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Background: High doses of doxorubicin put cancer patients at risk for developing dilated cardiomyopathy. Previously, we showed that doxorubicin treatment decreases SIRT3 (sirtuin 3), the main mitochondrial deacetylase and increases protein acetylation in rat cardiomyocytes. Here, we hypothesize that SIRT3 expression can attenuate doxorubicin induced dilated cardiomyopathy in vivo by preventing the acetylation of mitochondrial proteins. Methods: Nontransgenic, M3-SIRT3 (truncated SIRT3; short isoform), and M1-SIRT3 (full-length SIRT3; mitochondrial localized) transgenic mice were treated with doxorubicin for 4 weeks (8 mg/kg body weight per week). Echocardiography was performed to assess cardiac structure and function and validated by immunohistochemistry and immunofluorescence (n=4–10). Mass spectrometry was performed on cardiac mitochondrial peptides in saline (n=6) and doxorubicin (n=5) treated hearts. Validation was performed in doxorubicin treated primary rat and human induced stem cell derived cardiomyocytes transduced with adenoviruses for M3-SIRT3 and M1-SIRT3 and deacetylase deficient mutants (n=4–10). Results: Echocardiography revealed that M3-SIRT3 transgenic mice were partially resistant to doxorubicin induced changes to cardiac structure and function whereas M1-SIRT3 expression prevented cardiac remodeling and dysfunction. In doxorubicin hearts, 37 unique acetylation sites on mitochondrial proteins were altered. Pathway analysis revealed these proteins are involved in energy production, fatty acid metabolism, and oxidative stress resistance. Increased M1-SIRT3 expression in primary rat and human cardiomyocytes attenuated doxorubicin-induced superoxide formation, whereas deacetylase deficient mutants were unable to prevent oxidative stress. Conclusions: Doxorubicin reduced SIRT3 expression and markedly affected the cardiac mitochondrial acetylome. Increased M1-SIRT3 expression in vivo prevented doxorubicin-induced cardiac dysfunction, suggesting that SIRT3 could be a potential therapeutic target for mitigating doxorubicin-induced dilated cardiomyopathy.

Obesity often originates in early life, and is linked to excess sugar intake. Nonnutritive sweeteners (NNS) are widely consumed as “healthier” alternatives to sugar, yet recent evidence suggests NNS may adversely influence weight gain and metabolic health. The impact of NNS during critical periods of early development has rarely been studied. We investigated the effect of prenatal NNS exposure on postnatal adiposity and adipocyte development. In the CHILD birth cohort (N = 2298), we assessed maternal NNS beverage intake during pregnancy and child body composition at 3 years, controlling for maternal BMI and other potential confounders. To investigate causal mechanisms, we fed NNS to pregnant C57BL6J mice at doses relevant to human consumption (42 mg/kg/day aspartame or 6.3 mg/kg/day sucralose), and assessed offspring until 12 weeks of age for: body weight, adiposity, adipose tissue morphology and gene expression, glucose and insulin tolerance. We also studied the effect of sucralose on lipid accumulation and gene expression in cultured 3T3-L1 pre-adipocyte cells. In the CHILD cohort, children born to mothers who regularly consumed NNS beverages had elevated body mass index (mean z-score difference +0.23, 95% CI 0.05–0.42 for daily vs. no consumption, adjusted for maternal BMI). In mice, maternal NNS caused elevated body weight, adiposity, and insulin resistance in offspring, especially in males (e.g., 47% and 15% increase in body fat for aspartame and sucralose vs. controls, p

Doxorubicin (DOX) is a chemotherapeutic with dose-dependent cardiotoxic effects that limits its use in patients. Previously we showed that DOX decreases expression of the mitochondrial lysine deacetylase SIRT3 in the mouse heart. We hypothesize that DOX impairs cardiac function and energy production through reduced SIRT3 and altered mitochondrial acetylation. We further hypothesize that increased SIRT3 expression could attenuate DOX-induced cardiac dysfunction via alterations of protein acetylation to enzymes involved in lipid remodeling and metabolic processes. Mice expressing cardiac restricted full length M1-SIRT3 (mitochondrial localized), and short M3-SIRT3 (lacking localization signal) received saline or DOX injections of 8 mg/kg body weight for 4 weeks and compared to non-transgenic (Non-Tg) littermates. Transthoracic echocardiography was performed on all mice (n=10). Total cardiac lipids were isolated from DOX treated cardiac tissue by chloroform:methanol extraction and global lipid analysis was performed by QTRAP LC-MS/MS (n=6). Cardiac mitochondria were and an anti-acetylated lysine antibody was used to enrich for tryptic digested peptides containing Acetyl-K and analyzed by QTRAP LC-MS/MS (n=6). In non-Tg mice, DOX caused cardiac dysfunction and expression of M1-SIRT3 and M3-SIRT3 transgenes in the heart preserved left ventricular posterior wall thickness (P

Myocardin is a transcriptional co-activator required for cardiovascular development, but also promotes cardiomyocyte survival through an unclear molecular mechanism. Mitochondrial permeability transition is implicated in necrosis, while pore closure is required for mitochondrial maturation during cardiac development. We show that loss of myocardin function leads to subendocardial necrosis at E9.5, concurrent with elevated expression of the death gene Nix. Mechanistically, we demonstrate that myocardin knockdown reduces microRNA-133a levels to allow Nix accumulation, leading to mitochondrial permeability transition, reduced mitochondrial respiration, and necrosis. Myocardin knockdown elicits calcium release from the endo/sarcoplasmic reticulum with mitochondrial calcium accumulation, while restoration of microRNA-133a function, or knockdown of Nix rescues calcium perturbations. We observed reduced myocardin and elevated Nix expression within the infarct border-zone following coronary ligation. These findings identify a myocardin-regulated pathway that maintains calcium homeostasis and mitochondrial function during development, and is attenuated during ischemic heart disease. Given the diverse role of Nix and microRNA-133a, these findings may have broader implications to metabolic disease and cancer.

