Chapter 23 PATHOPHYSIOLOGY OF MUSCLE DISORDERS LINKED TO MUTATIONS IN THE SKELETAL MUSCLE RYANODINE RECEPTOR Robert T. Dirksen1 and Guillermo Avila2 1 Dept. of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, USA; 2 Dept. of Biochemistry, Cinvestav-IPN, Mexico City, Mexico INTRODUCTION Skeletal muscle excitation contraction (EC) coupling involves a unique, bi-directional mechanical interaction between two different types of calcium channels: a sarcolemmal voltage-gated L-type calcium channel (dihydropyridine receptor, DHPR) and the ryanodine receptor (RyR1), a ligand-gated intracellular release channel located in the sarcoplasmic reticulum (SR) (see Dirksen852 for review). In response to sarcolemma depolarization, the DHPR undergoes a conformational change that results in activation of nearby RyR1 release channels and subsequent massive release of SR into the myoplasm (see Melzer et al.169 for review). Thus, the DHPR and RyR1 proteins are essential components of the EC coupling machinery in skeletal muscle, and thus, play a central role in muscle homeostasis. Not surprisingly, mutations and/or deletions in the genes that encode the skeletal muscle DHPR and RyR1 proteins are linked to at least five different human diseases: Malignant hyperthermia (MH), hypokalemic periodic paralysis, central core disease (CCD), multiminicore disease (MmD) and nemaline rod myopathy (NM). Mutations in RyR1 result in MH, CCD, MmD and NM, whereas DHPR mutations are linked only to MH and hypokalemic periodic paralysis. The genetic bases of these diseases, as well as the cellular mechanisms involved, have been thoroughly reviewed elsewhere.853,854 This chapter will focus on the clinical manifestations and functional defects associated with human RyR1 disease mutations and how 230 Chapter 23 these defects might contribute to the pathophysiology of the skeletal muscle ryanodinopathies. CLINICAL FEATURES MH is a clinical syndrome in which genetically susceptible individuals respond to inhalation anesthetics (e.g. halothane) and muscle relaxants (e.g. succinylcholine) with attacks of high fever, skeletal muscle rigidity, hypermetabolism, lactic acidosis, hypoxia and tachycardia.172,174 MH episodes may be life threatening if not corrected immediately by suspension of administration of the triggering agent, treatment with dantrolene sodium, and hyperventilation with 100% The incidence of MH has been estimated to be ~1 in 15,000 anesthetized children and ~1 in 50,000-100,000 anesthetized adults. In nearly 50-80% of families with MH the disorder has be linked to mutations in the RyR1 gene (Table 23-1). An in vitro contracture test (IVCT) is used to detect MH susceptibility. This test determines the sensitivity of muscle biopsies to contractures induced by caffeine and halothane. If certain contracture thresholds are reached in the presence of low concentrations of caffeine and/or halothane, then a diagnosis of MH susceptibility is made. Patients with CCD are at risk for MH and are often diagnosed as MH susceptible by the IVCT.825 Muscle disorders linked to RyR1 mutations 231 232 Chapter 23 CCD, MmD and NM are congenital myopathies, a heterogeneous group of early-onset neuromuscular disorders that exhibit a number of shared characteristics. The most common symptoms observed for each of these myopathies are fetal hypotonia and proximal muscle weakness during infancy. Although the clinical severity for these disorders varies considerably (both within and between disorders), symptoms can at times be fatal during the first few months of life. A significant predominance and atrophy of type 1 skeletal muscle fibers is typically observed and diagnosis is made on the basis of identification of characteristic histochemical or structural abnormalities linked to each myopathy.855 CCD is the most frequently observed congenital myopathy and is associated to mutations in the RyR1 gene.171,856 Although CCD is primarily inherited in an autosomal dominant manner, recessive forms have also been confirmed.847,849,850,857 Diagnosis of CCD is based on histochemical identification of amorphous areas (cores), which lack mitochondria and oxidative enzyme activity in type 1 muscle fibers.848 Cores exhibit clearly circumscribed boundaries and can be located in central (Fig. 23-1 A), eccentric (Fig. 23-1 C), or multiple peripheral regions (Fig. 23-1 B) of individual type I muscle fibers. In addition, cores in CCD are often large and can run throughout the length of the muscle fiber (Fig. 23-2 A and B). CCD patients are often, but not always, found to be MH susceptible and may also exhibit foot/thoracic deformities and/or other skeletal defects.825 Muscle disorders linked to RyR1 mutations 233 Figure 23-1. Spectrum of histological phenotypes observed in