The effect of pharmacological modulation of the sarcoplasmic reticulum on left ventricular function in the isolated working rabbit heart

The work described in this thesis investigated the role of the sarcoplasmic reticulum (SR) in the regulation of normal mechanical function of the isolated heart, with the focus of the work resting on the calcium release channel, the ryanodine receptor (RyR) and the SR calcium pump, the sarco-endoplasmic reticulum calcium ATPase (SERCA). This was performed by characterising left ventricular mechanical function in an isolated working heart model in the face of pharmacological manipulation of the above intracellular SR proteins. The following were the aims of this project. 1. Characterisation of ex vivo working heart left ventricular mechanical function using miniaturised single and multi-segment pressure-conductance technology 2. Characterisation of working heart mechanical function under conditions where SR calcium cycling is altered; i) perfusion with elevated extracellular calcium and beta-adrenergic stimulation to induce SR calcium loading and ii) in a rabbit coronary artery ligation model of left ventricular dysfunction. Following this, the influence of acute perfusion of the compound K201 in healthy hearts and in the above experimental conditions was investigated. 3. Investigation of the specific contribution of SR calcium uptake to normal left ventricular contractility and relaxation by specific pharmacological blockade of SERCA in the isolated working heart. Hearts from male New Zealand White rabbits were cannulated via the aorta and a pulmonary vein ostium and perfused in working heart mode. Contractile function and changes in left ventricular volume during the cardiac cycle were assessed using a 3F pressure-conductance catheter inserted into the left ventricle via the aorta. The isolated working heart was found to produce a maximal stroke volume under identical loading conditions and pacing frequency using a volume of air within the compliance chamber of 15 ml of air. Using an atrial filling pressure of 7.4 mmHg and an afterload of 60 mmHg the preparation was also found to have stable function in working mode for 60-90 min with <10% decline in cardiac output. To assess the effect of the compound K201 on cardiac mechanical function isolated working hearts were perfused with increasing concentrations of K201 (0.3, 1.0 and 3.0) for 4 min at each concentration. When normalised to control hearts paced at equivalent rates 0.3 microM K201 led to a significant increase in end diastolic pressure (EDP) but did not alter other indices of mechanical function. 1.0 and 3.0 microM K201 led to a significant decline in a number of indices of cardiac contractility and relaxation. At 3.0 microM depression of mechanical function was of a sufficient extent as to cause cessation of forward flow (aortic flow) in 3 of the 8 hearts studied. In a subset of experiments where heart rate was kept constant via electrical stimulation of the right atrium, exposure to 1.0 microM K201 led to a decline in mechanical function that was of a similar degree to that of unpaced hearts. High calcium loading conditions were simulated by raising extracellular calcium concentrations from 2.5 mM to 4.5 mM, followed after 5 min by addition of 150 nM isoproterenol (ISO). Addition of ISO led to a transient improvement in all indices of mechanical function lasting approximately 20s. In hearts perfused with 4.5 mM extracellular calcium and ISO only (n=11) mechanical function declined thereafter such that by 600s post-ISO addition aortic flow had ceased in all but 5 of the 11 hearts in this group. In contrast, hearts perfused with K201 30s after addition of ISO (n=9) all maintained aortic flow and demonstrated significantly improved contractile function post-ISO in comparison to hearts not perfused with K201. K201 also led to a preservation of left ventricular peak pressure and stroke volume when applied 300s post-ISO. Despite being detrimental to mechanical function in healthy hearts K201 preserved function in a pharmacological model of high calcium loading. Left ventricular dysfunction was surgically induced in rabbits by coronary artery ligation. Ex vivo mechanical function was assessed 8 weeks post-op using a 3 segment multi-segmental pressure-conductance catheter inserted into the left ventricle with each segment delineating 3 discrete functional regions within the ventricular chamber; apex, mid and base. Hearts were paced at 200 beats/min throughout. 8 week ligation hearts exhibited a significantly reduced ejection fraction and higher end systolic volumes in comparison to sham operated controls. Assessment of regional mechanical function in 8 week ligation hearts revealed that a significantly greater proportion of total stroke volume and stroke work was performed by the mid segment vs. the apex, while regional function was similar between all segments in the sham operated controls. Treatment with 1.0 microM K201 led to a decline in systolic function and impairment of relaxation (decreased dP/dtmin and increased tau) in sham operated control hearts similar to that shown in chapter 3. In contrast, aside from dP/dtmax which was significantly reduced from baseline, K201 had no significant effect on mechanical function in 8 week ligation hearts. No region specific effects of K201 were detected, as the percentage contribution of each region to total stroke volume and stroke work remained unchanged in both groups after treatment with K201. The effects of K201 on mechanical function in hearts with left ventricular dysfunction appear to be altered in comparison to both healthy and sham operated hearts. SR calcium uptake was inhibited in an isolated working rabbit heart model using the specific pharmacological SERCA2a inhibitor thapsigargin (TG). Hearts were paced at 220 beats/min throughout the investigation. The experimental endpoint for TG treated hearts was reached when hearts no longer produced an aortic flow. Hearts perfused in working heart mode with the thapsigargin vehicle for 60 min acted as controls. At the end of each experiment left ventricular tissue samples were homogenised in a protease-phosphatase inhibitor cocktail and stored for subsequent analysis. A significant decline in systolic and diastolic functional parameters was evident 5 min after addition of TG, with no significant changes demonstrable in the vehicle control group at this timepoint. In TG treated hearts function had declined even further by the endpoint. In a subset of TG treated hearts end systolic and end diastolic volumes were shown to decline over time, indicative of imparied left ventricular filling and diastolic abnormalities. Analysis of SERCA uptake by Elliott et al. (2009) using tissue homogenates from the current investigation revealed that at the time of aortic flow cessation SERCA activity was <10%, suggesting that SR calcium uptake may decrease to very low levels before working heart function fails.