Performing Department
Veterinary & Biomedical Sciences
Non Technical Summary
By the year 2030, an estimated 8 million Americans are expected to be diagnosed with heart failure (HF), burdening the US healthcare system nearly $70 billion. Disease trajectory in HF patients is frequently confounded by pulmonary vascular remodeling leading to cardiac pulmonary hypertension (PH), a common hemodynamic complication of prolonged HF. There are no approved medical therapies specific to cardiac PH, and patient response to off-label drugs prescribed for treating other forms of PH are unpredictable or associated with worse outcomes. The number of patients developing end-stage HF will greatly exceed the availability of transplantable donor hearts, necessitating improved pharmacologics and mechanical support devices to enhance patient care and quality of life.More than 80% of promising drug therapies fail in human clinical trials, a problem shared across diseases including pulmonary hypertension (PH) and heart failure (HF). While rodent models have greatly informed our understanding of this devastating disease, their low fidelity to human pathology and failure to recapitulate the diversity in patient phenotypes pose a significant barrier to our mechanistic understanding and reproducibility of preclinical data, hindering development of therapies that translate into improved patient care and outcomes. The progressive and fatal trajectory of PH and HF prompts patients to enroll in promising yet unproven drug trials, excluding participation in trials more suited for disease phenotype.The PH community has cast doubt on the utility of current rodent models in predicting human responses to novel therapeutics, advocating for the development of larger animal models sharing similar size, cardiopulmonary anatomy, and physiology to humans. These models need to encompass all salient disease attributes and attendant co-morbidities, permitting the identification and optimization of novel therapeutic targets before commencement of costly clinical trials. Production animal species are commonly overlooked in experimental animal models of human cardiovascular and pulmonary diseases because of economic and logistical concerns, even though cardiopulmonary anatomic and physiologic dissimilarities regress with body and heart mass approaching the human correlate. Large animal species, particularly cattle, are already widely used as an experimental platform for development and testing of left ventricular assist devices (LVADs) to support the failing left ventricle in bridge-to-transplant and destination therapies (BTT, DT) and advancing continuous-flow total artificial heart (CFTAH) technology. Pre-clinical trials almost always use healthy animals to test drug and device safety, reliability, and biocompatibility, limiting their usefulness in predicting patient response and outcomes when patients are already in the end-stages of HF. Surgical approaches and cardiotoxins are commonly used to induce experimental HF in large animal models, however a major drawback is these techniques create acute, not chronic HF and therefore do not faithfully recapitulate the complexities of pulmonary vascular remodeling and PH in the context of chronic HF. Features of metabolic syndrome including obesity, systemic hypertension, hyperinsulinemia, insulin resistance, glucose intolerance, and coronary artery disease are not uncommon in PH and HF patients, enhancing phenotypic diversity among PH-HF patients and adding further impediment to development of suitable animal models that faithfully recreate human pathology. Another major constraint to dissecting the mechanisms associated with onset and progression of PH in the context of chronic HF is tissues are obtained at transplantation in human patients or from experimental animals at study termination when signaling pathways and protein expression are influenced by treatment and long-standing disease. This highlights the need for tissues from both untreated patients and experimental animals with early disease and during longitudinal interrogations of disease progression. Like human patients with PH in the context of chronic HF, a subset of fattening beef cattle develops HF with evidence of global cardiac and pulmonary remodeling leading to PH and right heart failure (RHF). These animals appear to recapitulate features of WHO Group 2 PH on left heart disease (LHD), the largest and fastest growing segment of the human PH population. Bovine congestive heart failure (CHF) is an important cause of mortality in North American feedyards, and independent of mortality, the condition negatively impacts productivity and carcass traits. For reasons that are poorly understood, a subset of cattle develops this invariably fatal condition in response to prototypical management and feeding regimens similarly used across the industry. Affected beef cattle faithfully recapitulate the complex spectrum of pathologic lesions in the cardiopulmonary axis observed in the human correlate, without surgical manipulation or administration of cardiotoxic compounds. Further, bovine cardiac lesions are reminiscent of chronic, not acute heart failure, potentially offering a more robust pre-clinical model of PH in the setting of prolonged HF for testing of medical therapies and mechanical support devices in relation to the failing heart. This should eliminate false positive targets identified in pre-clinical rodent studies, such that monetary resources can be appropriated towards clinical trials aimed at relevant therapeutic targets, saving millions of dollars and shortening the time to commercialization. Likewise, development of translationally-relevant pre-clinical bovine models of PH on chronic HF has the added benefit of enhancing our mechanistic understanding of signaling networks and protein expression involved in disease onset and evolution in beef cattle.Collaborations between physician and veterinary scientists can bridge the gap between basic science and clinical trials in Group 2 PH on LHD patients with the added benefit of developing a mechanistic understanding of bovine CHF. In turn this will inform the industry on how best to mitigate the impact of CHF, enhance cattle health and welfare, and improve sustainability of beef production. This can only be achieved in an environment that supports an integrative, multidisciplinary One Health approach to optimizing human health and the health of the production animal species that sustain global protein requirements. Using a multidisciplinary approach drawing upon the expertise of physician-scientists, pathologists, molecular biologists and beef cattle veterinarians with over 40 years of experience elucidating the complex multistep pathogenesis of bovine PH in the context of chronic environmental hypoxia, we aim to apply this knowledge and expertise to elucidate the pathophysiology of feedlot CHF. We aim to identify protein changes underlying health and disease in feedlot cattle by assessing potential biomarkers associated with adverse cardiopulmonary events in humans with chronic HF, allowing for characterization of cardiopulmonary risk phenotypes and prognostic stratification using combinations of mass spectrometry-based proteomic analysis and high throughput technologies employing targeted binding reagents in a multiplex manner permitting simultaneous measurement of thousands of high and low abundance proteins. Identification of prognostic proteins will also report on the biologically relevant pathways involved in bovine PH and CHF and suggest potential therapeutic targets. The overall goal is to create a multiprotein biomarker screening tool for identification and regular assessment of disease severity and clinical progression, informing clinical management decisions.
