Katja Gehmlich

Mutations in the Human Muscle LIM Protein Gene in Families With Hypertrophic Cardiomyopathy

Background—Muscle LIM protein (MLP) is an essential nuclear regulator of myogenic differentiation. Additionally, it may act as an integrator of protein assembly of the actin-based cytoskeleton. MLP-knockout mice develop a marked cardiac hypertrophy reaction and dilated cardiomyopathy (DCM). MLP is therefore a candidate gene for heritable forms of hypertrophic cardiomyopathy (HCM) and DCM in humans. Methods and Results—We analyzed 1100 unrelated individuals (400 patients with DCM, 200 patients with HCM, and 500 controls) for mutations in the human CRP3 gene that encodes MLP. We found 3 different missense mutations in 3 unrelated patients with familial HCM but detected no mutation in the DCM group or the controls. All mutations predicted an amino acid exchange at highly conserved residues in the functionally important LIM1 domain, which is responsible for interaction with &agr;-actinin and with certain muscle-specific transcription factors. Protein-binding studies indicate that mutations in the CRP3 gene lead to a decreased binding activity of MLP to &agr;-actinin. All 3 index patients were characterized by typical asymmetrical septal hypertrophy. Family studies revealed cosegregation of clinically affected individuals with the respective mutations in MLP. Conclusion—Here, we present evidence that mutations in the CRP3/MLP gene can cause HCM.

Beyond the sarcomere: CSRP3 mutations cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a frequent genetic cardiac disease and the most common cause of sudden cardiac death in young individuals. Most of the currently known HCM disease genes encode sarcomeric proteins. Previous studies have shown an association between CSRP3 missense mutations and either dilated cardiomyopathy (DCM) or HCM, but all these studies were unable to provide comprehensive genetic evidence for a causative role of CSRP3 mutations. We used linkage analysis and identified a CSRP3 missense mutation in a large German family affected by HCM. We confirmed CSRP3 as an HCM disease gene. Furthermore, CSRP3 missense mutations segregating with HCM were identified in four other families. We used a newly designed monoclonal antibody to show that muscle LIM protein (MLP), the protein encoded by CSRP3, is mainly a cytosolic component of cardiomyocytes and not tightly anchored to sarcomeric structures. Our functional data from both in vitro and in vivo analyses suggest that at least one of MLPs mutated forms seems to be destabilized in the heart of HCM patients harbouring a CSRP3 missense mutation. We also present evidence for mild skeletal muscle disease in affected persons. Our results support the view that HCM is not exclusively a sarcomeric disease and also suggest that impaired mechano-sensory stress signalling might be involved in the pathogenesis of HCM.

Decreased interactions of mutant muscle LIM protein (MLP) with N-RAP and α-actinin and their implication for hypertrophic cardiomyopathy

Abstract.Previous work has shown that mutations in muscle LIM protein (MLP) can cause hypertrophic cardiomyopathy (HCM). In order to gain an insight into the molecular basis of the disease phenotype, we analysed the binding characteristics of wild-type MLP and of the (C58G) mutant MLP that causes hypertrophic cardiomyopathy. We show that MLP can form a ternary complex with two of its previously documented myofibrillar ligand proteins, N-RAP and α-actinin, which indicates the presence of distinct, non-overlapping binding sites. Our data also show that, in comparison to wild-type MLP, the capacity of the mutated MLP protein to bind both N-RAP and α-actinin is significantly decreased. In addition, this single point mutation prevents zinc coordination and proper folding of the second zinc-finger in the first LIM domain, which consequently renders the protein less stable and more susceptible to proteolysis. The molecular basis for HCM-causing mutations in the MLP gene might therefore be an alteration in the equilibrium of interactions of the ternary complex MLP–N-RAP–α-actinin. This assumption is supported by the previous observation that in the pathological situation accompanied by MLP down regulation, cardiomyocytes try to compensate for the decreased stability of MLP protein by increasing the expression of its ligand N-RAP, which might finally result in the development of myocyte disarray that is characteristic of this disease.

