Alison M. Maggs

MyoD protein is differentially accumulated in fast and slow skeletal muscle fibres and required for normal fibre type balance in rodents

MyoD is a muscle-specific transcription factor involved in commitment of cells to myogenesis. MyoD mRNA levels differ between fast and slow muscles, suggesting that MyoD may regulate aspects of fibre type. Here we show that detectable MyoD protein becomes restricted during development to the nuclei of the fastest classes of fibres in fast muscles. myoDm1 mice, in which the myoD gene has been disrupted, show subtle shifts in fibre type of fast muscles toward a slower character, suggesting that MyoD is involved in the maintenance of the fast IIB/IIX fibre type. In contrast, slow muscle shifts to a faster phenotype in myoDm1. Moreover, MD6.0-lacZ transgenic mice with the myoD promoter driving lacZ, show highest beta-galactosidase activity in the fastest fibres of fast muscles, but also express low levels in slow fibres of slow, but not fast, muscles, suggesting distinct regulation of gene expression in slow fibres of fast and slow muscles.

The CKK Domain (DUF1781) Binds Microtubules and Defines the CAMSAP/ssp4 Family of Animal Proteins

We describe a structural domain common to proteins related to human calmodulin-regulated spectrin-associated protein1 (CAMSAP1). Analysis of the sequence of CAMSAP1 identified a domain near the C-terminus common to CAMSAP1 and two other mammalian proteins KIAA1078 and KIAA1543, which we term a CKK domain. This domain was also present in invertebrate CAMSAP1 homologues and was found in all available eumetazoan genomes (including cnidaria), but not in the placozoan Trichoplax adherens, nor in any nonmetazoan organism. Analysis of codon alignments by the sitewise likelihood ratio method gave evidence for strong purifying selection on all codons of mammalian CKK domains, potentially indicating conserved function. Interestingly, the Drosophila homologue of the CAMSAP family is encoded by the ssp4 gene, which is required for normal formation of mitotic spindles. To investigate function of the CKK domain, human CAMSAP1-enhanced green fluorescent protein (EGFP) and fragments including the CKK domain were expressed in HeLa cells. Both whole CAMSAP1 and the CKK domain showed localization coincident with microtubules. In vitro, both whole CAMSAP1-glutathione-s-transferase (GST) and CKK-GST bound to microtubules. Immunofluorescence using anti-CAMSAP1 antibodies on cerebellar granule neurons revealed a microtubule pattern. Overexpression of the CKK domain in PC12 cells blocked production of neurites, a process that requires microtubule function. We conclude that the CKK domain binds microtubules and represents a domain that evolved with the metazoa.

Evidence for differential post-translational modifications of slow myosin heavy chain during murine skeletal muscle development

The contractile properties of muscle fibres are, in part, determined by the myosin heavy chain (MyHC) isoforms they express. Using monoclonal antibodies, we show that at least three forms of slow twitch MyHC accumulate sequentially during mouse fetal development and that slow MyHC maturation in slow fibres occurs before expression of the adult fast MyHCs in fast fibres. Expression of deletion derivatives of β-cardiac MyHC cDNA shows that the slow MyHC epitopes that are detected in adult but not in young animals are located near the N-terminus. The same N-terminal region of various fast MyHC molecules contains a conserved epitope that can, on occasions, be observed when slow MyHC cDNA is expressed in non-muscle cells. The results raise the possibility that the N-terminal epitopes result from post-translational modification of the MyHC and that a sequence of slow and fast MyHC isoform post-translational modifications plays a significant role during development of murine muscle fibres.

Not just a plasma membrane protein: in cardiac muscle cells alpha-II spectrin also shows a close association with myofibrils.

Spectrin and its associated proteins are essential for the integrity of muscle cells and there is increasing evidence for their involvement in signalling pathways as well as having a structural function in mediating stress. Spectrin is a multigene family and it is essential to determine which isoforms are present and their location in the cell. In heart muscle, we have found that one spectrin isoform, αII-spectrin, is strongly represented and, using immunofluorescence, we show that it lies within the contractile fibres near the Z-disc as well as on the cardiomyocyte plasma membrane. Electron microscopy of immunogold-labelled cryosections reveals statistically significant clustering of gold particles near the Z-disc, within and close to the edge of myofibrils. βII-spectrin and ankyrin-R and G are both known to occupy this region. We suggest that αIIβII spectrin tetramers with ankyrin organise and/or stabilise cardiac muscle cell membrane components relative to the contractile apparatus.

Cardiac muscle cell cytoskeletal protein 4.1: analysis of transcripts and subcellular location--relevance to membrane integrity, microstructure, and possible role in heart failure.

