Zhu, Wuqiang

The regulation of the cardiomyocyte cell cycle is central to understanding heart regeneration. In adult mammals, cardiomyocytes withdraw from the cell cycle, limiting their ability to proliferate and repair heart tissue after injury. This process is controlled by intrinsic genetic programs and extrinsic signals, which together restrict the regenerative response in mature hearts. Understanding the mechanisms that regulate cardiomyocyte cell cycle activity is therefore critical for advancing cardiac regenerative medicine. In this review, we provide a comparative and integrated overview of cardiomyocyte cell cycle regulation in lower vertebrates and mammals, and discuss the major intrinsic factors that govern this process, including cyclin/CDK pathways, transcription factors and co-activators, oxygen and metabolic regulation, and epigenetic mechanisms. We also review the influence of hormones and growth factors, as well as the supportive roles of nonmyocyte populations in heart regeneration. By integrating findings across species, developmental stages, and regulatory levels, these findings highlight the complex regulatory network controlling cardiomyocyte proliferation and provide insight into potential therapeutic strategies for stimulating cardiac repair in the adult mammalian heart.

Background: Programmed Cell Death 4 (PDCD4) is a tumor suppressor known for promoting apoptosis and inhibiting growth and protein translation in cancer cells. However, the role of PDCD4 in the heart remains elusive. This study explores PDCD4's regulation and function in the heart through genetic manipulation. Methods: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), neonatal mouse cardiomyocytes, and transgenic mice with constitutive cardiomyocyte-specific PDCD4 overexpression were used in this study. In vitro, PDCD4 expression was manipulated by plasmid-based overexpression or siRNA-mediated knockdown, and confirmed by Western blot. Subcellular localization of PDCD4 was assessed by immunofluorescence staining. Apoptosis was evaluated using cleaved-PARP immunoblotting and TUNEL staining. Cardiac function in mice was assessed by echocardiography. Results: In hiPSC-CMs and isolated neonatal cardiomyocytes, expression of PDCD4 is predominantly found in the nucleus, suggesting a possible role in nuclear signaling or gene regulation. Treatment of hiPSC-CMs with 1 μM doxorubicin (DOX) for 3 days significantly reduced PDCD4 expression. Functionally, PDCD4 overexpression attenuated DOX-induced apoptosis, as evidenced by decreased cleaved-PARP levels and fewer TUNEL-positive cells, whereas PDCD4 knockdown increased cell death, indicating a protective role under chemotherapeutic stress. PDCD4 expression decreased in mouse hearts during postnatal development. Transgenic mice with cardiomyocyte-specific PDCD4 overexpression showed normal heart weight, size, and function, without induced apoptosis under baseline conditions. We are currently investigating the potential cardioprotection of PDCD4 transgene against DOX induced acute and chronic cardiotoxicities. Conclusion: PDCD4 has a pro-apoptotic function in cancer cells but demonstrates an anti-apoptotic role in cardiomyocytes during chemotherapeutic stress in vitro. These observations indicate a context-dependent role for PDCD4 in cardioprotection, presenting it as a potential therapeutic target for reducing cardiotoxicity during cancer treatment.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) possess significant therapeutic potential for myocardial regeneration. However, clinical application remains constrained due to the immature phenotype of these cells, which impacts their engraftment as well as their electrical and mechanical integration post-transplantation into the injured heart. In particular, proper alignment of transplanted hiPSC-CMs is essential for synchronous contraction and efficient electromechanical coupling with native tissue. To address this critical clinical challenge, we engineered a tissue patch incorporating aligned electrospun nanofibers designed to enhance hiPSC-CM alignment and promote their structural maturation. Poly(caprolactone) (PCL) nanofibers were fabricated by electrospinning a 10% w/v solution in 1,1,1,3,3,3-hexafluoro-2-propanol at a flow rate of 0.5 mL/h. A voltage of 14 kV was applied to the spinneret and 3kV to the collector, positioned 13 cm away, with additional electrodes to induce fiber alignment. Scanning electron microscopy and ImageJ analysis confirmed fiber diameter and orientation. hiPSC-CMs (1 x 10 6 cells) were seeded onto fiber scaffolds with varied diameters (1.0, 1.5, and 2.0 μm) and densities (low: 20%, moderate: 30%, high: 40%). Among these, 1.5 ± 0.2 μm diameter fibers with a density of 40 ± 3% provided the most effective topographical cues for promoting cell elongation and alignment. Immunofluorescence staining demonstrated that hiPSC-CMs cultured on aligned fibers exhibited enhanced cytoskeletal organization along the fiber axis. Morphological assessment over time showed progressive alignment by day 3. Importantly, hiPSC-CMs cultured on aligned fibers demonstrated stronger and more synchronized contractile activity compared to those cultured on flat glass surfaces. Together, these findings suggest that our aligned nanofiber-based cardiac patch promotes the structural and functional maturation of hiPSC-CMs, highlighting their potential as an advanced platform for improving cardiac cell therapy outcomes.

