Mapping cardiac drug transport: In vitro assessment of cardiac P-glycoprotein function with [18F]MC225 by using µ-engineered heart tissues

Figures

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  Figure 1. Function of P-gp transporter [18F]MC225 in vivo vs. in vitro. (A) The P-gp efflux transporters that are present in the cardiac endothelial cells in vivo pump its substrates out of the cells. [18F]MC225 acts as a substrate for the P-gp efflux transporter. (B) The chemical structure of [18F]MC225. (C) When P-gp transporters are inhibited in vitro, the efflux of [18F]MC225 is reduced, causing it to accumulate inside the cells. When P-gp transporters are induced in vitro, e.g. by cardiotoxic Doxorubicin, more [18F]MC225 is pumped out of the cells.

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  Figure 2. (A) Chip design and dimensions (adapted from18) and representation of the chip during culture. (B) Cell composition of tissue formation and drug administration before contraction force measurements. (C) A representative µ-EHT before and after drug administration during contraction force measurements for control and Doxorubicin (n = 1 showed). (D) Contraction force data (Control n = 12 tissues, Doxorubicin n = 8 tissues) in which the contraction force reduces significantly after 24 h of a single dose of Doxorubicin. P-values were determined by unpaired t-test: ***P < 0.0001, ****P < 0.001.

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  Figure 3. Immunohistochemistry for P-gp expression in µ-EHTs (red, P-gp; blue, nuclei). (A–D) P-gp expression in a 96-wells 2D-monolayer culture of (A) CMs (white, TroponinT, 3 × 104 cells), (B) ECs (white, VE-cadherin, 1 × 104 cells), (C) FBs (white, Vimentin, 1 × 104 cells), and (D) SMCs (white, SM22, 1 × 104 cells). (E, F) Quantification of P-gp expression in co-culture vs. CM, FB, SMC, EC is shown before and after Doxorubicin treatment. In (E) significant increase of P-gp expression after Doxorubicin induction is shown in co-culture as well as compared to CMs, FBs, ECs, and SMCs. In (F) significant increase of P-gp expression after Doxorubicin induction is shown in co-culture as well as compared to CMs, FBs, ECs, and SMCs. In (G) previously generated RNA sequencing data shows that Doxorubicin significantly increases P-gp gene expression after 24 h. Data are presented as the mean ± standard deviation, with P-values determined by unpaired t-test: ****P < 0.0001, ***P < 0.001, and *P < 0.05 (H, I) A 3D- µ-EHT (n = 1 tissue) in which the endothelial layer (H, white, VE-cadherin) is shown with P-gp expression inside the tissue, vs. the P-gp expression inside the CMs (I, white, troponin T).…

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  Figure 4. (A) Workflow for incubating and radioactivity detection (B) Relative uptake as percentages for the Tariquidar (n = 19 tissues), Doxorubicin (n = 20 tissues), and control groups (n = 24 tissues and n = 13 tissues, respectively) detected by γ-counters. (C) The relative uptake percentages for the Tariquidar (n = 11 tissues), Doxorubicin (n = 13 tissues), and control groups (n = 10 tissues and n = 10 tissues, respectively) were determined through autoradiography detection. (D) Display of radioactive imaging following treatment with Tariquidar, Doxorubicin, and control groups. Data are presented as the mean ± standard deviation, with P-values determined by unpaired t-test: ***P < 0.001, and *P < 0.05.