Role of Blood Oxygen Saturation During Post-Natal Human Cardiomyocyte Cell Cycle Activities

Abstract

Blood oxygen saturation (SaO₂) is one of the most important environmental factors in clinical heart protection. This study used human heart samples and human induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) to assess how SaO₂ affects human CM cell cycle activities. The results showed that there were significantly more cell cycle markers in the moderate hypoxia group (SaO₂: 75% to 85%) than in the other 2 groups (SaO₂ <75% or >85%). In iPSC-CMs 15% and 10% oxygen (O₂) treatment increased cell cycle markers, whereas 5% and rapid change of O₂ decreased the markers. Moderate hypoxia is beneficial to the cell cycle activities of post-natal human CMs.

Keywords: CHD, congenital heart disease; CM, cardiomyocytes; IF, immunofluorescence; LV, lentivirus; O2, oxygen; SaO2, blood oxygen saturation; TOF, tetralogy of Fallot; YAP1, yes-associated protein 1; blood oxygen saturation; cardiomyocyte; congenital heart disease; iPSC, induced pluripotent stem cell; pATM, phosphorylated ataxia telangiectasia mutated; pHH3, phospho-histone H3; pediatric patients; proliferation; qPCR, quantitative polymerase chain reaction; sh, short hairpin.

Graphical abstract

Figure 1

Higher Cell Cycle Activity of Human CMs in Moderate SaO₂ Conditions (A) Representative Ki67-positive cardiomyocytes (CMs) in group B; cardiac troponin-T (cTnT) (red), Ki67 (green), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) stainings are shown. The arrow indicates proliferating CMs. (B) Quantification of Ki67-positive CMs: 1-way analysis of variance (ANOVA), Student Newman Keuls (SNK), n = 10; ∗∗p < 0.01. (C) Representative phospho-histone H3 (pHH3)−positive CMs in group B; cTnT (red), pHH3 (green), and DAPI (blue) stainings are shown; the arrow indicates proliferating CMs and hatch sign indicates proliferating non-CMs. (D) Quantification of pHH3-positive CMs; 1-way ANOVA, SNK, n = 10; ∗∗p < 0.01. (E) Representative aurora B-positive CMs in group B; cTnT (red), aurora B (green), and DAPI (blue) stainings are shown; the arrow indicates proliferating CMs. (F) Quantification of aurora B-positive CMs; 2-way ANOVA, SNK, n = 10; ∗∗p < 0.01. We used quantitative polymerase chain reaction (qPCR) to analyze the expression of mRNA levels of (G) Ki67, (H) cyclin D2, and (I) AURKB in CMs treated with different levels of oxygen saturation (SaO₂). Our results indicated that Ki67, cyclin D2, and AURKB mRNA were significantly increased in the moderate SaO₂ group compared with the other 2 groups. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a control; 1-way ANOVA, SNK, n = 10; ∗∗p < 0.01.

Figure 2

Higher Cell Cycle Activity of Human iPSC-CMs in 10% and 15% O₂-Treated Conditions (A) Ki67-positive CMs, sarcomeric α-actinin (SAA) (red), Ki67 (green), and DAPI (blue) stainings are shown; the arrow indicates proliferating CMs, and the hatch sign indicates proliferating non-CMs. (B) Quantification of Ki67-positive CMs for each group: 1-way ANOVA, SNK; ∗∗p < 0.01, n = 10 fields for each group from 3 independent experiments. (C) pHH3-positive CM: SAA (red), pHH3 (green), and DAPI (blue) are shown; the arrow indicates proliferating CMs, and the hatch sign indicates proliferating non-CMs. (D) Quantification of pHH3-positive CMs for each group: 1-way ANOVA, SNK; ∗∗p < 0.01, n = 10 fields for each group from 3 independent experiments. (E) Aurora B-positive CM: SAA (red), aurora B (green), and DAPI (blue) stainings are shown; the arrow indicates proliferating CMs, and the hatch sign indicates proliferating non-CMs. (F) Quantification of aurora B-positive CMs for each group: 1-way ANOVA, SNK; ∗∗p < 0.01, n = 10 fields for each group from 3 independent experiments. We used qPCR to analyze mRNA levels of (G)Ki67, (H)cyclin D2, and (I) AURKB in CMs treated with different levels of oxygen (O₂). Our results indicated that Ki67, cyclin D2, and AURKB mRNA were significantly increased in 15% and 10% O₂-treated human induced pluripotent stem cell−cardiomyocytes (iPSC-CMs). GAPDH served as a control; 1-way ANOVA, SNK, n = 3; ∗p < 0.05; ∗∗p < 0.01. Abbreviations as in Figure 1.

