Tamoxifen treatment ameliorates contractile dysfunction of Duchenne muscular dystrophy stem cell-derived cardiomyocytes on bioengineered substrates

Fig. 1. Experimental design for chronic 4-hydroxytamoxifen treatment. a Cells are replated on day 10 (D10), then seeded onto our bioengineered platform on day 25 (D25). Cells are imaged and treated every 3 days from day 30 (D30). b Schematic of iPSC-CMs cultured in a monolayer (left) and as single cells on extracellular matrix (ECM) micropatterns with a length to width aspect ratio of 7:1 (right). c Motion-tracking analysis was used to measure the beating rate and beating velocity of the iPSC-CM monolayers and single cells. Videos of contracting cardiomyocytes are recorded, and analysis is performed to calculate speed of contraction (d/dt). The beating rate is defined as the number of contraction cycles per unit of time. The beating velocity is defined as the maximum contraction speed. d Ratiometric analysis of calcium flux in iPSC-CM single cells. Videos of contracting cardiomyocytes loaded with a ratiometric calcium dye are recorded, and analysis is performed to measure calcium bound dye and calcium-free dye relative to baseline (ΔF/F0). Time to peak is defined as the time between the local minimum at the start of the peak and the next local maximum. Transient amplitude is defined as…

Fig. 2. Characterization of iPSC-CMs. a–d RT-qPCR results from iPSCs and iPSC-CMs of cardiac lineage markers, MYBPC3, MYL2, MYH7, and TNNT2. Data represent mean ± SEM. Statistical significance determined by one-way ANOVA and Tukey test for post hoc comparison. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns (nonsignificant). n = 2–4 biological replicates. e 3D deconvoluted micrographs of iPSC-CMs stained for DAPI (blue), cTnT (red), and dystrophin (green) taken at ×63 magnification. Healthy1 and DMD1 iPSC-CMs were stained with MANEX1A. Healthy2 and DMD2 iPSC-CMs were stained with ab15277. Detection of the short Dp71 isoform of dystrophin likely accounts for the nuclear staining. f 3D deconvoluted micrographs of iPSC-CMs stained with DAPI (blue) and MYL2 (green) taken at ×63 magnification. g Micrograph of iPSC-CMs stained with ERα (yellow), cTnT (red), and DAPI (blue) immediately after treatment with 4-hydroxytamoxifen taken at ×40 magnification. Scale bars represent 20 μm. h Intensity of ERα staining over cell area in arbitrary units. Data represent mean ± SEM. Statistical significance determined by unpaired t test. *P < 0.05, ns (nonsignificant). n = 2–5 biological replicates. N…

Fig. 3. Video analysis software captures responses to pharmacological stimulation. a, b Representative phase contrast (top) and contractile heatmaps (bottom) for iPSC-CMs in monolayers and as single cells. Scale bars represent 25 μm. c, d Representative traces of speed over time at baseline (top) and after treatment with 1 nM isoproterenol (bottom) in Healthy1 iPSC-CMs cultured in monolayers and as single cells. Beating speed traces show peaks for both the contraction and relaxation cycles of contraction. Examples of peaks depicting contraction and relaxation are indicated by arrows. e, f Relative beating rate represented as fold change over pre-treatment of Healthy1 iPSC-CMs cultured in monolayers and as single cells in response to increasing concentrations of isoproterenol. Data represent mean ± SEM. Statistical significance determined by one-way ANOVA and Tukey test for post hoc comparison. *P < 0.05, ***P < 0.001, ns (nonsignificant). n = 3 biological replicates. N = 6–17 regions for monolayers and N = 8–24 cells for single cells.

Fig. 4. Treatment with 4-hydroxytamoxifen decreases the beating rate and improves cell survival in monolayers. a Relative beating rate represented as fold change over pre-treatment (day 30) to chronic 0.5 μM 4-hydroxytamoxifen or vehicle. n = 3 biological replicates, N = 9–22 regions. b Cell survival determined by Calcein Blue AM positive area on day 42 of differentiation relative to pre-treatment (day 30). n = 3–5 biological replicates. Cells were treated every 3 days beginning on day 30. Data represent mean ± SEM. Statistical significance determined by one-way ANOVA and Tukey test for post hoc comparison. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns (nonsignificant).

Fig. 5. Treatment with 4-hydroxytamoxifen prolongs cell survival, decreases beating rate, and increases beating velocity in micropatterned single cells. a Micrograph of a patterned iPSC-CM stained with F-actin (red) and Hoescht (blue) taken at ×40 magnification. Scale bar represents 20 μm. b Percentage of iPSC-CMs that retained the ability to spontaneous beat as individual cells. n = 3 biological replicates, N = 14–29 cells. Statistical significance determined by log-rank (Mantel–Cox) test with Bonferroni correction. c, d Relative beating rate and relative beating velocity on day 36 of differentiation represented as fold change over pre-treatment (day 30) to chronic 0.5 μM 4-hydroxytamoxifen or vehicle. Cells were treated every 3 days beginning on Day 30. Data represent mean ± SEM. Statistical significance determined by one-way ANOVA and Tukey test for post hoc comparison. *P < 0.05, ns (nonsignificant). n = 3 biological replicates, N = 9–18 cells.

Fig. 6. Treatment with 4-hydroxytamoxifen reduces the percentage of arrhythmic cells and lowers resting calcium ratio. a–c Representative calcium transients in iPSC-CMs on day 42 of differentiation. Calcium imaging was performed after cells were treated every 3 days beginning on day 30. d Percentage of cells exhibiting arrhythmic events on day 42. Pearson’s chi-square test was used to compare frequencies of arrhythmic cells. Z-test with Bonferroni correction was used for pairwise comparisons. n = 3 biological replicates, N indicates total number of cells analyzed. *P < 0.05. e, f Time to peak and resting calcium ratio on day 42. Cells were treated every 3 days beginning on day 30 of differentiation. Data represent mean ± SEM. Statistical significance determined by one-way ANOVA and Tukey test for post hoc comparison. *P < 0.05, **P < 0.01, ns (nonsignificant). n = 3 biological replicates, N = 32–75 cells.