Contractile proteins for BDS students. MSK system

Musculoskeletal System

1. Muscle C o ntraction, C o ntractile Proteins, Fuels for Skeletal Muscle Dr. Apeksha Niraula Assistant Professor C l inical Biochemistry Institute of Medicine TUTH Maharajgunj

2. Objectives C o ntractile Proteins Mechanism of Muscle Contraction Fuels for Skeletal Muscle Purine Nucleotide Cycle

3. Movement is an important property of life, especially of the members of the animal kingdom The organism may move as a whole (walking) or movement of cells may occur (diapedesis or sperm movement) or it may occur at the subcellular level (transfer and exocytosis of secretory proteins) The important contractile proteins are Actin and Myosin in muscles Beating of cilia or sperm is achieved by Tubulin and Dynein Tubulin, actin, microfilaments, kinesin, and intermediate filaments  are involved in the movement of secretory granules from their site of production to their release Introduction

4. Striated muscle is made up of multinucleated cells bound by a plasma membrane called Sarcolemma The sarcomere is the functional unit of the muscle Each muscle cell contains Myofibrils about 1 mm in diameter The myofibrils are immersed in a cytosol that is rich in glycogen, ATP, creatine phosphate, and glycolytic enzymes The functional unit of a myofibril is a sarcomere The dark A bands and light I bands alternate regularly MUSCLE PROTEINS

5. These bands are formed by a variable combination of thick and thin filaments The thick filaments have a diameter of about 150 Å whereas thin filaments have a diameter of about 70 Å The thick filament is primarily Myosin and the thin filament contains actin, tropomyosin, and troponin

6. The Z line contains 2 actin molecules and the M protein is located in the M line Thick and thin filaments slide past each other during the muscle contraction, so that the muscle shortens by as much as a third of its original length However, the lengths of the thick and thin filaments do not change during muscle contraction

7. A-band: Dark band seen as part of striation (Anisotropic band) I-band: Light band area between the ends of the Myosin (Isotropic Band) Z-Line: Membrane that separates Sarcomeres H-Zone: Area by which the two ends of the thin filaments fail to meet Sarcomere: Functional unit composed of two Myofilaments Thick Filaments (Myosin) Thin Filaments (Actin-Troponin- Tropomyosin)

8. Myosin has 3 different biological activities: a . Myosin molecules assemble into filaments b. Myosin acts as the enzyme ATPase c. Myosin binds to actin polymer which is the major component of the thin filaments Myosin molecules are large (about 540 kD), each with 6 polypeptide chains; 2 identical heavy chains and 4 light chains The myosin molecule has a double-headed globular end Myosin

9. Trypsin cleaves myosin into 2 parts; light meromyosin (LMM) and heavy meromyosin (HMM) types LMM can form filaments but has no enzymatic activity HMM has enzymatic activity and binds actin, but cannot form filaments HMM can further be split into the S1 fragments having the ATPase site plus the actin-binding site and the S2 subfragment

10. Major protein of the thin filaments Monomeric protein often referred to as G-actin due to its globular shape It can polymerize into a fibrous form, called F-actin , which is a helix of actin monomer Actin Filament: Composed of three protein components; major protein of the thin filaments Actin Tropomyosin Troponin The muscle contraction results from interaction of actin and myosin, to form actomyosin, with energy provided by ATP When the two thin filaments that bind the cross bridges of a thick filament are drawn towards each other, the distance between Z lines becomes shorter Actin

11. This could result in the process of contraction of muscle fibers This needs energy from the hydrolysis of ATP, affected by the ATPase activity of myosin The contractile force is generated by conformational changes, leading to the dissociation of actin and S1 heads of myosin There is a reversible attachment and detachment of myosin S1 head to actin

12. Actin participates in many important cellular processes : Muscle C o ntraction Cell Motility Cell Division Cytokinesis Vesicle and Organelle movement Cell Signalling Maintenance of cell junctions and cell shape

13. The muscle contraction is modulated by troponin and tropomyosin through Ca++ which is the physiological regulator of muscle contraction In the resting muscle, the Ca ++ is within the sarcoplasmic reticulum The nerve impulse releases Ca++ from the sarcoplasmic stores and increases its cytosolic concentration about 10 times (1 mM to 10 mM) The action of calcium is brought about by 2 proteins, troponin complex and tropomyosin located in the thin filament Troponins

14. The troponin complex has 3 different polypeptide chains Troponin-C (TnC, 18 kD) binds calcium Troponin-I (TnI, 21 kD) , otherwise called “Actomyosin-ATPase inhibitory element”, binds to actin and inhibits the binding of actin to myosin Troponin I is a marker for myocardial infarction Its level in serum is increased within 4 hours of myocardial infarction and remains high for about 7 days It is about 75% sensitive index for myocardial infarction

