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Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles.

Rossi AC, Mammucari C, Argentini C, Reggiani C, Schiaffino S - J. Physiol. (Lond.) (2009)

Bottom Line: During development, MYH14 is expressed at low levels in skeletal muscles, heart and all EO muscle fibres but disappears from most fibres, except the slow-tonic fibres, after birth.In contrast, MYH15 is absent in embryonic and fetal muscles and is first detected after birth in the orbital layer of EO muscles.The identification of the expression pattern of MYH14 and MYH15 brings to completion the inventory of the MYH isoforms involved in sarcomeric architecture of skeletal muscles and provides an unambiguous molecular basis to study the contractile properties of slow-tonic fibres in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy.

ABSTRACT
The mammalian genome contains three ancient sarcomeric myosin heavy chain (MYH) genes, MYH14/7b, MYH15 and MYH16, in addition to the two well characterized clusters of skeletal and cardiac MYHs. MYH16 is expressed in jaw muscles of carnivores; however the expression pattern of MYH14 and MYH15 is not known. MYH14 and MYH15 orthologues are present in frogs and birds, coding for chicken slow myosin 2 and ventricular MYH, respectively, whereas only MYH14 orthologues have been detected in fish. In all species the MYH14 gene contains a microRNA, miR-499. Here we report that in rat and mouse, MYH14 and miR-499 transcripts are detected in heart, slow muscles and extraocular (EO) muscles, whereas MYH15 transcripts are detected exclusively in EO muscles. However, MYH14 protein is detected only in a minor fibre population in EO muscles, corresponding to slow-tonic fibres, and in bag fibres of muscle spindles. MYH15 protein is present in most fibres of the orbital layer of EO muscles and in the extracapsular region of bag fibres. During development, MYH14 is expressed at low levels in skeletal muscles, heart and all EO muscle fibres but disappears from most fibres, except the slow-tonic fibres, after birth. In contrast, MYH15 is absent in embryonic and fetal muscles and is first detected after birth in the orbital layer of EO muscles. The identification of the expression pattern of MYH14 and MYH15 brings to completion the inventory of the MYH isoforms involved in sarcomeric architecture of skeletal muscles and provides an unambiguous molecular basis to study the contractile properties of slow-tonic fibres in mammals.

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Distribution of MYH14 in adult musclesA, Western blot analysis of homogenates from different rat striated muscles and chicken ALD muscle with anti-MYH14 antibody. The same blot was reacted with an antibody against α-actin to demonstrate equal loading of all lanes with the exception of ALD, that was about 50 times less loaded. B, Western blot of homogenates from human EO and vastus lateralis (VL) muscles with anti-MYH14 antibody. C, Western blot with human muscle myosins separated in glycerol gels, blotted and reacted with antibodies against MYH14 (upper panel), or MYH14 followed by anti-MYH-β/slow (lower panel). MYH14 corresponds to a band with lower electrophoretic mobility compared to MYH-β/slow. D, transverse sections of rat EO muscle reacted with antibodies specific for MYH14, showing few reactive fibres mostly localized in the orbital layer (left panel). These fibres correspond to slow-tonic fibres labelled by anti-ALD and S46 antibodies, as shown at high power in the right panels. Note that these fibres also co-express MYH15. Scale bar left panel, 100 μm; scale bar right panels, 25 μm. E, serial transverse sections of rat soleus muscle showing a muscle spindle cut through the intracapsular region, stained with anti-MYH14 or anti-ALD or examined by phase contrast microscopy (right panel). Note similar staining pattern of the two antibodies with stronger reactivity in one of the two bag fibres, corresponding to bag 2 fibre. Also note that surrounding extrafusal fibres are unstained. Scale bar, 20 μm. F, transverse sections of human EO muscle reacted with antibodies specific for MYH14, showing numerous reactive fibres. Scale bar, 500 μm.
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fig04: Distribution of MYH14 in adult musclesA, Western blot analysis of homogenates from different rat striated muscles and chicken ALD muscle with anti-MYH14 antibody. The same blot was reacted with an antibody against α-actin to demonstrate equal loading of all lanes with the exception of ALD, that was about 50 times less loaded. B, Western blot of homogenates from human EO and vastus lateralis (VL) muscles with anti-MYH14 antibody. C, Western blot with human muscle myosins separated in glycerol gels, blotted and reacted with antibodies against MYH14 (upper panel), or MYH14 followed by anti-MYH-β/slow (lower panel). MYH14 corresponds to a band with lower electrophoretic mobility compared to MYH-β/slow. D, transverse sections of rat EO muscle reacted with antibodies specific for MYH14, showing few reactive fibres mostly localized in the orbital layer (left panel). These fibres correspond to slow-tonic fibres labelled by anti-ALD and S46 antibodies, as shown at high power in the right panels. Note that these fibres also co-express MYH15. Scale bar left panel, 100 μm; scale bar right panels, 25 μm. E, serial transverse sections of rat soleus muscle showing a muscle spindle cut through the intracapsular region, stained with anti-MYH14 or anti-ALD or examined by phase contrast microscopy (right panel). Note similar staining pattern of the two antibodies with stronger reactivity in one of the two bag fibres, corresponding to bag 2 fibre. Also note that surrounding extrafusal fibres are unstained. Scale bar, 20 μm. F, transverse sections of human EO muscle reacted with antibodies specific for MYH14, showing numerous reactive fibres. Scale bar, 500 μm.