Heart disease continues to be the leading cause of death globally, even with the development and implementation of numerous pharmacological and surgical treatments. Transplanting cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) shows potential for repairing heart damage. However, tracking and quantifying these cells after transplantation in large animals remains challenging. This study aimed to develop a hiPSC line that overexpresses ferritin heavy chain (FHC) to enable tracking of transplanted hiPSC-CMs via magnetic resonance imaging (MRI). Using the CRISPR/dCas9 activation system, we increased FHC expression in hiPSCs and hiPSC-CMs. Both mRNA and protein levels of FHC were significantly elevated following transfection. Cell viability was unaffected by iron chloride concentrations between 0 μM and 2000 μM. hiPSCs and hiPSC-CMs overexpressing FHC (hiPSC-FHC OE and hiPSC-CM-FHC OE ) showed enhanced iron chloride uptake without changes in their electrophysiological properties compared to control hiPSC-CMs. Additionally, hiPSC-CM-FHCOE cells provided strong contrast and lower T 2 * values, indicating their effectiveness for cardiac MRI. When hiPSC-CM-FHCOE cells were injected into mouse hearts and imaged three days later, MRI revealed distinct signals from these cells with lower T 2 * values and rapid signal decay. Overall, endogenous FHC labeling in hiPSC-CMs could significantly improve the precision of cardiac MRI, marking a crucial advancement in assessing the success of hiPSC-CM transplantation in large animal models and enhancing cardiac regenerative therapies.

Introduction: Neonatal pig hearts exhibit remarkable regenerative capacity within two days post-birth. In this study, we propose that a comprehensive multiomics analysis encompassing circRNA, miRNA, and mRNA data from neonatal mouse hearts can unveil intricate circRNA-miRNA-mRNA relationships associated with cardiac regeneration. Our aim is to identify new novel circRNAs and miRNAs implicated in cardiomyocyte proliferation through leveraging multiomics analysis. Methods: Utilizing neonatal mouse hearts, we conducted circRNA, miRNA, and mRNA sequencing across four different postnatal days (P1, P3, P7, and P28; n=6, encompassing both males and females). We employed integrative bioinformatics analyses across distinct experimental designs (P1 vs P3, P1 vs P7, and P1 vs P28). Back splice junctions generated by CIRCexplorer2’s parse command was utilized to annotate sequenced circRNAs as known or novel. Validation of circRNA, miRNA, and mRNA expression levels was achieved through qRT-PCR, while protein expression levels were assessed via western blotting. Fluorescence in situ hybridization (FISH) technology was employed to determine circRNA localization. The functional significance of key genes involved in cardiomyocyte proliferation was assessed via siRNA-mediated knockdown of circRNAs and miRNAs, accompanied by cell cycle assessment using bioluminescence assay and immunostaining. Results: Intersection of differentially expressed target genes from circRNA, miRNA, and mRNA analyses at each experimental design facilitated the construction of circRNA-miRNA-mRNA regulatory networks, aiding in the development of cardiac regeneration-related regulatory networks and inference of novel relationships between circRNA, miRNA, and mRNA. Integrative bioinformatics analysis revealed significant alterations in circRNA-miRNA-mRNA expression profiles during postnatal cardiomyocyte maturation in mice. Pathway enrichment analysis identified several differentially expressed circRNAs enriched in cell division and proliferation-related pathways. qPCR and cell cycle studies identified 17 potential circRNAs associated with cardiomyocyte proliferation in mice, with knockdown of these circRNAs significantly impacting cardiomyocyte cell cycle. Conclusion: Our findings suggest a crucial role for circRNAs in the regulation of neonatal myocardial regeneration.

