Dyad Content is Reduced in Cardiac Myocytes of Mice with Impaired Calmodulin Regulation of RyR2

Abstract

Calmodulin (CaM), a small cytoplasmic calcium binding protein, regulates various proteins, including e-coupling components of cardiac muscle (CaV1.2; RyR2 and CaATPase). Three amino acid substitutions in the CaM binding site of RyR2 in mice (Ryr2ADA/ADA mice) strongly affect cardiac function, with impaired CaM inhibition of RyR2, reduction in SR calcium uptake, early cardiac hypertrophy and death (Yamaguchi et al., J Clin Invest 117:1344, 2007). We have examined the ultrastructure and RyR2 immunolocalization of mutant hearts fixed at ∼10 days after birth. Despite previously described obvious hypertrophy, the myocytes show no evidence of widespread structural damage, but fairly rare and limited areas of Z line streaming, some increase in intermyofibrillar spaces and occasional areas of disarray. Quantitative analysis however reveals the following significant changes. The average cross sectional areas of cells in the papillary muscle increases by ∼ 15% (from 43±17 to 49±20 µm2 mean±SD), but the myofibrils' diameters are on the average smaller (0.4±0.3 to 0.3±0.2 µm2) due to the presence of groups of small myofibrils in disarranged areas. A notable feature is the dramatic decrease in the frequency and size of dyads, without obvious alterations in location and general structure. The frequency of dyads (counted as number per intermyofibrillar spaces) declines from 0.4±0.1 to 0.2±0.1, and the lengths of the junctional contacts, where RyR2s are located, is also reduced (0.25±0.1 to 0.20±0.1 µm). Immunolabeling with a pan anti-RyR antibody confirmed that RyR-positive foci located at the level of the Z lines are less frequent and more randomly disposed in mutant hearts. The data indicate an overall reduction in RyR2 content, which may contribute to poor cardiac performance and early death of Ryr2ADA/ADA mice. (RO1 HL 48093 to CFA and R01 HL 073051 to GM).