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1.  Loss of mXinα, an intercalated disk protein, results in cardiac hypertrophy and cardiomyopathy with conduction defects 
The intercalated disk protein Xin was originally discovered in chicken striated muscle and implicated in cardiac morphogenesis. In the mouse, there are two homologous genes, mXinα and mXinβ. The human homolog of mXinα, Cmya1, maps to chromosomal region 3p21.2–21.3, near a dilated cardiomyopathy with conduction defect-2 locus. Here we report that mXinα-null mouse hearts are hypertrophied and exhibit fibrosis, indicative of cardiomyopathy. A significant upregulation of mXinβ likely provides partial compensation and accounts for the viability of the mXinα-null mice. Ultrastructural studies of mXinα-null mouse hearts reveal intercalated disk disruption and myofilament disarray. In mXinα-null mice, there is a significant decrease in the expression level of p120-catenin, β-catenin, N-cadherin, and desmoplakin, which could compromise the integrity of the intercalated disks and functionally weaken adhesion, leading to cardiac defects. Additionally, altered localization and decreased expression of connexin 43 are observed in the mXinα-null mouse heart, which, together with previously observed abnormal electrophysiological properties of mXinα-deficient mouse ventricular myocytes, could potentially lead to conduction defects. Indeed, ECG recordings on isolated, perfused hearts (Langendorff preparations) show a significantly prolonged QT interval in mXinα-deficient hearts. Thus mXinα functions in regulating the hypertrophic response and maintaining the structural integrity of the intercalated disk in normal mice, likely through its association with adherens junctional components and actin cytoskeleton. The mXinα-knockout mouse line provides a novel model of cardiac hypertrophy and cardiomyopathy with conduction defects.
PMCID: PMC2394510  PMID: 17766470
Xin repeat proteins; N-cadherin; β-catenin; p120-catenin; connexin 43
2.  Calcineurin Protects the Heart in a Murine Model of Dilated Cardiomyopathy 
Dilated cardiomyopathy (DCM) is a relatively common disease with a poor prognosis. Given that the only meaningful treatment for DCM is cardiac transplantation, investigators have explored the underlying molecular mechanisms of this disease in the hopes of identifying novel therapeutic targets. One such target is the serine-threonine phosphatase calcineurin, a Ca2+-activated signaling factor that is known to regulate the cardiac hypertrophic program, although its role in DCM is currently unknown.
In order to address this issue, we crossed muscle lim protein (MLP) knock-out mice - a murine model of DCM - with calcineurin Aβ ko mice, which lack the stress responsive isoform of calcineurin that critically regulates the cardiac hypertrophic response. Interestingly, the majority (73%) of the MLP/calcineurin Aβ double knock-out mice died within 20 days of birth with signs of cardiomyopathy. Ultrastructural examination revealed enhanced cardiomyocyte apoptosis and necrosis in the postnatal myocardium of these mice. The MLP/calcineurin Aβ double knock-out mice that survived until adulthood showed reduced left ventricular function, enhanced apoptotic and necrotic cardiomyocyte death and augmented myocardial fibrosis compared to various control groups. Antithetically, mild overexpression of activated calcineurin in the mouse heart improved function and adverse remodeling in MLP knock-out mice.
Collectively, these results reveal an important and previously unrecognized protective function of endogenous myocardial calcineurin in a mouse model of dilated cardiomyopathy.
PMCID: PMC2891089  PMID: 19854199
Dilated Cardiomyopathy; Calcineurin; Muscle Lim Protein; Apoptosis; Necrosis
3.  Genotype—phenotype relationships involving hypertrophic cardiomyopathy-associated mutations in titin, muscle LIM protein, and telethonin ☆ 
TTN-encoded titin, CSRP3-encoded muscle LIM protein, and TCAP-encoded telethonin are Z-disc proteins essential for the structural organization of the cardiac sarcomere and the cardiomyocyte’s stretch sensor. All three genes have been established as cardiomyopathy-associated genes for both dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). Here, we sought to characterize the frequency, spectrum, and phenotype associated with HCM-associated mutations in these three genes in a large cohort of unrelated patients evaluated at a single tertiary outpatient center.
DNA was obtained from 389 patients with HCM (215 male, left ventricular wall thickness of 21.6 ± 6 mm) and analyzed for mutations involving all translated exons of CSRP3 and TCAP and targeted HCM-associated exons (2, 3, 4, and 14) of TTN using polymerase chain reaction (PCR), denaturing high performance liquid chromatography (DHPLC), and direct DNA sequencing. Clinical data were extracted from patient records and maintained independent of the genotype.
Overall, 16 patients (4.1%) harbored a Z-disc mutation: 12 had a MLP mutation and 4 patients a TCAP mutation. No TTN mutations were detected. Seven patients were also found to have a concomitant myofilament mutation. Seven patients with a MLP-mutation were found to harbor the DCM-associated, functionally characterized W4R mutation. W4R-MLP was also noted in a single white control subject. Patients with MLP/TCAP-associated HCM clinically mimicked myofilament-HCM.
Approximately 4.1% of unrelated patients had HCM-associated MLP or TCAP mutations. MLP/TCAP-HCM phenotypically mirrors myofilament-HCM and is more severe than the subset of patients who still remain without a disease-causing mutation. The precise role of W4R-MLP in the pathogenesis of either DCM or HCM warrants further investigation.
