Myofibril Organization Research Articles

Coxsackievirus-adenovirus receptor (CAR) is essential for early embryonic cardiac development

The coxsackievirus-adenovirus receptor (CAR) is a cell contact protein on various cell types with unknown physiological function. It belongs to a subfamily of the immunoglobulin-superfamily of which some members are junctional adhesion molecules on epithelial and/or endothelial cells. CAR is dominantly expressed in the hearts and brains of mice until the newborne phase after which it becomes mainly restricted to various epithelial cells. To understand more about the physiological function of CAR, we have generated CAR-deficient mice by gene targeting. We found that these mice die between E11.5 and E13.5 of embryonal development. Ultrastructural analysis of cardiomyocytes revealed that the density of myofibrils was reduced and that their orientation and bundling was disorganized. In addition, mitochondria were enlarged and glycogen storage strongly enriched. In line with these defects, we observed pericardial edema formation as a clear sign of insufficient heart function. Developmental abnormalities likely to be secondary effects of gene ablation were the persistent singular cardial atrio-ventricular canal and dilatations of larger blood vessels such as the cardinal veins. The secondary nature of these defects was supported by the fact that CAR was not expressed on vascular cells or on cells of the vascular wall. No obvious signs for alterations of the histological organization of the placenta were observed. We conclude that CAR is required for embryonal heart development, most likely due to its function during the organization of myofibrils in cardiomyocytes.

RHO-ASSOCIATED KINASE MODULATES MYOCARDIAL INFLAMMATORY CYTOKINE RESPONSES

Rho, a Ser-Thr kinase identified as a member of the RAS GTPase super family, is highly expressed in the heart, and has been implicated in the development of heart failure. GTPase Rho is located downstream of Gq, and Rho and the associated kinase (Rho kinase) regulate myofibril organization, apoptosis, and myofibrillar sensitivity to calcium. Myocardial injury and dysfunction occur after major burn injury, and this phenomenon has been linked to cardiac myocyte synthesis and the secretion of proinflammatory cytokines. Whether Rho-associated kinase modulates any aspect of cardiomyocyte synthesis of inflammatory mediators, contributing to myocardial dysfunction, has not been studied and was the focus of this study. Hearts were collected at several times postburn to determine if an acute injury such as thermal trauma altered myocardial Rho kinase expression. In addition, cardiomyocytes were isolated (collagenase digestion) from adult control Sprague Dawley rats, plated (5 x 10 cells/microtiter well), incubated with medium alone or in the presence of burn serum (collected 24 h after burn over 40% total body surface area in rats) in a CO2 incubator at 37 degrees C in the presence/absence of specific Rho-kinase inhibitors (HA1077, 10 microM or Y27632, 10 microM). After 18 h, supernatants were collected to measure secreted cytokines (enzyme-linked immunoabsorbant assay), cells were loaded with Fura-2AM (2 microg) or sodium-binding benzofuran isophthalate (2 microg) for 45 min at 37 degrees C, and fluorescence was measured with an InCyt IM2 fluorescence imaging system to measure myocyte calcium and sodium. In parallel studies, cells were examined to determine if burn serum challenge increased Rho kinase in this cell population. In vivo burn injury or in vitro burn serum challenge of isolated myocytes increased Rho-kinase expression and promoted cardiomyocyte secretion of tumor necrosis factor-alpha, interleukin 1beta, and interleukin 6, and increased cardiomyocyte calcium and sodium levels compared with values measured when myocytes were incubated in medium alone (P < 0.05). Pretreating cardiomyocytes with Rho-kinase inhibitor (HA1077 or Y27632) prevented burn serum-related upregulation of Rho-kinase and attenuated the associated inflammatory cytokine responses, and attenuated myocyte calcium and sodium loading. Our data suggest that the Rho-kinase pathway is one potential upstream regulator of cardiac inflammatory response to burn injury.

