New roles of calsequestrin and triadin in cardiac muscle

Abstract

Cardiac calsequestrin (Casq2) and triadin are proteins located in specialized areas of the sarcoplasmic reticulum (SR) where the SR forms junctions with the sarcolemma (junctional SR). Casq2, triadin and junctin form a protein complex that is associated with cardiac ryanodine receptor 2 (RyR2) SR Ca(2+) release channels. This review highlights new insights of the roles of triadin and Casq2 derived from gene-targeted knock-out and knock-in mouse models that have recently become available. Characterization of the mouse models suggests that Casq2's contribution to SR Ca(2+) storage and release during excitation-contraction coupling is largely dispensable. Casq2's primary role appears to be in protecting the heart against premature Ca(2+) release and triggered arrhythmias. Furthermore, both cardiac Casq2 and triadin are important for the structural organization of the SR, which had previously not been recognized. In particular, ablation of triadin causes a 50% reduction in the extent of the junctional SR, which results in impaired excitation-contraction coupling at the level of the myocyte. While catecholamines could normalize contractile function by increasing I(Ca) and SR Ca(2+) content, it comes at the price of an increased risk for spontaneous Ca(2+) releases in triadin knock-out myocytes and catecholamine-induced ventricular arrhythmias in triadin knock-out mice.

Figure 1

Cartoon depicting the structural and protein changes that occur in ventricular myocytes after gene-targeted ablation of Casq2 or Trdn

Figure 2. Effect of reducing Casq2 on the relationship between SR Ca²⁺ content (load) and SR Ca²⁺ leak

Note that with progressive reduction in the endowment of cardiac muscle with Casq2, the relationship becomes progressively steeper. At low SR Ca²⁺ content, SR Ca²⁺ leak is not different between the three groups. Leak–load relationship was generated by pooling results from homozygous and heterozygous Casq2 mice (Knollmann et al. 2006; Chopra et al. 2007). Myocytes were loaded with fura-2 acetoxymethyl ester (Fura-2 AM). Excitation wavelengths of 360 and 380 nm were used to monitor the fluorescence signals of Ca²⁺-bound and Ca²⁺-free Fura-2. Intracellular Ca²⁺ concentration [Ca²⁺]_i is proportional to the fluorescence ratio at 360 nm and 380 nm excitation, albeit in a non-linear fashion (Grynkiewicz et al. 1985). Since Fura-2 AM compartmentalizes into intracellular organelles (Williford et al. 1990), calculating [Ca²⁺]_i from Fura-2 fluorescence ratios may not be accurate in intact cells. Thus, [Ca²⁺]_i measurements are reported as fluorescence ratios (F_ratio). The increase in the F_ratio induced by rapid caffeine applications was used as estimate of SR Ca²⁺ content. SR leak was estimated by the reduction in F_ratio after blocking RyR2 channels with tetracaine. For details see (Knollmann et al. 2006; Chopra et al. 2007). +/+, wild-type, +/−, Casq2 heterozygous, −/−, Casq2 null.

Figure 3. Relative changes in the SR protein composition (A) and SR structure (B) of Trdn null myocardium

The jSR extent (where RyR2 channels ‘feet’ are located) was obtained by multiplying the jSR contact length by the frequency of dyads (data not shown). The jSR extent was reduced by 56% in the Trdn⁻/⁻ myocardium. Bar graphs were generated from absolute data reported by Chopra et al. (2009). *P < 0.05 compared to wild-type, JP, junctophilin.