Urolithin A (UA) is a metabolite produced by gut bacteria from ellagitannin and ellagic acid, which is present in pomegranates, berries, and walnuts. Previous studies have shown that UA supplementation improves skeletal muscle health in both mice and humans. UA supplementation also appears to be beneficial to the heart, resulting in both systolic and diastolic functional improvement in old mice. This study aims to determine a mechanism for the improvements in the aging heart. We hypothesize that the mechanisms behind improved heart function from UA supplementation are related to the pathways that directly improves mitochondria, contractile properties and mitophagy. To investigate the mechanism behind improved heart function with UA supplementation, we split the treatments into two staggered cohorts with 20, 24-month-old mice per cohort (10 control/10 UA treatment). Both the control and the treatment group were kept on the control diet during week 1 for acclimation. In the following 8 weeks, the UA treatment group were moved to a UA 50 mpk supplemented diet. Echocardiography measurements were done pre and post supplementation. At the end of the study, all mice were euthanized under anesthesia after an overnight fast and sample collection was performed. Heart samples were collected from each mouse. Western blots of heart samples on mitophagy, mitochondrial and cardiac contractile protein markers indicate that those pathways remain unaffected by UA supplementation. In future work, we will explore other informatory pathways in order to determine the mechanism behind UA supplementation improvement of heart function.

Heart disease is the leading cause of death in the United States. Echocardiography is used clinically to highlight cardiac structures, wall motion, and contraction abnormalities to diagnose heart failure. Heart failure is classified into diastolic (relaxation) or systolic (contraction) heart failure using left ventricular ejection fraction (LVEF), a measurement of the amount of blood pumped during each contraction. Global Longitudinal Strain (GLS) analysis, which measures the stiffness and deformation of the heart wall during contraction, has recently emerged as a more sensitive metric of systolic function that predicts cardiovascular mortality in patients. Elamipretide (ELAM;SS-31) is a mitochondrial-targeted intervention that improves aging heart mitochondrial and diastolic function. In this study, we used an aging mouse model to compare sex-specific outcomes in systolic function by LVEF and GLS. We hypothesized that GLS would be a better predictor of systolic dysfunction in mice than LVEF, and that ELAM would repair aging systolic dysfunction. We compared young (4-6 mo) and old (25-26 mo) male and female mice using 2D echocardiography to obtain left ventricular parasternal short and long axis images. A cohort of aged male mice was imaged before and after 8-week ELAM treatment. These images were analyzed using Vevo LAB software using conventional echocardiography to measure LVEF and speckle-tracking echocardiography (STE) for GLS and LVEFStrain. Statistical analysis was performed using GraphPad Prism Software. Limited change in LVEF was observed by conventional echocardiography. Using STE, GLS and LVEFStrain declined with age. Treatment with ELAM restored GLS in aging mice to young levels. Here we show that the more sensitive STE analysis reveals that aging mice exhibit both systolic and diastolic dysfunction. The research supports our hypothesis that ELAM treatment improves systolic function in aging. Future treatments to target systolic dysfunction can be assessed in an aging mouse model using STE.

Evaluating the risk of developing breast cancer is an important aspect of cancer care as it can allow for more tailored screening strategies and preventative therapies. Clinicians use multiple measures to determine a patient’s risk of developing breast cancer, including breast density on mammography and genetic mutations. Background parenchymal enhancement (BPE) on magnetic resonance imaging (MRI) has shown promise to improve stratification of breast cancer risk in women at high-risk of cancer development. BPE is the increase in signal intensity of normal breast tissue on dynamic contrast-enhanced (DCE) MRI after the administration of contrast agent. Despite BPE having an association with an increased risk of breast cancer development, the biological basis of this increased enhancement is unknown. The aim of this study is to investigate what biologically drives BPE by connecting quantitative MRI measurements with pathological markers from normal breast tissue. Our study cohort includes women that received prophylactic mastectomies and DCE-MRI scans acquired ≤1 year before surgery. From mastectomy specimens, pathological measures of COX-2, VEGF, and Ki-67 are used to measure inflammation, vascular recruitment, and proliferation, respectively. To quantify BPE, I used in-house software to correct pre-contrast images using N4 bias field correction and segment the whole breast. I then applied the breast mask to the pre-contrast MRI and used fuzzy c-means clustering to automatically segment fibroglandular tissue (FGT) from surrounding fat, generating an FGT mask. This mask was then applied to the DCE-MRI series, which includes pre- and post-contrast images, to calculate BPE, which is the mean percent enhancement across FGT. As part of ongoing work, I will obtain more specific measurements in quadrants of the breast from which the pathology specimen was derived. I will then correlate BPE measurements to the pathology measures to determine if any associations exist between BPE and inflammation, vascular recruitment, and proliferation.

