Effect of ischemia and reperfusion on cardiac ryanodine receptors--sarcoplasmic reticulum Ca2+ channels.

Abstract

We investigated the effect of ischemia and reperfusion on the cardiac ryanodine receptor, which corresponds to the sarcoplasmic reticulum Ca2+ channel. Isolated working rat hearts were subjected to 10 to 30 minutes of global ischemia, followed or not by reperfusion. Ischemia produced significant reduction in the density of high-affinity 3H-ryanodine binding sites, determined either in whole-heart homogenate (Bmax, 220 +/- 22, 203 +/- 12, and 228 +/- 14 fmol/mg protein after 10, 20, and 30 minutes of ischemia versus 298 +/- 18 fmol/mg protein in the control condition; P < .01) or in a fraction enriched in sarcoplasmic reticulum (Bmax, 1.08 +/- 0.15 pmol/mg protein after 20 minutes of ischemia versus 1.69 +/- 0.08 pmol/mg protein in the control condition; P < .01). The Kd (1.5 +/- 0.1 nmol/L) and the Ca2+ dependence of high-affinity 3H-ryanodine binding were not affected by ischemia. The density of low-affinity 3H-ryanodine binding sites was also reduced after 20 minutes of ischemia (14.0 +/- 2.3 versus 34.0 +/- 8.2 pmol/mg protein in the sarcoplasmic reticulum fraction, P < .05), without significant changes in Kd (4.7 +/- 1.2 versus 2.4 +/- 1.0 mumol/L). All these changes persisted after 20 minutes of reperfusion. Analysis of tissue fractions showed that 55% of the ryanodine binding sites were retained in the pellet of a low-speed centrifugation ("nuclear pellet") and that the effects of ischemia concerned only the receptors released in the supernatant ("postnuclear supernatant"). In parallel experiments, we evaluated the effect of ryanodine on oxalate-supported Ca2+ uptake, which represents sarcoplasmic reticulum Ca2+ uptake. As expected, we found that high concentrations of ryanodine stimulated Ca2+ uptake, owing to channel blockade. The response to 900 mumol/L ryanodine was slightly reduced in crude homogenate and significantly reduced in postnuclear supernatant obtained from ischemic hearts. In conclusion, the number of ryanodine receptors is reduced after ischemia; this effect concerns a subpopulation of the receptors, persists after reperfusion, and might contribute to modify sarcoplasmic reticulum function.

Effects of Cardioplegic Arrest and Reperfusion on Rabbit Cardiac Ryanodine Receptors

“…This finding is in agreement with previous studies. [6][7][8]10 Daring et al observed a reduction in the Bmax for ryanodine after 60 min of ischemia, but could not conclude that the density of SR channels was affected by ischemia because of contamination by myofibrillar protein. 6 However, Zucchi et al reported that myofibrillar contamination, as estimated by K-ATPase activity, was not different in SR preparations obtained from controls or after 20 min of ischemia.…”

“…6 However, Zucchi et al reported that myofibrillar contamination, as estimated by K-ATPase activity, was not different in SR preparations obtained from controls or after 20 min of ischemia. 7 In the present study, we purified the enriched fraction of the SR vesicles for the binding assay because a previous study showed that this successfully avoided contamination by myofibrillar proteins. The smaller reduction in ryanodine binding seen in the CA group indicated that the number of receptors in the functional state was much better preserved during cardioplegic arrest and reperfusion than during global ischemia -reperfusion.…”

“…6 In addition, Zucchi and colleagues noted that in rat hearts the Bmax for high-affinity ryanodine binding to an enriched fraction of SR vesicles was reduced after 20 min of ischemia, and that the reduction persisted after reperfusion. 7 These investigators concluded that the number of RyR was reduced after ischemia, and that this effect might contribute to an alteration in SR function. Using a mature rabbit heart homogenate, Matsuda and colleagues observed that while the Bmax for high-affinity ryanodine binding was reduced after 30 min of ischemia, there was no difference between ischemia and non-ischemia in the expression levels of RyR mRNA or protein.…”

“…A number of reports indicate that ischemia depresses SR function; however, both the nature and cause of this effect remain controversial. [6][7][8][9][10] Darling and coworkers reported that the maximum number of binding sites (Bmax) for ryanodine binding to isolated dog SR vesicles was reduced after 60 min of ischemia. 6 In addition, Zucchi and colleagues noted that in rat hearts the Bmax for high-affinity ryanodine binding to an enriched fraction of SR vesicles was reduced after 20 min of ischemia, and that the reduction persisted after reperfusion.…”

Design, Synthesis, and Evaluation of Thyronamine Analogues as Novel Potent Mouse Trace Amine Associated Receptor 1 (mTAAR1) Agonists

“…After injection of 1000 U sodium heparin in the femoral vein, the heart was quickly excised and perfused according to the working heart technique, as described previously. 43 The height of the atrial and aortic cannulae was set at 20 and 100 cm, respectively. Unless otherwise specified, the perfusion buffer included (mM): 118 NaCl, 25 NaHCO 3 , 4.5 KCl, 1.2 KH 2 PO 4 , 1.2 MgSO 4 , 1.5 CaCl 2 , and 11 glucose.…”