Karin Eizema

Differential Expression of Equine Myosin Heavy-chain mRNA and Protein Isoforms in a Limb Muscle

The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei.

Changes in fibre type composition of gluteus medius and semitendinosus muscles of Dutch Warmblood foals and the effect of exercise during the first year postpartum

In order to obtain broader insights into the equine musculoskeletal system, we studied the fibre type composition of 2 locomotory muscles in biopsies from Dutch Warmblood foals taken at 3 different ages in the first year postpartum. The muscle fibre types were determined histochemically as well as immunohistochemically. ATPase-characterised IIB fibres appear to express either IId or type lIa plus IId myosin heavy chain (MHC). A high percentage of fibres classified as IIA with ATPase expressed both fast types of MHC. The type I classification by the 2 methods matched almost completely. There was an increase with age of fibres expressing I and IIa MHC in the gluteus medius. At the same time, there was a decrease of fibres expressing IId MHC and fibres co-expressing MHC IIa and IId. MHC expression of the semitendinosus muscle did not change over time at first, but from age 22-48 weeks there was a decrease in the percentage of type IId fibres. In general, the gluteus medius contained more type I fibres but fewer type IId fibres compared to the semitendinosus. At most ages the fibre type compositions of both muscles correlated with one another. To examine the effect of exercise, one-third of the foals were given box rest, one-third received training and one-third kept at pasture during the first 22 weeks of life. The 3 exercise groups differed in their fibre type composition; however, these differences could not be attributed to the effect of exercise.

Myosin heavy chain isoforms in equine gluteus medius muscle: comparison of mRNA and protein expression profiles.

The major structural protein in skeletal muscle, myosin heavy chain (MyHC), is primarily transcriptionally controlled. We compared the expression of MyHC isoforms on the mRNA and protein level in biopsies from the m. gluteus medius from adult untrained horses. In transverse sections, the majority of fibers showed qualitatively identical mRNA and protein expression patterns. However, coexpression of 2a and 2d/x MyHCs was substantially more common at the protein than at the mRNA level, suggesting a fine-tuning of these two genes in normal muscle not subjected to any training protocol. Because transverse sections give a limited sampling of mRNA expression in the case of uneven distribution of transcripts in a muscle fiber, we also analyzed longitudinal sections. We present, for the first time, evidence that expression of MyHC mRNA and protein was equal along the length of the fiber. Hence, mRNA expression is not regulated by differential expression of isoforms by separate myonuclei. It is concluded that the number of protein hybrid fibers in equine gluteus medius muscle is controlled by alteration of the transcription pattern uniformly along the fiber, rather than by simultaneous transcription of genes. The differences with the results in muscle of small animals and humans are discussed.

Differential Expression of Calcineurin and SR Ca2+ Handling Proteins in Equine Muscle Fibers During Early Postnatal Growth

During early postnatal development, the myosin heavy chain (MyHC) expression pattern in equine gluteus medius muscle shows adaptation to movement and load, resulting in a decrease in the number of fast MyHC fibers and an increase in the number of slow MyHC fibers. In the present study we correlated the expression of MyHC isoforms to the expression of sarcoplasmic(endo)reticulum Ca2+-ATPase 1 and 2a (SERCA), phospholamban (PLB), calcineurin A (CnA), and calcineurin B (CnB). Gluteus medius muscle biopsies were taken at 0, 2, 4, and 48 weeks and analyzed using immunofluorescence. Both SERCA isoforms and PLB were expressed in almost all fiber types at birth. From 4 weeks of age onward, SERCA1 was exclusively expressed in fast MyHC fibers and SERCA2a and PLB in slow MyHC fibers. At all time points, CnA and CnB proteins were expressed at a basal level in all fibers, but with a higher expression level in MyHC type 1 fibers. From 4 weeks onward, expression of only CnA was also higher in MyHC type 2a and 2ad fibers. We propose a double function of calcineurin in calcium homeostasis and maintenance of slow MyHC fiber type identity. Although equine muscle is already functional at birth, expression patterns of the monitored proteins still show adaptation, depending on the MyHC fiber type.

Presence of SERCA and Calcineurin during Fetal Development of Porcine Skeletal Muscle

Mechanisms involved in skeletal myofiber differentiation during fetal development of large animals are poorly understood. Studies in small animals suggest that the calcineurin (Cn) pathway is involved in myofiber differentiation. Neural activity is a prerequisite for Cn activity, implying maintenance of sustained low intracellular Ca2+ concentrations. To study the role of Cn in fetal myofiber differentiation, we monitored the temporal and spatial distribution of Cn subunits, sarcoplasmic reticulum Ca2+ ATPase (SERCA), phospholamban (PLB), and myosin heavy chain (MyHC) isoforms in relation to ingrowing nerves in porcine semitendinosus muscle (m. semitendinosus) at 55 and 75 days of gestation (dg) and at term. Immunofluorescence analysis revealed the presence of Cn subunits and SERCA isoforms at all analyzed stages. Cn distribution was not fiber-type specific, but expression became more prominent at term. At 75 dg, differential SERCA2 expression was accompanied by perinuclear PLB in primary fibers. SERCA1 was expressed in all fiber types at all stages. No specific MyHC isoform distribution was seen in relation to neuromuscular contacts, although neuromuscular contacts were present. From these results we speculate that in porcine m. semitendinosus differential SERCA2 expression precedes differential Cn expression. The question whether the Cn pathway is involved in prenatal myofiber differentiation needs further studies.

Effects of acute exercise and long-term exercise on total Na+,K+-ATPase content and Na+,K+-ATPase isoform expression profile in equine muscle.

OBJECTIVE To investigate the effects of acute exercise and long-term training on Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentration in equine muscle. ANIMALS 6 Standardbreds. PROCEDURES Horses performed a bout of exercise on a treadmill before and after 18 weeks of combined interval and endurance training. Muscle biopsy specimens were obtained from vastus lateralis muscle (VLM) and pectoralis descendens muscle (PDM) before and after exercise. The Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentrations were determined by use of [(3)H]ouabain binding, real-time PCR assay, and western blotting, respectively. RESULTS 6 Na(+),K(+)-ATPase mRNA isoforms were present in equine muscle, but only A2 and B1 proteins were detected. Exercise before training resulted in increases of mRNA isoforms A1, A2, A3, and B2 in VLM and A1 and B3 in PDM. Training increased resting values for mRNA isoforms A3 and B1 in VLM and B3 in PDM. The Na(+),K(+)-ATPase, [(3)H]ouabain binding, and proteins of mRNA A2 and B1 increased in VLM, whereas in PDM, only A2 protein increased as a result of training. After training, effects of strenuous exercise on mRNA expression were no longer detectable. CONCLUSIONS AND CLINICAL RELEVANCE Equine muscle contained all Na(+),K(+)-ATPase mRNA isoforms, but only A2 and B1 proteins could be detected. Expression of these isoforms changed as a result of strenuous exercise and long-term training, representing an adaptive response. Determination of Na(+),K(+)-ATPase gene expression may be relevant for understanding alterations in excitability during neuromuscular diseases.