Does timing matter in radiotherapy of hepatocellular carcinoma? An experimental study in mice.

This study investigates whether a chronotherapeutic treatment of hepatocellular carcinoma (HCC) may improve treatment efficacy and mitigate side effects on non-tumoral liver (NTL). HCC was induced in Per2::luc mice which were irradiated at four time points of the day. Proliferation and DNA-double strand breaks were analyzed in irradiated and nonirradiated animals by detection of Ki67 and γ-H2AX. Prior to whole animal experiments, organotypic slice cultures were investigated to determine the dosage to be used in whole animal experiments. Irradiation was most effective at the proliferation peaks in HCC at ZT02 (early inactivity phase) and ZT20 (late activity phase). Irradiation effects on NTL were minimal at ZT20. As compared with NTL, nonirradiated HCC revealed disruption in daily variation and downregulation of all investigated clock genes except Per1. Irradiation affected rhythmic clock gene expression in NTL and HCC at all ZTs except at ZT20 (late activity phase). Irradiation at ZT20 had no effect on total leukocyte numbers. Our results indicate ZT20 as the optimal time point for irradiation of HCC in mice at which the ratio between efficacy of tumor treatment and toxic side effects was maximal. Translational studies are now needed to evaluate whether the late activity phase is the optimal time point for irradiation of HCC in man.

Iron-regulatory proteins secure iron availability in cardiomyocytes to prevent heart failure.

Aims: Iron deficiency (ID) is associated with adverse outcomes in heart failure (HF) but the underlying mechanisms are incompletely understood. Intracellular iron availability is secured by two mRNA-binding iron-regulatory proteins (IRPs), IRP1 and IRP2. We generated mice with a cardiomyocyte-targeted deletion of Irp1 and Irp2 to explore the functional implications of ID in the heart independent of systemic ID and anaemia. Methods and results: Iron content in cardiomyocytes was reduced in Irp-targeted mice. The animals were not anaemic and did not show a phenotype under baseline conditions. Irp-targeted mice, however, were unable to increase left ventricular (LV) systolic function in response to an acute dobutamine challenge. After myocardial infarction, Irp-targeted mice developed more severe LV dysfunction with increased HF mortality. Mechanistically, the activity of the iron-sulphur cluster-containing complex I of the mitochondrial electron transport chain was reduced in left ventricles from Irp-targeted mice. As demonstrated by extracellular flux analysis in vitro, mitochondrial respiration was preserved at baseline but failed to increase in response to dobutamine in Irp-targeted cardiomyocytes. As shown by 31P-magnetic resonance spectroscopy in vivo, LV phosphocreatine/ATP ratio declined during dobutamine stress in Irp-targeted mice but remained stable in control mice. Intravenous injection of ferric carboxymaltose replenished cardiac iron stores, restored mitochondrial respiratory capacity and inotropic reserve, and attenuated adverse remodelling after myocardial infarction in Irp-targeted mice but not in control mice. As shown by electrophoretic mobility shift assays, IRP activity was significantly reduced in LV tissue samples from patients with advanced HF and reduced LV tissue iron content. Conclusions: ID in cardiomyocytes impairs mitochondrial respiration and adaptation to acute and chronic increases in workload. Iron supplementation restores cardiac energy reserve and function in iron-deficient hearts.

Impact of dietary nitrate on age-related diastolic dysfunction.

AIMS: Diastolic dysfunction is highly prevalent, and ageing is the main contributor due to impairments in active cardiac relaxation, ventriculo-vascular stiffening, and endothelial dysfunction. Nitric oxide (NO) affects cardiovascular functions, and NO bioavailability is critically reduced with ageing. Whether replenishment of NO deficiency with dietary inorganic nitrate would offer a novel approach to reverse age-related cardiovascular alterations was not known. METHODS AND RESULTS: A dietary nitrate supplementation was applied to young (6 month) and old (20 month) wild-type mice for 8 weeks and compared with controls. High-resolution ultrasound, pressure-volume catheter techniques, and isolated heart measurements were applied to assess cardiac diastolic and vascular functions. Cardiac manganese-enhanced magnetic resonance imaging was performed to study the effects of dietary nitrate on myocyte calcium handling. In aged mice with preserved systolic function, dietary nitrate supplementation improved LV diastolic function, arterial compliance, and coronary flow reserve. Mechanistically, improved cardiovascular functions were associated with an accelerated cardiomyocyte calcium handling and augmented NO/cyclic guanosine monophosphate/protein kinase G signalling, while enhanced nitrate reduction was related to age-related differences in the oral microbiome. CONCLUSION: Dietary inorganic nitrate reverses age-related LV diastolic dysfunction and improves vascular functions. Our results highlight the potential of a dietary approach in the therapy of age-related cardiovascular alterations.

Sexual dimorphism of lipid metabolism in very long-chain acyl-CoA dehydrogenase deficient (VLCAD-/-) mice in response to medium-chain triglycerides (MCT).

