On the Role of the Gap Junction Protein Cx43 (GJA1) in Human Cardiac Malformations with Fallot-Pathology. A Study on Paediatric Cardiac Specimen

<div><p>Introduction</p><p>Gap junction channels are involved in growth and differentiation. Therefore, we wanted to elucidate if the main cardiac gap junction protein connexin43 (GJA1) is altered in patients with Tetralogy of Fallot or double-outlet right ventricle of Fallot-type (62 patients referred to as Fallot) compared to other cardiac anomalies (21 patients referred to as non-Fallot). Patients were divided into three age groups: 0–2years, 2–12years and >12years. Myocardial tissue samples were collected during corrective surgery and analysis of cell morphology, GJA1- and N-cadherin (CDH2)-distribution, as well as GJA1 protein- and mRNA-expression was carried out. Moreover, <i>GJA1-</i>gene analysis of 16 patients and 20 healthy subjects was performed.</p><p>Results</p><p>Myocardial cell length and width were significantly increased in the oldest age group compared to the younger ones. GJA1 distribution changed significantly during maturation with the ratio of polar/lateral GJA1 increasing from 2.93±0.68 to 8.52±1.41. While in 0–2years old patients ∼6% of the lateral GJA1 was co-localised with CDH2 this decreased with age. Furthermore, the changes in cell morphology and GJA1-distribution were not due to the heart defect itself but were significantly dependent on age. Total GJA1 protein expression decreased during growing-up, whereas <i>GJA1</i>-mRNA remained unchanged. Sequencing of the <i>GJA1-</i>gene revealed only few heterozygous single nucleotide polymorphisms within the Fallot and the healthy control group.</p><p>Conclusion</p><p>During maturation significant changes in gap junction remodelling occur which might be necessary for the growing and developing heart. In our study point mutations within the <i>Cx43-</i>gene could not be identified as a cause of the development of TOF.</p></div

Histological analysis of patients with TOF or DORV of Fallot-type and patients with other cardiac malformations.

<p>Fallot refers to patients with TOF or DORV of Fallot-type, non-Fallot refers to patients with pulmonary atresia with or without ventricular septal defect, double chamber right ventricle, truncus arteriosus communis, pulmonary stenosis or subaortic stenosis (patient characteristics are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095344#pone-0095344-t001" target="_blank">table 1</a>). <b>A:</b> Cell morphology. Note significant changes between the two age groups but not between the Fallot and non-Fallot groups. All values of cell length and width (in µm) are given as means±SEM. Significant differences within the two age groups are indicated by asterisks (*p<0.05;**p<0.005). <b>B:</b> Cx43 (GJA1) cellular distribution. Note significant changes between the two age groups but not between Fallot and non-Fallot groups. All values expressed as the percentage of polar and lateral Cx43 (GJA1) distribution are given as means±SEM. Significant differences within the two age groups are indicated by asterisks (**p<0.005).</p

Cx43 (GJA1) expression and phosphorylation pattern in patients with TOF or DORV of Fallot-type.

<p><b>A:</b> Cx43 (GJA1) protein and mRNA expression All values of Cx43 (GJA1) protein and mRNA are given as means±SEM. Significant differences within the three age groups are indicated by asterisks (*p<0.05;**p<0.005). <b>B:</b> Cx43 (GJA1) protein phosphorylation. P0, P1 and P2 refer to the three protein bands of Cx43 (GJA1) analysed by Western blotting. P0 non-phosphorylated Cx43 (GJA1), P1 and P2 phosphorylated-Cx43 (GJA1) isoforms. All values of are given as means±SEM. Significant differences within the three age groups are indicated by asterisks (*p<0.05;**p<0.005). <b>C:</b> Ratio of polar Cx43 (GJA1) and lateral Cx43 (GJA1) in patients with TOF or DORV of Fallot-type compared to patients with other cardiac malformations. Depicted is the ratio of phosphorylated Cx43 (GJA1) to non-phosphorylated Cx43 (GJA1). Fallot refers to patients with TOF or DORV of Fallot-type, non-Fallot refers to patients with pulmonary atresia with or without ventricular septal defect, double chamber right ventricle, truncus arteriosus communis, pulmonary stenosis or subaortic stenosis (patient characteristics are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095344#pone-0095344-t001" target="_blank">table 1</a>). Note significant changes between the two age groups but not between Fallot and non-Fallot groups. All values of are given as means±SEM. Significant differences within the three age groups are indicated by asterisks (**p<0.005).</p