Mechanistic insights into arrhythmogenic right ventricular cardiomyopathy caused by desmocollin-2 mutations

Aims Recent immunohistochemical studies observed the loss of plakoglobin (PG) from the intercalated disc (ID) as a hallmark of arrhythmogenic right ventricular cardiomyopathy (ARVC), suggesting a final common pathway for this disease. However, the underlying molecular processes are poorly understood. Methods and results We have identified novel mutations in the desmosomal cadherin desmocollin 2 (DSC2 R203C, L229X, T275M, and G371fsX378). The two missense mutations (DSC2 R203C and T275M) have been functionally characterized, together with a previously reported frameshift variant (DSC2 A897fsX900), to examine their pathogenic potential towards PGs functions at the ID. The three mutant proteins were transiently expressed in various cellular systems and assayed for expression, processing, localization, and binding to other desmosomal components in comparison to wild-type DSC2a protein. The two missense mutations showed defects in proteolytic cleavage, a process which is required for the functional activation of mature cadherins. In both cases, this is thought to cause a reduction of functional DSC2 at the desmosomes in cardiac cells. In contrast, the frameshift variant was incorporated into cardiac desmosomes; however, it showed reduced binding to PG. Conclusion Despite different modes of action, for all three variants, the reduced ability to provide a ligand for PG at the desmosomes was observed. This is in agreement with the reduced intensity of PG at these structures observed in ARVC patients.

A novel desmocollin-2 mutation reveals insights into the molecular link between desmosomes and gap junctions

Background Cellular adhesion mediated by cardiac desmosomes is a prerequisite for proper electric propagation mediated by gap junctions in the myocardium. However, the molecular principles underlying this interdependence are not fully understood. Objective The purpose of this study was to determine potential causes of right ventricular conduction abnormalities in a patient with borderline diagnosis of arrhythmogenic right ventricular cardiomyopathy. Methods To assess molecular changes, the patients myocardial tissue was analyzed for altered desmosomal and gap junction (connexin43) protein levels and localization. In vitro functional studies were performed to characterize the consequences of the desmosomal mutations. Results Loss of plakoglobin signal was evident at the cell junctions despite expression of the protein at control levels. Although the distribution of connexin43 was not altered, total protein levels were reduced and changes in phosphorylation were observed. The truncation mutant in desmocollin-2a is deficient in binding plakoglobin. Moreover, the ability of desmocollin-2a to directly interact with connexin43 was abolished by the mutation. No pathogenic potential of the desmoglein-2 missense change was identified. Conclusion The observed abnormalities in gap junction protein expression and phosphorylation, which precede an overt cardiac phenotype, likely are responsible for slow myocardial conduction in this patient. At the molecular level, altered binding properties of the desmocollin-2a mutant may contribute to the changes in connexin43. In particular, the newly identified interaction between the desmocollin-2a isoform and connexin43 provides novel insights into the molecular link between desmosomes and gap junctions.

Novel missense mutations in exon 15 of desmoglein-2: Role of the intracellular cadherin segment in arrhythmogenic right ventricular cardiomyopathy?

Background The diagnosis of arrhythmogenic right ventricular cardiomyopathy can be challenging. Disease-causing mutations in desmosomal genes have been identified. A novel diagnostic feature, loss of immunoreactivity for plakoglobin from the intercalated disks, recently was proposed. Objective The purpose of this study was to identify two novel mutations in the intracellular cadherin segment of desmoglein-2 (G812S and C813R in exon 15). Co-segregation of the G812S mutation with disease expression was established in a large Caucasian family. Endomyocardial biopsies of two individuals showed reduced plakoglobin signal at the intercalated disk. Methods To understand the pathologic changes occurring in the diseased myocardium, functional studies on three mutations in exon 15 of desmoglein-2 (G812C, G812S, C813R) were performed. Results Localization studies failed to detect any differences in targeting or stability of the mutant proteins, suggesting that they act via a dominant negative mechanism. Binding assays were performed to probe for altered binding affinities toward other desmosomal proteins, such as plakoglobin and plakophilin-2. Although no differences were observed for the mutated proteins in comparison to wild-type desmoglein-2, binding to plakophilin-2 depended on the expression system (i.e., bacterial vs mammalian protein expression). In addition, abnormal migration of the C813R mutant protein was observed in gel electrophoresis. Conclusion Loss of plakoglobin immunoreactivity from the intercalated disks appears to be the endpoint of complex pathologic changes, and our functional data suggest that yet unknown posttranslational modifications of desmoglein-2 might be involved.

