The role of autophagy in microwave radiation induced toxicity in iPSC-derived cardiomyocytes

Figures

• 

  Fig. 1. S-band microwave radiation source schematic diagram.

• 

  Fig. 2. Experimental flowchart.

• 

  Fig. 3. Differentiation and structural identification of iPSC-CMs (Scale bar = 20 μm). A: Differentiation identification—DAPI (blue), TNNT2 (green), Nkx2-5 (red); Merge: overlay of all channels. B: α-Actinin immunofluorescence (red). C: Cx43 immunofluorescence (green). D: PKP-2 immunofluorescence (green). E: ZO-1 immunofluorescence (red). For panels B–E, DAPI (blue); Merge: overlay of two channels; Zoom-in: Partially enlarge the image, with an enlargement ratio of approximately 654%. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

• 

  Fig. 4. Ca2+ conduction and calcium transient changes in iPSC-CMs following microwave radiation. (n = 3). A: Calcium transient conduction activation map. Red indicates an earlier activation time, while blue indicates a later activation time; B: Conduction direction. It shows the direction of calcium conduction, indicated by arrows. The direction of the arrows represents the conduction direction, and the length indicates the conduction speed; C: APD30/80 heatmap. The colors represent the values of APD30/80. The red values are smaller while the blue values are larger, demonstrating the heterogeneity of APD30/80 among cells; D: CaTD90 heatmap. The numerical value representing the 90% duration of the calcium transient is shown. Red indicates a shorter duration, while blue indicates a longer duration; E: Amplitude heatmap. It reflects the amplitude distribution of calcium fluctuations, with the color indicating the intensity value of the calcium fluctuations. The red color represents a lower intensity, while the blue color represents a higher intensity.; F: Waveform diagram. It visually presents the comparison of calcium transient waveforms between Group C and Group R at different time points…

• 

  Fig. 5. Ultrastructure and mitochondrial functional analysis of iPSC-CMs at 6 h post-microwave exposure(n = 4). A-F: Ultrastructural observation of myocardial cells under electron microscope (bar = 300 nm). The red arrow indicates the area with severe damage. In C and F: green marks normal mitochondria, orange marks mitophagic mitochondria, blue indicates mitochondria with cristae loss.A: Intercalated disc (ID, control group),; B: Sarcomere/Z-line (control group); C: Mitochondria (control group); D: Intercalated disc (ID, radiation group); E: Sarcomere/Z-line (radiation group); F: Mitochondria (radiation group); G: Normal mitochondria/30 μm2. This statistical graph shows the number of normal mitochondria in the C group and the R group within an area of 30 μm2.; H: Mitochondria with autophagosomes/30 μm2. This statistical graph shows the number of autophagic mitochondria in the C group and the R group within an area of 30 μm2.; I: Mitochondria with cristae loss/30 μm2. This statistical graph shows the number of mitochondrial spindles lacking spines in group C and group R within an area of 30 μm2.; J: ATP statistics. This figure shows the statistical data of ATP concentrations in group C…

• 

  Fig. 6. Mitochondrial structural injury in iPSC-CMs at 6 h post-exposure. A: Mitochondrial vacuolation change; B: Mitochondrial cleft-like change; C: Formation of mitochondrial concentric circular structure; D: Mitochondrial myelin-like structure. E: Formation of an isolation membrane surrounding the mitochondria (indicated by red lines as the isolation membrane); F: Lysosome contacts the isolation membrane of the mitochondria (indicated by green lines as the lysosome); G: Fusion of mitochondrial autophagosomes with lysosomes (indicated by blue lines as the fused autophagosomes and green lines as the lysosomes); H: Formation and digestion of mitochondrial autophagosomes and isolated substances (indicated by orange lines as the digested substances and green lines as the lysosomes); I-L: Process of mitochondrial autophagosomes being digested; M−P: Instance of a single mitochondrial autophagy process containing multiple autophagosomes. Pink lines indicate the cells of key concern. The images are all from the R group 6 h after microwave radiation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

• 

  Fig. 7. Integrated transcriptomic and autophagy analysis post-microwave exposure. A: Overlap between transcriptome and autophagy genes; B: GO enrichment heatmap; C: Network clustering; D: P-value network; E: KEGG pathway bubble plot; F: Key gene map by Metascape; G: KEGG pathway heatmap for key genes.

• 

  Fig. 8. Assessment of autophagy at 6 h post-microwave in iPSC-CMs (Scale bar = 20 μm, n = 5). A: LC3B immunofluorescence; B: p62 immunofluorescence; C: LC3B fluorescence intensity statistics; D: p62 fluorescence intensity statistics; E: LC3I, LC3II, and p62 WB; F: p62 WB quantification; G: LC3II/I WB quantification. DAPI (blue), LC3B (green), p62 (green); Merge: two-channel overlay; Zoom-in: magnified local images. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

• 

  Fig. 9. Toxicity and autophagy modulation by AICAR(n = 5). A: Cell toxicity of different concentrations of AICAR was detected by flow cytometry. It is divided into three groups: C: Control group; A 0.5 mM: Group with 0.5 mM AICAR drug added; A 1 mM: Group that adds 1 mM AICAR drug. First, select the viable cells, and then analyze the proportion of dead cells among the viable cells; B: Toxicity quantification; C: LC3B immunofluorescence histogram; D: p62 immunofluorescence histogram; E: LC3B immunofluorescence staining; F: p62 LC3B immunofluorescence staining. DAPI (blue); LC3B (green); p62 (green); Merge: two-channel overlay; Zoom-in: local magnification. C group: Control group, the blue enlarged box; R group: Microwave radiation group, orange enlarged box; AC group: Control group + 0.5 mM AICAR group, green enlarged box; AR group: Microwave radiation group + 0.5 mM AICAR group, pink enlarged box. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

• 

  Fig. 10. Changes in calcium transients at 6 h post-radiation in iPSC-CMs(n = 6). A: Calcium transient heatmap (ms); B: Amplitude heatmap; C: APD30/80 heatmap; D: APD30/80 statistics; E: CaTD90 statistics; F: Amplitude statistics; G: Heatmap summary with star marks for the most significant changes and optimum recovery after drug treatment.