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Increasing age is the greatest risk factor for development and progression of calcific aortic valve disease (CAVD), and mitochondrial dysfunction has been implicated in the pathogenesis of cardiovascular calcification. Previous findings by our group suggested a significant reduction in expression of multiple sirtuin (SIRT) isoforms in normocholesterolemic aged mouse aortic valves, however, it is unclear if losses in SIRT3—a major mitochondrial SIRT isoform—modulates progression of aortic valve calcification. Therefore, we hypothesized that loss of SIRT3 in a hypercholesterolemic mouse model of CAVD will result in augmented calcium burden in aortic valve, increased osteogenic signaling, and impaired aortic valve function. To test this we used mice that were Ldlr -/- /ApoB 100/100 mice that were either wild-type (LA-SIRT3 +/+ ) or null for SIRT3 (LA-SIRT3 -/- ) and fed a western diet (TD88137) for 12 months. Alizarin red was used to quantitate calcium burden, qRT-PCR was used to measure changes in mRNA levels, and high-resolution echocardiography was used to assess aortic valve function (cusp separation distance). In line with our hypothesis, we observed a substantial increase in calcium burden in LA-SIRT3 -/- mice compared to their LA-SIRT3 +/+ littermates(11.7±4.0, 4.8±1.8; respectively). Interestingly, expression of Runx2 and osterix—classic markers of osteogenic differentiation—were decreased in LA-SIRT3 -/- mice compared to their LA-SIRT3 +/+ littermates. Expression of Msx2, were markedly increased in aortic valve tissue from LA-SIRT3-deficient mice. Despite these histological and molecular changes, SIRT3 deletion did not alter cusp separation distance (LA-SIRT3 +/+ = 0.81±0.04; LA-SIRT3 -/- = 0.81±0.02). Collectively, this data suggest losses in SIRT3 can contribute to accelerated valve calcification through unclear molecular mechanisms, but these changes are not sufficient to drive reductions in cusp separation distance. Additional experiments delineating the histopathological and molecular sequelae of SIRT3 deletion will be critical to understanding its role in the pathogenesis of CAVD.

Marfan syndrome is a connective tissue disorder frequently driven by mutations in the fibrillin-1 gene (Fbn1). This results in multiple aberrant cardiovascular phenotypes, including aortic aneurysm and mitral valve prolapse. While the molecular changes underlying aortic aneurysm formation have been extensively studied, the molecular underpinnings of mitral valve prolapse in this syndrome remain poorly understood. Therefore, we hypothesized that smad2/3 phosphorylation would be significantly increased in both aorta and mitral valve from Fbn1 +/1037G mice with Marfan syndrome and associated with impaired anti-inflammatory signaling in both tissues. We used young (3 months) mice that were wild-type (Fbn1 +/+ ) or fibrillin-1 mutant (Fbn1 +/1037G ) and fed a normal chow diet. qRT-PCR was used to measure mRNA levels in aortic arch and mitral valve tissue, and fluorescent immunohistochemistry to evaluate changes in canonical TGFβ signaling. Consistent with previous reports, TGF-β1 mRNA levels were increased in Fbn1 +/1037G compared to wild-type mice in both aorta and mitral valve. Interestingly, while p-SMAD2/3 protein levels were increased in aorta from Fbn1 +/1037G mice, we were surprised to find that p-SMAD2/3 protein levels were decreased in mitral valve from Fbn1 +/1037G mice compared their wild-type littermates. Given the emerging role of inflammatory signaling in accelerated development of Marfan phenotypes, we sought to determine whether there were compensatory or maladaptive changes in IL10 (a known inhibitor of p-SMAD2/3). In aorta, IL10 mRNA levels were decreased in Fbn1 +/1037G compared to Fbn1 +/+ mice, suggesting a maladaptive response. In contrast, IL10 levels in mitral valve tissue were significantly increased in Fbn1 +/1037G mice compared to their wild-type littermates, suggesting a compensatory/protective response. Collectively, these data suggest that tissue-specific changes in IL10 levels may modulate p-SMAD2/3 signaling in aorta and mitral valve, which may be a key permissive step in the onset of Marfan-related phenotypes. Future work to experimentally determine the role of IL10 in the regulation of canonical and non-canonical TGFβ signaling and the penetrance of Marfan-associated phenotypes is warranted.

