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Purpose of Review This study is aimed at reviewing the recent progress in Drp1 inhibition as a novel approach for reducing doxorubicin-induced cardiotoxicity and for improving cancer treatment. Recent Findings Anthracyclines (e.g. doxorubicin) are one of the most common and effective chemotherapeutic agents to treat a variety of cancers. However, the clinical usage of doxorubicin has been hampered by its severe cardiotoxic side effects leading to heart failure. Mitochondrial dysfunction is one of the major aetiologies of doxorubicin-induced cardiotoxicity. The morphology of mitochondria is highly dynamic, governed by two opposing processes known as fusion and fission, collectively known as mitochondrial dynamics. An imbalance in mitochondrial dynamics is often reported in tumourigenesis which can lead to adaptive and acquired resistance to chemotherapy. Drp1 is a key mitochondrial fission regulator, and emerging evidence has demonstrated that Drp1-mediated mitochondrial fission is upregulated in both cancer cells to their survival advantage and injured heart tissue in the setting of doxorubicin-induced cardiotoxicity. Summary Effective treatment to prevent and mitigate doxorubicin-induced cardiotoxicity is currently not available. Recent advances in cardio-oncology have highlighted that Drp1 inhibition holds great potential as a targeted mitochondrial therapy for doxorubicin-induced cardiotoxicity.

Patients with Type 2 diabetes mellitus (T2DM) frequently exhibit a distinctive cardiac phenotype known as diabetic cardiomyopathy. Cardiac complications associated with T2DM include cardiac inflammation, hypertrophy, fibrosis, and diastolic dysfunction in the early stages of the disease, which can progress to systolic dysfunction and heart failure. Effective therapeutic options for diabetic cardiomyopathy are limited and often have conflicting results. The lack of effective treatments for diabetic cardiomyopathy is due in part, to our poor understanding of the disease development and progression, as well as a lack of robust and valid preclinical human models that can accurately recapitulate the pathophysiology of the human heart. In addition to cardiomyocytes, the heart contains a heterogeneous population of non-myocytes including fibroblasts, vascular cells, autonomic neurons, and immune cells. These cardiac non-myocytes play important roles in cardiac homeostasis and disease, yet the effect of hyperglycaemia and hyperlipidaemia on these cell types is often overlooked in preclinical models of diabetic cardiomyopathy. The advent of human-induced pluripotent stem cells provides a new paradigm in which to model diabetic cardiomyopathy as they can be differentiated into all cell types in the human heart. This review will discuss the roles of cardiac non-myocytes and their dynamic intercellular interactions in the pathogenesis of diabetic cardiomyopathy. We will also discuss the use of sodium-glucose cotransporter 2 inhibitors as a therapy for diabetic cardiomyopathy and their known impacts on non-myocytes. These developments will no doubt facilitate the discovery of novel treatment targets for preventing the onset and progression of diabetic cardiomyopathy.

Mitochondrial dynamin-related protein 1 (Drp1) is a large GTPase regulator of mitochondrial dynamics and is known to play an important role in numerous pathophysiological processes. Despite being the most widely used Drp1 inhibitor, the specificity of Mdivi-1 towards human Drp1 has not been definitively proven and there have been numerous issues reported with its use including off-target effects. In our hands Mdivi-1 showed varying binding affinities toward human Drp1, potentially impacted by compound aggregation. Herein, we sought to identify a novel small molecule inhibitor of Drp1. From an initial virtual screening, we identified DRP1i27 as a compound which directly bound to the human isoform 3 of Drp1 via surface plasmon resonance and microscale thermophoresis. Importantly, DRP1i27 was found to have a dose-dependent increase in the cellular networks of fused mitochondria but had no effect in Drp1 knock-out cells. Further analogues of this compound were identified and screened, though none displayed greater affinity to human Drp1 isoform 3 than DRP1i27. To date, this is the first small molecule inhibitor shown to directly bind to human Drp1.

Due to a relative paucity of studies on human lymphatic assembly in vitro and subsequent in vivo transplantation, capillary formation and survival of primary human lymphatic (hLEC) and blood endothelial cells (hBEC) ± primary human vascular smooth muscle cells (hvSMC) were evaluated and compared in vitro and in vivo. hLEC ± hvSMC or hBEC ± hvSMC were seeded in a 3D porous scaffold in vitro, and capillary percent vascular volume (PVV) and vascular density (VD)/mm2 assessed. Scaffolds were also transplanted into a sub-cutaneous rat wound with morphology/morphometry assessment. Initially hBEC formed a larger vessel network in vitro than hLEC, with interconnected capillaries evident at 2 days. Interconnected lymphatic capillaries were slower (3 days) to assemble. hLEC capillaries demonstrated a significant overall increase in PVV ( p = 0.0083) and VD ( p = 0.0039) in vitro when co-cultured with hvSMC. A similar increase did not occur for hBEC + hvSMC in vitro, but hBEC + hvSMC in vivo significantly increased PVV ( p = 0.0035) and VD ( p = 0.0087). Morphology/morphometry established that hLEC vessels maintained distinct cell markers, and demonstrated significantly increased individual vessel and network size, and longer survival than hBEC capillaries in vivo, and established inosculation with rat lymphatics, with evidence of lymphatic function. The porous polyurethane scaffold provided advantages to capillary network formation due to its large (300–600 μm diameter) interconnected pores, and sufficient stability to ensure successful surgical transplantation in vivo. Given their successful survival and function in vivo within the porous scaffold, in vitro assembled hLEC networks using this method are potentially applicable to clinical scenarios requiring replacement of dysfunctional or absent lymphatic networks.

