Zhu, Wuqiang

Background: Nanoparticles (NPs) have been extensively utilized as a drug delivery system to control the release of therapeutic agents to treat cardiac injuries. However, despite the advantages of utilizing NP-based drug delivery for treating heart diseases, the current delivery system lacks specificity in targeting the cardiac tissue, thus limiting its application. Methods: We created three linear peptides, each consisting of 16–24 amino acids. These peptides were conjugated on the surface of NPs, resulting in the formation of cardiac targeting peptide (CTP)-NPs (designated as CTP-NP1, CTP-NP2, and CTP-NP3). To assess their effectiveness, we compared the binding efficiency of these three CTP-NPs to human and mouse cardiomyocytes. Additionally, we determined their distribution 24 h after injecting the CTP-NPs intravenously into adult C57BL/6J mice. Results: When compared to control NPs without CTP (Con-NPs), all three CTP-NPs exhibited significantly increased binding affinity to both human and mouse cardiomyocytes in vitro and enhanced retention in mouse hearts in vivo. A thorough assessment of the heart sections demonstrated that the binding specificity of CTP-NP3 to cardiomyocytes in vivo was significantly greater than that of Con-NPs. None of the three CTP-NPs were proven to cause cardiomyocyte apoptosis. Conclusions: Biocompatible and safe CTP-NP3 can target the heart via binding to cardiomyocytes. This approach of targeting specific molecules-coated NPs may help in delivering therapeutic compounds to cardiomyocytes for the treatment of heart diseases with high efficacy and low toxicity to other tissues.

An estimated 64 [...]

Introduction: Transplantation of human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) emerges as a promising approach for the treatment of heart diseases. However, it is an unmet challenge to precisely localize the hiPSC-CMs upon transplantation and quantify the graft size in the live large animals. To address this issue, we established a new approach to track grafted CMs in large animals using magnetic resonance imaging detectable reporter molecules, which allows the assessment of graft size in live animals at different time points. Methods: We employed a CRISPR/Cas9 system as a virus-free approach to overexpress ferritin in hiPSCs. Results: hiPSC cells were transfected with ferritin heavy chain CRISPR activation plasmid for 24 hours. Before antibiotic selection, we established a kill curve to determine the appropriate antibiotic concentration. Based on the kill curve, we conducted antibiotic selection using puromycin for 7 days and isolated transfected colonies for further culture. Next, we treated various concentrations of iron chloride (0-100 μM) to the FHC transfected hiPSC for 4 hours to examine a proper concentration of iron loading for MRI detection. There was no significant difference in cell viability between 0 and 25 μM of iron chloride. In addition, we confirmed transfection efficiency with higher ferritin expression in transfected cells compared to controls (non-transfected wild-type hiPSC). Following, we plan to differentiate ferritin overexpressing hiPSC into cardiomyocytes, inject the cells into porcine hearts, and conduct magnetic resonance imaging to detect the engrafted transgenic cells in live animals. Conclusion: The ferritin-expressing hiPSC line has the potential to provide a reliable measurement of the graft size in large animals.

Heart failure secondary to myocardial injuries is a leading cause of death worldwide. Recently, a growing number of novel therapies have emerged for injured myocardium repairment. However, delivering therapeutic agents specifically to the injured heart remains a significant challenge. Nanoparticles are the most commonly used vehicles for targeted drug delivery. Various nanoparticles have been synthesized to deliver drugs and other therapeutic molecules to the injured heart via passive or active targeting approaches, and their targeting specificity and therapeutic efficacies have been investigated. Here, we summarized nanoparticle-based, cardiac-specific drug delivery systems, their potency for treating heart diseases, and the mechanisms underlying these cardiac-targeting strategies. We also discussed the clinical studies that have employed nanoparticle-based cardiac-specific drug delivery.

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.

