Activation of Apoptotic Caspase Cascade During the Transition to Pressure Overload-Induced Heart Failure
Narain Moorjani, MRCS*,*,
Manzoor Ahmad, PhD ,
Pedro Catarino, FRCS*,
Robin Brittin, MSc ,
Danyah Trabzuni, BSc,
Futwan Al-Mohanna, PhD,
Navneet Narula, MD ,
Jagat Narula, MD, PhD and
Stephen Westaby, MS, PhD, FETCS*
* Department of Cardiothoracic Surgery, Oxford Heart Centre, John Radcliffe Hospital, Oxford, England
Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania
University of California, Irvine School of Medicine, Orange, California
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Figure 1 Variable aortic constriction device consisting of Gore-Tex cuff encaging the balloon of a Foley catheter. The proximal port of the catheter was brought out subcutaneously to allow gradual inflation and increase in pressure overload. RA = right atrium; LA = left atrium.
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Figure 2 Echocardiographic changes induced by gradually increasing afterload on left ventricle. Graphs show changes in (A) left ventricular mass index, (B) left ventricular internal diameter in diastole, and (C) fractional shortening caused by progressive pressure overload.
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Figure 3 Caspase activity during the transition to heart failure. Biopsy samples (50 mg, n = 10 from each stage) were taken at echocardiographically distinct stages of normal left ventricle (control), left ventricular hypertrophy (LVH), left ventricular dilation (LVD), and left ventricular failure (LVF). Activities of caspase-3, -8, and -9 were measured using specific fluorogenic substrates (DEVD-AFC for caspase-3, LETD-AFC for caspase-8, and LEHD-AFC for caspase-9) and expressed relative to control samples (mean ± SEM). *p < 0.05; p < 0.01 comparing stages with control (paired Student t test).
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Figure 4 Immunohistochemical staining for active caspase-3 during the transition to heart failure. Sheep lymph node tissue used as (a) positive control and (b) negative control with primary antibody omitted. Biopsy samples (n = 4 from each stage) were taken at echocardiographically distinct stages of (c) normal left ventricle (control), (d) left ventricular hypertrophy, (e) left ventricular dilation, and (f) left ventricular failure and probed with mouse monoclonal antibody specific for the active p11 fragment of caspase-3. Positivity for active caspase-3 is shown by brown cytoplasmic staining (magnification x20).
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Figure 5 Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in the transition to heart failure. Sheep lymph node tissue used as (a) positive control and (b) negative control with TdT enzyme omitted. Biopsy samples (n = 4 from each stage) were taken at echocardiographically distinct stages of (c) normal left ventricle (control), (d) left ventricular hypertrophy, (e) left ventricular dilation, and (f) left ventricular failure and subjected to peroxidase labeled TUNEL assay. The TUNEL-positivity is shown by dark brown staining nuclei (magnification x20).
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Figure 6 In situ oligoligation (ISOL) in the transition to heart failure. Sheep lymph node tissue used as (a) positive control and (b) negative control with T4 DNA ligase enzyme omitted. Biopsy samples (n = 4 from each stage) were taken at echocardiographically distinct stages of (c) normal left ventricle (control), (d) left ventricular hypertrophy, (e) left ventricular dilation, and (f) left ventricular failure and subjected to peroxidase-labeled ISOL assay. The ISOL positivity is shown by dark brown staining nuclei (magnification x20).
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