Is the fundamental pathology in Duchenne's muscular dystrophy caused by a failure of glycogenolysis-glycolysis in costameres?

Duchenne muscular dystrophy (DMD) is the most common form of progressive childhood muscular dystrophy associated with weakness of limbs, loss of ambulation, heart weakness and early death. The mutations causing either loss-of-expression or function of the full-length protein dystrophin (Dp427) from the DMD gene are responsible for the disease pathology. Dp427 forms a part of the large dystroglycan complex, called DAPC, in the sarcolemma, and its absence derails muscle contraction. Muscle biopsies from DMD patients show an overactivation of excitation-contraction-coupling (ECC) activable calcium incursion, sarcolemmal ROS production, NHE1 activation, IL6 secretion, etc. The signalling pathways, like Akt/PBK, STAT3, p38MAPK, and ERK1/2, are also hyperactive in DMD. These pathways are responsible for post-mitotic trophic growth and metabolic adaptation, in response to exercise in healthy muscles, but cause atrophy and cell death in dystrophic muscles. We hypothesize that the metabolic background of repressed glycolysis in DMD, as opposed to excess glycolysis seen in cancers or healthy contracting muscles, changes the outcome of these 'growth pathways'. The reduced glycolysis has been considered a secondary outcome of the cytoskeletal disruptions seen in DMD. Given the cytoskeleton-crosslinking ability of the glycolytic enzymes, we hypothesize that the failure of glycogenolytic and glycolytic enzymes to congregate is the primary pathology, which then affects the subsarcolemmal cytoskeletal organization in costameres and initiates the pathophysiology associated with DMD, giving rise to the tissue-specific differences in disease progression between muscle, heart and brain. The lacunae in the regulation of the key components of the hypothesized metabolome, and the limitations of this theory are deliberated. The considerations for developing future therapies based on known pathological processes are also discussed.

Subcellular Remodeling in Filamin C Deficient Mouse Hearts Impairs Myocyte Tension Development during Progression of Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is a life-threatening form of heart disease that is typically characterized by progressive thinning of the ventricular walls, chamber dilation, and systolic dysfunction. Multiple mutations in the gene encoding filamin C (FLNC), an actin-binding cytoskeletal protein in cardiomyocytes, have been found in patients with DCM. However, the mechanisms that lead to contractile impairment and DCM in patients with FLNC variants are poorly understood. To determine how FLNC regulates systolic force transmission and DCM remodeling, we used an inducible, cardiac-specific FLNC-knockout (icKO) model to produce a rapid onset of DCM in adult mice. Loss of FLNC reduced systolic force development in single cardiomyocytes and isolated papillary muscles but did not affect twitch kinetics or calcium transients. Electron and immunofluorescence microscopy showed significant defects in Z-disk alignment in icKO mice and altered myofilament lattice geometry. Moreover, a loss of FLNC induces a softening myocyte cortex and structural adaptations at the subcellular level that contribute to disrupted longitudinal force production during contraction. Spatially explicit computational models showed that these structural defects could be explained by a loss of inter-myofibril elastic coupling at the Z-disk. Our work identifies FLNC as a key regulator of the multiscale ultrastructure of cardiomyocytes and therefore plays an important role in maintaining systolic mechanotransmission pathways, the dysfunction of which may be key in driving progressive DCM.

Costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for Limb-girdle muscular dystrophy type 2H.

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of ßPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and ß-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration.

Focal adhesion kinase coordinates costamere-related JNK signaling with muscle fiber transformation after Achilles tenotomy and tendon reconstruction.

