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1.  Loss-of-Function of hNav1.5 by ZASP1-D117N Associated with Intraventricular Conduction Disturbances in Left Ventricular Noncompaction 
Defects of cytoarchitectural proteins can cause left ventricular noncompaction (LVNC), which is often associated with conduction system diseases. We have previously identified a p.D117N mutation in the LDB3-encoding Z-band Alternatively Spliced PDZ motif gene (ZASP) in a patient with LVNC and conduction disturbances. We sought to investigate a role of p.D117N mutation in the LBD3 NM_001080114.1 isoform (ZASP1-D117N) in the regulation of cardiac sodium channel (Nav1.5) that plays an important role in the cardiac conduction system.
Methods and Results
Effects of ZASP1-wt and ZASP1-D117N on Nav1.5 were studied in HEK-293 cells and neonatal rat cardiomyocytes (NRCMs). Patch-clamp study demonstrated that ZASP1-D117N significantly attenuated INa by 27% in HEK-293 cells and by 32% in NRCMs. In addition, ZASP1-D117N rightward shifted the voltage-dependent activation and inactivation in both systems. In silico simulation using Luo-Rudy phase 1 model demonstrated that altered Nav1.5 function can reduce cardiac conduction velocity by 28% compared to the control. Pull-down assays showed that both wt and ZASP1-D117N can complex with Nav1.5 and telethonin/T-Cap, which required intact PDZ domains. Immunohistochemical staining in NRCMs demonstrates that ZASP1-D117N did not significantly disturb the Z-line structure. Disruption of cytoskeletal networks with ML-7 and cytochalasin D abolished the effects of ZASP1-D117N on the Nav1.5.
ZASP1 can form protein complex with telethonin/T-Cap and Nav1.5. The LVNC-specific ZASP1 mutation can cause loss-of-function of Nav1.5 without significant alteration of the cytoskeletal protein complex. Our study suggests that electrical remodeling can occur in LVNC subject due to a direct effect of mutant ZASP on Nav1.5.
PMCID: PMC4331025  PMID: 22929165
ZASP; sodium channel; cardiac conduction disturbance; left ventricular noncompaction
2.  ZASP Interacts with the Mechanosensing Protein Ankrd2 and p53 in the Signalling Network of Striated Muscle 
PLoS ONE  2014;9(3):e92259.
ZASP is a cytoskeletal PDZ-LIM protein predominantly expressed in striated muscle. It forms multiprotein complexes and plays a pivotal role in the structural integrity of sarcomeres. Mutations in the ZASP protein are associated with myofibrillar myopathy, left ventricular non-compaction and dilated cardiomyopathy. The ablation of its murine homologue Cypher results in neonatal lethality. ZASP has several alternatively spliced isoforms, in this paper we clarify the nomenclature of its human isoforms as well as their dynamics and expression pattern in striated muscle. Interaction is demonstrated between ZASP and two new binding partners both of which have roles in signalling, regulation of gene expression and muscle differentiation; the mechanosensing protein Ankrd2 and the tumour suppressor protein p53. These proteins and ZASP form a triple complex that appears to facilitate poly-SUMOylation of p53. We also show the importance of two of its functional domains, the ZM-motif and the PDZ domain. The PDZ domain can bind directly to both Ankrd2 and p53 indicating that there is no competition between it and p53 for the same binding site on Ankrd2. However there is competition for this binding site between p53 and a region of the ZASP protein lacking the PDZ domain, but containing the ZM-motif. ZASP is negative regulator of p53 in transactivation experiments with the p53-responsive promoters, MDM2 and BAX. Mutations in the ZASP ZM-motif induce modification in protein turnover. In fact, two mutants, A165V and A171T, were not able to bind Ankrd2 and bound only poorly to alpha-actinin2. This is important since the A165V mutation is responsible for zaspopathy, a well characterized autosomal dominant distal myopathy. Although the mechanism by which this mutant causes disease is still unknown, this is the first indication of how a ZASP disease associated mutant protein differs from that of the wild type ZASP protein.
PMCID: PMC3960238  PMID: 24647531
3.  Telethonin deficiency is associated with maladaptation to biomechanical stress in the mammalian heart 
Circulation research  2011;109(7):758-769.
Telethonin (also known as titin-cap or t-cap) is a 19 kDa Z-disk protein with a unique β-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardio-mechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin’s in vivo function.
Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation.
