title: Impaired calcium-calmodulin-dependent inactivation of Cav1.2 contributes to loss of sarcoplasmic reticulum calcium release refractoriness in mice lacking calsequestrin 2.
abstract: OBJECTIVE
  In cardiac muscle, Ca(2+) release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca(2+) release refractoriness. We recently reported that the SR luminal Ca(2+) binding protein calsequestrin 2 (Casq2) contributes to release refractoriness in intact mouse hearts, but the underlying mechanisms remain unclear. Here, we further investigate the mechanisms responsible for physiological release refractoriness.
  
  
  RESULTS
  Gene-targeted ablation of Casq2 (Casq2 KO) abolished SR Ca(2+) release refractoriness in isolated mouse ventricular myocytes. Surprisingly, impaired Ca(2+)-dependent inactivation of L-type Ca(2+) current (ICa), which is responsible for triggering SR Ca(2+) release, significantly contributed to loss of Ca(2+) release refractoriness in Casq2 KO myocytes. Recovery from Ca(2+)-dependent inactivation of ICa was significantly accelerated in Casq2 KO compared to wild-type (WT) myocytes. In contrast, voltage-dependent inactivation measured by using Ba(2+) as charge carrier was not significantly different between WT and Casq2 KO myocytes. Ca(2+)-dependent inactivation of ICa was normalized by intracellular dialysis of excess apo-CaM (20 μM), which also partially restored physiological Ca(2+) release refractoriness in Casq2 KO myocytes.
  
  
  CONCLUSIONS
  Our findings reveal that the intra-SR protein Casq2 is largely responsible for the phenomenon of SR Ca(2+) release refractoriness in murine ventricular myocytes. We also report a novel mechanism of impaired Ca(2+)-CaM-dependent inactivation of Cav1.2, which contributes to the loss of SR Ca(2+) release refractoriness in the Casq2 KO mouse model and, therefore, may further increase risk for ventricular arrhythmia in vivo.
authors.affiliation: Department of Medicine, Vanderbilt University, Nashville, TN, USA.
authors.forename: Dmytro O
authors.initials: DO
authors.lastname: Kryshtal
authors.affiliation: Department of Medicine, Vanderbilt University, Nashville, TN, USA; Department of General Physiology of Nervous System, Bogomoletz Institute of Physiology, Kyiv, Ukraine.
authors.forename: Oleksiy
authors.initials: O
authors.lastname: Gryshchenko
authors.affiliation: Department of Medicine, Vanderbilt University, Nashville, TN, USA.
authors.forename: Nieves
authors.initials: N
authors.lastname: Gomez-Hurtado
authors.affiliation: Department of Medicine, Vanderbilt University, Nashville, TN, USA. Electronic address: bjorn.knollmann@vanderbilt.edu.
authors.forename: Bjorn C
authors.initials: BC
authors.lastname: Knollmann
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002155:Calsequestrin / Q000235:genetics* / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005260:Female; D006352:Heart Ventricles / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D032383:Myocytes, Cardiac / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
chemical_list: D020746:Calcium Channels, L-Type; D002147:Calmodulin; D002155:Calsequestrin; C419385:L-type calcium channel alpha(1C); C514388:casq2 protein, mouse; D002118:Calcium
keywords: Calcium release restitution; Calmodulin; Calsequestrin; L-type calcium channel; Sarcoplasmic reticulum