a haemodynamically assessed, coronary artery ligation model of heart failure at 8 and 15 weeks after ligation. Trabeculae were isolated from the right ventricle ...
Pflügers Arch – Eur J Physiol (1998) 436:436–442
© Springer-Verlag 1998
O R I G I N A L A RT I C L E
M.A. Denvir · N.G. MacFarlane · S.M. Cobbe D.J. Miller
Sarcoplasmic reticulum Ca2+ loading in rabbits 8 and 15 weeks after coronary artery ligation
Received: 21 January 1998 / Received after revision and accepted: 3 April 1998
Abstract Calcium uptake by cardiac sarcoplasmic reticulum (SR) is reported to be reduced in heart failure in the human and in a number of animal models. However, the majority of studies have examined end-stage heart failure in the human and few animal studies have taken account of the duration and severity of left ventricular dysfunction. In this study we have compared SR Ca2+ loading in a haemodynamically assessed, coronary artery ligation model of heart failure at 8 and 15 weeks after ligation. Trabeculae were isolated from the right ventricle and mounted for isometric tension measurement. They were treated with saponin to permeabilize the sarcolemma but retain SR function and bathed in a mock intracellular solution including adenosine triphosphate (ATP) and buffered Ca2+. Caffeine was used to release Ca2+ from the SR. The amplitude of the caffeine-induced contracture was used as a quantitative gauge of the Ca2+ content of the SR. Eight weeks after ligation, trabeculae demonstrated enhanced SR Ca2+ uptake as manifest by larger caffeine-induced contractures (e.g. 200 nM [Ca2+], 120 s loading – 38.2±9.2 versus 67.3±10.1% of maximum Ca2+-activated force, FCa, max, P=0.03). At 15 weeks, trabeculae from ligated hearts were not significantly different from controls with SR Ca2+ loading returning to control levels (e.g. 200 nM [Ca2+], 120 s loading – 47.3±9.6 versus 30.2±12.8% FCa, max, P=0.12). These data suggest that SR Ca2+ loading may increase in the early stages of heart failure and fall back to normal with an increasing duration of left ventricular dysfunction. Increased incidence of spontaneous Ca2+ release observed from the SR at 8 weeks and not at 15 weeks may represent an arrhythmogenic mechanism specific to the early phase of heart failure.
M.A. Denvir (✉) · SM Cobbe Department of Medical Cardiology, Glasgow Royal Infirmary, 10 Alexandra Parade, Glasgow G31 2ER, UK N.G. MacFarlane · D.J. Miller Institute of Biological and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
Key words Calcium · Chemically-skinned muscle · Heart failure · Myofilaments · Rabbit · Sarcoplasmic reticulum
Introduction There is now considerable evidence that Ca2+ uptake by the sarcoplasmic reticulum (SR) is reduced in heart failure [8, 12, 15, 19, 20]. Studies of human myocardium have been almost exclusively confined to patients with end-stage heart failure since this tissue becomes available at the time of cardiac transplantation [14]. Few studies have taken account of the severity and duration of heart failure. However, there are a few reports which suggest that SR Ca2+ uptake may increase in mild pressure overload hypertrophy [2, 16] and reduce with more severe overload hypertrophy [2]. We have reported previously that SR Ca2+ uptake was increased in isolated saponintreated cardiac trabeculae in the rabbit coronary artery ligation model of heart failure 8 weeks after ligation [10]. In studies of similar preparations from patients with a range of clinical left ventricular (LV) dysfunction, we reported that SR Ca2+ loading ability diminished with increasing severity of LV dysfunction [9]. However, we noted that trabeculae from patients with moderate heart failure could produce caffeine-induced contractures close to maximum Ca2+-activated force, indicating that the SR could load a considerable quantity of Ca2+. This suggests that these patients had well preserved myocardial SR Ca2+ loading. Therefore, although there appears to be a bimodal change in SR Ca2+ loading in pressure overload hypertrophy, there is little information in the literature to show that such changes occur in failing myocardium. We hypothesized that the findings at 8 weeks after ligation in the rabbit represent a period of compensation and that later, either as heart failure becomes more severe or with increasing duration of heart failure, SR Ca2+ uptake will decrease. This study compares SR Ca2+ loading in saponin-treated rabbit cardiac trabeculae at 8 weeks and 15
437
weeks after coronary ligation with detailed haemodynamic assessment at both time points.