PMCID: PMC2756511  PMID: 16352453
Genetics; Genes; Hypertrophy; Cardiomyopathy; Z-disc; Muscle LIM protein; Telethonin; TCAP; Titin
4.  Cardiomyocyte growth and sarcomerogenesis at the intercalated disc 
Cardiomyocytes grow during heart maturation or disease-related cardiac remodeling. We present evidence that the intercalated disc (ID) is integral to both longitudinal and lateral growth: increases in width are accommodated by lateral extension of the plicate tread regions and increases in length by sarcomere insertion within the ID. At the margin between myofibril and the folded membrane of the ID lies a transitional junction through which the thin filaments from the last sarcomere run to the ID membrane and it has been suggested that this junction acts as a proto Z-disc for sarcomere addition. In support of this hypothesis, we have investigated the ultrastructure of the ID in mouse hearts from control and dilated cardiomyopathy (DCM) models, the MLP-null and a cardiac-specific β-catenin mutant, cΔex3, as well as in human left ventricle from normal and DCM samples. We find that the ID amplitude can vary tenfold from 0.2 μm up to a maximum of ~2 μm allowing gradual expansion during heart growth. At the greatest amplitude, equivalent to a sarcomere length, A-bands and thick filaments are found within the ID membrane loops together with a Z-disc, which develops at the transitional junction position. Here, also, the tops of the membrane folds, which are rich in αII spectrin, become enlarged and associated with junctional sarcoplasmic reticulum. Systematically larger ID amplitudes are found in DCM samples. Other morphological differences between mouse DCM and normal hearts suggest that sarcomere inclusion is compromised in the diseased hearts.
PMCID: PMC3889684  PMID: 23708682
Heart structure; Dilated cardiomyopathy; Adherens junction; Electron microscopy; Transitional junction
5.  Assembly of the Cardiac Intercalated Disk during Pre- and Postnatal Development of the Human Heart 
PLoS ONE  2014;9(4):e94722.
In cardiac muscle, the intercalated disk (ID) at the longitudinal cell-edges of cardiomyocytes provides as a macromolecular infrastructure that integrates mechanical and electrical coupling within the heart. Pathophysiological disturbance in composition of this complex is well known to trigger cardiac arrhythmias and pump failure. The mechanisms underlying assembly of this important cellular domain in human heart is currently unknown.
We collected 18 specimens from individuals that died from non-cardiovascular causes. Age of the specimens ranged from a gestational age of 15 weeks through 11 years postnatal. Immunohistochemical labeling was performed against proteins comprising desmosomes, adherens junctions, the cardiac sodium channel and gap junctions to visualize spatiotemporal alterations in subcellular location of the proteins.
Changes in spatiotemporal localization of the adherens junction proteins (N-cadherin and ZO-1) and desmosomal proteins (plakoglobin, desmoplakin and plakophilin-2) were identical in all subsequent ages studied. After an initial period of diffuse and lateral labelling, all proteins were fully localized in the ID at approximately 1 year after birth. Nav1.5 that composes the cardiac sodium channel and the gap junction protein Cx43 follow a similar pattern but their arrival in the ID is detected at (much) later stages (two years for Nav1.5 and seven years for Cx43, respectively).
Our data on developmental maturation of the ID in human heart indicate that generation of the mechanical junctions at the ID precedes that of the electrical junctions with a significant difference in time. In addition arrival of the electrical junctions (Nav1.5 and Cx43) is not uniform since sodium channels localize much earlier than gap junction channels.
PMCID: PMC3986238  PMID: 24733085
6.  Coxsackievirus-Induced miR-21 Disrupts Cardiomyocyte Interactions via the Downregulation of Intercalated Disk Components 
PLoS Pathogens  2014;10(4):e1004070.
Intercalated disks (ICDs) are substantial connections maintaining cardiac structures and mediating signal communications among cardiomyocytes. Deficiency in ICD components such as desmosomes, fascia adherens and gap junctions leads to heart dysfunction. Coxsackievirus B3 (CVB3) infection induces cardiac failure but its pathogenic effect on ICDs is unclear. Here we show that CVB3-induced miR-21 expression affects ICD structure, i.e., upregulated miR-21 targets YOD1, a deubiquitinating enzyme, to enhance the K48-linked ubiquitination and degradation of desmin, resulting in disruption of desmosomes. Inhibition of miR-21 preserves desmin during CVB3 infection. Treatment with proteasome inhibitors blocks miR-21-mediated desmin degradation. Transfection of miR-21 or knockdown of YOD1 triggers co-localization of desmin with proteasomes. We also identified K108 and K406 as important sites for desmin ubiquintination and degradation. In addition, miR-21 directly targets vinculin, leading to disturbed fascia adherens evidenced by the suppression and disorientation of pan-cadherin and α-E-catenin proteins, two fascia adherens-components. Our findings suggest a new mechanism of miR-21 in modulating cell-cell interactions of cardiomyocytes during CVB3 infection.
Author Summary
Coxsackievirus B3 (CVB3) is one of most common causes of heart inflammation and failure. However, the mechanism by which CVB3 induces cardiac damage has not been fully elucidated. Particularly, the involvement of microRNAs (miRNAs), a family of small RNAs controlling the progression of a wide range of diseases, in CVB3 infection is still unclear. These small RNAs are essential to understand the CVB3-caused heart muscle cell injury and have great potential to serve therapeutic purposes. Here, we systematically analyzed the miRNA changes during CVB3 infection and found that miR-21 is increased by viral infection. We further demonstrated that the CVB3-induced miR-21 triggers heart muscle cell damage by interfering with the cell-cell interactions. miR-21 suppresses the levels of components in cell-cell interactions by either promoting the degradation of those proteins or directly inhibiting the protein production. Inhibition of miR-21 can reduce the host injury caused by CVB3 infection. Our findings will shed new lights on the pathogenesis of CVB3-induced heart failure.
PMCID: PMC3983067  PMID: 24722419
7.  Molecular basis for clinical heterogeneity in inherited cardiomyopathies due to myopalladin mutations 
Human Molecular Genetics  2012;21(9):2039-2053.
Abnormalities in Z-disc proteins cause hypertrophic (HCM), dilated (DCM) and/or restrictive cardiomyopathy (RCM), but disease-causing mechanisms are not fully understood. Myopalladin (MYPN) is a Z-disc protein expressed in striated muscle and functions as a structural, signaling and gene expression regulating molecule in response to muscle stress. MYPN was genetically screened in 900 patients with HCM, DCM and RCM, and disease-causing mechanisms were investigated using comparative immunohistochemical analysis of the patient myocardium and neonatal rat cardiomyocytes expressing mutant MYPN. Cardiac-restricted transgenic (Tg) mice were generated and protein–protein interactions were evaluated. Two nonsense and 13 missense MYPN variants were identified in subjects with DCM, HCM and RCM with the average cardiomyopathy prevalence of 1.66%. Functional studies were performed on two variants (Q529X and Y20C) associated with variable clinical phenotypes. Humans carrying the Y20C-MYPN variant developed HCM or DCM, whereas Q529X-MYPN was found in familial RCM. Disturbed myofibrillogenesis with disruption of α-actinin2, desmin and cardiac ankyrin repeat protein (CARP) was evident in rat cardiomyocytes expressing MYPNQ529X. Cardiac-restricted MYPNY20C Tg mice developed HCM and disrupted intercalated discs, with disturbed expression of desmin, desmoplakin, connexin43 and vinculin being evident. Failed nuclear translocation and reduced binding of Y20C-MYPN to CARP were demonstrated using in vitro and in vivo systems. MYPN mutations cause various forms of cardiomyopathy via different protein–protein interactions. Q529X-MYPN causes RCM via disturbed myofibrillogenesis, whereas Y20C-MYPN perturbs MYPN nuclear shuttling and leads to abnormal assembly of terminal Z-disc within the cardiac transitional junction and intercalated disc.
PMCID: PMC3315208  PMID: 22286171
8.  Role of an Alternatively Spliced Form of αII-Spectrin in Localization of Connexin 43 in Cardiomyocytes and Regulation by Stress-Activated Protein Kinase 
Decreases in the expression of connexin 43 and the integrity of gap junctions in cardiac muscle, induced by the constitutive activation of the c-Jun N-terminal kinase (JNK) signaling pathway, have been linked to conduction defects and sudden cardiac failure in mice [16,17]. We examined the membrane cytoskeletal protein, αII-spectrin, which associates with connexin 43, to learn if changes in its association with connexin 43 are linked to the instability of gap junctions. Several forms of αII-spectrin are expressed in heart, including one, termed αII-SH3i, which contains a 20-amino acid sequence next to the SH3 domain of repeat 10. In adult mouse heart, antibodies to all forms of αII-spectrin labeled the sarcolemma, transverse (“t-”) tubules and intercalated disks of cardiomyocytes. In contrast, antibodies specific for αII-SH3i labeled only gap junctions and transverse tubules. In transgenic hearts, in which the JNK pathway was constitutively activated, αII-SH3i was lost specifically from gap junctions but not from t-tubules while other isoforms of αII-spectrin were retained at intercalated disks. Immunoprecipitations confirmed the decreased association of αII-SH3i with connexin 43 in transgenic hearts compared to controls. Furthermore, activation of JNK in neonatal myocytes blocked the formation of gap junctions by exogenously expressed Cx43-GFP fusion protein. Similarly, over-expression of the SH3i fragment in the context of repeats 9-11 of αII–spectrin specifically caused the accumulation of Cx43-GFP in the perinuclear region and inhibited its accumulation at gap junctions. These results support a critical role for the αII-SH3i isoform of spectrin in intracellular targeting of Cx43 to gap junctions and implicates αII-SH3i as a potential target for stress signaling pathways that modulate intercellular communication.
PMCID: PMC1983066  PMID: 17276456
spectrin; connexin 43; gap junctions; cytoskeleton
9.  Muscle LIM Proteins Are Associated with Muscle Sarcomeres and Require dMEF2 for Their Expression during Drosophila Myogenesis 
Molecular Biology of the Cell  1999;10(7):2329-2342.
A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis of Drosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and α-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.
PMCID: PMC25449  PMID: 10397768
10.  Targeted disruption of the mouse Csrp2 gene encoding the cysteine- and glycine-rich LIM domain protein CRP2 result in subtle alteration of cardiac ultrastructure 
The cysteine and glycine rich protein 2 (CRP2) encoded by the Csrp2 gene is a LIM domain protein expressed in the vascular system, particularly in smooth muscle cells. It exhibits a bimodal subcellular distribution, accumulating at actin-based filaments in the cytosol and in the nucleus. In order to analyze the function of CRP2 in vivo, we disrupted the Csrp2 gene in mice and analysed the resulting phenotype.