Achilles tendon rupture necessitates rapid tendon reattachment to reinstate plantar flexion before affected muscles deteriorate through muscle fiber atrophy and transformation. The implicated process may involve alterations in sarcolemmal sites of myofibril attachment (costameres), which control myofibrillogenesis via a mechano-regulated mechanism through integrin-associated focal adhesion kinase (FAK). We assessed the contribution of FAK to alterations in fiber type composition and expression of costamere-associated structural proteins, the phosphorylation status of Y397-FAK and downstream mTOR/JNK-P70S6K hypertrophy signaling in rat soleus muscle after Achilles tenotomy and tendon repair. Achilles tenotomy induced a profound deterioration of muscle composition 14 days, but not 4 days, following tendon release, comprising specifically increased area percentages of fast type fibers, fibers with internal nuclei, and connective tissue. Concomitantly, expression of costameric proteins FAK and meta-vinculin, and phosphorylation of T421/S424-P70S6K and T183/Y185-JNK was elevated, all of which was mitigated by tendon reattachment immediately after release. Overexpression of FAK in soleus muscle fibers and reattachment corrected the expression of meta- and gamma-vinculin isoforms to the lower levels in mock controls while further enhancing T183/Y185-JNK phosphorylation and levels of FAK C-terminus-related inhibitory proteins. Co-overexpression of the FAK inhibitor, FRNK, lowered FAK-overexpression driven Y397-FAK phosphorylation and T183/Y185-JNK phosphorylation. FAK levels correlated to molecular and cellular hallmarks of fiber degeneration. The findings demarcate the window between 4 and 14 days after tenotomy as costamere-dependent muscle transformation process, and expose that FAK overexpression prevents molecular aspects of the pathology which within the study limitations does not result in the mitigation of muscle fiber degeneration.250 words.

Evidence for skeletal muscle fiber type-specific expressions of mechanosensors.

Mechanosensors govern muscle tissue integrity and constitute a subcellular structure known as costameres. Costameres physically link the muscle extracellular matrix to contractile and signaling 'hubs' inside muscle fibers mainly via integrins and are localized beneath sarcolemmas of muscle fibers. Costameres are the main mechanosensors converting mechanical cues into biological events. However, the fiber type-specific costamere architecture in muscles is unexplored. We hypothesized that fiber types differ in the expression of genes coding for costamere components. By coupling laser microdissection to a multiplex tandem qPCR approach, we demonstrate that type 1 and type 2 fibers indeed show substantial differences in their mechanosensor complexes. We confirmed these data by fiber type population-specific protein analysis and confocal microscopy-based localization studies. We further show that knockdown of the costamere gene integrin-linked kinase (Ilk) in muscle precursor cells results in significantly increased slow-myosin-coding Myh7 gene, while the fast-myosin-coding genes Myh1, Myh2, and Myh4 are downregulated. In parallel, protein synthesis-enhancing signaling molecules (p-mTORSer2448, p < 0.05; p-P70S6KThr389, tendency with p < 0.1) were reduced upon Ilk knockdown. However, overexpression of slow type-inducing NFATc1 in muscle precursor cells did not change Ilk or other costamere gene expressions. In addition, we demonstrate fiber type-specific costamere gene regulation upon mechanical loading and unloading conditions. Our data imply that costamere genes, such as Ilk, are involved in the control of muscle fiber characteristics. Further, they identify costameres as muscle fiber type-specific loading management 'hubs' and may explain adaptation differences of muscle fiber types to mechanical (un)loading.

Costamere protein expression and tissue composition of rotator cuff muscle after tendon release in sheep.

Previous studies suggested that degradation of contractile tissue requires cleavage of the costamere, a structural protein complex that holds sarcomeres in place. This study examined if costamere turnover is affected by a rotator cuff tear in a previously established ovine model. We found the activity of focal adhesion kinase (FAK), a main regulator of costamere turnover, was unchanged at 2 weeks but decreased by 27% 16 weeks after surgical release of the infraspinatus tendon. This was accompanied by cleavage of the costamere protein talin into a 190 kDa fragment while full length talin remained unchanged. At 2 weeks after tendon release, muscle volume decreased by 17 cm3 from an initial 185 cm3 , the fatty tissue volume was halved, and the contractile tissue volume remained unchanged. After 16 weeks, the muscle volume decreased by 36 cm3 , contractile tissue was quantitatively lost, and the fat content increased by 184%. Nandrolone administration mitigated the loss of contractile tissue by 26% and prevented fat accumulation, alterations in FAK activity, and talin cleavage. Taken together, these findings imply that muscle remodeling after tendon release occurs in two stages. The early decrease of muscle volume is associated with reduction of fat; while, the second stage is characterized by substantial loss of contractile tissue accompanied by massive fat accumulation. Regulation of costamere turnover is associated with the loss of contractile tissue and seems to be impacted by nandrolone treatment. Clinically, the costamere may represent a potential intervention target to mitigate muscle loss after a rotator cuff tear. © 2017 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 36:272-281, 2018.

