EXERCISE-INDUCED CHANGES IN SINGLE MUSCLE FIBER FUNCTION

我们的长期目标是了解肌肉的细胞机制 收缩，并确定如何定期计划的运动训练后， 肌肉功能和提高工作能力。 我们目前的工作重点是 横纹肌（四肢和呼吸），但未来的工作将评估 规律性运动对提高心功能的作用 平滑肌 关于定期锻炼如何影响细胞的知识 骨骼肌、心肌和平滑肌的特性将导致更好的 运动项目在康复医学中的理解和应用 预防医学 本提案的主要目的是确定 耐力和短跑运动训练计划的方式和程度 影响慢和快横纹肌纤维的功能能力， 评估这两种不同的锻炼计划是否会引起不同的细胞 应答 我们还将确定特定纤维类型是否具有相同的 功能特性，无论其来源（快速或 慢颤、肢体或呼吸肌），并评估功能 快速型IIa和IIb纤维类型之间的差异。 去皮和冻干的单纤维制剂将用于 阐明耐力和短跑运动训练对 肢体（比目鱼肌和 腓肠肌）和呼吸（横膈膜）肌肉。 将从选定的肌肉中分离出单层皮肤纤维并安装 力和位置传感器与以下收缩 测定的性能：1）峰值张力（Po）; 2）峰值张力率 施加松弛后的再发展（dP/dt）; 3最大缩短 速度（Vmax）; 4）纤维ATP酶; 5）pCa-力关系;和6） 力-速度关系 Vmax将通过松弛测试确定 方法和纤维ATP酶通过荧光技术测量。 这 该过程涉及ADP生产与氧化的酶促偶联 的NADH，并允许同时测量Po和肌原纤维ATP酶。 在生理学研究之后，每个纤维的片段将被 溶解在缓冲液中并在SDS-PAGE和焦磷酸凝胶上电泳， 测定肌球蛋白重链（MHC）和轻链以及异肌球蛋白含量。 这些研究将直接检验以下假设：1）运动训练 诱导纤维Vmax和ATP酶的纤维类型特异性变化， 与改变的MHC化学计量有关; 2）耐力和短跑 锻炼计划产生根本不同的功能变化， 快速（IIa和IIb型）纤维类型。 此外，我们假设， 运动项目将导致pCa力的显著功能变化， 以及快、慢两种纤维类型的力-速度关系。 将采用复溶实验来评估相对 MHC、LC 2和肌钙蛋白-C在介导运动性心肌损伤中的作用 功能变化。 冷冻干燥的单纤维制剂将用于评估效果 耐力和短跑运动训练的具体活动， 选择的氧化和糖酵解代谢的标记酶。 在 特别是，我们预测，这两种类型的运动训练将改变 糖酵解酶磷酸果糖激酶和乳酸的活性 脱氢酶，并且特定纤维类型的变化方向 将始终反映肌原纤维ATP酶和Vmax的观察结果。 最后，拟议中的研究将检验一个假设，即与肢体不同， 肌肉中，来自隔膜的单纤维的功能特性是 不受锻炼计划的影响。

Our long-term objectives are to understand the cellular mechanisms of muscle contraction, and determine how regular programs of exercise-training after muscle function and improve work capacity. Our current work is focused on striated (limb and respiratory) muscle, but future work will evaluate the role of regular exercise in improving the functional capacity of cardiac and smooth muscle. The knowledge of how regular exercise affects the cellular properties of skeletal, cardiac, and smooth muscle will lead to a better understanding and application of exercise programs in rehabilitative and preventive medicine. The primary objective of this proposal is to determine how and to what extent endurance and sprint exercise-training programs affect the functional capacity of slow and fast striated muscle fibers, and assess if these two different exercise programs elicit different cellular responses. We will also determine if a specific fiber type has the same functional properties regardless of its source of origin (fast- or slow-twitch, limb or respiratory muscle), and assess the functional differences between the fast type IIa and IIb fiber types. The skinned and freeze-dried single fiber preparations will be utilized to elucidate the effects of endurance and sprint exercise-training on the functional properties of fast and slow fibers of limb (soleus and gastrocnemius) and respiratory (diaphragm) muscles of adult male rats. Single skinned fibers will be isolated from the selected muscles and mounted between force and position transducers and the following contractile properties determined: 1) peak tension (Po); 2) peak rate of tension redevelopment (dP/dt) following an imposed slack; 3 maximal shortening velocity (Vmax); 4) fiber ATPase; 5) pCa-force relationship; and 6) force-velocity relationship. Vmax will be determined by the slack test method and fiber ATPase measured by a fluorometric technique. This procedure involves enzymatic coupling of the ADP production to the oxidation of NADH, and allows simultaneous measurement of Po and myofibrillar ATPase. Following the physiological studies, segments of each fiber will be solubilized in buffer and run on SDS-PAGE and pyrophosphate gels for determination of myosin heavy (MHC) and light chain and isomyosin content. These studies will directly test the hypotheses that: 1) exercise-training induces fiber type-specific changes in fiber Vmax and ATPase which are linked to an altered MHC stoichiometry; and 2) the endurance and sprint exercise programs produce fundamentally different functional changes in the fast (type IIa and IIb) fiber types. Additionally, we hypothesize that the exercise programs will cause significant functional changes in the pCa-force and force-velocity relationship of both fast and slow fiber types. Reconstitution experiments will be employed to assess the relative importance of MHC, LC2 and troponin-C in mediating the exercise-induced functional changes. The freeze-dried single fiber preparation will be used to assess the effects of endurance and sprint exercise-training on the specific activity of selected marker enzymes of oxidative and glycolytic metabolism. In particular, we predict that both types of exercise-training will alter the activity of the glycolytic enzymes phosphofructokinase and lactate dehydrogenase, and that the direction of change in a particular fiber type will always mirror that observed for the myofibrillar ATPase and Vmax. Finally, the proposed studies will test the hypothesis that, unlike limb muscle, the functional properties of single fibers from the diaphragm are not altered by programs of exercise-training.