Spatial Gene Expression Changes in the Mouse Heart After Base-Targeted Irradiation.

PURPOSE: Radiation cardiotoxicity (RC) is a clinically significant adverse effect of treatment for patients with thoracic malignancies. Clinical studies in lung cancer have indicated that heart substructures are not uniformly radiosensitive, and that dose to the heart base drives RC. In this study, we aimed to characterize late changes in gene expression using spatial transcriptomics in a mouse model of base regional radiosensitivity. METHODS AND MATERIALS: An aged female C57BL/6 mouse was irradiated with 16 Gy delivered to the cranial third of the heart using a 6 × 9 mm parallel opposed beam geometry on a small animal radiation research platform, and a second mouse was sham-irradiated. After echocardiography, whole hearts were collected at 30 weeks for spatial transcriptomic analysis to map gene expression changes occurring in different regions of the partially irradiated heart. Cardiac regions were manually annotated on the capture slides and the gene expression profiles compared across different regions. RESULTS: Ejection fraction was reduced at 30 weeks after a 16 Gy irradiation to the heart base, compared with the sham-irradiated controls. There were markedly more significant gene expression changes within the irradiated regions compared with nonirradiated regions. Variation was observed in the transcriptomic effects of radiation on different cardiac base structures (eg, between the right atrium [n = 86 dysregulated genes], left atrium [n = 96 dysregulated genes], and the vasculature [n = 129 dysregulated genes]). Disrupted biological processes spanned extracellular matrix as well as circulatory, neuronal, and contractility activities. CONCLUSIONS: This is the first study to report spatially resolved gene expression changes in irradiated tissues. Examination of the regional radiation response in the heart can help to further our understanding of the cardiac base's radiosensitivity and support the development of actionable targets for pharmacologic intervention and biologically relevant dose constraints.

PLAC8-Mediated Activation of NOX4 Signalling Restores Angiogenic Function of Endothelial Colony-Forming Cells in Experimental Hypoxia.

Ischaemic cardiovascular disease is associated with tissue hypoxia as a significant determinant of angiogenic dysfunction and adverse remodelling. While cord blood-derived endothelial colony-forming cells (CB-ECFCs) hold clear therapeutic potential due to their enhanced angiogenic and proliferative capacity, their impaired functionality within the disease microenvironment represents a major barrier to clinical translation. The aim of this study was to define the specific contribution of NOX4 NADPH oxidase, which we previously reported as a key CB-ECFC regulator, to hypoxia-induced dysfunction and its potential as a therapeutic target. CB-ECFCs exposed to experimental hypoxia demonstrated downregulation of NOX4-mediated reactive oxygen species (ROS) signalling linked with a reduced tube formation, which was partially restored by NOX4 plasmid overexpression. siRNA knockdown of placenta-specific 8 (PLAC8), identified by microarray analysis as an upstream regulator of NOX4 in hypoxic versus normoxic CB-ECFCs, enhanced tube formation, NOX4 expression and hydrogen peroxide generation, and induced several key transcription factors associated with downstream Nrf2 signalling. Taken together, these findings indicated that activation of the PLAC8-NOX4 signalling axis improved CB-ECFC angiogenic functions in experimental hypoxia, highlighting this pathway as a potential target for protecting therapeutic cells against the ischaemic cardiovascular disease microenvironment.

Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation.

Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca2+, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with ß-alanine buildup. Raising ß-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.

Cardiac sub-volume targeting demonstrates regional radiosensitivity in the mouse heart.

BACKGROUND AND PURPOSE: Radiation-induced cardiac toxicity (RICT) remains one of the most critical dose limiting constraints in radiotherapy. Recent studies have shown higher doses to the base of the heart are associated with worse overall survival in lung cancer patients receiving radiotherapy. This work aimed to investigate the impact of sub-volume heart irradiation in a mouse model using small animal image-guided radiotherapy. MATERIALS AND METHODS: C57BL/6 mice were irradiated with a single fraction of 16 Gy to the base, middle or apex of the heart using a small animal radiotherapy research platform. Cone beam CT and echocardiography were performed at baseline and at 10 week intervals until 50 weeks post-treatment. Structural and functional parameters were correlated with mean heart dose (MHD) and volume of heart receiving 5 Gy (V5). RESULTS: All irradiated mice showed a time dependent increase in left ventricle wall thickness in diastole of ~0.2 mm detected at 10 weeks post-treatment, with the most significant and persistent changes occurring in the heart base-irradiated animals. Similarly, statistically different functional effects (p < 0.01) were observed in base-irradiated animals which showed the most significant decreases compared to controls. The observed functional changes did not correlate with MHD and V5 (R2 < 0.1), indicating that whole heart dosimetry parameters do not predict physiological changes resulting from cardiac sub-volume irradiation. CONCLUSIONS: This is the first report demonstrating the structural and functional consequences of sub-volume targeting in the mouse heart and reverse translates clinical observations indicating the heart base as a critical radiosensitive region.

NOX4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes post-ischaemic revascularization.

AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function. METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4, which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery, which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs up-regulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair. CONCLUSION: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective post-ischaemic revascularization.

Reactive oxygen species signalling in the diabetic heart: emerging prospect for therapeutic targeting.

Despite being first described 45 years ago, the existence of a distinct diabetic cardiomyopathy remains controversial. Nonetheless, it is widely accepted that the diabetic heart undergoes characteristic structural and functional changes in the absence of ischaemia and hypertension, which are independently linked to heart failure progression and are likely to underlie enhanced susceptibility to stress. A prominent feature is marked collagen accumulation linked with inflammation and extensive extracellular matrix changes, which appears to be the main factor underlying cardiac stiffness and subclinical diastolic dysfunction, estimated to occur in as many as 75% of optimally controlled diabetics. Whether this characteristic remodelling phenotype is primarily driven by microvascular dysfunction or alterations in cardiomyocyte metabolism remains unclear. Although hyperglycaemia regulates multiple pathways in the diabetic heart, increased reactive oxygen species (ROS) generation is thought to represent a central mechanism underlying associated adverse remodelling. Indeed, experimental and clinical diabetes are linked with oxidative stress which plays a key role in cardiomyopathy, while key processes underlying diabetic cardiac remodelling, such as inflammation, angiogenesis, cardiomyocyte hypertrophy and apoptosis, fibrosis and contractile dysfunction, are redox sensitive. This review will explore the relative contributions of the major ROS sources (dysfunctional nitric oxide synthase, mitochondria, xanthine oxidase, nicotinamide adenine dinucleotide phosphate oxidases) in the diabetic heart and the potential for therapeutic targeting of ROS signalling using novel pharmacological and non-pharmacological approaches to modify specific aspects of the remodelling phenotype in order to prevent and/or delay heart failure development and progression.