Materials and methods Animal model and assessment of heart failure The technique of coronary artery ligation has been reported previously [9] but is described here in brief. Coronary ligation was performed under general anaesthesia and mechanical ventilation via a left thoracotomy. The marginal branch of the left coronary artery was ligated at the midpoint between the atrio-ventricular groove and the cardiac apex. Animals were allowed to develop chronic heart failure over the subsequent 8 weeks (n=12, LIG8) or 15 weeks (n=12, LIG15). At the end of these time periods echocardiography was performed on the lightly sedated animal 2 days before invasive haemodynamic assessment. For invasive assessment a left ventricular catheter was placed via the right carotid artery and a thermodilution catheter placed in the descending aorta via the left femoral artery under general anaesthesia. The rabbit was allowed to recover over the next 24 h and then measurements of LV systolic and diastolic pressures were made from the conscious animal. Cardiac output was measured using a previously validated thermodilution technique involving the injection of 1 ml cold saline into the left ventricle [26]. Measurement of these haemodynamic parameters was repeated after a standardized fluid challenge (15 ml/kg of intra-ventricular saline over 1 min). Sham-operated animals (SH) were treated in an identical manner without ligation of the coronary artery and were caged under identical conditions for the same period of time for each group (8 week shams, SH8, n=7, 15 week shams, SH15, n=7). Sham animals also underwent identical assessment of LV function. All procedures were performed in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986.
from LV trabeculae due to difficulties in consistently obtaining suitable preparations for the experiments. The skinned fibre technique is described here in brief but in more detail elsewhere [22]. The trabecula was snared using nylon monofilaments to two stainless steel tubes – one fixed, the other attached to an isometric force transducer. The trabecula was immersed in one of a series of chambers, each containing approximately 5 ml of solution. The chambers were switched under microcomputer control to effect the solution change. The preparation was initially exposed to solution B (Table 1) with 50 µgml–1 saponin (Sigma) added for 30 min which permeabilizes the sarcolemma leaving the SR functionally intact. The sarcomere length was set at 2.1–2.2 µm by adjusting preparation length whilst viewed by differential interference contrast light microscopy, and preparation size (width and depth) was measured. All experiments were performed at room temperature (20–21°C) and at pH 7.0. The SR was allowed to load Ca2+ in solution A with aliquots of calcium chloride (10 mM) added to provide a range of [Ca 2+] in the submicromolar range. The SR loading protocol included five loading times ranging from 30 to 150 s. Caffeine-induced contractures were evoked by solution A (Table 2) containing caffeine (10 mM, Fig. 1). The SR loading protocol was followed stringently for LIG and SH animals. At the end of the SR loading protocol, the trabeculae were treated with Triton X-100 to disrupt all cell membranes leaving only the myofilaments intact. An assessment of maximum Ca2+activated force (FCa, max) in solution C (Table 1) and myofilament Ca2+ sensitivity (pCa50= –log[Ca2+] required for 50% activation) was then made for each preparation. The Ca2+ sensitivity curves are plotted according to the Hill equation and are fitted by a least mean square procedure (Fig. 5). To avoid distortions produced by pooling experimental data, curves were fitted for individual experiments and the pCa50 and Hill coefficient calculated on an individual basis. The curves shown represent those fitted to the mean Hill coefficient and mean pCa50 for the four groups of animals.
Isolated trabeculae experiments Right ventricular trabeculae were isolated for assessment of SR and myofilament function in both groups. No data were obtained Table 1 Composition of solutions (in mM, pH 7.0, room temperature) Soln K+a Mgb Ca A B C
140 7.0 140 7.0 140 7.0
pCa
0.02d 7.29 0.02 9.03 10.0 4.20
ATP CrP 5.0 5.0 5.0
Nac
EGTA HEPES
15.0 40.0 0.2e 15.0 40.0 10.0 15.0 40.0 10.0
25.0 25.0 25.0
a b
Potassium ions added as KCl and KOH Magnesium added as 1 M MgCl2; free Mg2+=2.1–2.5 mM in all solutions c Sodium ions from the salts Na ATP and Na CrP 2 2 d Estimated Ca2+ contamination level. Total chloride concentrations varied from about 110 to 120 mM e 1,6 Diaminohexane N, N, N′, N′-tetraacetic acid (9.8 mM) was added to this solution to maintain equivalent ionic strength
Fig. 1 Sarcoplasmic reticulum (SR) loading protocol used to assess Ca2+ loading ability. Arrow heads represent a 5-s application of 10 mM caffeine causing release of Ca 2+ from the SR
Table 2 Haemodynamic data from all 4 groups (LVEDD Left ventricular end-diastolic diameter, LVEDP left ventricular end diastolic pressure, CO cardiac output)
Ejection fraction (%) LVEDD (cm) LVEDP (mmHg) baseline CO [ml/(kg·min)] Peak CO [ml/(kg·min)] Body weight (kg)
Sh8 (n=7)
LIG8 (n=12)
P
SH15 (n=7)
LIG15 (n=12)
P
76.7±3.2 1.44±0.08 6.7±3.0 343.6±38.9 526.6±29.9 3.78±0.39
41.9±9.5 1.86±0.19 15.1±6.9 275.2±63.1 332.8±37.9 3.75±0.46
0.02 0.01 0.03 0.05 0.03 0.30
80.2±4.1 1.53±0.1 7.6±2.6 328.6±29.3 513.7±38.5 3.86±0.31
51.3±7.6 2.0±0.26 15.5±4.9 260.2±45.3 343.8±69.1 3.80±0.24
0.01 0.01 0.03 0.04 0.04 0.90
438 Statistics Values are expressed as mean ± standard deviation unless otherwise stated. An unpaired t-test was used to assess the statistical significance of the difference between groups. Significance was accepted at P
