Induction Of Cardiomyocyte Proliferation Research Articles

Abstract P1002: Elucidating The Role Of Dyrk1a Kinase In Controlling Cardiomyocyte Proliferation

Despite the development of new pharmacological and device-based therapies, no approaches currently exist to compensate for the irreversible loss of cardiomyocytes occurring after myocardial infarction. In a search for novel treatments that induce cardiomyocyte proliferation, we performed a high-content, fluorescence-microscopy-based, high-throughput screening in neonatal rat cardiomyocytes, using a library of FDA/EMA-approved drug library compounds. The screening, which was based on EdU incorporation as an S-phase marker, identified harmine as the most powerful drug enhancing proliferation in combination with miR-199a-3p and miR-590-3p, the most effective microRNAs that the laboratory had previously identified. Harmine treatment of CD1 mice showed an improved cardiac outcome upon harmine treatment compared to controls. Harmine is a potent inhibitor of the Dyrk1 family of protein kinases, with high specificity for the Dyrk1a isoform. Dyrk1a downregulation experiments in neonatal rat ventricular cardiomyocytes showed increased proliferation, consistent with a negative role of this kinase. The simultaneous knockdown of YAP1 blunted the effect of Dyrk1a downregulation. Of note, the YAP total protein levels were unchanged upon either Dyrk1a silencing or overexpression, but there was a marked modification of YAP subcellular localisation, being YAP1 nuclear upon knockdown and mostly cytoplasmic upon overexpression. Dyrk1a downregulation and harmine treatment activated YAP-controlled gene expression. In the course of these experiments, we also noticed a reciprocal regulation of YAP onto DYRK1a, as YAP downregulation markedly decreased Dyrk1a protein levels. This finding was reinforced by the observations that, in cardiomyocytes, the YAP-TEAD inhibitor verteporfin also decreased Dyrk1a protein levels. Of note, this downregulation was not rescued by the proteasome inhibitor MG132, thus indicating that the level of regulation is most likely transcriptional. These results support the role of Dyrk1a as an anti-proliferative factor in cardiomyocytes and highlight a novel feedback connection between Dyrk1a and YAP.

Abstract P3079: Hypoxia Stimulates Neonatal Hypoxia-inducible Factor 1α Dependent Macrophage Secretion Of Cardiomyocyte Mitogens

Background: The neonatal murine heart can regenerate after injury. This regenerative capacity is dependent on macrophages and lost shortly after birth. How macrophages facilitate regeneration remains vague, and the neonatal myeloid response to hypoxia as well as the role of myeloid Hypoxia-Inducible Factors (HIFs) in cardiac regeneration are unknown. We hypothesized that post-ischemic regulation of regenerative programs are confined to neonatal cardiac macrophages and are lost in the adult. We also hypothesized hypoxia is a stimulus for neonatal macrophages to secrete cardiomyocyte mitogens in a HIF dependent manner. Methods: Single-cell sequencing was performed on cardiac myeloid cells post myocardial infarction (MI) in wildtype neonatal and adult mice to explore differences in regenerative vs reparative responses to injury. Neonatal cardiomyocytes harvested from wildtype mice were co-cultured with supernatant from hypoxia stimulated neonatal bone marrow-derived macrophages from myeloid HIF-1 α expressing vs HIF-1 α genetically depleted mice. Immunofluorescent staining with antibodies to Troponin T, Ki67, and DAPI was performed to quantify cardiomyocyte proliferation. Results: High-resolution clustering identified 3 distinct macrophage populations present in neonatal and adult mice post MI. Gene Ontology identified significantly upregulated genes in biological pathways associated with regeneration and HIF-1α stabilization in Timd4+Mrc1+ macrophages in the neonate but not the adult. Immunofluorescent staining of neonatal cardiomyocytes at steady state revealed an average of 6.0% Ki67+ cardiomyocytes compared to 35.4% Ki67+ cardiomyocytes exposed to supernatant from hypoxia stimulated HIF-1 α + macrophages (N=10/group; SEM + 1.96; p value <0.0001) and 10.1% Ki67+ cardiomyocytes exposed to supernatant from hypoxia stimulated HIF-1 α - macrophages (N=10/group; SEM + 1.54; p value 0.0162). Conclusions: We have newly identified enrichment of regenerative and HIF-1α stabilization pathways specific to neonatal resident cardiac macrophages after ischemic injury. We also newly conclude hypoxia is a stimulus for neonatal macrophages to secrete HIF-1 α dependent mitogenic factors that induce cardiomyocyte proliferation.

