Biochemistry of the Muscle PDF

BIOCHEMISTR Y OF THE MUSCLE BY PROF MR S J E IKEKPEAZU  BIOCHEMISTR Y OF THE MUSCLE The muscle is an aggregate of proteins involved in contraction. The musculature(a system of muscles in the body eg the biceps and triceps in the arm are part of the musculature of t...

BIOCHEMISTR Y OF THE MUSCLE BY PROF MR S J E IKEKPEAZU  BIOCHEMISTR Y OF THE MUSCLE The muscle is an aggregate of proteins involved in contraction. The musculature(a system of muscles in the body eg the biceps and triceps in the arm are part of the musculature of the arm) is what makes movement possible. Muscle is a major biochemical transducer Converts potential (chemical) energy into kinetic (mechanical) energy.  Muscle They comprise the largest group of tissues in body There are 3 types of muscle Skeletal muscle: Make up muscular system They are the ones that make up musculoskeletal system Cardiac muscle: Found only in the heart Smooth muscle: Appears throughout the body systems as components of hollow organs and tubes Classified as: Striated/unstriated and voluntary/involuntary (see below)  CLASSIFICATION OF MUSCLES They are classified into 3 basic types: Skeletal muscle Cardiac muscle Smooth muscle They are also divided into 2 based on electron microscopic appearance into Striated muscles eg cardiac and skeletal Non striated muscles eg smooth muscles  Classification continued Divided based on control from the CNS into Voluntary muscles eg skeletal muscles Involuntary muscles eg cardiac and smooth muscles.  Muscle Action The controlled muscle contraction allows : Purposeful movement of the whole body or parts of the body Manipulation of external objects Propulsion of contents through various hollow internal organs Emptying of contents of certain organs to external environment eg gall bladder and emptying of bile  In all of these types of muscle, contraction is based on the interplay between two kinds of protein filaments: actin (called the thin filament) and myosin (called the thick filament).  ORGANISATION OF THE SKELETAL MUSCLE The skeletal muscle is striated and consists of parallel bundles of muscle fibers connected to tendons at both ends.  Structure of Skeletal Muscle Muscle consists of a number of muscle fibers lying parallel to one another and held together by connective tissue Single skeletal muscle cell is known as a muscle fiber Multinucleated Large, elongated, and cylindrically shaped Fibers usually extend entire length of muscle  Each muscle fiber is composed of several myofibrils arranged in parallel. Myofibrils are surrounded by an electrically excitable membrane called the sarcolemma. The myofibrils are immersed in a cytosol (Sarcoplasm). Sarcoplasm is rich in glycogen, ATP, creatine phosphate and glycolytic enzymes. Many regularly spaced mitochondria are found in the more active muscles.  Myofibrils exhibit a longitudinally long repeating structure called the sarcomere, which is the functional unit of a myofibril. Sarcolemmal invaginations gives rise to small membranous folds, known as the transverse tubules (T-tubules) which extend from the sarcolemma and surround each myofibril at the Z line-see later. The sarcoplasmic reticulum is a sheath of flattened vessicles that surround the myofibrils (like a net) and stores large quantities of calcium at its terminal cisternae.  Electron micrograph of the sarcomere The sarcomere is characterized by the appearance of several distinct bands. The less optically dense band is referred to as the I band, and the denser one as the A band. A dense line appears in the centre of the 1 band, called the Z line or disc, and a dense narrow band, somewhat similar in appearance also occurs in the centre of A band, called the M line. Adjacent to the M line are regions of the A band that appear lense dense than the remainder and termed the H zone.  Transverse sections of the sarcomere reveal that the above pattern result from the interdigitation of the two sets of protein filament- the thin (ACTIN) and the thick (MYOSIN) filaments. The 1 band consists of the thin filaments, while the H zone consists of thick filament but the A band shows a regularly packed array of interdigitating thick and thin filaments-see below.  Organizational details of a typical striated skeletal muscle. a: Representation of each muscle fiber showing the parallel bundles called myofibrils. b: Myofibrils are a series of sarcomeres separated by Z discs or lines which contain thick and thin filaments. c: Thick filaments are myosin bundles that span the A band and are bound to proteins of the M line and to the Z discs across the I bands by the large protein called titin-see below. d: Transmission electron micrograph (TEM) showing the molecular organization of the sarcomere. e: TEM of an oblique section showing the hexagonal organization between the thick and thin myofilaments.  The organization of individual contractile proteins making up a sarcomere is a key feature of the sliding filament model. Each sarcomere is composed of hundreds of filamentous protein aggregates, each known as a myofilament. Two kinds of myofilaments are identifiable on the basis of their diameter and protein composition (see image above). Thick myofilaments are composed of several hundred molecules of one of several different fibrous proteins known as myosin.  Thin myofilaments are composed of two helically interwound, linear polymers of globular proteins known as actin. Thin and thick filaments also contain accessory proteins as described below. Proteins of the Z disc, primarily the α-actinins (see later), serve as an embedding matrix or anchor for one end of the thin filaments, which extend toward the center of sarcomeres on either side of the Z disc. The Z disc proteins often appear continuous across the width of a muscle fiber and seem to act to keep the myofibrils within a myofiber.  