Danielle R. Bruns

Nrf2 Signaling and the Slowed Aging Phenotype: Evidence from Long-Lived Models

Studying long-lived animals provides novel insight into shared characteristics of aging and represents a unique model to elucidate approaches to prevent chronic disease. Oxidant stress underlies many chronic diseases and resistance to stress is a potential mechanism governing slowed aging. The transcription factor nuclear factor (erythroid-derived 2)-like 2 is the “master regulator” of cellular antioxidant defenses. Nrf2 is upregulated by some longevity promoting interventions and may play a role in regulating species longevity. However, Nrf2 expression and activity in long-lived models have not been well described. Here, we review evidence for altered Nrf2 signaling in a variety of slowed aging models that accomplish lifespan extension via pharmacological, nutritional, evolutionary, genetic, and presumably epigenetic means.

Long-lived crowded-litter mice have an age-dependent increase in protein synthesis to DNA synthesis ratio and mTORC1 substrate phosphorylation

Increasing mouse litter size [crowded litter (CL)] presumably imposes a transient nutrient stress during suckling and extends lifespan through unknown mechanisms. Chronic calorically restricted and rapamycin-treated mice have decreased DNA synthesis and mTOR complex 1 (mTORC1) signaling but maintained protein synthesis, suggesting maintenance of existing cellular structures. We hypothesized that CL would exhibit similar synthetic and signaling responses to other long-lived models and, by comparing synthesis of new protein to new DNA, that insight may be gained into the potential preservation of existing cellular structures in the CL model. Protein and DNA synthesis was assessed in gastroc complex, heart, and liver of 4- and 7-mo CL mice. We also examined mTORC1 signaling in 3- and 7-mo aged animals. Compared with controls, 4-mo CL had greater DNA synthesis in gastroc complex with no differences in protein synthesis or mTORC1 substrate phosphorylation across tissues. Seven-month CL had less DNA synthesis than controls in heart and greater protein synthesis and mTORC1 substrate phosphorylation across tissues. The increased new protein-to-new DNA synthesis ratio suggests that new proteins are synthesized more so in existing cells at 7 mo, differing from 4 mo, in CL vs. controls. We propose that, in CL, protein synthesis shifts from being directed toward new cells (4 mo) to maintenance of existing cellular structures (7 mo), independently of decreased mTORC1.

Long-lived Snell dwarf mice display increased proteostatic mechanisms that are not dependent on decreased mTORC1 activity

Maintaining proteostasis is thought to be a key factor in slowed aging. In several growth‐restricted models of long‐life, we have shown evidence of increased proteostatic mechanisms, suggesting that proteostasis may be a shared characteristic of slowed aging. The Snell dwarf mouse is generated through the mutation of the Pit‐1 locus causing reductions in multiple hormonal growth factors and mTORC1 signaling. Snell dwarfs are one of the longest lived rodent models of slowed aging. We hypothesized that proteostatic mechanisms would be increased in Snell compared to control (Con) as in other models of slowed aging. Using D2O, we simultaneously assessed protein synthesis in multiple subcellular fractions along with DNA synthesis in skeletal muscle, heart, and liver over 2 weeks in both sexes. We also assessed mTORC1‐substrate phosphorylation. Skeletal muscle protein synthesis was decreased in all protein fractions of Snell compared to Con, varied by fraction in heart, and was not different between groups in liver. DNA synthesis was lower in Snell skeletal muscle and heart but not in liver when compared to Con. The new protein to new DNA synthesis ratio was increased threefold in Snell skeletal muscle and heart compared to Con. Snell mTORC1‐substrate phosphorylation was decreased only in heart and liver. No effect of sex was seen in this study. Together with our previous investigations in long‐lived models, we provide evidence further supporting proteostasis as a shared characteristic of slowed aging and show that increased proteostatic mechanisms may not necessarily require a decrease in mTORC1.

Mitochondrial Integrity in a Neonatal Bovine Model of Right Ventricular Dysfunction

Right ventricular (RV) function is a key determinant of survival in patients with both RV and left ventricular (LV) failure, yet the mechanisms of RV failure are poorly understood. Recent studies suggest cardiac metabolism is altered in RV failure in pulmonary hypertension (PH). Accordingly, we assessed mitochondrial content, dynamics, and function in hearts from neonatal calves exposed to hypobaric hypoxia (HH). This model develops severe PH with concomitant RV hypertrophy, dilation, and dysfunction. After 2 wk of HH, pieces of RV and LV were obtained along with samples from age-matched controls. Comparison with control assesses the effect of hypoxia, whereas comparison between the LV and RV in HH assesses the additional impact of RV overload. Mitochondrial DNA was unchanged in HH, as was mitochondrial content as assessed by electron microscopy. Immunoblotting for electron transport chain subunits revealed a small increase in mitochondrial content in HH in both ventricles. Mitochondrial dynamics were largely unchanged. Activity of individual respiratory chain complexes was reduced (complex I) or unchanged (complex V) in HH. Key enzymes in the glycolysis pathway were upregulated in both HH ventricles, alongside upregulation of hypoxia-inducible factor-1α protein. Importantly, none of the changes in expression or activity were different between ventricles, suggesting the changes are in response to HH and not RV overload. Upregulation of glycolytic modulators without chamber-specific mitochondrial dysfunction suggests that mitochondrial capacity and activity are maintained at the onset of PH, and the early RV dysfunction in this model results from mechanisms independent of the mitochondria.