Figure 3

Oxidative DNA Damage Was Significantly Reduced in Moderate Hypoxia Human CMs (A) We used qPCR to analyze mitochondrial DNA (mtDNA) levels in the hearts of patients with tetralogy of Fallot (TOF). Our results indicated that mtDNA was significantly decreased in the moderate SaO₂ group compared with the other 2 groups: 1-way ANOVA, SNK, n = 10 samples; ∗∗p < 0.01. (B) 8-oxoguanine (8-oxoG) in mild, moderate, and severe hypoxia heart samples; cTnT (white), 8-oxoG (red), and DAPI (blue) stainings are shown. (C) Quantification of 8-oxoG immunofluorescence (IF) intensity in mild, moderate, and severe hypoxia heart samples: 1-way ANOVA, SNK, n = 10 samples, ∗∗p < 0.01, compared with group B (moderate hypoxia). (D) Phosphorylated ataxia telangiectasia mutated (pATM) in mild, moderate, and severe hypoxia heart samples; cTnT (green), pATM (red), and DAPI (blue) staining are shown. (E) Quantification of pATM IF intensity in mild, moderate, and severe hypoxia heart samples. One-way ANOVA, SNK, n = 10 samples; ∗∗p < 0.01, compared with group B (moderate hypoxia). (F) IF graph of Pitx2 in mild, moderate, and severe hypoxia heart samples; cTnT (red), Pitx2 (green), and DAPI (blue) staining are shown. (G) Quantification of Pitx2-positive CMs in mild, moderate, and severe hypoxia heart samples. One-way ANOVA, SNK, n = 10 samples; ∗∗p < 0.01, compared with group B (moderate hypoxia). (H) Western blot graph of Pitx2 in mild, moderate, and severe hypoxia heart samples. (I) Quantification of Pitx2 densitometry in mild, moderate, and severe hypoxia heart samples. One-way ANOVA, SNK, n = 10 samples; ∗∗p < 0.01, compared with group B (moderate hypoxia). Abbreviations as in Figures 1 and 2.

Figure 4

Oxidative DNA Damage Was Significantly Reduced in 10% and 15% O₂-Treated Human iPSC-CMs (A) 8-oxoG in normal and 15% O₂-Treated Human iPSC-CMs; cTnT (red), 8-oxoG (green), and DAPI (blue) stainings are shown. (B) Quantification of 8-oxoG IF intensity in differently O₂-treated human iPSC-CMs; 1-way ANOVA, SNK; ∗p < 0.05; ∗∗p < 0.01, n = 10 fields from 3 independent experiments, compared with normal. (C) Quantification of mtDNA content in differently O₂-treated human iPSC-CMs; 1-way ANOVA, SNK; ∗∗p < 0.01, n = 3 replicates compared with normal. (D) pATM in normal, 10%, and 5% O₂-treated human iPSC-CMs; cTnT (green), pATM (red), and DAPI (blue) stainings are shown. (E) Quantification of pATM IF intensity in differently O₂-treated human iPSC-CMs; 1-way ANOVA, SNK; ∗p < 0.05; ∗∗p < 0.01, n = 10 fields from 3 independent experiments compared with normal. (F) IF graph of Pitx2 in normal, 10% hypoxia, and 5% hypoxia iPSC-CMs; cTnT (green), Pitx2 (red), and DAPI (blue) staining are shown. (G) Quantification of Pitx2 positive CMs in normal, 10% hypoxia, and 5% hypoxia iPSC-CMs. One-way ANOVA, SNK, n = 10 fields from 3 independent experiments; ∗∗p < 0.01, compared with group B (moderate hypoxia). (H) Western blot graph of Pitx2 in normal, 10% hypoxia, and 5% hypoxia iPSC-CMs. (I) Quantification of Pitx2 densitometry in normal, 10% hypoxia, and 5% hypoxia iPSC-CMs. One-way ANOVA, SNK, n = 3 samples; ∗∗p < 0.01, compared with group B (moderate hypoxia). Abbreviations as Figures 1, 2, and 3.