Abstract P1004: Macrophage Apoptotic Cell Receptor Mertk Triggers The Synthesis Of Arachidonic Acid Derivatives To Promote Cardiac Regeneration

Introduction: Macrophages (MΦs) are indispensable for regeneration of the mammalian neonatal heart. However, the underlying mechanisms by which neonatal MΦs sense the injured cardiac milieu and promote regeneration remain obscure. Utilizing single-cell transcriptomics, we previously observed a regenerative neonatal cardiac MΦ population that distinctly upregulates MerTK, an apoptotic cell receptor, compared to adult cardiac MΦs. Objective: We hypothesized that MerTK is required to program neonatal cardiac MΦs to secrete specialized pro-“regenerative” lipids to induce cardiomyocyte proliferation leading to heart regeneration. Methods: Postnatal day 1 and adult mice were subjected to cardiac injury via ligation of the left anterior descending artery. Single-cell transcriptomics, lipid-omics, and molecular approaches were utilized to determine the mechanisms by which neonatal MΦs regulate cardiac regeneration. Results: To determine the necessity of MΦ MerTK for cardiac regeneration, we employed a myeloid-specific knockout of MerTK ( mMertk -/- ) which failed to regain homeostatic cardiac function. We next performed single-cell transcriptomics on Mertk +/+ and Mertk -/- neonates to determine how MerTK activation reprograms neonatal MΦs for regeneration. Mertk +/+ MΦs significantly upregulated genes associated with arachidonic acid metabolism. Hypothesizing that arachidonic acid derivatives may be key signaling molecules for regeneration, we performed a targeted lipid-omics screen which revealed an increase in Thromboxane B 2 (TXB 2 ) in regenerating hearts but not within non-regenerative Mertk -/- hearts. Furthermore, addition of a TXA 2 agonist to neonatal cardiomyocytes in vitro significantly increases their proliferation by regulating the YAP/TAZ signaling. Alternatively, inhibition of thromboxane synthase in neonatal MΦs or thromboxane receptor on cardiomyocytes by small molecule inhibitors significantly abrogated the increase in cardiomyocyte proliferation by efferocytic Mertk +/+ neonatal MΦs. Conclusions: Altogether, our data suggest that MerTK programs neonatal cardiac MΦs to secrete TXA 2 to induce cardiomyocyte proliferation via the HIPPO-YAP/TAZ pathway prompting neonatal cardiac regeneration.

CircRNA Samd4 induces cardiac repair after myocardial infarction by blocking mitochondria-derived ROS output

Reactive oxygen species (ROS) derived from oxygen-dependent mitochondrial metabolism are the essential drivers of cardiomyocyte (CM) cell-cycle arrest in adulthood. Mitochondria-localized circular RNAs (circRNAs) play important roles in regulating mitochondria-derived ROS production, but their functions in cardiac regeneration are still unknown. Herein, we investigated the functions and underlying mechanism of mitochondria-localized circSamd4 in cardiac regeneration. We found that circSamd4 was selectively expressed in fetal and neonatal CMs. The transcription factor Nrf2 controlled circSamd4 expression by binding to the promoter of circSamd4 host gene. CircSamd4 overexpression reduced while circSamd4 silenced increased mitochondrial oxidative stress and subsequent oxidative DNA damage. Moreover, circSamd4 overexpression induced CM proliferation and prevented CM apoptosis, which reduced the size of the fibrotic area and improved cardiac function after myocardial infarction (MI). Mechanistically, circSamd4 reduced oxidative stress generation and maintained mitochondrial dynamics by inducing the mitochondrial translocation of the Vcp protein, which downregulated Vdac1 expression and prevented the mitochondrial permeability transition pore (mPTP) from opening. Our findings suggest that circSamd4 is a novel therapeutic target for heart failure after MI.

