Uremic cardiomyopathy is characterised by loss of the cardioprotective effects of insulin

Chronic kidney disease is associated with a unique cardiomyopathy, characterised by a combination of structural and cellular remodelling, and an enhanced susceptibility to ischaemia-reperfusion injury. This may represent dysfunction of the reperfusion injury salvage kinase pathway, due to insulin resistance. Aims: The susceptibility of the uraemic heart to ischaemia-reperfusion injury and the cardioprotective effects of insulin and rosiglitazone were investigated. Methods and Results: Uraemia was induced in Sprague-Dawley rats by subtotal nephrectomy. Functional recovery from ischaemia was investigated in vitro in control and uraemic hearts ±insulin ±rosiglitazone. The response of myocardial oxidative metabolism to insulin was determined by 13C NMR spectroscopy. Activation of reperfusion injury salvage kinase pathway intermediates (Akt and GSK3β) were assessed by SDS-PAGE and immuno-precipitation. Insulin improved post-ischaemic rate pressure product in control but not uraemic hearts, (recovered rate pressure product (%), control 59.6±10.7 vs 88.9±8.5, p<0.05; uraemic 19.3±4.6 vs 28.5±10.4, p=ns). Rosiglitazone resensitised uraemic hearts to insulin-mediated cardio-protection (recovered rate pressure product (%) 12.7±7.0 vs. 61.8±15.9, p<0.05). Myocardial carbohydrate metabolism remained responsive to insulin in uraemic hearts. Uraemia was associated with increased phosphorylation of Akt (1.00±0.08 vs. 1.31±0.11, p<0.05) in normoxia, but no change in post-ischaemic phosphorylation of Akt or GSK3β. Akt2 isoform expression was decreased post-ischaemia in uraemic hearts (p<0.05). Conclusion: Uraemia is associated with enhanced susceptibility to ischaemia-reperfusion injury and a loss of insulin-mediated cardio-protection, which can be restored by administration of rosiglitazone. Altered Akt2 expression in uraemic hearts post ischaemia-reperfusion and impaired activation of reperfusion injury salvage kinase pathway may underlie these findings

Multi-Parametric Analysis and Modeling of Relationships between Mitochondrial Morphology and Apoptosis

Mitochondria exist as a network of interconnected organelles undergoing constant fission and fusion. Current approaches to study mitochondrial morphology are limited by low data sampling coupled with manual identification and classification of complex morphological phenotypes. Here we propose an integrated mechanistic and data-driven modeling approach to analyze heterogeneous, quantified datasets and infer relations between mitochondrial morphology and apoptotic events. We initially performed high-content, multi-parametric measurements of mitochondrial morphological, apoptotic, and energetic states by high-resolution imaging of human breast carcinoma MCF-7 cells. Subsequently, decision tree-based analysis was used to automatically classify networked, fragmented, and swollen mitochondrial subpopulations, at the single-cell level and within cell populations. Our results revealed subtle but significant differences in morphology class distributions in response to various apoptotic stimuli. Furthermore, key mitochondrial functional parameters including mitochondrial membrane potential and Bax activation, were measured under matched conditions. Data-driven fuzzy logic modeling was used to explore the non-linear relationships between mitochondrial morphology and apoptotic signaling, combining morphological and functional data as a single model. Modeling results are in accordance with previous studies, where Bax regulates mitochondrial fragmentation, and mitochondrial morphology influences mitochondrial membrane potential. In summary, we established and validated a platform for mitochondrial morphological and functional analysis that can be readily extended with additional datasets. We further discuss the benefits of a flexible systematic approach for elucidating specific and general relationships between mitochondrial morphology and apoptosis

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment