Development of a CRISPRi Human Retinal Pigmented Epithelium Model for Functional Study of Age-Related Macular Degeneration Genes.

Age-related macular degeneration (AMD) is a blinding disease characterised by dysfunction of the retinal pigmented epithelium (RPE) which culminates in disruption or loss of the neurosensory retina. Genome-wide association studies have identified >60 genetic risk factors for AMD; however, the expression profile and functional role of many of these genes remain elusive in human RPE. To facilitate functional studies of AMD-associated genes, we developed a human RPE model with integrated CRISPR interference (CRISPRi) for gene repression by generating a stable ARPE19 cell line expressing dCas9-KRAB. We performed transcriptomic analysis of the human retina to prioritise AMD-associated genes and selected TMEM97 as a candidate gene for knockdown study. Using specific sgRNAs, we showed that knockdown of TMEM97 in ARPE19 reduced reactive oxygen species (ROS) levels and exerted a protective effect against oxidative stress-induced cell death. This work provides the first functional study of TMEM97 in RPE and supports a potential role of TMEM97 in AMD pathobiology. Our study highlights the potential for using CRISPRi to study AMD genetics, and the CRISPRi RPE platform generated here provided a useful in vitro tool for functional studies of AMD-associated genes.

Mitochondrial Dynamin-Related Protein Drp1: a New Player in Cardio-oncology.

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. 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.

Scalable Generation of Nanovesicles from Human-Induced Pluripotent Stem Cells for Cardiac Repair.

Extracellular vesicles (EVs) from stem cells have shown significant therapeutic potential to repair injured cardiac tissues and regulate pathological fibrosis. However, scalable generation of stem cells and derived EVs for clinical utility remains a huge technical challenge. Here, we report a rapid size-based extrusion strategy to generate EV-like membranous nanovesicles (NVs) from easily sourced human iPSCs in large quantities (yield 900× natural EVs). NVs isolated using density-gradient separation (buoyant density 1.13 g/mL) are spherical in shape and morphologically intact and readily internalised by human cardiomyocytes, primary cardiac fibroblasts, and endothelial cells. NVs captured the dynamic proteome of parental cells and include pluripotency markers (LIN28A, OCT4) and regulators of cardiac repair processes, including tissue repair (GJA1, HSP20/27/70, HMGB1), wound healing (FLNA, MYH9, ACTC1, ILK), stress response/translation initiation (eIF2S1/S2/S3/B4), hypoxia response (HMOX2, HSP90, GNB1), and extracellular matrix organization (ITGA6, MFGE8, ITGB1). Functionally, NVs significantly promoted tubule formation of endothelial cells (angiogenesis) (p < 0.05) and survival of cardiomyocytes exposed to low oxygen conditions (hypoxia) (p < 0.0001), as well as attenuated TGF-ß mediated activation of cardiac fibroblasts (p < 0.0001). Quantitative proteome profiling of target cell proteome following NV treatments revealed upregulation of angiogenic proteins (MFGE8, MYH10, VDAC2) in endothelial cells and pro-survival proteins (CNN2, THBS1, IGF2R) in cardiomyocytes. In contrast, NVs attenuated TGF-ß-driven extracellular matrix remodelling capacity in cardiac fibroblasts (ACTN1, COL1A1/2/4A2/12A1, ITGA1/11, THBS1). This study presents a scalable approach to generating functional NVs for cardiac repair.

Amorphous SiO2 nanoparticles promote cardiac dysfunction via the opening of the mitochondrial permeability transition pore in rat heart and human cardiomyocytes.

