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Cardiac micro-engineered tissue for high-throughput screening

Periodic Reporting for period 2 - CAMEOS (Cardiac micro-engineered tissue for high-throughput screening)

Reporting period: 2018-07-01 to 2019-06-30

Heart disease is the most significant cause of morbidity and mortality in the industrialized world, and the cause of 4 million deaths each year within the European Union. The prevalence of the disease is a huge burden on society estimated to cost the EU economy 60 billion annually on drug therapy, patient care, and loss in productivity. Nonetheless, despite the latest advances in research much remains to be learn about pharmacological treatments in cardiovascular disease. Recently, the ability to produce an unlimited supply of human cardiomyocytes (CMs) from induced-pluripotent stem cells (iPSC) has allowed researcher to study human heart disease in an unprecedented manner. The scalability of iPSC-CMs makes them amendable for high throughput screening (HTS) applications, in which the role of proteins and signaling pathway can be unraveled in context of CM physiological function. Furthermore, the creation of iPSC line from patients allows for the creation of ‘patient-in-a-dish” models. In this setting, HTS approach can applied to find novel therapeutic interventions and help in the development of individualized medicine. However, the limitation of the current format centers on the lack of phenotypic cardiomyocytes maturity (the iPSC-cardiomyocyte do not behave like cardiomyocyte present in the heart). Consequently, these in vitro models often fail to recapitulate relevant physiological traits. The aim of CAMEOS is to develop a culture media to enhance the physiological relevance for in vitro high-throughput screening application. This will be achieved by re-formulating key component of culture media, to offer the cells a more physiological relevant environment in order to promote their maturation. After the development of optimized culture condition, I will apply the developed media to model a complex disease mutation, phospholamban (PLN) R14del, for which the pathology is poorly understood and no cure is available. Finally, I will perform HTS in attempt to find novel therapeutic targets in the PLN-R14del patient iPSC-CMs.
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.
Although the frequency of PLN-encoded mutations is on average very low (~0.2%) in the cardiomyopathy population, in the Netherlands the PLN R14del mutation accounts for 13% of diagnosed dilated cardiomyopathy patients (a serious unmet clinical need). At the University Medical Center Utrecht, a special focus has been put on this patient population in order to unravel the complex underlying mechanism behind this phenotype. In this context, the utilization of my developed maturation media has helped set into motion a new therapeutic avenue to tackle the disease. Furthermore, my training at Stanford University has enabled me to setup a new pipeline in Utrecht with a focus on patient iPSC disease modeling in combination with high throughput functional assays.
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