Cross Talk between Cell–Cell and Cell–Matrix Adhesion Signaling Pathways during Heart Organogenesis: Implications for Cardiac Birth Defects

The anterior-posterior and dorsal-ventral progression of heart organogenesis is well illustrated by the patterning and activity of two members of different families of cell adhesion molecules: the calcium-dependent cadherins, specifically N-cadherin, and the extracellular matrix glycoproteins, fibronectin. N-cadherin by its binding to the intracellular molecule beta-catenin and fibronectin by its binding to integrins at focal adhesion sites, are involved in regulation of gene expression by their association with the cytoskeleton and through signal transduction pathways. The ventral precardiac mesoderm cells epithelialize and become stably committed by the activation of these cell-matrix and intracellular signaling transduction pathways. Cross talk between the adhesion signaling pathways initiates the characteristic phenotypic changes associated with cardiomyocyte differentiation: electrical activity and organization of myofibrils. The development of both organ form and function occurs within a short interval thereafter. Mutations in any of the interacting molecules, or environmental insults affecting either of these signaling pathways, can result in embryonic lethality or fetuses born with severe heart defects. As an example, we have defined that exposure of the embryo temporally to lithium during an early sensitive developmental period affects a canonical Wnt pathway leading to beta-catenin stabilization. Lithium exposure results in an anterior-posterior progression of severe cardiac defects.

Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

Anti‐sense‐mediated inhibition of expression of the novel striated tropomyosin isoform TPM1κ disrupts myofibril organization in embryonic axolotl hearts

Striated muscle tropomyosin (TM) is described as containing ten exons; 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b. Exon 9a/b has critical troponin binding domains and is found in striated muscle isoforms. We have recently discovered a smooth (exon 2a)/striated (exons 9a/b) isoform expressed in amphibian, avian, and mammalian hearts, designated as an isoform of the TPM1 gene (TPM1kappa). TPM1kappa expression was blocked in whole embryonic axolotl heart by transfection of exon-specific anti-sense oligonucleotide. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed lower transcript expression of TPM1kappa and in vitro analysis confirmed the specificity of the TPM1kappa anti-sense oligonucleotide. Altered expression of the novel TM isoform disrupted myofibril structure and function in embryonic hearts.

Diminished Myofibril Organization in Mutant Axolotl Hearts Transfected With Site-Directed Mutants of Sarcomeric Tropomyosins

We used a model lacking endogenous sarcomeric tropomyosin, the cardiac mutant of the Mexican axolotl, to examine the effect of mutant tropomyosins on sarcomeric myofibril formation. Previous studies have introduced wild-type mouse alpha-tropomyosin into mutant hearts in organ culture with subsequent for-mation of organized myofibrils. This study examines the predominant embry-onic axolotl TPM-4 type tropomyosin (TPM4alpha), containing a conservative re- placement of glutamic acid for aspartic acid at the clinically important 175 site. In this study, ATmC-3 (TPM4alpha) promoted formation of organized myofibrils in hearts without endogenous tropomyosin. Site-directed mutagenesis of 175 glutamic acid with 175 glutamine or 175 lysine was toxic to the formation of organized myofibrils in mutant hearts in the absence of endogenous tropo-myosin. Cationic liposome co-transfection of both wild-type tropomyosin and Glu175Gln.TPM4alpha cDNA formed organized myofibrils in mutant hearts. A construct with GFP.Glu175Gln.TPM4alpha cDNA was used to confirm expression of the mutant fusion protein. Mutation at the 175 site in TPM4alpha type or TPM1alpha (striated muscle isoform of the TPM1 gene) was sufficient to alter the protein such that organized myofibrils would not form in ventricles of mutant hearts without endogenous tropomyosin.

