MUSCLE RYANODINE RECEPTOR 23

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 Dirksen

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.

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.

This chapter will focus on the clinical manifestations and

functional defects associated with human RyR1 disease mutations and how

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.

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.

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.

CCD is the most frequently observed congenital myopathy and is associated to mutations in the RyR1 gene.

Although CCD is primarily inherited in an autosomal dominant manner, recessive forms have also been confirmed.

Diagnosis of CCD is based on histochemical

identification of amorphous areas (cores), which lack mitochondria and

oxidative enzyme activity in type 1 muscle fibers.

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.

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.

; figures E-F from Monnier et al.

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

(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,

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.

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.

;

and panels C-D are adapted from Monnier et al.

Panel B was kindly provided by Dr. Joël

Lunardi.

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).

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.

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,

whereas MH/CCD regions 1 and 2 are located in the large cytosolic aspect of RyR1. For certain RyR1 mutations, individuals exhibit both MH and CCD (MH+CCD mutants), whereas others appear to result in either a pure MH phenotype (MH-only) or CCD in the apparent absence of enhanced MH-susceptibility (CCD-only). Because MH and CCD are inherited primarily in an autosomal dominant manner (except for instances of MmD and CCD coincidence), it is widely believed that the majority of these mutations produce “gain-of-function” or “change-in- function” effects on the activity of the SR release channel. In fact, as outlined below, functional studies have provided compelling evidence in support of this prediction for at least some of the disease mutations in RyR1.

Initial pioneering studies designed to characterize the functional effects

of MH-linked mutations were conducted using muscle samples from pigs

carrying the R615C RyR1 mutation (equivalent to the human R614C

mutation). These studies found that MH-susceptible muscle exhibited higher specific ryanodine binding, an increased sensitivity to activation by micromolar and a higher resistance to inactivation. In addition, MH-susceptible muscle exhibits increased sensitivity to activation by caffeine and 4-chloro-m-cresol (4-cmc) (see for reviews

). These abnormalities may be potentiated by inhalation anesthetics and depolarizing skeletal muscle relaxants, and thus, result in supersensitive or overactive SR

release channels.

The increased sensitivity of RyR1 release channels to activation by exogenous agents (including caffeine, halothane, and 4-cmc) also extends to the physiologic trigger. In skeletal muscle bundles and myotubes obtained from MH pigs, contractions exhibit enhanced sensitivity to activation via sarcolemmal depolarization.

The increased contractile sensitivity to depolarization arises from a hyperpolarizing shift in the voltage dependence of SR release that occurs in the absence of an effect on the magnitude of voltage-gated SR release or the magnitude/voltage-dependence of DHPR L-type currents.

Thus, the porcine MH mutation (R615C) results in an enhanced release channel sensitivity to activation by the voltage sensor (i.e.

a leftward shift in the release versus voltage relationship).

Many RyR1 mutations, particularly those in MH/CCD regions 1 and 2, result in the co-occurrence of both MH and CCD (Table 23-1). Several studies have provided evidence that the MH+CCD mutations in regions 1 and 2 result in the formation of “overactive” or “leaky” SR release channels. In line with this view, heterologous expression in HEK 293 cells of RyR1 mutations in regions 1 and 2 that result in co-occurrence of both MH and CCD lead to varying degrees of both a reduction in luminal endoplasmic reticulum (ER) content and an increase in resting levels.

Similarly, homologous expression of the same mutant RyR1 proteins in RyR1-deficient (dyspedic) skeletal myotubes also results in SR depletion and elevations in resting levels that follow a similar rank order as that observed in HEK 293 cells.

In contrast, SR depletion does not appear to occur following expression of RyR1 mutants that only result in MH and not CCD in either HEK 293 cells

or dyspedic myotubes.

Apparently, significant ER/SR leak only occurs for RyR1 disease mutants that result in coincidence of MH and CCD.

Interestingly, similar to that observed for the porcine MH mutation, the

MH+CCD mutations in MH/CCD regions 1 and 2 also reduce the threshold

for voltage activation of SR release in the absence of a change in L-type

channel activity.

In addition, voltage sensitivity of SR release is

also increased for a number of MH-only mutations in RyR1.

However, in

contrast to MH-only mutations, maximal amplitude of voltage-gated SR

release is reduced following expression of MH+CCD mutations,

terminus of RyR1 (I4898T) that results in an unusually severe and highly penetrant form of CCD, but did not appear to result in clinical MH. More recent genetic studies (reviewed in Lueck et al.

and Dirksen and Avila

) have identified an additional 26 CCD mutations in the C-terminal region of RyR1 (MH/CCD region 3), demonstrating that MH/CCD region 3 represents a primary molecular hot-spot for CCD. In addition, several MH-selective mutations have now been identified in this region (Table 23-1). Functional measurements of the I4897T mutation (the human I4898T mutation engineered into the analogous position of the rabbit RyR1) expressed in HEK 293 cells suggested that this mutation also promotes leak and subsequent store depletion.

