<p> We hypothesized that acute inhibition of the contraction-induced expansion of the muscle TCA cycle intermediate (TCAI) pool via would not adversely effect metabolism or contractile function. Forty rats were anaesthetized and the gastrocnemius muscle (GAS) from one leg was vascularly isolated and perfused with saline (CON) or a red cell media containing DL-cycloserine (CYCLO; Sigma C-7005; dose=0.05 mg/g), an inhibitor of alanine aminotransferase (AAT). After 1h of perfusion, the GAS muscle was either snap frozen (CON-Rest, n=11; CYCLO-Rest, n=9) or stimulated to contract for 10 min (1Hz, 0.3 ms, 2 V) with blood flow fixed at 30 ml min-1 100g-1 and then snap frozen (CON-Stim, n=10; CYCLO-Stim, n=10). The maximal activity of AAT was lower (P≤0.05) at both CYCLO-Rest (0.61±0.02 mmol·kg-1w.w./min; mean± SEM) and CYCLO-Stim (0.63±0.01 mmol·kg-1w.w./min) vs CON-Rest (3.56±0.16 mmol·kg-1w.w./min) and CON-Stim (3.92±0.29 mmol·kg-1w.w./min). Consistent with lower net flux through AAT, muscle [alanine] was lower (P≤0.05) after CYCLO-Stim (6.97±0.26 mmol·kg-1 dw) compared to CON-Stim (8.55±0.56 mmol·kg-1 dw) and not different vs CON-Rest (6.79±0.41 mmol·kg-1 dw). The sum of five measured TCAI (malate, fumarate, citrate, isocitrate, and 2-oxoglutarate) was higher (P≤0.05) at both CON-Rest (2.10± 0.09 mmol·kg-1 dw) and CON-Stim (2.48± 0.11 mmol·kg-1 dw) vs CYCLO-Rest (1.56± 0.11 mmol·kg-1 dw) and CYCLO-Stim (1.88± 0.15 mmol·kg-1 dw) respectively. Despite the reduction in [TCAI] following CYCLO treatment, there was no difference between conditions in muscle lactate accumulation or phosphocreatine degradation after 10 min of stimulation. Contractile function was not different (P≤0.05) between conditions at either rest or stimulation and the decline in force production over ten minutes of stimulation was identical (~60%) between CON-Stim and CYCLO-Stim respectively. We conclude that flux through AAT was reduced after cycloserine treatment, however the acute inhibition of TCAI expansion did not compromise aerobic energy provision. These data support the hypothesis that the contraction-induced increase in muscle [TCAI] is not causally linked to oxidative energy delivery.</p> / Thesis / Master of Science (MSc)

This study investigated the effect of the mouse estrous cycle on myocyte contractile function. Female mice displayed irregular estrous cycles unless induced to cycle though exposure to bedding collected from cages housing male mice. Fractional shortening and Ca2+ transient amplitudes were significantly larger in myocytes isolated from mice in estrus. The effect of the estrous cycle was preserved even when cells were paced at a more physiological frequency and in the presence of ?-adrenergic stimulation. Myofilament Ca2+ sensitivity was also modified by the estrous cycle, as myofilaments isolated from the hearts of mice in estrus were least sensitive to Ca2+. However, acute application of either 17?-estradiol or the G protein-coupled estrogen receptor (GPER) agonist, G-1, had no effect on contractions or Ca2+ transients, regardless of the estrous stage. Thus, physiological fluctuations in sex hormone levels modify myocyte contractions, Ca2+ release, and myofilament Ca2+ sensitivity.

