Transient secretion of VEGF protein from transplanted hiPSC-CMs enhances engraftment and improves rat heart function post MI.

Cell-based therapies offer an exciting and novel treatment for heart repair following myocardial infarction (MI). However, these therapies often suffer from poor cell viability and engraftment rates, which involve many factors, including the hypoxic conditions of the infarct environment. Meanwhile, vascular endothelial growth factor (VEGF) has previously been employed as a therapeutic agent to limit myocardial damage and simultaneously induce neovascularization. This study took an approach to transiently overexpress VEGF protein, in a controlled manner, by transfecting human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with VEGF mRNA prior to transplantation. The conditioning of iPSC-CMs with VEGF mRNA ultimately led to greater survival rates of the transplanted cells, which promoted a stable vascular network in the grafted region. Furthermore, bulk RNA transcriptomics data and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) and AGE-RAGE signaling pathways were significantly upregulated in the VEGF-treated iPSC-CMs group. The over-expression of VEGF from iPSC-CMs stimulated cell proliferation and partially attenuated the hypoxic environment in the infarcted area, resulting in reduced ventricular remodeling. This study provides a valuable solution for the survival of transplanted cells in tissue-engineered heart regeneration and may further promote the application of modified mRNA (modRNA) in the field of tissue engineering.

Engineering a conduction-consistent cardiac patch with rGO/PLCL electrospun nanofibrous membranes and human iPSC-derived cardiomyocytes.

The healthy human heart has special directional arrangement of cardiomyocytes and a unique electrical conduction system, which is critical for the maintenance of effective contractions. The precise arrangement of cardiomyocytes (CMs) along with conduction consistency between CMs is essential for enhancing the physiological accuracy of in vitro cardiac model systems. Here, we prepared aligned electrospun rGO/PLCL membranes using electrospinning technology to mimic the natural heart structure. The physical, chemical and biocompatible properties of the membranes were rigorously tested. We next assembled human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on electrospun rGO/PLCL membranes in order to construct a myocardial muscle patch. The conduction consistency of cardiomyocytes on the patches were carefully recorded. We found that cells cultivated on the electrospun rGO/PLCL fibers presented with an ordered and arranged structure, excellent mechanical properties, oxidation resistance and effective guidance. The addition of rGO was found to be beneficial for the maturation and synchronous electrical conductivity of hiPSC-CMs within the cardiac patch. This study verified the possibility of using conduction-consistent cardiac patches to enhance drug screening and disease modeling applications. Implementation of such a system could one day lead to in vivo cardiac repair applications.

Engineering a conduction-consistent cardiac patch with graphene oxide modified butterfly wings and human pluripotent stem cell-derived cardiomyocytes.

Engineering a conduction-consistent cardiac patch has direct implications to biomedical research. However, there is difficulty in obtaining and maintaining a system that allows researchers to study physiologically relevant cardiac development, maturation, and drug screening due to the issues around inconsistent contractions of cardiomyocytes. Butterfly wings have special nanostructures arranged in parallel, which could help generate the alignment of cardiomyocytes to better mimic the natural heart tissue structure. Here, we construct a conduction-consistent human cardiac muscle patch by assembling human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on graphene oxide (GO) modified butterfly wings. We also show this system functions as a versatile model to study human cardiomyogenesis by assembling human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSC-CPCs) on the GO modified butterfly wings. The GO modified butterfly wing platform facilitated the parallel orientation of hiPSC-CMs, enhanced relative maturation as well as improved conduction consistency of the cardiomyocytes. In addition, GO modified butterfly wings enhanced the proliferation and maturation characteristics of the hiPSC-CPCs. In accordance with data obtained from RNA-sequencing and gene signatures, assembling hiPSC-CPCs on GO modified butterfly wings stimulated the differentiation of the progenitors into relatively mature hiPSC-CMs. These characteristics and capabilities of GO modified butterfly wings make them an ideal platform for heart research and drug screening.

Large-scale microRNA functional high-throughput screening identifies miR-515-3p and miR-519e-3p as inducers of human cardiomyocyte proliferation.

Ischemic cardiomyopathy, driven by loss of cardiomyocytes and inadequate proliferative response, persists to be a major global health problem. Using a functional high-throughput screening, we assessed differential proliferative potential of 2019 miRNAs after transient hypoxia by transfecting both miR-inhibitor and miR-mimic libraries in human iPSC-CM. Whereas miR-inhibitors failed to enhance EdU uptake, overexpression of 28 miRNAs substantially induced proliferative activity in hiPSC-CM, with an overrepresentation of miRNAs belonging to the primate-specific C19MC-cluster. Two of these miRNAs, miR-515-3p and miR-519e-3p, increased markers of early and late mitosis, indicative of cell division, and substantially alter signaling pathways relevant for cardiomyocyte proliferation in hiPSC-CM.

