Muscle weakness and mitochondrial stress occur before metastasis in a novel mouse model of ovarian cancer cachexia.

Objectives: A high proportion of women with advanced epithelial ovarian cancer (EOC) experience weakness and cachexia. This relationship is associated with increased morbidity and mortality. EOC is the most lethal gynecological cancer, yet no preclinical cachexia model has demonstrated the combined hallmark features of metastasis, ascites development, muscle loss and weakness in adult immunocompetent mice. Methods: Here, we evaluated a new model of ovarian cancer-induced cachexia with the advantages of inducing cancer in adult immunocompetent C57BL/6J mice through orthotopic injections of EOC cells in the ovarian bursa. We characterized the development of metastasis, ascites, muscle atrophy, muscle weakness, markers of inflammation, and mitochondrial stress in the tibialis anterior (TA) and diaphragm ~45, ~75 and ~90 days after EOC injection. Results: Primary ovarian tumour sizes were progressively larger at each time point while robust metastasis, ascites development, and reductions in body, fat and muscle weights occurred by 90 Days. There were no changes in certain inflammatory (TNFα), atrogene (MURF1 and Atrogin) or GDF15 markers within both muscles whereas IL-6 was increased at 45 and 90 Day groups in the diaphragm. TA weakness in 45 Day preceded atrophy and metastasis that were observed later (75 and 90 Day, respectively). The diaphragm demonstrated both weakness and atrophy in 45 Day. In both muscles, this pre-metastatic muscle weakness corresponded with considerable reprogramming of gene pathways related to mitochondrial bioenergetics as well as reduced functional measures of mitochondrial pyruvate oxidation and creatine-dependent ADP/ATP cycling as well as increased reactive oxygen species emission (hydrogen peroxide). Remarkably, muscle force per unit mass at 90 days was partially restored in the TA despite the presence of atrophy and metastasis. In contrast, the diaphragm demonstrated progressive weakness. At this advanced stage, mitochondrial pyruvate oxidation in both muscles exceeded control mice suggesting an apparent metabolic super-compensation corresponding with restored indices of creatine-dependent adenylate cycling. Conclusion: This mouse model demonstrates the concurrent development of cachexia and metastasis that occurs in women with EOC. The model provides physiologically relevant advantages of inducing tumour development within the ovarian bursa in immunocompetent adult mice. Moreover, the model reveals that muscle weakness in both TA and diaphragm precedes metastasis while weakness also precedes atrophy in the TA. An underlying mitochondrial bioenergetic stress corresponded with this early weakness. Collectively, these discoveries can direct new research towards the development of therapies that target pre-atrophy and pre-metastatic weakness during EOC in addition to therapies targeting cachexia.

Therapy-naïve malignancy causes cardiovascular disease: A state-of-the-art cardio-oncology perspective.

Cardiovascular disease (CVD) and cancer are the leading causes of mortality worldwide. Although generally thought of as distinct diseases, the intersectional overlap between CVD and cancer is increasingly evident in both causal and mechanistic relationships. The field of cardio-oncology is largely focused on cardiotoxic effects of cancer therapies (e.g., chemotherapy, radiation). Further, the cumulative effects of cardiotoxic therapy exposure and the prevalence of CVD risk factors in cancer patients leads to long-term morbidity and poor quality of life in this patient population-even when patients are cancer-free. Evidence from cancer patients and animal models demonstrates that the presence of malignancy itself, independent of cardiotoxic therapy exposure or CVD risk factors, negatively impacts cardiac structure and function. As such, the primary focus of this review is the cardiac pathophysiologic and molecular features of therapy-naïve cancer. We also summarize the strengths and limitations of preclinical cancer models for cardio-oncology research and discuss therapeutic strategies that have been tested experimentally for the treatment of cancer-induced cardiac atrophy and dysfunction. Finally, we explore an adjacent area of interest, called 'reverse cardio-oncology', where the sequelae of heart failure augment cancer progression. Here, we emphasize the cross-disease communication between malignancy and the injured heart, and discuss the importance of chronic low-grade inflammation and endocrine factors in the progression of both diseases.

Novel roles of cardiac-derived erythropoietin in cardiac development and function.