A ~17.3 kbp fragment of the murine Csrp2 gene containing exon 3 through 6 was isolated. Using this construct we confirmed the recently determined chromosomal localization (Chromosome 10, best fit location between markers D10Mit203 proximal and D10Mit150 central). A gene disruption cassette was cloned into exon 4 and a mouse strain lacking functional Csrp2 was generated. Mice lacking CRP2 are viable and fertile and have no obvious deficits in reproduction and survival. However, detailed histological and electron microscopic studies reveal that CRP2-deficient mice have subtle alterations in their cardiac ultrastructure. In these mice, the cardiomyocytes display a slight increase in their thickness, indicating moderate hypertrophy at the cellular level. Although the expression of several intercalated disc-associated proteins such as β-catenin, N-RAP and connexin-43 were not affected in these mice, the distribution of respective proteins was changed within heart tissue.
We conclude that the lack of CRP2 is associated with alterations in cardiomyocyte thickness and hypertrophy.
PMCID: PMC2529283  PMID: 18713466
11.  Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly 
The Journal of Cell Biology  1995;129(3):683-695.
Tropomodulin is a pointed end capping protein for tropomyosin-coated actin filaments that is hypothesized to play a role in regulating the precise lengths of striated muscle thin filaments (Fowler, V. M., M. A. Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120:411-420; Weber, A., C. C. Pennise, G. G. Babcock, and V. M. Fowler. 1994, J. Cell Biol. 127:1627-1635). To gain insight into the mechanisms of thin filament assembly and the role of tropomodulin therein, we have characterized the temporal appearance, biosynthesis and mechanisms of assembly of tropomodulin onto the pointed ends of thin filaments during the formation of striated myofibrils in primary embryonic chick cardiomyocyte cultures. Our results demonstrate that tropomodulin is not assembled coordinately with other thin filament proteins. Double immunofluorescence staining and ultrastructural immunolocalization demonstrate that tropomodulin is incorporated in its characteristic sarcomeric location at the pointed ends of the thin filaments after the thin filaments have become organized into periodic I bands. In fact, tropomodulin assembles later than all other well characterized myofibrillar proteins studied including: actin, tropomyosin, alpha-actinin, titin, myosin and C-protein. Nevertheless, at steady state, a significant proportion (approximately 39%) of tropomodulin is present in a soluble pool throughout myofibril assembly. Thus, the absence of tropomodulin in some striated myofibrils is not due to limiting quantities of the protein. In addition, kinetic data obtained from [35S]methionine pulse-chase experiments indicate that tropomodulin assembles more slowly into myofibrils than does tropomyosin. This observation, together with results obtained using a novel permeabilized cell model for thin filament assembly, indicate that tropomodulin assembly is dependent on the prior association of tropomyosin with actin filaments. We conclude that tropomodulin is a late marker for the assembly of striated myofibrils in cardiomyocytes; its assembly appears to be linked to their maturity. We propose that tropomodulin is involved in maintaining and stabilizing the final lengths of thin filaments after they are assembled.
PMCID: PMC2120443  PMID: 7730404
12.  Two muscle-specific LIM proteins in Drosophila 
The Journal of Cell Biology  1996;134(5):1179-1195.
The LIM domain defines a zinc-binding motif found in a growing number of eukaryotic proteins that regulate cell growth and differentiation during development. Members of the cysteine-rich protein (CRP) family of LIM proteins have been implicated in muscle differentiation in vertebrates. Here we report the identification and characterization of cDNA clones encoding two members of the CRP family in Drosophila, referred to as muscle LIM proteins (Mlp). Mlp60A encodes a protein with a single LIM domain linked to a glycine-rich region. Mlp84B encodes a protein with five tandem LIM-glycine modules. In the embryo, Mlp gene expression is spatially restricted to somatic, visceral, and pharyngeal muscles. Within the somatic musculature, Mlp84B transcripts are enriched at the terminal ends of muscle fibers, whereas Mlp60A transcripts are found throughout the muscle fibers. The distributions of the Mlp60A and Mlp84B proteins mirror their respective mRNA localizations, with Mlp84B enrichment occurring at sites of muscle attachment. Northern blot analysis revealed that Mlp gene expression is developmentally regulated, showing a biphasic pattern over the course of the Drosophila life cycle. Peaks of expression occur late in embryogenesis and during metamorphosis, when the musculature is differentiating. Drosophila Mlp60A and Mlp84B, like vertebrate members of the CRP family, have the ability to associate with the actin cytoskeleton when expressed in rat fibroblast cells. The temporal expression and spatial distribution of muscle LIM proteins in Drosophila are consistent with a role for Mlps in myogenesis, late in the differentiation pathway.
PMCID: PMC2120973  PMID: 8794860
13.  The Spatiotemporal Development of Intercalated Disk in Three-Dimensional Engineered Heart Tissues Based on Collagen/Matrigel Matrix 
PLoS ONE  2013;8(11):e81420.