Die vorliegende Untersuchung sollte die kontraktile Funktion von humanen Vorhoftrabekeln und linksventrikulären Papillarmuskeln der Ratte während Hypoxie / Reoxygenierung als Hauptkomponenten von Ischämie / Reperfusion charakterisieren. Weitere Merkmale der Ischämie wurden durch erhöhte extrazelluläre K+-Konzentration und Azidose simuliert. Einblicke in die zelluläre Ca2+-Regulation ergaben sich aus Aktionspotential-(AP)-messungen, der SR- Ca2+-ATPase-Aktivität und Kraft-Intervall- Beziehungen. Die Rolle des Energiestoffwechsels und der endogenen antioxidativen Kapazität für die kontraktile Funktion von infarktbedingt hypertrophiertem Rattenmyokard während Hypoxie / Reoxygenierung ist durch Messung der Kreatinkinase-(CK)-Aktivität, ihrer Isoenzymverteilung und der Aktivitäten von Superoxiddismutase (SOD) und Glutathionperoxidase (GSH-Px) charakterisiert worden. Der Einsatz der Radikalfänger Histidin und Butylhydroxytoluen während Hypoxie und schneller Reoxygenierung an Rattenpapillarmuskeln sollte zur Protektion gegen den toxischen Effekt unterschiedlicher reaktiver Sauerstoffspezies dienen. In den durchgeführten Experimenten zeigte sich eine geringere Empfindlichkeit des humanen Vorhofmyokards gegenüber reduzierter O2-Versorgung und Reoxygenierung als im Rattenmyokard. Die während simulierter Ischämie im humanen Myokard auftretende Azidose hat einen günstigen Effekt auf die Wiederherstellung der isometrischen Kontraktionskraft nach Reoxygenierung, was jedoch mit einer gestörten Regulation der kontraktilen Funktion verbunden ist. Hypertrophiertes Myokard in der chronischen Phase nach Infarkt zeigt eine verminderte Empfindlichkeit gegenüber Hypoxie / Reoxygenierung, was auf adaptive Veränderungen im Energiestoffwechsel (erhöhte CK-MB und CK-BB Isoenzyme mit kleinerem Km-Wert für Kreatinphosphat), in der endogenen antioxidativen Kapazität (Erhöhung der Aktivitäten von SOD und GSH-Px um 40% bzw. 50%) und in der Regulation der kontraktilen Funktion (verminderte SR Ca2+-ATPase-Aktivität und Isomyosinverschiebung von V1 nach V3) zurückgeführt werden kann. Eine bessere Erholung der kontraktilen Funktion nach Reoxygenierung kann durch schnellen pO2- Wiederanstieg erreicht werden. Der Einsatz von Pharmaka mit verschiedenen Angriffspunkten im Radikalstoffwechsel und besonders deren Kombination während Hypoxie / Reoxygenierung ermöglicht zusätzlich eine verbesserte Kardioprotektion. / This study characterizes the contractile function of human atrial trabeculae and rat left ventricular papillary muscles during hypoxia / reoxygenation as the major components of ischemia / reperfusion. Further characteristics of ischemia were simulated by increased extracellular K+ concentration and acidosis during hypoxia. Insights into the cellular Ca2+ regulation were obtained from action potential recordings, from measurements of sarcoplasmic reticulum (SR) Ca2+ transport, and from force-interval relations. We examined changes in SR calcium transport, creatine kinase (CK) system, the antioxidant enzymes glutathionperoxidase (GSH-Px) and superoxiddismutase (SOD) 6 wks. after infarction (MI) due to coronary ligation in rats. Phenotypic modifications vs. sham operation (SHAM) were related to the contractile response of hypertrophied papillary muscle to hypoxia / reoxygenation. The oxygen radical scavengers histidine and butylhydroxytoluene were applied during hypoxia and rapid reoxygenation to protect the myocardium against oxygen radical damage. Generally, human atrial trabeculae were less sensitive to reduced oxygen supply and reoxygenation when compared to rat papillary muscles. In human atrial trabeculae, isometric peak force development recovered better after simulated ischemia than after hypoxia but the regulation of contractile function was clearly disturbed. In rat papillary muscles, rapid reoxygenation caused a better recovery of contractile function after hypoxia. Application of the oxygen radical scavengers histidine, butylhydroxytoluene, and especially their combination during hypoxia / reoxygenation had additional cardioprotective effects. In MI vs. SHAM we observed under aerobic control conditions: decreses in isometric contraction and relaxation rate, a reduced Vmax-equivalent of sarcomeric shortening, a faster twitch-to- twitch decay of post-rest potentiation (PRC) which correlated closely to the decrease in SR Ca2+ uptake (-25%), a decrease in CK activity (-20%), reduced CK-MI and CK-MM, increased CK-MB and CK-BB, and enhanced activities of SOD (+40%) and GSH-Px (+50%). During hypoxia, an initial increase in peak force (PF) was followed by a slower PF decline in MI vs. SHAM. During reoxygenation, rates of contraction and relaxation recovered better in MI. In SHAM but not MI, twitch-to-twitch decay of PRC was accelerated after reoxygenation vs. aerobic control. The results suggest that adaptive changes in SR Ca2+ handling, CK isoenzymes, and antioxidant enzymes may contribute to higher resistance against reduced O2 supply and reoxygenation in hypertrophy due to MI.