Targeting calcineurin induces cardiomyocyte proliferation in adult mice.

The mammalian neonatal heart can regenerate for 1 week after birth, after which, the majority of cardiomyocytes exit the cell cycle. Recent studies demonstrated that calcineurin mediates cell-cycle arrest of postnatal cardiomyocytes, partly through induction of nuclear translocation of the transcription factor Hoxb13 (a cofactor of Meis1). Here we show that inducible cardiomyocyte-specific deletion of calcineurin B1 in adult cardiomyocytes markedly decreases cardiomyocyte size and promotes mitotic entry, resulting in increased total cardiomyocyte number and improved left ventricular (LV) systolic function after myocardial infarction (MI). Similarly, pharmacological inhibition of calcineurin activity using FK506 promotes cardiomyocyte proliferation in vivo and increases cardiomyocyte number; however, FK506 administration after MI in mice failed to improve LV systolic function, possibly due to inhibition of vasculogenesis and blunting of the post-MI inflammatory response. Collectively, our results demonstrate that loss of calcineurin activity in adult cardiomyocytes promotes cell cycle entry; however, the effects of the calcineurin inhibitor FK506 on other cell types preclude a significant improvement of LV systolic function after MI.

Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration.

Cardiac injury leads to the loss of cardiomyocytes, which are rapidly replaced by the proliferation of the surviving cells in zebrafish, but not in mammals. In both the regenerative zebrafish and non-regenerative mammals, cardiac injury induces a sustained macrophage response. Macrophages are required for cardiomyocyte proliferation during zebrafish cardiac regeneration, but the mechanisms whereby macrophages facilitate this crucial process are fundamentally unknown. Using heartbeat-synchronized live imaging, RNA sequencing, and macrophage-null genotypes in the larval zebrafish cardiac injury model, we characterize macrophage function and reveal that these cells activate the epicardium, inducing cardiomyocyte proliferation. Mechanistically, macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium, which upregulates vegfaa expression to induce cardiomyocyte proliferation. Our data suggest that epicardial Vegfaa augments a developmental cardiac growth pathway via increased endocardial notch signaling. The identification of this macrophage-dependent mechanism of cardiac regeneration highlights immunomodulation as a potential strategy for enhancing mammalian cardiac repair.

FUCCI-Based Live Imaging Platform Reveals Cell Cycle Dynamics and Identifies Pro-proliferative Compounds in Human iPSC-Derived Cardiomyocytes.

One of the major goals in cardiac regeneration research is to replace lost ventricular tissue with new cardiomyocytes. However, cardiomyocyte proliferation drops to low levels in neonatal hearts and is no longer efficient in compensating for the loss of functional myocardium in heart disease. We generated a human induced pluripotent stem cell (iPSC)-derived cardiomyocyte-specific cell cycle indicator system (TNNT2-FUCCI) to characterize regular and aberrant cardiomyocyte cycle dynamics. We visualized cell cycle progression in TNNT2-FUCCI and found G2 cycle arrest in endoreplicating cardiomyocytes. Moreover, we devised a live-cell compound screening platform to identify pro-proliferative drug candidates. We found that the alpha-adrenergic receptor agonist clonidine induced cardiomyocyte proliferation in vitro and increased cardiomyocyte cell cycle entry in neonatal mice. In conclusion, the TNNT2-FUCCI system is a versatile tool to characterize cardiomyocyte cell cycle dynamics and identify pro-proliferative candidates with regenerative potential in the mammalian heart.

A small-molecule cocktail promotes mammalian cardiomyocyte proliferation and heart regeneration.