Background: Cellular transplantation has emerged as promising approach for treating cardiac diseases. However, a poor engraftment rate limits our understanding on how transplanted cardiomyocytes contribute to cardiac function in the recipient’s heart. Methods: The CRISPR/Cas9 technique was employed for stable and constitutive gene expression in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). Myocardial infarction was induced in adult immunodeficient mice, followed by intramyocardial injection of hiPSC-CMs expressing either CCND2/channelrhodopsin 2 (hiPSC-CCND2OE/ChR2OECMs) or CCND2/luciferase (hiPSC-CCND2OE/LuciOECMs). Six months later, hemodynamics and intramural electrocardiogram were recorded upon blue light illuminations in anesthetized, open-chest mice. Results: Blue light resets automaticity of spontaneously beating hiPSC-CCND2OE/ChR2OECMs in culture, but not that of hiPSC-CCND2OE/LuciOECMs. Response to blue light was also observed in mice carrying large (>106 cells) intracardiac grafts of hiPSC-CCND2OE/ChR2OECM but not in mice carrying hiPSC-CCND2OE/LuciOECMs. The former exhibited single premature ventricular contractions upon light illumination or ventricular quadrigeminy upon second-long illuminations. At the onset of premature ventricular contractions, maximal systolic ventricular pressure decreased while ventricular volume rose concomitantly. Light-induced changes reversed upon resumption of sinus rhythm. Conclusions: We established an in vivo model for optogenetic-based modulation of the excitability of donor cardiomyocytes in a functional, reversible, and localized manner. This approach holds unique value for studying electromechanical coupling and molecular interactions between donor cardiomyocytes and recipient hearts in live animals.

Background: Human induced pluripotent stem cells with normal (wild-type) or upregulated (overexpressed) levels of CCND2 (cyclin D2) expression were differentiated into cardiomyocytes (CCND2 WT CMs or CCND2 OE CMs, respectively) and injected into infarcted pig hearts. Methods: Acute myocardial infarction was induced by a 60-minute occlusion of the left anterior descending coronary artery. Immediately after reperfusion, CCND2 WT CMs or CCND2 OE CMs (3×10 7 cells each) or an equivalent volume of the delivery vehicle was injected around the infarct border zone area. Results: The number of the engrafted CCND2 OE CMs exceeded that of the engrafted CCND2 WT CMs from 6- to 8-fold, rising from 1 week to 4 weeks after implantation. In contrast to the treatment with the CCND2 WT CMs or the delivery vehicle, the administration of CCND2 OE CM was associated with significantly improved left ventricular function, as revealed by magnetic resonance imaging. This correlated with reduction of infarct size, fibrosis, ventricular hypertrophy, and cardiomyocyte apoptosis, and increase of vascular density and arterial density, as per histologic analysis of the treated hearts. Expression of cell proliferation markers (eg, Ki67, phosphorylated histone 3, and Aurora B kinase) was also significantly upregulated in the recipient cardiomyocytes from the CCND2 OE CM-treated than from the CCND2 WT CM-treated pigs. The cell proliferation rate and the hypoxia tolerance measured in cultured human induced pluripotent stem cell cardiomyocytes were significantly greater after treatment with exosomes isolated from the CCND2 OE CMs (CCND2 OE Exos) than from the CCND2 WT CMs (CCND2 WT Exos). As demonstrated by our study, CCND2 OE Exos can also promote the proliferation activity of postnatal rat and adult mouse cardiomyocytes. A bulk miRNA sequencing analysis of CCND2 OE Exos versus CCND2 WT Exos identified 206 and 91 miRNAs that were significantly upregulated and downregulated, respectively. Gene ontology enrichment analysis identified significant differences in the expression profiles of miRNAs from various functional categories and pathways, including miRNAs implicated in cell-cycle checkpoints (G2/M and G1/S transitions), or the mechanism of cytokinesis. Conclusions: We demonstrated that enhanced potency of CCND2 OE CMs promoted myocyte proliferation in both grafts and recipient tissue in a large mammal acute myocardial infarction model. These results suggest that CCND2 OE CMs transplantation may be a potential therapeutic strategy for the repair of infarcted hearts.