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

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.

Figure 1. Syntenic organization of MYH14/7b and MYH15 genes and amino acid sequence of MYH14 and MYH15 loops 1 and 2

A, physical maps showing syntenic organization of MYH14/7b and MYH15 genes in fish, frog, chicken and mammals. The microRNA, miR-499, is contained within the MYH14 gene in all species. Spacing of markers does not reflect actual scale. Gene organization information was obtained by NCBI and Ensembl Genome Browser databases (see Table S1 for gene ID). B, amino acid sequence of MYH14 and MYH15 loop 1 and loop 2 in different vertebrate species. The structure of the two surface loops in other human sarcomeric MYHs is also shown for comparison. The dog sequence is shown for MYH16, because human MYH16 is a pseudogene.

Figure 2. Quantitative RT-PCR analysis of different rat striated muscles with probes specific for MYH14 and MYH15 gene transcripts

The expression pattern of MYH7, coding for β/slow MYH, is shown for comparison. Transcripts levels are normalized to housekeeping genes and expressed as the percentage of the tissue with the highest expression level. Note that the distribution of miR-499 is similar to that of MYH14. EO: extraocular muscles; SOL: slow-twitch soleus muscle; TA: fast-twitch tibialis anterior muscle. Data are means ±s.e.m.; n= 6 (MYH14 and MYH15), n= 3 (MYH7 and mir-499).

Figure 3. Distribution of MYH15 in adult muscles

A, Western blot analysis of homogenates from different rat striated muscles with anti-MYH15 antibody. Note specific reaction of this antibody with MYH (M) from EO muscles. The same blot was reacted with an antibody against α-actin (A) to demonstrate equal loading of all lanes. Emb: embryonic day 20 (E20) hindlimb skeletal muscles; Neo: neonatal hindlimb muscles; SOL: adult slow soleus muscle; EDL: adult fast extensor ditorum longus muscle. B, myosins from different rat muscles were separated by SDS-PAGE using glycerol gels, a procedure which allows separation of different MYH isoforms, blotted and reacted with antibodies against MYH15 (upper panel), MYH15 followed by anti-β/slow MYH (middle panel), or MYH15 followed by an antibody reactive with fast 2A- and 2X-MYH and developing MYHs (lower panel). Note that MYH15 co-migrates with MYH-β/slow. C, serial transverse sections of rat EO muscle reacted with antibodies specific for MYH15, chicken ALD MYH (slow-tonic) or embryonic MYH. Note that MYH15 protein, like embryonic MYH, is expressed in most fibres of the orbital layer (O) and rare fibres of the global layer (G), whereas slow-tonic MYH is present in a very minor fibre population mostly localized in the orbital layer. Scale bar, 100 μm. D, serial transverse sections of rat soleus muscle showing two muscle spindles, one cut through the intracapsular region and one through the polar extracapsular region, stained with antibodies specific for MYH15, chicken ALD MYH (slow-tonic) and embryonic MYH or examined by phase contrast microscopy (right panel). Note selective expression of MYH15 in the polar region of the two bag intrafusal fibres (fibres 1 and 2), whereas embryonic MYH is expressed in the chain fibres (fibres 3 and 4) and slow-tonic MYH is present in the intracapsular region of two bag fibres (fibres 5 and 6) and in the polar region of one bag fibre (fibre 1). Extrafusal fibres are unstained. Scale bar, 20 μm.

Figure 4. Distribution of MYH14 in adult muscles

A, 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.

Figure 5. Serial sections of developing rat EO muscles stained for embryonic MYH (left panels), MYH15 (middle panels) or MYH14 (right panels)

Note that MYH15 is undetectable in fetal muscles (E20), is barely visible in the orbital layer at postnatal day 7 (P7) and is clearly expressed at P14. In contrast, MYH14 is expressed in all fibres in fetal EO muscles and disappears in most fibres except the slow-tonic fibres during early postnatal stages. Scale bar, 100 μm.

Figure 6. Scheme illustrating the evolutionary relationships among sarcomeric MYH genes in mammals, with the corresponding protein products and their expression pattern

The phylogenetic tree on the left is modified from that of the human MYH head domain (see McGuigan et al. 2004; Ikeda et al. 2007). Spacing and length of the branches do not reflect actual scale in this simplified scheme.