Expression of myosin heavy chain isoforms in myogenic clones obtained from developing quail breast muscle

Summary Monoclonal antibodies specifically recognizing cardiac and extraocular muscle myosin heavy chains of the quail (Coturnix coturnix japonica) were used to determine the patterns of expression of these isoforms in clonal cultures of embryonic quail myoblasts. Myoblasts prepared from 9 day embryonic pectoralis are virtually homogeneous in their ability to form clones expressing both cardiac and extraocular isoforms. The majority of myoblasts obtained from day 5 embryos also formed clones which co-express the cardiac and extraocular isoforms, but a small percentage of the clones expressed only cardiac isoforms.     

An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression

The monitoring of gene expression is fundamental for understanding developmental biology. Here we report a successful experimental protocol for in situ hybridization in both whole-mount and sectioned planarian embryos. Conventional in situ hybridization techniques in developmental biology are used on whole-mount preparations. However, given that the inherent lack of external morphological markers in planarian embryos hinders the proper interpretation of gene expression data in whole-mount preparations, here we used sectioned material. We discuss the advantages of sectioned versus whole-mount preparations, namely, better probe penetration, improved tissue preservation, and the possibility to interpret gene expression in relation to internal morphological markers such as the epidermis, the embryonic and definitive pharynges, and the gastrodermis. Optimal fixatives and embedding methods for sectioning are also discussed. A. Cardona and J. Fernández have contributed equally to this work.     

Sequential activation of three myogenic regulatory genes during somite morphogenesis in quail embryos.

We report the cloning of two new quail myogenic cDNAs, quail myogenic factor 2 (qmf2) and qmf3, which encode helix-loop-helix proteins homologous to mammalian myogenic factors myogenin and myf-5. In situ hybridization has been used to investigate the developmental expression of qmf2 and qmf3, as well as qmf1, the quail homologue to mammalian MyoD1, during the formation of the brachial somites. These studies show that qmf1 and qmf3 are activated sequentially in medially localized somite cells, immediately following somite formation but prior to myotome formation. qmf1, qmf2, and qmf3 are expressed in the myotome of compartmentalized somites. These findings suggest that determination of the myogenic cell lineage in quail somites is a progressive process controlled by influences of the neural tube on the expression of the qmf regulatory genes in newly forming somites.     

Distinct myogenic programs of embryonic and fetal mouse muscle cells: expression of the perinatal myosin heavy chain isoform in vitro.

Early embryonic and late fetal mouse myogenic cells showed distinct patterns of perinatal myosin heavy chain (MHC) isoform expression upon differentiation in vitro. In cultures of somite or limb muscle cells isolated from Day 9 to Day 12 embryos, differentiated cells that expressed perinatal MHC were rare and perinatal MHC was not detectable by immunoblotting. In cultures of limb muscle cells isolated from Day 13 to Day 18 fetuses, in contrast, the perinatal MHC isoform was easily detected and was expressed in a substantial percentage of myocytes and myotubes. Analyses of clonally derived muscle colonies and cytosine arabinoside-treated fetal muscle cell cultures suggested that different fetal muscle cell nuclei initiated perinatal MHC expression at different times. In both embryonic and fetal cell cultures, the embryonic MHC isoform was expressed by all differentiated cells examined. A small number of myotubes in fetal muscle cell cultures showed a mosaic distribution of MHC isoform accumulation in which the perinatal MHC isoform accumulated in a restricted region of the myotube near particular nuclei, whereas the embryonic MHC isoform accumulated throughout the myotube. Thus, the myogenic program of fetal, but not embryonic, mouse myogenic cells includes expression of the perinatal MHC isoform upon differentiation in culture.     

Role of myosin light chain kinase and myosin light chain phosphatase in the resistance arterial myogenic response to intravascular pressure

The intrinsic ability of vascular smooth muscle cells (VSMCs) within arterial resistance vessels to respectively contract and relax in response to elevation and reduction of intravascular pressure is essential for appropriate blood flow autoregulation. This fundamental mechanism, referred to as the myogenic response, is dependent on apposite control of myosin regulatory light chain (LC₂₀) phosphorylation, a prerequisite for force generation, through the coordinated activity of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Here, we highlight the molecular basis of the smooth muscle contractile mechanism and review the regulatory pathways demonstrated to participate in the control of LC₂₀ phosphorylation in the myogenic response, with a focus on the Ca²⁺-dependent and Rho-associated kinase (ROK)-mediated regulation of MLCK and MLCP, respectively.     

Unexpectedly low mutation rates in beta-myosin heavy chain and cardiac myosin binding protein genes in Italian patients with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. Fourteen sarcomeric and sarcomere‐related genes have been implicated in HCM etiology, those encoding β‐myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3) reported as the most frequently mutated: in fact, these account for around 50% of all cases related to sarcomeric gene mutations, which are collectively responsible for approximately 70% of all HCM cases. Here, we used denaturing high‐performance liquid chromatography followed by bidirectional sequencing to screen the coding regions of MYH7 and MYBPC3 in a cohort (n = 125) of Italian patients presenting with HCM. We found 6 MHY7 mutations in 9/125 patients and 18 MYBPC3 mutations in 19/125 patients. Of the three novel MYH7 mutations found, two were missense, and one was a silent mutation; of the eight novel MYBPC3 mutations, one was a substitution, three were stop codons, and four were missense mutations. Thus, our cohort of Italian HCM patients did not harbor the high frequency of mutations usually found in MYH7 and MYBPC3. This finding, coupled to the clinical diversity of our cohort, emphasizes the complexity of HCM and the need for more inclusive investigative approaches in order to fully understand the pathogenesis of this disease. J. Cell. Physiol. 226: 2894–2900, 2011. © 2011 Wiley‐Liss, Inc.     

Mapping of actin-binding sites on the heavy chain of myosin subfragment 1

When the rigor complex of actin and myosin subfragment 1 (S1) was treated with a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, covalently linked complexes of actin and S1 heavy chain with apparent molecular weights of 165,000 and 175,000 were generated. Measurements of the molar ratio of actin to S1 heavy chain in the 165K and 175K products showed that they were 1:1 complexes of actin and S1 heavy chain. Chemical cleavages of the cross-linked products followed by peptide mappings revealed that two distinct segments of S1 heavy chain spanning the 18K-20K region and the 27K-35K region from its C terminus participated in cross-linking with actin. Cross-linking of actin to the former site generated the 165K peptide while the latter site was responsible for generating the 175K peptide.     

Diversity of native myosin and myosin heavy chain in fish skeletal muscles

• 1.1. Polymorphism of native myosin and myosin heavy chain (MHC) of fish skeletal muscles was analysed by pyrophosphate and SDS-gel electrophoreses.
• 2.2. Depending on the species, three or four myosin isoforms were detected in the white muscle, one or two isoforms in the pure red muscle, and four isomyosins were found in the red muscle composed of red and pink (intermediate) fibres.
• 3.3. It is suggested that all main types of fish muscle fibre (red, intermediate and white) differ in myosin isoform content.
• 4.4. Myosin heavy chain of the red muscle is a distinct protein from that of the white muscle. However, structural differences between these proteins vary among species.

     

Analysis of myosin heavy chain functionality in the heart

Comparison of mammalian cardiac alpha- and beta-myosin heavy chain isoforms reveals 93% identity. To date, genetic methodologies have effected only minor switches in the mammalian cardiac myosin isoforms. Using cardiac-specific transgenesis, we have now obtained major myosin isoform shifts and/or replacements. Clusters of non-identical amino acids are found in functionally important regions, i.e. the surface loops 1 and 2, suggesting that these structures may regulate isoform-specific characteristics. Loop 1 alters filament sliding velocity, whereas Loop 2 modulates actin-activated ATPase rate in Dictyostelium myosin, but this remains untested in mammalian cardiac myosins. Alpha --> beta isoform switches were engineered into mouse hearts via transgenesis. To assess the structural basis of isoform diversity, chimeric myosins in which the sequences of either Loop 1+Loop 2 or Loop 2 of alpha-myosin were exchanged for those of beta-myosin were expressed in vivo. 2-fold differences in filament sliding velocity and ATPase activity were found between the two isoforms. Filament sliding velocity of the Loop 1+Loop 2 chimera and the ATPase activities of both loop chimeras were not significantly different compared with alpha-myosin. In mouse cardiac isoforms, myosin functionality does not depend on Loop 1 or Loop 2 sequences and must lie partially in other non-homologous residues.