The spectrin-based cytoskeleton assembly has emerged as a major player in heart functioning; however, cardiac protein 4.1, a key constituent, is uncharacterized. Protein 4.1 evolved to protect cell membranes against mechanical stresses and to organize membrane microstructure. 4.1 Proteins are multifunctional and, among other activities, link integral/signaling proteins on the plasma and internal membranes with the spectrin-based cytoskeleton. Four genes, EPB41, EPB41L1, EPB41L2, and EPB41L3 encode proteins 4.1R, 4.1N, 4.1G, and 4.1B, respectively. All are extensively spliced. Different isoforms are expressed according to tissue and developmental state, individual function being controlled through inclusion/exclusion of interactive domains. We have defined mouse and human cardiac 4.1 transcripts; other than 4. 1B in humans, all genes show activity. Cardiac transcripts constitutively include conserved FERM and C-terminal domains; both interact with membrane-bound signaling/transport/cell adhesion molecules. Variable splicing within and adjacent to the central spectrin/actin-binding domain enables regulation of cytoskeleton-binding activity. A novel heart-specific exon occurs in human 4.1G, but not in mouse. Immunofluorescence reveals 4.1 staining within mouse cardiomyocytes; thus, both at the plasma membrane and, interdigitated with sarcomeric myosin, across myofibrils in regions close to the sarcoplasmic reticulum. These are all regions to which spectrin locates. 4.1R in human heart shows similar distribution; however, there is limited plasma membrane staining. We conclude that cardiac 4.1s are highly regulated in their ability to crosslink plasma/integral cell membranes with the spectrin-actin cytoskeleton. We speculate that over the repetitive cycles of heart muscle contraction and relaxation, 4.1s are likely to locate, support, and coordinate functioning of key membrane-bound macromolecular assemblies.

Nerve‐dependent changes in skeletal muscle myosin heavy chain after experimental denervation and cross‐reinnervation and in a demyelinating mouse model of Charcot–Marie–Tooth disease type 1A

Innervation regulates the contractile properties of vertebrate muscle fibers, in part through the effect of electrical activity on expression of distinct myosins. Herein we analyze the role of innervation in regulating the accumulation of the general, maturational, and adult forms of rodent slow myosin heavy chain (MyHC) that are defined by the presence of distinct antigenic epitopes. Denervation increases the number of fibers that express general slow MyHC, but it decreases the adult slow MyHC epitope. Cross‐reinnervation of slow muscle by a fast nerve leads to an increase in the number of fibers that express fast MyHC. In both cases, there is an increase in the number of fibers that express slow and fast IIA MyHCs, but without the adult slow MyHC epitope. The data suggest that innervation is required for maturation and maintenance of diversity of both slow and fast fibers. The sequence of slow MyHC epitope transitions is a useful biomarker, and it may play a significant role during nerve‐dependent changes in muscle fiber function. We applied this detailed muscle analysis to a transgenic mouse model of human motor and sensory neuropathy IA, also known as Charcot–Marie–Tooth disease type 1A (CMT1A), in which electrical conduction in some motor nerves is poor due to demyelination. The mice display atrophy of some muscle fibers and changes in slow and fast MyHC epitope expression, suggestive of a progressive increase in innervation of muscle fibers by fast motor neurons, even at early stages. The potential role of these early changes in disease pathogenesis is assessed. Muscle Nerve 38: 1572–1584, 2008

Isoforms of protein 4.1 are differentially distributed in heart muscle cells: Relation of 4.1R and 4.1G to components of the Ca2+ homeostasis system

The 4.1 proteins are cytoskeletal adaptor proteins that are linked to the control of mechanical stability of certain membranes and to the cellular accumulation and cell surface display of diverse transmembrane proteins. One of the four mammalian 4.1 proteins, 4.1R (80 kDa/120 kDa isoforms), has recently been shown to be required for the normal operation of several ion transporters in the heart (Stagg MA et al. Circ Res, 2008; 103: 855-863). The other three (4.1G, 4.1N and 4.1B) are largely uncharacterised in the heart. Here, we use specific antibodies to characterise their expression, distribution and novel activities in the left ventricle. We detected 4.1R, 4.1G and 4.1N by immunofluorescence and immunoblotting, but not 4.1B. Only one splice variant of 4.1N and 4.1G was seen whereas there are several forms of 4.1R. 4.1N, like 4.1R, was present in intercalated discs, but unlike 4.1R, it was not localised at the lateral plasma membrane. Both 4.1R and 4.1N were in internal structures that, at the level of resolution of the light microscope, were close to the Z-disc (possibly T-tubules). 4.1G was also in intracellular structures, some of which were coincident with sarcoplasmic reticulum. 4.1G existed in an immunoprecipitable complex with spectrin and SERCA2. 80 kDa 4.1R was present in subcellular fractions enriched in intercalated discs, in a complex resistant to solubilization under non-denaturing conditions. At the intercalated disc 4.1R does not colocalise with the adherens junction protein, β-catenin, but does overlap with the other plasma membrane signalling proteins, the Na/K-ATPase and the Na/Ca exchanger NCX1. We conclude that isoforms of 4.1 proteins are differentially compartmentalised in the heart, and that they form specific complexes with proteins central to cardiomyocyte Ca(2+) metabolism.