BACKGROUND: Silica nanoparticles (nanoSiO2) are promising systems that can deliver biologically active compounds to tissues such as the heart in a controllable manner. However, cardiac toxicity induced by nanoSiO2 has been recently related to abnormal calcium handling and energetic failure in cardiomyocytes. Moreover, the precise mechanisms underlying this energetic debacle remain unclear. In order to elucidate these mechanisms, this article explores the ex vivo heart function and mitochondria after exposure to nanoSiO2. RESULTS: The cumulative administration of nanoSiO2 reduced the mechanical performance index of the rat heart with a half-maximal inhibitory concentration (IC50) of 93 µg/mL, affecting the relaxation rate. In isolated mitochondria nanoSiO2 was found to be internalized, inhibiting oxidative phosphorylation and significantly reducing the mitochondrial membrane potential (ΔΨm). The mitochondrial permeability transition pore (mPTP) was also induced with an increasing dose of nanoSiO2 and partially recovered with, a potent blocker of the mPTP, Cyclosporine A (CsA). The activity of aconitase and thiol oxidation, in the adenine nucleotide translocase, were found to be reduced due to nanoSiO2 exposure, suggesting that nanoSiO2 induces the mPTP via thiol modification and ROS generation. In cardiac cells exposed to nanoSiO2, enhanced viability and reduction of H2O2 were observed after application of a specific mitochondrial antioxidant, MitoTEMPO. Concomitantly, CsA treatment in adult rat cardiac cells reduced the nanoSiO2-triggered cell death and recovered ATP production (from 32.4 to 65.4%). Additionally, we performed evaluation of the mitochondrial effect of nanoSiO2 in human cardiomyocytes. We observed a 40% inhibition of maximal oxygen consumption rate in mitochondria at 500 µg/mL. Under this condition we identified a remarkable diminution in the spare respiratory capacity. This data indicates that a reduction in the amount of extra ATP that can be produced by mitochondria during a sudden increase in energy demand. In human cardiomyocytes, increased LDH release and necrosis were found at increased doses of nanoSiO2, reaching 85 and 48%, respectively. Such deleterious effects were partially prevented by the application of CsA. Therefore, exposure to nanoSiO2 affects cardiac function via mitochondrial dysfunction through the opening of the mPTP. CONCLUSION: The aforementioned effects can be partially avoided reducing ROS or retarding the opening of the mPTP. These novel strategies which resulted in cardioprotection could be considered as potential therapies to decrease the side effects of nanoSiO2 exposure.

Cardiomyocyte functional screening: interrogating comparative electrophysiology of high-throughput model cell systems.

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 ß-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.

Generation of a nitric oxide signaling pathway in mesenchymal stem cells promotes endothelial lineage commitment.

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 ß-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.

Role of lysophosphatidic acid in the retinal pigment epithelium and photoreceptors.

The human retina is a complex structure of organised layers of specialised cells that support the transmission of light signals to the visual cortex. The outermost layer of the retina, the retinal pigment epithelium (RPE), forms part of the blood retina barrier and is implicated in many retinal diseases. Lysophosphatidic acid (LPA) is a bioactive lipid exerting pleiotropic effects in various cell types, during development, normal physiology and disease. Its producing enzyme, AUTOTAXIN (ATX), is highly expressed by the pigmented epithelia of the human eye, including the RPE. Using human pluripotent stem cell (hPSC)-derived retinal cells, we interrogated the role of LPA in the human RPE and photoreceptors. hPSC-derived RPE cells express and synthesize functional ATX, which is predominantly secreted apically of the RPE, suggesting it acts in a paracrine manner to regulate photoreceptor function. In RPE cells, LPA regulates tight junctions, in a receptor-dependent mechanism, with an increase in OCCLUDIN and ZONULA OCCLUDENS (ZO)-1 expression at the cell membrane, accompanied by an increase in the transepithelial resistance of the epithelium. High concentration of LPA decreases phagocytosis of photoreceptor outer segments by the RPE. In hPSC-derived photoreceptors, LPA induces morphological rearrangements by modulating the actin myosin cytoskeleton, as evidenced by Myosin Light Chain l membrane relocation. Collectively, our data suggests an important role of LPA in the integrity and functionality of the healthy retina and blood retina barrier.

Cardiac Repair With a Novel Population of Mesenchymal Stem Cells Resident in the Human Heart.

Cardiac resident stem cells (CRSCs) hold much promise to treat heart disease but this remains a controversial field. Here, we describe a novel population of CRSCs, which are positive for W8B2 antigen and were obtained from adult human atrial appendages. W8B2(+) CRSCs exhibit a spindle-shaped morphology, are clonogenic and capable of self-renewal. W8B2(+) CRSCs show high expression of mesenchymal but not hematopoietic nor endothelial markers. W8B2(+) CRSCs expressed GATA4, HAND2, and TBX5, but not C-KIT, SCA-1, NKX2.5, PDGFRα, ISL1, or WT1. W8B2(+) CRSCs can differentiate into cardiovascular lineages and secrete a range of cytokines implicated in angiogenesis, chemotaxis, inflammation, extracellular matrix remodeling, cell growth, and survival. In vitro, conditioned medium collected from W8B2(+) CRSCs displayed prosurvival, proangiogenic, and promigratory effects on endothelial cells, superior to that of other adult stem cells tested, and additionally promoted survival and proliferation of neonatal rat cardiomyocytes. Intramyocardial transplantation of human W8B2(+) CRSCs into immunocompromised rats 1 week after myocardial infarction markedly improved cardiac function (∼40% improvement in ejection fraction) and reduced fibrotic scar tissue 4 weeks after infarction. Hearts treated with W8B2(+) CRSCs showed less adverse remodeling of the left ventricle, a greater number of proliferating cardiomyocytes (Ki67(+) cTnT(+) cells) in the remote region, higher myocardial vascular density, and greater infiltration of CD163(+) cells (a marker for M2 macrophages) into the border zone and scar regions. In summary, W8B2(+) CRSCs are distinct from currently known CRSCs found in human hearts, and as such may be an ideal cell source to repair myocardial damage after infarction.