The role of erythropoietin (EPO) has extended beyond hematopoiesis to include cytoprotection, inotropy, and neurogenesis. Extra-renal EPO has been reported for multiple tissue/cell types, but the physiological relevance remains unknown. Although the EPO receptor is expressed by multiple cardiac cell types and human recombinant EPO increases contractility and confers cytoprotection against injury, whether the heart produces physiologically meaningful amounts of EPO in vivo is unclear. We show a distinct circadian rhythm of cardiac EPO mRNA expression in adult mice and increased mRNA expression during embryogenesis, suggesting physiological relevance to cardiac EPO production throughout life. We then generated constitutive, cardiomyocyte-specific EPO knockout mice driven by the Mlc2v promoter (EPOfl/fl:Mlc2v-cre+/-; EPOΔ/Δ-CM). During cardiogenesis, cardiac EPO mRNA expression and cellular proliferation were reduced in EPOΔ/Δ-CM hearts. However, in adult EPOΔ/Δ- CM mice, total heart weight was preserved through increased cardiomyocyte cross-sectional area, indicating the reduced cellular proliferation was compensated for by cellular hypertrophy. Echocardiography revealed no changes in cardiac dimensions, with modest reductions in ejection fraction, stroke volume, and tachycardia, whereas invasive hemodynamics showed increased cardiac contractility and lusitropy. Paradoxically, EPO mRNA expression in the heart was elevated in adult EPOΔ/Δ-CM, along with increased serum EPO protein content and hematocrit. Using RNA fluorescent in situ hybridization, we found that Epo RNA colocalized with endothelial cells in the hearts of adult EPOΔ/Δ-CM mice, identifying the endothelial cells as a cell responsible for the EPO hyper-expression. Collectively, these data identify the first physiological roles for cardiomyocyte-derived EPO. We have established cardiac EPO mRNA expression is a complex interplay of multiple cell types, where loss of embryonic cardiomyocyte EPO production results in hyper-expression from other cells within the adult heart.

Dietary Nitrate and Corresponding Gut Microbiota Prevent Cardiac Dysfunction in Obese Mice.

Impaired heart function can develop in individuals with diabetes in the absence of coronary artery disease or hypertension, suggesting mechanisms beyond hypertension/increased afterload contribute to diabetic cardiomyopathy. Identifying therapeutic approaches that improve glycemia and prevent cardiovascular disease are clearly required for clinical management of diabetes-related comorbidities. Since intestinal bacteria are important for metabolism of nitrate, we examined whether dietary nitrate and fecal microbial transplantation (FMT) from nitrate-fed mice could prevent high-fat diet (HFD)-induced cardiac abnormalities. Male C57Bl/6N mice were fed a low-fat diet (LFD), HFD, or HFD+Nitrate (4 mmol/L sodium nitrate) for 8 weeks. HFD-fed mice presented with pathological left ventricle (LV) hypertrophy, reduced stroke volume, and increased end-diastolic pressure, in association with increased myocardial fibrosis, glucose intolerance, adipose inflammation, serum lipids, LV mitochondrial reactive oxygen species (ROS), and gut dysbiosis. In contrast, dietary nitrate attenuated these detriments. In HFD-fed mice, FMT from HFD+Nitrate donors did not influence serum nitrate, blood pressure, adipose inflammation, or myocardial fibrosis. However, microbiota from HFD+Nitrate mice decreased serum lipids, LV ROS, and similar to FMT from LFD donors, prevented glucose intolerance and cardiac morphology changes. Therefore, the cardioprotective effects of nitrate are not dependent on reducing blood pressure, but rather mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis. ARTICLE HIGHLIGHTS: Identifying therapeutic approaches that prevent cardiometabolic diseases are clearly important, and nitrate represents one such potential compound given its multifactorial metabolic effects. We aimed to determine whether nitrate could prevent high-fat diet (HFD)-induced cardiac abnormalities and whether this was dependent on the gut microbiome. Dietary nitrate attenuated HFD-induced pathological changes in cardiac remodelling, left ventricle reactive oxygen species, adipose inflammation, lipid homeostasis, glucose intolerance, and gut dysbiosis. Fecal microbial transplantation from nitrate-fed mice also prevented serum dyslipidemia, left ventricle reactive oxygen species, glucose intolerance, and cardiac dysfunction. Therefore, the cardioprotective effects of nitrate are related to mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis.

