In order to validate our approach we designed a study to directly compare key physiological cardiomyocyte parameters in hiPSC-CM cultured in either standard media versus or the new “maturation” media. For example, calcium ions play an integral role in the excitation-contraction coupling in CMs. In adult CMs, calcium is mostly recycled to an intracellular organelle called the sarcoplasmic reticulum (SR). However, in iPSC-CM calcium is mostly released outside the cells and not recycled to the SR. In this regard, culture of iPSC-CM in our maturation media led to a dramatic shift towards SR-dependent calcium fluxes. Since calcium is critical for the contraction of the heart muscle, we also noticed that the force produced by the cells was affectedly higher after culture in the maturation media. Another aspect of maturation we studied was the metabolism of the iPSC-CM. We know that adult cardiomyocyte heavily depend upon lipids as a fuel source. We noticed that maturation media pushed the cells towards an aerobic metabolism, a key feature of lipid depend metabolism.
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.