Growing vascularized heart tissue from stem cells.

The promise of stem cells to repair the heart after damage or heart attack has not been realized because most such cells are lost after transplantation. A new approach is to grow substantial viable pieces of cardiac tissue from human stem cells by cardiac tissue engineering. Such constructs must be fully vascularized and perfused to ensure the viability of clinically relevant volumes of tissue. This requires careful choice of cells, culture conditions, a biomaterial to act as scaffold, and crucial strategies for vascularization. Autologous stem cells with high plasticity, which would avoid the need for antirejection therapies after transplantation, are an attractive source of both cardiomyocytes and vascular cells. Most stem cells also have inherent paracrine activity, releasing cytoprotective factors and growth-promoting cytokines that can further stimulate tissue regeneration and neovascularization through recruitment of endogenous stem and progenitor cells. Current advances for growing vascularized and functional cardiac constructs with human stem cells are described, bringing us a step closer to the engineering of complex cardiac tissues such as pacemaker, conducting tissue, or contractile myocardial flaps ideal for transplantation. From studies in rats successful transplantation of thin constructs to the ventricle has been reported, but there remain further issues to resolve before larger human constructs will be available to test in the clinic.

Cellular interplay between cardiomyocytes and non-myocytes in diabetic cardiomyopathy.

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.

Cardiomyocyte intercellular signalling increases oxidative stress and reprograms the global- and phospho-proteome of cardiac fibroblasts.

Pathological reprogramming of cardiomyocyte and fibroblast proteome landscapes drive the initiation and progression of cardiac fibrosis. Although the secretome of dysfunctional cardiomyocytes is emerging as an important driver of pathological fibroblast reprogramming, our understanding of the downstream molecular players remains limited. Here, we show that cardiac fibroblast activation (αSMA+) and oxidative stress mediated by the secretome of TGFß-stimulated cardiomyocytes is associated with a profound reprogramming of their proteome and phosphoproteome landscape. Within the fibroblast global proteome there was a striking dysregulation of proteins implicated in extracellular matrix, protein localisation/metabolism, KEAP1-NFE2L2 pathway, lysosomes, carbohydrate metabolism, and transcriptional regulation. Kinase substrate enrichment analysis of phosphopeptides revealed potential role of kinases (CK2, CDK2, PKC, GSK3B) during this remodelling. We verified upregulated activity of casein kinase 2 (CK2) in secretome-treated fibroblasts, and pharmacological CK2 inhibitor TBB (4,5,6,7-Tetrabromobenzotriazole) significantly abrogated fibroblast activation and oxidative stress. Our data provides molecular insights into cardiomyocyte to cardiac fibroblast crosstalk, and the potential role of CK2 in regulating cardiac fibroblast activation and oxidative stress.

Knockout of AMD-associated gene POLDIP2 reduces mitochondrial superoxide in human retinal pigment epithelial cells.

Genetic and epidemiologic studies have significantly advanced our understanding of the genetic factors contributing to age-related macular degeneration (AMD). In particular, recent expression quantitative trait loci (eQTL) studies have highlighted POLDIP2 as a significant gene that confers risk of developing AMD. However, the role of POLDIP2 in retinal cells such as retinal pigment epithelium (RPE) and how it contributes to AMD pathology are unknown. Here we report the generation of a stable human RPE cell line ARPE-19 with POLDIP2 knockout using CRISPR/Cas, providing an in vitro model to investigate the functions of POLDIP2. We conducted functional studies on the POLDIP2 knockout cell line and showed that it retained normal levels of cell proliferation, cell viability, phagocytosis and autophagy. Also, we performed RNA sequencing to profile the transcriptome of POLDIP2 knockout cells. Our results highlighted significant changes in genes involved in immune response, complement activation, oxidative damage and vascular development. We showed that loss of POLDIP2 caused a reduction in mitochondrial superoxide levels, which is consistent with the upregulation of the mitochondrial superoxide dismutase SOD2. In conclusion, this study demonstrates a novel link between POLDIP2 and SOD2 in ARPE-19, which supports a potential role of POLDIP2 in regulating oxidative stress in AMD pathology.

Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro.

