Browsing by Subject "skeletal"
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Item Pyruvate-Enriched Ringer's Solution Protects Hindlimb and Myocardial Tissue During Hemorrhagic Shock and Hindlimb Ischemia(2011-07-22) Gurji, Hunaid Adam; Mallet, Robert T.; Olivencia-Yurvati, Albert; Raven, Peter B.Gurji, HA. Pyruvate-Enriched Ringer’s Solution Protects Hindlimb and Myocardial Tissue During Hemorrhagic Shock and Hindlimb Ischemia. Doctor of Philosophy (Integrative Physiology), July 22, 2011, 111 pp, 1 table, 23 figures, 209 references, 142 titles. Copious blood loss is the leading cause of death in military combat. Extreme exsanguination following traumatic injury causes hypotension which may culminate in hemorrhagic shock, multiple open organ failure, and death. Currently, the only available strategy to treat hemorrhage is to apply tourniquets and administer resuscitative fluids. Although necessary to limit blood loss, protracted tourniquet application imposes ischemia on distal tissues. Revascularization of the injured limb reintroduces oxygenated blood into the ischemic zone, forming toxic reactive oxygen species. These highly reactive compounds can inactivate key metabolic enzymes, hamper ATP production, and cause end organ dysfunction. Fluid resuscitation provides crucial hemodynamic support, and affords an opportunity to treat the deleterious effects of hemorrhagic shock and ischemia-reperfusion. In order to mitigate the harmful effects of hemorrhagic shock and ischemia-reperfusion of tourniqueted extremities, a fluid resuscitant should contain agents capable of suppressing the formation of reactive oxygen and nitrogen species, thus protecting cellular metabolic function; stabilizing tissue energetics; and safeguarding end organic function. Pyruvate, an endogenous energy substrate, possesses strong antioxidative properties. This study tested whether substituting pyruvate for lactate in a Ringer’s solution would be effective at mitigating reactive oxygen species formation, protect key ATP-generating and ATP-shuttling enzymes from inactivation, bolster skeletal and cardiac muscle phosphorylation potentials, and stabilize cardiac electrical function in goats subjected to hemorrhagic shock and hindlimb ischemia-reperfusion. Isoflurane-anesthetize goats were subjected to a controlled hemorrhaged to reduce the mean arterial pressure to c. 50 mmHg. After reaching this target pressure, hindlimb ischemia (HLI) was imposed for a total of 90 min by femoral artery crossclamp and tourniquet application around the hindlimb. After 30 min of hindlimb ischemia, pyruvate- (PR) or lactate- enriched (LR) Ringer’s solution was infused intravenously (10mL/min) for 90 min. Time control (TC) goats were neither hemorrhaged nor subjected to hindlimb ischemia. At the conclusion of- and 3.5 h after- fluid resuscitation, the left ventricle and the right gastrocnemius were biopsied and flash-frozen for biochemical analysis of metabolites, enzymes, and markers of oxidative stress. In addition, custom-written software was developed to analyze QT interval variability- a marker of electrical instability- from the lead II electrocardiogram. The first phase of this project tested the hypothesis that resuscitation with PR vs. LR effectively protects cardiac metabolism and preserves cardiac electrical performance during hemorrhagic shock and hindlimb ischemia. Resuscitation with PR effectively suppressed the formation of myocardial tissue 8-isoprostane vs. goats resuscitated with LR during the acute and subacute phases of the protocol. In addition, myocardial creatine kinase (CK) activity fell after LR administration vs. TC; however, PR preserved CK activity better than LR during fluid resuscitation and 4 h after hindlimb ischemia reperfusion. PR administration augmented myocardial phosphocreatine phosphorylation potential during fluid administration and 3.5 h later to values significantly higher than those in LR-resuscitated goats. Pro-arrhythmic QTc variability was markedly increased in LR vs. PR and TC during both phases of the protocol. The second phase of this project tested the hypothesis that resuscitation with PR preserves tissue energetics in the reperfused gastrocnemius during hemorrhagic shock and hindlimb ischemia. Resuscitation with PR vs. LR effectively protected the gastrocnemius from oxidative stress in both protocols, as evidenced by the suppression of 8-isoprostane formation. PR prevented CK and aconitase inactivation vs. LR during the acute phase of reperfusion, and this enzyme protection persisted at least 3.5 h after completing fluid resuscitation. Additionally, PR augmented muscle phosphocreatine phosphorylation potential vs. TC and LR during the acute phase of