Muscle Contraction Lab Report

Muscle adaptations to the increase in energy demands at the start of exercise

Introduction

The transition from rest to exercise is associated with a huge upsurge in energy expenditure, due primarily to skeletal muscle contractions (Connett & Sahlin, 1996). Contractions require energy in the form of adenosine tri-phosphate (ATP). ATP stores in muscle are around 8mmol/l and are exhausted within 2s of exercise (Connett & Sahlin, 1996). To continue exercise and maintain ATP homeostasis, ATP production must increase rapidly. The adaptations that occur are tailored to suit the energy requirements of the exercise, therefore the adaptations during marathon running are different to those seen during sprinting. Breakdown of phosphocreatine
(PC) is the first adaptation to increased energy demand and has been called the alactic energy system because it does not result in lactate formation. As this fails the process of glycolysis is turned on and glucose utilization begins. Anaerobic respiration at the onset of exercise was traditionally attributed to insufficient oxygen supply to muscle. However the finding that fully oxygenated muscles still utilize glycolysis at the onset of exercise put this theory out of contention (Conley et al, 1998). Today the mechanisms by which glycolysis are activated are better understood and a review of current understandings will ensue. Fat oxidation is also increased during exercise and adaptations leading to this also need attention.
Muscle Contraction
Muscle contraction is the result of a complex chain of command stemming from the motor cortex in the brain (Connett & Sahlin, 1996).
In the muscle fibre the immediate steps leading to contraction involve action potentials and calcium. T tubules conduct action potentials into the interior of the fibre. Dihydropyridine (DHP) receptors on the T tubule membrane release calcium when an action potential arrives. This stimulates the Ryanodine receptors (RyR) on the sacroplasmic reticulum to release larger amounts of calcium. Troponin is wrapped around actin and prevents myosin from binding to it. Calcium diffuses into the myofibrils where it binds to troponin causing a conformational shape change, revealing the myosin-binding site and facilitating myosin-actin interaction (Astrand & Rodahl, 2003).
The bound myosin head requires ATP in order to detach from actin. Once detached the myosin head hydrolyses ATP and the products adenosine diphosphate (ADP) and inorganic phosphate (Pi) remain in the myosin head. Hydrolysis of ATP initiates a change in the shape of the myosin head promoting myosin-actin interaction. Once rebound the myosin releases Pi, causing the head to swivel and drawing the actin in. At the end of its range the myosin head releases ADP and ATP is once again needed in order for myosin to detach from actin.
Released ADP can be used to create small amounts of adenosine mononphosphate (AMP), which is an important signal transducer(Astrand & Rodahl, 2003). The enzyme adenylate
cyclase catalyses the reaction:
2ADP ATP + AMP
Thus resynthesizing ATP and helping to maintain cellular homeostasis. A second mechanism is:
ADP AMP + Pi
Here splitting the high energy phosphate bond in ADP releases energy that can be used in cellular processes. During muscle contraction there are hence elevated levels of CA2+, ADP, AMP and Pi.
Creatine Kinase action
ATP is rapidly resynthesized by the creatine kinase enzyme. This converts:
ADP + CP + H+ ATP + C
As indicated by the arrow this reaction goes both ways and it is the high ADP created by contractions that drives this reaction towards ATP production (Nelson & Cox, 2000). Furthermore this reaction requires hyrdrogen ions which become available during ATP hydrolysis. Concentration of CP in the muscle is roughly 3-4 times greater than the ATP stores. However this is still relatively minute compared to glycogen stores. Thus CP is rapidly used up (within 10s) and glycolysis must increase to maintain ATP concentrations.
The rise in ADP and fall in pH facilitate the use of CP as a fuel.

Activation of muscle glycogen breakdown

Glycogen storage avoids the predicament of a large osmotic pressure that would be created by a similar amount of stored free glucose (Connett & Sahlin, 1996). The highly branched structure includes both α1-4 and α1-6 linkages, this provides high substrate exposure and facilitates rapid metabolism. Glycolytic flux at rest is extremely small, this stands to reason since ATP requirements are in skeletal muscle are equally small. As CP levels are depleted glycolytic flux must increase in order to maintain ATP levels and cellular homeostasis. This requires an increased flux in the three steps glycogenolysis, glycolysis and mitochondrial oxidation of pyruvate. Flux through all three steps must increase for total glycolytic flux to increase.
Glycogenolysis
The process is activated by the accumulation of Pi, which binds to glycogen and enables it to interact with glycogen phosphorylase (GP), the primary enzyme in glycogenolysis (Connett & Sahlin, 1996). This enzyme splits α1-4 bonds cleaving glucose 1 phosphate from the glycogen molecule. Debranching enzyme facilitates this action by breaking down α1-6 bonds, which untangles glycogen and gives GP greater access.
GP activity is controlled via two separate mechanisms; 1. Conversion between active and inactive forms 2. Allosteric regulation

GP conversion

The convesion between forms is controlled by the counterbalanced roles of the active phosphoylase kinase (PKa) and the phosphoprotein phosphatise (see below).
Adrenaline plays an important role in this through β-adrenergic stimulation resulting in increased cAMP (see below). Adrenaline is released via two mechanisms. Before exercise the anticipated exertion results in high sympathetic activity. When action potentials from the sympathetic pathway reach the adrenal medulla they cause it to release adrenaline into the blood (Connett & Sahlin, 1996). The second mechanism occurs in high intensity exercise such as a 100m sprint. Here the accumulation of potassium (k) and the low pH stimulates type 3 afferents which increase adrenaline secretion via the adrenal medulla (Astrand and Rodahl,
2003).
[pic]
Fig-1 – schematic showing how adrenaline leads to increased cAMP
Increased cAMP activates a cAMP dependant protein kinase which then phosphorylates PKb converting it to PKa. PKa uses ATP to phosphorylase inactive GP (GPb) making it active GP (GPa). Phosphoprotein phosphatase (PP) removes the phosphate from both GPa and Pka returning both to there inactive forms (Crowther et al, 2002).
A second action of the cAMP dependant protein kinase is the phosphorylation of one site on the PP resulting in its inactivity. The duel action of adrenaline in both activating the kinase and inhibiting the phosphatase is thought to be responsible for the huge glycogen utilisation during high intensity exercise (Nelson & Cox, 2000).

[pic]
Fig2- Overview of phoshorylase regulation created using
Allosteric activation
In low intensity exercise for instance marathon running, adrenaline release is lower due to less K accumulation. In exercise such as this the allosteric mechanisms of activating GP become increasingly important. Allosteric activation is the binding of a ligand to a site other than the active site causing a conformational change in shape. This change facilitates interaction between a substrate molecule and the active site and thus increases the rate of the reaction (Nelson & Cox, 2000). NMR and X ray crystallagrophy revealed that adenosine monophosphate (AMP) has a specific binding site on GP and can activate both the GPb and GPa (Nelson & Cox, 2000). When adrenaline is high very little GP is found in the GPb form, conversely the low adrenaline during a marathon means some GPb is present. Accumulation of enough AMP has been seen to override its inactive status and enable GPb to breakdown glycogen. GPa activity although independent of AMP can be augmented by low concentrations of AMP (