Signalling in sarcomeres in development and disease

Sarcomeres are the smallest contractile units of heart and skeletal muscles and are essential for generation and propagation of mechanical force in these striated muscles. During the last decades it has become increasingly clear that components of sarcomeres also play a fundamental role in signal transduction in physiological and pathophysiological conditions. Mutations or misexpression of both sarcomeric contractile and non-contractile proteins have been associated with a variety of cardiac diseases. Moreover, re-expression of foetal sarcomeric proteins or isoforms during cardiac disease can be observed, emphasising the importance of understanding signalling in sarcomeres in both development and disease. The prospective of pharmacological intervention at the level of the sarcomere is now emerging and may lead to novel therapeutic strategies for the treatment of cardiac and skeletal muscle diseases. These aspects will be discussed in this brief review and recent findings, which led to novel insights into the role of the sarcomeric cytoskeleton in muscle development and disease, will be highlighted.     

Evolutionary implications of three novel members of the human sarcomeric myosin heavy chain gene family

Sarcomeric myosin heavy chain (MyHC) is the major contractile protein of striated muscle. Six tandemly linked skeletal MyHC genes on chromosome 17 and two cardiac MyHC genes on chromosome 14 have been previously described in the human genome. We report the identification of three novel human sarcomeric MyHC genes on chromosomes 3, 7, and 20, which are notable for their atypical size and intron-exon structure. Two of the encoded proteins are structurally most like the slow-beta MyHC, whereas the third one is closest to the adult fast IIb isoform. Data from pairwise comparisons of aligned coding sequences imply the existence of ancestral genomes with four sarcomeric genes before the emergence of a dedicated smooth muscle MyHC gene. To further address the evolutionary relationships of the distinct sarcomeric and nonsarcomeric rod sequences, we have identified and further annotated human genomic DNA sequences corresponding to 14 class-II MyHCs. An extensive analysis provides a timeline for intron gain and loss, gene contraction and expansion, and gene conversion among genes encoding class-II myosins. One of the novel human genes is found to have introns at positions shared only with the molluscan catchin/MyHC gene, providing evidence for the structure of a pre-Cambrian ancestral gene.     

An Integrated Strategy to Study Muscle Development and Myofilament Structure in Caenorhabditis elegans

A crucial step in the development of muscle cells in all metazoan animals is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, many of the critical proteins involved in this process have been uncovered through mutational screens focusing on uncoordinated movement and embryonic arrest phenotypes. We propose that additional sarcomeric proteins exist for which there is a less severe, or entirely different, mutant phenotype produced in their absence. We have used Serial Analysis of Gene Expression (SAGE) to generate a comprehensive profile of late embryonic muscle gene expression. We generated two replicate long SAGE libraries for sorted embryonic muscle cells, identifying 7,974 protein-coding genes. A refined list of 3,577 genes expressed in muscle cells was compiled from the overlap between our SAGE data and available microarray data. Using the genes in our refined list, we have performed two separate RNA interference (RNAi) screens to identify novel genes that play a role in sarcomere assembly and/or maintenance in either embryonic or adult muscle. To identify muscle defects in embryos, we screened specifically for the Pat embryonic arrest phenotype. To visualize muscle defects in adult animals, we fed dsRNA to worms producing a GFP-tagged myosin protein, thus allowing us to analyze their myofilament organization under gene knockdown conditions using fluorescence microscopy. By eliminating or severely reducing the expression of 3,300 genes using RNAi, we identified 122 genes necessary for proper myofilament organization, 108 of which are genes without a previously characterized role in muscle. Many of the genes affecting sarcomere integrity have human homologs for which little or nothing is known.     

Comparative sequence analysis of the complete human sarcomeric myosin heavy chain family: implications for functional diversity.

The conventional myosin motor proteins that drive mammalian skeletal and cardiac muscle contraction include eight sarcomeric myosin heavy chain (MyHC) isoforms. Six skeletal MyHCs are encoded by genes found in tightly linked clusters on human and mouse chromosomes 17 and 11, respectively. The full coding regions of only two out of six mammalian skeletal MyHCs had been sequenced prior to this work. In an effort to assess the extent of sequence diversity within the human MyHC family we present new full-length coding sequences corresponding to four additional human genes: MyHC-IIb, MyHC-extraocular, MyHC-IIa and MyHC-IIx/d. This represents the first opportunity to compare the full coding sequences of all eight sarcomeric MyHC isoforms within a vertebrate organism. Sequence variability has been analyzed in the context of available structure/function data with an emphasis on potential functional diversity within the family. Results indicate that functional diversity among MyHCs is likely to be accomplished by having small pockets of sequence diversity in an otherwise highly conserved molecule.     

