Book Text

PATHOPHYSIOLOGY AND MOLECULAR BIOLOGY

MH is a myopathy, usually subclinical, that features an acute loss of control of intracellular calcium ions (Ca²⁺ ). Normal muscle contraction is initiated at the neuromuscular junction (i.e., the motor end plate). Acetylcholine is released from the terminals of motor neurons and diffuses a short distance to the postsynaptic membrane, where binding to nicotinic cholinergic receptors triggers a wave of depolarization referred to as an excitatory postsynaptic potential (EPSP) that leads to action potentials that propagate to transverse tubules (T tubules). The T tubules act as conduits to bring action potentials deep within the myofibrils, where their excitatory signal is

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transduced to the junctional face of sarcoplasmic reticulum (SR) within the muscle cells to initiate release of Ca²⁺ stored within the SR terminal cisternae. In skeletal muscle, the release of SR Ca²⁺ is an essential step for contraction. The whole process, from T-tubule depolarization to release of SR Ca²⁺ , is called excitation-contraction (EC) coupling. Knowledge of the molecular events contributing to EC coupling is essential to understanding the cause of MH.

Skeletal EC coupling begins deep within the T-tubule membrane at high-density, voltage-gated L-type Ca²⁺ channels, historically labeled dihydropyridine receptors (DHPRs). The DHPR possesses an integral membrane voltage sensor whose function is to respond to T-tubule depolarization and initiate long-range conformational changes within the Ca²⁺ channel complex. Voltage-dependent activation of DHPR opens an integral Ca²⁺ -selective conductance path that permits entry of small amounts of Ca²⁺ into the muscle cell. The entry of Ca²⁺ into the skeletal myotube is not necessary for engaging skeletal-type EC coupling. Rather, one of the DHPR subunits (α_1S -subunit) provides physical links between DHPRs within T tubules and Ca²⁺ release channels within junctional SR. Skeletal muscle expresses a specific type of Ca²⁺ release channel called the skeletal isoform, or RYR1. Skeletal EC coupling is the result of physical coupling between α_1S -subunits and RYR1 at specialized "triadic" regions where the T-tubule membrane comes in close apposition to junctional SR and does not depend on the influx of Ca²⁺ . After EC coupling is initiated, the free, ionized, unbound intracellular Ca²⁺ concentration within

Figure 29-1 The key ion channels involved in neuromuscular transmission and excitation contraction coupling. Nerve impulses arriving at the nerve terminal activate voltage-gated Ca²⁺ channels (1). The resulting increase in cytoplasmic Ca²⁺ concentration is essential in exocytosis of acetylcholine. Binding of acetylcholine to postsynaptic nicotinic cholinergic receptors activates an integral nonselective cation channel, which depolarizes the sarcolemmal membrane (2). Depolarizing the sarcolemma to threshold activates voltage-gated Na⁺ channels (3), which propagate action potential impulses deep into the muscle through the transverse tubule system. Within the transverse tubule system, L-type voltage-gated Ca²⁺ channels sense membrane depolarization and undergo a conformational change (4). A physical link between the α₁ -subunit and the ryanodine receptor is thought to transfer the signal to sarcoplasmic reticulum to induce the release of stored Ca²⁺ (5). (Adapted from Alberts B, Bray D, Lewis J, et al: Molecular Biology of the Cell, 3rd ed. New York, Garland Press, 1994.)

the relaxed muscle cell increases from 10^-7 M to about 5 × 10⁻⁵ M. The increase in Ca²⁺ removes the troponin inhibition from the contractile proteins, resulting in muscle contraction. Intracellular Ca²⁺ pumps (i.e., sarcoplasmic/endoplasmic reticulum Ca²⁺ -ATPase [SERCA] pumps) rapidly reaccumulate Ca²⁺ back into the SR, and relaxation occurs when the concentration is restored to less than mechanical threshold. Contraction and relaxation require adenosine triphosphate (ATP); both are energy-related processes that consume ATP ( Fig. 29-1 ).

Clinical and laboratory data for swine and humans indicate decreased control of intracellular Ca²⁺ , resulting in a release of free, unbound, ionized Ca²⁺ from storage sites that normally maintain muscle relaxation. Aerobic and anaerobic forms of metabolism increase to provide added ATP to drive the Ca²⁺ pumps that maintain Ca²⁺ homeostasis in SR and mitochondria and across the sarcolemma in extracellular fluid. Virtually all of these reactions are exothermic (i.e., they produce heat). Rigidity occurs when unbound myofibrillar Ca²⁺ approaches the contractile threshold. Dantrolene is therapeutic because it reduces Ca²⁺ release from the SR without altering Ca²⁺ reuptake.