Gestational diabetes mellitus (GDM) is the most common complication of pregnancy. Children exposed to GDM are at an increased risk of developing cardiometabolic diseases later in life, though the mechanisms responsible are unknown. We hypothesize that fetal exposure to GDM induces alterations in cardiomyocyte metabolism and concomitant left ventricular (LV) dysfunction with age. GDM was induced in female rats with a high fat (45% kcal) and sucrose diet prior to mating, throughout pregnancy and lactation. Lean control females received a low fat (10% kcal) diet. Fetal rat ventricular cardiomyocytes (FRVC) were isolated from e20.5 offspring for U-13C glucose metabolic flux analysis and mitochondrial respiration. Serum metabolites and cardiac transcriptome profiles were measured in 3-month old offspring. LV morphology and function was assessed through the life course of the offspring (e18 to 12-months of age) by transthoracic ultrasound. Offspring exposed to GDM exhibited increased LV posterior wall thickness across their life course (fetal to 12-months of age; p

Overweight and obesity affect over 20% of children worldwide. Emerging evidence shows that nonnutritive sweeteners (NNS) could adversely influence weight gain and metabolic health, particularly during critical periods of development. Thus, we aimed to investigate the impact of prenatal NNS exposure on postnatal growth and adiposity. Among 2298 families participating in the CHILD cohort study, children born to mothers who regularly consumed NNS during pregnancy had elevated body mass index and adiposity at 3 years of age. In a complementary study designed to eliminate confounding by human lifestyle factors and investigate causal mechanisms, we exposed pregnant mice and cultured adipocytes to NNS (aspartame or sucralose) at doses relevant to human consumption. In mice, maternal NNS exposure caused elevated body weight, adiposity and insulin resistance in offspring, especially in males. Further, in 3T3-L1 pre-adipocyte cells, sucralose exposure during early stages of differentiation caused increased lipid accumulation and expression of adipocyte differentiation genes (e.g. C/EBP-α, FABP4, FAS). The same genes were upregulated in the adipose tissue of male mouse offspring born to sucralose-fed dams. Together, these clinical and experimental findings provide evidence suggesting that maternal NNS consumption induces obesity risk in the offspring through effects on adiposity and adipocyte differentiation.One Sentence SummaryMaternal consumption of non-nutritive sweeteners during pregnancy stimulates adipocyte differentiation, insulin resistance, weight gain, and adiposity in mouse and human offspring.

Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electron transfer resulting in the production of reactive oxygen species (ROS). Sirtuin-3 (SIRT3) is a class III lysine deacetylase that is localized to the mitochondria and regulates mitochondrial respiration and oxidative stress resistance enzymes such as superoxide dismutase-2 (SOD2). The purpose of this study was to determine whether SIRT3 prevents DOX-induced mitochondrial ROS production. Administration of DOX to mice suppressed cardiac SIRT3 expression, and DOX induced a dose-dependent decrease in SIRT3 and SOD2 expression in H9c2 cardiomyocytes. SIRT3-null mouse embryonic fibroblasts produced significantly more ROS in the presence of DOX compared with wild-type cells. Overexpression of wild-type SIRT3 increased cardiolipin levels and rescued mitochondrial respiration and SOD2 expression in DOX-treated H9c2 cardiomyocytes and attenuated the amount of ROS produced following DOX treatment. These effects were absent when a deacetylase-deficient SIRT3 was expressed in H9c2 cells. Our results suggest that overexpression of SIRT3 attenuates DOX-induced ROS production, and this may involve increased SOD2 expression and improved mitochondrial bioenergetics. SIRT3 activation could be a potential therapy for DOX-induced cardiac dysfunction.

Myocardin is a transcriptional co-activator required for cardiovascular development, but also promotes cardiomyocyte survival through an unclear molecular mechanism. Mitochondrial permeability transition is implicated in necrosis, while pore closure is required for mitochondrial maturation during cardiac development. We show that loss of myocardin function leads to subendocardial necrosis at E9.5, concurrent with elevated expression of the death gene Nix. Mechanistically, we demonstrate that myocardin knockdown reduces microRNA-133a levels to allow Nix accumulation, leading to mitochondrial permeability transition, reduced mitochondrial respiration, and necrosis. Myocardin knockdown elicits calcium release from the endo/sarcoplasmic reticulum with mitochondrial calcium accumulation, while restoration of microRNA-133a function, or knockdown of Nix rescues calcium perturbations. We observed reduced myocardin and elevated Nix expression within the infarct border-zone following coronary ligation. These findings identify a myocardin-regulated pathway that maintains calcium homeostasis and mitochondrial function during development, and is attenuated during ischemic heart disease. Given the diverse role of Nix and microRNA-133a, these findings may have broader implications to metabolic disease and cancer.