Abstract

Background. The recent advances in the pluripotent stem cell biology now make it possible to generate human cardiomyocytes in vitro from both healthy individuals and from patients with cardiac abnormalities. This offers unprecedented opportunities to study cardiac disease development in a very controlled setting and establish novel platforms for drug discovery. However, to date, there are almost no studies evaluating the role of mitochondrial function and dynamics in the metabolism of human induced pluripotent stem cells (iPSC) and how these processes affect cardiomyocyte commitment. Mitochondria are highly dynamic organelles that undergo a continuous process of fission and fusion events. Perturbations in normal mitochondrial dynamics in either direction can lead to the accumulation of damaged and inefficient organelles. Altered mitochondrial enzyme activities, elevated reactive oxygen species and defective mitochondrial DNA repair have been reported in Down’s syndrome cultures and tissues. However, the underlying cause of this defective mitochondrial respiration is still unclear. Furthermore, the frequency of heart disease is very high in infants with Down’s syndrome. The prevalence of these malformations in patients with trisomy 21 is near 50% and cardiac anomalies are the main cause of death in the first two years of life. RCAN1, an endogenous feedback inhibitor of calcineurin, is located on chromosome 21 in the region of 21q22.1, the minimal candidate region for the Down’s syndrome phenotype. Preliminary studies using disomic (normal) versus trisomic (Down’s syndrome) iPSC showed that the trisomic cell lines present increased RCAN1 protein levels together with increased mitochondrial volume, indicative of mitochondrial fusion. The protein phosphatase calcineurin promotes mitochondrial fission through the dephosphorylation and activation of the fission protein DRP1. We will study the contribution of mitochondrial dynamics and function on the commitment to the cardiac linage of normal and Down’s syndrome iPSC. Hypothesis: Trisomy of RCAN1 inhibits DRP1-dependent mitochondrial fission producing mitochondrial dysfunction and altering the differentiation of human iPSC to cardiomyocytes. Specific aims: 1. To evaluate the role of RCAN1 on mitochondrial dynamics and function in disomic (normal) and trisomic (Down’s syndrome) human iPSC. 2. To evaluate the effect of RCAN1 on cardiomyocyte differentiation from disomic and trisomic human iPSC. 3. To evaluate the impact of RCAN1-dependent mitochondrial dynamics and function on cardiomyocyte differentiation from disomic and trisomic human iPSC. Experimental design and methodologies: Human Trisomy 21 iPSCs and disomic isogenic controls have been obtained through the PCBC Disease Lines resource (NHLBI Progenitor Cell Biology Consortium, NIH, USA). The following analyses will be carried out both at the pluripotent state and following differentiation toward a cardiac lineage. • Mitochondrial dynamics (number, size and subcellular distribution) will be analyzed in live cells labeled with Mitotracker green FM and imaged on a Zeiss LSM-5, Pascal 5 confocal microscope, as previously described by our group. Z-stacks of thresholded-images will be volume-reconstituted and quantified. • Mitochondrial potential, mitochondrial mass, and ROS production will be measured by flow cytrometry using tetramethyl-rhodamine methyl ester, Mitotracker green FM or MitoSox, respectively. • Oxygen consumption and mitochondrial coupling will be measured using an oxygen sensor. • Changes in the capacity for mitochondrial calcium uptake will be assessed using Rhod-FF, whereas cytoplasmic calcium will be quantified using Fluo3-AM. • siRNA will be used to deplete RCAN1 (or other genes on chromosome 21) and the analysis of mitochondrial function will be assessed. Expected results: we expect that Down syndrome iPSCs will show increased mitochondrial fusion and over-sufficient mitochondrial function. If these changes are directly attributed by trisomy of RCAN1, then siRNA depletion of RCAN1 should help to normalize mitochondrial morphology and function. These studies will be the first to use iPSC to define a functional link between specific genes on chromosome 21 and changes in mitochondrial dynamics and function associated with cardiomyocyte differentiation.