Background: Cardiomyocytes derived from pluripotent stem cells are immature. Maturation of cardiomyocytes is a multifactorial dynamic process that involves various factors in vivo that cannot be fully recapitulated in vitro. Here, we report a novel tissue engineering chamber with an integrated electrical stimulator and electrodes that will allow wireless electrical stimulation of cardiac tissue in vivo. Materials and Methods: Immunocompromised rats were implanted with tissue engineering chambers containing the stimulator and electrodes, and control chambers (chambers with electrical stimulator but without the electrodes) in the contralateral limb. Each chamber contained cardiomyocytes derived from human induced pluripotent stem cells (iPSCs). After 7 days of chamber implantation, the electrical stimulators were activated for 4 h per day, for 21 consecutive days. Results: At 4 weeks postimplantation, cardiomyocytes derived from human iPSCs survived, were assembled into compact cardiac tissue, and were perfused and vascularized by the host neovessels. Conclusion: This proof-of-principle study demonstrates the biocompatibility of the tissue engineering chamber with integrated electrical stimulator and electrodes. This could be utilized to study the influence of continuous electrical stimulation on vascularized cardiac or other tissues in vivo.

To assess the transcriptomic profile of disease-specific cell populations, fibroblasts from patients with primary open-angle glaucoma (POAG) were reprogrammed into induced pluripotent stem cells (iPSCs) before being differentiated into retinal organoids and compared to those from healthy individuals. We performed single-cell RNA-sequencing of a total of 330,569 cells and identified cluster-specific molecular signatures. Comparing the gene expression profile between cases and controls, we identified novel genetic associations for this blinding disease. Expression quantitative trait mapping identified a total of 2,235 significant loci across all cell types, 58 of which are specific to the retinal ganglion cell subpopulations, which ultimately degenerate in POAG. Transcriptome-wide association analysis identified genes at loci previously associated with POAG, and analysis, conditional on disease status, implicated 54 statistically significant retinal ganglion cell-specific expression quantitative trait loci. This work highlights the power of large-scale iPSC studies to uncover context-specific profiles for a genetically complex disease.

SummaryDiabetic heart disease is highly prevalent and characterized by diastolic dysfunction. The mechanisms of diabetic heart disease are poorly understood and no targeted therapies are available. Here we show that the diabetic myocardium (type 1 and type 2) is characterized by marked glycogen elevation and ectopic cellular localization - a paradoxical metabolic pathology given suppressed cardiomyocyte glucose uptake in diabetes. We demonstrate involvement of a glycogen-selective autophagy pathway (‘glycophagy’) defect in mediating this pathology. Genetically manipulated deficiency of Gabarapl1, an Atg8 autophagy homologue, induces cardiac glycogen accumulation and diastolic dysfunction. Stbd1, the Gabarapl1 cognate autophagosome partner is identified as a unique component of the early glycoproteome response to hyperglycemia in cardiac, but not skeletal muscle. Cardiac-targeted in vivo Gabarapl1 gene delivery normalizes glycogen levels, diastolic function and cardiomyocyte mechanics. These findings reveal that cardiac glycophagy is a key metabolic homeostatic process perturbed in diabetes that can be remediated by Gabarapl1 intervention.

Human induced pluripotent stem cells (iPSCs) can be differentiated in vitro into bona fide cardiomyocytes for disease modelling and personalized medicine. Mitochondrial morphology and metabolism change dramatically as iPSCs differentiate into mesodermal cardiac lineages. Inhibiting mitochondrial fission has been shown to promote cardiac differentiation of iPSCs. However, the effect of hydrazone M1, a small molecule that promotes mitochondrial fusion, on cardiac mesodermal commitment of human iPSCs is unknown. Here, we demonstrate that treatment with M1 promoted mitochondrial fusion in human iPSCs. Treatment of iPSCs with M1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. The pro-fusion and pro-cardiogenic effects of M1 were not associated with changes in expression of the α and β subunits of adenosine triphosphate (ATP) synthase. Our findings demonstrate for the first time that hydrazone M1 is capable of promoting cardiac differentiation of human iPSCs, highlighting the important role of mitochondrial dynamics in cardiac mesoderm lineage specification and cardiac development. M1 and other mitochondrial fusion promoters emerge as promising molecular targets to generate lineages of the heart from human iPSCs for patient-specific regenerative medicine.