Zebrafish and mammalian neonates possess robust cardiac regeneration via the induction of endogenous cardiomyocyte (CM) proliferation, but adult mammalian hearts have very limited regenerative potential. Developing small molecules for inducing adult mammalian heart regeneration has had limited success. We report a chemical cocktail of five small molecules (5SM) that promote adult CM proliferation and heart regeneration. A high-content chemical screen, along with an algorithm-aided prediction of small-molecule interactions, identified 5SM that efficiently induced CM cell cycle re-entry and cytokinesis. Intraperitoneal delivery of 5SM reversed the loss of heart function, induced CM proliferation, and decreased cardiac fibrosis after rat myocardial infarction. Mechanistically, 5SM potentially targets α1 adrenergic receptor, JAK1, DYRKs, PTEN, and MCT1 and is connected to lactate-LacRS2 signaling, leading to CM metabolic switching toward glycolysis/biosynthesis and CM de-differentiation before entering the cell-cycle. Our work sheds lights on the understanding CM regenerative mechanisms and opens therapeutic avenues for repairing the heart.

Yes-associated protein activation potentiates glycogen synthase kinase-3 inhibitor-induced proliferation of neonatal cardiomyocytes and iPS cell-derived cardiomyocytes.

Because mammalian cardiomyocytes largely cease to proliferate immediately after birth, the regenerative activity of the heart is limited. To date, much effort has been made to clarify the regulatory mechanism of cardiomyocyte proliferation because the amplification of cardiomyocytes could be a promising strategy for heart regenerative therapy. Recently, it was reported that the inhibition of glycogen synthase kinase (GSK)‐3 promotes the proliferation of neonatal rat cardiomyocytes (NRCMs) and human iPS cell‐derived cardiomyocytes (hiPSC‐CMs). Additionally, Yes‐associated protein (YAP) induces cardiomyocyte proliferation. The purpose of this study was to address the importance of YAP activity in cardiomyocyte proliferation induced by GSK‐3 inhibitors (GSK‐3Is) to develop a novel strategy for cardiomyocyte amplification. Immunofluorescent microscopic analysis using an anti‐Ki‐67 antibody demonstrated that the treatment of NRCMs with GSK‐3Is, such as BIO and CHIR99021, increased the ratio of proliferative cardiomyocytes. YAP was localized in the nuclei of more than 95% of cardiomyocytes, either in the presence or absence of GSK‐3Is, indicating that YAP was endogenously activated. GSK‐3Is increased the expression of β‐catenin and promoted its translocation into the nucleus without influencing YAP activity. The knockdown of YAP using siRNA or pharmacological inhibition of YAP using verteporfin or CIL56 dramatically reduced GSK‐3I‐induced cardiomyocyte proliferation without suppressing β‐catenin activation. Interestingly, the inhibition of GSK‐3 also induced the proliferation of hiPSC‐CMs under sparse culture conditions, where YAP was constitutively activated. In contrast, under dense culture conditions, in which YAP activity was suppressed, the proliferative effects of GSK‐3Is on hiPSC‐CMs were not detected. Importantly, the activation of YAP by the knockdown of α‐catenin restored the proproliferative activity of GSK‐3Is. Collectively, YAP activation potentiates the GSK‐3I‐induced proliferation of cardiomyocytes. The blockade of GSK‐3 in combination with YAP activation resulted in remarkable amplification of cardiomyocytes.

Abstract 10754: Disrupted N-Cadherin Expression Leads to Sarcomeric Disassembly and Cell Cycle Activation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Introduction: Induction of cardiomyocyte proliferation holds great promise for cardiac regenerative therapy. We showed previously that in the presence of Wnt signaling agonist, the removal of cell-cell contact between cardiomyocytes is the key to drive massive expansion of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the molecular mechanism regulating cell contact-mediated suppression of hiPSC-CM proliferation remains unknown. Hypothesis: We hypothesized that N-cadherin, the major adherens junction molecule expressed in cardiomyocytes, mediates contact inhibition of hiPSC-CM proliferation. Methods/Results: By qPCR and Western blotting, we showed comparable transcript and protein expression level of N-cadherin when hiPSC-CMs were cultured either densely (with cell contact) or sparsely (without cell contact), despite the observed N-cadherin localization at intercellular junction in densely cultured cells only. We disrupted N-cadherin expression with transient transfection of siRNA and found that the loss of N-cadherin expression resulted in 1.75-fold increase in Ki67 expression in densely but not sparsely cultured day 30 hiPSC-CM, suggesting that junction-localized N-cadherin may affect hiPSC-CM proliferation. Furthermore, we found that the loss of N-cadherin expression led to wide-spread sarcomeric disassembly. To examine whether sarcomeric disassembly alone is sufficient to stimulate cell cycle activity in densely cultured hiPSC-CMs, we knocked down α-actinin expression which led to sarcomere disruption and an increase in Ki67+ expression by 1.6-fold in day 30 hiPSC-CMs. Conclusions: Taken together, our results suggest that N-cadherin mediates the previously observed cell-cell contact inhibition of hiPSC-CM proliferation and the disruption of N-cadherin expression in densely cultured hiPSC-CMs leads to sarcomeric disassembly and hiPSC-CM proliferation. Furthermore, we showed that sarcomeric disassembly alone stimulates hiPSC-CM proliferation. These findings support the need for inducing cardiomyocyte dedifferentiation to achieve in situ cardiomyocyte proliferation for developing novel regenerative therapy to treat congenital and adult heart diseases in the future.