Biochemical and cell biological analysis of actin in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has long been a useful model organism for muscle research. Its body wall muscle is obliquely striated muscle and exhibits structural similarities with vertebrate striated muscle. Actin is the core component of the muscle thin filaments, which are highly ordered in sarcomeric structures in striated muscle. Genetic studies have identified genes that regulate proper organization and function of actin filaments in C. elegans muscle, and sequence of the worm genome has revealed a number of conserved candidate genes that may regulate actin. To precisely understand the functions of actin-binding proteins, such genetic and genomic studies need to be complemented by biochemical characterization of these actin-binding proteins in vitro. This article describes methods for purification and biochemical characterization of actin from C. elegans. Although rabbit muscle actin is commonly used to characterize actin-binding proteins from many eukaryotic organisms, we detect several quantitative differences between C. elegans actin and rabbit muscle actin, highlighting that use of actin from an appropriate source is important in some cases. Additionally, we describe probes for cell biological analysis of actin in C. elegans.     

An adhesion molecule involved in muscular dystrophies: structural and functional analysis of dystroglycan domains

Dystroglycan (DG) plays a pivotal role within the dystrophin-glycoprotein complex (DGC) which represents a major factor for muscle fibre stability upon contraction. It has been shown that many muscular dystrophy phenotypes are caused by mutations of proteins belonging to or being associated with the DGC. Due to its prominent role for muscle stability, the detailed knowledge of DG structural and functional aspects should be considered of primary importance in order to develop new treatments for neuromuscular diseases.     

Familial hypertrophic cardiomyopathy: diagnostic and therapeutic implications of recent genetic studies

Familial hypertrophic cardiomyopathy is the first primary cardiomyopathy to have yielded to the techniques of modern molecular genetics. In the past few years, four genes responsible for this disease have been identified, all of which code for sarcomeric structural proteins. In addition, structure—function analysis and genotype—phenotype correlation studies have shed significant light on the molecular basis of this disease. It is hoped that within the next few years the application of molecular genetic tools will not only facilitate the diagnosis of hypertrophic cardiomyopathy but will also provide prognostic and therapeutic stratification for more definitive therapy.     

The pathogenesis of familial hypertrophic cardiomyopathy: early and evolving effects from an alpha-cardiac myosin heavy chain missense mutation

Familial hypertrophic cardiomyopathy (FHC) is a genetic disorder resulting from mutations in genes encoding sarcomeric proteins. This typically induces hyperdynamic ejection, impaired relaxation, delayed early filling, myocyte disarray and fibrosis, and increased chamber end-systolic stiffness. To better understand the disease pathogenesis, early (primary) abnormalities must be distinguished from evolving responses to the genetic defect. We did in vivo analysis using a mouse model of FHC with an Arg403Gln alpha-cardiac myosin heavy chain missense mutation, and used newly developed methods for assessing in situ pressure-volume relations. Hearts of young mutant mice (6 weeks old), which show no chamber morphologic or gross histologic abnormalities, had altered contraction kinetics, with considerably delayed pressure relaxation and chamber filling, yet accelerated systolic pressure rise. Older mutant mice (20 weeks old), which develop fiber disarray and fibrosis, had diastolic and systolic kinetic changes similar to if not slightly less than those of younger mice. However, the hearts of older mutant mice also showed hyperdynamic contraction, with increased end-systolic chamber stiffness, outflow tract pressure gradients and a lower cardiac index due to reduced chamber filling; all 'hallmarks' of human disease. These data provide new insights into the temporal evolution of FHC. Such data may help direct new therapeutic strategies to diminish disease progression.     