Weight loss (WL) is recommended for people with obesity to mitigate cardiometabolic risk, but its effect becomes limited when reaching a WL-plateau (WL-PL), when the rate of WL becomes minimal despite efforts to continue losing weight. The biological basis for the WL-PL is not fully understood. The goal of this study is 1) to test two diet-regimens in the development of a WL-PL in mice with diet-induced obesity (DIO), and 2) to identify subsequent changes in mitochondrial function. We hypothesized that despite similarities in caloric intake, higher fat content will make high-fat diet (HFD-CR20) mice protect their adiposity and reach a WL-PL sooner than low fat diet (LFD-CR20) mice. We also hypothesized that mitochondrial function will be reduced in mice that have reached a WL-PL. To test hypothesis 1, individually housed DIO mice were divided into two groups, and were provided with 80% of their ad libitum caloric intake with either a high-fat, or low-fat diet daily for ~2.5 weeks. Body weights were recorded daily. A WL-PL was identified by weight stability (<0.5% change BW/day) following weight loss. HFD-CR20 mice reached a WL-PL phase after 10 days of caloric restriction. LFD-CR20 mice did not achieve a plateau within the study time frame. LFD-CR20 mice lost more weight than HFD-CR20 (-10.8% ± 2.2 vs. -5.2% ± 1.8, p<0.05 respectively), which was attributed to a greater loss in adiposity, measured by an EchoMRI, (-23.3 g ± 6.0 vs. -3.3 g ± 3.0, p<0.05). To test hypothesis 2, mitochondrial function was assessed by high resolution respirometry at the study endpoint. We will further analyze this data to identify differences in mitochondrial function attributed to the WL-PL. This work will improve our understanding on the biological mechanisms behind resistance to weight loss to help advance obesity treatments in humans.

Despite decades of research very little is known about how mitochondria control stress responses. Therefore, new and innovative models are needed to understand the mechanisms of mitochondrial stress response. We developed a new mouse model of skeletal muscle mitochondrial stress to mimic the aging process in young animals to determine if mitochondrial oxidative stress replicates age-related skeletal muscle and mitochondrial dysfunction. We generated a mouse model to induce skeletal muscle mitochondrial redox stress to mimic skeletal muscle aging by knocking down superoxide dismutase 2 (SOD2). My project was to determine whether this model works in vivo. I fed animals a Doxycycline (DOX) chow diet (0.625g/kg) to induce SOD2 knockdown (KD). After 3-week DOX feeding, tissues were collected and processed for western blotting (WB). WB's were run for SOD2 in gastrocnemius, quadriceps, liver, kidney, heart and brain tissue. Normally, SOD2 is expressed in all tissues that contain mitochondria, so comparing SOD2 expression levels across tissues in KD animals validates tissue specificity. HNE adducts, a marker of oxidative stress, were measured by WB to confirm increases in oxidative stress associated with SOD2 KD. Three-week DOX feeding showed significant decreases in SOD2 protein in gastrocnemius (p<0.001) and quadriceps muscles (p<0.0001) compared to unfed littermate controls of the same genotype. There were no differences in SOD2 protein in heart, brain, liver or kidneys between DOX and control groups. HNE protein adducts were also significantly increased in skeletal muscle of DOX compared to controls (p<0.05). SOD2 is knocked down in skeletal muscle in response to DOX feeding. The increase in HNE adducts confirms that the knockdown of SOD2 causes an increase in oxidative stress. This model can now be used to explore the physiological mechanisms of inducing mitochondrial redox stress in young animals to recapitulate the effects of aging in a controlled manner. 

