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Abnormal connexin43 in arrhythmogenic right ventricular cardiomyopathy caused by plakophilin-2 mutations.

Fidler LM, Wilson GJ, Liu F, Cui X, Scherer SW, Taylor GP, Hamilton RM - J. Cell. Mol. Med. (2008)

Bottom Line: Missense and frameshift mutations of the PKP-2 gene were found in four patients with biopsy material available for analysis.Immunofluorescent studies showed PKP-2 localization to the intercalated disk despite mutations, but associated with decreased connexin43 expression and abnormal colocalization.Reduced connexin43 expression and localization to the intercalated disk occurs in heterozygous human PKP-2 mutations, potentially explaining the delayed conduction and propensity to develop arrhythmias seen in this disease.

View Article: PubMed Central - PubMed

Affiliation: Heart Centre-Cardiology Division, The Hospital for Sick Children, Toronto, ON, Canada.

ABSTRACT
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disorder of cardiomyocyte intercalated disk proteins causing sudden death. Heterozygous mutations of the desmosomal protein plakophilin-2 (PKP-2) are the commonest genetic cause of ARVC. Abnormal gap junction connexin43 expression has been reported in autosomal dominant forms of ARVC (Naxos and Carvajal disease) caused by homozygous mutations of desmosomal plakoglobin and desmoplakin. In tissue culture, suppression of PKP-2 results in decreased expression of connexin43. We sought to characterize the expression and localization of connexin43 in patients with ARVC secondary to heterozygous PKP-2 mutations. Complete PKP-2 gene sequencing of 27 ARVC patients was utilized to identify mutant genotypes. Endomyocardial biopsies of identified carriers were then assessed by immunofluorescence to visualize intercalated disk proteins. N-cadherin was targeted to highlight intercalated disks, followed by counterstaining for PKP-2 or connexin43 using confocal double immunofluorescence microscopy. Immunofluorescence was quantified using an AdobeA Photoshop protocol, and colocalization coefficients were determined. PKP-2 siRNA experiments were performed in mouse cardiomyocyte (HL1) cell culture with Western blot analysis to assess connexin43 expression following PKP-2 suppression. Missense and frameshift mutations of the PKP-2 gene were found in four patients with biopsy material available for analysis. Immunofluorescent studies showed PKP-2 localization to the intercalated disk despite mutations, but associated with decreased connexin43 expression and abnormal colocalization. PKP-2 siRNA in HL1 culture confirmed decreased connexin43 expression. Reduced connexin43 expression and localization to the intercalated disk occurs in heterozygous human PKP-2 mutations, potentially explaining the delayed conduction and propensity to develop arrhythmias seen in this disease.

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Diagrammatic representation of the human PKP-2 protein. (i) Representation of the full length, wild-type form of PKP-2 showing its HR2 domain within the amino terminal head domain and 8 arm-repeat domains. Letters A–H represent each arm-repeat domain, which encompass amino acids 341-383, 385-424, 427-467, 571-616, 671-711, 719-758, 763-804 and 807-849, respectively. The amino terminal HR2 domain spans residues 29-60. Two isoforms of PKP-2 exist. The yellow bar beneath the C domain represents residues 460-503, which are absent in the PKP-2a configuration. Patients 1 through 4 are represented as ii, iii, iv and v, respectively. Arrows indicate the precise location of particular mutations, while carboxy terminal numbers indicate the length of the resulting protein in amino acids.
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fig01: Diagrammatic representation of the human PKP-2 protein. (i) Representation of the full length, wild-type form of PKP-2 showing its HR2 domain within the amino terminal head domain and 8 arm-repeat domains. Letters A–H represent each arm-repeat domain, which encompass amino acids 341-383, 385-424, 427-467, 571-616, 671-711, 719-758, 763-804 and 807-849, respectively. The amino terminal HR2 domain spans residues 29-60. Two isoforms of PKP-2 exist. The yellow bar beneath the C domain represents residues 460-503, which are absent in the PKP-2a configuration. Patients 1 through 4 are represented as ii, iii, iv and v, respectively. Arrows indicate the precise location of particular mutations, while carboxy terminal numbers indicate the length of the resulting protein in amino acids.

Mentions: Patient 1 was found to be heterozygous for a G to A transition at the upstream splice site border of intron 5 in the PKP-2 gene (Fig. 1.ii). This mutation disrupts the splice site consensus sequence, resulting in a truncated protein that is 464 residues in length (Fig. 1.ii). This mutation has not been previously reported in ARVC patients. Patient 2 showed an eight-nucleotide insertion (TTGACTCA) starting at nucleotide 1756 in the PKP-2 gene, which led to a prematurely truncated protein of 658 amino acids (Fig. 1.iii). This genotype has been previously described in ARVC pathogenesis [9]. A missense mutation, R635W, was elucidated in patient 3 (Fig. 1.iv). The underlying cause of this genotype is a C to T transition at nucleotide 1903. This novel mutation was not located in any recognized domains, but does target an evolutionarily conserved residue. Another previously recognized missense mutation, Q62K, caused by a C to A transversion at nucleotide 184, was discovered in Patient 4 to be the underlying cause of ARVC (Fig. 1.v) [22].