Figure 5

Higher Expression of YAP1 in Moderate SaO₂ Human CMs and 10% O₂-Treated Human iPSC-CMs; Overexpression of YAP1 Protected the Proliferative Potential of Human iPSC-CMs From Oxidative DNA Damage (A) Representative yes-associated protein 1 (YAP1) IF in human heart samples; cTnT (red), YAP1 (green), and DAPI (blue) stainings are shown. (B) Quantification of YAP1 IF intensity in human CMs; 1-way ANOVA, SNK; ∗∗p < 0.01, n = 30 slides, compared with the moderate group. (C) Higher expression of YAP1 in 10% O₂-treated group as measured by Western blot. (D) Quantification of YAP1 expression; ∗p < 0.05; n = 3 replicates. (E) Higher expression and nuclear location of YAP1 in 10% O2-treated group as measured by IF. (F) YAP1 overexpression (Op) in the 5% O₂-treated group as verified by the Western blot. (G) Quantification of YAP1 expression; ∗∗p < 0.01; n = 3 replicates. (H) YAP1 OP in the 5% O₂-treated group as verified by IF; cTnT (red), YAP1 (green), and DAPI (blue) stainings are shown. (I) Ki67-, pHH3-, and aurora B−positive human iPSC-CMs in 5% O₂-treated and YAP1 OP groups. (J) Quantification of Ki67-, pHH3-, and aurora B−positive CMs in the 10% O₂-treated, 5% O₂-treated, 5% O₂-treated and YAP1 OP group; 1-way ANOVA, SNK; ∗∗p < 0.01, n = 10 fields from 3 independent experiments compared with the 5% O₂ group. (K) YAP1 knockout (KO) in the 10% O₂-treated group as verified by Western blotting. (L) Quantification of YAP1 expression; ∗∗p < 0.01, n = 3 replicates. (M) YAP1 KO in the 10% O₂-treated group as verified by IF; cTnT (white), YAP1 (green), and DAPI (blue) staining are shown. (N) Representative Ki67-, pHH3-, and aurora B−positive human iPSC-CMs in 10% O₂-treated and YAP1 KO groups. (O) Quantification of Ki67-, pHH3-, and aurora B−positive CMs in the 10% O₂-treated, 10% O₂-treated, and YAP1 KO group; 1-way ANOVA, SNK; ∗∗p < 0.01, n = 10 fields from 3 independent experiments compared with the 10% O₂ group. Abbreviations as in Figures 1, 2, 3, and 4.

Figure 6

Reduced Oxidative DNA Damage After OP of YAP1 in 5% O₂-Treated Human iPSC-CMs (A) Representative 8-oxoG IF in the 5% O₂, 10% O₂, 5% O₂-treated, and YAP1 OP group; cTnT (red), 8-oxoG (green), and DAPI (blue) stainings are shown. (B) Quantification of 8-oxoG IF intensity; 1-way ANOVA, SNK; ∗∗p < 0.01, n = 10 fields from 3 independent experiments compared with the 5% O₂ group. (C) Reduced mtDNA content in YAP1 OP group; 1-way ANOVA, SNK; ∗∗p < 0.01, n = 5 replicates compared with the 5% O₂ group. Abbreviations as in Figures 1, 2, 3, 4, and 5.