The heart ofSparus auratus: a reappraisal of cardiac functional morphology in teleosts

This morphodynamic study provides an insight on how the architecture of the heart ventricle of the gilthead seabream (Sparus auratus) is designed to accomplish the functional performance typical of an active teleost species. Using an in vitro working heart preparation, mechanical performance was analyzed under loading (i.e., preload and afterload) challenges. The hearts were very sensitive to filling pressure increases. Maximum cardiac output (CO: 55.66+/-4.54 ml/min/kg body weight; mean+/-SEM) and maximum stroke volume (VS: 0.42+/-0.027 ml/kg body weight; mean+/-SEM) were obtained at an input pressure of 1 kPa. When exposed to output pressure (OP) changes, the hearts maintained constant CO and SV up to about 4 kPa; further increases of afterload significantly compromised mechanical performance. Surprisingly, this "athletic" pumping performance was achieved by an entirely trabeculated pyramidal ventricle. The ventricular architecture was characterized by a system of small luminae and trabecular sheets radiating outward from the central lumen. The most peripheral part of the ventricular chamber contained single trabeculae and the corresponding lacunary spaces. The ventricular cavity was bounded by an outer myocardial monolayer "shell" to which the peripheral trabeculae were attached. Myofibril organization differed in the trabeculae and in the outer monolayer. The structural features challenge common beliefs regarding the typical "athletic" teleost heart design.

Cardiac Myofibril Formation Is Not Affected by Modification of Both N- and C-Termini of Sarcomeric Tropomyosin

Although the role of tropomyosin is well-defined in striated muscle, the precise mechanism of how tropomyosin functions is still unclear. It has been shown that extension of either N- or C-terminal ends of sarcomeric tropomyosin do not affect cardiac myofibrillogenesis, but it is not known whether simultaneous extension of both ends affects the process. For studying structural/functional relationships of sarcomeric tropomyosin, we have chosen the Ambystoma mexicanum because cardiac mutant hearts are deficient in sarcomeric tropomyosin. In this study, we have made an expression construct, pEGFP.TPM4alpha.E-L-FLAG, that, on transfection into normal and mutant axolotl hearts in organ culture, expresses GFP.TPM4alpha.E-L-FLAG fusion protein in which both the N- and C-termini of TPM4alpha are being extended. TPM4alpha is one of the three tropomyosins expressed in normal axolotl hearts. Both confocal and electron microscopic analyses show that this modified sarcomeric tropomyosin can form organized myofibrils in axolotl hearts.

Cardiomyocyte Apoptosis Triggered by RAFTK/pyk2 via Src Kinase Is Antagonized by Paxillin

Altered cellular adhesion and apoptotic signaling in cardiac remodeling requires coordinated regulation of multiple constituent proteins that comprise cytoskeletal focal adhesions. One such protein activated by cardiac remodeling is related adhesion focal tyrosine kinase (RAFTK, also known as pyk2). Adenoviral-mediated expression of RAFTK in neonatal rat cardiomyocytes involves concurrent increases in phosphorylation of Src, c-Jun N-terminal kinase, and p38 leading to characteristic apoptotic changes including cleavage of poly(ADP-ribose) polymerase, caspase-3 activation, and increased DNA laddering. DNA laddering was decreased by mutation of the Tyr(402) Src-binding site in RAFTK, suggesting a central role for Src activity in apoptotic cell death that was confirmed by adenoviral-mediated Src expression. Multiple apoptotic signaling cascades are recruited by RAFTK as demonstrated by prevention of apoptosis using caspase-3 inhibitor IV (caspase-3 specific inhibitor), PP2 (Src-specific kinase inhibitor), or Csk (cellular negative regulator for Src), as well as dominant negative constructs for p38beta or MKP-1. These RAFTK-mediated phenotypic characteristics are prevented by concurrent expression of wild-type or a phosphorylation-deficient paxillin mutated at Tyr(31) and Tyr(118). Wild-type or mutant paxillin protein accumulation in the cytoplasm has no overt effect upon cell structure, but paxillin accumulation prevents losses of myofibril organization as well as focal adhesion kinase, vinculin, and paxillin protein levels mediated by RAFTK. Apoptotic signaling cascade inhibition by paxillin indicates interruption of signaling proximal to but downstream of RAFTK activity. Chronic RAFTK activation in cardiac remodeling may represent a maladaptive reactive response that can be modulated by paxillin, opening up novel possibilities for inhibition of cardiomyocyte apoptosis and structural degeneration in heart failure.