An Evaluation of Cardiac Health in the Spontaneously Hypertensive Rat Colony: Implications of Evolutionary Driven Increases in Concentric Hypertrophy.

BACKGROUND: The Spontaneously Hypertensive Rat (SHR) Colony was established in 1963 and is the most commonly used rodent model for studying heart failure (HF). Ideally, animal models should recapitulate the clinical disease as closely as possible. Any drift in a genetic model may create a new model that no longer adequately represents the human pathology. Further, instability overtime may lead to conflicting data between laboratories and/or irreproducible results. While systolic blood pressure (SBP) is closely monitored during inbreeding, the sequelae of HF (e.g., cardiac hypertrophy) are not. Thus, the object of this review was to investigate whether the hypertension-induced sequelae of HF in the SHR have remained stable after decades of inbreeding. METHODS: A systematic review was performed to evaluate indices of cardiovascular health in the SHR over the past 60 years. For post hoc statistical analyses, studies were separated into 2 cohorts: Initial (mid to late 1900s) and Current (early 2000s to present) Colony SHRs. Wistar-Kyoto rats (WKY) were used as controls. RESULTS: SBP was consistent between Initial and Current Colony SHRs. However, Current Colony SHRs presented with increased concentric hypertrophy (i.e., elevated heart weight and posterior wall thickness) while cardiac output remained consistent. Since these changes were not observed in the WKY controls, cardiac-derived changes in Current Colony SHRs were unlikely due to differences in environmental conditions. CONCLUSIONS: Together, these data firmly establish a cardiac-based phenotypic shift in the SHR model and provide important insights into the beneficial function of concentric hypertrophy in hypertension-induced HF.

A new approach to improve the hemodynamic assessment of cardiac function independent of respiratory influence.

Cardiovascular and respiratory systems are anatomically and functionally linked; inspiration produces negative intrathoracic pressures that act on the heart and alter cardiac function. Inspiratory pressures increase with heart failure and can exceed the magnitude of ventricular pressure during diastole. Accordingly, respiratory pressures may be a confounding factor to assessing cardiac function. While the interaction between respiration and the heart is well characterized, the extent to which systolic and diastolic indices are affected by inspiration is unknown. Our objective was to understand how inspiratory pressure affects the hemodynamic assessment of cardiac function. To do this, we developed custom software to assess and separate indices of systolic and diastolic function into inspiratory, early expiratory, and late expiratory phases of respiration. We then compared cardiac parameters during normal breathing and with various respiratory loads. Variations in inspiratory pressure had a small impact on systolic pressure and function. Conversely, diastolic pressure strongly correlated with negative inspiratory pressure. Cardiac pressures were less affected by respiration during expiration; late expiration was the most stable respiratory phase. In conclusion, inspiration is a large confounding influence on diastolic pressure, but minimally affects systolic pressure. Performing cardiac hemodynamic analysis by accounting for respiratory phase yields more accuracy and analytic confidence to the assessment of diastolic function.

Hemodynamic assessment of diastolic function for experimental models.

Traditionally, the evaluation of cardiac function has focused on systolic function; however, there is a growing appreciation for the contribution of diastolic function to overall cardiac health. Given the emerging interest in evaluating diastolic function in all models of heart failure, there is a need for sensitivity, accuracy, and precision in the hemodynamic assessment of diastolic function. Hemodynamics measure cardiac pressures in vivo, offering a direct assessment of diastolic function. In this review, we summarize the underlying principles of diastolic function, dividing diastole into two phases: 1) relaxation and 2) filling. We identify parameters used to comprehensively evaluate diastolic function by hemodynamics, clarify how each parameter is obtained, and consider the advantages and limitations associated with each measure. We provide a summary of the sensitivity of each diastolic parameter to loading conditions. Furthermore, we discuss differences that can occur in the accuracy of diastolic and systolic indices when generated by automated software compared with custom software analysis and the magnitude each parameter is influenced during inspiration with healthy breathing and a mild breathing load, commonly expected in heart failure. Finally, we identify key variables to control (e.g., body temperature, anesthetic, sampling rate) when collecting hemodynamic data. This review provides fundamental knowledge for users to succeed in troubleshooting and guidelines for evaluating diastolic function by hemodynamics in experimental models of heart failure.