Mentions: Next, we examined the expression of MYH14 protein. Western blots show no reactivity with different rat or mouse muscles, but give a strong reaction with chicken ALD MYH (Fig. 4A). In contrast, a specific reaction with MYH from human EO but not vastus lateralis muscles was easily detected (Fig. 4B). Glycerol gel analysis shows that the MYH band recognized by anti-MYH14 antibody migrates slightly slower than human β/slow MYH (Fig. 4C). By immunofluorescence, MYH14 is detected in a minor fibre population present in the orbital layer and occasional global layer fibres of rat EO muscles: these fibres correspond to slow-tonic fibres labelled by anti-ALD myosin and S46 antibodies and co-express MYH15 (Fig. 4D). In rat hindlimb muscles anti-MYH14 antibody reacts with different intensities with the two bag fibres of muscle spindles, strongly with bag 2 and weakly with bag 1 fibres, a pattern similar to that obtained with anti-ALD (Fig. 4E). In human EO muscles, MYH14 positive fibres are more numerous than in rat, which might explain the different results in Western blotting, and widespread in both orbital and global layers (Fig. 4F). MYH14 positive fibres correspond to slow-tonic fibres stained by anti-ALD (Fig. S4). Anti-MYH14 antibody stains specifically the sarcomere A-band in longitudinal sections of EO muscles (Fig. S5).


Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles.

Rossi AC, Mammucari C, Argentini C, Reggiani C, Schiaffino S - J. Physiol. (Lond.) (2009)

Distribution of MYH14 in adult musclesA, Western blot analysis of homogenates from different rat striated muscles and chicken ALD muscle with anti-MYH14 antibody. The same blot was reacted with an antibody against α-actin to demonstrate equal loading of all lanes with the exception of ALD, that was about 50 times less loaded. B, Western blot of homogenates from human EO and vastus lateralis (VL) muscles with anti-MYH14 antibody. C, Western blot with human muscle myosins separated in glycerol gels, blotted and reacted with antibodies against MYH14 (upper panel), or MYH14 followed by anti-MYH-β/slow (lower panel). MYH14 corresponds to a band with lower electrophoretic mobility compared to MYH-β/slow. D, transverse sections of rat EO muscle reacted with antibodies specific for MYH14, showing few reactive fibres mostly localized in the orbital layer (left panel). These fibres correspond to slow-tonic fibres labelled by anti-ALD and S46 antibodies, as shown at high power in the right panels. Note that these fibres also co-express MYH15. Scale bar left panel, 100 μm; scale bar right panels, 25 μm. E, serial transverse sections of rat soleus muscle showing a muscle spindle cut through the intracapsular region, stained with anti-MYH14 or anti-ALD or examined by phase contrast microscopy (right panel). Note similar staining pattern of the two antibodies with stronger reactivity in one of the two bag fibres, corresponding to bag 2 fibre. Also note that surrounding extrafusal fibres are unstained. Scale bar, 20 μm. F, transverse sections of human EO muscle reacted with antibodies specific for MYH14, showing numerous reactive fibres. Scale bar, 500 μm.
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getmorefigures.php?uid=PMC2821527&req=5

