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Basics of skeletal muscle electrophysiology Tóth András, PhD Topics • Structure • Contraction and relaxation • Activation • Excitation-contraction coupling • Action potential • Ion channels* • Calcium homeostasis Structure Skeletal muscle Cardiac muscle In skeletal muscle the SR is greatly enlarged at the terminal cisternae, the diameter of the T-tubules is relatively narrow. In cardiac muscle T-tubules are much larger in diameter and the SR is more sparse, but includes junctional couplings with the external sarcolemma, as well, as the T-tubules. Myofibrils are also more irregular. Mitochondria are plentiful. Schematic diagram of skeletal and cardiac muscles Skeletal muscle fibre types • Voluntary muscles contain a variety of fibre types which are specialised for particular tasks. Most muscles contain a mixture of fibre types although one type may predominate. The pattern of gene expression within each voluntary muscle cell is governed by the firing pattern of its single motor neurone. Motor neurones branch within their target muscle and thereby control several muscle fibres, called a motor unit. The high precision eye muscles have only a few fibres in each motor unit, but the muscles in your back have thousands. All the cells in a motor unit contract in unison and they all belong to the same fibre type: • Type 1 or slow oxidative fibres have a slow contraction speed and a low myosin ATPase activity. These cells are specialised for steady, continuous activity and are highly resistant to fatigue. Their motor neurones are often active, with a low firing frequency. These cells are thin (high surface to volume ratio) with a good capillary supply for efficient gas exchange. They are rich in mitochondria and myoglobin which gives them a red colour. They are built for aerobic metabolism and prefer to use fat as a source of energy. These are the marathon runner's muscle fibres. • Type 2A or fast oxidative-glycolytic fibres have a fast contraction speed and a high myosin ATPase activity. They are progressively recruited when additional effort is required, but are still very resistant to fatigue. Their motor neurones show bursts of intermittent activity. These cells are thin (high surface to volume ratio) with a good capillary supply for efficient gas exchange. They are rich in mitochondria and myoglobin which gives them a red colour. They are built for aerobic metabolism and can use either glucose or fats as a source of energy. These are general purpose muscle fibres which give the edge in athletic performance, but they are more expensive to operate than type 1. • Type 2B or fast glycolytic fibres have a fast contraction speed and a high myosin ATPase activity. They are only recruited for brief maximal efforts and are easily fatigued. Their motor neurones transmit occasional bursts of very high frequency impulses. These are large cells with a poor surface to volume ratio and their limited capillary supply slows the delivery of oxygen and removal of waste products. They have few mitochondria and little myoglobin, resulting in a white colour (e.g. chicken breast). They generate ATP by the anaerobic fermentation of glucose to lactic acid. These are sprinter's muscle fibres, no use for sustained performance. Contraction - relaxation The length-tension relationship in skeletal & cardiac muscle The contractile force in skeletal muscle is determined by A) Contraction summation (tetanus) B) Sarcomer length (myofilament overlap) C) Activation of further fibers (recruitment) The contractile force in cardiac muscle is determined by A) Intracellular Ca concentration (analog) (intrinsic regulation) B) Sarcomer length (myofilament overlap) (extrinsic regulation) The regulation of the contractile force in skeletal & cardiac muscle Activation Comparison of motor nerve terminal, the neuromuscular junction and a skeletal muscle fibre, showing ion channels (numbered 1–8) whose mutation leads to disease. Mutations in several AChR subunits (1) cause myasthenia (muscle weakness). Autoimmune channelopathies are indicated by binding of antibodies (stars), which leads to internalization and downregulation of channel number. Loss of presynaptic K+-channel function (KV1.1, KCNA1) (2) leads to increased transmitter release and enhanced muscle contraction, whereas downregulation of presynaptic Ca2+ channels (3) or AChR (1) function causes myasthenia, by preventing neurotransmitter release, or binding, respectively. Gain-of-function mutations in the muscle Na+ channel (4, SCN4A), and loss-of-function mutations wild-type and mutant (red trace) Na+ current showing the persistent inward current produced by mutations associated with myotonia and/or periodic paralysis. c, Unlike normal muscle (above), action-potential firing in myotonic muscle (below) continues after stimulation has ceased, producing a sustained contraction. The „restricted space” located between junctional sarcosplasmic reticulum (JSR) and the junctional sarcolemma (JSL) forms a local intracellular compartment which, has a very special role in both EC coupling and calcium homeostasis. In this space changes in Na+, K+ & Ca2+ concentrations are significantly greater than in all other compartments of the cytosol. L-type Ca channel & NCX protein densities in the junctional sarcolemma are also much higher than in any other regions of the sarcolemma. The „restricted space” Excitation-contraction coupling (ECC) A) Isolated rat ventricular myocyte B) Frog skeletal muscle fiber In contrast to skeletal muscle in cardiac myocytes external Ca influx is essential for activation of contraction In contrast to skeletal muscle, where DHPRs are found in very regular structure, the DHPRs in the heart cells are sparse and less aligned. Structural differences in skeletal & cardiac T-tubule junctions In skeletal muscle The physical link between DHPR & RyR1 is critical for VDCR Influx of external Ca (ICa) is not required In cardiac muscle The physical link between DHPR & RyR2 is not critical for CICR Influx of external Ca (ICa) is crucial Comparison of EC-coupling in skeletal & cardiac muscle A) Two Ca sparks (2D confocal fluorescence) B) Single Ca spark (line-scan image) C) [Ca]i computed from the image D) Surface plot of [Ca]i during a Ca spark Fusion of a large number of sparks leads to Ca-transient & contraction !!! The elementary event of Ca-release from the SR is the local „spark”, which often occurs during rest in a stochastic manner. 