title: 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 micrograms 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. Non-specific binding was determined in the presence of a high concentration (10 microM) 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) microM (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) microM (DCM, n = 10) or 0.28 (0.25-0.31) microM (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 Ca(2+)-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.
  
  
  CONCLUSIONS
  Caffeine and AMP-PCP sensitize, whereas Mg2+ desensitizes the myocardial Ca2+ release channel to Ca2+. The determination of Ca(2+)-dependent 3H-ryanodine binding to the human myocardial Ca2+ release channel is a useful tool to investigate its open probability. Furthermore, the Ca(2+)-sensitivity and the pharmacological behavior of the human SR Ca2+ release channel are similar in failing and nonfailing myocardium.
authors.affiliation: Medical Clinic I, University Hospital Aachen. usch@pcserver.mk1.rwth-aachen.de
authors.forename: U
authors.initials: U
authors.lastname: Schotten
authors.forename: C
authors.initials: C
authors.lastname: Schumacher
authors.forename: V
authors.initials: V
authors.lastname: Conrads
authors.forename: V
authors.initials: V
authors.lastname: Braun
authors.forename: F
authors.initials: F
authors.lastname: Schöndube
authors.forename: M
authors.initials: M
authors.lastname: Voss
authors.forename: P
authors.initials: P
authors.lastname: Hanrath
mesh_terms: D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000502:physiology*; D009202:Cardiomyopathies / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D006321:Heart / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating; D009200:Myocardial Contraction / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism / Q000648:ultrastructure; D012433:Ryanodine; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D012433:Ryanodine; D002118:Calcium