Proteins of the myofibril – Thin and thick filaments. The thin filaments are composed of three proteins Actin Troponin. Tropomyosin Actin is the major constituent (protein) of thin filaments. Comprises 25% of muscle protein by weight.  The monomer of Actin is called the G-actin (because of its globular shape). G-actin polymerizes as ionic strength increase to physiological level into a fibrous form called F-actin. Each actin filament (F-actin) consists of two stands of actin twisted into an -helical pattern. An F-actin fibre looks (micrographycally) like two strings of beads wound around each other in an  helical form. Polymerization of G-Actin to F-Actin Sequence of polymerization of G- Actin FORMATION AND ASSEMBLY OF THE THIN FILAMENT F-Actin showing exposed binding site usually covered by tropomyosin- role of calcium ions.  Tropomyosin (TM) is a filamentous protein containing two subunits wound in an -helical form. It lies in the groove on either side of the F- actin filament It mediates access to the site on actin monomers unto which myosin head binds.  Present in cells and muscle-like structures  Troponin is a complex of three non- identical subunits: TN-C (a calcium binding subunit), TN-T (a TM-binding subunit) and TN-1 (an “inhibitory” subunit). Two molecules of troponin (TN) bind to the actin filament at each helical repeat. Troponin T binds to tropomyosin and together with troponin I, inhibits the interaction of actin and myosin.  Troponin I binds actin and inhibits the binding of actin to myosin ie has a strong affinity for actin. In other words, it inhibits F-actin –myosin interactions Binds to other troponin molecules Troponin C a calcium binding protein (similar to calmodulin in function) has a binding site for Ca2+ Binds 4 molecules of calcium when calcium is bound, actin and myosin interaction is promoted.  NOTE: Troponin is found only in striated muscle cells. The thick and thin filaments interact via cross bridges Interaction between this cross bridges and actin filament cause contraction The filaments slide pass one another during contraction.  The thick filaments-MYOSIN The thick filaments are composed of myosin molecules which are long fibrous structures composed of six subunits (a hexamer): two heavy chains and four light chains. Contributes 55% of muscle protein by weight Form the thick filament Has a fibrous portion consisting of 2 intertwined helices Each myosin heavy chain consists of a globular head and a fibrous tail.  The two fibrous tails of the heavy chains are twisted into a double helical structure, forming regions where myosin molecules interact to form filaments. The globular head of the myosin molecule contain ATpase activity as well as site for binding to actin filaments. The helical coils therefore form the backbone of the thick filaments. They also form an arm that can provide a flexible extension or hinge connecting the globular head to the body of the thick filament. DETAILED STRUCTURE OF MYOSIN  Summary of notes on myosin and actin Myosin Component of thick filament Protein molecule consisting of two identical subunits shaped somewhat like a golf club racket Tail ends are intertwined around each other Globular heads project out at one end-see structure above. Tails oriented toward center of filament and globular heads protrude outward at regular intervals on each filament Heads form cross bridges between thick and thin filaments Cross bridge has 2 important sites critical to contractile process An actin-binding site A myosin ATPase (ATP-splitting) site  Actin Primary structural component of thin filaments. Spherical in shape Thin filament also has 2 other proteins Tropomyosin Troponin Each actin molecule has special binding site for attachment with myosin cross bridge Binding results in contraction of muscle fiber  Summary of notes on myosin and actin CTD Actin and Myosin pls note these: Actin and myosin are often called contractile proteins. Actin and myosin are not unique to muscle cells, but are more abundant and more highly organized in muscle cells Tropomyosin and Troponin Often called regulatory proteins Tropomyosin Thread-like molecules that lie end to end alongside groove of actin spiral In this position, covers actin binding sites, blocking interaction of actin and myosin that should lead to muscle contraction  Troponin Made of 3 polypeptide units One binds to tropomyosin(TNT) One binds to actin(TNI) One can bind with Ca2+(TNC) Tropomyosin and Troponin Troponin When not bound to Ca2+ Troponin stabilizes tropomyosin in blocking position over actin’s cross-bridge binding sites When Ca2+ binds to troponin Tropomyosin moves away from blocking position With tropomyosin out of the way, actin and myosin bind, interact at cross-bridges Muscle contraction results  ENZYMATIC HYDROLYSIS OF MYOSIN Myosin can be cleaved enzymatically into functional fragments that retain some of the activities of the intact molecule. Myosin is split by trypsin into two fragments called light meromyosin (LMM) and heavy meromyosin (HMM). LMM like myosin tail forms filament but lack ATpase activity and does not combine with actin. HMM catalyzes the hydrolysis of ATP and binds to actin, but does not form filaments.  The LMM is a two stranded  helical rod They are insoluble alpha-helical fibres HMM consists of a short rod attached to a double headed globular region can be split by papain into two globular fragments (called S1) and one rod-shaped subfragment (called S2). S2 fragment is fibrous Each S1 fragment contain an ATpase active site and a binding site for actin. The light chains of myosin are bound to the S 1 fragments.  GENERAL MECHANISM OF MUSCLE CONTRACTION The major biochemical events occurring during one cycle of muscle contraction and relaxation can be represented in the five steps shown below: (1) In the relaxation phase of muscle contraction, the S-1 head of myosin hydrolyzes ATP to ADP and Pi but these products remain bound. The resultant ADP-Pi-myosin complex has been energized and is in high-energy conformation.  (2) When contraction of muscle is stimulated (via events involving Ca2+, troponin, tropomyosin, and actin, which are described below later), actin becomes accessible. The S-1 head of myosin finds it and binds to it forms the actin-myosin-ADP-Pi complex – so called actinomyosin-ADP-Pi complex.  (3) Formation of this complex promotes the release of Pi, which initiates the power stroke. This step which is followed by release of ADP is accompanied by a large conformational change in the head of myosin in relation to its tail , pulling actin about 10 nm toward the centre of the sarcomere. This is the power stroke. The myosin is now in a low-energy state, indicated as actin-myosin.  (4) Another molecule of ATP binds to the S-1 head, forming an actin-myosin-ATP complex. (5) Myosin-ATP has a low affinity for actin, and actin is thus released. This last step is a key component of relaxation and is dependent upon the binding of ATP to the actin-myosin complex.  DETAILS STEPS IN MUSCLE CONTRACTION sliding filament model of muscle contraction. 1. The process of muscular contraction entails a sliding of the thick and thin filaments past each other. The movement of myosin (thick filaments) along the actin ( thin filament) is ATP dependent. This contraction is a cyclical process. 2. The myosin head binds ATP, which is hydrolyzed to ADP and Pi by myosin ATpase. ADP and Pi remain bound to the ATpase site.  3. The myosin head moves to an adjacent thin (actin) filament (the binding site) and as it makes contact with the actin binding site, Pi is released. The release of pi is the rate-limiting step in muscular contraction. 4. Strong cross-bridges form between actin and myosin which is followed by a structural alteration (conformational change) in the myosin molecules and an effective translocation of the thick filament relative to the thin filament.  ie large conformational change in the head of myosin in relation to its tail , pulling actin about 10 nm toward the center of the sarcomere. During this process, the ADP is released. 5. After the translocation step, the bridge structure is broken by the binding of ATP, which is hydrolysed to ADP and pi. Hydrolysis of ATP result in the myosin head resuming its original conformation and is then ready for another cycle further along.  The contraction process therefore involves the breakage and reformation of cross bridges between the actin and myosin molecules in a reaction that requires the expenditure of ATP. Each thick filament has about 500 myosin heads and each head cycles about five times per second in the course of a rapid contraction.  In a fully contracted myofibril, the actin and myosin filament show a maximum overlap with each. However there is no change in the length of either types of filament (see diagrams below). In other words during contraction 1. The Z lines approach one another 2. The sarcomere get smaller and 3. The A band, does not change size.  Note: In a relaxed muscle, the actin filament extend approximately halfway over the myosin filament-see above diagram. During contraction, the actin filaments slide towards each other, past the myosin filaments resulting in the shortening of the sarcomere, the myofibrils and the length of the muscle. This is known as the sliding filament model of muscle contraction.  The sliding filament cross-bridge model is the foundation of current thinking about muscle contraction. The basis of this model is that the interdigitating filaments slide past one another during contraction and cross-bridges between myosin and actin generate and sustain the tension. The hydrolysis of ATP is therefore used to drive movement of the filaments.  REGULATION OF MUSCLE CONTRACTION: ROLE OF TROPONIN AND TROPOMYOSIN IN MUSCULAR CONTRACTION (ACTIN –BASED REGULATION). This takes place manly in striated muscles (skeletal and cardiac) Control of muscle contraction is provided by troponin, tropomyosin, and a change in intracellular calcium concentration. When the calcium ion concentration is low (10-7M), troponin I and troponin T are strongly bound to tropomyosin, holding it in the actin groove so that it blocks/covers the myosin binding site on the actin monomers. Troponin C is either loosely associated with tropomyosin or is bound to troponin I and troponin T.  When calcium concentration increase above 10-7M in the cell, troponin C binds calcium and undergoes change in conformation, forcing troponin I and troponin T to move. The movement exposes the myosin binding site on the actin by the removal of tropomyosin, permitting interaction between the two filaments to occur. This however does not necessarily result in contraction; contraction can result only if ATP is present.  CONTROL OF INTRACELLULAR CALCIUM CONCENTRATIONS. Small membranous folds, known as T-tubules extend from the sarcolemma and surround each myofibril at the Z line-see earlier. The T-tubules act as a communication system for the depolarization of the plasma membrane that occurs when a nervous stimulation signals the muscle to contract. Depolarization of the sarcolemma is relayed to the sarcoplasmic reticulum (SR ), a sheath of flattened vesicles that surround the myofibrils and store large quantities of calcium at the terminal cisterna.  Normally, extracellular calcium concentrations are high (10-3M) whereas those in the cytosol are low (10- 7 M). Upon depolarization, the calcium level rise from 10 -7 to greater than 10-5m during contraction. The increase is due primarily to the movement of calcium ions through ca2+ release channels in the membrane of the sarcoplasmic reticulum. The change in calcium concentration occurs rapidly and relieves the inhibition of myosin binding to actin.  