Avaliou-se os efeitos dos ácidos linoleico e oleico na regeneração do músculo gastrocnêmio lacerado em ratos e em mioblastos, miotubos e fibroblastos em cultura. Houve regeneração incompleta e recuperação parcial da função contrátil do músculo lesionado. O ácido linoleico diminuiu a massa, a atividade contrátil, a área de secção tranversa das fibras, aumentou tecido fibroso no músculo lesionado e elevou a expressão de PCNA, colágeno e fibronectina nos fibroblastos. O ácido oleico aboliu as alterações na atividade contrátil e o aumento de tecido fibroso e elevou a expressão de MyoD em mioblastos e desmina em miotubos e reduziu de PCNA, colágeno e fibronectina em fibroblastos. O ácido linoleico comprometeu a regeneração enquanto que o oleico otimizou a capacidade regenerativa e a função contrátil do músculo lesionado. / The effects of linoleic and oleic acids on regeneration of lacerated gastrocnemius muscle in rats and on cultured myoblasts, myotubes and fibroblasts were examined. There was incomplete regeneration and partial recovery of the contractile function of the injured muscle. Linoleic acid reduced the mass, contratile activity and cross-sectional area of the fibers, raised the fibrous area and reduced the expression of PCNA, collagen and fibronectin in fibroblasts. Oleic acid abolished the changes in contratile activity and the increase in the fibrous area, raised the expressions of MyoD in myoblasts, desmin in myotubes and inhibited the expressions of PCNA, collagen and fibronectin in fibroblasts. Linoleic acid impaired regeneration whereas oleic acid optimized the regenerative capacity and contractile function of the injured muscle.

Avaliou-se os efeitos dos ácidos linoleico e oleico na regeneração do músculo gastrocnêmio lacerado em ratos e em mioblastos, miotubos e fibroblastos em cultura. Houve regeneração incompleta e recuperação parcial da função contrátil do músculo lesionado. O ácido linoleico diminuiu a massa, a atividade contrátil, a área de secção tranversa das fibras, aumentou tecido fibroso no músculo lesionado e elevou a expressão de PCNA, colágeno e fibronectina nos fibroblastos. O ácido oleico aboliu as alterações na atividade contrátil e o aumento de tecido fibroso e elevou a expressão de MyoD em mioblastos e desmina em miotubos e reduziu de PCNA, colágeno e fibronectina em fibroblastos. O ácido linoleico comprometeu a regeneração enquanto que o oleico otimizou a capacidade regenerativa e a função contrátil do músculo lesionado. / The effects of linoleic and oleic acids on regeneration of lacerated gastrocnemius muscle in rats and on cultured myoblasts, myotubes and fibroblasts were examined. There was incomplete regeneration and partial recovery of the contractile function of the injured muscle. Linoleic acid reduced the mass, contratile activity and cross-sectional area of the fibers, raised the fibrous area and reduced the expression of PCNA, collagen and fibronectin in fibroblasts. Oleic acid abolished the changes in contratile activity and the increase in the fibrous area, raised the expressions of MyoD in myoblasts, desmin in myotubes and inhibited the expressions of PCNA, collagen and fibronectin in fibroblasts. Linoleic acid impaired regeneration whereas oleic acid optimized the regenerative capacity and contractile function of the injured muscle.