Introduction: Idiopathic Dilated (DCM) and hypertrophic cardiomyopathies (HCM) are leading causes of sudden cardiac death. The incidence and prevalence of both DCM and HCM were originally estimated in Olmsted County, MN over 1975-1984. Incidence was 6.0 and prevalence was 36.5 per 100,000 person-years, for DCM; with incidence and prevalence rates of 2.5 and 19.7 per 100,000 person-years for HCM. This study aims to determine the incidence and mortality rates of individuals diagnosed with HCM and DCM in Olmsted County, MN from 1984 to 2016. Methods: The Rochester Epidemiology Project is a validated long term medical record linkage system that collects and links medical information for the approximately 145000 residents of Olmsted County, Minnesota. Using this database and the Electronic Health Record systems at Mayo Clinic and Olmsted Medical Center in Rochester, MN, we manually reviewed and validated the medical records of 4440 patients. This cohort includes all patients with any mention of a cardiomyopathy based on ICD and HICDA codes during the period 1984 to 2016. Data were summarized with mean and standard deviation, frequency and percentage and compared between groups using two sample t-test for continuous data and chi-square for proportions. Survival was computed using Kaplan-Meier methods and compared to expected survival based on age and gender. Incidence was computed using Olmsted County population numbers during the time period and adjusted to the US 1980 white population to allow for comparison with the previous study. Results: The calculated crude incidence rate for DCM and HCM are 10.1 and 7.8 per 100,000 respectively. Incidence rates adjusted for comparison to US 1980 white population (age and sex adjusted) are 10.4 (95% CI: 9.3-11.4) and 8.0 (95% CI: 7.1-8.8) per 100,000 person-years, respectively. The observed number of deaths in this cohort (HCM and DCM) was 420 vs 194 expected. Conclusions: Calculated incidence, both crude and adjusted rates are higher than previously estimated rates. The crude incidence rate for HCM is almost 4-fold higher than that for DCM is 2.4 times higher than the original estimate by Codd et al. Individuals with cardiomyopathies were found to have a higher overall mortality rate than the general population with an observed to expected hazard ratio of 1.73 (95% CI 1.49-2.01, p<0.001) for DCM and 1.44 (95% CI 1.21-1.71, p<0.001) for HCM. Our findings suggest that previous studies may have underestimated the true burden of HCM and DCM in the population which has important implications for healthcare provision and cost.

Previous studies have reported increases in oxidative stress due to reductions in antioxidant enzymes in calcified regions of stenotic human aortic valves. Whether reductions in antioxidant enzymes are adaptive, maladaptive, or an epiphenomenon in calcifying aortic valves remains unclear. Thus, the purpose of this study was to test whether overexpression of catalase reduces valvular calcification and slows progression of aortic valve stenosis in mice. Wild type (WT) mice with calcific aortic valve disease (ldlr-deficient, apoB100-only) were crossed with transgenic mice that overexpress human catalase (TG). All mice were fed a western diet (TD88137; Harlan Teklad). Functional echocardiogram data was collected and analyzed at early (3 months) and advanced (12 months) stages of valve disease, and valves were OCT embedded, sectioned, and stained for calcium levels (Alizarin Red). The transgenic valves show a slight increase in calcification compared to wild type counterparts. While there were no differences in valve function between genotypes at the early time point, cusp separation was decreased in the transgenic mice (0.74 ± 0.02 mm) compared to their wild-type littermates (0.84 ± 0.03 mm) at the 12 month time point (p < 0.05). In a subset of animals, valve calcification also tended to increase in transgenic mice at both time points compared to their wild-type littermates. In conclusion, overexpression of catalase resulted in a surprising finding of accelerated valvular calcification and stenosis. Interestingly, this finding aligns with the findings from several negative clinical trials testing antioxidant therapies in humans with atherosclerosis, suggests that the net impact of changing redox balance on disease progression are highly context dependent, and that increasing antioxidant capacity may not be a viable strategy to slow progression of calcific aortic valve disease in humans.