Desminopathies in muscle disease

A recently identified class of myopathies is produced by abnormal desmin, and is characterized by a disorganization of the desmin filament network, the accumulation of insoluble desmin-containing aggregates, and destructive changes in the sarcomeric organization of striated muscles. The desmin filaments interact with various other cytoskeletal proteins. The distinct clinical phenotypes are heterogeneous, with progressive skeletal myopathy, cardiomyopathy, and respiratory insufficiency as the most prominent features. Most of the desmin mutations are autosomal dominant. Identification of the causal genetic mutations shows that the desmin gene is not the only gene implicated in desminopathies; other genes encoding desmin-associated proteins, such as alpha-B-crystallin, and synemin may also be involved. Patients with mutations in their alpha-B-crystallin gene, which produce similar skeletal and cardiac myopathies, also have opaque lenses. Knockout mice have helped to reveal the fundamental role of desmin filaments in cell architecture, sarcomere alignment, myofibril organization, and the distribution of mitochondria. Transgenic mice, which accumulate aggregates of desmin and associated proteins in their muscles, show that the loss of desmin intermediate function as a result of mutations in desmin itself, or in the desmin-associated constituents, is important for disease progression.

Mutations in the motor domain modulate myosin activity and myofibril organization.

We have investigated the functional impact on cardiac myofibril organization and myosin motor activity of point mutations associated with familial hypertrophic cardiomyopathies (FHC). Embryonic chicken cardiomyocytes were transfected with vectors encoding green fluorescent protein (GFP) fused to a striated muscle myosin heavy chain (GFP-myosin). Within 24 hours of transfection, the GFP-myosin is found co-assembled with the endogenous myosin in striated myofibrils. The wild-type GFP-myosin had no effect on the organization of the contractile cytoskeleton of the cardiomyocytes. However, expression of myosin with the R403Q FHC mutation resulted in a small but significant decrease in myofibril organization, and the R453C and G584R mutations caused a more dramatic increase in myofibril disarray. The embryonic cardiomyocytes beat spontaneously in culture and this was not affected by expression of the wild-type or mutant GFP-myosin. For the biochemical analysis of myosin motor activity, replication defective adenovirus was used to express the wild-type and mutant GFP-myosin in C2C12 myotubes. The R403Q mutation enhanced actin filament velocity but had no effect on the myosin duty ratio. The R453C and G584R mutations impaired actin filament movement and both increased the duty ratio. The effects of these mutations on myosin motor activity correlate with changes in myofibril organization of live cardiomyocytes. Thus, mutations associated with hypertrophic cardiomyopathies that alter myosin motor activity can also impair myofibril organization.

Contribution of Myosin Rod Protein to the Structural Organization of Adult and Embryonic Muscles in Drosophila

Myosin rod protein (MRP) is a naturally occurring 155 kDa protein in Drosophila that includes the myosin heavy chain (MHC) rod domain, but contains a unique 77 amino acid residue N-terminal region that replaces the motor and light chain-binding domains of S1. MRP is a major component of myofilaments in certain direct flight muscles (DFMs) and it is present in other somatic, cardiac and visceral muscles in adults, larvae and embryos, where it is coexpressed and polymerized into thick filaments along with MHC. DFM49 has a relatively high content of MRP, and is characterized by an unusually disordered myofibrillar ultrastructure, which has been attributed to lack of cross-bridges in the filament regions containing MRP. Here, we characterize in detail the structural organization of myofibrils in adult and embryonic Drosophila muscles containing various MRP/MHC ratios and in embryos carrying a null mutation for the single MHC gene. We examined MRP in embryonic body wall and intestinal muscles as well as in DFMs with consistent findings. In DFMs numbers 49, 53 and 55, MRP is expressed at a high level relative to MHC and is associated with disorder in the positioning of thin filaments relative to thick filaments in the areas of overlap. Embryos that express MRP in the absence of MHC form thick filaments that participate in the assembly of sarcomeres, suggesting that myofibrillogenesis does not depend on strong myosin–actin interactions. Further, although thick filaments are not well ordered, the relative positioning of thin filaments is fairly regular in MRP-only containing sarcomeres, confirming the hypothesis that the observed disorder in MRP/MHC containing wild-type muscles is due to the combined action between the functional behavior of MRP and MHC myosin heads. Our findings support the conclusion that MRP has an active function to modulate the contractile activity of muscles in which it is expressed.