Medium-chain triglycerides (MCT) are widely applied in the treatment of long-chain fatty acid oxidation disorders. Previously it was shown that long-term MCT supplementation strongly affects lipid metabolism in mice. We here investigate sex-specific effects in mice with very-long-chain-acyl-CoA dehydrogenase (VLCAD) deficiency in response to a long-term MCT modified diet. We quantified blood lipids, acylcarnitines, glucose, insulin and free fatty acids, as well as tissue triglycerides in the liver and skeletal muscle under a control and an MCT diet over 1 year. In addition, visceral and hepatic fat content and muscular intramyocellular lipids (IMCL) were assessed by in vivo(1)H magnetic resonance spectroscopy (MRS) techniques. The long-term application of an MCT diet induced a marked alteration of glucose homeostasis. However, only VLCAD-/- female mice developed a severe metabolic syndrome characterized by marked insulin resistance, dyslipidemia, severe hepatic and visceral steatosis, whereas VLCAD-/- males seemed to be protected and only presented with milder insulin resistance. Moreover, the highly saturated MCT diet is associated with a decreased hepatic stearoyl-CoA desaturase 1 (SCD1) activity in females aggravating the harmful effects of a saturated MCT diet. Long-term MCT supplementation deeply affects lipid metabolism in a sexual dimorphic manner resulting in a severe metabolic syndrome only in female mice. These findings are striking since the first signs of insulin resistance already occur in female VLCAD-/- mice during their reproductive period. How these metabolic adaptations are finally regulated needs to be determined. More important, the relevance of these findings for humans under these dietary modifications needs to be investigated.

Development and pathomechanisms of cardiomyopathy in very long-chain acyl-CoA dehydrogenase deficient (VLCAD(-/-)) mice.

Hypertrophic cardiomyopathy is a typical manifestation of very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), the most common long-chain ß-oxidation defects in humans; however in some patients cardiac function is fully compensated. Cardiomyopathy may also be reversed by supplementation of medium-chain triglycerides (MCT). We here characterize cardiac function of VLCAD-deficient (VLCAD(-/-)) mice over one year. Furthermore, we investigate the long-term effect of a continuous MCT diet on the cardiac phenotype. We assessed cardiac morphology and function in VLCAD(-/-) mice by in vivo MRI. Cardiac energetics were measured by (31)P-MRS and myocardial glucose uptake was quantified by positron-emission-tomography (PET). Metabolic adaptations were identified by the expression of genes regulating glucose and lipid metabolism using real-time-PCR. VLCAD(-/-) mice showed a progressive decrease in heart function over 12 months accompanied by a reduced phosphocreatine-to-ATP-ratio indicative of chronic energy deficiency. Long-term MCT supplementation aggravated the cardiac phenotype into dilated cardiomyopathy with features similar to diabetic heart disease. Cardiac energy production and function in mice with a ß-oxidation defect cannot be maintained with age. Compensatory mechanisms are insufficient to preserve the cardiac energy state over time. However, energy deficiency by impaired ß-oxidation and long-term MCT induce cardiomyopathy by different mechanisms. Cardiac MRI and MRS may be excellent tools to assess minor changes in cardiac function and energetics in patients with ß-oxidation defects for preventive therapy.

Disrupted fat distribution and composition due to medium-chain triglycerides in mice with a ß-oxidation defect.

BACKGROUND: Because of the enhanced recognition of inherited long-chain fatty acid oxidation disorders by worldwide newborn screening programs, an increasing number of asymptomatic patients receive medium-chain triglyceride (MCT) supplements to prevent the development of cardiomyopathy and myopathy. OBJECTIVE: MCT supplementation has been recognized as a safe dietary intervention, but long-term observations into later adulthood are still not available. We investigated the consequences of a prolonged MCT diet on abdominal fat distribution and composition and on liver fat. DESIGN: Mice with very-long-chain acyl-coenzyme A dehydrogenase deficiency (VLCAD(-/-)) were supplemented for 1 y with a diet in which MCTs replaced long-chain triglycerides without increasing the total fat content. The dietary effects on abdominal fat accumulation and composition were analyzed by in vivo (1)H- and (13)C-magnetic resonance spectroscopy (9.4 Tesla). RESULTS: After 1 y of MCT supplementation, VLCAD(-/-) mice accumulated massive visceral fat and had a dramatic increase in the concentration of serum free fatty acids. Furthermore, we observed a profound shift in body triglyceride composition, ie, concentrations of physiologically important polyunsaturated fatty acids dramatically decreased. (1)H-Magnetic resonance spectroscopy analysis and histologic evaluation of the liver also showed pronounced fat accumulation and marked oxidative stress. CONCLUSION: Although the MCT-supplemented diet has been reported to prevent the development of cardiomyopathy and skeletal myopathy in fatty acid oxidation disorders, our data show that long-term MCT supplementation results in a severe clinical phenotype similar to that of nonalcoholic steatohepatitis and the metabolic syndrome.