Once the stimulation of the nerves cease, calcium is pumped back into the sarcoplasmic reticulum by the action of a SR- specific ATpase. The calcium is bound in the SR calsequesterin, an acidic protein with a high density of aspartate and glutamate residues.  Sequence of events in contraction and relaxation of skeletal muscle (starting with the events at the neuromuscular junction). Steps in contraction (1) Discharge of motor neuron (2) Release of transmitter (acetylcholine) at motor endplate (3) Binding of acetylcholine to nicotinic acetylcholine receptors (4) Increased Na+ and K+ conductance in endplate membrane (5) Generation of endplate potential (6) Generation of action potential in muscle fibers  (7) Inward spread of depolarization along T tubules (8) Release of Ca2+ from terminal cisternae of sarcoplasmic reticulum and diffusion to thick and thin filaments (9) Binding of Ca2+ to troponin C, uncovering myosin binding sites of actin (10) Formation of cross-linkages between actin and myosin and sliding of thin on thick filaments, producing shortening  Steps in relaxation (1) Ca2+ pumped back into sarcoplasmic reticulum (2) Release of Ca2+ from troponin (3) Cessation of interaction between actin and myosin The details of the other events between actin and myosin are already described.  ACTIN-BINDING DRUGS Can interfere with the polymerization- depolymerization cycle of microfilaments. Cytochalasin B inhibits the assembly of actin filaments by binding to one end of the filament and preventing the addition of actin molecules to the filament. Processes such as endocytosis, cytokinesis, cytoplasmic and amoeboid movements are all inhibited by cytochalasin B. Phallodin binds along the length of actin filament, preventing depolymerization. ACCESSOR Y PROTEINS OF THE MYOFIBRIL.  ACCESSORY PROTEINS OF THE MYOFIBRIL. Several other proteins are associated with the sarcomere. Their location and functions are as follows: Titin - summary Giant, highly elastic protein (largest in body) Extends in both directions from M line along length of thick filament to Z lines at opposite ends of sarcomere 2 important roles: Helps stabilize position of thick filaments in relation to thin filaments Greatly augments muscle’s elasticity by acting like a spring It plays a role in relaxation of muscle. May regulate assembly and lenght of filaments  Nebulin is a large fibrous protein attached to the Z-line and extends the length of the thin filament. It stabilizes the highly ordered structure of the myofibril by regulating the assembly of the actin filaments. α-Actinin Anchors actin to Z lines. Stabilizes actin filaments.  Desmin is found in Z line, where it holds myofibril in place. It gives the muscle its “Striated” appearance. Attaches to plasma membrane (sarcolemma). Myosin- binding protein C Arranged transversely in sarcomere A-bands Binds myosin and titin Plays a role in maintaining the structural integrity of the sarcomere. Myomesin- cross-links adjacent filaments at the M-line (the centre of the sarcomere).  Calcineurin Present in the Cytosol (sarcoplasm) A calmodulin-regulated protein phosphatase. May play important roles in cardiac hypertrophy and in regulating amounts of slow and fast twitch muscles. Dystrophin Attached to sarcolemma through dystroglycans Deficient in Duchenne muscular dystrophy. Mutations of its gene can also cause dilated cardiomyopathy.  The Dystrophin Complex The dystrophin-glycoprotein complex (DGC) is a multi-subunit complex within and across the membranes of cardiac and skeletal muscle cells as well as vascular smooth muscle cells. The complex serves both a mechanical stabilizing and a signaling role in these various cell types. The complex mediates interactions between the cytoskeleton, membrane, and the extracellular matrix. The complex is composed of up to 15 different proteins dependent upon its location. These proteins include, extracellular , transmembrane, and intracellular subunits.  Within the DGC there is the protein dystrophin, two dystroglycan proteins (α- dystroglycan, α-DG and β-dystroglycan, β- DG), the syntrophins (α and β), the dystrobrevins (α and β), caveolin-3, sarcospan, and a sub-complex composed of members of the sarcoglycan family. There are six known sarcoglycans designated α-, β-, γ-, δ-, ε (epsilon)-, and ζ (zeta)-sarcoglycan. An additional component of the DGC is neuronal nitric oxide synthase (nNOS, NOS- 1). The major intracellular protein is the large dystrophin protein. Organizational details of a typical dystrophin- glycoprotein complex.  The dystrophin proteins serves to link the extracellular portions of the DGC with the actin filaments within the cell. Dystrophin physically interacts with the cytoplasmic tail of β-DG, via a C-terminal domain in dystrophin, and γ-actin in actin filaments (F-actin) via an N-terminal domain. The clinical significance of the dystrophin protein relates to the fact that mutations in the DMD gene are the cause of various muscular dystrophies (see below): Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and X-linked dilated cardiomyopathy (XLDCM). See later  CONTRACTION OF SMOOTH MUSCLES Smooth muscle differs from skeletal muscle in various ways: Smooth muscles are found, for example, in blood vessel walls and in the walls of the intestine. In smooth-muscle cells, which are usually spindle- shaped, the contractile proteins are arranged in a less regular pattern than in striated muscle.  The smooth muscle contain actin filament attached to the plasma membrane, but the fibrils are not aligned, thus when myosin slides along the actin filaments, the cell contracts in all direction.  Contraction in this type of muscle is usually not stimulated by nerve impulses, but occurs in a largely spontaneous way. Ca2+ (in the form of Ca2+-calmodulin); also activates contraction in smooth muscle. In this case, however, it does not bind to troponin, but activates a protein kinase that phosphorylates the light chains in myosin and thereby increase myosin’s ATPase activity. Most of the Ca2+ are from the ECF and there is no troponin.  