reperfusion, and, like CK and aconitase activities, this augmented energy state persisted 3.5 h after the end of fluid resuscitation. We conclude that 1) Pyruvate Ringer’s resuscitation during hemorrhagic shock and hindlimb ischemia provides antioxidative protection in skeletal and cardiac muscle during fluid resuscitation; 2) Pyruvate-fortified fluid resuscitation prevents inactivation of enzymes involved in production and shuttling of ATP; 3) PR augments cardiac and muscle phosphorylation potentials during fluid resuscitation; and 4) Resuscitation with PR effectively protects cardiac electrical rhythm in the face of hemorrhagic shock and hindlimb ischemia. These investigations demonstrate the powerful antioxidative protection imposed by pyruvate, its positive effects on muscle and cardiac metabolism and energy state and its role in stabilizing cardiac electrical function during hemorrhagic shock and hindlimb ischemia.Item Studies in Molecular Mechanisms of Skeletal Muscle Contraction: Applications to Transgenic Mice with Inherited Cardiomyopathies(2013-05-01) Midde, Krishna; Julian BorejdoMuscular contraction impacts virtually every physiological function in a human body. Nearly 50% of the body weight is contributed by muscle. Skeletal muscle contraction is responsible for performing every day general functions such as moving, grabbing things and lifting weights. Cardiac muscle contraction is responsible for blood circulation in the body and smooth muscle contraction is involved in the contraction of hollow organs such as lungs, stomach and kidneys. It also maintains body temperature. Central to these events is the transformation of energy derived by hydrolysis of ATP to mechanical force. The force generating steps of muscle contraction is thought to result from the interaction of actin and myosin proteins. Although much of the general knowledge of the mechanism of contraction has been known for over 50 years, emerging advanced techniques have identified some of the key intermediate steps and regulating parameters. My doctoral research involves utilizing one such high resolution technique – single molecule fluorescence spectroscopy. I have used it to discern the motion and conformation of myosin cross-bridges in ex-vivo muscle. When studying the dynamic behavior of actin and myosin it is essential to reduce the number of molecules under observation because 1. The local concentration of actin and myosin is dense and the information obtained by averaging trillions of molecules doesn’t reflect the true character of the process. 2. The trajectory of an enzyme catalyzed reaction cannot be followed and the associated kinetics of actomyosin interaction is lost. 3. The situation becomes worse when studying mutations in sarcomeric proteins with low expression. 4. Heterogeneity between neighboring sarcomeres can be reduced. An important goal of muscle research is to measure the rate of the power stroke. Therefore, part of my thesis is focused on characterizing the pre- and post- power stroke states of muscle contraction. While the extent of force generated during muscle contraction is proportional to the extent of Ca2+ released into the muscle, some of the recent studies have shown that the phosphorylation of the regulatory light chain (RLC) of myosin also modulates contraction. Considering the importance of the phosphorylation of RLC, I investigated the distribution of orientation and kinetics of myosin cross-bridges in phosphorylated and de-phosphorylated muscle. Lastly, I have applied our fluorescence polarization technique with single molecule sensitivity to unravel the deranged contractile properties of muscle in people afflicted with Familial Hypertrophic Cardiomyopathy (FHC) disease. Some of the significant conclusions drawn from my project include evidence for the existence of distinct pre- and post- power stroke states of myosin cross-bridges during contraction in Ex Vivo muscle, Regulatory Light Chain phosphorylation disturbs cross-bridge organization and enhances the power stroke state of contraction and FHC induced by mutations in Troponin-T protein impairs myosin cross-bridge interaction with actin and alters cross-bridge kinetics. Clinically, drugs can be developed to modulate power stroke and enhance muscle performance in myopathies. Site targeted small molecules (peptides) can now be screened to correct for hypo-contractile or hyper-contractile properties associated with FHC. Our technique may also serve as a diagnosis tool for early identification of FHC disease. Finally increasing the basal ATPase activity of resting muscle by RLC phosphorylation is of therapeutic importance in treating individuals with Obesity, Type II Diabetes and Metabolic syndrome.