Tissue flaps are used to cover large/poorly healing wounds, but involve complex surgery and donor site morbidity. In this study a tissue flap is assembled using the mammalian body as a bioreactor to functionally connect an artery and vein to a human capillary network assembled from induced pluripotent stem cell-derived endothelial cells (hiPSC ECs). In vitro: Porous NovoSorb™ scaffolds (3 mm × 1.35 mm) were seeded with 200,000 hiPSC ECs ± 100,000 human vascular smooth muscle cells (hvSMC), and cultured for 1-3 days, with capillaries formed by 24 h which were CD31+, VE-Cadherin+, EphB4+, VEGFR2+ and Ki67+, whilst hvSMCs (calponin+) attached abluminally. In vivo: In SCID mice, bi-lateral epigastric vascular pedicles were isolated in a silicone chamber for a 3 week 'delay period' for pedicle capillary sprouting, then reopened, and two hiPSC EC ± hvSMCs seeded scaffolds transplanted over the pedicle. The chamber was either resealed (Group 1), or removed and surrounding tissue secured around the pedicle + scaffolds (Group 2), for 1 or 2 weeks. Human capillaries survived in vivo and were CD31+, VE-Cadherin+ and VEGFR2+. Human vSMCs remained attached, and host mesenchymal cells also attached abluminally. Systemically injected FITC-dextran present in human capillary lumens indicated inosculation to host capillaries. Human iPSC EC capillary morphometric parameters at one week in vivo were equal to or higher than the same parameters measured in human abdominal skin. This 'proof of concept' study has demonstrated that bio-engineering an autologous human tissue flap based on hiPSC EC could minimize the use of donor flaps and has potential applications for complex wound coverage. STATEMENT OF SIGNIFICANCE: Tissue flaps, used for surgical reconstruction of wounds, require complex surgery, often associated with morbidity. Bio-engineering a simpler alternative, we assembled a human induced pluripotent stem cell derived endothelial cell (hiPSC ECs) capillary network in a porous scaffold in vitro, which when transplanted over a mouse vascular pedicle in vivo formed a functional tissue flap with mouse blood flow in the human capillaries. Therefore it is feasible to form an autologous tissue flap derived from a hiPSC EC capillary network assembled in vitro, and functionally connect to a vascular pedicle in vivo that could be utilized in complex wound repair for chronic or acute wounds.

Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1, and NEUROD1) in human cells, whereas the rat cells exhibit more variable and less-efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular- and cell-biology disciplines. Keywords: CRISPR, cloning free, gene activation, CRISPR activation, SpCas9, SaCas9, multiplex gene activation, transcriptional activator

Background Human bone marrow-derived mesenchymal stem/stromal cells (hBM MSCs) have multiple functions, critical for skeletal formation and function. Their functional heterogeneity, however, represents a major challenge for their isolation and in developing potency and release assays to predict their functionality prior to transplantation. Additionally, potency, biomarker profiles and defining mechanisms of action in a particular clinical setting are increasing requirements of Regulatory Agencies for release of hBM MSCs as Advanced Therapy Medicinal Products for cellular therapies. Since the healing of bone fractures depends on the coupling of new blood vessel formation with osteogenesis, we hypothesised that a correlation between the osteogenic and vascular supportive potential of individual hBM MSC-derived CFU-F (colony forming unit-fibroblastoid) clones might exist. Methods We tested this by assessing the lineage (i.e. adipogenic (A), osteogenic (O) and/or chondrogenic (C)) potential of individual hBM MSC-derived CFU-F clones and determining if their osteogenic (O) potential correlated with their vascular supportive profile in vitro using lineage differentiation assays, endothelial-hBM MSC vascular co-culture assays and transcriptomic (RNAseq) analyses. Results Our results demonstrate that the majority of CFU-F (95%) possessed tri-lineage, bi-lineage or uni-lineage osteogenic capacity, with 64% of the CFU-F exhibiting tri-lineage AOC potential. We found a correlation between the osteogenic and vascular tubule supportive activity of CFU-F clones, with the strength of this association being donor dependent. RNAseq of individual clones defined gene fingerprints relevant to this correlation. Conclusions This study identified a donor-dependent correlation between osteogenic and vascular supportive potential of hBM MSCs and important gene signatures that support these functions that are relevant to their bone regenerative properties. Electronic supplementary material The online version of this article (10.1186/s13287-018-1095-7) contains supplementary material, which is available to authorized users.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterized by degeneration of dorsal root ganglia (DRG) sensory neurons, which is due to low levels of the mitochondrial protein Frataxin. To explore cell replacement therapies as a possible approach to treat FRDA, we examined transplantation of sensory neural progenitors derived from human embryonic stem cells (hESC) and FRDA induced pluripotent stem cells (iPSC) into adult rodent DRG regions. Our data showed survival and differentiation of hESC and FRDA iPSC‐derived progenitors in the DRG 2 and 8 weeks post‐transplantation, respectively. Donor cells expressed neuronal markers, including sensory and glial markers, demonstrating differentiation to these lineages. These results are novel and a highly significant first step in showing the possibility of using stem cells as a cell replacement therapy to treat DRG neurodegeneration in FRDA as well as other peripheral neuropathies., This study is the first to describe transplantation of dorsal root ganglia (DRG) sensory neural progenitors derived from human pluripotent stem cells (hPSC) into adult rodent DRG tissue. Donor cells differentiated to neurons and glia in vivo and expressed DRG sensory markers. These findings are highly significant in supporting stem cell‐based therapies for treating peripheral DRG neuropathies, including Friedreich ataxia. Figure created using BioRender.com

