Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth

Abstract

To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (alpha-GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I. The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4-6 weeks of age, which coincides with the increased physical activity of puppies.

Fig. 1

The activity of mATPase after acid pre-incubation at pH 4.4 at prenatal days F50 (A), F55 (B) and F60 (C), and postnatal days 1 (D), 5 (E), 15 (F), 28 (G), 42 (H) and 62 (I) in the longissimus dorsi muscle. Arrows indicate the primary myotubes and arrowheads the secondary myotubes. In C–F the slow fibres are bright; in G, the intermediate phase, the undifferentiated fibres are the darkest; and in H and I the slow and undifferentiated fibres are dark. Scale bar, 50 µm.

Fig. 2

Immunohistochemical reactions with the MHC-D antibody directed against MHC-emb in the diaphragm (left column) and the triceps brachii muscle (right column) on days F60 (A,B), 5 (C,D), 22 (E,F) and 42 (G,H). Note the weakening of the MHC-emb expression and the increase in fibre diameter; the most reactive fibres are, however, the smallest. Magnification 200×.

Fig. 3

Immunohistochemical reactions with the F158.4C10 antibody directed against MHC-neo in the diaphragm (left column) and the triceps brachii muscle (right column) on days 1 (A,B), 15 (C,D), 28 (E,F) and 60 (G,H). Note the weakening of the MHC-neo expression and the increase in fibre diameter. Magnification 200×.

Fig. 4

Proportion of fibres expressing different isoforms in the diaphragm, triceps brachii, rhomboideus and extensor carpi radialis muscles during postnatal development. There was a prominent switch in the high expression rates of the developmental isoforms (MHC-emb and MHC-neo) and the adult MHC-I and MHC-IIa between days 28 and 42, i.e. the fourth and sixth weeks of age. The decrease in developmental isoforms and increase in adult fast isoforms occurred earlier in the diaphragm. The line for MHC-IIa also includes hybrid IIA/IIX fibres, which react intensively with the SC-71 antibody. The strong expression of MHC-IIx (pure IIX fibres or hybrid with a predominance of MHC-IIx) was assessed by negative BF-35 staining and fibre-to-fibre comparison of SC-71 and A4.74 reactions. The rhomboideus muscle exhibited the highest percentage of slow fibres in neonates. The number of slow fibres gradually increased during postnatal development in all muscles. After the initial neonatal increase, the number of MHC-IIa fibres decreased in the slow rhomboideus muscle.

Fig. 5

Immunohistochemical demonstration of MHC isoforms in the cranial tibial muscle in a newborn puppy. Immunostaining with A4.74 for MHC-IIa/x (A), F158.4C10 for MHC-neo (B), MHC-s for MHC-I (C) and MHC-D for MHC-emb (D). All muscle fibres express developmental isoforms MHC-emb and MHC-neo. Primary fibres (p) have a weaker reaction with antibodies against MHC-emb (D) and a very strong expression of the slow isoform (C). Secondary fibres (s) have a varied expression of adult fast isoforms (A). Scale bar, 50 µm.

Fig. 6

Muscle fibres in the cranial tibial muscle at 3 weeks of age. Activity of mATPase after pre-incubation at pH 4.5 (A); and immunohistochemical demonstration of MHC-I (B), MHC-emb (C) and MHC-neo (D). The mATPase staining is most intensive in fibres with a prominent MHC-emb expression (arrows). Primary slow fibres (I) have a medium staining intensity in the mATPase reaction; besides MHC-emb they also ceased to express MHC-neo. The mATPase staining intensity of the secondary slow fibres (arrowheads) varied according to their MHC-emb content, i.e. fibres with more prominent MHC-emb expression are darker. Scale bar, 50 µm.

Fig. 7

The reaction for the glycolytic enzyme α-GPDH (A) and oxidative enzyme SDH (B), and immunohistochemical demonstration of fast fibres (MHC-IIa and MHC-IIx) (C) and slow fibres (MHC-I) (D) in the rectus femoris muscle of a 2-month-old dog. Slow fibres (I) and hybrid MHC-I/IIa (IIC) fibres have a high SDH activity and slightly weaker α-GPDH. The strongest reaction to the oxidative enzyme was noted in some fast fibres (x). Scale bar, 100 µm.