Periodic Reporting for period 2 - CAMEOS (Cardiac micro-engineered tissue for high-throughput screening)
Okres sprawozdawczy: 2018-07-01 do 2019-06-30
After clearly establishing an improved physiological maturity of iPSC-CM in the newly developed media, we wanted to validate if these culture conditions would lead to increased relevance of the iPSC-disease modeling potential. We decided to try the media in two different cardiac disease background; channelopathy (defect in electrophysiology) and cardiomyopathy (defect in contraction). For the channelopahty we chose to work in a LQT3 (sodium channel defect) patient line, since we knew from the physiology data that the sodium channel function was improved in maturation media condition. Remarkably, we were able to see the functional manifestation of the LQT3 disease in the maturation media, which was completely absent in standard conditions. For modeling a cardiomyopathy disorder, we chose the RBM20 mutation that causes problems with a SR calcium channel; ryanodine receptor (RyR2). In this experiment, we were able to establish dramatic contractile defect in the RBM20-mutant line after culture in maturation media. Overall, our newly designed media has shown great potential to improve the physiology of iPSC-CM and increase the relevance of iPSC disease models.
Next, I worked on with another familial cardiomyopathy mutation PLN-Arg14del which particularly affects the Dutch population. I created a patient iPSC-line carrying the PLN-R14del mutation and used maturation media to attempt to recapitulate a cardiomyopathy phenotype. Although we did not detect a abnormal calcium fluxes in the cells, we did observe decreased contractile performance after maturation. Interestingly, on the molecular level we noticed elevation of the unfolded protein response (UPR) in PLN-R14del iPSC-CMs eluding that the mutation causes PLN to become a misfolded protein. Blocking key players in the UPR pathway had deleterious effect on the contractility of the PLN-R14del cells showing that increased activity of the UPR plays a protective effects. In this regard, we hypothesized that increasing UPR activity could be beneficial for the disease. We setup a phenotypic screen to test +60 compounds known to increase UPR activity and are currently following up several interesting drug candidates in confirmatory assays.