Animal Health Component
100%
Research Effort Categories
Basic
40%
Applied
50%
Developmental
10%
Goals / Objectives
HYPOTHESIS: We propose to test the hypothesis that bovine congestive heart failure (CHF) is characterized by a specific metabolic/lipidomic and inflammatory signature that may be used to identify cattle with CHF. The overall goal is to determine a precise signature of bovine obesity-induced CHF in plasma using high throughput mass spectrometry and proteomic approaches, and to correlate this signature to gene and protein expression in cardiac tissues.OBJECTIVE 1: Dissect the metabolic and lipidomic profile discriminating cattle with CHF incomparison to healthy cattle matched for age and sex across the production cycle.OBJECTIVE 2: Determine the links between obesity, CHF, and inflammation todiscriminate cattle with CHF from healthy cattle matched for age and sex across the productioncycle.OBJECTIVE 3: Evaluate the contribution of epicardial adipose tissue (EAT) to the metabolic, lipidomic, and inflammatory signatures established in Objectives 1 and 2 for cattle with obesity-associated CHF vs. non-obese controls.OBJECTIVE 4: Evaluate the contribution of the ventricular myocardium independent of epicardial adipose tissue, to the metabolic and inflammatory profile identified in Objectives 1 and 2 for cattle with obesity-associated CHF vs. non-obese controls.
Project Methods
We have previously utilized the approaches described herein to study obesity-induced CHF in humans with chronic obstructive pulmonary disease (COPD), with and without PH. We were challenged with trying to differentiate the subgroup of patients with PH from those without, as the former have a much higher morbidity and mortality. Our search for plasma biomarkers is beginning to pay dividends in that it appears we can measure plasma metabolites and lipids that differentiate the PH phenotype. Comprehensive metabolomic analysis of plasma samples from patients with COPD and COPD with PH, together with principal component analysis, confirmed there are very significant differences. We are now concentrating on 1-2 sets of metabolites that may be useful in screening large patient populations at a reasonable cost. Also consistent with our preliminary data are changes in serum uric acid, which appears to discriminate patients with PH versus those without. Interestingly, this plasma marker also appears to be associated with mortality.In this project we propose to search for similar biomarkers in cattle. Development of such technology will greatly improve functional clinical assessments based on the presence of signs including intermandibular, sternal and ventral edema, jugular pulse, and distention. The gold standard for diagnosing PH in cattle remains right heart catheterization, and while commonly employed in the research setting, this invasive technique is fraught with logistical challenges and risks, precluding its routine use in the feedlot setting. High throughput protein screens performed on bovine cardiopulmonary tissues, plasma, and cultured cells will also report on the biological processes and signaling pathways involved in adipogenesis, lipid metabolism, and disease evolution revealing therapeutic strategies for obesity and its associated diseases. This project will also provide an ex-vivo platform for screening potential interventional therapies.Our experimental approach is summarized as follows:Retrospective Study Analysis: Archived blood and tissue samples, from cattle originating from the Colorado Beef Improvement Center (2,150 m elevation) that were selected for tolerance to environmental hypoxia and finished at moderate (1,420 m) or high elevation (2,150) with varied lengths of post-weaning grazing, will be used to address the specific study objectives. Steers were assigned to two separate management groups: 1) traditional stocker and finishing system (i.e. grown and finished at 1,420 m beginning at 10 months of age=12 steers) or 2) grass finished (2,150 m) >24 months of age=10 steers.Sequential venipuncture for assessment of blood-based biomarkers and pulmonary arterial pressure (PAP) testing was conducted at 4 different time points throughout the production cycle=168 total blood samples for analysis. Traditional stocker and finisher steers segregated into 3 distinct phenotypes throughout the production cycle and at harvest-low PAP=6 steers, high PAP=5 steers, high PAP with CHF=1 steer.OBJECTIVE 1: Dissect the metabolic and lipidomic profile discriminating cattle with CHF incomparison