Sources of calcium ions for smooth muscle contraction The phosphorylation of membrane-bound PI produces phosphatidylinositol 4,5-bisphosphate (PIP2). This compound is degraded by phospholipase C β in response to the binding of various neurotransmitters, hormones, and growth factors to membrane G protein– coupled receptors. The products of this degradation, inositol 1,4,5- trisphosphate (IP3) and DAG, mediate the mobilization of intracellular calcium from the sarcoplasmic reticulum. There is also opening of the calcium ion channels at the cell membrane that allows inflow of ca2+ from the ECF.  Smooth Muscle contraction - Details While the sliding filament model adequately describes the basic mechanism of contraction in all muscle types, there are significant differences between striated (skeletal and cardiac) and smooth muscle. Although smooth muscle lacks the troponin complex, its contractile activity is still regulated by cytoplasmic calcium levels.  A Ca2+/calmodulin (CaCM) binding protein known as caldesmon regulates the movement of smooth muscle tropomyosin on and off the myosin binding sites of thin filaments. Alterations in smooth muscle cytosolic calcium levels occur via: 1. voltage-dependent activation processes and by 2. receptor-mediated processes. The voltage-mediated processes involve the activation of plasma membrane voltage- gated calcium channels of the L-type- see above.  The principal receptor-mediated activation of smooth muscle contractile activity occurs in response to α1-adrenergic receptor activation. The α1 adrenergic receptor is coupled to a Gq-type G-protein which activates PLCβ leading to production of inositol-1,4,5- trisphosphate (IP3) and diacylglycerol, DAG from P1P2. The IP3 binds to receptors on the endoplasmic reticulum leading to release of stored Ca2+, the consequences of which are smooth muscle contraction.  In contrast to most of the vasculature, the smooth muscle cells of the vessels in skeletal muscle tissues possess predominantly the β2 adrenergic receptor. The β2 adrenergic receptor is coupled to a Gs type G-protein, the activation of which results in increased cAMP production and activation of PKA. Both PKA and cAMP interfere with smooth muscle contraction leading, instead, to relaxation of the vessels. This allows skeletal muscle cells access to increased nutrients and oxygen in response to stress.  Both the voltage-mediated and the receptor- mediated smooth muscle activation processes lead to increased intracellular calcium levels which lead to increased levels of active CaCM which, in turn, binds caldesmon, removing it from its site on thin filaments. Calmodulin is also a component of the myosin light- chain kinases (MLCK or MYLK) and the activation of calmodulin (CM or CaM) in these enzyme complexes results in phosphorylation of myosin light-chains (MLC or MYL) on serine 19 (S19). Concurrently, tropomyosin is observed to change its location in the helical grooves of F-actin, and the ATPase activity of the actin-myosin complex (actomyosin) is stimulated. Each of these events results in contraction of smooth muscle cells.  Myosin Light Chain Kinases When calcium is depleted, the CaCM complex dissociates and caldesmon is released from its complex with calmodulin and re-associates with thin filaments while simultaneously, MLCK (MYLK) activity is reduced. The actomyosin complex ATPase activity is correspondingly inhibited. In essence, caldesmon in smooth muscle cells replaces the troponin complex as a calcium (CaCM)-dependent regulator of the location of the tropomyosin complex within the context of the thin filaments.  Smooth muscle relaxation is effected via activation of β2-adrenergic receptors which are coupled to Gs- type G-proteins. Activation of β2-adrenergic receptors leads to increased production of cAMP via activation of adenylate cyclase. The increased cAMP levels activate PKA which in turn phosphorylates MLCK (MYLK) resulting in inhibition of MLCK-mediated phosphorylation of myosin light chains. In addition, cAMP itself will inhibit the activity of MLCK (MYLK).  Another path to smooth muscle relaxation initiated via β2 receptor activation is the PKA-mediated phosphorylation of a membrane potassium channel (KATP) which results in depolarization of the cell and closure of the plasma membrane Ca2+ channels. Closure of the Ca2+ channel results in reduced intracellular Ca2+ and thus, reduced levels of active MLCK (MYLK). Activation of α2-adrenergic receptors can interfere with the effects of β2 receptor activation since α2-adrenergic receptors are coupled to Gi-type G-proteins that inhibit the activity of adenylate cyclase.  Myosin Light Chain Phosphatases Following removal of the initiating stimulus for smooth muscle contraction, all of the events that participated in the activation of contraction must be reversed. With respect to the myosin proteins this entails dephosphorylation of S19 of the myosin light chains. Phosphate removal from myosin light chains is catalyzed by an enzyme called simply, myosin phosphatase. The catalytic activity of myosin phosphatase is a protein that is a member of the protein phosphatase 1 (PP1) family of phosphatases.  