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.