Abstract 10747: Genetic Enhancement of Epicardial Paracrine Signalling for Cardiac Regeneration

After myocardial infarction (MI), paracrine signalling has been shown to contribute to heart regeneration. The epicardium, as an important signalling centre, is crucial for heart development and it is re-activated after MI, indicating its involvement in the response to injury. This project aims to accomplish in situ transfection of the epicardium to stimulate the overexpression of growth factors that will enhance regenerative signalling pathways after MI, with cardiomyocyte proliferation as the primary target. Selective epicardial transfection can potentially be achieved via a surface-mediated delivery. Therefore, we developed a novel transfection approach consisting of the deposition of bioreducible cationic polymeric polyplexes onto flat and nanostructured silicon surfaces by freeze-drying. Recombinant growth factors (rGF) were used in single and combinatorial proliferation assays to select for genes to be delivered to the epicardium. The proliferative effects were assessed on cardiomyocytes, cardiac mesenchymal stromal cells, endothelial cells and cardiac fibroblasts via high-content image analysis. Neuregulin-1 led consistently to higher S-phase proliferation marker in IMR90- and H7-derived cardiomyocytes and higher cardiomyocyte cell numbers (fold increase 1.28 ± 0.12), quantified by flow cytometry. Hepatocyte Growth Factor induced proliferation of cardiac mesenchymal stromal cells (fold increase 2.75 ± 0.31) and angiogenesis (fold increase 1.47 ± 0.34). Follistatin-like 1, from animal and bacterial sources, was also tested but no proliferative effect was detected. The relevant rGF for each cell type maintained its positive effect in combination with other rGFs. Furthermore, preliminary results from in vitro transfection of epicardial cells with these GF genes indicate the proliferative advantage of enriched epicardial paracrine signalling over the incubation of cardiac cells with the rGF . This study remains to be a first-in-field potential material-based gene delivery strategy specific to the epicardium that explores a combinatorial treatment via the delivery of more than one gene for induction of cardiomyocyte proliferation and other regenerative mechanisms for MI treatment.

Human Erbb2-induced Erk activity robustly stimulates cycling and functional remodeling of rat and human cardiomyocytes.

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte (CM) proliferation have been identified as potential candidates for functional heart repair following myocardial infarction. However, it is unclear whether the effects of these mitogens are species-specific and how they directly compare in the same cardiac setting. Here, we examined how CM-specific lentiviral expression of various candidate mitogens affects human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) in vitro. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues generated from NRVMs, a constitutively active mutant form of the human gene Erbb2 (cahErbb2) was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM proliferation, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of Erk signaling. These results suggest transient activation of Erbb2/Erk axis in CMs as a potential strategy for regenerative heart repair.