Adrenergic Receptors in Muscle Functions The catecholamines, norepinephrine and epinephrine, exert potent effects on cardiac, skeletal, and smooth muscle cells. These effects are exerted in response to binding to one or more of the different types of adrenergic receptors – Read up these receptors types (α1,α2,β1,β2,β3), their expression profile and functions. Acetylcholine and Receptors in Muscle Functions Also read up acetylcholine and its receptors and their role in muscle functions. Receptor Type Gene Symbol(s) Expression Profile Functions / Comments three subtypes: α1A, α1B, α1D; coupled to Gq-type G-proteins, ADRA1A vasoconstrictor for coronary arteries and veins, decreases GI predominates in heart, blood vessels, and α1 ADRA1B smooth muscle cell motility, induces contraction of smooth kidneys, also expressed in adipose tissue ADRA1D muscle in uterus, urethral sphincter, vas deferens, and ureter, modulates glycolysis and gluconeogenesis three subtypes: α2A, α2B, α2C; coupled to Gi-type G-proteins, acts within the CNS to decrease blood pressure and exert bradycardic effects, exerts a hypothermic effect, arterial and ADRA2A central nervous system (widely distributed); venous vasoconstriction, inhibits insulin release and stimulates α2 ADRA2B vessels, adipose tissue, kidneys, and platelets glucagon secretion, modulates gluconeogenesis and glycolysis, ADRA2C inhibits gastric acid secretion and gastric motility, inhibits release of norepinephrine and acetylcholine, involved in thrombus stabilization by inducing platelet aggregation  coupled to Gs-type G-protein, exerts inotropic (contraction strength) and chronotropic (heart rate) heart, kidney, skeletal muscle, lung, β1 effects on the heart, increases fat mobilization from ADRB1 colon, liver, thyroid gland, adipocytes adipose tissue, increases renin release from kidneys, (preadipocytes only in BAT) enhances sensation of hunger through release of ghrelin by the stomach coupled to Gs-type G-protein, bronchodilator and adipose tissue but not brown adipocytes, vasodepressor, induces relaxation of smooth muscle in bronchioles, skeletal muscle, smooth β2 ADRB2 bronchus, bronchioles, uterus, and detrusor muscle, inhibits muscle, lung, kidney, colon, liver, thyroid release of insulin, stimulates lipolysis, glycolysis, and gland, heart gluconeogenesis abundant in adipocytes of BAT and omental fat, gallbladder and bladder, is coupled to Gs-type G-protein, regulation of lipolysis, β3 ADRB3 not expressed in the heart, skeletal principal norepinephrine receptor in BAT, increase lipolysis muscle, liver, kidneys, lung, or thyroid in BAT and plays major role in adaptive thermogenesis gland  NEUROMUSCULAR DISORDERS 1. Myasthenia Gravis (MG) This is a particularly devastating disease that results from defects in the overall processes of neuromuscular nerve transmission. MG is a very serious disorder that is often times fatal. The characteristic features of the disease are weakened skeletal muscles that tire with very little exertion. MG is an auto-immune disease associated with antibodies to the nAChR of the neuromuscular junction. Binding of the antibodies to the receptor results in receptor destruction as well as receptor cross-  In most patients with MG there is a 70%–90% reduction in motor end plate nicotinic receptor number. Two major forms of MG exist, one in which the extraocular muscles are the ones primarily affected and in the other form there is a generalized skeletal muscle involvement. In the latter form of MG, the muscles of the diaphragm become affected resulting in respiratory failure which contributes to the mortality of MG. Treatment of MG involves numerous approaches including the use of acetylcholinesterase inhibitors. The use of these types of drugs allows for enhanced levels of ACh at the motor end plate during repeated muscle stimulation.  2. The Muscular Dystrophies The muscular dystrophies represent a group of nine characterized disorders, all of which are associated with some level of loss of muscle function along with atrophy of muscle tissue. These diseases are Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), myotonic dystrophy (DM, for dystrophia myotonica), distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, fascioscapulohumeral muscular dystrophy, and congenital muscular dystrophy.  The muscular dystrophies that are associated with defects in the gene encoding the intracellular protein, dystrophin (gene symbol, DMD), are known as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Because the dystrophin gene is found on the X chromosome, both of these diseases are inherited in an X- linked manner. Both DMD and BMD are caused by mutations in the DMD gene. The primary clinical differences between DMD and BMD are due to the fact that the mutations in the DMD gene that cause Duchene muscular dystrophy result in virtually no functional dystrophin protein being made. With Becker muscular dystrophy the mutations result in some functional dystrophin protein ranging from 10%– 40% of normal.  Duchenne Muscular Dystrophy, DMD Duchenne muscular dystrophy (DMD) represents the most severe form of nine characterized muscular dystrophies. DMD is an X-linked recessive disorder and, therefore, primarily manifests in males. The disease is inherited with a frequency of approximately 1 in 3,600. DMD is a rapidly progressing, fatal form of muscular dystrophy. Symptoms of DMD usually begin to appear within the first 6-months of life but can also be seen at birth in some afflicted infants.  The symptoms of DMD are characterized by progressive muscle degeneration and weakness, eventually resulting in death. The average life span for DMD patients is around 25 years of age. The early signs that are characteristic in all DMD patients is a progressive proximal muscle weakness evident in the legs and pelvis.  These symptoms are associated with, and the result of, a loss of muscle mass. Although muscle loss is characteristic of DMD, very early in the disease the calf muscles hypertrophy. As the disease progresses the muscle loss and weakness spreads to the arms, neck, and other areas of the body. As the disease progresses, the loss of muscle tissue is replaced fatty tissue and fibrotic tissue.  