Aims To establish pre-clinical proof of concept that sustained subcutaneous delivery of the secretome of human cardiac stem cells (CSCs) can be achieved in vivo to produce significant cardioreparative outcomes in the setting of myocardial infarction. Methods and results Rats were subjected to permanent ligation of left anterior descending coronary artery and randomized to receive subcutaneous implantation of TheraCyte devices containing either culture media as control or 1 × 106 human W8B2+ CSCs, immediately following myocardial ischaemia. At 4 weeks following myocardial infarction, rats treated with W8B2+ CSCs encapsulated within the TheraCyte device showed preserved left ventricular ejection fraction. The preservation of cardiac function was accompanied by reduced fibrotic scar tissue, interstitial fibrosis, cardiomyocyte hypertrophy, as well as increased myocardial vascular density. Histological analysis of the TheraCyte devices harvested at 4 weeks post-implantation demonstrated survival of human W8B2+ CSCs within the devices, and the outer membrane was highly vascularized by host blood vessels. Using CSCs expressing plasma membrane reporters, extracellular vesicles of W8B2+ CSCs were found to be transferred to the heart and other organs at 4 weeks post-implantation. Furthermore, mass spectrometry-based proteomic profiling of extracellular vesicles of W8B2+ CSCs identified proteins implicated in inflammation, immunoregulation, cell survival, angiogenesis, as well as tissue remodelling and fibrosis that could mediate the cardioreparative effects of secretome of human W8B2+ CSCs. Conclusions Subcutaneous implantation of TheraCyte devices encapsulating human W8B2+ CSCs attenuated adverse cardiac remodelling and preserved cardiac function following myocardial infarction. The TheraCyte device can be employed to deliver stem cells in a minimally invasive manner for effective secretome-based cardiac therapy.

Aims: The glucose-driven enzymatic modification of myocardial proteins by the sugar moiety, β-N-acetylglucosamine (O-GlcNAc), is increased in pre-clinical models of diabetes, implicating protein O-GlcNAc modification in diabetes-induced heart failure. Our aim was to specifically examine cardiac manipulation of the two regulatory enzymes of this process on the cardiac phenotype, in the presence and absence of diabetes, utilising cardiac-targeted recombinant-adeno-associated viral-vector-6 (rAAV6)-mediated gene delivery. Methods and results: In human myocardium, total protein O-GlcNAc modification was elevated in diabetic relative to non-diabetic patients, and correlated with left ventricular (LV) dysfunction. The impact of rAAV6-delivered O-GlcNAc transferase (rAAV6-OGT, facilitating protein O-GlcNAcylation), O-GlcNAcase (rAAV6-OGA, facilitating de-O-GlcNAcylation) and empty vector (null) were determined in non-diabetic and diabetic mice. In non-diabetic mice, rAAV6-OGT was sufficient to impair LV diastolic function and induce maladaptive cardiac remodelling, including cardiac fibrosis and increased Myh-7 and Nppa pro-hypertrophic gene expression, recapitulating characteristics of diabetic cardiomyopathy. In contrast, rAAV6-OGA (but not rAAV6-OGT) rescued LV diastolic function and adverse cardiac remodelling in diabetic mice. Molecular insights implicated impaired cardiac PI3K(p110α)-Akt signalling as a potential contributing mechanism to the detrimental consequences of rAAV6-OGT in vivo. In contrast, rAAV6-OGA preserved PI3K(p110α)-Akt signalling in diabetic mouse myocardium in vivo and prevented high glucose-induced impairments in mitochondrial respiration in human cardiomyocytes in vitro. Conclusion: Maladaptive protein O-GlcNAc modification is evident in human diabetic myocardium, and is a critical regulator of the diabetic heart phenotype. Selective targeting of cardiac protein O-GlcNAcylation to restore physiological O-GlcNAc balance may represent a novel therapeutic approach for diabetes-induced heart failure. Translational perspective: There remains a lack of effective clinical management of diabetes-induced cardiac dysfunction, even via conventional intensive glucose-lowering approaches. Here we reveal that the modification of myocardial proteins by O-GlcNAc, a glucose-driven process, is not only increased in human diabetic myocardium, but correlates with reduced cardiac function in affected patients. Moreover, manipulation of the two regulatory enzymes of this process exert opposing influences on the heart, whereby increasing O-GlcNAc transferase (OGT) is sufficient to replicate the cardiac phenotype of diabetes (in the absence of this disease), while increasing O-GlcNAc-ase (OGA) rescues diabetes-induced impairments in both cardiac dysfunction and remodelling. Cardiac O-GlcNAcylation thus represents a novel therapeutic target for diabetes-induced heart failure.