to healthy cattle matched for age and sex across the production cycle.Ultra high pressure liquid chromatography mass spectrometry (UHPLC-MS) techniques will be used to characterize metabolic and lipidomic profiles of cattle with CHF in comparison to healthy cattle matched for age and sex across the production cycle.OBJECTIVE 2: Determine the links between obesity, CHF, and inflammation todiscriminate cattle with CHF from healthy cattle matched for age and sex across the production cycle.Inflammatory protein expression in cattle with CHF, as opposed to healthy cattle, matched for age and sex across the production cycle will be characterized using multiplex immunolabeling assays. This work will be complemented by analyses of the complete plasma proteome using UHPLC-MS in a subset of cattle.At harvest, sections of heart fat, LV, and RV myocardium were collected from each steer=22 total LV specimens, 22 total RV specimens, 22 total heart fat specimens. Tissue expression of proteins in cardiac fat and muscle tissue specimens obtained at harvest will be paired with circulating proteins identified in blood obtained from the last venipuncture collection (approximately 3 wks. pre-harvest).OBJECTIVE 3: Evaluate the contribution of epicardial adipose tissue (EAT) to the metabolic, lipidomic, and inflammatory signatures established in Objectives 1 and 2 for cattle with obesity-associated CHF vs. non-obese controls.We will use mass spectrometry-based lipidomics and proteomics, as well as gene expression (using a novel product for targeted gene sequencing called TempO-Seq [BioSpyder, Carlsbad, CA] to enable analysis of thousands of genes in high throughput) to analyze epicardial adipose tissue dissected from cattle with obesity-associated CHF vs. non-obese controls.OBJECTIVE 4: Evaluate the contribution of the ventricular myocardium independent of epicardial adipose tissue, to the metabolic and inflammatory profile identified in Objectives 1 and 2 for cattle with obesity-associated CHF vs. non-obese controls.Evaluate the contribution of the ventricular myocardium independent of epicardial adipose tissue, to the metabolic and inflammatory profile identified in Objectives 1 and 2 using mass spectrometry-based metabolomics and proteomics and gene expression (TempO-Seq) to analyze the ventricles from cattle with obesity-associated CHF vs. non-obese controls.PROSPECTIVE STUDY ANALYSIS: Proteins predictive of cardiovascular risk and pathways indicative of inflammatory activation, as well as metabolic and adipose tissue dysfunction identified in retrospective study analysis can be validated and further dissected using steers enrolled in the USDA funded study, Metabolism and Inflammation Predict Cardiopulmonary Disease Outcomes in Fattening Beef Cattle (18-7727A). The calves enrolled in this study are from an altitude naïve northeast Colorado cow-calf operation (1,420 m) that does not select for high elevation performance and has a history of CHF during the fattening phase. The calves entered the Eastern Colorado Research Center (ECRC) feeding facility in mid-October 2018, weighing approximately 600 lb. On December 10, the calves were screened for weight, blood-based biomarker analysis, and PAP scores. Calves averaged 750 lb and none had been treated for BRD since their arrival. In response to early fattening, three distinct phenotypes have emerged: low PAP (mPAP<38 mm Hg, n=22 steers); moderate PAP (mPAP 39-47mm Hg, n=59 steers), and high PAP (mPAP 48-91, n=24 steers). It is apparent that early in the fattening phase of the production cycle, a subset of cattle are susceptible to systemic inflammatory activation and metabolic and lipidomic dysfunction associated with fattening, arising from as yet unknown genetic variation. Three weeks prior to harvest, BRSV intra-nasal inoculation will be conducted in 10 high PAP and 10 low PAP steers because we hypothesize that cattle with underlying cardiopulmonary disease are predisposed to viral pneumonia with secondary opportunistic bacterial colonization, accelerating clinical progression and outcomes associated with bovine CHF. Like the retrospective study, LV, RV, and heart fat specimens will be collected at harvest from the 40 steers (40 total LV, 40 total RV and 40 total heart fat). The outcomes of this analysis will have broader implications for the cattle industry because the overwhelming majority of cattle originate at low to moderate elevations and are fattened in High Plains feed yards situated at similarly low to moderate elevations.