Molecular Events in Excitation-Contraction Coupling

Understanding the mechanisms underlying MH requires a more detailed description of the process of EC coupling, the process by which skeletal muscle transforms a chemical signal in the form of a neurotransmitter at the surface

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of the fiber into muscle contraction.^[17] ^[18] ^[19] Figure 29-1 depicts a neuromuscular junction in which an efferent motor neuron synapses with muscle fibers to form a motor end plate. Membrane depolarization at the nerve terminal activates voltage-dependent Ca²⁺ channels on the presynaptic membrane. Most members of the family of voltage-dependent Ca²⁺ channels are composed of five subunits: α₁ , α₂ , β, γ, and δ. Although subunits α₂ , β, γ, and δ contribute important membrane targeting and modulatory functions, it is the larger α₁ -subunit that performs essential functions of voltage sensing and conduction of Ca²⁺ . Specifically, it is the N-type channel (i.e., Ca_V 2.1 in International Union of Pharmacology nomenclature, α_1A in alternate nomenclature) that is primarily responsible for depolarized induced Ca²⁺ entry into motor nerve terminals. Rise of cytosolic Ca²⁺ concentrations within the nerve terminals initiates a process of vesicle migration and fusion that leads to exocytosis of acetylcholine stored within the synaptic vesicles. Simultaneous release of thousands of quanta of acetylcholine results in EPSPs. Acetylcholine binds to nicotinic acetylcholine receptors, which are nonselective cation channels, and activates inward current (primarily carried by sodium ions), thereby depolarizing the muscle cells. When EPSPs sum to threshold, action potentials are propagated from the sarcolemma to the T tubule. Acetylcholinesterase in the synaptic cleft catalyzes rapid breakdown of acetylcholine; rapid removal from the cleft enables the motor unit to be ready for another stimulus within a few milliseconds.

Within the T-tubule membrane of skeletal muscle, a highly homologous relative of the N-type channels, the L-type voltage-gated Ca²⁺ channel (Ca_V 1.1 or α_1S ), or DHPR, is enriched within the T-tubule membrane. Three chemical

Figure 29-2 Schematic representation of the triad junction of skeletal muscle shows the junctional foot protein (ryanodine receptor [RyR1]) and its associated proteins. In skeletal muscle, the α_1S -subunit of the dihydropyridine receptor (DHPR) participates in excitation-contraction coupling. These physical links transmit essential signals across the narrow gap of the triadic junction that activate RyR1 and release Ca²⁺ from the sarcoplasmic reticulum. (Adapted from Pessah IN, Lynch C III, Gronert GA: Complex pharmacology of malignant hyperthermia. Anesthesiology 84:1275, 1996.)

classes of drugs used for controlling cardiovascular function—dihydropyridines, phenylalkylamines, and benzothiazepines—are also capable of blocking DHPRs by direct interaction with α₁ -subunits. In skeletal muscle, it is α_1S -DHPR that participates in EC coupling. Unique to skeletal muscle is the highly ordered arrangement of α_1S -DHPRs into linear arrays of clustered tetrads. Electron microscopic and immunocytochemical analyses indicate that each α_1S -DHPR tetrad is close to (superimposed above) a single RYR1 within the junctional face of SR terminal cisternae ( Fig. 29-2 ). Because each functional RYR1 channel is composed of a tetramer of four identical subunits, each α_1S -DHPR overlies a single RYR1 subunit. The relative restrictions imposed by the dimensions of α_1S -DHPR tetrads and RYR1 tetramers permit only alternate RYR1 channels to pair with α_1S -DHPR tetrads. In common with other voltage-gated ion channels, each α_1S -DHPR possesses a stretch of amphipathic amino acids within the fourth α-helix (S4) of each of four transmembrane domains that functions as a voltage sensor within the T-tubule membrane. Membrane depolarization induces a discrete movement of charge within the S4 segment of the α_1S -DHPR. A mechanical signal, thought to be in the form of a conformational transition, is transmitted to the cytoplasmic loop between repeats II and III of the α_1S -DHPR. Significant evidence exists for a direct physical coupling between the II-III loop of α_1S -DHPR and multiple noncontiguous regions within the large, hydrophilic cytoplasmic domain of RYR1. Such physical links transmit essential signals across the narrow gap of the triadic junction that activate RYR1 and release Ca²⁺ from SR. This conformational coupling model is consistent with the nature of skeletal muscle EC coupling,

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which is independent of extracellular Ca²⁺ . However, after the initial signal activates Ca²⁺ release from SR, Ca²⁺ -induced Ca²⁺ release appears to play a role in regulating the temporal and quantitative characteristics of RYR1 activation. RYR1 also sends a retrograde signal to α_1S -DHPR, enhancing its Ca²⁺ entry function. However, unlike Ca²⁺ entry mediated by α_1S -DHRP, which is essential for cardiac EC coupling, Ca²⁺ entry through α_1S -DHPR is neither essential nor needed for engaging skeletal type EC coupling. Bidirectional signaling between α_1S -DHPR and RYR1 in skeletal muscle appears to represent a fundamental mechanism involving conformational coupling between sarcoplasmic/endoplasmic reticulum Ca²⁺ release channels and voltage- or store-operated Ca²⁺ entry (SOC) channels within the surface membrane in a variety of mammalian cells.

After release into the sarcoplasm, Ca²⁺ is rapidly removed through active transport by SERCA pumps located on junctional and longitudinal SR. Calsequestrin inside the lumen of SR binds to Ca²⁺ and further enhances Ca²⁺ loading within SR. Cytosolic Ca²⁺ is typically brought back to basal nanomolar concentration within 30 msec of muscle contraction. The rapid removal of cytosolic Ca²⁺ is essential for normal muscle relaxation and requires rapid termination of Ca²⁺ efflux from SR. Aberrant termination of RYR1 activity has emerged as a key underlying mechanism in MH susceptibility.