Dimitra K. Georgiou

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are responsible for the release of Ca(2+) from intracellular stores during excitation-contraction coupling in both cardiac and skeletal muscle. RyRs are the largest known ion channels (> 2MDa) and exist as three mammalian isoforms (RyR 1-3), all of which are homotetrameric proteins that interact with and are regulated by phosphorylation, redox modifications, and a variety of small proteins and ions. Most RyR channel modulators interact with the large cytoplasmic domain whereas the carboxy-terminal portion of the protein forms the ion-conducting pore. Mutations in RyR2 are associated with human disorders such as catecholaminergic polymorphic ventricular tachycardia whereas mutations in RyR1 underlie diseases such as central core disease and malignant hyperthermia. This chapter examines the current concepts of the structure, function and regulation of RyRs and assesses the current state of understanding of their roles in associated disorders.

AICAR prevents heat-induced sudden death in RyR1 mutant mice independent of AMPK activation

Mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation that is associated with malignant hyperthermia in humans, die when exposed to short periods of temperature elevation (≥37 °C). We show here that treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevents this heat-induced sudden death in this mouse model. The protection by AICAR is independent of AMP-activated protein kinase (AMPK) activation and results from a newly identified action of the compound on mutant Ryr1 to reduce Ca2+ leak from the sarcoplasmic reticulum to the sarcoplasm. AICAR thus prevents Ca2+-dependent increases in the amount of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) that act to further increase resting Ca2+ concentrations. If unchecked, the temperature-driven increases in resting Ca2+ concentrations and the amounts of ROS and RNS create an amplifying cycle that ultimately triggers sustained muscle contractions, rhabdomyolysis and death. Although antioxidants are effective in reducing this cycle in vitro, only AICAR prevents heat-induced death in vivo. Our findings suggest that AICAR is probably effective in prophylactic treatment of humans with enhanced susceptibility to exercise- and/or heat-induced sudden death associated with RYR1 mutations.

Determinants in CaV1 Channels That Regulate the Ca2+ Sensitivity of Bound Calmodulin

Calmodulin binds to IQ motifs in the α1 subunit of CaV1.1 and CaV1.2, but the affinities of calmodulin for the motif and for Ca2+ are higher when bound to CaV1.2 IQ. The CaV1.1 IQ and CaV1.2 IQ sequences differ by four amino acids. We determined the structure of calmodulin bound to CaV1.1 IQ and compared it with that of calmodulin bound to CaV1.2 IQ. Four methionines in Ca2+-calmodulin form a hydrophobic binding pocket for the peptide, but only one of the four nonconserved amino acids (His-1532 of CaV1.1 and Tyr-1675 of CaV1.2) contacts this calmodulin pocket. However, Tyr-1675 in CaV1.2 contributes only modestly to the higher affinity of this peptide for calmodulin; the other three amino acids in CaV1.2 contribute significantly to the difference in the Ca2+ affinity of the bound calmodulin despite having no direct contact with calmodulin. Those residues appear to allow an interaction with calmodulin with one lobe Ca2+-bound and one lobe Ca2+-free. Our data also provide evidence for lobe-lobe interactions in calmodulin bound to CaV1.2.

Ca2+ permeation and/or binding to CaV1.1 fine-tunes skeletal muscle Ca2+ signaling to sustain muscle function

BackgroundCa2+ influx through CaV1.1 is not required for skeletal muscle excitation-contraction coupling, but whether Ca2+ permeation through CaV1.1 during sustained muscle activity plays a functional role in mammalian skeletal muscle has not been assessed.MethodsWe generated a mouse with a Ca2+ binding and/or permeation defect in the voltage-dependent Ca2+ channel, CaV1.1, and used Ca2+ imaging, western blotting, immunohistochemistry, proximity ligation assays, SUnSET analysis of protein synthesis, and Ca2+ imaging techniques to define pathways modulated by Ca2+ binding and/or permeation of CaV1.1. We also assessed fiber type distributions, cross-sectional area, and force frequency and fatigue in isolated muscles.ResultsUsing mice with a pore mutation in CaV1.1 required for Ca2+ binding and/or permeation (E1014K, EK), we demonstrate that CaV1.1 opening is coupled to CaMKII activation and refilling of sarcoplasmic reticulum Ca2+ stores during sustained activity. Decreases in these Ca2+-dependent enzyme activities alter downstream signaling pathways (Ras/Erk/mTORC1) that lead to decreased muscle protein synthesis. The physiological consequences of the permeation and/or Ca2+ binding defect in CaV1.1 are increased fatigue, decreased fiber size, and increased Type IIb fibers.ConclusionsWhile not essential for excitation-contraction coupling, Ca2+ binding and/or permeation via the CaV1.1 pore plays an important modulatory role in muscle performance.

Ca2+ Binding/Permeation via Calcium Channel, CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein, CD36, and Fatty Acid Metabolism

Background: Ca2+ binding and/or permeation via CaV1.1 in skeletal muscle activates CaMKII. Results: Mice with a Ca2+ binding/permeation defect in CaV1.1 have increased body fat, reduced fatty acid metabolism, and altered CD36 distribution. Conclusion: CaV1.1 regulates CD36 distribution and fatty acid metabolism. Significance: New therapeutic targets are identified to increase skeletal muscle energy expenditure. Ca2+ permeation and/or binding to the skeletal muscle L-type Ca2+ channel (CaV1.1) facilitates activation of Ca2+/calmodulin kinase type II (CaMKII) and Ca2+ store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α1 subunit of CaV1.1) gene that abolishes Ca2+ binding within the CaV1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that CaV1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Cav1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized CaV1.1-mediated pathway that regulates energy utilization in skeletal muscle.

A chemical chaperone improves muscle function in mice with a RyR1 mutation

Mutations in the RYR1 gene cause severe myopathies. Mice with an I4895T mutation in the type 1 ryanodine receptor/Ca2+ release channel (RyR1) display muscle weakness and atrophy, but the underlying mechanisms are unclear. Here we show that the I4895T mutation in RyR1 decreases the amplitude of the sarcoplasmic reticulum (SR) Ca2+ transient, resting cytosolic Ca2+ levels, muscle triadin content and calsequestrin (CSQ) localization to the junctional SR, and increases endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and mitochondrial ROS production. Treatment of mice carrying the I4895T mutation with a chemical chaperone, sodium 4-phenylbutyrate (4PBA), reduces ER stress/UPR and improves muscle function, but does not restore SR Ca2+ transients in I4895T fibres to wild type levels, suggesting that decreased SR Ca2+ release is not the major driver of the myopathy. These findings suggest that 4PBA, an FDA-approved drug, has potential as a therapeutic intervention for RyR1 myopathies that are associated with ER stress.