The human adult cardiomyocyte phenotype.

Determination of the phenotype of adult human atrial and ventricular myocytes based on gene expression and morphology. Atrial and ventricular cardiomyocytes were obtained from patients undergoing cardiac surgery using a modified isolation procedure. Myocytes were isolated and cultured with or without serum. The relative cell attachment promoting efficiency of several reagents was evaluated and compared. Morphological changes during long-term culture were assessed with phase contrast microscopy, morphometric analysis and immunocytochemistry or RT-PCR of sarcomeric markers including alpha-actinin, myosin light chain-2 (MLC-2) and the adhesion molecule, cadherin. The isolation method produced viable rod-shaped atrial (16.6+/-6.0%, mean+/-S.E.; n=5) and ventricular cells (22.4+/-8.0%, mean+/-S.E.; n=5) in addition to significant numbers of apoptotic and necrotic cells. Cell dedifferentiation was characterized by the loss of sarcomeric structure, condensation and extrusion of sarcomeric proteins. Cells cultured with low serum recovered and assumed a flattened, spread form with two distinct morphologies apparent. Type I cells were large, had extensive sarcolemmal spreading, with stress fibers and nascent myofibrils, whilst type II cells appeared smaller, with more mature myofibril organisation and focal adhesions. Characterization of the redifferentiation capabilities of cultured adult cardiac myocytes in culture, provides an important system for comparing cardiomyocytes differentiating from human stem cells and provides the basis for an in vitro transplantation model to study interaction and communication between primary adult and stem cell-derived cardiomyocytes.

A point mutation in bioactive RNA results in the failure of mutant heart correction in Mexican axolotls.

Ambystoma mexicanum is an intriguing animal model for studying heart development because it carries a mutation in gene c. Hearts of homozygous recessive (c/c) mutant embryos do not contain organized myofibrils and fail to beat. The defect can be corrected by organ-culturing the mutant heart in the presence of RNA from anterior endoderm or endoderm/mesoderm-conditioned medium. By screening a cDNA library made of total conditioned medium RNA from normal axolotl embryonic endoderm, we isolated a single clone (MIR), the synthetic RNA from which corrects the mutant heart defect by promoting myofibrillogenesis and thus was named MIR (myofibrillogenesis inducing RNA). In the present study, we have examined MIR gene expression in mutant axolotl hearts at early pre-heart-beat developmental stages and found its quantitative expression, as detected by RT-PCR, to be the same as in normal hearts. However, careful analysis of sequence data revealed a G-->U point mutation in the mutant MIR RNA. Further computational analyses, using GENEBEE software to compare normal and mutant MIR RNAs show a significant alteration in RNA secondary structure of the point-mutated MIR RNA. The results from bioassay and confocal microscopy immunofluorescent studies demonstrate that, unlike MIR RNA derived from normal embryos, the mutated MIR RNA does not promote myofibrillogenesis in mutant embryonic hearts and fails to rescue/correct the mutant heart defect.

Identification, characterization, and expression of a novel alpha-tropomyosin isoform in cardiac tissues in developing chicken.