Intercalated disk (ID), which electromechanically couples cardiomyocytes into a functional syncitium, is closely related to normal morphology and function of engineered heart tissues (EHTs), but the development mode of ID in the three-dimensional (3D) EHTs is still unclear. In this study, we focused on the spatiotemporal development of the ID in the EHTs constructed by mixing neonatal rat cardiomyocytes with collagen/Matrigel, and investigated the effect of 3D microenvironment provided by collagen/Matrigel matrix on the formation of ID. By histological and immmunofluorescent staining, the spatiotemporal distribution of ID-related junctions was detected. Furthermore, the ultra-structures of the ID in different developmental stages were observed under transmission electron microscope. In addition, the expression of the related proteins was quantitatively analyzed. The results indicate that accompanying the re-organization of cardiomyocytes in collagen/Matrigel matrix, the proteins of adherens junctions, desmosomes and gap junctions redistributed from diffused distribution to intercellular regions to form an integrated ID. The adherens junction and desmosome which are related with mechanical connection appeared earlier than gap junction which is essential for electrochemical coupling. These findings suggest that the 3D microenvironment based on collagen/Matrigel matrix could support the ordered assembly of the ID in EHTs and have implications for comprehending the ordered and coordinated development of ID during the functional organization of EHTs.
PMCID: PMC3829928  PMID: 24260578
14.  Mechanical stress-strain sensors embedded in cardiac cytoskeleton: Z disk, titin, and associated structures 
Cardiac muscle is equipped with intricate intrinsic mechanisms to regulate adaptive remodeling. Recent and extensive experimental findings powered by novel strategies for screening protein-protein interactions, improved imaging technologies, and versatile transgenic mouse methodologies reveal that Z disks and titin filaments possess unexpectedly complicated sensory and modulatory mechanisms for signal reception and transduction. These mechanisms employ molecules such as muscle-enriched LIM domain proteins, PDZ-LIM domain proteins, myozenin gene family members, titin-associated ankyrin repeat family proteins, and muscle-specific ring finger proteins, which have been identified as potential molecular sensor components. Moreover, classic transmembrane signaling processes, including mitogen-activated kinase, protein kinase C, and calcium signaling, also involve novel interactions with the Z disk/titin network. This compartmentalization of signaling complexes permits alteration of receptor-dependent transcriptional regulation by direct sensing of intrinsic stress. Newly identified mechanical stress sensors are not limited to Z-disk region and to I-band and M-band regions of titin but are also embedded in muscle-specific membrane systems such as the costamere, intercalated disks, and caveolae-like microdomains. This review summarizes current knowledge of this rapidly developing area with focus on how the heart adjusts physiological remodeling process to meet with mechanical demands and how this process fails in cardiac pathologies.
PMCID: PMC3241960  PMID: 16537787
heart; actin cytoskeleton; transmembrane signaling; proteosome
15.  Myocyte remodelling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein 
CSRP3 or Muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein and a mechanosensor in cardiac myocytes. MLP regulation and function was studied in cultured neonatal rat myocytes treated with pharmacological or mechanical stimuli. Either verapamil or BDM decreased nuclear MLP while phenylephrine and cyclic strain increased it. These results suggest that myocyte contractility regulates MLP subcellular localization. When RNA polymerase II was inhibited with α-amanitin, nuclear MLP was reduced by 30%. However, when both RNA polymerase I & II were inhibited with actinomycin D, there was a 90% decrease in nuclear MLP suggesting that its nuclear translocation is regulated by both nuclear and nucleolar transcriptional activity. Using cell permeable synthetic peptides containing the putative nuclear localization signal (NLS) of MLP, nuclear import of the protein in cultured rat neonatal myocytes was inhibited. The NLS of MLP also localizes to the nucleolus. Inhibition of nuclear translocation prevented the increased protein accumulation in response to phenylephrine. Furthermore, cyclic strain of myocytes after prior NLS treatment to remove nuclear MLP resulted in disarrayed sarcomeres. Increased protein synthesis and brain natriuretic peptide expression were also prevented suggesting that MLP is required for remodeling of the myofilaments and gene expression. These findings suggest that nucleocytoplasmic shuttling MLP plays an important role in the regulation of the myocyte remodeling and hypertrophy and is required for adaptation to hypertrophic stimuli.
PMCID: PMC2739242  PMID: 19376126
hypertrophy; sarcomere remodeling; nucleocytoplasmic shuttling; mechanosensing; mechanotransduction
16.  Cell-Cell Connection to Cardiac Disease 
Trends in cardiovascular medicine  2009;19(6):182-190.
Intercalated disks (ICDs) are highly organized cell-cell adhesion structures, which connect cardiomyocytes to one another. They are composed of three major complexes: desmosomes, fascia adherens, and gap junctions. Desmosomes and fascia adherens junction are necessary for mechanically coupling and reinforcing cardiomyocytes, whereas gap junctions are essential for rapid electrical transmission between cells. Because human genetics and mouse models have revealed that mutations and/or deficiencies in various ICD components can lead to cardiomyopathies and arrhythmias, considerable attention has focused on the biologic function of the ICD. This review will discuss recent scientific developments related to the ICD and focus on its role in regulating cardiac muscle structure, signaling, and disease.
PMCID: PMC3601820  PMID: 20211433
17.  Mutations with pathogenic potential in proteins located in or at the composite junctions of the intercalated disk connecting mammalian cardiomyocytes: a reference thesaurus for arrhythmogenic cardiomyopathies and for Naxos and Carvajal diseases 
Cell and Tissue Research  2012;348(2):325-333.