AIMS Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission. METHODS AND RESULTS Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6 ± 1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated ischaemia/reperfusion injury (IRI), hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7 ± 2.5% vs control 34.1 ± 1.5%, P = 0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6 ± 6.5% vs vehicle control 54.1 ± 4.9%, P = 0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9 ± 3.0% vs vehicle control 58.2 ± 3.8%, P

Human induced pluripotent stem cells (iPSCs) are a valuable tool for studying the cardiac developmental process in vitro, and cardiomyocytes derived from iPSCs are a putative cell source for personalized medicine. Changes in mitochondrial morphology have been shown to occur during cellular reprogramming and pluripotent stem cell differentiation. However, the relationships between mitochondrial dynamics and cardiac mesoderm commitment of iPSCs remain unclear. Here we demonstrate that changes in mitochondrial morphology from a small granular fragmented phenotype in pluripotent stem cells to a filamentous reticular elongated network in differentiated cardiomyocytes are required for cardiac mesodermal differentiation. Genetic and pharmacological inhibition of the mitochondrial fission protein, Drp1, by either small interfering RNA or Mdivi-1, respectively, increased cardiac mesoderm gene expression in iPSCs. Treatment of iPSCs with Mdivi-1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. Furthermore, Drp1 gene silencing was accompanied by increased mitochondrial respiration and decreased aerobic glycolysis. Our findings demonstrate that shifting the balance of mitochondrial morphology toward fusion by inhibition of Drp1 promoted cardiac differentiation of human iPSCs with a metabolic shift from glycolysis towards oxidative phosphorylation. These findings suggest that Drp1 may represent a new molecular target for future development of strategies to promote the differentiation of human iPSCs into cardiac lineages for patient-specific cardiac regenerative medicine.

Cybrid technology was used to replace Leber hereditary optic neuropathy (LHON) causing mitochondrial DNA (mtDNA) mutations from patient-specific fibroblasts with wildtype mtDNA, and mutation-free induced pluripotent stem cells (iPSCs) were generated subsequently. Retinal ganglion cell (RGC) differentiation demonstrates increased cell death in LHON-RGCs and can be rescued in cybrid corrected RGCs.

Cardiac arrhythmias of both atrial and ventricular origin are an important feature of cardiovascular disease. Novel antiarrhythmic therapies are required to overcome current drug limitations related to effectiveness and pro-arrhythmia risk in some contexts. Cardiomyocyte culture models provide a high-throughput platform for screening antiarrhythmic compounds, but comparative information about electrophysiological properties of commonly used types of cardiomyocyte preparations is lacking. Standardization of cultured cardiomyocyte microelectrode array (MEA) experimentation is required for its application as a high-throughput platform for antiarrhythmic drug development. The aim of this study was to directly compare the electrophysiological properties and responses to isoproterenol of three commonly used cardiac cultures. Neonatal rat ventricular myocytes (NRVMs), immortalized atrial HL-1 cells, and custom-generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured on microelectrode arrays for 48–120 h. Extracellular field potentials were recorded, and conduction velocity was mapped in the presence/absence of the β-adrenoceptor agonist isoproterenol (1 µM). Field potential amplitude and conduction velocity were greatest in NRVMs and did not differ in cardiomyocytes isolated from male/female hearts. Both NRVMs and hiPSC-CMs exhibited longer field potential durations with rate dependence and were responsive to isoproterenol. In contrast, HL-1 cells exhibited slower conduction and shorter field potential durations and did not respond to 1 µM isoproterenol. This is the first study to compare the intrinsic electrophysiologic properties of cultured cardiomyocyte preparations commonly used for in vitro electrophysiology assessment. These findings offer important comparative data to inform methodological approaches in the use of MEA and other techniques relating to cardiomyocyte functional screening investigations of particular relevance to arrhythmogenesis.

Enhancing differentiation of mesenchymal stem cells (MSCs) to endothelial cells may improve their ability to vascularize tissue and promote wound healing. This study describes a novel role for nitric oxide (NO) in reprogramming MSCs towards an endothelial lineage and highlights the role of Wnt signaling and epigenetic modification by NO. Rat MSCs were transduced with lentiviral vectors expressing endothelial nitric oxide synthase (pLV-eNOS) and a mutated caveolin gene (pLV-CAV-1F92A ) to enhance NO generation resulting in increased in vitro capillary tubule formation and endothelial marker gene expression. An exogenous source of NO could also stimulate CD31 expression in MSCs. NO was associated with an arterial-specific endothelial gene expression profile of Notch1, Dll4, and Hey2 and significantly reduced expression of venous markers. Wnt signaling associated with NO was evident through increased gene expression of Wnt3a and β-catenin protein, and expression of the endothelial marker Pecam-1 could be significantly reduced by treatment with the Wnt signaling inhibitor Dkk-1. The role of NO as an epigenetic modifier was evident with reduced gene expression of the methyltransferase, DNMT1, and bisulfite sequencing of the endothelial Flt1 promoter region in NO-producing MSCs showed significant demethylation compared to control cells. Finally, subcutaneous implantation of NO-producing MSCs seeded in a biomaterial scaffold (NovoSorb®) resulted in survival of transplanted cells and the formation of blood vessels. In summary, this study describes, NO as a potent endothelial programming factor which acts as an epigenetic modifier in MSCs and may provide a novel platform for vascular regenerative therapy.

