Lisa M. Godsel

Desmosomal Dysfunction due to Mutations in Desmoplakin Causes Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by progressive degeneration of the right ventricular myocardium, ventricular arrhythmias, fibrous-fatty replacement, and increased risk of sudden death. Mutations in 6 genes, including 4 encoding desmosomal proteins (Junctional plakoglobin (JUP), Desmoplakin (DSP), Plakophilin 2, and Desmoglein 2), have been identified in patients with ARVD/C. Mutation analysis of 66 probands identified 4 variants in DSP; V30M, Q90R, W233X, and R2834H. To establish a cause and effect relationship between those DSP missense mutations and ARVD/C, we performed in vitro and in vivo analyses of the mutated proteins. Unlike wild-type (WT) DSP, the N-terminal mutants (V30M and Q90R) failed to localize to the cell membrane in desomosome-forming cell line and failed to bind to and coimmunoprecipitate JUP. Multiple attempts to generate N-terminal DSP (V30M and Q90R) cardiac-specific transgenes have failed: analysis of embryos revealed evidence of profound ventricular dilation, which likely resulted in embryonic lethality. We were able to develop transgenic (Tg) mice with cardiac-restricted overexpression of the C-terminal mutant (R2834H) or WT DSP. Whereas mice overexpressing WT DSP had no detectable histologic, morphological, or functional cardiac changes, the R2834H-Tg mice had increased cardiomyocyte apoptosis, cardiac fibrosis, and lipid accumulation, along with ventricular enlargement and cardiac dysfunction in both ventricles. These mice also displayed interruption of DSP-desmin interaction at intercalated discs (IDs) and marked ultra-structural changes of IDs. These data suggest DSP expression in cardiomyocytes is crucial for maintaining cardiac tissue integrity, and DSP abnormalities result in ARVD/C by cardiomyocyte death, changes in lipid metabolism, and defects in cardiac development.

Flagellar protein localization mediated by a calcium- myristoyl/palmitoyl switch mechanism

The mechanisms by which proteins are targeted to flagella and cilia are poorly understood. We set out to determine the basis for the specific localization of a 24 kDa flagellar calcium‐binding protein (FCaBP) expressed in all life cycle stages of Trypanosoma cruzi. Through the study of trypanosome transfectants expressing various FCaBP deletion mutants, we found that the N‐terminal 24 amino acids of the protein are necessary and sufficient for flagellar localization. Subsequent experiments revealed that FCaBP is myristoylated and palmitoylated and, in fact, is one of very few proteins in the cell possessing these acyl modifications. Both fatty acids are required for flagellar localization, suggesting that FCaBP localization may be mediated through association with the flagellar plasma membrane. Indeed, FCaBP associates with the flagellar membrane in a calcium‐dependent manner, reminiscent of the recoverin family of calcium–myristoyl switch proteins. Thus, FCaBP is a novel member of the calcium–acyl switch protein family and is the only member described to date that requires two fatty acid modifications for specific membrane association. Its unique localization mechanism is the first described for any flagellar protein. The existence of such a protein in this protozoan suggests that acylation and calcium switch mechanisms for regulated membrane association are conserved among eukaryotes.

Intermediate filament–membrane attachments function synergistically with actin-dependent contacts to regulate intercellular adhesive strength

By tethering intermediate filaments (IFs) to sites of intercellular adhesion, desmosomes facilitate formation of a supercellular scaffold that imparts mechanical strength to a tissue. However, the role IF–membrane attachments play in strengthening adhesion has not been directly examined. To address this question, we generated Tet-On A431 cells inducibly expressing a desmoplakin (DP) mutant lacking the rod and IF-binding domains (DPNTP). DPNTP localized to the plasma membrane and led to dissociation of IFs from the junctional plaque, without altering total or cell surface distribution of adherens junction or desmosomal proteins. However, a specific decrease in the detergent-insoluble pool of desmoglein suggested a reduced association with the IF cytoskeleton. DPNTP-expressing cell aggregates in suspension or substrate-released cell sheets readily dissociated when subjected to mechanical stress whereas controls remained largely intact. Dissociation occurred without lactate dehydrogenase release, suggesting that loss of tissue integrity was due to reduced adhesion rather than increased cytolysis. JD-1 cells from a patient with a DP COOH-terminal truncation were also more weakly adherent compared with normal keratinocytes. When used in combination with DPNTP, latrunculin A, which disassembles actin filaments and disrupts adherens junctions, led to dissociation up to an order of magnitude greater than either treatment alone. These data provide direct in vitro evidence that IF–membrane attachments regulate adhesive strength and suggest furthermore that actin- and IF-based junctions act synergistically to strengthen adhesion.

Plakophilins: multifunctional scaffolds for adhesion and signaling

Armadillo family proteins known as plakophilins have been characterized as structural components of desmosomes that stabilize and strengthen adhesion by enhancing attachments with the intermediate filament cytoskeleton. However, plakophilins and their close relatives are emerging as versatile scaffolds for multiple signaling and metabolic processes that not only facilitate junction dynamics but also more globally regulate diverse cellular activities. While perturbation of plakophilin functions contribute to inherited diseases and cancer pathogenesis, the functional significance of the multiple PKP isoforms and the mechanisms by which their behaviors are regulated remain to be elucidated.

Desmoplakin assembly dynamics in four dimensions: Multiple phases differentially regulated by intermediate filaments and actin

The intermediate filament (IF)–binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell–cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 μm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell–cell contact and regulated by actin and DP–IF interactions.