Tropomyosins are present in various muscle (skeletal, cardiac, and smooth) and non-muscle cells with different isoforms characteristic of specific cell types. We describe here a novel smooth/striated chimeric isoform that was expressed in developing chick heart in addition to the classically described TM-4 type. This novel alpha-Tm tropomyosin isoform, designated as alpha-Tm-2, contains exon 2a (in place of exon 2b). The known striated muscle isoform (alpha-Tm-1) was also expressed in embryonic hearts along with the striated muscle isoform of TM-4. In adult heart, TM-4 was expressed, however, expression of both alpha-Tm-1 and alpha-Tm-2 isoforms was drastically reduced or downregulated. Interestingly, we were unable to detect the expression of alpha-Tm-2 in embryonic and adult skeletal muscle, however, the alpha-Tm-1 isoform is expressed in embryonic and adult skeletal muscle. Examination of other possible isoforms of the alpha-TM gene, i.e., alpha-smooth muscle tropomyosin (alpha-Sm), alpha-Fibroblast-1 (alpha-F1), and alpha-Fibroblast-2 (alpha-F2) revealed expression in embryonic hearts and a significant reduction of each of these isoforms in adult heart. In order to elucidate the role of the newly discovered tropomyosin isoform in chicken, we ectopically expressed the GFP fusion protein of alpha-Tm-1 and alpha-Tm-2 separately into cardiomyocytes isolated from neonatal rats. Each isoform was incorporated into organized myofibrils. Our results suggest that the alpha-TM gene may undergo both positive and negative transcriptional control in chicken hearts during development.

Cadherin-mediated adhesion is essential for myofibril continuity across the plasma membrane but not for assembly of the contractile apparatus.

The strong coordinated contraction of heart muscle is dependent on the correct alignment and connection of the myofibrils across the plasma membrane. Previous studies indicate that N-cadherin is involved in cardiac myocyte adhesion and myofibrillogenesis. To investigate whether N-cadherin is specifically required for normal myocyte structure and function, we cultured myocytes from wild-type, N-cadherin-null and mutant embryos expressing the epithelial cadherin E-cadherin. In contrast to previous studies in chicken using N-cadherin-perturbing antibodies, our in vitro studies with mouse cells demonstrate that N-cadherin is not required for myofibrillogenesis, but is critical for myofibril organization. That is, N-cadherin-deficient myocytes beat and myofibrils were well formed; however, alignment of the myofibrils through regions of cell-cell contact was lost, resulting in their random orientation. Gap junctions were perturbed in the N-cadherin-null myocytes. By contrast, focal contacts appeared normal in the mutant cells. Furthermore, E-cadherin restored normal cell morphology and behavior to the N-cadherin-deficient myocytes, including proper alignment of the myofibrils. We conclude that a different adhesive system, most probably integrin, is responsible for myofibrillogenesis in the N-cadherin-null myocytes.

Protein kinase C-mediated desmin phosphorylation is related to myofibril disarray in cardiomyopathic hamster heart.

The cardiomyopathic (CM) Syrian golden hamster (strain UM-X7.1) exhibits a hereditary cardiomyopathy, which causes premature death resulting from congestive heart failure. The CM animals show extensive cardiac myofibril disarray and myocardial calcium overload. The present study has been undertaken to examine the role of desmin phosphorylation in myofibril disarray observed in CM hearts. The data from skinned myofibril protein phosphorylation assays have shown that desmin can be phosphorylated by protein kinase C (PKC). There is no significant difference in the content of desmin between CM and control hamster hearts. However, the desmin from CM hearts has a higher phosphorylation level than that of the normal hearts. Furthermore, we have examined the distribution of desmin and myofibril organization with immunofluorescent microscopy and immunogold electron microscopy in cultured cardiac myocytes after treatment with the PKC-activating phorbol ester, 12-O-tetradecanylphorbol-13-acetate (TPA). When the cultured normal hamster cardiac cells are treated with TPA, desmin filaments are disassembled and the myofibrils become disarrayed. The myofibril disarray closely mimics that observed in untreated CM cultures. These results suggest that disassembly of desmin filaments, which could be caused by PKC-mediated phosphorylation, may be a factor in myofibril disarray in cardiomyopathic cells and that the intermediate filament protein, desmin, plays an important role in maintaining myofibril alignment in cardiac cells.

Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis.