Enhancement of engineered cardiac tissues by promotion of hiPSC-cardiomyocyte proliferation

Abstract Background/Introduction Cardiac tissue engineering is a promising strategy to generate human cardiac tissues for modelling cardiac diseases, screening for therapeutic drugs, and repairing the injured heart. Yet, several issues remain to be resolved including the generation of tissues with high cardiomyocyte density. Purpose Determining the effects of the induction of human-induced pluripotent stem cell-derived (hiPSC) cardiomyocyte proliferation post-fabrication. Methods hiPSCs were differentiated into cardiomyocytes, embedded with or without CHIR990121 at three concentrations in a collagen pre-gel, and cast. The engineered cardiac tissues were then cultured in the absence or presence of CHIR99021 for up to 35 days. Hydrogels and engineered cardiac tissues were analysed utilizing rheology and assays to determine viability, proliferation, calcium flow, and contractility. Results Here, we show that the integration of CHIR99021 in collagen I hydrogels promotes proliferation of hiPSC-cardiomyocytes post-fabrication improving contractility of and calcium flow in engineered cardiac tissues. Presence of CHIR99021 has no effect on the gelation kinetic or the mechanical properties of collagen I hydrogels. Analysis of cell density and proliferation based on Ki-67 staining indicates that integration of CHIR99021 together with external CHIR99021 stimulation increases hiPSC-cardiomyocyte number by ∼2-fold within 7 days post-fabrication. Analysis of the contractility of engineered cardiac tissues after another 3 days in the absence of external CHIR99021 shows that CHIR99021-induced hiPSC-cardiomyocyte proliferation results in synchronized calcium flow, rhythmic beating, increases speed of contraction and contraction amplitude, and reduces peak-to-peak time. The CHIR99021-stimulated engineered cardiac tissues exhibited spontaneous rhythmic contractions for at least 35 days. Conclusion Collectively, our data demonstrate the potential of induced cardiomyocyte proliferation to enhance engineered cardiac tissues by increasing cardiomyocyte density and reducing arrhythmia. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Metformin accelerates zebrafish heart regeneration by inducing autophagy

Metformin is one of the most widely used drugs for type 2 diabetes and it also exhibits cardiovascular protective activity. However, the underlying mechanism of its action is not well understood. Here, we used an adult zebrafish model of heart cryoinjury, which mimics myocardial infarction in humans, and demonstrated that autophagy was significantly induced in the injured area. Through a systematic evaluation of the multiple cell types related to cardiac regeneration, we found that metformin enhanced the autophagic flux and improved epicardial, endocardial and vascular endothelial regeneration, accelerated transient collagen deposition and resolution, and induced cardiomyocyte proliferation. Whereas, when the autophagic flux was blocked, then all these processes were delayed. We also showed that metformin transiently enhanced the systolic function of the heart. Taken together, our results indicate that autophagy is positively involved in the metformin-induced acceleration of heart regeneration in zebrafish and suggest that this well-known diabetic drug has clinical value for the prevention and amelioration of myocardial infarction.

Direct coculture of human pluripotent stem cell-derived cardiac progenitor cells with epicardial cells induces cardiomyocyte proliferation and reduces sarcomere organization

Epicardial cells (EpiCs) are necessary for myocardium formation, yet little is known about crosstalk between EpiCs and cardiomyocytes (CMs) during development and the potential impact of EpiCs on CM maturation. To investigate the effects of EpiCs on CM commitment and maturation, we differentiated human pluripotent stem cells (hPSCs) to cardiac progenitor cells (CPCs) and EpiCs, and cocultured EpiCs and CPCs for two weeks. When EpiCs were allowed to form epicardial-derived cells, we observed increased expression of cTnI in developing CMs. In the presence of the TGFβ inhibitor A83-01, EpiCs remained in the epicardial state and induced CM proliferation, increased MLC2v expression, and led to less organized sarcomeres. These effects were not observed if CPCs were treated with EpiC-conditioned medium or if CPCs were indirectly cocultured with EpiCs. Finally, single cell RNA sequencing identified that EpiC-CPC coculture had bi-directional effects on transcriptional programs in EpiCs and CMs, and biased EpiC lineages from a SFRP2-enriched population to a DLK1- or C3-enriched population. This work suggests important crosstalk between EpiCs and CMs during differentiation which can be used to influence cell fate and improve the ability to generate cardiac cells and tissues for in vitro models and development of cardiac cellular therapies.

