Centrosome Integrity On Maturation Of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human cardiovascular diseases remain high prevalence and morbidity worldwide,and the adult human heart is incapable of regeneration after heart injury,leading to heart remodeling and failure,which has no effective treatment other than heart transplantation.Human pluripotent stem cells derived cardiomyocytes（hPSC-CMs）provide an unlimited source for biomedical application including regenerating the injured heart,but they remain developmentally immature in almost all aspects including cell morphology,energy metabolism,excitation-contraction coupling and gene expression.It is important to achieve hPSC-CM maturation in vitro for simulating phenotypes of diseases or as pharmacological models,as well as a source for efficient cell transplantation.Although multiple methods have been found to promote maturation of hPSC-CMs,there is still no method with sufficient effectiveness due to our incomprehensive understanding of the underlying molecular mechanism.PCM1,a centriole satellite protein,localizes at the centrosome in embryonic day15 mouse cardiomyocytes,and the nuclear envelope in postnatal day 3cardiomyocytes.This shift is accompanied by cell cycle arrest and loss of centrosome integrity,suggesting that centrosome integrity and subcellular localization of PCM1are associated with cardiomyocyte maturation and may be used to identify novel molecules and pathways regulating complex biological processes of cardiomyocyte maturation.In addition,mammalian cardiomyocytes lose the ability to proliferate and regenerate during the maturation process with cell cycle exit,and the underlying mechanism has not been fully elucidated.Cell proliferation is highly dependent on functional centrosomes,suggesting that centrosome integrity may regulate the proliferation as well as maturation of cardiomyocytes.Moreover,although nuclear envelope localization of PCM1 has been used to identify cardiomyocyte maturation,the function of PCM1 in cardiomyocyte maturation remains unknown.Thus,we conducted High Content Screening（HCS）of small molecules based on PCM1localization in nuclear envelope and Ed U incorporation for measuring cardiomyocyte maturation and proliferation respectively,and investigated the mechanism of small molecules that can promote cardiomyocyte maturation and inhibit cardiomyocyte proliferation.The function and mechanism of small molecules that reduce the distribution of PCM1 in nuclear membrane and promote proliferation of cardiomyocytes were also investigated.The effects of PCM1 and centrosome integrity on cardiomyocyte maturation were studied in vitro and in vivo.We found that PCM1 is located in centrosome,throughout the cytoplasm,or absent in multiple non-cardiomyocyte cell types,while it is mainly distributed around the nucleus in neonatal rat ventricular cardiomyocytes（NRVCs）and mouse embryonic stem cell-derived cardiomyocytes（m ESC-CMs）.In hPSC-CMs generated by Gi Wi protocol,PCM1 is observed in centrosome before day 15 and then migrate to the nuclear envelope and nucleus at day 18,concurrent with well-developed mitochondria and organized sarcomere structures.Image analysis based on Cell Profiler showed that the perinuclear localization of PCM1 could be used as a quantifiable marker of the maturity of hPSC-CMs for high content screen assays.Therefore,we performed HCS screens based on PCM1 subcellular localization and Ed U incorporation against a library of 1693 compounds,with DMSO and Triiodothyronine/Dexamethasone combination as negative and positive controls respectively.We identified 93 compounds that can enhance maturation of hPSC-CMs.Among the most active compounds,pathways such as cell cycle,epigenetics,and protease are enriched targets.Cell cycle inhibitors Centrinone and Volasertib,targeting polo-like kinases（PLK）,decreased hPSC-CM proliferation and increased cell area,mitochondrial membrane potential,calcium releasing capacity,contraction frequency and velocity,and expression of oxidative phosphorylation related genes.RNA-Seq analysis revealed that PI3K-AKT signaling pathway was the most significantly downregulated pathway upon Centrinone and Volasertib treatment.Akt agonist VO-Ohpic can blunt the effect of inhibiting PLK on hPSC-CM maturation,suggesting that PLK inhibitors Volasertib and Centrinone promote hPSC-CM maturation via suppressing AKT signaling pathway in vitro.To dissect the relationship between PCM1,centrosome integrity and cardiomyocyte proliferation,we studied the intersection of small molecules that can promote cardiomyocyte proliferation and reduce PCM1 distribution in nuclear envelope.We found that L-type calcium channel blockers Nimodipine robustly reduced perinuclear PCM1 expression and enhanced cardiomyocyte proliferation.RNA-Seq analysis revealed that canonical Wnt signaling pathway was affected by Nimodipine,which was confirmed by Luciferase assays andβ-catenin translocation to the nucleus.Inhibiting Wnt signaling blunted the proliferative effect of Nimodipine and nuclear translocation ofβ-catenin.Wnt Co-receptor LRP5 is required for nuclear translocation ofβ-catenin and cardiomyocyte proliferation induced by Nimodipine,thus is likely to be a mediator between LTCC and canonical Wnt signaling pathway in cardiomyocytes.Importantly,Nimodipine treatment for 7 days after myocardial infarction（MI）significantly improved mouse cardiac contractile function and enhanced resident cardiomyocyte proliferation in vivo.Our data suggested that L-type calcium channel blockers may be potentially used in the treatment of MI and heart failure to promote cardiac regeneration.In this study,we also found that sarcoplasmic reticulum Ca²⁺-ATPase（SERCA）inhibitor Thapsigargin significantly promoted the distribution of PCM1 in the nuclear membrane of cardiomyocytes.While Blebbistatin,a myosin Ⅱ inhibitor that discrupts the cardiomyocyte cytoskeleton,can promote the proliferation of cardiomyocytes and reduce distribution of perinuclear PCM1.However,the molecular mechanisms require further exploration.To study the function of PCM1 in cardiomyocyte maturation,we knocked down PCM1 gene using small interfering RNA（si RNA）in hPSC-CMs and NRVCs.Proliferation was significantly decreased in hPSC-CMs but increased in NRVCs upon PCM1 knockdown,which may be due to the different developmental stages of hPSC-CMs and NRVCs.Pcm1 knockout mice on postnatal day 1 showed enhanced cardiomyocyte proliferation,consistent with that of NRVCs.Transverse aortic constriction（TAC）surgery was performed in adult Pcm1 knockout mice and wild-type mice.Cardiac function of Pcm1 knockout mice was significantly improved8 weeks after surgery,suggesting that Pcm1 deficiency in adult mice can prevent cardiac hypertrophy and maintain cardiac homeostasis.RNA-Seq data suggested that the underlying mechanism might be related to PI3K-AKT signaling pathway.In summary,the studies in this thesis investigated the effect and mechanism of centrosome integrity on the maturation of human induced pluripotent stem cell-derived cardiomyocytes.We performed high content screening based on PCM1subcellular localization and Ed U incorporation and found that Volasertib and Centrinone promoted the maturation of hPSC-CMs by regulating PI3K-AKT signaling pathway,which provides a theoretical basis and a feasible scheme for generating mature cardiomyocytes in vitro.We also found that L-type calcium channel blockers Nimodipine can reduce perinuclear PCM1 and enhance CM proliferation.Nimodipine treatment after MI significantly improved cardiac contractile function and promoted resident cardiomyocyte regeneration,providing theoretical foundation and possible novel methods to treat cardiovascular diseases.The function of PCM1 gene in cardiomyocyte proliferation and maturation was explored,and we found that deletion of Pcm1 can inhibit stress-induced cardiac hypertrophy in vivo,providing experimental basis for dissecting the relationship between centrosome integrity and cardiomyocyte proliferation and maturation.