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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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Immunoflourescence studies staining with specific antibodies for N-cadherin, connexin43, PKP-2 and DSP in right ventricle tissue samples of Patient 1 and control. All tissue samples were stained using the same primary and secondary antibodies and were incubated for the same period of time. Blue signal represents cardiomyocyte cell nuclei, visualized with DAPI staining. The white bar corresponds to 10 μm.
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fig02: Immunoflourescence studies staining with specific antibodies for N-cadherin, connexin43, PKP-2 and DSP in right ventricle tissue samples of Patient 1 and control. All tissue samples were stained using the same primary and secondary antibodies and were incubated for the same period of time. Blue signal represents cardiomyocyte cell nuclei, visualized with DAPI staining. The white bar corresponds to 10 μm.

Mentions: Immunofluorescent studies were used to visualize expression and localization of intercalated disk proteins in patients identified to have mutations of PKP-2. N-cadherin, connexin43 and PKP-2 proteins were visualized in both patients and controls. Patient 1 showed normal N-cadherin and PKP-2 positioning and expression, however, there was a noticeable decrease in connexin43 quantity (Fig. 2). While it was more difficult to visually discern decreased intercalated disk expression of connexin43 in the other three patients, increased ectopic expression of connexin43 was evident in patients 2–4 (Fig. 3). Patients 3 and 4 demonstrated abnormal connexin43 organization at the intercalated disk, where a punctate pattern of gap junction protein, rather than a continuous localization pattern was recognized (Fig. 3).


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)

Immunoflourescence studies staining with specific antibodies for N-cadherin, connexin43, PKP-2 and DSP in right ventricle tissue samples of Patient 1 and control. All tissue samples were stained using the same primary and secondary antibodies and were incubated for the same period of time. Blue signal represents cardiomyocyte cell nuclei, visualized with DAPI staining. The white bar corresponds to 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig02: Immunoflourescence studies staining with specific antibodies for N-cadherin, connexin43, PKP-2 and DSP in right ventricle tissue samples of Patient 1 and control. All tissue samples were stained using the same primary and secondary antibodies and were incubated for the same period of time. Blue signal represents cardiomyocyte cell nuclei, visualized with DAPI staining. The white bar corresponds to 10 μm.
Mentions: Immunofluorescent studies were used to visualize expression and localization of intercalated disk proteins in patients identified to have mutations of PKP-2. N-cadherin, connexin43 and PKP-2 proteins were visualized in both patients and controls. Patient 1 showed normal N-cadherin and PKP-2 positioning and expression, however, there was a noticeable decrease in connexin43 quantity (Fig. 2). While it was more difficult to visually discern decreased intercalated disk expression of connexin43 in the other three patients, increased ectopic expression of connexin43 was evident in patients 2–4 (Fig. 3). Patients 3 and 4 demonstrated abnormal connexin43 organization at the intercalated disk, where a punctate pattern of gap junction protein, rather than a continuous localization pattern was recognized (Fig. 3).

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