CHIR99021 Promotes hiPSC-Derived Cardiomyocyte Proliferation in Engineered 3D Microtissues.

Cardiac tissue engineering is a promising strategy to generate human cardiac tissues for modeling cardiac diseases, screening for therapeutic drugs, and repairing the injured heart. Yet, several issues remain to be resolved including the generation of tissues with high cardiomyocyte density. Here, it is shown that the integration of the glycogen synthase kinase-3β inhibitor CHIR99021 in collagen I hydrogels promotes proliferation of human-induced pluripotent stem cell-derived (hiPSC) cardiomyocytes post-fabrication improving contractility of and calcium flow in engineered 3D cardiac microtissues. CHIR99021 has no effect on the gelation kinetics or the mechanical properties of collagen I hydrogels. Analysis of cell density and proliferation based on Ki-67 staining indicates that integration of CHIR99021 together with external CHIR99021 stimulation increases hiPSC-cardiomyocyte number by ≈2-fold within 7 d post-fabrication. Analysis of the contractility of engineered cardiac tissues after another 3 d in the absence of external CHIR99021 shows that CHIR99021-induced hiPSC-cardiomyocyte proliferation results in synchronized calcium flow, rhythmic beating, increased speed of contraction and contraction amplitude, and reduced peak-to-peak time. The CHIR99021-stimulated engineered cardiac microtissues exhibit spontaneous rhythmic contractions for at least 35 d. Collectively, the data demonstrate the potential of induced cardiomyocyte proliferation to enhance engineered cardiac microtissues by increasing cardiomyocyte density.

Abstract P457: Systems Analysis To Understand The Impact Of The CDK Module On Cardiomyocyte Proliferation

Rationale: Heart failure is caused by the inability of adult mammalian hearts to overcome the loss of cardiomyocytes (CMs). This is due partly to the limited proliferative capacity of CMs, which exit the cell cycle and do not undergo cell division. Current knowledge in cardiac regeneration lacks an understanding of the molecular regulatory networks that determine whether CMs will progress through the cell cycle to proliferate. Our goal is to use computational modeling to understand the expression and activation levels of the core cell cycle network, specifically cyclins and cyclin-cyclin-dependent kinase (CDK) complexes. Methods: A model of core cell cycle dynamics was curated using previously published studies of CM proliferation regulators. This model incorporates those regulators known to stimulate G1/S and G2/M transitions through the core CDKs. The activity of each of the 22 network nodes (22 reactions) was predicted using a logic-based differential equation approach. The CDK model was then coupled with a minimal ODE model of cell cycle phase distributions and validated based on descriptions and experimental data from the literature. To prioritize key nodes for experimental validation, we performed a sensitivity analysis by stimulating individual knockdown for every node in the network, measuring the fractional activity of all nodes. Results: Our model confirmed that the knockdown of p21 and Rb protein and the overexpression of E2F transcription factor and cyclinD-cdk4 showed an increase in cells going through DNA synthesis and entering mitosis. A combined knockdown of p21 and p27 showed an increase of cells entering mitosis. Cyclin D-cdk4 and p21 overexpression showed a decrease and increase of Rb expression, respectively. Of the 14 model predictions, 12 agreed with experimental data in the literature. A comprehensive knockdown of the model nodes suggests that E2F (a key transcription factor driving DNA synthesis) is positively regulated by cyclin D while negatively regulated by GSK3B, SMAD3, and pRB. Conclusion: This model enables us to predict how cardiomyocytes respond to stimuli in the CDK network and identify potential therapeutic regulators that induce cardiomyocyte proliferation.

