individuals were ten males and one female ages 38−57
(average ejection fraction, 16%) (Table). We obtained
left ventricular heart samples and quantified activated calcineurin levels
by calmodulin co-immunoprecipitation (Fig.). To control for variability in
the immunoprecipitation procedure and to normalize calcineurin levels, we
also probed the same blot with calmodulin antibody. Calcineurin is activated
by an average of 400 19% in the failed left ventricular samples,
compared with an average of 100 18% in the non-failed samples (
P < 0.0001). Separate GAPDH (Fig.) and calmodulin (not shown) western
blots demonstrated similar integrities for each of the protein extracts and
equal levels of endogenous calmodulin protein (Fig.). The data were quantified
on a PhosphorImager (Molecular Dynamics, Sunnyvale, California) by detecting
fluorescence emission generated with the ECF western blot kit (Amersham).
Table 1. Patient information of left ventricular tissue used in the calcineurin
activation assay
One variable that might affect the observed profile of activated calcineurin
in this study is drug treatment regimen. Each heart failure patient was on
some combination of diuretics, angiotensin converting enzyme (ACE) inhibitors,
inotropic agonists or anti-arrythmia agents (Table).
However, although three of the five normal ventricular samples were obtained
from patients receiving either a diuretic or inotropic agonist (
Table, patients 1−3), calcineurin was not substantially activated
in these samples compared with the samples from patients with heart failure.
Other variables such as age or sex of the patients did not seem to affect
the levels of activated calcineurin substantially. These data indicate that
calcineurin may play a critical part in the progressive nature of human heart
failure, consistent with the known profile of altered calcium handling during
heart failure.
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Molkentin, J.D. et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell93, 215-228 (1998). | Article | PubMed | ISI | ChemPort |
Sussman, M.A. et al. Prevention of Cardiac Hypertrophy in Mice by Calcineurin Inhibition. Science281, 1690-1693 (1998). | Article | PubMed | ISI | ChemPort |
s.e.m. Significance was demonstrated by an unpaired
t-test with software from Instat (GraphPad, San Diego, California).
