Central Core Disease (CCD), the most common congenital myopathy in humans, is characterized by fetal hypotonia, proximal muscle weakness, and inert core regions that lack functional mitochondria and oxidative enzyme activity in type 1 skeletal muscle fibers. Although CCD is known to be caused by mutations in the skeletal muscle Ca2+ release channel (RyR1), diagnosis still depends on painful and invasive muscle biopsies and there are currently no effective treatments. The long-term goal of this project is to characterize the pathophysiological mechanisms of muscle weakness in CCD and to develop novel therapeutic and diagnostic strategies. Motivated by this goal, we aim to drive discovery of novel and fundamental aspects of altered Ca2+ signaling in CCD, as well as test the validity of potential therapeutic (allele-specific gene silencing) and diagnostic (superoxide flashes as a CCD biomarker) strategies.
Aim #1 will test the hypothesis that CCD mutations in different structural regions of RyR1 alter Ca2+ release during muscle contraction via fundamentally distinct molecular and biophysical mechanisms. Experiments will determine if CCD mutations in the M6 and pore/TM10 transmembrane regions of RyR1 alter Ca2+ permeation, channel gating, and/or regulation by specific RyR1 binding proteins.
Aim #2 will assess the utility of allele-specific gene silencing approaches to correct defects in Ca2+ homeostasis, Ca2+ release, and mitochondrial function observed in knock-in mice heterozygous for either the Y522S or I4895T CCD mutations in RyR1. An important conceptual innovation of this project is to use shRNA-mediated mutant RyR1 allele-specific knockdown to reverse toxic gain-of-function effects produced by CCD mutations in RyR1 in heterozygous Y522S and I4895T knock-in mice. An important technological innovation is to take advantage of our recently developed mitochondrial-targeted superoxide sensor (mt-cpYFP) to assess changes in mitochondrial function (reflected in the incidence and properties of mitochondrial superoxide flashes) in normal and RyR1 knock-in mice. The fundamental pathophysiologic mechanisms identified and discoveries made as a result of this venture will provide extraordinary promise for the development of novel approaches, therapeutics, and diagnostics not only for CCD, but also other disorders of Ca2+ dysregulation and altered oxidative stress in related clinical arenas including cardiac arrhythmias, heart failure, hypertension, diabetes, neuro-degeneration and stroke.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR044657-15
Application #
7906021
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
1997-09-01
Project End
2012-08-31
Budget Start
2010-09-01
Budget End
2011-08-31
Support Year
15
Fiscal Year
2010
Total Cost
$312,779
Indirect Cost
Name
University of Rochester
Department
Pharmacology
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Wei-LaPierre, Lan; Gong, Guohua; Gerstner, Brent J et al. (2013) Respective contribution of mitochondrial superoxide and pH to mitochondria-targeted circularly permuted yellow fluorescent protein (mt-cpYFP) flash activity. J Biol Chem 288:10567-77
Kraeva, Natalia; Zvaritch, Elena; Rossi, Ann E et al. (2013) Novel excitation-contraction uncoupled RYR1 mutations in patients with central core disease. Neuromuscul Disord 23:120-32
Loy, Ryan E; Lueck, John D; Mostajo-Radji, Mohammed A et al. (2012) Allele-specific gene silencing in two mouse models of autosomal dominant skeletal myopathy. PLoS One 7:e49757
Wei, Lan; Dirksen, Robert T (2012) Perspectives on: SGP symposium on mitochondrial physiology and medicine: mitochondrial superoxide flashes: from discovery to new controversies. J Gen Physiol 139:425-34
Liu, X; Yu, L; Wang, Q et al. (2012) Expression of ecto-ATPase NTPDase2 in human dental pulp. J Dent Res 91:261-7
Jhun, Bong Sook; O-Uchi, Jin; Wang, Weiye et al. (2012) Adrenergic signaling controls RGK-dependent trafficking of cardiac voltage-gated L-type Ca2+ channels through PKD1. Circ Res 110:59-70
Tang, Zhen Zhi; Yarotskyy, Viktor; Wei, Lan et al. (2012) Muscle weakness in myotonic dystrophy associated with misregulated splicing and altered gating of Ca(V)1.1 calcium channel. Hum Mol Genet 21:1312-24
Loy, Ryan E; Orynbayev, Murat; Xu, Le et al. (2011) Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum. J Gen Physiol 137:43-57
Ryu, Shin-Young; Beutner, Gisela; Kinnally, Kathleen W et al. (2011) Single channel characterization of the mitochondrial ryanodine receptor in heart mitoplasts. J Biol Chem 286:21324-9
Tae, HanShen; Wei, Lan; Willemse, Hermia et al. (2011) The elusive role of the SPRY2 domain in RyR1. Channels (Austin) 5:148-60

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