Biochemistry of muscles Seminar No. 14 Thick filament is the myosin aggregate of cca 350 monomers Myosin monomer Describe the thin filament Thin filament – Actin • globular monomer (G-actin) makes a double helix (F-actin) • F-actin has other accessory proteins attached: • tropomyosin (smaller double helix) • troponin C – binds calcium ions • troponin I – inhibits interaction actin-myosin • troponin T – binds to tropomyosin and other troponins Q. 10 Q. 11 A. 11 The functions of ATP and calcium are antagonistic: • ATP – separates actin from myosin • Calcium ion – joins actin with myosin Rigor mortis Rigor mortis is a recognizable sign of death (L. mors, mortis, f.) that is caused by a chemical change in the muscles, causing the limbs of the corpse to become stiff (L. rigor, oris, m.) and difficult to move or manipulate. Assuming mild temperatures, rigor usually sets in about 3-4 hours after clinical death, with full rigor being in effect at about 12 hours. ATP supply from metabolic reactions is exhausted, the muscles remain contracted for ever. Red and white filaments Q. 16 Calcium concentrations in sarcoplasm Q. 17 Calcium concentrations in body fluids Q. 19 Events on neuromuscular junctions • junction consists from nerve terminal separated from postsynaptic region by the synaptic cleft • acetylcholine is released from presynaptic vesicles and binds to nicotinic receptors in muscle cell membrane Þ depolarization of membrane and T-tubules • T-tubules are connected with sarcoplastic reticulum (SR)  Ca^2+ ions are released from SR (where are associated with calsequestrin protein) • calcium ions then bind to troponin C  contraction Q. 20 Inhibitors of skeletal muscle contraction Skeletal muscle relaxants bind to nicotinic receptor, but are not hydrolyzed by acetylcholinesterase • Botulinum toxin is produced by bacterium Clostridium botulinum. The toxin is a two-chain polypeptide with a heavy chain joined by a disulphide bond to a light chain. • The light chain is a protease that attacks one of the fusion proteins at a neuromuscular junction, preventing vesicles from anchoring to the membrane to release acetylcholine. By inhibiting acetylcholine release, the toxin interferes with nerve impulses and causes paralysis of muscles (botulism). • no action potential is generated Þ permanent relaxation Medical uses of botulinum toxin • Currently, Botox (= trade name) is finding enormous potential in several therapeutic areas including the treatment of migraine headaches, cervical dystonia (a neuromuscular disorder involving the head and neck), blepharospasm (involuntary contraction of the eye muscles), and severe primary axillary hyperhidrosis (excessive sweating). • Other uses of botulinum toxin include urinary incontinence, anal fissure, spastic disorders associated with injury or disease of the central nervous system including trauma, stroke, multiple sclerosis, or cerebral palsy and focal dystonias affecting the limbs, face, jaw etc. Bungarotoxin is the antagonist of nicotinic receptor (blocks opening the Na^+/K^+ channel) Cardiac muscle: Three sources of calcium • Extracellular Ca^2+ (~ 10 %) enters by voltage operated channels (VOC) • This influx of calcium triggers the release of calcium ions from SR and mitochondria (~ 90 %) Cardiac muscles - Contraction ^• In sarcoplasm, Ca^2+ ions bind to:^ Cardiac muscles - Relaxation • Ca^2+ ions are liberated from troponin C and removed from sarcoplasm • there are four systems how to vanish Ca^2+ in sarcoplasm • Ca^2+-ATPase in SR • Ca^2+-ATPase in sarcolemma • Na^+/Ca^2+ antiport in sarcolemma • Ca^2+ re-entry to mitochondria ^ Autoregulation in cardiac muscle (scheme p. 4) • intracellular calcium is in the complex with protein calmodulin: CM-4Ca^2+ • Ca^2+-CM stimulates all Ca^2+-pumps (some by phosphorylation) which decrease the Ca^2+ concentration in sarcoplasm • the increase of intracellular [Ca^2+] triggers contraction but, at the same