Calreticulin (crt) is an ubiquitously expressed and multifunctional Ca2+-binding protein that regulates diverse vital cell functions, including Ca2+ storage in the ER and protein folding. Calreticulin deficiency in mice is lethal in utero due to defects in heart development and function. Herein, we used crt − / − embryonic stem (ES) cells differentiated in vitro into cardiac cells to investigate the molecular mechanisms underlying heart failure of knockout embryos. After 8 d of differentiation, beating areas were prominent in ES-derived wild-type (wt) embryoid bodies (EBs), but not in ES-derived crt − / − EBs, despite normal expression levels of cardiac transcription factors. Crt − / − EBs exhibited a severe decrease in expression and a lack of phosphorylation of ventricular myosin light chain 2 (MLC2v), resulting in an impaired organization of myofibrils. Crt − / − phenotype could be recreated in wt cells by chelating extracellular or cytoplasmic Ca2+ with EGTA or BAPTA, or by inhibiting Ca2+/calmodulin-dependent kinases (CaMKs). An imposed ionomycin-triggered cystolic-free Ca2+ concentration ([Ca2+]c) elevation restored the expression, phosphorylation, and insertion of MLC2v into sarcomeric structures and in turn the myofibrillogenesis. The transcription factor myocyte enhancer factor C2 failed to accumulate into nuclei of crt − / − cardiac cells in the absence of ionomycin-triggered [Ca2+]c increase. We conclude that the absence of calreticulin interferes with myofibril formation. Most importantly, calreticulin deficiency revealed the importance of a Ca2+-dependent checkpoint critical for early events during cardiac myofibrillogenesis.

A novel striated tropomyosin incorporated into organized myofibrils of cardiomyocytes in cell and organ culture

Striated muscle tropomyosin is classically described as consisting of 10 exons, 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b, in both skeletal and cardiac muscle. A novel isoform found in embryonic axolotl heart maintains exon 9a/b of striated muscle but also has a smooth muscle exon 2a instead of exon 2b. Translation and subsequent incorporation into organized myofibrils, with both isoforms, was demonstrated with green fluorescent protein fusion protein construct. Mutant axolotl hearts lack sufficient tropomyosin in the ventricle and this smooth/straited chimeric tropomyosin was sufficient to replace the missing tropomyosin and form organized myofibrils.

Remodeling the intercalated disc leads to cardiomyopathy in mice misexpressing cadherins in the heart.

The contractile force of the cardiomyocyte is transmitted through the adherens junction, a component of the intercalated disc, enabling the myocardium to function as a syncytium. The cadherin family of cell adhesion receptors, located in the adherens junction, interact homophilically to mediate strong cell-cell adhesion. Ectopic expression of cadherins is associated with changes in tumor cell behavior and pathology. To examine the effect of cadherin specificity on cardiac structure and function, we expressed either the epithelial cadherin, E-cadherin, or N-cadherin in the heart of transgenic mice. E-cadherin was localized to the intercalated disc structure in these animals similar to endogenous N-cadherin. Both N- and E-cadherin transgenic animals developed dilated cardiomyopathy. However, misexpression of E-cadherin led to earlier onset and increased mortality compared with N-cadherin mice. A dramatic decrease in connexin 43 was associated with the hypertrophic response in E-cadherin transgenic mice. Myofibril organization appeared normal although, vinculin, which normally localizes to the intercalated disc, was redistributed to the cytoplasm in the E-cadherin transgenic mice. Furthermore, E-cadherin induced cyclin D1, nuclear reduplication, and karyokinesis in the absence of cytokinesis, resulting in myocytes with two closely opposed nuclei. By contrast, N-cadherin overexpressing transgenic mice did not exhibit an increase in cyclin D1, suggesting that E-cadherin may provide a specific growth signal to the myocyte. This study demonstrates that modulation of cadherin-mediated adhesion can lead to dilated cardiomyopathy and that E-cadherin can stimulate DNA replication in myocytes normally withdrawn from the cell cycle.