Mechanical Regulation of Cardiac Aging in Model Systems.

Unlike diet and exercise, which individuals can modulate according to their lifestyle, aging is unavoidable. With normal or healthy aging, the heart undergoes extensive vascular, cellular, and interstitial molecular changes that result in stiffer less compliant hearts that experience a general decline in organ function. Although these molecular changes deemed cardiac remodeling were once thought to be concomitant with advanced cardiovascular disease, they can be found in patients without manifestation of clinical disease. It is now mostly acknowledged that these age-related mechanical changes confer vulnerability of the heart to cardiovascular stresses associated with disease, such as hypertension and atherosclerosis. However, recent studies have aimed at differentiating the initial compensatory changes that occur within the heart with age to maintain contractile function from the maladaptive responses associated with disease. This work has identified new targets to improve cardiac function during aging. Spanning invertebrate to vertebrate models, we use this review to delineate some hallmarks of physiological versus pathological remodeling that occur in the cardiomyocyte and its microenvironment, focusing especially on the mechanical changes that occur within the sarcomere, intercalated disc, costamere, and extracellular matrix.

TGF-ß receptor type II costameric localization in cardiomyocytes and host cell TGF-ß response is disrupted by Trypanosoma cruzi infection.

Transforming growth factor beta (TGF-ß) cytokine is involved in Chagas disease establishment and progression. Since Trypanosoma cruzi can modulate host cell receptors, we analysed the TGF-ß receptor type II (TßRII) expression and distribution during T. cruzi - cardiomyocyte interaction. TßRII immunofluorescent staining revealed a striated organization in cardiomyocytes, which was co-localized with vinculin costameres and enhanced (38%) after TGF-ß treatment. Cytochalasin D induced a decrease of 45·3% in the ratio of cardiomyocytes presenting TßRII striations, demonstrating an association of TßRII with the cytoskeleton. Western blot analysis showed that cytochalasin D significantly inhibited Smad 2 phosphorylation and fibronectin stimulation after TGF-ß treatment in cardiomyocytes. Trypanosoma cruzi infection elicited a decrease of 79·8% in the frequency of cardiomyocytes presenting TßRII striations, but did not interfere significantly in its expression. In addition, T. cruzi-infected cardiomyocytes present a lower response to exogenous TGF-ß, showing no enhancement of TßRII striations and a reduction of phosphorylated Smad 2, with no significant difference in TßRII expression when compared to uninfected cells. Together, these results suggest that the co-localization of TßRII with costameres is important in activating the TGF-ß signalling cascade, and that T. cruzi-derived cytoskeleton disorganization could result in altered or low TGF-ß response in infected cardiomyocytes.

Costamere proteins and their involvement in myopathic processes.

Muscle fibres are very specialised cells with a complex structure that requires a high level of organisation of the constituent proteins. For muscle contraction to function properly, there is a need for not only sarcomeres, the contractile structures of the muscle fibre, but also costameres. These are supramolecular structures associated with the sarcolemma that allow muscle adhesion to the extracellular matrix. They are composed of protein complexes that interact and whose functions include maintaining cell structure and signal transduction mediated by their constituent proteins. It is important to improve our understanding of these structures, as mutations in various genes that code for costamere proteins cause many types of muscular dystrophy. In this review, we provide a description of costameres detailing each of their constituent proteins, such as dystrophin, dystrobrevin, syntrophin, sarcoglycans, dystroglycans, vinculin, talin, integrins, desmin, plectin, etc. We describe as well the diseases associated with deficiency thereof, providing a general overview of their importance.