Abstract MP213: Activation Of Biosynthetic Pathways Regulates Cardiomyocyte Cell Cycle Induction In Human Cardiomyocyte

Induction of cardiomyocyte proliferation is a promising therapeutic approach to treat heart failure. Several studies have identified metabolism as an important regulator of myocyte proliferation; however, the changes in metabolism during cardiomyocyte division remain unclear. Here, we use ectopic expression of cyclin B1, Cyclin D1, CDK1, and CDK4 (termed 4F) as a tool for understanding how metabolism influences cardiomyocyte proliferation. Mature hiPS-CMs stimulated to proliferate by 4F expression showed significant downregulation of oxidative phosphorylation genes, decreased glucose oxidation, and upregulation of genes that regulate biosynthetic pathways of glucose metabolism such as those involved in NAD(P) + synthesis ( NAMPT, NADK1, NNT ), the hexosamine biosynthetic pathway (HBP) and protein O-GlcNAcylation ( GFPT1 , OGT, OGA ), and the serine biosynthesis pathway (SBP; PHGDH , PSAT1 , SHMT2 ). In 4F-expressing hiPSC-CMs, stable isotope tracing indicated higher enrichment of glucose-derived 13 C in pentose phosphate intermediates, UDP-hexose, phospholipid precursors, NAD + , pyrimidines, UDP-HexNAc, and products of the serine biosynthesis pathway and one-carbon metabolism, suggesting that cell cycle induction activates biosynthetic pathways in cardiomyocytes. Knocking down nicotinamide phosphoribosyltransferase (NAMPT), a critical enzyme in the NAD + salvage pathway, 2 days before 4F overexpression significantly inhibited cell cycle progression in 4F-transduced hiPS-CMs. OGA overexpression, which catalyzes the hydrolytic cleavage of O-GlcNAc from post-transitionally modified proteins, completely abolished 4F-mediated cell cycle induction. Furthermore, NCT503, an inhibitor of the rate-limiting step in the serine biosynthesis pathway, abolished 4F-mediated increases in cell cycle markers. In a gain-of-function approach, we overexpressed phosphoenolpyruvate carboxykinase 2 (PCK2), which can drive carbon from the Krebs cycle to the glycolytic intermediate pool. PCK2 overexpression significantly augmented 4F-mediated cell cycle entry. These findings suggest that a metabolic shift from catabolic to anabolic activity is a critical step for cardiomyocyte cell cycle entry and is required to facilitate proliferation.

Alternative Splicing of Pericentrin Contributes to Cell Cycle Control in Cardiomyocytes.

Induction of cardiomyocyte proliferation is a promising option to regenerate the heart. Thus, it is important to elucidate mechanisms that contribute to the cell cycle arrest of mammalian cardiomyocytes. Here, we assessed the contribution of the pericentrin (Pcnt) S isoform to cell cycle arrest in postnatal cardiomyocytes. Immunofluorescence staining of Pcnt isoforms combined with SiRNA-mediated depletion indicates that Pcnt S preferentially localizes to the nuclear envelope, while the Pcnt B isoform is enriched at centrosomes. This is further supported by the localization of ectopically expressed FLAG-tagged Pcnt S and Pcnt B in postnatal cardiomyocytes. Analysis of centriole configuration upon Pcnt depletion revealed that Pcnt B but not Pcnt S is required for centriole cohesion. Importantly, ectopic expression of Pcnt S induced centriole splitting in a heterologous system, ARPE-19 cells, and was sufficient to impair DNA synthesis in C2C12 myoblasts. Moreover, Pcnt S depletion enhanced serum-induced cell cycle re-entry in postnatal cardiomyocytes. Analysis of mitosis, binucleation rate, and cell number suggests that Pcnt S depletion enhances serum-induced progression of postnatal cardiomyocytes through the cell cycle resulting in cell division. Collectively, our data indicate that alternative splicing of Pcnt contributes to the establishment of cardiomyocyte cell cycle arrest shortly after birth.

Induced Cardiomyocyte Proliferation: A Promising Approach to Cure Heart Failure.

Unlike some lower vertebrates which can completely regenerate their heart, the human heart is a terminally differentiated organ. Cardiomyocytes lost during cardiac injury and heart failure cannot be replaced due to their limited proliferative capacity. Therefore, cardiac injury generally leads to progressive failure. Here, we summarize the latest progress in research on methods to induce cardiomyocyte cell cycle entry and heart repair through the alteration of cardiomyocyte plasticity, which is emerging as an effective strategy to compensate for the loss of functional cardiomyocytes and improve the impaired heart functions.