Elsevier

Heart Rhythm

Volume 5, Issue 12, December 2008, Pages 1715-1723
Heart Rhythm

Original-experimental
Characterization of the molecular phenotype of two arrhythmogenic right ventricular cardiomyopathy (ARVC)-related plakophilin-2 (PKP2) mutations

https://doi.org/10.1016/j.hrthm.2008.09.009Get rights and content

Background

Arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to mutations in desmosomal proteins, including plakophilin-2 (PKP2). Little is known about the changes in cellular function and structure that follow expression of ARVC-relevant PKP2 mutations.

Objective

The purpose of this study was to investigate the function and distribution of an ARVC-relevant PKP2 mutant where arginine at position 79 was replaced by a stop codon (R79x).

Methods

Results were compared with those obtained with mutation 179fs (frameshift at position 179). Mutant constructs were introduced by adenoviral infection into neonatal rat ventricular myocytes in culture.

Results

Both mutant proteins failed to preferentially localize to sites of cell-cell apposition. Their expression did not disrupt localization of endogenous PKP2, connexin-43 (Cx43), or desmoplakin (DP). However, we observed reduced abundance of Cx43 after R79x expression. Early truncation of PKP2 at position 79 also prevented its physical interaction with both DP and Cx43. Finally, R79x expression correlated with loss of expression of HSP90, a protein relevant to cardiomyocyte apoptosis.

Conclusion

These results provide the first observations of the cellular/molecular phenotype consequent to these PKP2 mutations and give insight into the possible cellular substrates that lead to ARVC.

Introduction

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disease characterized by fibrofatty infiltration of right ventricular predominance, a high incidence of arrhythmias, and sudden death.1, 2, 3 ARVC has been linked to mutations in desmosomal proteins, including plakophilin-2 (PKP24, 5, 6). Little is known about the cellular/molecular changes that follow the expression of ARVC-relevant PKP2 mutations.

In the vast majority of cases of PKP2-related ARVC mutations are found in only one allele. As such, two conditions are present: decreased gene dose for the wild-type protein and expression of a mutant form. Recently, we demonstrated that loss of PKP2 expression leads to remodeling of connexin-43 (Cx437). The latter is consistent with results obtained in the study of Naxos disease8 and Carvajal syndrome.9 Here we explored for the first time the structural/functional consequences of expression of two ARVC-related PKP2 mutations in neonatal rat ventricular myocytes (NRVMs).

Over 50 ARVC-related PKP2 mutations have been reported. We focused on two N-terminal mutations, R79x (stop-codon replacing arginine 794, 5, 6) and 179fs (a frameshift in that position6). It has been suggested that the plakophilin-desmoplakin (DP) interaction involves the amino termini of both proteins. Thus R79x and 179fs allowed us to explore the minimal PKP2 sequence necessary for DP binding. We extended this suggestion to the previously reported PKP2-Cx43 interaction.7 Mutation R79x has been detected in a number of independent probands,4, 5, 6 suggesting important functional effects upon expression of the mutated protein. Our data support the notion that mutant R79x plays an active role in determining the ARVC phenotype and provide the first characterization of a cellular phenotype related to ARVC-relevant PKP2 mutations.

Section snippets

Generation of cDNA constructs

Full length (“wild-type”) human PKP2a (p915 PKP2a-pFLAG-cmv5a) and DP (p1140 DP.FLAG) C-terminal FLAG constructs were gifts from Dr. Kathleen Green (Northwestern University). Construct p915 served as a template to generate PKP2a mutants. Mutation 179fs was generated by inserting bases C and T at nucleotides 534–535, respectively.6 A C235T substitution produced mutant R79x. A FLAG tag was placed at the C-terminus of both mutants. To generate adenoviruses, each p915 construct was used as template

Expression of exogenous constructs

We used immunochemical techniques to assess expression and distribution of exogenous, C-terminus FLAG-tagged proteins R79x-FLAG, 179fs-FLAG, and wtPKP2-FLAG in NRVMs. Genetic material was transferred by adenoviral infection (see the Methods section). Concentration of viral particles was adjusted for each case (12.5, 50, and 12.5 Multiplicity of infection (MOI) for wtPKP2, R79x, and 179fs, respectively) to ensure maximum expression of the exogenous product while limiting cell damage due to the

Discussion

A number of PKP2 mutations have been associated with ARVC. However, a correlate as to the effects of disease-associated mutations on cell/molecular function is still lacking. We have shown that loss of PKP2 expression leads to Cx43 remodeling and decreased intercellular coupling.7 Here we show that mutants R79x and 179fs failed to localize to the cell membrane but did not alter the localization of endogenous PKP2, DP, or Cx43. The well-known interaction of PKP2 with DP was preserved for mutant

Acknowledgments

The authors thank April Lazarus for generating the viruses and Li Gao and Christine Burrer for assistance with experiments.

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    The first two authors contributed equally.

    Supported by National Institutes of Health grant nos. HL39707, HL087226, and GM57691 and by a Predoctoral Fellowship from the American Heart Association (to E.O.).

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