Myopathic changes in murine skeletal muscle lacking synemin.

Diseases of striated muscle linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus and its links to costameres, which connect the sarcomeres to the cell membrane. Here we study the role in skeletal muscle of synemin, a type IV IF protein, by examining mice null for synemin (synm-null). Synm-null mice have a mild skeletal muscle phenotype. Tibialis anterior (TA) muscles show a significant decrease in mean fiber diameter, a decrease in twitch and tetanic force, and an increase in susceptibility to injury caused by lengthening contractions. Organization of proteins associated with the contractile apparatus and costameres is not significantly altered in the synm-null. Elastimetry of the sarcolemma and associated contractile apparatus in extensor digitorum longus myofibers reveals a reduction in tension consistent with an increase in sarcolemmal deformability. Although fatigue after repeated isometric contractions is more marked in TA muscles of synm-null mice, the ability of the mice to run uphill on a treadmill is similar to controls. Our results suggest that synemin contributes to linkage between costameres and the contractile apparatus and that the absence of synemin results in decreased fiber size and increased sarcolemmal deformability and susceptibility to injury. Thus synemin plays a moderate but distinct role in fast twitch skeletal muscle.

Distribution of formins in cardiac muscle: FHOD1 is a component of intercalated discs and costameres.

The formin homology domain-containing protein1 (FHOD1) suppresses actin polymerization by inhibiting nucleation, but bundles actin filaments and caps filament barbed ends. Two polyclonal antibodies against FHOD1 were generated against (i) its N-terminal sequence (residues 1-339) and (ii) a peptide corresponding the sequence from position 358-371, which is unique for FHOD1 and does not occur in its close relative FHOD3. After affinity purification both antibodies specifically stain purified full length FHOD1 and a band of similar molecular mass in homogenates of cardiac muscle. The antibody against the N-terminus of FHOD1 was used for immunostaining cells of established lines, primary neonatal (NRC) and adult (ARC) rat cardiomyocytes and demonstrated the presence of FHOD1 in HeLa and fibroblastic cells along stress fibers and within presumed lamellipodia and actin arcs. In NRCs and ARCs we observed a prominent staining of presumed intercalated discs (ICD). Immunostaining of sections of hearts with both anti-FHOD1 antibodies confirmed the presence of FHOD1 in ICDs and double immunostaining demonstrated its colocalisation with cadherin, plakoglobin and a probably slightly shifted localization to connexin43. Similarly, immunostaining of isolated mouse or pig ICDs corroborated the presence of FHOD1 and its colocalisation with the mentioned cell junctional components. Anti-FHOD1 immunoblots of isolated ICDs demonstrated the presence of an immunoreactive band comigrating with purified FHOD1. Conversely, an anti-peptide antibody specific for FHOD3 with no cross-reactivity against FHOD1 immunostained on sections of cardiac muscle and ARCs the myofibrils in a cross-striated pattern but not the ICDs. In addition, the anti-peptide-FHOD1 antibody stained the lateral sarcolemma of ARCs in a banded pattern. Double immunostaining with anti-cadherin and -integrin-ß1 indicated the additional localization of FHOD1 in costameres. Immunostaining of cardiac muscle sections or ARCs with antibodies against mDia3-FH2-domain showed colocalisation with cadherin along the lateral border of cardiomyocytes suggesting also its presence in costameres.

The Src homology and collagen A (ShcA) adaptor protein is required for the spatial organization of the costamere/Z-disk network during heart development.

Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca(2+)/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere.

Early changes in costameric and mitochondrial protein expression with unloading are muscle specific.