Myosin binding protein C： Structural abnormalities in familial hypertrophic cardiomyopathy

The muscle protein myosin binding protein C (MyBPC) is a large multi-domain protein whose role in the sarcomere is complex and not yet fully understood. Mutations in MyBPC are strongly associated with the heart disease familial hypertrophic cardiomyopathy (FHC) and these experiments of nature have provided some insight into the intricate workings of this protein in the heart. While some regions of the MyBPC molecule have been assigned a function in the regulation of muscle contraction, the interaction of other regions with various parts of the myosin molecule and the sarcomeric proteins, actin and titin, remain obscure. In addition, several intra-domain interactions between adjacent MyBPC molecules have been identified. Although the basic structure of the molecule (a series of immunoglobulin and fibronectin domains) has been elucidated, the assembly of MyBPC in the sarcomere is a topic for debate. By analysing the MyBPC sequence with respect to FHC-causing mutations it is possible to identify individual residues or regions of each domain that may be important either for binding or regulation. This review looks at the current literature, in concert with alignments and the structural models of MyBPC, in an attempt to understand how FHC mutations may lead to the disease state.     

Quantitative proteomics profiling of sarcomere associated proteins in limb and extraocular muscle allotypes

The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only allow the analysis of a small fraction of proteins at a time. Here we introduce a quantitative label-free, shotgun proteomics approach to differentially quantitate sarcomeric proteins from microgram quantities of muscle tissue in a fast and reliable manner by liquid chromatography and mass spectrometry. The high sequence similarity of some sarcomeric proteins poses a problem for shotgun proteomics because of limitations in subsequent database search algorithms in the exclusive assignment of peptides to specific isoforms. Therefore multiple sequence alignments were generated to improve the identification of isoform specific peptides. This methodology was used to compare the sarcomeric proteome of the extraocular muscle allotype to limb muscle. Extraocular muscles are a unique group of highly specialized muscles with distinct biochemical, physiological, and pathological properties. We were able to quantitate 40 sarcomeric proteins; although the basic sarcomeric proteins in extraocular muscle are similar to those in limb muscle, key proteins stabilizing the connection of the Z-bands to thin filaments and the costamere are augmented in extraocular muscle and may represent an adaptation to the eccentric contractions known to normally occur during eye movements. Furthermore, a number of changes are seen that closely relate to the unique nature of extraocular muscle.     

Intermediate filaments in cardiomyopathy

Intermediate filament (IF) proteins are critical regulators in health and disease. The discovery of hundreds of mutations in IF genes and posttranslational modifications has been linked to a plethora of human diseases, including, among others, cardiomyopathies, muscular dystrophies, progeria, blistering diseases of the epidermis, and neurodegenerative diseases. The major IF proteins that have been linked to cardiomyopathies and heart failure are the muscle-specific cytoskeletal IF protein desmin and the nuclear IF protein lamin, as a subgroup of the known desminopathies and laminopathies, respectively. The studies so far, both with healthy and diseased heart, have demonstrated the importance of these IF protein networks in intracellular and intercellular integration of structure and function, mechanotransduction and gene activation, cardiomyocyte differentiation and survival, mitochondrial homeostasis, and regulation of metabolism. The high coordination of all these processes is obviously of great importance for the maintenance of proper, life-lasting, and continuous contraction of this highly organized cardiac striated muscle and consequently a healthy heart. In this review, we will cover most known information on the role of IFs in the above processes and how their deficiency or disruption leads to cardiomyopathy and heart failure.     

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

Many genetic mutations in sarcomeric proteins, including the cardiac myosin regulatory light chain (RLC) encoded by the MYL2 gene, have been implicated in familial cardiomyopathies. Yet, the molecular mechanisms by which these mutant proteins regulate cardiac muscle mechanics in health and disease remain poorly understood. Evidence has been accumulating that RLC phosphorylation has an influential role in striated muscle contraction and, in addition to the conventional modulation via Ca²⁺ binding to troponin C, it can regulate cardiac muscle function. In this review, we focus on RLC mutations that have been reported to cause cardiomyopathy phenotypes via compromised RLC phosphorylation and elaborate on pseudo-phosphorylation rescue mechanisms. This new methodology has been discussed as an emerging exploratory tool to understand the role of phosphorylation as well as a genetic modality to prevent/rescue cardiomyopathy phenotypes. Finally, we summarize structural effects post-phosphorylation, a phenomenon that leads to an ordered shift in the myosin S1 and RLC conformational equilibrium between two distinct states.     

Structural analysis of the titin gene in hypertrophic cardiomyopathy: identification of a novel disease gene.

