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Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitrocan expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro(i.e., 100- to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3β inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.

Introduction:Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. Mutations in genes encoding sarcomere proteins are the most common cause (sarcomere mutation positive: SMP), although in a group of patients no mutation is found (sarcomere mutation negative: SMN). We have still only limited knowledge about the pathomechanism leading to disease outcome.Hypothesis:Here, we applied an unbiased proteomics approach in cardiac samples from a clinically well-characterized HCM patient cohort to understand HCM pathophysiology as well as gain a better understanding of the genotype-phenotype relationship.Methods:We analyzed the proteome of 51 HCM samples (40 SMP, 11 SMN) obtained during septal myectomy by unlabeled LC-MS/MS mass spectrometry and compared it to 8 non-failing controls. Protein expression was quantified by peptide counts and protein-protein interaction networks of differentially expressed proteins were obtained from String database. Gene ontology analysis was performed with ClusterOne and Bingo. A new HCM mouse model was used to validate microtubule remodeling as a HCM disease modulator.Results:Next to a general downregulation of metabolic pathways and an upregulation of extracellular matrix proteins that have been found in all patient samples, we found an increase of tubulin and an altered posttranslational modification pattern in SMP patients. In an HCM mouse model we showed that inhibition of tubulin detyrosination normalizes contraction and relaxation of isolated cardiomyocytes.Conclusions:Mechanistically, these findings indicate that the tubulin network plays a role in the pathomechanism of individuals carrying a sarcomere mutation and that it might represent a potential treatment target to improve cardiac function.