Back to square one: what do we know about the functions of Muscle LIM Protein in the heart?

Muscle LIM Protein (MLP) is small, just 198 amino acid long protein, which is specifically expressed in slow skeletal muscle and cardiac tissues. This article will focus on the cardiac functions of MLP: the current knowledge about localisation data, binding partners and animal models for the protein will be summarised, and the role of MLP in maintaining a healthy heart be discussed. This review will furthermore attempt to identify gaps in our knowledge—and hence future research potential—with a special focus on MLP’s role in cardiac mechano-signalling.

Molecular changes in the heart of a severe case of arrhythmogenic right ventricular cardiomyopathy caused by a desmoglein-2 null allele

INTRODUCTIONnArrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder caused by mutations in desmosomal genes. It is often associated with life-threatening arrhythmias. Some affected individuals develop progressive heart failure and may require cardiac transplantation.nnnMETHODSnThe explanted heart of a young adult with end-stage heart failure due to a null allele in desmoglein-2 was studied at macroscopic, microscopic, and molecular level. Myocardial samples were probed for junctional localization of desmosomal components and the gap junction protein connexin43 by immunohistochemical staining. In addition, the protein content of desmosomal and adherens junction markers as well as connexin43 was assessed by Western blotting.nnnRESULTSnHistological analysis confirmed ARVC. Despite the loss of specific immunoreactive signal for desmosomal components at the cardiac intercalated disks (shown for plakoglobin, desmoplakin, and plakophilin-2), these proteins could be detected by Western blotting. Only for desmoglein-2, desmocollin-2, and plakoglobin were reduced protein levels observed. Adherens junction proteins were not affected. Lower phosphorylation levels were observed for connexin43; however, localization of the gap junction protein displayed regional differences. At the molecular level, disease progression was more severe in the right ventricle compared to the left ventricle.nnnCONCLUSIONnOur data suggest that, in the ARVC heart, plakoglobin is mainly redistributed from the junctions to other cellular pools and that protein degradation only plays a secondary role. Homogenous changes in the phosphorylation status of connexin43 were observed in multiple ARVC samples, suggesting that this might be a general feature of the disease.

Constitutive upregulations of titin-based signalling proteins in KY deficient muscles.

An increase in the expression of stretch/stress response elements in fast and slow muscles has been previously described in a transcriptional profiling of KY deficient muscles. Here, we have characterized the induction of this titin-based family of signalling proteins in ky/ky muscles at the protein level. Changes in expression of MLP, MARP2 and Xin have been related to the onset of dystrophic and adaptive changes that operate in ky/ky muscles. Our results indicate that induction of this set of genes is an early consequence of the interference caused by the absence of the KY protein. A search of muscle profiles of mouse models revealed such molecular hallmark only in muscles subjected to a single bout of eccentric contractions and specific titin mutants. Based on the role of this family as titin-based stress response molecules, it is suggested that titin structural/signalling instability is common to ky and titin mouse mutants and eccentric contractions.

Ponsin interacts with Nck adapter proteins: implications for a role in cytoskeletal remodelling during differentiation of skeletal muscle cells

Skeletal muscle differentiation is a complex process: It is characterised by changes in gene expression and protein composition. Simultaneously, a dramatic remodelling of the cytoskeleton and associated cell-matrix contacts, the costameres, occurs. The expression and localisation of the protein ponsin at cell-matrix contacts marks the establishment of costameres. In this report we show that skeletal muscle cells are characterised by a novel ponsin isoform, which contains a large insertion in its carboxy-terminus. This skeletal muscle-specific module binds the adapter proteins Nck1 and Nck2, and increased co-localisation of ponsin with Nck2 is observed at remodelling cell-matrix contacts of differentiating skeletal muscle cells. Since this ponsin insertion can be phosphorylated, it may adjust the interaction affinity with Nck adapter proteins. The novel ponsin isoform and its interaction with Nck1/2 provide exciting insight into the convergence of signalling pathways at the costameres, and its crucial role for skeletal muscle differentiation and re-generation.