Hypertrophic cardiomyopathy (HCM) is characterized by ventricular hypertrophy accompanied by myofibrillar disarrays. Molecular genetic analyses have revealed that mutations in 8 different genes cause HCM. Mutations in these disease genes, however, could be found in about half of HCM patients, suggesting that there are other unknown disease gene(s). Because the known disease genes encode sarcomeric proteins expressed in the cardiac muscle, we searched for a disease-associated mutation in the titin gene in 82 HCM patients who had no mutation in the known disease genes. A G to T transversion in codon 740, from CGC to CTC, replacing Arginine with Leucine was found in a patient. This mutation was not found in more than 500 normal chromosomes and increased the binding affinity of titin to alpha-actitin in the yeast two-hybrid assay. These observations suggest that the titin mutation may cause HCM in this patient via altered affinity to alpha-actinin.     

Genetic variation in angiotensin-converting enzyme 2 gene is associated with extent of left ventricular hypertrophy in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy, a common, inherited cardiac muscle disease, is primarily caused by mutations in sarcomeric protein-encoding genes and is characterized by overgrowth of ventricular muscle that is highly variable in extent and location. This variability has been partially attributed to locus and allelic heterogeneity of the disease-causing gene, but other factors, including unknown genetic factors, also modulate the extent of hypertrophy that develops in response to the defective sarcomeric functioning. Components of the renin-angiotensin-aldosterone system are plausible candidate hypertrophy modifiers because of their role in controlling blood pressure and biological effects on cardiomyocyte hypertrophy.     

Molecular Modeling of Disease Causing Mutations in Domain C1 of cMyBP-C

Cardiac myosin binding protein-C (cMyBP-C) is a multi-domain (C0–C10) protein that regulates heart muscle contraction through interaction with myosin, actin and other sarcomeric proteins. Several mutations of this protein cause familial hypertrophic cardiomyopathy (HCM). Domain C1 of cMyBP-C plays a central role in protein interactions with actin and myosin. Here, we studied structure-function relationship of three disease causing mutations, Arg177His, Ala216Thr and Glu258Lys of the domain C1 using computational biology techniques with its available X-ray crystal structure. The results suggest that each mutation could affect structural properties of the domain C1, and hence it’s structural integrity through modifying intra-molecular arrangements in a distinct mode. The mutations also change surface charge distributions, which could impact the binding of C1 with other sarcomeric proteins thereby affecting contractile function. These structural consequences of the C1 mutants could be valuable to understand the molecular mechanisms for the disease.     

Degradation of sarcomeric and cytoskeletal proteins in cultured skeletal muscle cells

The goal of this research was to evaluate the roles of calpains and their interactions with the proteasome and the lysosome in degradation of individual sarcomeric and cytoskeletal proteins in cultured muscle cells. Rat L8-CID muscle cells, in which we expressed a transgene calpain inhibitor (CID), were used in the study. L8-CID cells were grown as myotubes after which the relative roles of calpain, proteasome and lysosome in total protein degradation were assessed during a period of serum withdrawal. Following this, the roles of proteases in degrading cytoskeletal proteins (desmin, dystrophin and filamin) and of sarcomeric proteins (alpha-actinin and tropomyosin) were assessed. Total protein degradation was assessed by release of radioactive tyrosine from pre-labeled myotubes in the presence and absence of protease inhibitors. Effects of protease inhibitors on concentrations of individual sarcomeric and cytoskeletal proteins were assessed by Western blotting. Inhibition of calpains, proteasome and lysosome caused 20, 62 and 40% reductions in total protein degradation (P<0.05), respectively. Therefore, these three systems account for the bulk of degradation in cultured muscle cells. Two cytoskeletal proteins were highly-sensitive to inhibition of their degradation. Specifically, desmin and dystrophin concentrations increased markedly when calpain, proteasome and lysosome activities were inhibited. Conversely, sarcomeric proteins (alpha-actinin and tropomyosin) and filamin were relatively insensitive to the addition of protease inhibitors to culture media. These data demonstrate that proteolytic systems work in tandem to degrade cytoskeletal and sarcomeric protein complexes and that the cytoskeleton is more sensitive to inhibition of degradation than the sarcomere. Mechanisms, which bring about changes in the activities of the proteases, which mediate muscle protein degradation are not known and represent the next frontier of understanding needed in muscle wasting diseases and in muscle growth biology.