A similar conclusion was reached in experiments utilizing human B-lymphocytes (which express functional RyR1 release channels) obtained from patients possessing the I4898T CCD mutation.

However, neither HEK 293 cells nor B-lymphocytes contain all of the many triadic proteins (e.g. DHPR, calsequestrin, triadin junctin, FKBP12) known to regulate RyR1 channel activity in skeletal muscle. In particular, these systems lack RyR1 regulatory control imparted by the skeletal muscle DHPR, which acts as an activator at depolarized membrane potentials and a key negative modulator at resting membrane potentials.

For these reasons, the impact of the I4897T CCD mutation on release channel function was also characterized following homologous expression in dyspedic skeletal myotubes.

In contrast to that observed in non-muscle cells, expression of I4897T-containing release channels within a skeletal muscle environment did not promote SR leak and store depletion (no change in resting or releasable SR content).

Nevertheless, voltage-gated release was either absent or reduced by

50% following expression in dyspedic myotubes of I4897T alone or in

combination with wild-type RyR1, respectively. Thus, the I4897T mutation

exerts a dominant-negative reduction in voltage-gated SR release,

consistent with the autosomal-dominant nature of this disorder. Based on

these results, it was concluded that the I4897T mutation reduces release

during skeletal muscle EC coupling via a mechanism distinct from that of

SR leak. Rather, the mutation appears to produce a functional uncoupling of excitation from the efficient release of SR (termed EC uncoupling, Fig. 23-3).

Figure 23-3. A conceptual model to account for two distinct mechanisms by which CCD mutations in RyR1 alter SR release channel function. A. In the domain-switch model (see Ikemoto et al.

and Chapter 6 for details), the II-III loop of the skeletal muscle DHPR interacts with a binding site in RyR1 that is functionally coupled to two domains (MH/CCD regions 1 and 2) that interact and regulate the opening of the release channel pore. Under resting conditions, the DHPR promotes interactions between domains 1 and 2 (“zipped”

state), which stabilize the resting closed state of the release channel. B. During skeletal muscle EC coupling, membrane depolarization causes a voltage-driven conformational change in the DHPR that disrupts the interaction between domains 1 and 2 (“unzipped” state), and subsequently leads to rapid opening of the release channel. C. Mutations in MH/CCD regions 1 and 2 alter this interdomain interaction, and thus, cause a partial unzipping of the domains such that release channel sensitivity to activation by endogenous (i.e. voltage sensor) and exogenous (e.g. caffeine, halothane, 4-cmc) triggers is increased. CCD mutations in regions 1 and 2 are shown to cause a degree of unzipping that is sufficient to cause leak through the channel at resting membrane potentials. D. CCD mutations in the selectivity filter/pore-lining region of the channel disrupt permeation through the channel without affecting voltage sensor unzipping of domains 1 and 2.

Interestingly, single channel measurements of mutations in I4897 and

neighboring amino acids have provided strong evidence that this region of

the protein comprises the pore-lining region of the channel.

Thus,

an important cellular mechanism for muscle weakness in CCD, then other mutations in the pore-lining/selectivity filter of the channel would also be expected to result in EC uncoupling. Consistent with this notion, all of the CCD mutations tested so far that are located in the pore-lining region of the channel (including G4890R, R4892W, I4897T, G4898E, G4898R, A4905V, and R4913G) lead to varying degrees of EC uncoupling. Specifically, mutations to the key “GIGD” selectivity filter residues (e.g. I4897T, G4898E, and G4898R) result in complete EC uncoupling (under homozygous conditions), while mutations to more peripheral pore-lining residues (e.g. R4892W and A4905V) can result in lesser degrees of EC uncoupling.

TWO DISTINCT CELLULAR MECHANISMS FOR MUSCLE WEAKNESS IN CCD

Experiments investigating the effects of CCD mutations on RyR1

function

have lead to the proposal of two distinct cellular

mechanisms by which mutations in RyR1 contribute to muscle weakness in

CCD (see Fig. 23-3). The conceptual model presented in Fig. 23-3 combines

information gleaned from the functional measurements described above as

applied to the “domain-switch” hypothesis proposed by Ikemoto and

colleagues (see Ikemoto et al.

and Chapter 6 for further details). The

domain-switch hypothesis predicts that under resting conditions the closed

state of the release channel is stabilized by strong interactions between

MH/CCD regions 1 and 2 (the “zipped” state). Moreover, agents that

promote unzipping of regions 1 and 2 cause a conformational change in the

complex that results in rapid activation and opening of the release channel

pore (“unzipped” state). According to this model, both release channel

triggers (e.g. DHPR, caffeine, halothane, 4-cmc) and mutations that promote

unzipping result in a destabilization of the channel closed state that lowers

the energy required to open the channel.