Genetic ablation of interleukin-18 does not attenuate hypobaric hypoxia-induced right ventricular hypertrophy

Interleukin-18 (IL-18), a proinflammatory cytokine, has been implicated in pathologic left ventricular hypertrophy and is elevated in plasma of heart failure patients. However, IL-18 blockade strategies have been conflicting. The purpose of these experiments was to determine whether genetic ablation of IL-18 would protect mice against hypobaric hypoxia (HH)-induced right ventricular (RV) hypertrophy, a condition in which chamber-specific inflammation is prominent. We hypothesized that IL-18 knockout (KO) mice would be protected while wild-type (WT) mice would demonstrate RV hypertrophy in response to HH exposure. KO and WT mice were exposed to HH for 7 wk, and control mice were exposed to normoxic ambient air. Following echocardiography, the RV was dissected and flash-frozen for biochemical analyses. HH exposure increased IL-18 mRNA (P = 0.08) in RV from WT mice. Genetic ablation of IL-18 mildly attenuated RV hypertrophy as assessed by myocyte size. However, IL-18 KO mice were not protected against HH-induced organ-level remodeling, as evidenced by higher RV weights, elevated RV systolic pressure, and increased RV anterior wall thickness compared with normoxic KO mice. These RV changes were similar to those seen in HH-exposed WT mice. Compensatory upregulation of other proinflammatory cytokines IL-2 and stromal cell-derived factor-1 was seen in the HH-KO animals, suggesting that activation of parallel inflammatory pathways might mitigate the effect of IL-18 KO. These data suggest targeted blockade of IL-18 alone is not a viable therapeutic strategy in this model.

Differential Effects of Vitamin C or Protandim on Skeletal Muscle Adaptation to Exercise

Maintaining proteostasis is a key mechanism for preserving cell function. Exercise-stimulated proteostasis is regulated, in part, by redox-sensitive signaling. Several studies suggest that supplementation with exogenous antioxidants blunts exercise-induced cellular adaptations, although this conclusion lacks consensus. Our group uses a fundamentally different approach to maintain redox balance by treatment with bioactive phytochemicals to activate the transcription factor nuclear factor (erythroid-derived 2)-like 2 and downstream endogenous antioxidant pathways. We hypothesized that vitamin C (VitC) would interfere with redox-sensitive proteostatic mechanisms in skeletal muscle, whereas phytochemical treatment would permit proteostatic maintenance. We measured protein and DNA synthesis in skeletal muscle from high-volume voluntary wheel-running rats. Whereas phytochemical treatment permitted mitochondrial and other proteostatic adaptations to exercise, VitC treatment did not. During an in vitro oxidative challenge, phytochemical treatment helped maintain proteostasis, including the mitochondrial fraction while VitC did not. Our findings support the conclusion that VitC can blunt some of the beneficial adaptations to exercise. We propose that regulation of endogenous antioxidants represents a novel approach to maintain redox balance while still permitting redox-sensitive proteostatic adaptations. NEW & NOTEWORTHY Whether vitamin C blocks aerobic exercise adaptions lacks consensus, perhaps because of approaches that only assess markers of mitochondrial biogenesis. By directly measuring mitochondrial biogenesis, we demonstrate that vitamin C blunts exercise-induced adaptations. Furthermore, we show that treatment with Protandim, a purported nuclear factor (erythroid-derived 2)-like 2 activator that upregulates endogenous antioxidants, permits mitochondrial biogenesis. We confirm that vitamin C blunts aerobic exercise adaptions, whereas Protandim does not, suggesting targeting the endogenous antioxidant network facilitates adaptations to exercise.

Interleukin-19 is cardioprotective in dominant negative cyclic adenosine monophosphate response-element binding protein-mediated heart failure in a sex-specific manner

AIM To investigate the role of interleukin-19 (IL-19) in a murine model of female-dominant heart failure (HF). METHODS Expression of one copy of a phosphorylation-deficient cyclic adenosine monophosphate response-element binding protein (dnCREB) causes HF, with accelerated morbidity and mortality in female mice compared to males. We assessed expression of IL-19, its receptor isoforms IL-20R α/β, and downstream IL-19 signaling in this model of female-dominant HF. To test the hypothesis that IL-19 is cardioprotective in dnCREB-mediated HF, we generated a novel double transgenic (DTG) mouse of dnCREB and IL-19 knockout and assessed cardiac morbidity by echocardiography and survival of male and female mice. RESULTS IL-19 is expressed in the murine heart with decreased expression in dnCREB female compared to male mice. Further, the relative expression of the two IL-19 receptor isoforms manifests differently in the heart by sex and by disease. Male DTG mice had accelerated mortality and cardiac morbidity compared to dnCREB males, while female DTG mice showed no additional detriment, supporting the hypothesis that IL-19 is cardioprotective in this model. CONCLUSION Together, these data suggest IL-19 is an important cytokine mediating sex-specific cardiac (dys) function. Ongoing investigations will elucidate the mechanism(s) of sex-specific IL-19 mediated cardiac remodeling.