time, stimulates relaxation processes Q. 25 Modulatory effect of cAMP Modulatory effect of cAMP on cardiac muscles • cAMP is the second messenger produced after the activation of G[s]-protein-linked-receptors (β-adrenergic receptors) • such receptors are activated by catecholamines (nor/adrenaline) • cAMP activates protein kinase A • protein kinase A catalyzes the phosphorylation of: calciductin of VOC Þ influx of Ca^2+  contraction Ca^2+-ATPase in sarcolemma  eflux of Ca^2+  relaxation Ca^2+-ATPase in SR  eflux of Ca^2+  relaxation troponin I  conformation change - contact of actin-myosin  contraction Q. 26 Compare Chapter 9, p. 8 Metabolic background of MI • ischemia (lack of oxygen in tissues) leads to anaerobic metabolism Þ glucose is converted to lactate • lactate accumulates in ICF and alters intracellular environment  prolonged acidosis causes irreversible cell damage (necrosis) • permeability of cell membrane increases  cytoplasmatic/mitochondrial/contractile proteins are released into ECF • the best markers of MI are: myoglobin, CK-MB, cardial troponins (T or I) – this triple combination is recommended • LD isoforms are no longer used Smooth muscles - Contraction • source of Ca^2+: ECF (VOC, ROC), SR • there is no troponine C, but two other regulatory proteins binding calcium – calmodulin + caldesmon • calcium-calmodulin complex (Ca^2+-CM) activates MLCK (myosin light chain kinase) • activated MLCK catalyzes the phosphorylation of myosin • phosphorylated myosin is capable to make complex with actin Þ contraction Smooth muscles - Relaxation Two relaxing processes occur: • Removing intracellular Ca^2+ from ICF (like in cardiac m.) • MLC-phosphatase catalyzes the hydrolysis of phosphorylated myosin: MLC-P + H[2]O ® P[i] + MLC MLC does not bind to actin  relaxation The influence of cAMP on smooth muscles • cAMP activates protein kinase A (PK-A) • PK-A phosphorylates MLC-kinase: MLCK ® MLCK-P • MLCK-P is inactive, does not phosphorylates MLC  no interaction between actin and myosin  relaxation Compare: Influence of cAMP on muscles Q. 30 Different actions mediated through different adrenergic receptors Q. 32 A. 32 • nitric oxide (NO) is a relaxant of smooth muscles (e.g. arterial myocytes) [• ]activates guanylate cyclase in cytosol: GTP ® cGMP + PP[i] • cGMP activates protein kinase G (PK-G) • PK-G phosphorylates MLC-kinase: MLCK ® MLCK-P • MLCK-P is inactive, does not phosphorylate MLC  no interaction between actin and myosin  relaxation Q. 33 NO releasing compounds • Endogenous: L-arginine (the imino nitrogen of guanidine part) • Exogenous: organic nitrates = esters of nitric acid (R-O-NO[2]) organic nitrites = esters of nitrous acid (R-O-N=O) sodium nitroprusside = a complex of Fe^3+ with CN^- and NO NO originates from imino nitrogen of L-arginine Organic nitrates (alkyl nitrates) Organic nitrites (alkyl nitrites) Other NO releasing compounds Other metabolic pathways of NO Q. 34 ! Different actions of the same signal molecule Maximal intesity of muscle work • anaerobic phase • 30 sec – 2 min • muscles use glucose Þ metabolized to lactate • lactate goes to liver Þ substrate of gluconeogenesis • small portion of lactate becomes metabolic fuel for resting muscles and myocardium Prolonged muscle work/exercise • working muscles are adapted to aerobic metabolism of glucose and FA • resting muscles utilize FA and KB • glycerol from lipolysis is the substrate for liver gluconeogenesis Q. 35 A. 35 Q. 38 A. 38 • in the first 10 sec – ATP itself and creatine phosphate currently present in muscle cell • After 30 sec – mainly anaerobic glycolysis glucose ® 2 lactate + 2 ATP • After 10 min – aerobic oxidation of glucose glucose ® 2 pyruvate  2 acetyl-CoA  38 ATP • After 2 hours – aerobic oxidation of FA stearic acid  9 acetyl-CoA  146 ATP palmitic acid  8 acetyl-CoA  129 ATP Credit test (30 Q / 35 min) • all seminar chapters • all practical chapters • reference values: YES • calculations: NO