Methods and Results
By using a variety of different genetically altered animal models and biophysical experiments we show that, contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin-titin crosslinks via α-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the pro-apoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis (“mechanoptosis”). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect which may contribute to enhanced rates of apoptosis found in these hearts.
Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect which may also play a role in human heart failure.
PMCID: PMC3664427  PMID: 21799151
Genetics; Mechanosensation; Mechanotransduction; Cardiomyopathy; Heart failure
4.  Multi-Tasking Role of the Mechanosensing Protein Ankrd2 in the Signaling Network of Striated Muscle 
PLoS ONE  2011;6(10):e25519.
Ankrd2 (also known as Arpp) together with Ankrd1/CARP and DARP are members of the MARP mechanosensing proteins that form a complex with titin (N2A)/calpain 3 protease/myopalladin. In muscle, Ankrd2 is located in the I-band of the sarcomere and moves to the nucleus of adjacent myofibers on muscle injury. In myoblasts it is predominantly in the nucleus and on differentiation shifts from the nucleus to the cytoplasm. In agreement with its role as a sensor it interacts both with sarcomeric proteins and transcription factors.
Methodology/Principal Findings
Expression profiling of endogenous Ankrd2 silenced in human myotubes was undertaken to elucidate its role as an intermediary in cell signaling pathways. Silencing Ankrd2 expression altered the expression of genes involved in both intercellular communication (cytokine-cytokine receptor interaction, endocytosis, focal adhesion, tight junction, gap junction and regulation of the actin cytoskeleton) and intracellular communication (calcium, insulin, MAPK, p53, TGF-β and Wnt signaling). The significance of Ankrd2 in cell signaling was strengthened by the fact that we were able to show for the first time that Nkx2.5 and p53 are upstream effectors of the Ankrd2 gene and that Ankrd1/CARP, another MARP member, can modulate the transcriptional ability of MyoD on the Ankrd2 promoter. Another novel finding was the interaction between Ankrd2 and proteins with PDZ and SH3 domains, further supporting its role in signaling. It is noteworthy that we demonstrated that transcription factors PAX6, LHX2, NFIL3 and MECP2, were able to bind both the Ankrd2 protein and its promoter indicating the presence of a regulatory feedback loop mechanism.
In conclusion we demonstrate that Ankrd2 is a potent regulator in muscle cells affecting a multitude of pathways and processes.
PMCID: PMC3189947  PMID: 22016770
5.  Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle 
Future cardiology  2006;2(4):467-476.
The heart is a force-generating organ that responds to self-generated electrical stimuli from specialized cardiomyocytes. This function is modulated by sympathetic and parasympathetic activity.
In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle, cardiomyocytes depend on their highly evolved and specialized cytoskeletal apparatus. Defects in components of the cytoskeleton, in the long term, affect the ability of the cell to compensate at both functional and structural levels. In addition to the structural remodeling, the myocardium becomes increasingly susceptible to altered electrical activity leading to arrhythmogenesis. The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive. Here I will review the current knowledge of the molecular and functional relationships between the cytoskeleton and ion channels and, I will discuss the future impact of new data on molecular cardiology research and clinical practice.
PMCID: PMC2747736  PMID: 19774097
Arrhythmias; Ion Channel; SCN5A; Dystrophin; DGC; Caveolin-3; Cytoskeleton; Syntrophin; Potassium Channels
6.  A Class III PDZ Binding Motif in the Myotilin and FATZ Families Binds Enigma Family Proteins: a Common Link for Z-Disc Myopathies▿  
Molecular and Cellular Biology  2008;29(3):822-834.
Interactions between Z-disc proteins regulate muscle functions and disruption of these interactions results in muscle disorders. Mutations in Z-disc components myotilin, ZASP/Cypher, and FATZ-2 (calsarcin-1/myozenin-2) are associated with myopathies. We report here that the myotilin and the FATZ (calsarcin/myozenin) families share high homology at their final C-terminal five amino acids. This C-terminal E[ST][DE][DE]L motif is present almost exclusively in these families and is evolutionary conserved. We show by in vitro and in vivo studies that proteins from the myotilin and FATZ (calsarcin/myozenin) families interact via this novel type of class III PDZ binding motif with the PDZ domains of ZASP/Cypher and other Enigma family members: ALP, CLP-36, and RIL. We show that the interactions can be modulated by phosphorylation. Calmodulin-dependent kinase II phosphorylates the C terminus of FATZ-3 (calsarcin-3/myozenin-3) and myotilin, whereas PKA phosphorylates that of FATZ-1 (calsarcin-2/myozenin-1) and FATZ-2 (calsarcin-1/myozenin-1). This is the first report of a binding motif common to both the myotilin and the FATZ (calsarcin/myozenin) families that is specific for interactions with Enigma family members.