We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS) and subjected to fibre typing and measures for costameric (FAK and FRNK), mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1), and MHCI protein and RNA content. Mean cross-sectional area (MCSA) of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05 ≤ P < 0.10). FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P = 0.029). SOL muscle demonstrated a specific reduction in UCP3 content (-23%; P = 0.012). Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

Actin scaffolding by clathrin heavy chain is required for skeletal muscle sarcomere organization.

The ubiquitous clathrin heavy chain (CHC), the main component of clathrin-coated vesicles, is well characterized for its role in intracellular membrane traffic and endocytosis from the plasma membrane (PM). Here, we demonstrate that in skeletal muscle CHC regulates the formation and maintenance of PM-sarcomere attachment sites also known as costameres. We show that clathrin forms large coated lattices associated with actin filaments and the muscle-specific isoform of α-actinin at the PM of differentiated myotubes. Depletion of CHC in myotubes induced a loss of actin and α-actinin sarcomeric organization, whereas CHC depletion in vivo induced a loss of contractile force due to the detachment of sarcomeres from the PM. Our results suggest that CHC contributes to the formation and maintenance of the contractile apparatus through interactions with costameric proteins and highlight an unconventional role for CHC in skeletal muscle that may be relevant to pathophysiology of neuromuscular disorders.

Unc45b is essential for early myofibrillogenesis and costamere formation in zebrafish.

Despite the prevalence of developmental myopathies resulting from muscle fiber defects, the earliest stages of myogenesis remain poorly understood. Unc45b is a molecular chaperone that mediates the folding of thick-filament myosin during sarcomere formation; however, Unc45b may also mediate specific functions of non-muscle myosins (NMMs). unc45b Mutants have specific defects in striated muscle development, which include myocyte detachment indicative of dysfunctional adhesion complex formation. Given the necessity for non-muscle myosin function in the formation of adhesion complexes and premyofibril templates, we tested the hypothesis that the unc45b mutant phenotype is not mediated solely by interaction with muscle myosin heavy chain (mMHC). We used the advantages of a transparent zebrafish embryo to determine the temporal and spatial patterns of expression for unc45b, non-muscle myosins and mMHC in developing somites. We also examined the formation of myocyte attachment complexes (costameres) in wild-type and unc45b mutant embryos. Our results demonstrate co-expression and co-regulation of Unc45b and NMM in myogenic tissue several hours before any muscle myosin heavy chain is expressed. We also note deficiencies in the localization of costamere components and NMM in unc45b mutants that is consistent with an NMM-mediated role for Unc45b during early myogenesis. This represents a novel role for Unc45b in the earliest stages of muscle development that is independent of muscle mMHC folding.

Transcriptional networks regulating the costamere, sarcomere, and other cytoskeletal structures in striated muscle.

Structural abnormalities in striated muscle have been observed in numerous transcription factor gain- and loss-of-function phenotypes in animal and cell culture model systems, indicating that transcription is important in regulating the cytoarchitecture. While most characterized cytoarchitectural defects are largely indistinguishable by histological and ultrastructural criteria, analysis of dysregulated gene expression in each mutant phenotype has yielded valuable information regarding specific structural gene programs that may be uniquely controlled by each of these transcription factors. Linking the formation and maintenance of each subcellular structure or subset of proteins within a cytoskeletal compartment to an overlapping but distinct transcription factor cohort may enable striated muscle to control cytoarchitectural function in an efficient and specific manner. Here we summarize the available evidence that connects transcription factors, those with established roles in striated muscle such as MEF2 and SRF, as well as other non-muscle transcription factors, to the regulation of a defined cytoskeletal structure. The notion that genes encoding proteins localized to the same subcellular compartment are coordinately transcriptionally regulated may prompt rationally designed approaches that target specific transcription factor pathways to correct structural defects in muscle disease.

Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity.

Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and ß-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of ß-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity.

Cardiac-specific deletion of the microtubule-binding protein CENP-F causes dilated cardiomyopathy.