fig04: Distribution of MYH14 in adult musclesA, Western blot analysis of homogenates from different rat striated muscles and chicken ALD muscle with anti-MYH14 antibody. The same blot was reacted with an antibody against α-actin to demonstrate equal loading of all lanes with the exception of ALD, that was about 50 times less loaded. B, Western blot of homogenates from human EO and vastus lateralis (VL) muscles with anti-MYH14 antibody. C, Western blot with human muscle myosins separated in glycerol gels, blotted and reacted with antibodies against MYH14 (upper panel), or MYH14 followed by anti-MYH-β/slow (lower panel). MYH14 corresponds to a band with lower electrophoretic mobility compared to MYH-β/slow. D, transverse sections of rat EO muscle reacted with antibodies specific for MYH14, showing few reactive fibres mostly localized in the orbital layer (left panel). These fibres correspond to slow-tonic fibres labelled by anti-ALD and S46 antibodies, as shown at high power in the right panels. Note that these fibres also co-express MYH15. Scale bar left panel, 100 μm; scale bar right panels, 25 μm. E, serial transverse sections of rat soleus muscle showing a muscle spindle cut through the intracapsular region, stained with anti-MYH14 or anti-ALD or examined by phase contrast microscopy (right panel). Note similar staining pattern of the two antibodies with stronger reactivity in one of the two bag fibres, corresponding to bag 2 fibre. Also note that surrounding extrafusal fibres are unstained. Scale bar, 20 μm. F, transverse sections of human EO muscle reacted with antibodies specific for MYH14, showing numerous reactive fibres. Scale bar, 500 μm.
Mentions: Next, we examined the expression of MYH14 protein. Western blots show no reactivity with different rat or mouse muscles, but give a strong reaction with chicken ALD MYH (Fig. 4A). In contrast, a specific reaction with MYH from human EO but not vastus lateralis muscles was easily detected (Fig. 4B). Glycerol gel analysis shows that the MYH band recognized by anti-MYH14 antibody migrates slightly slower than human β/slow MYH (Fig. 4C). By immunofluorescence, MYH14 is detected in a minor fibre population present in the orbital layer and occasional global layer fibres of rat EO muscles: these fibres correspond to slow-tonic fibres labelled by anti-ALD myosin and S46 antibodies and co-express MYH15 (Fig. 4D). In rat hindlimb muscles anti-MYH14 antibody reacts with different intensities with the two bag fibres of muscle spindles, strongly with bag 2 and weakly with bag 1 fibres, a pattern similar to that obtained with anti-ALD (Fig. 4E). In human EO muscles, MYH14 positive fibres are more numerous than in rat, which might explain the different results in Western blotting, and widespread in both orbital and global layers (Fig. 4F). MYH14 positive fibres correspond to slow-tonic fibres stained by anti-ALD (Fig. S4). Anti-MYH14 antibody stains specifically the sarcomere A-band in longitudinal sections of EO muscles (Fig. S5).

Bottom Line: During development, MYH14 is expressed at low levels in skeletal muscles, heart and all EO muscle fibres but disappears from most fibres, except the slow-tonic fibres, after birth.In contrast, MYH15 is absent in embryonic and fetal muscles and is first detected after birth in the orbital layer of EO muscles.The identification of the expression pattern of MYH14 and MYH15 brings to completion the inventory of the MYH isoforms involved in sarcomeric architecture of skeletal muscles and provides an unambiguous molecular basis to study the contractile properties of slow-tonic fibres in mammals.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy.

ABSTRACT
The mammalian genome contains three ancient sarcomeric myosin heavy chain (MYH) genes, MYH14/7b, MYH15 and MYH16, in addition to the two well characterized clusters of skeletal and cardiac MYHs. MYH16 is expressed in jaw muscles of carnivores; however the expression pattern of MYH14 and MYH15 is not known. MYH14 and MYH15 orthologues are present in frogs and birds, coding for chicken slow myosin 2 and ventricular MYH, respectively, whereas only MYH14 orthologues have been detected in fish. In all species the MYH14 gene contains a microRNA, miR-499. Here we report that in rat and mouse, MYH14 and miR-499 transcripts are detected in heart, slow muscles and extraocular (EO) muscles, whereas MYH15 transcripts are detected exclusively in EO muscles. However, MYH14 protein is detected only in a minor fibre population in EO muscles, corresponding to slow-tonic fibres, and in bag fibres of muscle spindles. MYH15 protein is present in most fibres of the orbital layer of EO muscles and in the extracapsular region of bag fibres. During development, MYH14 is expressed at low levels in skeletal muscles, heart and all EO muscle fibres but disappears from most fibres, except the slow-tonic fibres, after birth. In contrast, MYH15 is absent in embryonic and fetal muscles and is first detected after birth in the orbital layer of EO muscles. The identification of the expression pattern of MYH14 and MYH15 brings to completion the inventory of the MYH isoforms involved in sarcomeric architecture of skeletal muscles and provides an unambiguous molecular basis to study the contractile properties of slow-tonic fibres in mammals.

Show MeSH
Related in: MedlinePlus