Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC21
Correlation of whole heart function and SERCA activity in a rabbit working heart model
E. B. Elliott1, A. Kelly2, G. L. Smith2, C. M. Loughrey1
1. Faculty of Veterinary Medicine, University of Glasgow, Glasgow, United Kingdom. 2. Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Alterations of SERCA activity are common to all models of heart failure yet the dependence of whole heart function on SERCA activity remains unclear. This study aimed to correlate SERCA activity with whole heart function in an ex vivo working heart model. Adult male New Zealand white rabbits (2.5-3.5kg) were killed (pentabarbitone, 100mg/kg, i.v.), hearts removed and rapidly cannulated onto a working heart rig. Pre-load and after-loads were set to 7 and 63mm Hg respectively. Hearts were perfused with physiological Tyrodes solution (37oC, 2.5mmol/L [Ca2+]) and a pressure-volume catheter was placed in the left ventricular (LV) cavity; hearts were paced at 200-220 beats/min. SERCA inhibition was achieved via addition of thapsigargin (TG; 2.8μmol/L) and vehicle time-matched hearts (0.09% DMSO) served as controls. Following the perfusion period a transmural sample of LV tissue was removed, snap frozen and homogenised in a buffer containing protease and phosphatase inhibitors. Total protein content was determined and SERCA activity was assessed in homogenates via an oxalate-facilitated Ca2+ uptake assay. In the continued presence of TG, the working heart sustained forward flow function for 31±11min (mean±S.E.M., n=6). At this endpoint, function (as % of pre TG) was significantly altered - peak pressure: 100.56±0.63% vs. 96.12±0.74%, dP/dtmax: 100.89±1.72% vs. 87.14±1.59%, dP/dtmin: 98.83±2.06% vs. 87.71±2.29% and tau: 98.56±1.08% vs. 112.88±2.85% (control vs. TG; mean±S.E.M.; P<0.05 in all by Student’s unpaired t-test). Coronary flow was constant throughout. Analysis of homogenate samples revealed SERCA activity had fallen below detectable limits (<5% of control) in all endpoint hearts. In a separate set of experiments, modest alterations in whole heart function were obtained using short exposure times to TG (80-100s) yielding hearts with undetectable SERCA activity (n=4) and hearts with detectable SERCA activity (n=5). In the latter group, SERCA activity was quantified by fitting gradients to the decline phase of free [Ca2+] at 1μM and 300nM. These decreased in parallel at both points in TG hearts suggesting no alteration in SERCA Kd. When plotted against whole heart functional parameters there was a strong negative correlation of dP/dtmax with SERCA activity (P<0.05). When SERCA activity was undetectable (<5%), dP/dtmax was reduced to 80% of control levels. Tau was increased by 110.59±1.87% when SERCA fell to 45.27±2.12% of control. These data indicate that the rabbit working heart model can maintain function for a sustained period of time when SERCA levels are below 5% of control. SERCA activity falls rapidly (<2min) upon exposure to thapsigargin with only a modest drop in whole heart function (to 80% of control). The point of pump failure (after~31min) occurs by an as yet unidentified mechanism.
Where applicable, experiments conform with Society ethical requirements