Yohimbine Directly Induces Cardiotoxicity on Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Yohimbine is a highly selective and potent α2-adrenoceptor antagonist, which is usually treated as an adjunction for impotence, as well for weight loss and natural bodybuilding aids. However, it was recently reported that Yohimbine causes myocardial injury and controversial results were reported in the setting of cardiac diseases. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to explore electrophysiologic characterization after exposure to Yohimbine. HiPSC-CMs were differentiated by employment of inhibitory Wnt compounds. For analysis of electrophysiological properties, conventional whole-cell patch-clamp recording was used. Specifically, spontaneous action potentials, pacemaker currents (If), sodium (Na+) channel (INa), and calcium (Ca++) channel currents (ICa) were assessed in hiPSC-CMs after exposure to Yohimbine. HiPSC-CMs expressed sarcomeric-α-actinin and MLC2V proteins, as well as exhibited ventricular-like spontaneous action potential waveform. Yohimbine inhibited frequency of hiPSC-CMs spontaneous action potentials and significantly prolonged action potential duration in a dose-dependent manner. In addition, rest potential, threshold potential, amplitude, and maximal diastolic potential were decreased, whereas APD50/APD90 was prolonged. Yohimbine inhibited the amplitude of INa in low doses (IC50 = 14.2 µM, n = 5) and inhibited ICa in high doses (IC50 = 139.7 µM, n = 5). Whereas Yohimbine did not affect the activation curves, treatment resulted in left shifts in inactivation curves of both Na+ and Ca++ channels. Here, we show that Yohimbine induces direct cardiotoxic effects on spontaneous action potentials of INa and ICa in hiPSC-CMs. Importantly, these effects were not mediated by α2-adrenoceptor signaling. Our results strongly suggest that Yohimbine directly and negatively affects electrophysiological properties of human cardiomyocytes. These findings are highly relevant for potential application of Yohimbine in patients with atrioventricular conduction disorder.

Dexmedetomidine exhibits antiarrhythmic effects on human-induced pluripotent stem cell-derived cardiomyocytes through a Na/Ca channel-mediated mechanism.

BACKGROUND: Ventricular-like human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibit the electrophysiological characteristics of spontaneous beating. Previous studies demonstrated that dexmedetomidine (DMED), a highly selective and widely used α2-adrenoceptor agonist for sedation, analgesia, and stress management, may induce antiarrhythmic effects, especially ventricular tachycardia. However, the underlying mechanisms of the DMED-mediated antiarrhythmic effects remain to be fully elucidated. METHODS: A conventional patch-clamp recording method was used to investigate the direct effects of DMED on spontaneous action potentials, pacemaker currents (I f), potassium (K+) channel currents (I K1 and I Kr), sodium (Na+) channel currents (I Na), and calcium (Ca2+) channel currents (I Ca) in ventricular-like hiPSC-CMs. RESULTS: DMED dose-dependently altered the frequency of ventricular-like spontaneous action potentials with a half-maximal inhibitory concentration (IC50) of 27.9 µM (n=6) and significantly prolonged the action potential duration at 90% repolarization (APD90). DMED also inhibited the amplitudes of the I Na and I Ca without affecting the activation and inactivation curves of these channels. DMED decreased the time constant of the Na+ and Ca2+ channel activation at potential -40 to -20 mv, and -20 mv. DMED increased the time constant of inactivation of the Na+ and Ca2+ channels. However, DMED did not affect the I K1, I Kr, I f, and their current-voltage relationship. The ability of DMED to decrease the spontaneous action potential frequency and the Na+ and Ca2+ channel amplitudes, were not blocked by yohimbine, idazoxan, or phentolamine. CONCLUSIONS: DMED could inhibit the frequency of spontaneous action potentials and decrease the I Na and I Ca of hiPSC-CMs via mechanisms that were independent of the α2-adrenoceptor, the imidazoline receptor, and the α1-adrenoceptor. These inhibitory effects on hiPSC-CMs may contribute to the antiarrhythmic effects of DMED.

Intrinsic Color Sensing System Allows for Real-Time Observable Functional Changes on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Stem-cell based in vitro differentiation for disease modeling offers great value to explore the molecular and functional underpinnings driving many types of cardiomyopathy and congenital heart diseases. Nevertheless, one major caveat in the application of in vitro differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) involves the immature phenotype of the CMs. Most of the existing methods need complex apparatus and require laborious procedures in order to monitor the cardiac differentiation/maturation process and often result in cell death. Here we developed an intrinsic color sensing system utilizing a microgroove structural color methacrylated gelatin film, which allows us to monitor the cardiac differentiation process of hiPSC-derived cardiac progenitor cells in real time. Subsequently this system can be employed as an assay system to live monitor induced functional changes on hiPSC-CMs stemming from drug treatment, the effects of which are simply revealed through color diversity. Our research shows that early intervention of cardiac differentiation through simple physical cues can enhance cardiac differentiation and maturation to some extent. Our system also simplifies the previous complex experimental processes for evaluating the physiological effects of successful differentiation and drug treatment and lays a solid foundation for future transformational applications.