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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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Colocalization plots for connexin43 and N-cadherin from both ARVC patient and control tissues. The letters A, B, C and D correspond to the colocalization plots for Control patient, Patient 2, Patient 3 and Patient 4, respectively. Numerically, these plots correspond to values of 0.897, 0.601, 0.445 and 0.125. Calculations were made using the Velocity 3.0 program on a Macintosh PC. The above plots were calculated from the pictures shown in Fig. 3. Red signal indicates connexin43, green signal represents N-cadherin and yellow shows the overlap of the two.
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fig05: Colocalization plots for connexin43 and N-cadherin from both ARVC patient and control tissues. The letters A, B, C and D correspond to the colocalization plots for Control patient, Patient 2, Patient 3 and Patient 4, respectively. Numerically, these plots correspond to values of 0.897, 0.601, 0.445 and 0.125. Calculations were made using the Velocity 3.0 program on a Macintosh PC. The above plots were calculated from the pictures shown in Fig. 3. Red signal indicates connexin43, green signal represents N-cadherin and yellow shows the overlap of the two.

Mentions: Abnormal connexin43 expression and colocalization to the intercalated disk were each quantified by assessing luminescence (Adobe Photoshop) and colocalization coefficients (Volocity), respectively. The percentage values of connexin43 luminescence in comparison to N-cadherin for patients 1–4 corresponded to values of 12.90%, 46.85%, 30.25% and 23.33%, respectively (Fig. 4). Thus, all patients showed decreased connexin43 luminescence in comparison to controls. Patient 1 showed a 48.46% reduction in connexin43 luminescence, while patients 2, 3 and 4 demonstrated reductions of 4.74%, 21.34% and 28.26% connexin43 luminescence with respect to control tissue (Fig. 4). Luminescence percentages for both controls 1 and 2 were calculated as 61.36% and 51.59% respectively (Fig. 4). Examining signal overlap of N-cadherin and connexin43 in merged images from patients 2–4 and a control sample allowed for the calculation of colocalization coefficients. These values are visually represented as yellow signal in Figure 5. The colocalization plot for the control patient showed a diagonal clustering pattern, representing a relatively high degree of protein overlap. The colocalization plots of patients 2, 3 and 4 were spread diffusely across the grid, indicating an increase in protein distribution and less colocalization. The control slide was determined to have a colocalization coefficient of 0.897. Patients 2, 3 and 4 showed progressively decreasing colocalization coefficients of 0.601, 0.445 and 0.125, respectively. Colocalization could not be performed for patient 1 because the image was not properly formatted for manipulation in the Volocity program, however, it can be inferred that connexin43 colocalization would be minimal, since connexin43 quantity was notably diminished.


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)

Colocalization plots for connexin43 and N-cadherin from both ARVC patient and control tissues. The letters A, B, C and D correspond to the colocalization plots for Control patient, Patient 2, Patient 3 and Patient 4, respectively. Numerically, these plots correspond to values of 0.897, 0.601, 0.445 and 0.125. Calculations were made using the Velocity 3.0 program on a Macintosh PC. The above plots were calculated from the pictures shown in Fig. 3. Red signal indicates connexin43, green signal represents N-cadherin and yellow shows the overlap of the two.
© Copyright Policy
Related In: Results  -  Collection

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

fig05: Colocalization plots for connexin43 and N-cadherin from both ARVC patient and control tissues. The letters A, B, C and D correspond to the colocalization plots for Control patient, Patient 2, Patient 3 and Patient 4, respectively. Numerically, these plots correspond to values of 0.897, 0.601, 0.445 and 0.125. Calculations were made using the Velocity 3.0 program on a Macintosh PC. The above plots were calculated from the pictures shown in Fig. 3. Red signal indicates connexin43, green signal represents N-cadherin and yellow shows the overlap of the two.
Mentions: Abnormal connexin43 expression and colocalization to the intercalated disk were each quantified by assessing luminescence (Adobe Photoshop) and colocalization coefficients (Volocity), respectively. The percentage values of connexin43 luminescence in comparison to N-cadherin for patients 1–4 corresponded to values of 12.90%, 46.85%, 30.25% and 23.33%, respectively (Fig. 4). Thus, all patients showed decreased connexin43 luminescence in comparison to controls. Patient 1 showed a 48.46% reduction in connexin43 luminescence, while patients 2, 3 and 4 demonstrated reductions of 4.74%, 21.34% and 28.26% connexin43 luminescence with respect to control tissue (Fig. 4). Luminescence percentages for both controls 1 and 2 were calculated as 61.36% and 51.59% respectively (Fig. 4). Examining signal overlap of N-cadherin and connexin43 in merged images from patients 2–4 and a control sample allowed for the calculation of colocalization coefficients. These values are visually represented as yellow signal in Figure 5. The colocalization plot for the control patient showed a diagonal clustering pattern, representing a relatively high degree of protein overlap. The colocalization plots of patients 2, 3 and 4 were spread diffusely across the grid, indicating an increase in protein distribution and less colocalization. The control slide was determined to have a colocalization coefficient of 0.897. Patients 2, 3 and 4 showed progressively decreasing colocalization coefficients of 0.601, 0.445 and 0.125, respectively. Colocalization could not be performed for patient 1 because the image was not properly formatted for manipulation in the Volocity program, however, it can be inferred that connexin43 colocalization would be minimal, since connexin43 quantity was notably diminished.

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