Accelerated Growth, Differentiation, and Ploidy with Reduced Proliferation of Right Ventricular Cardiomyocytes in Children with Congenital Heart Defect Tetralogy of Fallot

Figures

• 

  Figure 1. Distribution of CMCs of different ploidy classes in patients with TF and the control group. Comparison of ploidy (2c:4c:8c:16c:32c) of patients with TF and the control group (A). The ploidy of the CMCs in children with TF was significantly higher than the CMCs in the control groups (median, Mann-Whitney test, p < 0.05) (B). Comparison of CMCs ploidy classes in patients with TF and controls (C). Change in the ratio of different ploidy classes in patients with TF in different age groups: up to six months, 7–12 months, more than 13 months (D). Comparison of the proportion of binucleated CMCs in the group of children with TF and the control group (median, Mann-Whitney test, p < 0.05) (E). Inverse correlation of CMCs ploidy in children with TF with Nakata index (r = −0.44; p = 0.034) (F). In the myocardium of children under six months of age with TF: CMCs ploidy correlated inversely with age (r = −0.73; p = 0.007) (G). The myocardium of patients with a high percentage of diploid CMCs differentiated faster—the number of CMCs filled with myofibrils increased in it (r = 0.68; p = 0.015) (H) and the number of CMCs not filled with myofibrils decreased (r = −0.68; p = 0.014) (I).

• 

  Figure 2. RV myocardium of patients with TF and in the control group. In the myocardium of patients with TF: significant variability of the CMCs diameter was revealed: on average, from 7.5 ± 2.4 μm (patient with TF, 10 months) (A) and 10.0 ± 1.5 μm (patient with TF, 5 months) (B) up to 10.8 ± 1.5 μm (patient with TF, 8 months) (C). In the control group: the diameter was 4.7–7.7 microns (D,E). Hematoxylin and eosin (×400) bar 20 µm. The diameter of the CMCs of children with TF significantly exceeded Table 1. (F). In children with TF over 6 months of age: The proportion of CMCs filled with myofibrils increased with age (r = 0.49; p = 0.003) (G), a high-pressure gradient on the pulmonary artery provoked accelerated differentiation with a decrease in the number of CMCs with sarcoplasm not filled with myofibrils (r = −0.39; p = 0.04) (H).

• 

  Figure 3. Connexin-43-containing (Cx43) gap junctions (GJs) in RV CMCs of patients with TF and the control group. In the myocardium of children with TF: Cx43-containing GJs were located both in the intercalated discs (A) and on the lateral sides, along the perimeter of the sarcolemma of the CMCs (B); a large number of binucleated CMCs. Immunohistochemical staining (×200), bar 10 µm. At the ultrastructural level, lateral GJs were found at the border of two CMCs in the form of concentric structures, following the bends of the CMCs sarcolemma (arrows) (C). In the control group: point Cx43-containing GJs were diffusely located along the perimeter of the sarcolemma (D). In the differentiated CMCs filled with myofibrils, the Cx43-containing GJs were localized predominantly in the intercalated discs (E). Immunohistochemical staining (×200), bar 10 µm. The relative length of lateral Cx43-containing GJs was significantly higher in the myocardium of patients with TF than in controls (Mann-Whitney test, p < 0.05) (F). The relative length of Cx43-containing GJs on the lateral surfaces of the CMCs decreased with age (r = −0.45; p = 0.0003) (G). The lateral arrangement of Cx43-containing GJs was…

• 

  Figure 4. Ultrastructure of immature RV CMCs of children with TF. CMCs of an immature phenotype with electron-transparent sarcoplasm, not filled with myofibrils, with many small mitochondria and vacuoles in the sarcoplasm. Myofibrils were located in a thin layer along the periphery of the sarcoplasm, bar 5 µm (A). In the CMCs of a more differentiated phenotype, myofibrils filled most of the sarcoplasm, in the perinuclear zone there were mitochondria, glycogen granules, single lipofuscin granules, bar 5 µm (B). Assembly of myofibrils in the sarcoplasm of the CMC, bar 1 µm. (C). T-system channels at the level of Z-bands of organizing myofibrils, bar 0,5 µm (D). Paired centrioles, cisterns, and vesicles of the Golgi apparatus in the perinuclear zone of the CMC, bar 0.5 µm (E). Myelin figures, vacuole in the perinuclear zone of the CMC, bar 1 µm. (F). In children with TF under six months of age: the assembly of myofibrils was more common in the CMCs of patients with a low gradient on the pulmonary artery (r = −0.52; p = 0.01) (G); signs of dystrophic changes in ultrastructure (myelin figures, phagosomes) were found in the CMCs of children with low hemoglobin levels (r = −0.54; p = 0.025) (H),…

• 

  Figure 5. Interstitial tissue of the RV myocardium of children with TF and the control group. An insignificant proportion of interstitial tissue in the perivascular zone of the myocardium in children with TF (A,B) and the control group (C,D). Masson trichrome (×200), bar 10 µm. The proportion of interstitial tissue in the myocardium of children with TF and the control group did not differ significantly (E) (median, Mann-Whitney test, p > 0.05). CD34-positive capillaries and small vessels in the myocardial interstitium of a patient with TF. Immunohistochemical staining (×400), bar 20 µm (F). In children with TF over 6 months of age: the proportion of interstitial tissue correlated with age (r = 0.38; p = 0.03) (G) and increased with an increase in the number of differentiated CMCs, the sarcoplasm of which was filled with myofibrils (myofibril-free zones were 10%)(r = 0.64; p = 0.0001) (H). The density of CD34-positive small vessels and capillaries directly correlated with LVEF (r = 0.64; p = 0.003) (I).

• 

  Figure 6. Proliferative-active Ki67+/Sarc α-actin+ CMC in the RV myocardium of patients with TF. In multinucleated CMC, all nuclei were Ki67-positive (arrows) (A). Alexa 488—anti-rabbit antibody to Ki67 (B); Alexa 546—anti-mouse antibody to Sarcomeric α-Actin (C); DAPI nuclei staining (D). Immunoconfocal microscopy, bar 10 мкм. Patient with TF, five months. Ki67-positive CMCs were detected in the myocardium with a large proportion of interstitial tissue (r = 0.33; p = 0.27) (E); in the myocardium with a smaller diameter of the CMCs (r = −0.31; p = 0.02) (F); in the myocardium with a greater relative length of lateral Cx43-containing GJs in the CMCs (r = 0.29; p = 0.02) (G).