serial transverse sections of skeletal muscle biopsies obtained from individuals possessing disease mutations in RyR1. A. Classic unique central cores shown using NADH tetrazolium staining of a skeletal muscle section obtained from a patient with autosomal dominant CCD (L4793P). B-D. Succinate dehydrogenase staining of skeletal muscle obtained from patients with autosomal dominant CCD showing unique cores in small fibers and multiple cores in large fibers (B, D4214-4216), autosomal dominant CCD with unique eccentric cores near the sarcolemma (C, R4893W), and autosomal recessive multiple minicores (D, P3527S). E- F. Gomori staining (E) and succinate dehydrogenase staining (F) of muscle samples obtained from a patient exhibiting coincidence of autosomal dominant CCD with rods (Y4796C). Figures A-D are adapted from Monnier et al.848; figures E-F from Monnier et al.858 Multiminicore disease (MmD), or minicore myopathy, is morphologically characterized by the presence of multiple small core-like areas (minicores), which lack mitochondria and oxidative activity (Fig. 23-1 D and 23-2 C). In contrast to conventional cores observed in CCD patients, minicores are poorly circumscribed, multi-focal, and found in both type 1 and type 2 muscle fibers. The longitudinal length of minicores represents another major difference between minicores (Fig. 23-2 D) and classic cores 234 Chapter 23 (Fig. 23-2 B). Clinical features of MmD are widely variable and include at least three distinct subgroups. The most common or classical phenotype (including an ophthalmoplegic subgroup associated with a severe facial weakness) exhibits axial muscle weakness, neonatal hypotonia, scoliosis and respiratory failure. The second group represents an early onset form and arthogryposis (persistent joint contracture). The third group is a slowly progressive form with hand involvement. MmD clinically overlaps with other neuromuscular disorders including CCD,855,856,859 Interestingly, MmD is genetically heterogeneous and, in contrast to CCD, typically exhibits an autosomal-recessive mode of inheritance. The recent identification of recessively inherited mutations in RyR1 linked to MmD provides a genetic explanation for the clinical overlap between CCD and a subset of MmD patients.847,849,850,857 Figure 23-2. Histological comparison of classic central cores and multiple minicores. A- B. Transverse (A) and longitudinal (B) sections showing classic central cores following staining with NADH tetrazolium. Note that central cores are large, exhibit clearly circumscribed boundaries, and run throughout the length of the fiber. C. NADH tetrazolium staining of a transverse muscle section exhibiting multiple minicores. D. Minicores observed in two adjacent fibers in a longitudinal semithin section stained with toluidine blue. Minicores are characterized by multiple short regions of diffuse negative staining and are coincident with a disruption of the normal sarcomeric pattern. Panel A is adapted from Monnier et al.848; and panels C-D are adapted from Monnier et al.858 Panel B was kindly provided by Dr. Joël Lunardi. Muscle disorders linked to RyR1 mutations 235 NM is a congenital neuromuscular disorder affecting 1 in every 50,000 live births. The clinical spectrum of NM is wide and ranges from a severe fatal neonatal form to only mildly affected adults. The most common symptoms of NM are congenital hypotonia and generalized skeletal muscle weakness, predominantly affecting facial and axial muscles. NM is also characterized by the presence of nemaline bodies (or rods), visualized using Gomori’s trichrome stain,858,860 composed primarily of and other Z-disc proteins arranged in irregular clusters in the periphery and/or at the Z- line (Fig. 23-1 E). Nemaline myopathy typically arises from mutations in genes encoding thin filament proteins including (TPM3,), (ACTA1), nebulin (NEB), (TPM2) and troponin T (TNNT1).855,856,859 However, studies have also reported the simultaneous occurrence of both central cores and nemaline rods in the same muscle biopsy, suggesting a “core-rod myopathy” that represents a clinical overlap between CCD and NM. In some cases, core-rod myopathy has been linked to mutations in the RyR1 gene.845,858,860 In these families, both central cores and nemaline rods can be found within the same muscle biopsy (Fig. 23-1 E and F). FUNCTIONAL DEFECTS OF RYR1 DISEASE MUTANTS Most disease-linked mutations in RyR1 are distributed among three distinct regions of the RyR1 protein, known as MH/CCD region 1 (amino acids 35-614), MH/CCD region 2 (amino acids 2129-2458), and MH/CCD region 3 (C-terminal domain) (Table 23-1). MH/CCD region 3 contains all of the putative transmembrane (TM) segments including the selectivity filter/pore-lining region,8 whereas