Background Adipose-derived stem cells (ASCs) have applications in regenerative medicine based on their therapeutic potential to repair and regenerate diseased and damaged tissue. They are commonly subject to oxidative stress during harvest and transplantation, which has detrimental effects on their subsequent viability. By functioning as an antioxidant against free radicals, melatonin may exert cytoprotective effects on ASCs. Methods We cultured human ASCs in the presence of varying dosages of hydrogen peroxide and/or melatonin for a period of 3 hours. Cell viability and apoptosis were determined with propidium iodide and Hoechst 33342 staining under fluorescence microscopy. Results Hydrogen peroxide (1–2.5 mM) treatment resulted in an incremental increase in cell death. 2 mM hydrogen peroxide was thereafter selected as the dose for co-treatment with melatonin. Melatonin alone had no adverse effects on ASCs. Co-treatment of ASCs with melatonin in the presence of hydrogen peroxide protected ASCs from cell death in a dose-dependent manner, and afforded maximal protection at 100 µM (n=4, one-way analysis of variance P Conclusions Melatonin possesses cytoprotective properties against oxidative stress in human ASCs and might be a useful adjunct in fat grafting and cell-assisted lipotransfer.

Reprogramming of somatic cells into a pluripotent state is known to be accompanied by extensive restructuring of mitochondria and switch in metabolic requirements. Here we utilized Leber's hereditary optic neuropathy (LHON) as a mitochondrial disease model to study the effects of homoplasmic mtDNA mutations and subsequent oxidative phosphorylation (OXPHOS) defects in reprogramming. We obtained fibroblasts from a total of 6 LHON patients and control subjects, and showed a significant defect in complex I respiration in LHON fibroblasts by high-resolution respiratory analysis. Using episomal vector reprogramming, our results indicated that human induced pluripotent stem cell (hiPSC) generation is feasible in LHON fibroblasts. In particular, LHON-specific OXPHOS defects in fibroblasts only caused a mild reduction and did not significantly affect reprogramming efficiency, suggesting that hiPSC reprogramming can tolerate a certain degree of OXPHOS defects. Our results highlighted the induction of genes involved in mitochondrial biogenesis (TFAM, NRF1), mitochondrial fusion (MFN1, MFN2) and glycine production (GCAT) during reprogramming. However, LHON-associated OXPHOS defects did not alter the kinetics or expression levels of these genes during reprogramming. Together, our study provides new insights into the effects of mtDNA mutation and OXPHOS defects in reprogramming and genes associated with various aspects of mitochondrial biology.

The benefits of adult stem cells for repair of the heart have been attributed to the repertoire of salutary paracrine activities they appear to exert. We previously isolated human W8B2+ cardiac stem cells (CSCs) and found they powerfully influence cardiomyocytes and endothelial cells to collectively promote cardiac repair and regeneration. Here, the complexity of the W8B2+ CSC secretomes was characterised and examined in more detail. Using ion exchange chromatography to separate soluble proteins based on their net surface charge, the secreted factors responsible for the pro-survival activity of W8B2+ CSCs were found within the low and medium cation fractions. In addition to the soluble proteins, extracellular vesicles generated from W8B2+ CSCs not only exhibited pro-survival and pro-angiogenic activities, but also promoted proliferation of neonatal cardiomyocytes. These extracellular vesicles contain a cargo of proteins, mRNA and primary microRNA precursors that are enriched in exosomes and are capable of modulating collectively many of the cellular pathways involved in protein metabolism, cell growth, as well as cellular responses to stress and organisation of the extracellular matrix. Thus the W8B2+ CSC secretome contains a multitude of bioactive paracrine factors we have now characterised, that might well be harnessed for therapeutic application for cardiac repair and regeneration.

The benefits of adult stem cells for repair of the heart have been attributed to the repertoire of salutary paracrine activities they appear to exert. We previously isolated human W8B2+ cardiac stem cells (CSCs) and found they powerfully influence cardiomyocytes and endothelial cells to collectively promote cardiac repair and regeneration. Here, the complexity of the W8B2+ CSC secretomes was characterised and examined in more detail. Using ion exchange chromatography to separate soluble proteins based on their net surface charge, the secreted factors responsible for the pro-survival activity of W8B2+ CSCs were found within the low and medium cation fractions. In addition to the soluble proteins, extracellular vesicles generated from W8B2+ CSCs not only exhibited pro-survival and pro-angiogenic activities, but also promoted proliferation of neonatal cardiomyocytes. These extracellular vesicles contain a cargo of proteins, mRNA and primary microRNA precursors that are enriched in exosomes and are capable of modulating collectively many of the cellular pathways involved in protein metabolism, cell growth, as well as cellular responses to stress and organisation of the extracellular matrix. Thus the W8B2+ CSC secretome contains a multitude of bioactive paracrine factors we have now characterised, that might well be harnessed for therapeutic application for cardiac repair and regeneration.