In the past decade, an avalanche of findings and reports has correlated arrhythmogenic ventricular cardiomyopathies (ARVC) and Naxos and Carvajal diseases with certain mutations in protein constituents of the special junctions connecting the polar regions (intercalated disks) of mature mammalian cardiomyocytes. These molecules, apparently together with some specific cytoskeletal proteins, are components of (or interact with) composite junctions. Composite junctions contain the amalgamated fusion products of the molecules that, in other cell types and tissues, occur in distinct separate junctions, i.e. desmosomes and adherens junctions. As the pertinent literature is still in an expanding phase and is obviously becoming important for various groups of researchers in basic cell and molecular biology, developmental biology, histology, physiology, cardiology, pathology and genetics, the relevant references so far recognized have been collected and are presented here in the following order: desmocollin-2 (Dsc2, DSC2), desmoglein-2 (Dsg2, DSG2), desmoplakin (DP, DSP), plakoglobin (PG, JUP), plakophilin-2 (Pkp2, PKP2) and some non-desmosomal proteins such as transmembrane protein 43 (TMEM43), ryanodine receptor 2 (RYR2), desmin, lamins A and C, striatin, titin and transforming growth factor-β3 (TGFβ3), followed by a collection of animal models and of reviews, commentaries, collections and comparative studies.
PMCID: PMC3349860  PMID: 22450909
Arrhythmogenic ventricular cardiomyopathy; Carvajal disease; Composite junction; Desmosomes; Intercalated disk; Naxos disease
18.  Further Analysis of the Lens Phenotype in Lim2-Deficient Mice 
Lim2 is one of the most abundant proteins of the lens fiber cell membrane. A comparative analysis of lenses from wild-type and Lim2-deficient mice reveals roles for Lim2 in the maintenance of cytoskeletal integrity, cell morphology, and intercellular communication.
Lim2 (MP20) is the second most abundant integral protein of lens fiber cell membranes. A comparative analysis was performed of wild-type and Lim2-deficient (Lim2Gt/Gt) mouse lenses, to better define the anatomic and physiologic roles of Lim2.
Scanning electron microscopy (SEM) and confocal microscopy were used to assess the contribution of Lim2 to lens tissue architecture. Differentiation-dependent changes in cytoskeletal composition were identified by mass spectrometry and immunoblot analysis. The effects on cell–cell communication were quantified using impedance analysis.
Lim2-null lenses were grossly normal. At the cellular level, however, subtle structural alterations were evident. Confocal microscopy and SEM analysis revealed that cortical Lim2Gt/Gt fiber cells lacked the undulating morphology that characterized wild-type fiber cells. On SDS-PAGE analysis the composition of cortical fiber cells from wild-type and Lim2-null lenses appeared similar. However, marked disparities were evident in samples prepared from the lens core of the two genotypes. Several cytoskeletal proteins that were abundant in wild-type core fiber cells were diminished in the cores of Lim2Gt/Gt lenses. Electrophysiological measurements indicated a small decrease in the membrane potential of Lim2Gt/Gt lenses and a two-fold increase in the effective intracellular resistivity. In the lens core, this may have reflected decreased expression levels of the gap junction protein connexin 46 (Cx46). In contrast, increased resistivity in the outer cell layers of Lim2Gt/Gt lenses could not be attributed to decreased connexin expression and may reflect the absence of cell fusions in Lim2Gt/Gt lenses.
Comparative analysis of wild-type and Lim2-deficient lenses has implicated Lim2 in maintenance of cytoskeletal integrity, cell morphology, and intercellular communication.
PMCID: PMC3183970  PMID: 21775657
19.  Cardiac-Specific NRAP Overexpression Causes Right Ventricular Dysfunction in Mice 
Experimental cell research  2011;317(8):1226-1237.
The muscle-specific protein NRAP is concentrated at cardiac intercalated disks, plays a role in myofibril assembly, and is upregulated early in mouse models of dilated cardiomyopathy. Using a tet-off system, we developed novel transgenic lines exhibiting cardiac-specific NRAP overexpression ~ 2.5 times greater than normal. At 40-50 weeks, NRAP overexpression resulted in dilation and decreased ejection fraction in the right ventricle, with little effect on the left ventricle. Expression of transcripts encoding brain natriuretic peptide and skeletal α-actin was increased by cardiac-specific NRAP overexpression, indicative of a cardiomyopathic response. NRAP overexpression did not alter the levels or organization of N-cadherin and connexin-43. The results show that chronic NRAP overexpression in the mouse leads to right ventricular cardiomyopathy by 10 months, but that the early NRAP upregulation previously observed in some mouse models of dilated cardiomyopathy is unlikely to account for the remodeling of intercalated disks and left ventricular dysfunction observed in those cases.
PMCID: PMC3070810  PMID: 21276443
NRAP; cardiomyopathy; heart; intercalated disk; transgenic; tetracycline-controlled transactivator
20.  MLP (muscle LIM protein) as a stress sensor in the heart 
Pflugers Archiv  2011;462(1):135-142.