The domain-switch model provides a simple conceptual framework for interpreting the functional effects of RyR1 MH and CCD mutations on EC coupling and homeostasis discussed above. At resting membrane potentials, the II-III loop of the skeletal muscle DHPR is shown to interact with a specific DHPR binding core formed by disparate regions of the cytoplasmic aspect of the release channel (Fig. 23-3 A). This interaction between the DHPR and RyR1 stabilizes the closed state of the release channel at hyperpolarized membrane potentials.

During EC coupling, voltage-driven conformational changes in the skeletal muscle DHPR promote the unzipping of MH/CCD domains 1 and 2, and resulting in rapid release channel activation, opening, and a massive release of into the myoplasm (Fig. 23-3 B). According to this model, CCD mutations in regions 1 and 2 promote SR leak (and subsequent store depletion) by destabilizing the critical interactions between MH/CCD regions 1 and 2 in a manner sufficient to cause a partial unzipping and increased channel opening even under resting conditions (Fig. 23-3 C). MH-selective mutations in regions 1 and 2 may disrupt this regulatory domain-domain interaction to a lesser degree, and thus cause increased sensitivity to activation by all RyR1 triggers (e.g. voltage sensor, caffeine, halothane, 4-cmc) without enhanced SR leak. In contrast, mutations in the selectivity filter/pore-lining region of the channel act downstream of the interdomain regulatory mechanism. Although voltage-driven unzipping may be unaffected by the pore mutations, permeation through the pore of the activated channel is nevertheless disrupted (Fig. 23-3 D). Thus, both leaky and EC uncoupled release channels would lead to reduced release during EC coupling and muscle weakness, although via distinct cellular mechanisms (SR leak/store depletion vs. reduced permeation). The ability of the I4897T pore mutation to abolish severe leak and store depletion caused by the Y523S mutation in MH/CCD region 1 is entirely consistent with this model.

CONCLUDING REMARKS

It will be important for future work to characterize the effects of CCD

mutations in MH/CCD region 3 that lie outside the putative selectivity

filter/pore-lining region of the channel. Interestingly, a mutation in

MH/CCD region 3 (Y4796C) was recently shown to exhibit all the

hallmarks of a leaky channel following expression in dyspedic myotubes

(including elevated resting store depletion, increased sensitivity to

activation by voltage, and reduced maximal voltage-gated release).

Thus, this portion of the C-terminal region of RyR1 may function as hinge

Mutations that alter permeation of the pore might not be the only mechanism of EC uncoupling. In fact, it is conceivable that EC uncoupling (reduced release during EC coupling in the absence of store depletion) could also result from mutations that dramatically reduce DHPR or RyR1 expression, perturb RyR1 or DHPR junctional targeting, alter the proper formation of DHPR tetrads or RyR1 arrays, or disrupt functional coupling between properly targeted DHPR and RyR1 proteins.

In fact, a recently discovered, cryptic splice-site mutation in the RyR1 gene (14646+2.99 kb, see Table 23-1) results in recessive MmD/CCD and an ~90% reduction in skeletal muscle RyR1 transcript/protein levels. Thus, this splicing mutation would be expected to result in EC uncoupling via a mechanism involving markedly reduced RyR1 expression.

Finally, because of the very recent association of MmD and NM to alterations in the RyR1 gene, almost nothing is known regarding how MmD- or NM-linked RyR1 mutations modify SR release channel activity and/or trigger MmD- and NM-specific phenotypes. In addition, as RyR1 release channels are also expressed in B-lymphocytes and certain neuronal cells (including cerebellar Purkinje cells, dentate gyrus of the hippocampus, CA1 and CA3 cells of the Ammons’ horn and the olfactory bulb),

it seems likely that future work may identify specific clinical manifestations of the RyR1 ryanodinopathies that extend to certain non- muscle related phenotypes/disorders. In any event, these unanswered questions provide substantial fertile ground for future mechanistic studies regarding the molecular mechanisms and pathophysiology that underlie the skeletal muscle ryanodinopathies.

ACKNOWLEDGEMENTS

The authors thank Dr. J. Lunardi for providing micrographs of the cores

and nemaline rods depicted in Figs. 23-1 and 23-2 and to Dr. N. Ikemoto for

helpful discussions regarding intramolecular RyR1 domain-domain

interactions. This work is supported by research grants from the National

Institutes of Health (AR44657) and the Muscular Dystrophy Association.