We sought to identify the impacts of Friedreich's ataxia (FRDA) on cardiomyocytes. FRDA is an autosomal recessive degenerative condition with neuronal and non-neuronal manifestations, the latter including progressive cardiomyopathy of the left ventricle, the leading cause of death in FRDA. Little is known about the cellular pathogenesis of FRDA in cardiomyocytes. Induced pluripotent stem cells (iPSCs) were derived from three FRDA individuals with characterized GAA repeats. The cells were differentiated into cardiomyocytes to assess phenotypes. FRDA iPSC- cardiomyocytes retained low levels of FRATAXIN (FXN) mRNA and protein. Electrophysiology revealed an increased variation of FRDA- cardiomyocyte beating rates which was prevented by addition of nifedipine, suggestive of a calcium handling deficiency. Finally, calcium imaging was performed and we identified small amplitude, diastolic and systolic calcium transients confirming a deficiency in calcium handling. We defined a robust FRDA cardiac-specific electrophysiological profile in patient-derived iPSCs which could be used for high throughput compound screening. This cell-specific signature will contribute to the identification and screening of novel treatments for this life-threatening disease.

Cybrid technology was used to replace Leber hereditary optic neuropathy (LHON) causing mitochondrial DNA (mtDNA) mutations from patient-specific fibroblasts with wildtype mtDNA, and mutation-free induced pluripotent stem cells (iPSCs) were generated subsequently. Retinal ganglion cell (RGC) differentiation demonstrates increased cell death in LHON-RGCs and can be rescued in cybrid corrected RGCs.

Human induced pluripotent stem (iPS) cells are a promising source of autologous cardiomyocytes to repair and regenerate myocardium for treatment of heart disease. In this study, we have identified a novel strategy to enhance cardiac differentiation of human iPS cells by treating embryoid bodies (EBs) with a histone deacetylase inhibitor, trichostatin A (TSA), together with activin A and bone morphogenetic protein 4 (BMP4). Over a narrow window of concentrations, TSA (1 ng/ml) directed the differentiation of human iPS cells into a cardiomyocyte lineage. TSA also exerted an additive effect with activin A (100 ng/ml) and BMP4 (20 ng/ml). The resulting cardiomyocytes expressed several cardiac-specific transcription factors and contractile proteins at both gene and protein levels. Functionally, the contractile EBs displayed calcium cycling and were responsive to the chronotropic agents isoprenaline (0.1 μM) and carbachol (1 μM). Implanting microdissected beating areas of iPS cells into tissue engineering chambers in immunocompromised rats produced engineered constructs that supported their survival, and they maintained spontaneous contraction. Human cardiomyocytes were identified as compact patches of muscle tissue incorporated within a host fibrocellular stroma and were vascularized by host neovessels. In conclusion, human iPS cell-derived cardiomyocytes can be used to engineer functional cardiac muscle tissue for studying the pathophysiology of cardiac disease, for drug discovery test beds, and potentially for generation of cardiac grafts to surgically replace damaged myocardium.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Mitochondria are morphologically dynamic organelles constantly undergoing processes of fission and fusion that maintain integrity and bioenergetics of the organelle: these processes are vital for cell survival. Disruption in the balance of mitochondrial fusion and fission is thought to play a role in several pathological conditions including ischemic heart disease. Proteins involved in regulating the processes of mitochondrial fusion and fission are therefore potential targets for pharmacological therapies. Mdivi‐1 is a small molecule inhibitor of the mitochondrial fission protein Drp1. Inhibiting mitochondrial fission with Mdivi‐1 has proven cytoprotective benefits in several cell types involved in a wide array of cardiovascular injury models. On the other hand, Mdivi‐1 can also exert antiproliferative and cytotoxic effects, particularly in hyperproliferative cells. In this review, we discuss these divergent effects of Mdivi‐1 on cell survival, as well as the potential and limitations of Mdivi‐1 as a therapeutic agent.

Background.Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes.Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days.Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression ofACTC1,TNNT2,MYH7, andMYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner.Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used.

Ischemic preconditioning (IPC) is a potent and effective means of protecting cells against ischemic injury. The protection has been demonstrated to involve release of paracrine factors that promote cell survival and angiogenesis, factors important for successful tissue engineering. The aim of the present study was to determine whether IPC of a vascular bed in vivo is an effective strategy to prepare it for tissue engineering with implanted cells. To test this hypothesis, an in vivo vascularized tissue engineering approach was employed, whereby polyacrylic chambers were placed around the femoral vessels of adult Sprague-Dawley rats. IPC was induced by 3 cycles of 5 min femoral artery occlusion interspersed with 5-min periods of reperfusion. Rats subjected to IPC generated bigger tissue constructs at 7 and 28 days postimplantation of empty chambers (∼50% increase in weight and volume, p

To grow more robust cardiac tissue for implantation in vivo, strategies to improve survival of implanted stem cells are required. Here we report the protective effects of hypoxic preconditioning (HPC) and identify mechanisms for improving survival of adipose-derived stem cells (ASC) in vitro. Human ASC were preconditioned for 24 h with hypoxia and then exposed to simulated ischemia for a further 24 h. HPC significantly increased ASC viability, and reduced cell injury and apoptosis compared with non-preconditioned cells under ischemic conditions, as shown by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase-release, and caspase activity assays. Preconditioned ASC increased levels of hypoxia-inducible factor-1 alpha and secreted significantly more of the downstream target vascular endothelial growth factor (VEGF-A; 13-fold) compared with control during the 24 h. Exogenous VEGF (50 ng/mL) increased phosphorylation of Akt without affecting ERK1/2, JNK, or p38 MAPK protein levels. Phospho-Akt was also increased in preconditioned ASC compared with non-preconditioned ASC, an effect that may be mediated via VEGF-A. Importantly, the protective effects of HPC were abolished by a neutralizing antibody against VEGF-A and the phosphoinositol 3-kinase inhibitor LY294002, demonstrating the importance of VEGF-A and Akt in hypoxia-induced ASC survival. Importantly, we showed that media derived from hypoxic preconditioned ASC support endothelial cell survival and endothelial tube formation in vitro. Our in vitro findings indicate that HPC may be a promising strategy to improve survival of ASC and promote angiogenesis in ischemic environments.