CENP-F is a large multifunctional protein with demonstrated regulatory roles in cell proliferation, vesicular transport and cell shape through its association with the microtubule (MT) network. Until now, analysis of CENP-F has been limited to in vitro analysis. Here, using a Cre-loxP system, we report the in vivo disruption of CENP-F gene function in murine cardiomyocytes, a cell type displaying high levels of CENP-F expression. Loss of CENP-F function in developing myocytes leads to decreased cell division, blunting of trabeculation and an initially smaller, thin-walled heart. Still, embryos are born at predicted mendelian ratios on an outbred background. After birth, hearts lacking CENP-F display disruption of their intercalated discs and loss of MT integrity particularly at the costamere; these two structures are essential for cell coupling/electrical conduction and force transduction in the heart. Inhibition of myocyte proliferation and cell coupling as well as loss of MT maintenance is consistent with previous reports of generalized CENP-F function in isolated cells. One hundred percent of these animals develop progressive dilated cardiomyopathy with heart block and scarring, and there is a 20% mortality rate. Importantly, although it has long been postulated that the MT cytoskeleton plays a role in the development of heart disease, this study is the first to reveal a direct genetic link between disruption of this network and cardiomyopathy. Finally, this study has broad implications for development and disease because CENP-F loss of function affects a diverse array of cell-type-specific activities in other organs.

The Mef2A transcription factor coordinately regulates a costamere gene program in cardiac muscle.

The Mef2 family of transcription factors regulates muscle differentiation, but the specific gene programs controlled by each member remain unknown. Characterization of Mef2A knock-out mice has revealed severe myofibrillar defects in cardiac muscle indicating a requirement for Mef2A in cytoarchitectural integrity. Through comprehensive expression analysis of Mef2A-deficient hearts, we identified a cohort of dysregulated genes whose products localize to the peripheral Z-disc/costamere region. Many of these genes are essential for costamere integrity and function. Here we demonstrate that these genes are directly regulated by Mef2A, establishing a mechanism by which Mef2A controls the costamere. In an independent model system, acute knockdown of Mef2A in primary neonatal cardiomyocytes resulted in profound malformations of myofibrils and focal adhesions accompanied by adhesion-dependent programmed cell death. These findings indicate a role for Mef2A in cardiomyocyte survival through regulation of costamere integrity. Finally, bioinformatics analysis identified over-represented transcription factor-binding sites in this network of costamere promoters that may provide insight into the mechanism by which costamere genes are regulated by Mef2A. The global control of costamere gene expression adds another dimension by which this essential macromolecular complex may be regulated in health and disease.

Phosphoinositide 3-kinase (PI3K(p110alpha)) directly regulates key components of the Z-disc and cardiac structure.

Maintenance of cardiac structure and Z-disc signaling are key factors responsible for protecting the heart in a setting of stress, but how these processes are regulated is not well defined. We recently demonstrated that PI3K(p110α) protects the heart against myocardial infarction. The aim of this study was to determine whether PI3K(p110α) directly regulates components of the Z-disc and cardiac structure. To address this question, a unique three-dimensional virtual muscle model was applied to gene expression data from transgenic mice with increased or decreased PI3K(p110α) activity under basal conditions (sham) and in a setting of myocardial infarction to display the location of structural proteins. Key findings from this analysis were then validated experimentally. The three-dimensional virtual muscle model visually highlighted reciprocally regulated transcripts associated with PI3K activation that encoded key components of the Z-disc and costamere, including melusin. Studies were performed to assess whether PI3K and melusin interact in the heart. Here, we identify a novel melusin-PI3K interaction that generates lipid kinase activity. The direct impact of PI3K(p110α) on myocyte structure was assessed by treating neonatal rat ventricular myocytes with PI3K(p110α) inhibitors and examining the myofiber morphology of hearts from PI3K transgenic mice. Results demonstrate that PI3K is critical for myofiber maturation and Z-disc alignment. In summary, PI3K regulates the expression of genes essential for cardiac structure and Z-disc signaling, interacts with melusin, and is critical for Z-disc alignment.