Rewarming shock following accidental hypothermia is a potentially fatal scenario involving cardiac dysfunction. Previously our lab demonstrated that rewarming shock results in a decrease in Ca 2+ sensitivity of contraction in cardiac muscle due to an increase in cTnI phosphorylation. In this study, we hypothesized that rewarming shock initiates a cascade of intracellular events including reactive oxygen species (ROS) formation, endoplasmic reticulum (ER) stress, and mitochondrial disruption. Isolated cardiomyocytes from Sprague‐Dawley rats were exposed to a hypothermia/rewarming protocol (cooled for 1 h, held at 17 o C for 30 min, rewarmed for 1 h) that results in contractile dysfunction indicated by an increase in cytosolic Ca 2+ and reduced cell shortening measured using an IonOptix system. Rewarming shock increased ROS levels measured by an HPLC‐based dihydroethidium assay and by MitoSox fluorescence. Hypothermia/rewarming was also associated with ER stress indicated by increased expression of ER stress markers (Grp78, PERK, and CHOP) measured by Western blot. Finally, rewarming shock was associated with mitochondrial disruption evidenced by mitochondrial membrane depolarization (decreased in TMRM fluorescence) and by an increase in cytosolic cytochrome c (measured by Western blot). Our results support our overall hypothesis and suggest that these ER and mitochondrial intracellular events may underlie myocardial contractile dysfunction following hypothermia/rewarming. Supported by NIH Grant 5T32HL105355‐02 (GCS)

Type I hypersensitivity reactions (HSR) to dabrafenib are rare but have been previously described. We present a case where a 72-year-old woman with recurrent, metastatic BRAF-mutated melanoma developed a type I HSR to dabrafenib. We, therefore, developed a desensitization protocol with encorafenib, a similar class agent, to allow the patient to continue with treatment. Patients with a history of HSR to dabrafenib may be considered for encorafenib desensitization when other therapeutic options are limited.

Nutrient excess and physical inactivity are primary drivers of numerous age‐related chronic diseases. A growing body of evidence has implicated cellular senescence, a process in which cells lose the ability to divide and concurrently damage neighboring cells by the factors they secrete, as an underlying mechanism of aging and its associated conditions. However, the impact of lifestyle choices on the age‐associated accumulation of senescent cells remains poorly understood. Thus, we examined the effects of a fast food diet (FFD) and a voluntary exercise intervention on multiple parameters of healthspan and cellular senescence using a transgenic mouse model that expresses green fluorescent protein (GFP) in p16 Ink4a ‐positive senescent cells. As expected, 12‐month old male mice that had received the FFD diet for 5 months demonstrated significantly greater gains in body weight and fat mass compared to mice fed a standard chow diet. Moreover, mice fed the FFD exhibited hyperinsulinemia, impaired glucose tolerance, reduced exercise capacity, and diastolic dysfunction. Impressively, providing an exercise wheel (26.9 ± 6.9 km/wk) in parallel with the FFD nullified the deleterious effects of the FFD on body weight and composition, whole‐body metabolism, physical performance and cardiac function. Strikingly, our data further suggest that exercise markedly lowers the number of cells in visceral fat depots that express GFP and stain positively for senescent‐associated β‐galactosidase; markers of senescence, that are highly abundant in sedentary mice fed the FFD. Correspondingly, FFD‐induced expression of senescence‐associated inflammatory factors was also diminished by the exercise intervention. Collectively, our data suggest that lifestyle choices may affect the abundance of senescence cells with advancing age, and exercise may partly mediate its salutary effects on multiple organ systems by preventing their accumulation and/or mediating their removal.

