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. 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. 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. Administration of cardiac targeted rAAV-OGT to the non-diabetic myocardium induced cardiac fibrosis, increased hypertrophic gene expression and reduced cardiac function, mimicking the diabetic heart. In contrast, administration of rAAV-OGA to the diabetic heart limits cardiac remodelling and dysfunction associated with diabetic cardiomyopathy. O-GlcNAcylation of the PI3K-Akt axis has been attributed to the cardiac pathological effects, while the opposite (de-GlcNAcylation) associated with cardioprotective.

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.

Metabolism is central to embryonic stem cell (ESC) pluripotency and differentiation, with distinct profiles apparent under different nutrient milieu, and conditions that maintain alternate cell states. The significance of altered nutrient availability, particularly oxygen, and metabolic pathway activity has been highlighted by extensive studies of their impact on preimplantation embryo development, physiology, and viability. ESC similarly modulate their metabolism in response to altered metabolite levels, with changes in nutrient availability shown to have a lasting impact on derived cell identity through the regulation of the epigenetic landscape. Further, the preferential use of glucose and anaplerotic glutamine metabolism serves to not only support cell growth and proliferation but also minimise reactive oxygen species production. However, the perinuclear localisation of spherical, electron-poor mitochondria in ESC is proposed to sustain ESC nuclear-mitochondrial crosstalk and a mitochondrial-H₂O₂presence, to facilitate signalling to support self-renewal through the stabilisation of HIFα, a process that may be favoured under physiological oxygen. The environment in which a cell is grown is therefore a critical regulator and determinant of cell fate, with metabolism, and particularly mitochondria, acting as an interface between the environment and the epigenome.

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. 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. 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.

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.

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. 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). 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.