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.

Abstract Histones variant composition and post-translational modifications (PTMs) of core and linker histones jointly orchestrate chromatin architecture and tissue-specific gene regulation. However, capturing the full complexity of the “histone code” in native human tissues remains challenging. Here we present a human cardiac histone proteoform atlas—comprising intact histone variants with combinatorial PTMs—enabled by a streamlined top‑down proteomics workflow. In a single one‑dimensional reversed‑phase liquid chromatography (LC)-mass spectrometry (MS) run, we achieve baseline separation of all four core histone families (H2A, H2B, H3, and H4) together with the linker histone H1 directly from human myocardium. Notably, this intact-protein analysis preserves the connectivity of co-occurring PTMs to individual histone molecules to reveal the combinatorial histone code, enabling proteoform‑level quantification of histone‑variant composition and PTM stoichiometry at the chromatographic scale. Targeted MS/MS directly resolves co-occurring PTM combinations and distinguishes highly homologous histone isoforms. Using this approach, we assemble the first comprehensive cardiac histone proteoform atlas and uncover previously unobserved histone proteoforms. More broadly, this top-down proteomics workflow provides a robust framework for proteoform-resolved analysis of histone variants and PTMs in complex biological systems.

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.

Introduction: Designer receptors exclusively activated by designer drugs (DREADDs) are a robust chemogenetic tool for the interrogation of cellular electrical activity. Particularly, hM4Di activates the Gi signaling pathway, and can induce cellular hyperpolarization, leading to decreased electrical activity. Our aim is to engineer human induced pluripotent stem cell-derived cardiomyocytes (hiSPC-CMs) with controllable electromechanical properties to elucidate the role of transplanted hiPSC-CMs in cardiac regeneration. We hypothesize that activating hM4Di in transgenic hiSPC-CMs will inhibit their electrical activity. Methods: We developed a new hiPSC line expressing DREADD using the PiggyBac system (PB). The PB transposon included the hM4Di gene under the cTnT promoter, with mCherry and puromycin resistance genes as transfection markers ( Fig 1A ). After establishing the line, we differentiated these hiPSCs into DREADD-expressing cardiomyocytes (hiPSC-CM M4Di ). We activated hM4Di using clozapine N-oxide (CNO) and determined the optimal concentration through a concentration swipe. Optical mapping was used to analyze the electrical activity of hiPSC-CM M4Di . Results: After transfection, we observed the expression of mCherry in hiPSCs ( Fig 1B ). Additionally, we confirmed that these cells retained their pluripotency by evaluating the stemness markers Sox2 and Nanog ( Fig 1C ). Then, we performed directed cardiac differentiation. The cells showed spontaneous beating after day 8 of differentiation. We determined that the optimal concentration for hiPSC-CM M4Di inhibition was 200µM CNO, based on concentration swipe. Furthermore, our optical mapping data showed that spontaneous beating rate was decreased 30 minutes after adding CNO and completely stopped at 60 minutes. The spontaneous beating recovered after CNO was removed. CNO did not affect the electrical behavior of wild type cells (Fig 1D) . Conclusions: We developed a transgenic hiPSC line expressing DREADD. The produced hiPSC-CM M4Di responded positively to the CNO ligand, allowing inhibition of their electromechanical activity. This cell line holds potential for investigating the electrophysiology of grafted hiPSC-CMs, and for application as disease model in vitro.

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.

Background: Doxorubicin (DOX) is an effective chemotherapy but limited by severe cardiotoxicity. Chemokine receptors CXCR4 and CXCR7 are involved in regulating cardiac survival, inflammation, and fibrosis, yet their specific roles in DOX-induced cardiotoxicity (DICT) remain unclear. Objective: We investigated the cardioprotective effects of TC14012, a dual-target CXCR4 antagonist and CXCR7 agonist, against DICT. Methods: Male C57BL/6J mice received chronic or acute DOX administration with twice-weekly TC14012 treatment. Cardiac function was evaluated by echocardiography. Markers of cardiac fibrosis and inflammation were assessed. RNA-seq analysis was performed to explore associated molecular pathways. Key pathways were validated in cardiac cells and bone marrow-derived mast cells. Results: TC14012 significantly improved cardiac function, indicated by preservation of ejection fraction and fractional shortening, and increased survival rates optimally at 5 mg/kg, which were accompanied with reductions in cardiac collagen deposition and infiltration of macrophages and neutrophils. RNA-seq revealed that TC14012 protecting against DICT was associated with a remarkable stimulation of signals involving in cardiac contraction and energy reserve metabolism processes, particularly glycogen and glucan metabolism that are crucial for meeting the cardiac energy demand in response to DOX induction. These signals could be further enriched into AMPK signalling pathway and validated by enhancement of cardiac AMPK phosphorylation both in vivo and in vitro . Meanwhile, TC14012’s cardioprotective effects were accompanied by a reduction in signals involving in cardiac mast cell activation and degranulation, which could be enriched into Rap1 signalling pathway and validated by TC14012 inhibiting mast cell activation and Rap1-GTP activity in vitro . Importantly, TC14012 did not compromise DOX’s antitumor efficacy in EL-4 lymphoma-bearing mice. Conclusions: TC14012 may prevent DICT by activating CXCR7-AMPK signalling to reprogram cardiomyocyte metabolism and antagonizing CXCR4-Rap1 signalling to blunt mast cell-mediated sentinel effects without compromising the anticancer efficacy DOX.

Biomaterials like gold nanorod-embedded gelatin methacrylate (GelMA-GNR) hydrogels support the delivery of regenerative cells such as human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), enhancing EHT performance.

Abstract Mesenchymal stem cells (MSCs) offer significant therapeutic potential, but traditional 2D culture on rigid substrates results in progressive cell enlargement and senescence, reducing proliferative capacity and therapeutic potency. This poses a major challenge for widespread clinical application. We explored a novel strategy for placenta-derived MSCs combining 2D expansion with 3D spheroid culture to address these limitations. Our research shows that culturing MSCs as 3D spheroids significantly reduces individual cell size and size distribution compared to 2D culture. Spheroid formation is feasible in chemically defined media with minimal cell death, and is enhanced by extracellular matrix protein supplementation. Although MSCs do not proliferate in 3D suspension, an alternating 2D/3D culture protocol, transitioning cells between 2D flasks and 3D spheroids after each passage, effectively slows MSC enlargement and senescence over long periods. This alternating method also preserves MSC immunomodulatory function, unlike continuous 2D culture which leads to its loss. For scalability, we developed an RGD-functionalized alginate hydrogel tube (AlgTube) system that mimics this alternating environment, supporting both adherent growth and chemically triggered spheroid formation. This alternating 2D/3D culture strategy and the scalable AlgTube platform provide a foundation for developing next-generation MSC manufacturing technologies to meet future clinical demands.