Assessment of human bioengineered cardiac tissue function in hypoxic and re-oxygenized environments to understand functional recovery in heart failure

Figures

• 

  Fig. 1. Assessment of the purity of iPSC-CMs after exposure to puromycin and schematic of the experimental design. (A) Representative flow cytometry plots demonstrating the presence of cTnT-positive and myosin heavy chain-positive cardiomyocytes after puromycin treatment. (B) Upper panel: photographs showing the contractile force measurement system. Lower panel: schematic illustration of the contractile measurement system. (C) Cardiomyocytes were subjected to three different experimental conditions: continuous normoxia (20% O2) for 12 days; hypoxia (1% O2) for 4 days followed by normoxia (20% O2) for 8 days; or continuous hypoxia (1% O2) for 12 days. Functional assessments were made on day 0; functional, molecular biological and morphological investigations were carried out on day 4 and day 12; functional assessments were made on day 8.

• 

  Fig. 2. Effects of hypoxia on the contractile function of iPSC-derived bioengineered cardiac tissue. (A) Representative contractile force traces recorded from a cardiac cell sheet. (B) Cardiac tissues were incubated in normoxic (20% O2) or hypoxic (1% O2) conditions for 4 days. Data for contractile force, beating rate, contraction time, relaxation time, maximum contraction velocity and maximum relaxation velocity are shown for day 0 (D0) and day 4 (D4). (C) Comparison of contractile force, beating rate, contraction time, relaxation time, maximum contraction velocity and maximum relaxation velocity on day 4 between the normoxic group (N4) and hypoxic group (H4). Data in (B) and (C) are presented as the mean ± SD of a minimum of 7 preparations. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

• 

  Fig. 3. Morphological and molecular biological changes induced by hypoxia. (A–E) hiPSC-CMs were incubated in normoxic (20% O2; N4) or hypoxic (1% O2; H4) conditions for 4 days. (A) Representative images of cTnT-positive cardiomyocytes. Nuclei were labeled with Hoechst. Bars, 200 μm. (B) The number of cTnT-positive cells in 49 fields (7 × 7) was calculated for each time point. The number of cTnT-positive cells in each group was normalized to the mean value of the N4 group (10 biological repeats). (C) Representative images showing sarcomere structure. Cells were stained with α-actinin, and nuclei were labeled with Hoechst. Bars, 40 μm. (D) The mRNA expressions of various genes in cardiomyocytes. Data are shown as the mean ± SD of 3 biological repeats and are normalized to the mean value of the N4 group. (E) Cellular ATP levels in cardiomyocytes. Data are shown as the mean ± SD of 14 biological repeats. RLU: relative light units. (F, G) Cardiac spheroids were cultured for the assigned periods. (F) Representative Ca2+ transients recorded from cardiac spheroids. (G) The amplitudes of Ca2+ transients measured in cells loaded with fluo-8. Data for F/F0 (peak fluorescence divided by baseline…

• 

  Fig. 4. Effects of continuous normoxia, hypoxia/re-oxygenation and continuous hypoxia on the contractile function of iPSC-derived bioengineered cardiac tissue. Cardiac tissues were maintained under three conditions: continuous normoxia (20% O2) for 12 days; hypoxia (1% O2) for 4 days followed by normoxia (20% O2) for 8 days; or continuous hypoxia (1% O2) for 12 days. (A) Representative contractile force traces for day 4 (D4), day 8 (D8) and day 12 (D12). (B) Comparison of contraction time, relaxation time, contractile force, beating rate, maximum contraction velocity and maximum relaxation velocity between day 4 (D4), day 8 (D8) and day 12 (D12) for each experimental condition. Data are presented as the mean ± SD of 6 preparations. (C) Comparison of contractile force, beating rate, contraction time, relaxation time, maximum contraction velocity and maximum relaxation velocity on day 12 between the normoxia group (NN12) and hypoxia/normoxia group (HN12). Data are presented as the mean ± SD of 6 preparations. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

• 

  Fig. 5. Morphological and molecular biological changes induced by hypoxia/re-oxygenation. (A–E) hiPSC-CMs were incubated under normoxic conditions (20% O2) for 12 days (NN12) or hypoxic conditions (1% O2) for 4 days followed by normoxic conditions (20% O2) for 8 days (HN12). (A) Representative images of cTnT-positive cardiomyocytes. Nuclei were labeled with Hoechst. Bars, 200 μm. (B) The number of cTnT-positive cells in 49 fields (7 × 7) was calculated for each time point. The number of cardiomyocytes in each group was normalized to the mean value of the NN12 group. (C) Representative images showing sarcomere structure. Cells were stained with α-actinin, and nuclei were labeled with Hoechst. Bars, 40 μm. (D) The mRNA expressions of various genes in cardiomyocytes. Data are shown as the mean ± SD and are normalized to the mean value of the NN12 group. (E) Cellular ATP levels in cardiomyocytes. Data are shown as the mean ± SD. RLU: relative light units. (F–I) Cardiac spheroids were cultured for the assigned periods. (F) Representative Ca2+ transients recorded from cardiac spheroids on day 12. (G) The amplitudes of Ca2+ transients measured from cells loaded with fluo-8 and exposed to…