Abstract
Triadin, a protein of the sarcoplasmic reticulum (SR) of striated muscles, anchors the calcium-storing protein calsequestrin to calcium release RyR channels at the junction with t-tubules, and modulates these channels by conformational effects. Triadin ablation induces structural SR changes and alters the expression of other proteins. Here we quantify alterations of calcium signaling in single skeletal myofibers of constitutive triadin-null mice. We find higher resting cytosolic and lower SR-luminal [Ca2+], 40% lower calsequestrin expression, and more CaV1.1, RyR1 and SERCA1. Despite the increased CaV1.1, the mobile intramembrane charge was reduced by ~20% in Triadin-null fibers. The initial peak of calcium release flux by pulse depolarization was minimally altered in the null fibers (revealing an increase in peak calcium permeability). The “hump” phase that followed, attributable to calcium detaching from calsequestrin, was 25% lower, a smaller change than expected from the reduced calsequestrin content and calcium saturation. The exponential decay rate of calcium transients was 25% higher, consistent with the higher SERCA1 content. Recovery of calcium flux after a depleting depolarization was faster in triadin-null myofibers, consistent with the increased uptake rate and lower SR calsequestrin content. In sum, the triadin knockout determines an increased RyR1 channel openness, which depletes the SR, a substantial loss of calsequestrin and gains in other couplon proteins. Powerful functional compensations ensue: activation of SOCE that increases [Ca2+]cyto; increased SERCA1 activity, which limits the decrease in [Ca2+]SR and a restoration of SR calcium storage of unknown substrate. Together, they effectively limit the functional loss in skeletal muscles.
Highlights
In the excitation-contraction (EC) coupling process of striated muscles, action potentials command the transient release of Ca2+ into the myoplasm, enabling muscle contraction
Comparing Western blots of total FDB fractions (Fig 1) we found statistically significant increases of multiple couplon proteins in Tr-null myofibers, namely SERCA1: 40%, Stim1: 71%, CaV1.1: 74%, and ryanodine receptor 1 (RyR1): 62% (Table 1 for summary)
Two other proteins had lower densities; Jph1 was reduced by 35%, and Casq1 by 46%, a difference consistent with the 40% deficit reported by Boncompagni et al (2012) [51], but less than the 73% loss reported by Oddoux et al (2009) [11]
Summary
In the excitation-contraction (EC) coupling process of striated muscles, action potentials command the transient release of Ca2+ into the myoplasm, enabling muscle contraction. The crucial device in this process is the couplon, a physical continuum of proteins that includes the dihydropyridine receptor (DHPR, CaV1.1), the ryanodine receptor 1 (RyR1), FKBP12, junctophilin 1 (JPh1), stac, junctin (Jct), triadin (Tr) and calsequestrin (Casq1), among other components [1, 2]. A number of myopathies have their origin in structural and functional alterations caused by mutations in proteins of the couplon [3]. The Ca2+ signaling deficit in Tr-null muscles funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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