Background— We explored the role that inhibitory heterotrimeric G-proteins play in ventricular arrhythmia. Methods and Results— Mice with global genetic deletion of Gα i2 [Gα i2 (−/−)] were studied and found, based on telemetry, to have a prolonged QT interval on surface ECG when awake. In vivo electrophysiology studies revealed that the Gα i2 (−/−) mice have a reduced ventricular effective refractory period and a predisposition to ventricular tachycardia when challenged with programmed electrical stimulation. Neither control nor combined global deletion of Gα i1 and Gα i3 mice showed these abnormalities. There was no evidence for structural heart disease at this time point in the Gα i2 (−/−) mice as assessed by cardiac histology and echocardiography. The absence of Gα i2 thus leads to a primary electrical abnormality, and we explored the basis for this finding. With patch clamping, single isolated ventricular cells showed that Gα i2 (−/−) mice had a prolonged ventricular action potential duration (APD) but steeper action potential shortening as the diastolic interval was reduced in restitution studies. Gene expression studies showed increased expression of L-type Ca 2+ channel subunits, and patch clamping revealed an increase in these currents in Gα i2 (−/−) mice. There were no changes in K + currents. Conclusions— The absence of inhibitory G-protein signaling mediated through Gα i2 is a substrate for ventricular arrhythmias.

Background— Whether alterations in mitochondrial morphology affect the susceptibility of the heart to ischemia/reperfusion injury is unknown. We hypothesized that modulating mitochondrial morphology protects the heart against ischemia/reperfusion injury. Methods and Results— In response to ischemia, mitochondria in HL-1 cells (a cardiac-derived cell line) undergo fragmentation, a process that is dependent on the mitochondrial fission protein dynamin-related protein 1 (Drp1). Transfection of HL-1 cells with the mitochondrial fusion proteins mitofusin 1 or 2 or with Drp1 K38A , a dominant-negative mutant form of Drp1, increased the percentage of cells containing elongated mitochondria (65±4%, 69±5%, and 63±6%, respectively, versus 46±6% in control: n=80 cells per group; P P P P =0.023). Finally, treatment of adult murine cardiomyocytes with mitochondrial division inhibitor-1 reduced cell death and inhibited mitochondrial permeability transition pore opening after simulated ischemia/reperfusion injury, and in vivo treatment with mitochondrial division inhibitor-1 reduced myocardial infarct size in mice subject to coronary artery occlusion and reperfusion (21.0±2.2% with mitochondrial division inhibitor-1 versus 48.0±4.5% in control; n=6 animals per group; P Conclusion— Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury, suggesting a novel pharmacological strategy for cardioprotection.

Aims It has been suggested that mitochondrial reactive oxygen species (ROS), Akt and Erk1/2 and more recently the mitochondrial permeability transition pore (mPTP) may act as mediators of ischaemic preconditioning (IPC), although the actual interplay between these mediators is unclear. The aim of the present study is to determine whether the cyclophilin-D (CYPD) component of the mPTP is required by IPC to generate mitochondrial ROS and subsequently activate Akt and Erk1/2. Methods and results Mice lacking CYPD (CYPD−/−) and B6Sv129 wild-type (WT) mice were used throughout. We have demonstrated that under basal conditions, non-pathological mPTP opening occurs (indicated by the percent reduction in mitochondrial calcein fluorescence). This effect was greater in WT cardiomyocytes compared with CYPD−/− ones (53 ± 2% WT vs. 17 ± 3% CYPD−/−; P < 0.01) and was augmented by hypoxic preconditioning (HPC) (70 ± 9% WT vs. 56 ± 1% CYPD−/−; P < 0.01). HPC reduced cell death following simulated ischaemia–reperfusion injury in WT (23.2 ± 3.5% HPC vs. 43.7 ± 3.2% WT; P < 0.05) but not CYPD−/− cardiomyocytes (19.6 ± 1.4% HPC vs. 24.4 ± 2.6% control; P > 0.05). HPC generated mitochondrial ROS in WT (four-fold increase; P < 0.05) but not CYPD−/− cardiomyocytes. HPC induced significant Akt phosphorylation in WT cardiomyocytes (two-fold increase; P < 0.05), an effect which was abrogated by ciclosporin-A (a CYPD inhibitor) and N-2-mercaptopropionyl glycine (a ROS scavenger). Finally, in vivo IPC of adult murine hearts resulted in significant phosphorylation of Akt and Erk1/2 in WT but not CYPD−/− hearts. Conclusion The CYPD component of the mPTP is required by IPC to generate mitochondrial ROS and phosphorylate Akt and Erk1/2, major steps in the IPC signalling pathway.