Clinical data of patients with TOF and DORV of Fallot-type and with non-Fallot cardiac malformations.

<p>TOF Tetralogy of Fallot, DORV double-outlet right ventricle, Re-OP re-operation.</p><p>PA+VSD pulmonary atresia with ventricular septal defect, DCRV double chamber right ventricle.</p><p>PA+IVS pulmonary atresia with intact ventricular septum, TAC truncus arteriosus communis.</p><p>PS pulmonary stenosis.</p

Original histological specimen of patients with TOF or DORV of Fallot type.

<p><b>A:</b> Representative specimen immuno-stained for Cx43 (GJA1) (green fluorescence) and troponin I (TNNI3) (red fluorescence), nuclei are counter-stained in blue. The specific Cx43 (GJA1) staining is indicated by white arrows. Note the strict Cx43 (GJA1) polarisation in specimens of patient 18 (TOF, age 8.67 years) and patient 8 (TOF, age 21.7 years) in contrast to the polar and lateral Cx43 (GJA1) distribution in patient 4 (TOF, age 0.57years) and 10 (TOF, age 0.41 years). <b>B:</b> Co-localisation of Cx43 (GJA1) (green fluorescence) and N- cadherin (CDH2) (red fluorescence), nuclei are counter- stained in blue. Original specimen of patient 4 (TOF, age 0.57 years) is shown. White arrows show the specific Cx43 (GJA1) or N-cadherin (CDH2) staining. Yellow arrow heads point to polar co-localised Cx43 (GJA1) and N-cadherin (CDH2) in the merged picture (downright of each of the four images), white arrow heads point towards lateral Cx43 (GJA1) staining (without N-cadherin (CDH2) and red arrow heads point to lateral Cx43 (GJA1) and N-cadherin (CDH2) staining (co-localisation). <b>C:</b> 3D reconstructions of representative cells from TOF patients (A) <2 years, (B) 2–12 years, and (C) >12 years. Cells show group- typical distribution patterns of Cx43 (GJA1) (green), N-cadherin (CDH2) (red) and their co-localisation (yellow). Arrows indicate cell orientation. Arrow length corresponds to 10 µm. It can be seen that co-localisation of lateral Cx43 (GJA1) with N- cadherin (CDH2) (co-localised protein = yellow) progressively declines from young (<2years, A) to elder (<12years, C) patients.</p

Histological analysis of patients with TOF or DORV of Fallot type.

<p><b>A:</b> Cell morphology. All values of cell length and width (in µm) are given as means±SEM. Significant differences within the three age groups are indicated by asterisks (*p<0.05;**p<0.005). <b>B:</b> Cx43 (GJA1) cellular distribution. All values expressed as the percentage of polar and lateral Cx43 (GJA1) distribution are given as means±SEM. Significant differences within the three age groups are indicated by asterisks (**p<0.005). <b>C:</b> Ratio of polar Cx43 (GJA1) and lateral Cx43 (GJA1). All values expressed as the ratio of polar Cx43 (GJA1)/lateral Cx43 (GJA1) are given as means±SEM. Significant differences within the three age groups are indicated by asterisks (**p<0.005). <b>D:</b> Co-localisation of Cx43 (GJA1) and N-cadherin (CDH2). All values expressed as the percentage of co-localised Cx43 (GJA1) and N-cadherin (CDH2) are given as means±SEM. Significant differences within the three age groups are indicated by asterisks (*p<0.05;**p<0.005).</p