Diffusion-weighted imaging (DWI) shows great potential for improving breast cancer detection and diagnosis. Primary findings from the ECOG-ACRIN A6702 multi-site, multi-vendor clinical trial indicate that DWI apparent diffusion coefficient (ADC) values may help reduce false positives and unnecessary biopsies. Gradient nonlinearity (GNL) correction was previously found to improve the accuracy of ADC mapping within and across MRI vendor systems. In this study, we evaluated the impact of GNL correction on breast lesion ADC measures in the A6702 dataset. The dataset comprised 81 suspicious breast lesions (28/81 malignant) in 67 women. Standardized DWI scans were acquired across 9 different MRI scanners. ADC maps were created from DWI scans, and ADC values were measured for each lesion. Direction-averaged GNL correction maps were constructed based on scanner-specific gradient specifications. ADC map correction was then performed through pixel-wise scaling by the GNL correction maps using custom software developed in MATLAB. Lesion ADCs before and after GNL correction were compared using a two-tailed z-test. ADC diagnostic performance (benign vs. malignant) was evaluated using area under the receiver-operating-characteristic-curve (AUC), and optimal ADC cutoffs were chosen to maximize specificity while maintaining 100% sensitivity. GNL-corrected lesion ADCs were significantly lower than uncorrected ADCs (1.12±0.29 vs 1.17±0.30x10-3mm2/s, p<0.001). GNL error in lesion ADCs varied across gradient systems (mean ∆ADCvendorA=0.14±0.08, ∆ADCvendorB=0.03±0.02, ∆ADCvendorC =0.004±0.01, p<0.001). GNL correction produced a slightly lower optimal ADC cutoff (1.33 vs. 1.35x10-3mm2/sec). However, no overall difference in diagnostic performance was detected: AUCuncorrected=0.78 (95% CI 0.68-0.88), AUCcorrected=0.79 (95% CI:0.69-0.89), p=0.22, and 18% potential biopsy reduction for both. This study showed GNL substantially affects lesion ADC measures, with significant variability across different vendor platforms. These findings suggest that GNL correction should be implemented to ensure uniformity and consistency in diagnostic breast lesion ADC measures across MRI platforms, especially for multi-center clinical studies.

Obesity is a condition characterized by excessive fat accumulation, resulting in increased risk for chronic diseases like cardiovascular disease and diabetes. Weight loss can effectively reduce the burden of cardiometabolic risk factors, but weight loss maintenance is difficult to achieve. Mitochondria are key organelles within cells that are responsible for the breakdown of substrates to produce energy. Mitochondrial dysfunction is implicated in obesity, but little is known about the role of mitochondrial dysfunction in weight loss maintenance. Additionally, females are often underrepresented in obesity research, partly attributed to female mice being more resistant to develop obesity compared to males. The aim of this study is to compare mitochondrial function in liver, adipose tissue, and skeletal muscle, following weight loss in female mice with diet-induced obesity (DIO). We hypothesized that obesity would result in a reduction of mitochondrial function across tissues, and weight loss to further reduce it. We provided CB6F1 female mice with a high fat diet, where 87% of them developed DIO. DIO mice were separated into two groups: one underwent 20% caloric restriction for 4 weeks (HFD-CR), and the other group remained on ad libitum high fat diet for the same intervention (HFD-AL). A healthy weight control group was maintained on a regular chow diet. Their weight and food intake were recorded daily. Body composition was assessed twice, before and after the 4-week intervention period. We had an unexpected finding, where mice lost 5-10% of their body weight prior to the intervention period. HFD-AL mice regained lost weight at study endpoint, while the weight of HFD-CR mice remained weight reduced until study endpoint. CR mice had lower adipose tissue mass compared to HFD- AL mice. Future analyses will include comparisons of mitochondrial content and function in different tissues. Findings will provide more insight into the effects of weight maintenance and regain on mitochondrial function.

Obesity is associated with mitochondrial dysfunction. A study previously conducted in our laboratory produced preliminary data suggesting a reduction in T-cell mitochondrial function in subjects with obesity relative to healthy weight controls. The purpose of this present study is to determine whether changes in T-cell mitochondrial function (MITO) reflect MITO changes occurring in liver and skeletal muscle, which are known for having greater influence in glucose homeostasis and energy expenditure. We hypothesize that mice with diet-induced obesity (DIO) will exhibit reduced MITO in T-cells that will correlate to a decline in MITO in liver and skeletal muscle. Adult male C57Bl/6J mice were divided into two groups–the control group was fed a standard chow diet whereas the experimental group was fed a high-fat diet for fifteen weeks. Body weight and food intake were measured every week. Body composition was performed at the endpoint. MITO was measured via high-resolution respirometry in permeabilized liver tissue, skeletal muscle fibers, and splenic T-cells. DIO mice had higher body mass than standard CHOW [52.8g±1.8 vs. 35.1g±2.6] that was explained by an increase in fat mass [19.9g±0.9 vs. 6.1g±1.8] and lean mass [31.5g±1.7 vs. 26.1g±1.1]. We found that differences between DIO and CHOW in mitochondrial leak respiration, maximal oxidative capacity, maximal electron transport chain, and ADP sensitivity were not the same across all tissues. We will proceed to determine which aspects of MITO are correlated between different tissues and assess if variations in respiration are associated with differences in mitochondrial content. An improved understanding of how DIO affects different types of cells regarding oxidative capacity might provide key insights into the development of therapeutics and other preventative approaches to improve immunity and cardiovascular fitness in obesity.