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

Abstract Background:

Altered Ca²⁺ metabolism of the sarcoplasmic reticulum results in changes of the contractile behavior in failing human myocardium. The ryanodine-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 ³H-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 towards Ca²⁺, which acts as the trigger signal of channel activation and thereby initiates contraction, has not yet been investigated in failing and nonfailing myocardium.

Methods: Homogenates (100 μg protein) from hearts with dilated (DCM, n = 10) or ischemic (ICM, n = 9) cardiomyopathy were incubated with a saturating concentration of ³H-ryanodine (12 nM) in the presence of different Ca²⁺ concentrations 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 μM) of unlabeled ryanodine. Results: ³H-ryanodine binding to the Ca²⁺ release channel showed a bell-shaped pattern with an increase in specific binding at submicromolar Ca²⁺ concentrations and a decrease at higher Ca²⁺ concentrations than 0.5 mM, whereas nonspecific binding was not influenced by different Ca²⁺ concentrations. In nonfailing myocardium, maximal ³H-ryanodine binding (Bmax) was 85.2 ± 3.1 fmol/mg protein and half-maximal binding was reached at a free Ca²⁺ concentration of 0.25 (0.22 – 0.30)μM (EC₅₀). Neither EC₅₀ values nor maximal specific ³H-ryanodine binding differed between nonfailing and failing myocardium of both etiologies. EC₅₀ values were 0.24 (0.23 – 0.26)μM (DCM, n = 10) or 0.28 (0.25 – 0.31)μM (ICM, n = 9), respectively. Caffeine (2 mM) and the ATP-analogon AMP-PCP (1 mM) led to a shift towards lower Ca²⁺ concentrations consistent with an activation of the channel by these compounds, whereas Mg²⁺ (0.7 mM) shifted the Ca²⁺-dependence of ³H-ryanodine binding towards higher Ca²⁺ concentrations indicating inhibition of channel opening. After activation of the Ca²⁺ release channel by caffeine or AMP-PCP as well as after the inhibition with MG²⁺ EC₅₀ values were the same in failing and nonfailing myocardium.

Conclusion: Caffeine and AMP-PCP sensitize, whereas Mg²⁺ desensitizes the myocardial Ca²⁺ release channel to Ca²⁺. The determination of Ca²⁺-dependent ³H-ryanodine binding to the human myocardial Ca²⁺ release channel is a useful tool to investigate its open probability. Furthermore, the Ca²⁺-sensitivity and the pharmacological behavior of the human SR Ca²⁺ release channel are similar in failing and nonfailing myocardium.