15. TnC has calmodulin-like properties In the resting muscle, only the high-affinity sites are occupied by Calcium, but when Ca ++ is released from the sarcoplasmic reticulum, low-affinity sites are also occupied by Ca ++ This results in a conformational change that is transmitted to tropomyosin This shift in the position of tropomyosin alters the binding of actin to S1 The events may be depicted as: Ca ++ → Troponin→ Tropomyosin→ Actin→ Myosin

16. Troponin-T (TnT, 37 kD) binds to tropomyosin Two isoforms of cardiac TnT, called TnT1 and TnT2 are present in adult human cardiac tissue Serum levels of TnT2 increases within 4 hours of myocardial infarction, and remains high for up to 14 days

17. The amount of ATP in muscle is sufficient to sustain contractile activity for less than one second The reservoir of high energy phosphate in skeletal muscle is Creatine phosphate The reaction (Lohman’s reaction) is catalysed by Creatine Kinase (CK) CK Creatine phosphate + ADP -------→ ATP + Creatine Transduction of Chemical Energy to Mechanical Energy

18. The ∆Go’of creatine phosphate is -10.3 kilocalories per mol, whereas that for ATP is only -7.3 kilocalories Resting muscle has a high concentration of the creatine phosphate (25 mM) when compared to ATP (4 mM) The creatine phosphate provide a high ATP concentration during muscle contraction ( In athletes, it is the major source of energy during the first 4 seconds of a short sprint )

19. During muscle contraction, the ATP level remains high as long as creatine phosphate is present But following contractile activity, the levels of ADP and Pi rise The reduced energy charge of active muscle stimulates glycogen breakdown, glycolysis, TCA cycle and oxidative phosphorylation The red striated muscle has an active aerobic metabolism compared to white muscle

20. Process of Muscle Excitation Muscle: Excitable Tissue When Stimulates shows response: Electrical Response : Production of Action Potential Mechanical Response: Contraction

21. Process of Excitation- Contraction Coupling When End Plate Potential reaches threshold level It produces action potential Which propagates over muscle fibre and through it along transverse(T-tubules) Action Potential initiated in plasma membrane spread to surface and into muscle fibre through T tubules When reached the tip of T tubules activates the voltage gated Dihydropyridine receptors

22. Activated Dihydropyridine receptors triggers the opening of calcium release channels on terminal cisterns ( Ryanodine receptors) Calcium diffuses into cytoplasm and ICF Ca increases (upto 2000 fold) Calcium ion gets attached to Troponin C and starts chain of events Hence, Calcium acts as a link between excitation and contraction process

23. Sarcoplasmic reticulum (SR) regulates intracellular levels of calcium in skeletal muscle In the resting state, calcium ions are pumped into the SR through Ca-ATPase Inside the SR, calcium ions are bound with a specific calcium-binding protein, called Calsequestrin When nerve impulse excites the sarcolemma, the calcium channel is opened, calcium ions are released from SR into the sarcoplasm Calcium and Muscle Contraction

24. The calcium ion concentration in the cytoplasm is increased The calcium binding sites of TnC are now saturated with calcium The TnC-4Ca++ complex attaches with TnI and TnT, which then interacts with tropomyosin

25. Fuels for Skeletal Muscle ATP: Currency Immediate source: Phosphocreatine Ultimate sources: Carbohydrate, Fats and Proteins ATP Regeneration Dephosphorylation of Creatine Phosphate Anaerobic Glycolysis Aerobic Oxidation of glucose and fatty acids

26. Purine Nucleotide Cycle Purine nucleotide cycle  is a metabolic pathway in which ammonia and fumarate are generated from aspartate and inosine monophosphate (IMP) Functions: T o regulate the levels of adenine nucleotides To facilitate the liberation of ammonia from amino acids This cycle occurs in skeletal muscle (Cytosol) Purine nucleotide cycle occurs during strenuous exercise, fasting or starvation when ATP reservoirs run low

27. The cycle comprises three enzyme catalyzed reactions: First stage: Deamination of the purine  nucleotide   adenosine monophosphate  (AMP) to form  inosine monophosphate  (IMP) , catalyzed by the enzyme  AMP deaminase : AMP + H 2 O + H +  → IMP + NH 3 S econd stage is the formation of  adenylosuccinate  from IMP and the amino acid  aspartate , which is coupled to the energetically favorable hydrolysis of  GTP , and catalyzed by the enzyme  adenylosuccinate synthetase : Aspartate + IMP + GTP → Adenylosuccinate +  GDP  +  P i

28. Finally, A denylosuccinate is cleaved by the enzyme  A denylosuccinate L yase   to release fumarate and regenerate the starting material of AMP: Adenylosuccinate → AMP + Fumarate A recent study showed that activation of HIF-1 α allows cardiomyocytes to sustain mitochondrial membrane potential during anoxic stress by utilizing fumarate produced by adenylosuccinate lyase as an alternate terminal electron acceptor in place of oxygen: Mechanism should help provide protection in the ischemic heart

29. Thank You