6-20 RyRs contribute to a single spark, which starts at the T-tubule and increases [Ca2+]i in ∼ 10 ms to a peak value of 200-300 nmol. The reason for its time dependent decrease is Ca diffusion and Ca reuptake. Calcium sparks Similarities between skeletal muscle and cardiac EC coupling Both muscle types are striated & contain T-tubules and highly developed intracellular SR networks APs provide the excitation stimulus used to activate plasma membrane DHPRs (or Ca2+ channels ) Activated DHPRs (or Ca2+ channels) trigger the opening of SR Ca2+ release channels Resulting elevation in intracellular Ca2+ activates the contractile machinery Differences between skeletal muscle and cardiac EC coupling The skeletal muscle contains highly developed, cardiac muscle contains a less developed T-tubule and SR system The skeletal muscle does not contain, while the heart contains specialized excitatory tissues (SA node) and conductive fibers (Purkinje fibers) Each skeletal muscle fiber is independent, while the heart is a syncytium of many cells electrically connected at intercalated discs by gap junctions The skeletal muscle AP is 100x shorter (2.5 ms) than that of the ventricular myocardium Ca-sensitizer agents Positive inotropic agents Hypoxia – ischemia Factors which may alter Ca-sensitivity Conclusion A) In a simplified manner the 3 muscle types can serve as models for the 3 major mechanisms of SR Ca-release (VDCR: skeletal muscle; CICR: cardiac muscle; IP3ICR: smooth muscle) This is an oversimpliplification since all 3 mechanism may be present and functional in all 3 muscle types. B) In skeletal muscle VDCR seems to be the crucial initiating process, however, CICR may be very important in recruiting RyRs (∼ 50%) which are not physically coupled to T-tubule tetrads. IP3 can also induce Ca release (IP3ICR), but its significance is not yet clear. C) In cardiac muscle CICR is the essential EC-coupling mechanism. IP3 may also modulate cardiac Ca release. There is some evidence for a functional direct link between the SL and the SR (and possibly VDCR). The significance of this link is not yet clear. D) In smooth muscle there is evidence for both IP3ICR & CICR. There is also evidence for that the IP3ICR interacts with a different plasma membrane Ca channel (TRP), involved in CCE where the signal is retrograde from IP3R to TRP. Action potential Calcium homeostasis in skeletal muscle INFLUX = EFFLUX ICa + + INCX ↓↓ + TRIGGER - Ca-transient CDI 1 EFFLUX ≠ ICa↑ + TRIGGER INFLUX = EFFLUX ICa + TRIGGER CDI “Empty” cell INCX ↓ + Ca-transient CDI 2 Steady-state INFLUX 3 EFFLUX ≠ ICa ↑↑ INCX + TRIGGER INFLUX INCX + Ca-transient During “refill” CDI 4 Ca-transient Steady-state Principles of “autoregulation” of SR Ca2+ content in the heart This mechanism does not work in skeletal muscle Modes of regulated Ca2+ entry across the plasma membrane. Ca2+ can enter cells by any of several general classes of channels, including voltage-operated channels (VOC), second messenger-operated channels (SMOC), store-operated channels (SOC), and receptor-operated channels (ROC). VOCs are activated by membrane depolarization, and SMOCs are activated by any of a number of small messenger molecules, the most common being inositol phosphates, cyclic nucleotides, and lipid-derived messengers (diacylglycerol and arachidonic acid and its metabolites). SOCs are activated by depletion of intracellular Ca2+ stores, and ROCs are activated by direct binding of a neurotransmitter or hormone agonist (Ag). In addition, under some conditions, Ca2+ can enter cells via the Na-Ca2+ exchanger (NCX) operating in reverse mode. Model of SOCE in skeletal muscle. SOCE in skeletal muscle displays rapid kinetics compared to nonexcitable cells. STIM1 localization may account for these kinetic differences. Electron micrographs of skeletal muscle from STIM1 gene trapped mice revealed STIM1 protein aggregates located in membranes of the terminal cisternae and the para-junctional SR. The junctional STIM1 pool is located near or complexed with Orai1 and can respond rapidly to store depletion. Although recent studies have shown that STIM1 activation by store depletion suppresses L-type voltageoperated calcium (Cav1.2) channels, whether STIM1 plays a similar role in the regulation of L-type channels in skeletal muscle which expresses the Cav1.1 isoform is currently unknown. STIM & ORAI: two major players in the SOC(cer) field Structure and distribution of STIM proteins. (A) Comparison of the sequence domains of STIM1 and STIM2 proteins including the EF-hand Ca2+ binding regions, sterile-alpha motifs (SAM), glycosylation sites (hexagons), transmembrane domains (TM), coiled-coil regions, ezrin–radixin–moesin-like domains (ERM), and proline-rich domains (P). (B) Domain topologies for STIM1 and STIM2—a number of potential serine/threonine phosphorylation sites existing on the N-terminal domains of the proteins are shown (red P). Lower right panel: distribution of STIM1 and STIM2 in the PM and ER. While STIM1 can be observed in both the ER and PM, the STIM2 protein appears to be exclusively in the ER. Models for the coupling between STIM1 and Orai1 in the activation of store-operated Ca2+ entry. (A) Simplest model in which aggregated STIM1 in the ER interacts directly with Orai1 in the PM. (B) STIM1 in the PM is the primary site of a interaction of aggregated STIM1 in the ER; the plasma membrane STIM1 protein then mediates interaction with and activation of Orai1in the PM. (C) STIM1 aggregated in the ER activates PM Orai1 channels by direct activation as in (A) but STIM1 in the PM exerts a modulatory role on this channel activation by interacting with aggregated STIM1 in the ER.