Calcium-sensitivity of the SR calcium release channel in failing and nonfailing human myocardium

Background: Altered Ca2+ metabolism of the sarcoplasmic reticulum results i n changes of the contractile behavior in failing human myocardium. The ryan odine-sensitive Ca2+ release channel of the sarcoplasmic reticulum plays a key role in the intracellular Ca2+ handling in cardiac myocytes. Recently, we showed that the density of H-3-ryanodine binding sites which correspond to the SR Ca2+ release channel in human myocardial homogenates is unchanged in failing human myocardium. However, the sensitivity of the channel towar ds Ca2+, which acts as the trigger signal of channel activation and thereby initiates contraction, has not yet been investigated in failing and nonfai ling myocardium. Methods: Homogenates (100 mu g protein) from hearts with dilated (DCM, n = 10) or ischemic (ICM, n = 9) cardiomyopathy were incubated with a saturatin g concentration of H-3-ryanodine (12 nM) in the presence of different Ca2concentrations ranging from 1 nM to 10 mM. For comparison, myocardium of 8 nonfailing hearts which could not be transplanted for technical reasons was investigated. Nonspecific binding was determined in the presence of a high concentration (10 mu M) of unlabeled ryanodine. Results: H-3-ryanodine binding to the Ca2+ release channel showed a bell-sh aped pattern with an increase in specific binding at submicromolar Ca2+ con centrations and a decrease at higher Ca2+ concentrations than 0.5 mM, where as nonspecific binding was not influenced by different Ca2+ concentrations. In nonfailing myocardium, maximal H-3-ryanodine binding (Bmax) was 85.2 +/ - 3.1 fmol/mg protein and half-maximal binding was reached at a free Ca2+ c oncentration of 0.25 (0.22 - 0.30) mu M (EC50). Neither EC50 values nor max imal specific H-3-ryanodine binding differed between nonfailing and failing myocardium of both etiologies. EC50 values were 0.24 (0.23 - 0.26) mu M (D CM, n = 10) or 0.28 (0.25 - 0.31) mu M (ICM, n = 9), respectively. Caffeine (2 mM) and the ATP-analogon AMP-PCP (I mM) led to a shift towards lower Ca 2+ concentrations consistent with an activation of the channel by these com pounds, whereas Mg2+ (0.7 mM) shifted the Ca2+-dependence of H-3-ryanodine binding towards higher Ca2+ concentrations indicating inhibition of channel opening. After activation of the Ca2+ release channel by caffeine or AMP-P CP as well as after the inhibition with Mg2+ EC50 values were the same in f ailing and nonfailing myocardium. Conclusion: Caffeine and AMP-PCP sensitize, whereas Mg2+ desensitizes the m yocardial Ca2+ release channel to Ca2+. The determination of Ca2+-dependent H-3-ryanodine binding to the human myocardial Ca2+ release channel is a us eful tool to investigate its open probability. Furthermore, the Ca2+-sensit ivity and the pharmacological behavior of the human SR Ca2+ release channel are similar in failing and nonfailing myocardium.