Mechanisms of Plakoglobin-dependent Adhesion DESMOSOME-SPECIFIC FUNCTIONS IN ASSEMBLY AND REGULATION BY EPIDERMAL GROWTH FACTOR RECEPTOR

Plakoglobin (PG) is a member of the Armadillo family of adhesion/signaling proteins that can be incorporated into both adherens junctions and desmosomes. Loss of PG results in defects in the mechanical integrity of heart and skin and decreased adhesive strength in keratinocyte cultures established from the skin of PG knock-out (PG-/-) mice, the latter of which cannot be compensated for by overexpressing the closely related β-catenin. In this study, we examined the mechanisms of PG-regulated adhesion in murine keratinocytes. Biochemical and morphological analyses indicated that junctional incorporation of desmosomal, but not adherens junction, components was impaired in PG-/- cells compared with PG+/- controls. Re-expression of PG, but not β-catenin, in PG-/- cells largely reversed these effects, indicating a key role for PG in desmosome assembly. Epidermal growth factor (EGF) receptor activation resulted in Tyr phosphorylation of PG, which was accompanied by a loss of desmoplakin from desmosomes and decreased adhesive strength following 18-h EGF treatment. Importantly, introduction of a phosphorylation-deficient PG mutant into PG null cells prevented the EGF receptor-dependent loss of desmoplakin from junctions, attenuating the effects of long term EGF treatment on cell adhesion. Therefore, PG is essential for maintaining and regulating adhesive strength in keratinocytes largely through its contributions to desmosome assembly and structure. As a target for modulation by EGF, regulation of PG-dependent adhesion may play an important role during wound healing and tumor metastasis.

Captopril Prevents Experimental Autoimmune Myocarditis

Captopril, an angiotensin-converting enzyme inhibitor, is widely used in the treatment of a variety of cardiomyopathies, but its effect on autoimmune myocarditis has not been addressed experimentally. We investigated the effect of captopril on myosin-induced experimental autoimmune myocarditis. A/J mice, immunized with syngeneic cardiac myosin, were given 75 mg/L of captopril in their drinking water. Captopril dramatically reduced the incidence and severity of myocarditis, which was accompanied by a reduction in heart weight to body weight ratio and heart weight. Captopril specifically interfered with cell-mediated immunity as myosin delayed-type hypersensitivity (DTH) was reduced, while anti-myosin Ab production was not affected. Captopril-treated, OVA-immunized mice also exhibited a decrease in OVA DTH. In myosin-immunized, untreated mice, injection of captopril directly into the test site also suppressed myosin DTH. Interestingly, captopril did not directly affect Ag-specific T cell responsiveness because neither in vivo nor in vitro captopril treatment affected the proliferation, IFN-γ secretion, or IL-2 secretion by Ag-stimulated cultured splenocytes. These results indicate that captopril ameliorates experimental autoimmune myocarditis and may act, at least in part, by interfering with the recruitment of cells to sites of inflammation and the local inflammatory environment.

Plakophilin 2 Couples Actomyosin Remodeling to Desmosomal Plaque Assembly via RhoA

The desmosomal armadillo protein plakophilin 2 (PKP2) regulates cell contact-initiated cortical actin remodeling through the regulation of RhoA localization and activity to couple adherens junction maturation with desmosomal plaque assembly.

Angiotensin Converting Enzyme Inhibitors and Angiotensin II Receptor Antagonists in Experimental Myocarditis

Myocarditis is a disease whose pathogenesis is not completely understood and whose prevalence is likely underestimated. Individuals afflicted with this condition may be treated with agents that relieve symptoms arising from inflammation and concurrent cellular damage. One class of drugs commonly used in the treatment of myocarditis includes the angiotensin converting enzyme inhibitors, such as captopril, enalapril and lisinopril, and the angiotensin Pi receptor antagonists, such as L-158,809 and losartan. The effects of these drugs on cardiomyopathy have been studied using a variety of animal models of heart failure and hypertension. However, less research has been done in the area of animal models of frank myocarditis. Here we review the use of angiotensin converting enzyme inhibitors and angiotensin Pi receptor antagonists in animal models of myocarditis. We extend the implications of that published work by correlation with results from studies of other disease models and in vitro experiments that highlight the immunomodulatory potential of these compounds. The literature strongly suggests that aggressive therapy employing angiotensin converting enzyme inhibition and/or blockade of angiotensin Pi receptors is beneficial. Treatment is useful not only for reducing complications associated with myocarditis, but also for downregulating the potential autoimmune component of disease without increasing the levels of the infectious agent that may initiate the myocarditis.

Prevention of autoimmune myocarditis through the induction of antigen-specific peripheral immune tolerance.

Background—Autoimmunity to cardiac antigens, in particular cardiac myosin, has been observed in humans with myocarditis and in animals with experimental inflammatory heart disease. Current treatments for myocarditis are in many cases immunosuppressive and might lead to increased cardiac damage by reducing host defenses against infectious agents. Therefore, we sought to develop an antigen-specific approach to inhibit autoimmunity in mice with myosin-induced experimental autoimmune myocarditis. Methods and Results—Syngeneic splenocytes, coupled with cardiac myosin by use of ethylene carbodiimide, were administered intravenously before disease induction, and the effects of this peripheral tolerization on myosin-induced myocarditis were assessed. This antigen-specific immunotherapy significantly reduced both the incidence and severity of myocarditis, with the prevention of myocyte necrosis, mononuclear cell infiltration, and fibrosis. Myosin-specific delayed-type hypersensitivity and antibody production were significantly reduced, demonstrating that peripheral tolerance affected both T- and B-cell responsiveness to the autoantigen. Conclusions—These results suggest that the induction of antigen-specific peripheral immune tolerance may be an effective approach for the treatment of myocarditides with autoimmune involvement.