Abnormal connexin43 in arrhythmogenic right ventricular cardiomyopathy caused by plakophilin-2 mutations.

Fidler LM, Wilson GJ, Liu F, Cui X, Scherer SW, Taylor GP, Hamilton RM - J. Cell. Mol. Med. (2008)

Diagrammatic representation of the human PKP-2 protein. (i) Representation of the full length, wild-type form of PKP-2 showing its HR2 domain within the amino terminal head domain and 8 arm-repeat domains. Letters A–H represent each arm-repeat domain, which encompass amino acids 341-383, 385-424, 427-467, 571-616, 671-711, 719-758, 763-804 and 807-849, respectively. The amino terminal HR2 domain spans residues 29-60. Two isoforms of PKP-2 exist. The yellow bar beneath the C domain represents residues 460-503, which are absent in the PKP-2a configuration. Patients 1 through 4 are represented as ii, iii, iv and v, respectively. Arrows indicate the precise location of particular mutations, while carboxy terminal numbers indicate the length of the resulting protein in amino acids.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4496128&req=5

fig01: Diagrammatic representation of the human PKP-2 protein. (i) Representation of the full length, wild-type form of PKP-2 showing its HR2 domain within the amino terminal head domain and 8 arm-repeat domains. Letters A–H represent each arm-repeat domain, which encompass amino acids 341-383, 385-424, 427-467, 571-616, 671-711, 719-758, 763-804 and 807-849, respectively. The amino terminal HR2 domain spans residues 29-60. Two isoforms of PKP-2 exist. The yellow bar beneath the C domain represents residues 460-503, which are absent in the PKP-2a configuration. Patients 1 through 4 are represented as ii, iii, iv and v, respectively. Arrows indicate the precise location of particular mutations, while carboxy terminal numbers indicate the length of the resulting protein in amino acids.
Mentions: Patient 1 was found to be heterozygous for a G to A transition at the upstream splice site border of intron 5 in the PKP-2 gene (Fig. 1.ii). This mutation disrupts the splice site consensus sequence, resulting in a truncated protein that is 464 residues in length (Fig. 1.ii). This mutation has not been previously reported in ARVC patients. Patient 2 showed an eight-nucleotide insertion (TTGACTCA) starting at nucleotide 1756 in the PKP-2 gene, which led to a prematurely truncated protein of 658 amino acids (Fig. 1.iii). This genotype has been previously described in ARVC pathogenesis [9]. A missense mutation, R635W, was elucidated in patient 3 (Fig. 1.iv). The underlying cause of this genotype is a C to T transition at nucleotide 1903. This novel mutation was not located in any recognized domains, but does target an evolutionarily conserved residue. Another previously recognized missense mutation, Q62K, caused by a C to A transversion at nucleotide 184, was discovered in Patient 4 to be the underlying cause of ARVC (Fig. 1.v) [22].

Bottom Line: Missense and frameshift mutations of the PKP-2 gene were found in four patients with biopsy material available for analysis.Immunofluorescent studies showed PKP-2 localization to the intercalated disk despite mutations, but associated with decreased connexin43 expression and abnormal colocalization.Reduced connexin43 expression and localization to the intercalated disk occurs in heterozygous human PKP-2 mutations, potentially explaining the delayed conduction and propensity to develop arrhythmias seen in this disease.

View Article: PubMed Central - PubMed

Affiliation: Heart Centre-Cardiology Division, The Hospital for Sick Children, Toronto, ON, Canada.

ABSTRACT
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disorder of cardiomyocyte intercalated disk proteins causing sudden death. Heterozygous mutations of the desmosomal protein plakophilin-2 (PKP-2) are the commonest genetic cause of ARVC. Abnormal gap junction connexin43 expression has been reported in autosomal dominant forms of ARVC (Naxos and Carvajal disease) caused by homozygous mutations of desmosomal plakoglobin and desmoplakin. In tissue culture, suppression of PKP-2 results in decreased expression of connexin43. We sought to characterize the expression and localization of connexin43 in patients with ARVC secondary to heterozygous PKP-2 mutations. Complete PKP-2 gene sequencing of 27 ARVC patients was utilized to identify mutant genotypes. Endomyocardial biopsies of identified carriers were then assessed by immunofluorescence to visualize intercalated disk proteins. N-cadherin was targeted to highlight intercalated disks, followed by counterstaining for PKP-2 or connexin43 using confocal double immunofluorescence microscopy. Immunofluorescence was quantified using an AdobeA Photoshop protocol, and colocalization coefficients were determined. PKP-2 siRNA experiments were performed in mouse cardiomyocyte (HL1) cell culture with Western blot analysis to assess connexin43 expression following PKP-2 suppression. Missense and frameshift mutations of the PKP-2 gene were found in four patients with biopsy material available for analysis. Immunofluorescent studies showed PKP-2 localization to the intercalated disk despite mutations, but associated with decreased connexin43 expression and abnormal colocalization. PKP-2 siRNA in HL1 culture confirmed decreased connexin43 expression. Reduced connexin43 expression and localization to the intercalated disk occurs in heterozygous human PKP-2 mutations, potentially explaining the delayed conduction and propensity to develop arrhythmias seen in this disease.

Show MeSH
Related in: MedlinePlus