Long-term culture of primary neonatal rat cardiomyocytes is limited by the loss of spontaneous contractile phenotype within weeks in culture. This may be due to loss of contractile cardiomyocytes from the culture or overgrowth of the non-cardiomyocyte population. Using the mitochondria specific fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), we showed that neonatal rat cardiomyocytes enriched by fluorescence-activated cell sorting can be maintained as contractile cultures for long periods (24-wk culture vs. 2 wk for unsorted cardiomyocytes). Long-term culture of this purified cardiomyocyte (TMRM high) population retained the expression of cardiomyocyte markers, continued calcium cycling, and displayed cyclic electrical activity that could be regulated pharmacologically. These findings suggest that non-cardiomyocyte populations can negatively influence contractility of cardiomyocytes in culture and that by purifying cardiomyocytes, the cultures retain potential as an experimental model for longitudinal studies of cardiomyocyte biology in vitro.

In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation.

Pluripotent stem cells are a potential source of autologous cells for cell and tissue regenerative therapies. They have the ability to renew indefinitely while retaining the capacity to differentiate into all cell types in the body. With developments in cell therapy and tissue engineering these cells may provide an option for treating tissue loss in organs which do not repair themselves. Limitations to clinical translation of pluripotent stem cells include poor cell survival and low cell engraftment in vivo and the risk of teratoma formation when the cells do survive through implantation. In this study, implantation of human induced-pluripotent stem (hiPS) cells, suspended in Matrigel, into an in vivo vascularized tissue engineering chamber in nude rats resulted in substantial engraftment of the cells into the highly vascularized rat tissues formed within the chamber. Differentiation of cells in the chamber environment was shown by teratoma formation, with all three germ lineages evident within 4 weeks. The rate of teratoma formation was higher with partially differentiated hiPS cells (as embryoid bodies) compared to undifferentiated hiPS cells (100% versus 60%). In conclusion, the in vivo vascularized tissue engineering chamber supports the survival through implantation of human iPS cells and their differentiated progeny, as well as a novel platform for rapid teratoma assay screening for pluripotency.

Cellular pathophysiology of Friedreich's ataxia cardiomyopathy.

Friedreich's ataxia (FRDA) is a hereditary neuromuscular disorder. Cardiomyopathy is the leading cause of premature death in FRDA. FRDA cardiomyopathy is a complex and progressive disease with no cure or treatment to slow its progression. At the cellular level, cardiomyocyte hypertrophy, apoptosis and fibrosis contribute to the cardiac pathology. However, the heart is composed of multiple cell types and several clinical studies have reported the involvement of cardiac non-myocytes such as vascular cells, autonomic neurons, and inflammatory cells in the pathogenesis of FRDA cardiomyopathy. In fact, several of the cardiac pathologies associated with FRDA including cardiomyocyte necrosis, fibrosis, and arrhythmia, could be contributed to by a diseased vasculature and autonomic dysfunction. Here, we review available evidence regarding the current understanding of cellular mechanisms for, and the involvement of, cardiac non-myocytes in the pathogenesis of FRDA cardiomyopathy.

Engineering transplantable human lymphatic and blood capillary networks in a porous scaffold.

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.

A soft and ultrasensitive force sensing diaphragm for probing cardiac organoids instantaneously and wirelessly.

Time-lapse mechanical properties of stem cell derived cardiac organoids are important biological cues for understanding contraction dynamics of human heart tissues, cardiovascular functions and diseases. However, it remains difficult to directly, instantaneously and accurately characterize such mechanical properties in real-time and in situ because cardiac organoids are topologically complex, three-dimensional soft tissues suspended in biological media, which creates a mismatch in mechanics and topology with state-of-the-art force sensors that are typically rigid, planar and bulky. Here, we present a soft resistive force-sensing diaphragm based on ultrasensitive resistive nanocracked platinum film, which can be integrated into an all-soft culture well via an oxygen plasma-enabled bonding process. We show that a reliable organoid-diaphragm contact can be established by an 'Atomic Force Microscope-like' engaging process. This allows for instantaneous detection of the organoids' minute contractile forces and beating patterns during electrical stimulation, resuscitation, drug dosing, tissue culture, and disease modelling.

A novel small molecule inhibitor of human Drp1.

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.

Fine-tuning the cardiac O-GlcNAcylation regulatory enzymes governs the functional and structural phenotype of the diabetic heart.

AIMS: The glucose-driven enzymatic modification of myocardial proteins by the sugar moiety, ß-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.

Hydralazine protects the heart against acute ischaemia/reperfusion injury by inhibiting Drp1-mediated mitochondrial fission.

AIMS: Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) 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 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<0.001). CONCLUSION: We show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.