Background It has been reported that metabolic switch from fatty acid oxidation to glycolysis is associated with cardiomyocyte cell cycle induction. However, it is unknown if increased glycolysis and ATP production are indispensable for cardiomyocyte cell cycle induction. Here, we aim to address this question. Methods Cell cycle was induced by overexpression of CCND2 gene or treatment of FGF1 (100 ng/mL) and CHIR99021 (5 µM) on human induced pluripotent stem cells‐derived cardiomyocytes (hiPSC‐CMs). Contraction inhibition was induced by treatment of verapamil (0.5 μM). Cell cycle was evaluated by immunostaining using antibodies against cell cycle markers. Real‐time ATP production and mitochondrial oxidative phosphorylation were determined by Seahorse assay. Metabolic profile was assessed by liquid chromatography–mass spectrometry. Results Treatment of FGF1 and CHIR99021 induces cell cycle of hiPSC‐CMs, reduced carbohydrate metabolism and real‐time ATP production from both mitochondria and cytoplasm. Data from RNA Sequencing showed this treatment increased expression of CCND2 and CDK4 and decreased expression of sarcomeric contractile related genes. Overexpression of CCND2 in hiPSC‐CMs also induces cell cycle, increased aerobic glycolysis (Warburg effect) and no change of real‐time ATP production. Inhibition of contraction via treatment of verapamil resulted in reduced overall metabolism and real‐time ATP production. Conclusion Our data suggest that cardiomyocyte cell cycle may be induced without increased glycolysis or real‐time ATP production.

Background Cardiomyocytes in postnatal hearts undergo hypertrophy to adapt to increased blood pressure and volume. The process of cardiomyocyte maturation is associated with a cessation from cell cycle and a series of metabolic switches. Prior research has shown metabolic switches in developing mouse hearts. However, metabolic changes in postnatal pig hearts have not been reported. Methods We evaluated the metabolic profile in pig hearts at postnatal day 1 (P1), day 3 (P3), day 7 (P7), and day 28 (P28) (n=6 each, both males and females; Yorkshire‐Landrace Cross background) using a targeted liquid chromatography tandem mass spectrometry assay. Expression of lactate dehydrogenase isoforms was evaluated by western blotting. Results There is a clear separation of the detected metabolites in P1 versus P28 hearts. Active anabolisms of nucleotide and proteins were observed in P1 hearts when cardiomyocytes retain high cell cycle activity. Abundance of the intermediate metabolites in the pathways of glycolysis was decreased in P28 comparing to P1 hearts, which implicates an overall reduced level of glycolysis. The decreased abundance of intermediates in TCA cycle and increased abundance of lactic acid in P28 hearts suggested that glycolysis rather than glucose oxidation is the main form of glucose metabolism in mature pig hearts. Reduced abundance of GLCN6P was observed in P28 hearts compared to P1 hearts. Increased abundance of hydroxyproline was observed in P1, P3, and P7 hearts compared to P28 heart. Conclusion Postnatal cardiomyocyte maturation is associated with metabolic switch from glucose to fatty acids, active posttranslational protein modification and ECM remodeling.