SDH and actomyosin ATPase activities of different fiber types in rat diaphragm muscle

Abstract

In the rat diaphragm muscle, the histochemical classification of type I, IIa, IIb, or IIx fibers was correlated with myosin heavy chain (MHC) immunoreactivity. Expression of MHC isoforms in single dissected fibers was also assessed electrophoretically. Most fibers (approximately 86%) expressed a single MHC isoform, and when present, coexpression of MHC-2X and MHC-2B isoforms was most prevalent. Type I and IIa fibers were the smallest, type IIb fibers were the largest, and type IIx fibers were intermediate. Succinate dehydrogenase (SDH) and calcium-activated myosin adenosinetriphosphatase (actomyosin ATPase) activities were measured with quantitative histochemical procedures. Type I and IIa fibers had the highest SDH activities, followed in rank order by type IIx and IIb fibers. Type I fibers had the lowest actomyosin ATPase activity, followed in rank order by type IIa, IIx, and IIb fibers. Across all fibers, there was an inverse relationship between fiber SDH activity and cross-sectional area and a positive correlation between fiber actomyosin ATPase activity and cross-sectional area. The SDH and actomyosin ATPase activities of muscle fibers were also inversely correlated. These phenotypic differences in SDH and ATPase activities may be important in determining the contractile and fatigue properties of different fiber types in the rat diaphragm muscle.

Changes in actomyosin ATP consumption rate in rat diaphragm muscle fibers during postnatal development

“…The quantitative histochemical procedure for measuring V max ATPase in typeidentified muscle fibers has been previously described (1,31). Muscle segments were stretched to optimal length (1.5 times resting excised muscle length) (21) and then rapidly frozen in isopentane cooled by liquid nitrogen.…”

“…In previous studies, we determined that the actomyosin ATPase reaction is linear for at least a 9-min period (1,31). A single endpoint reaction time of 4 min was selected as well as the 10-m section thickness to limit OD measurements to Ͻ1.0 OD units and thereby reduce measurement errors.…”

“…Muscle cross sections were reacted with mouse primary antibodies against different MHC isoforms as previously described (31). Briefly, to detect MHC isoform coexpression, pairs of mouse IgG or IgM primary antibodies were used, e.g., anti-MHCSlow (Novocastra, IgG; 1:100), anti-MHC 2A (Blau A4.74, IgG; 1:1), anti-MHC2B (BFF3, IgM, purified from mouse hybridoma, German Collection of Microorganisms and Cell Culture; 1:1), and antiMHCNeo (Novocastra; 1:10).…”

“…myosin heavy chain; fiber types; skeletal muscle; immunohistochemistry IN SKELETAL MUSCLES SUCH AS the diaphragm muscle (Dia m ), myosin heavy chain (MHC) functions as a molecular motor, converting chemical (ATP) into mechanical energy, thus driving muscle contraction (5). In adult skeletal muscle, several MHC isoforms exist, and the singular expression of these MHC isoforms in muscle fibers forms the basis for histochemical classification of different fiber types (18,24,25,31). In the neonatal rat Dia m , a neonatal isoform of MHC (MHC Neo ) is predominantly expressed, most often in combination with MHC Slow or MHC 2A isoforms (10,16,18,30,33).…”

“…In previous studies (14,27,31), our laboratory employed a quantitative histochemical procedure to determine the maximum velocity of the actomyosin ATPase reaction (V max ATPase) in muscle fibers expressing different MHC isoforms. In the rat Dia m , we found that fibers expressing MHC 2X and MHC 2B isoforms display significantly higher V max ATPase compared with fibers expressing MHC Slow and MHC 2A isoforms.…”