Muscle LIM protein (MLP, also known as cysteine rich protein 3 (CSRP3, CRP3)) is a muscle-specific-expressed LIM-only protein. It consists of 194 amino-acids and has been described initially as a factor involved in myogenesis (Arber et al. Cell 79:221–231, 1994). MLP soon became an important model for experimental cardiology when it was first demonstrated that MLP deficiency leads to myocardial hypertrophy followed by a dilated cardiomyopathy and heart failure phenotype (Arber et al. Cell 88:393–403, 1997). At this time, this was the first genetically altered animal model to develop this devastating disease. Interestingly, MLP was also found to be down-regulated in humans with heart failure (Zolk et al. Circulation 101:2674–2677, 2000) and MLP mutations are able to cause hypertrophic and dilated forms of cardiomyopathy in humans (Bos et al. Mol Genet Metab 88:78–85, 2006; Geier et al. Circulation 107:1390–1395, 2003; Hershberger et al. Clin Transl Sci 1:21–26, 2008; Knöll et al. Cell 111:943–955, 2002; Knöll et al. Circ Res 106:695–704, 2010; Mohapatra et al. Mol Genet Metab 80:207–215, 2003). Although considerable efforts have been undertaken to unravel the underlying molecular mechanisms—how MLP mutations, either in model organisms or in the human setting cause these diseases are still unclear. In contrast, only precise knowledge of the underlying molecular mechanisms will allow the development of novel and innovative therapeutic strategies to combat this otherwise lethal condition. The focus of this review will be on the function of MLP in cardiac mechanosensation and we shall point to possible future directions in MLP research.
PMCID: PMC3114083  PMID: 21484537
Muscle stretch; Mechanoreceptor; Mechanosensitivity; Gene expression; Cardiac function; Cardiac muscle; Cardiac myocytes; Cardiac sarcomere; Cardiomyocyte; Cardiovascular control
21.  Electromechanical Coupling between Skeletal and Cardiac Muscle 
The Journal of Cell Biology  2000;149(3):731-740.
Skeletal myoblasts form grafts of mature muscle in injured hearts, and these grafts contract when exogenously stimulated. It is not known, however, whether cardiac muscle can form electromechanical junctions with skeletal muscle and induce its synchronous contraction. Here, we report that undifferentiated rat skeletal myoblasts expressed N-cadherin and connexin43, major adhesion and gap junction proteins of the intercalated disk, yet both proteins were markedly downregulated after differentiation into myo-tubes. Similarly, differentiated skeletal muscle grafts in injured hearts had no detectable N-cadherin or connexin43; hence, electromechanical coupling did not occur after in vivo grafting. In contrast, when neonatal or adult cardiomyocytes were cocultured with skeletal muscle, ∼10% of the skeletal myotubes contracted in synchrony with adjacent cardiomyocytes. Isoproterenol increased myotube contraction rates by 25% in coculture without affecting myotubes in monoculture, indicating the cardiomyocytes were the pacemakers. The gap junction inhibitor heptanol aborted myotube contractions but left spontaneous contractions of individual cardiomyocytes intact, suggesting myotubes were activated via gap junctions. Confocal microscopy revealed the expression of cadherin and connexin43 at junctions between myotubes and neonatal or adult cardiomyocytes in vitro. After microinjection, myotubes transferred dye to neonatal cardiomyocytes via gap junctions. Calcium imaging revealed synchronous calcium transients in cardiomyocytes and myotubes. Thus, cardiomyocytes can form electromechanical junctions with some skeletal myotubes in coculture and induce their synchronous contraction via gap junctions. Although the mechanism remains to be determined, if similar junctions could be induced in vivo, they might be sufficient to make skeletal muscle grafts beat synchronously with host myocardium.
PMCID: PMC2174851  PMID: 10791985
skeletal myocytes; cardiomyocytes; electromechanical coupling; N-cadherin; connexin43
22.  Transmembrane protein PERP is a component of tessellate junctions and of other junctional and non-junctional plasma membrane regions in diverse epithelial and epithelium-derived cells 
Cell and Tissue Research  2013;353(1):99-115.
Protein PERP (p53 apoptosis effector related to PMP-22) is a small (21.4 kDa) transmembrane polypeptide with an amino acid sequence indicative of a tetraspanin character. It is enriched in the plasma membrane and apparently contributes to cell-cell contacts. Hitherto, it has been reported to be exclusively a component of desmosomes of some stratified epithelia. However, by using a series of newly generated mono- and polyclonal antibodies, we show that protein PERP is not only present in all kinds of stratified epithelia but also occurs in simple, columnar, complex and transitional epithelia, in various types of squamous metaplasia and epithelium-derived tumors, in diverse epithelium-derived cell cultures and in myocardial tissue. Immunofluorescence and immunoelectron microscopy allow us to localize PERP predominantly in small intradesmosomal locations and in variously sized, junction-like peri- and interdesmosomal regions (“tessellate junctions”), mostly in mosaic or amalgamated combinations with other molecules believed, to date, to be exclusive components of tight and adherens junctions. In the heart, PERP is a major component of the composite junctions of the intercalated disks connecting cardiomyocytes. Finally, protein PERP is a cobblestone-like general component of special plasma membrane regions such as the bile canaliculi of liver and subapical-to-lateral zones of diverse columnar epithelia and upper urothelial cell layers. We discuss possible organizational and architectonic functions of protein PERP and its potential value as an immunohistochemical diagnostic marker.
PMCID: PMC3691483  PMID: 23689684
Adhering junctions; Desmosomes; Tessellate junctions; PERP; Tetraspanins; Immunocytochemistry
23.  Muscle Lim Protein Interacts with Cofilin 2 and Regulates F-Actin Dynamics in Cardiac and Skeletal Muscle▿  
Molecular and Cellular Biology  2009;29(22):6046-6058.
