Contraction pressure analysis using optical imaging in normal and MYBPC3-mutated hiPSC-derived cardiomyocytes grown on matrices with tunable stiffness.

Current in vivo disease models and analysis methods for cardiac drug development have been insufficient in providing accurate and reliable predictions of drug efficacy and safety. Here, we propose a custom optical flow-based analysis method to quantitatively measure recordings of contracting cardiomyocytes on polydimethylsiloxane (PDMS), compatible with medium-throughput systems. Movement of the PDMS was examined by covalently bound fluorescent beads on the PDMS surface, differences caused by increased substrate stiffness were compared, and cells were stimulated with ß-agonist. We further validated the system using cardiomyocytes treated with endothelin-1 and compared their contractions against control and cells incubated with receptor antagonist bosentan. After validation we examined two MYBPC3-mutant patient-derived cell lines. Recordings showed that higher substrate stiffness resulted in higher contractile pressure, while beating frequency remained similar to control. ß-agonist stimulation resulted in both higher beating frequency as well as higher pressure values during contraction and relaxation. Cells treated with endothelin-1 showed an increased beating frequency, but a lower contraction pressure. Cells treated with both endothelin-1 and bosentan remained at control level of beating frequency and pressure. Lastly, both MYBPC3-mutant lines showed a higher beating frequency and lower contraction pressure. Our validated method is capable of automatically quantifying contraction of hiPSC-derived cardiomyocytes on a PDMS substrate of known shear modulus, returning an absolute value. Our method could have major benefits in a medium-throughput setting.

No acceleration of UV-induced skin carcinogenesis from evenly spread dietary intake of cyclosporine in contrast to oral bolus dosages.

BACKGROUND: Organ transplant recipients using the immunosuppressant cyclosporine have an increased risk for developing nonmelanoma skin cancer. Disparate effects of cyclosporine have, however, been reported on UV-induced skin carcinogenesis in mouse experiments. Therefore, we set out to compare three experimental protocols using mice, with the aim to emulate most closely the increased skin cancer risk in organ transplant recipients. METHODS: UV carcinogenesis was performed in hairless SKH-1 mice by three protocols: dietary cyclosporine and daily UV exposures, dietary cyclosporine after a period of UV exposures, and bolus dosing cyclosporine by gavage and repeated UV exposures. RESULTS: Using chronic UV exposure, continuous dietary administration of cyclosporine was shown to inhibit tumor formation. Dietary cyclosporine after a period of UV exposures did not affect ensuing UV carcinogenesis. However, in contrast with dietary cyclosporine, bolus dosages of cyclosporine by gavage, resulting in strongly varying blood levels of cyclosporine, increased tumor development in chronically UV-exposed mice. There was no difference in tumor development between mice UV-irradiated during peak or trough levels of cyclosporine in the blood. Time-averaged levels in these mice were similar to those with cyclosporine in the diet. CONCLUSIONS: Cyclosporine in bolus doses appears to increase skin cancer development, whereas cyclosporine administration more evenly spread over time does not. Extrapolation to transplant patients suggests that the mode of administrating cyclosporine may be crucial for the increased skin cancer risk and that this risk might be lowered with a more steady release of cyclosporine in the body.

Dietary immunosuppressants do not enhance UV-induced skin carcinogenesis, and reveal discordance between p53-mutant early clones and carcinomas.

Immunosuppressive drugs are thought to cause the dramatically increased risk of carcinomas in sun-exposed skin of organ transplant recipients. These drugs differ in local effects on skin. We investigated whether this local impact is predictive of skin cancer risk and may thus provide guidance on minimizing the risk. Immunosuppressants (azathioprine, cyclosporine, tacrolimus, mycophenolate mofetil, and rapamycin) were assessed on altering the UV induction of apoptosis in human skin models and of p53 mutant cell clones (putative tumor precursors) and ensuing skin carcinomas (with mutant p53) in the skin of hairless mice. Rapamycin was found to increase apoptosis (three-fold), whereas cyclosporine decreased apoptosis (three-fold). Correspondingly, a 1.5- to five-fold reduction (P = 0.07) or a two- to three-fold increase (P < 0.001) was found in cell clusters overexpressing mutant p53 in chronically UV-exposed skin of mice that had been fed rapamycin or cyclosporine, respectively. Deep sequencing showed, however, that the allelic frequency (∼5%) of the hotspot mutations in p53 (codons 270 and 275) remained unaffected. The majority of cells with mutated p53 seemed not to overexpress the mutated protein. Unexpectedly, none of the immunosuppressants admixed in high dosages to the diet accelerated tumor development, and cyclosporine even delayed tumor onset by approximately 15% (P < 0.01). Thus, in contrast to earlier findings, the frequency of p53-mutant cells was not predictive of the incidence of skin carcinoma. Moreover, the lack of any accelerative effect on tumor development suggests that immunosuppressive medication is not the sole cause of the dramatic increase in skin cancer risk in organ transplant recipients.