Becker Muscular Dystrophy, BMD Becker muscular dystrophy (BMD) represents a milder form of muscular dystrophy caused by mutations in the dystrophin gene (gene symbol, DMD). Whereas in the case of Duchenne muscular dystrophy where no functional dystrophin protein is made, BMD is associated with some functional protein. For this later reason the symptoms of BMD are much less severe, begin manifesting later in life than for DMD, and the disorder is not lethal as in the case of DMD.  Because BMD is caused by mutations in the same gene, the location of symptoms is very similar to that in the case of DMD but just manifest much later and with less severity. Symptoms of BMD usually begin to appear between 5 and 15 years of age. Initial symptoms include calf muscle enlargement followed by the same fatty tissue and fibrotic tissue deposition as in DMD. The muscle weakness in BMD is very slowly progressing and many afflicted individuals can continue to walk, though with difficulties, well into adulthood. In many cases, patients with BMD die in the 4th decade of life, but many individuals have also lived a normal life span.  MUSCLE METABOLISM 1 The metabolic profile of the muscle The major fuels for muscle are glucose, fatty acids and Ketone bodies. Muscle has a large store of glycogen (1,200Kcal). About 3 /4 of all the glycogen in the body is stored in muscle. Muscle contraction is associated with a high level of ATP consumption. Without constant resynthesis, the amount of ATP available in the resting state would be used up in less than I second of contraction.  A. Energy metabolism in the white and red muscle fibers Muscles contain two types of fibers, the proportions of which vary from one type of muscle to another. Red fibers (type I or slow fibers) are suitable for prolonged effort. Their metabolism is mainly aerobic and therefore depends on an adequate supply of O2. White fibers (types 11 or fast fibers) are better suited for fast, strong contractions. These fibers are able to form sufficient ATP even when there is little O2 available  With appropriate training, athletes and sports participants are able to change the proportions of the two fiber types in the musculature This prepares them for the physiological demands of their disciplines in a targeted fashion. The expression of functional muscle proteins can also change during the course of the training.  Red fibers provide for their ATP requirements mainly (but not exclusively) from fatty acids, which are broken down via β-oxidation, the tricarboxylic acid cycle, and the respiratory chain. The red color in these fibers is due to the monomeric heme protein myogobin which they use as an O2 reserve. Myoglobin has a much higher affinity for 02 than hemoglobin and therefore only releases its O2 when there is a severe drop in O2 partial pressure.  At a high level of muscular effort-e.g, during weightlifting or in very fast contractions such as those carried out by the eye muscles-the 02 supply from the blood quickly becomes inadequate to maintain the aerobic metabolism. White fibers therefore mainly obtain ATP from anaerobic glycolysis. They have supplies of glycogen from which they can quickly release glucose-1- phosphate when needed.  By isomerization, this gives rise to glucose-6- phosphate, the substrate for glycolysis. The NADH+H+ formed during glycolysis has to be reoxidized into NAD+ in order to maintain glucose degradation and thus ATP formation. If there is a lack of O2, this is achieved by the formation of lactate, which is released into the blood and is resynthesized into glucose in the liver (cori cycle) when the cell resumes aerobic metabolism.  Muscle-specific auxiliary reactions for ATP synthesis: These exist in order to provide additional ATP in case of emergency. 1.Creatine phosphate acts as a buffer for the ATP level-see below. 2.Another ATP-supplying reaction is catalysed by adenylate kinase. This disproportionates two molecules of ADP into ATP and AMP. The AMP is deaminated by AMP deaminase into IMP in a subsequent reaction in order to shift the balance of the reversible reaction in the direction of ATP formation.  B. creatine metabolism Creatine (N-methylguanidoacetic acid) and its phosphorylated form creatine phosphate (a guanidophosphate) serve as an ATP buffer in muscle metabolism. In creatine phosphate, the phosphate residue is at a similarly high chemical potential as in ATP and is therefore easily transferred to ADP. Conversely when there is an excess of ATP, creatine phosphate can arise from ATP and creatine (during the resting phase of contraction). Both processes are catalyzed by creatine kinase.  In resting muscle, creatine phosphate forms due to the high level of ATP. If there is a risk of a severe drop in the ATP level during contraction, the level can be maintained for a short time by synthesis of ATP from creatine phosphate and ADP. In a nonenzymatic reaction, small amounts of creatine and creatine phosphate cyclizes constantly to form creatinine which can no longer be phosphorylated and is therefore excreted with the urine.  Creatine does not derive from the muscles themselves, but is synthesized in two steps in the kidneys and liver. Initially, the guanidino group of arginine is transferred to glycine in the kidneys, yielding guanidino acetate. In the liver, N-methylation of guanidino acetate leads to the formation of creatine from this. The coenzyme in this reaction is S-adenosyl methionine (SAM). SYNTHESIS OF CREATINE AND CREATININE  MUSCLE METABOLISM 11 A. cori and alanine cycle White muscle fibers mainly obtain ATP from anaerobic glycolysis-i.e, they convert glucose into lactate. The lactate arising in muscle and, in smaller quantities, its precursor pyruvate are released into the blood and transported to the liver, where lactate and pyruvate are resynthesize into glucose again via gluconeogenesis, with ATP being consumed in the process.  