The muscle LIM protein (MLP) and cofilin 2 (CFL2) are important regulators of striated myocyte function. Mutations in the corresponding genes have been directly associated with severe human cardiac and skeletal myopathies, and aberrant expression patterns have often been observed in affected muscles. Herein, we have investigated whether MLP and CFL2 are involved in common molecular mechanisms, which would promote our understanding of disease pathogenesis. We have shown for the first time, using a range of biochemical and immunohistochemical methods, that MLP binds directly to CFL2 in human cardiac and skeletal muscles. The interaction involves the inter-LIM domain, amino acids 94 to 105, of MLP and the amino-terminal domain, amino acids 1 to 105, of CFL2, which includes part of the actin depolymerization domain. The MLP/CFL2 complex is stronger in moderately acidic (pH 6.8) environments and upon CFL2 phosphorylation, while it is independent of Ca2+ levels. This interaction has direct implications in actin cytoskeleton dynamics in regulating CFL2-dependent F-actin depolymerization, with maximal depolymerization enhancement at an MLP/CFL2 molecular ratio of 2:1. Deregulation of this interaction by intracellular pH variations, CFL2 phosphorylation, MLP or CFL2 gene mutations, or expression changes, as observed in a range of cardiac and skeletal myopathies, could impair F-actin depolymerization, leading to sarcomere dysfunction and disease.
PMCID: PMC2772566  PMID: 19752190
24.  Chronic Treatment with Clenbuterol Modulates Endothelial Progenitor Cells and Circulating Factors in a Murine Model of Cardiomyopathy 
The purpose of this study was to determine the effects of chronic treatment with the beta 2 adrenergic receptor agonist clenbuterol on endothelial progenitor cells (EPC) in a well-characterized model of heart failure, the muscle LIM protein knockout (MLP−/−) mouse. MLP−/−mice were treated daily with clenbuterol (2 mg/kg) or saline subcutaneously for 6 weeks. Clenbuterol led to a 30% increase in CD31+ cells in the bone marrow of MLP−/− heart failure mice (p<0.004). Clenbuterol did not improve ejection fraction. Clenbuterol treatment in MLP−/− mice was associated with significant changes in the following circulating factors: tissue inhibitor of metalloproteinase-type 1, leukemia inhibitory factor 1, C-reactive protein, apolipoprotein A1, fibroblast growth factor 2, serum glutamic oxaloacetic transaminase, macrophage-derived chemokine, and monocyte chemoattractant protein-3. Clen-buterol treatment in the MLP−/− model of heart failure did not rescue heart function, yet did increase CD31+ cells in the bone marrow. This is the first evidence that a beta 2 agonist increases EPC proliferation in the bone marrow in a preclinical model of heart failure.
PMCID: PMC4066445  PMID: 20559986
Clenbuterol; Heart Failure; Muscle LIM Protein; Beta 2 Adrenergic Receptor; Endothelial Progenitor Cell
25.  Injection of Vessel-Derived Stem Cells Prevents Dilated Cardiomyopathy and Promotes Angiogenesis and Endogenous Cardiac Stem Cell Proliferation in mdx/utrn−/− but Not Aged mdx Mouse Models for Duchenne Muscular Dystrophy 
It was hypothesized that mesoangioblast stem cells (aorta-derived mesoangioblasts [ADMs]) would restore dystrophin and alleviate or prevent dilated cardiomyopathy (DCM) in animal models of Duchenne muscular dystrophy (DMD). It was found that ADMs delay or prevent development of DCM in dystrophin-deficient heart, resulting in dystrophin expression, angiogenesis, stimulation of endogenous cardiac stem cell division, and the appearance of nestin+ cardiomyocytes of host origin. It was also found that timing of stem cell transplantation may be critical for achieving benefit with cell therapy in DMD cardiac muscle.
Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy. DMD patients lack dystrophin protein and develop skeletal muscle pathology and dilated cardiomyopathy (DCM). Approximately 20% succumb to cardiac involvement. We hypothesized that mesoangioblast stem cells (aorta-derived mesoangioblasts [ADMs]) would restore dystrophin and alleviate or prevent DCM in animal models of DMD. ADMs can be induced to express cardiac markers, including Nkx2.5, cardiac tropomyosin, cardiac troponin I, and α-actinin, and adopt cardiomyocyte morphology. Transplantation of ADMs into the heart of mdx/utrn−/− mice prior to development of DCM prevented onset of cardiomyopathy, as measured by echocardiography, and resulted in significantly higher CD31 expression, consistent with new vessel formation. Dystrophin-positive cardiomyocytes and increased proliferation of endogenous Nestin+ cardiac stem cells were detected in ADM-injected heart. Nestin+ striated cells were also detected in four of five mdx/utrn−/− hearts injected with ADMs. In contrast, when ADMs were injected into the heart of aged mdx mice with advanced fibrosis, no functional improvement was detected by echocardiography. Instead, ADMs exacerbated some features of DCM. No dystrophin protein, increase in CD31 expression, or increase in Nestin+ cell proliferation was detected following ADM injection in aged mdx heart. Dystrophin was observed following transplantation of ADMs into the hearts of young mdx mice, however, suggesting that pathology in aged mdx heart may alter the fate of donor stem cells. In summary, ADMs delay or prevent development of DCM in dystrophin-deficient heart, but timing of stem cell transplantation may be critical for achieving benefit with cell therapy in DMD cardiac muscle.
PMCID: PMC3659745  PMID: 23283493
Cellular therapy; Muscular dystrophy; Angiogenesis; Cardiac; Cellular proliferation; Neural stem cell; Cell transplantation

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