Recovery from accidental hypothermia followed by rewarming (H/R) is often unsuccessful due to heart failure. Although the causes for H/R‐induced heart failure remain elusive, studies at the cellular level suggest alterations in both cytoplasmic and mitochondrial Ca 2+ regulation. We hypothesize that H/R is associated with mitochondrial Ca 2+ overload, which leads to: 1) excessive reactive oxygen species (ROS) generation, 2) mitochondrial membrane depolarization, and 3) mitochondrial cytochrome c release. To test this hypothesis, isolated cardiomyocytes from adult male Sprague‐Dawley rats were subjected to an in vitro H/R protocol (1 h cooling to 15 o C held for 30 min followed by 1 h rewarming to 37 o C). Cytoplasmic (CM‐H 2 DCFDA fluorescence) and mitochondrial (MitoSOX fluorescence) ROS generation increased during cooling and remained elevated throughout the H/R protocol. Mitochondrial membrane potential (TMRM fluorescence) depolarized during rewarming suggesting mitochondrial permeability transition pore opening. After rewarming, cytochrome c content (Western blot) in cytoplasmic to mitochondrial fractions increased indicating release. These results suggest that Ca 2+ overload associated with H/R induces ROS generation leading to mitochondrial disruption. Grant Funding Source : Supported by NIH Grant 5T32HL105355‐02 (GCS)

Since its emergence at the end of 2019, SARS-CoV-2, the causative agent of COVID-19, has caused over 100 million infections and 2.4 million deaths worldwide. Recently, countries have begun administering approved COVID-19 vaccines, which elicit strong immune responses and prevent disease in most vaccinated individuals.

11040 Background: Although women outnumber men in US medical school enrollment, women constitute < 50% of faculty in academic oncology. This study aimed to determine factors affecting women oncologists’ decisions to pursue academic versus non-academic oncology in Hematology/Oncology (HO), Pediatric Oncology (PO), Radiation Oncology (RO), and Surgical Oncology (SO), and to characterize the challenges women oncologists face. Methods: A survey was designed to collect cross-sectional data on factors affecting career choices among US women oncologists. The survey was distributed via email and social media. Data were collected anonymously and analyzed using t-tests for continuous variables and Chi-squared tests for categorical variables. Results: Six hundred sixty-seven women oncologists responded: 245 (45.3%) specialized in HO, 173 (25.9%) RO, 88 (13.2%) PO, 56 (8.4%) SO, and 48 (7.2%) other. Four-hundred twenty-two (63.2%) women identified as an academic oncologist (AO); 245 (36.8%) women identified as a non-academic oncologist (non-AO). Approximately ¼ of women oncologists reported their partner (156, 23.5%) or family (176, 26.4%) extremely or moderately impacted their decision whether to pursue academic practice. There was no difference in the timing of childbearing between AO and non-AO. AO perceived the biggest sacrifice of pursuing academics to be time with loved ones (181, 42.9%). Non-AO perceived the biggest sacrifice for AO to be pressure for academic promotion (102, 41.6%), which was the third most common response (44, 18.0%) among AO. Thirty-three (7.9%) AO and 5 (2.0%) non-AO reported feeling that they rarely or never have a sense of belonging in their work environment (p < 0.01). AO and non-AO had significantly different perceptions on how their gender impacted their ability to obtain a chosen job (p < 0.01), with 100 (23.8%) AO and 52 (21.2%) non-AO reporting a negative or somewhat negative impact, and 116 (27.6%) AO and 101 (41.2%) non-AO reporting a positive or somewhat positive impact. More than half of women surveyed (230 AO, 54.6%; 123 non-AO, 50.6%; p = 0.61) felt that they were somewhat or much less likely to be promoted compared to male colleagues. The majority of women reported they would choose the same career path again (71% of AO, 69% of non-AO); however, 92 (21.9%) of AO responded that they were likely or very likely to pursue a career outside academics in the next 5 years. Conclusions: While partners/family have a substantial impact on 1 in 4 women oncologists, this does not differ between AO and non-AO. Significantly more non-AO find their gender to have positively impacted their ability to obtain their chosen job. In contrast, a meaningful number of AO report a poor sense of belonging and perceived discrimination in obtaining jobs and being promoted; 1 in 5 are considering leaving academia. Academic oncology remains at high risk for continued gender inequality if the culture is not addressed.