Fiber type populations and Ca²⁺-activation properties of single fibers in soleus muscles from SHR and WKY rats

Abstract

Electrophoretic analyses of muscle proteins in whole muscle homogenates and single muscle fiber segments were used to examine myosin heavy chain (MHC) and myosin light chain 2 (MLC2) isoform composition and fiber type populations in soleus muscles from spontaneously hypertensive rats (SHRs) and their age-matched normotensive controls [Wistar-Kyoto (WKY) rats], at three stages in the development of high blood pressure (4 wk, 16 wk, and 24 wk of age). Demembranated (chemically skinned with 2% Triton X-100), single fiber preparations were used to determine the maximum Ca2+-activated force per cross-sectional area, calcium sensitivity, and degree of cooperativity of the contractile apparatus and Ca2+-regulatory system with respect to Ca2+. The results show that, at all ages examined, 1) SHR soleus contained a lower proportion of MHCI and MLC2 slow (MLC2s) and a higher proportion of MHCIIa, MHCIId/x, and MLC2 fast (MLC2f ) isoforms than the age-matched controls; 2) random dissection of single fibers from SHR and WKY soleus produced four populations of fibers: type I (expressing MHCI), type IIA (expressing MHCIIa), hybrid type I+IIA (coexpressing MHCI and MHCIIa), and hybrid type IIA+IID (coexpressing MHCIIa and MHCIId/x); and 3) single fiber dissection from SHR soleus yielded a lower proportion of type I fibers, a higher proportion of fast-twitch fibers (types IIA and IIA+IID), and a higher proportion of hybrid fibers (types I+IIA and IIA+IID) than the homologous muscles from the age-matched WKY rats. Because the presence of hybrid fibers is viewed as a marker of muscle transformation, these data suggest that SHR soleus undergoes transformation well into adulthood. Our data show also that, for a given fiber type, there are no significant differences between SHR and WKY soleus muscles with respect to any of the Ca2+-activation properties examined. This finding indicates that the lower specific tensions reported in the literature for SHR soleus muscles are not due to strain- or hypertension-related differences in the function of the contractile apparatus or regulatory system.

Morphological and biochemical alterations of skeletal muscles from the genetically obese (ob/ob) mouse

“…Single muscle fibre segments were isolated and their length and diameter measured (for calculation of single fibre CSA) using a dissecting microscope (Olympus SZPT, Shibuya, Tokyo, Japan), video monitoring system (Panasonic WV-BP100 CCD camera; Panasonic WV-BM900 monitor, www.panasonic.net/), and precision calipers (Mitutoyo, Japan), as described in Bortolotto et al 20 Muscle homogenisation for biochemical analyses and protein determination in muscle homogenates Whole muscles were placed in 6 volumes (note: 1 volume (ml) E1 g wet muscle mass) of relaxing solution (containing 90 mM HEPES, 126 mM K þ , 36 mM Na þ , 1 mM Mg 2 þ , 50 mM EGTA total , 8 mM ATP total , 10 mM CrP (pH 7.10) and homogenised manually, on ice, with a glass/glass homogeniser.…”

“…The protocol described by Bortolotto et al 20 was used for the preparation of whole muscle homogenate and single fibre samples. The final concentration was 0.5 mg protein/ml solubilisation buffer for whole muscle homogenate samples and 0.4 nl fibre volume/ml solubilisation buffer for single fibre samples.…”

“…An MHC i marker containing MHC I, MHC IIa, MHC IIb, and MHC IId isoforms (produced in the laboratory from SM, EDL, and SOL muscles of mouse, as described by Bortolotto et al 20 ) was also applied to gels to allow for the identification of the individual MHC isoforms in each sample when stained for visualisation. Electrophoresis was carried out at constant voltage (150 V) at 51 C for 27 h.…”

O treinamento físico aeróbio corrige a rarefação capilar e as alterações nas proporções dos tipos de fibra muscular esquelética em ratos espontaneamente hipertensos

“…The type II fibers are characterized as being fast twitch with predominance of glycolytic metabolism, while the type I fibers are slow twitch ones with predominance of oxidative metabolism 11,30 . There is a lot of evidence in the literature shows that the skeletal muscle of hypertensive individuals, as well as of SHR, contains higher percentage of type II fast twitch, glycolytic fibers compared with their normotensive control [7][8][9][10][11] . Interestingly, the results obtained in the composition of the fiber types of the soleus skeletal muscle, the muscle investigates in this study, which presents an average of 90% of type I fibers and 10% of type II fibers, performed both by histochemical myosin ATPase reaction and SDS-PAGE gel electrophoresis for detection of myosins of heavy chain (MHC) for each type of fiber, were positively correlated regardless of the technique applied 10 .…”

“…Experimental and clinical studies show that dysfunction in the vasomotor tonus and alterations in the microvascular structure are the primary processes in the HTN pathogenesis [3][4][5][6] . Many studies have shown capillary rarefaction in the skeletal muscle of animals and hypertensive patients [3][4][5][6] , with increase in the percentage of fast twitch fibers, which present predominance of glycolytic metabolism 8,12 and classified as type II fibers [7][8][9][10][11] . The skeletal muscle presents high plasticity and suffers transition of fiber type due to the alterations in the isoforms of myosin of heavy chain (MHC) in many conditions, such as: disuse, growth, aging, electrical stimulus, exposure to microgravity, physical exercises and CVD 8,11 .…”