PMCID: PMC2630697  PMID: 19047374
7.  Muscle Research and Gene Ontology: New standards for improved data integration 
The Gene Ontology Project provides structured controlled vocabularies for molecular biology that can be used for the functional annotation of genes and gene products. In a collaboration between the Gene Ontology (GO) Consortium and the muscle biology community, we have made large-scale additions to the GO biological process and cellular component ontologies. The main focus of this ontology development work concerns skeletal muscle, with specific consideration given to the processes of muscle contraction, plasticity, development, and regeneration, and to the sarcomere and membrane-delimited compartments. Our aims were to update the existing structure to reflect current knowledge, and to resolve, in an accommodating manner, the ambiguity in the language used by the community.
The updated muscle terminologies have been incorporated into the GO. There are now 159 new terms covering critical research areas, and 57 existing terms have been improved and reorganized to follow their usage in muscle literature.
The revised GO structure should improve the interpretation of data from high-throughput (e.g. microarray and proteomic) experiments in the area of muscle science and muscle disease. We actively encourage community feedback on, and gene product annotation with these new terms. Please visit the Muscle Community Annotation Wiki .
PMCID: PMC2657163  PMID: 19178689
8.  Targeted Deletion of the Muscular Dystrophy Gene myotilin Does Not Perturb Muscle Structure or Function in Mice▿  
Molecular and Cellular Biology  2006;27(1):244-252.
Myotilin, palladin, and myopalladin form a novel small subfamily of cytoskeletal proteins that contain immunoglobulin-like domains. Myotilin is a thin filament-associated protein localized at the Z-disk of skeletal and cardiac muscle cells. The direct binding to F-actin, efficient cross-linking of actin filaments, and prevention of induced disassembly of filaments are key roles of myotilin that are thought to be involved in structural maintenance and function of the sarcomere. Missense mutations in the myotilin-encoding gene cause dominant limb girdle muscular dystrophy type 1A and spheroid body myopathy and are the molecular defect that can cause myofibrillar myopathy. Here we describe the generation and analysis of mice that lack myotilin, myo−/− mice. Surprisingly, myo−/− mice maintain normal muscle sarcomeric and sarcolemmal integrity. Also, loss of myotilin does not cause alterations in the heart or other organs of newborn or adult myo−/− mice. The mice develop normally and have a normal life span, and their muscle capacity does not significantly differ from wild-type mice even after prolonged physical stress. The results suggest that either myotilin does not participate in muscle development and basal function maintenance or other proteins serve as structural and functional compensatory molecules when myotilin is absent.
PMCID: PMC1800670  PMID: 17074808
9.  Zasp 
The Journal of Cell Biology  1999;146(2):465-476.
PDZ motifs are modular protein–protein interaction domains, consisting of 80–120 amino acid residues, whose function appears to be the direction of intracellular proteins to multiprotein complexes. In skeletal muscle, there are a few known PDZ-domain proteins, which include neuronal nitric oxide synthase and syntrophin, both of which are components of the dystrophin complex, and actinin-associated LIM protein, which binds to the spectrin-like repeats of α-actinin-2. Here, we report the identification and characterization of a new skeletal muscle protein containing a PDZ domain that binds to the COOH-terminal region of α-actinin-2. This novel 31-kD protein is specifically expressed in heart and skeletal muscle. Using antibodies produced to a fragment of the protein, we can show its location in the sarcomere at the level of the Z-band by immunoelectron microscopy. At least two proteins, 32 kD and 78 kD, can be detected by Western blot analysis of both heart and skeletal muscle, suggesting the existence of alternative forms of the protein. In fact, several forms were found that appear to be the result of alternative splicing. The transcript coding for this Z-band alternatively spliced PDZ motif (ZASP) protein maps on chromosome 10q22.3-10q23.2, near the locus for infantile-onset spinocerebellar ataxia.
PMCID: PMC3206570  PMID: 10427098
skeletal muscle; sarcomeres; muscle proteins; immunoelectron microscopy; alternative splicing

Results 1-9 (9)