The glucose newly formed by the liver returns via the blood to the muscles, where it can be used as an energy source again. This circulation system is called the cori cycle. There is also a very similar cycle for erythrocytes, which do not have mitochondria and therefore produce ATP by anaerobic glycolysis.  The muscle themselves are not capable of gluconeogenesis nor would this be useful, as gluconeogenesis requires much more ATP than is supplied by glycolysis. As 02 deficiencies do not arise in the liver even during intensive muscle work , there is always sufficient energy there, available for gluconeogenesis. There is also a corresponding cycle for the amino acid alanine. The alanine cycle in the liver not only provide alanine as a precursor for gluconeogenesis, but also transports the amino nitrogen arising in muscles during protein degradation to the liver. In the liver, it is incorporated into urea for excretion.  Most of the amino acids that arise in muscle during proteolysis are converted into glutamate and -keto acids by transamination. Again by transamination, glutamate and pyruvate give rise to -ketoglutarate and alanine, (which after glutamate/glutamine), is the second important form of transport for amino nitrogen in the blood. In the liver, alanine and ketoglutarate are resynthesized into pyruvate and glutamate. Glutamate supplies ammonia to the urea cycle, while pyruvate is available for gluconeogenesis.  B. protein and amino acid metabolism The skeletal muscle is the most important site for degradation of the branched-chain amino acids (val, leu, lle), but other amino acids are also broken down in the muscles. Alanine and glutamate are resynthesiszed from the components and released into the blood. They transport the nitrogen that arises during amino acid breakdown to the liver (alanine cycle) and to the kidneys (as glutamine).  During periods of hunger, muscle proteins serve as an energy reserve for the body. They are broken down into amino acids, which are transported to the liver. In the liver, carbon skeletons of the amino acids are converted into intermediates in the tricarboxylic acid cycle (or into acetoacetyl-CoA). These amphibolic metabolites are then available for energy metabolism and for gluconeogenesis. After prolonged starvation, the brain switches to using ketone bodies (from acetyl-CoA produced from fatty acids β-oxidation) in order to save muscle protein.  The synthesis and degradation of muscle proteins are regulated by hormones. Cortisol leads to muscle degradation, while testosterone stimulates protein formation. Synthetic anabolics with a testosterone-like effect have repeatedly been used for doping purposes or for intensive muscle-building. Note-In general for energy provision, red muscles use fatty acids; ketone bodies also serve as fuel especially for the heart muscles; white muscles use mainly glucose and in resting muscle, fatty acids are the major fuel.  OXYGEN DEBT In actively contracting skeletal muscle, the rate of glycolysis far exceeds that of TCA cycle. Much of the pyrurate formed under these conditions is reduced to lactate, which flows to the liver, where it is converted into glucose (via gluconeogenesis). These interchanges, known as the cori cycle shifts the metabolic burden of the muscle to the liver.  After a period of maximal muscular exertion, such as a sprint, during which lactate appears in the blood in large amount, an animal will continue to breath, in excess of the normal resting state and consumes considerable extra oxygen. The extra oxygen so consumed during the recovery period is called the “oxygen debt” and correspond to the oxidation of some or all the excess lactate formed during maximal muscular contraction.  Tetany and Rigor Mortis Tetany, a condition of hyper-contracted muscle that sometimes follows a prolonged period of repetitive, summed muscle stimulation, is caused by the depletion of ATP and other high-energy phosphates that help maintain normal ATP levels. The latter include other nucleoside triphosphates (NTPs), creatine phosphate (CP), and ADP, as illustrated in the three equations below. The three reactions are carried out by nucleoside diphosphokinase, creatine kinase and adenylate kinase, respectively. NTP + ADP → NDP + ATP CP + ADP → Creatine +ATP ADP + ADP → AMP + ATP  CONSEQUENCES OF TETANY Since tetanic stimulation raises sarcoplasmic calcium and depletes ATP, the end result is a highly contracted muscle with calcium bound to TnC and no ATP available to re- sequester calcium into the cisternae of the SR, nor to break actomyosin cross-bridges. Under these conditions, mitochondria will preferentially pump calcium into the mitochondrial matrix, ultimately removing calcium bound to TnC, obscuring myosin binding sites on thin filaments, and, allowing the muscle to assume a flaccid state. However, the absence of ATP results in myosin remaining in its low-energy conformational state, with the result that new cycles of muscle stimulation will result in only limited ability of the muscle to generate contractile activity. Muscles in this physiological state are said to be fatigued.  RIGOR MORTIS In death, all reactions tend towards equilibrium. Among the first of these processes is that of ion equilibration across all compartments of the body as ion pumps loose their energy supplies. In the case of muscle, this results in cisternal and extracellular calcium leaking into the sarcoplasm, raising calcium concentrations to high levels. The calcium induces conformational changes in the troponin-tropomyosin complex, exposing myosin binding sites on thin filaments. The resulting uncontrolled contractile activity hastens the total exhaustion of ATP supplies and ends with all or nearly all myosin molecules in cross-linked actomyosin complexes. The rigid state of muscles that develops shortly after death is due to this highly cross-linked state of thin and thick filaments and is known as rigor mortis.