Background: Recently, our lab was the first to identify the functional presence of transient receptor potential (TRP) channels of the ankyrin 1 subtype (TRPA1) in mouse cardiomyocytes (CMs). However, the extent to which TRPA1 mediates signaling events in cardiac tissue has yet to be fully determined. Based upon investigations completed in other tissue types in the body, we hypothesized that TRPA1 activation will lead to Akt and eNOS activation in CMs.

Methods: CMs were untreated or treated with the TRPA1-specific agonist, allyl isothiocyanate (AITC), in the presence of absence of Akt inhibitor (LY294002) or eNOS inhibitor (L-NAME) and prepared for immunoblot analysis. Antibodies recognizing Akt phosphorylation at serine 473 (pAkt-S 473 ) and eNOS phosphorylation at serine 1177 (peNOS-S 1177 ) were utilized, as these sites are regularly used as indicators of protein activation. Results were obtained using an ImageQuant and quantified utilizing ImageJ software.

Results: Wild-type CMs treated with AITC demonstrated a significant increase in phosphorylated Akt and eNOS compared to the untreated control. These effects were absent in CMs obtained from TRPA1 null mice. Furthermore, CMs pretreated with LY294002 before AITC addition demonstrated a significant lowering of peNOS-S 1177 ., whereas pretreatment of CMs with L-NAME had no effect on the AITC-induced increase in pAkt-S 473 levels.

Conclusions: Herein, we identify a novel signaling cascade in adult mouse cardiomyocytes whereby TRPA1 stimulation elicits Akt phosphorylation followed by eNOS phosphorylation. As Akt and eNOS activation have direct implications in a myriad of cardiovascular physiological and pathophysiological events, the current results have potential for significant clinical applications.

Cocaine use creates a long term lengthening of circadian period (tau), which could underlie the significant health issues of cocaine addiction. Additionally the rewarding effects of paternal cocaine are transgenerational. We hypothesize that the tau lengthening effect of cocaine may also be transgenerational, causing altered subjective cocaine reward response in offspring. Male mice were exposed to forced cocaine-water (0.5 mg/ml; experimental) or water (control) and harem-mated with cocaine naïve females. Offspring were behaviorally phenotyped for drug or sucrose preference and circadian behaviors were analyzed in individual mice. RNA was isolated from the nucleus accumbens of cocaine naïve F1 males and a HiSeq RNA analysis was run. A long-term lengthening of tau after drug withdrawal was evident in sires with forced cocaine (p=0.041). Tau, alpha, and subjective daytime bouts were not altered in the F1’s. Cocaine preference was decreased in cocaine-sired F1 males compared to those from control sires (p=0.006); however, no difference was found in females. There were no differences in sucrose preference or ethanol preference. RNAseq and analysis revealed a significant correlation between genes upregulated in CocSire Males and genes upregulated by the expression of CREB and short-term ΔFosB. These data reveal that there is no transgenerational transmission of a cocaine-lengthened tau phenotype. However, paternal cocaine exposure significantly altered F1 preference for cocaine, but not sucrose or ethanol, suggesting a selective effect on cocaine reward mechanisms. Thus, cocaine addiction can be influenced by transgenerational paternal mode(s) of inheritance that alters gene expression.

Osteoarthritis is a severe joint disease that affects millions of people. At this time, the current treatment for osteoarthritis is total joint reconstruction surgery. GPNMB/Osteoactivin plays a key role in bone remodeling and bone growth. Data from our lab suggested that GPNMB/Osteoactivin is positive regulator of osteoblastogenesis and a negative regulator of osteoclastogenesis. Furthermore, the role of GPNMB/Osteoactivin in cartilage has not been investigated before. Treatment of human chondrocytes with GPNMB/Osteoactivin results in a significant increase in collagen Type II and Sox9 expression. In this study, we examined whether recombinant GPNMB/Osteoactivin has an anabolic effect on a model of post-traumatic osteoarthritis. The DMM (destabilization of the medial meniscus) in mice has been found to be an excellent model for studying post-traumatic osteoarthritis . We performed the DMM surgery on 28 C57/BL6 mice. Moderate to severe osteoarthritis develops around six to eight weeks with this model. We will inject pharmaceutical grade recombinant GPNMB/Osteoactivin into the joint capsule of the mice at six weeks. The experiment will be terminated at twelve weeks, and the joints will be collected for microCT analysis and immunohistochemistry. The serum from the blood will be analyzed via Elisa. Based on preliminary studies performed in our lab, we expect cartilage degeneration to be dramatically decreased in response to the therapeutic effects of GPNMB/Osteoactivin. A protective factor against osteoarthritis progression, osteoactivin-injected mice should have remarkably better articular cartilage in comparison to the control group, proving GPNMB/Osteoactivin a promising therapy in lieu of total joint reconstruction.

The greater metabolic cost associated with concentric muscle contractions, compared to eccentric muscle contractions, is well known. However, the influence this metabolic cost difference has on post exercise hypotension and arterial stiffness is unknown. Thus the purpose of this investigation was to determine changes in blood pressure and arterial stiffness following a session of concentric only and eccentric only resistance exercise. Twelve subjects performed 2 exercise training sessions that consisted of 6 separate lower and upper body resistance exercises that were comprised of either eccentric only or concentric only muscle contractions. Mean arterial pressure, pulse wave velocity, heart rate and blood lactate were assessed at baseline as well as immediately, 30 and 90 minutes post. The VO2 requirement across the whole concentric exercise session was 50.0% greater than eccentric exercise. MAP following the bout of eccentric exercise was elevated by 5% (p

Microglia are highly motile, phagocytic cells that are capable of releasing various signaling molecules including inflammatory cytokines (e.g. IL-1, TNF-alpha, and IL-6). Recent studies have indicated that stress has profound impacts on microglia morphology, density, proliferation, and activation state, implicating their role in certain pathologies (Tyan et al). Our lab has previously shown that exposure to chronic stress induces anhedonic behavioral responses in Fischer-344 rats after a 4-day chronic mild stress paradigm (Camp et al). To examine the activation state of microglia after this chronic mild stress protocol, microglia were isolated from the hippocampus and placed in culture to measure pro-inflammatory cytokines and their phagocytic capacity. Following 4h in culture basal IL-1, TNF-alpha, and IL-6 protein production were not altered in microglia collected from stressed animals compared to microglia collected from controls. After LPS stimulation IL-1 production was attenuated in microglia collected from stressed animals while there were no changes in the production of TNF-alpha or IL-6. Microglia stimulated with LPS and treated with the highest dose of norepinephrine resulted in an increase in IL-1 production and a decrease in TNF-alpha production in both control and stressed animals. Evidence has implicated that following stress exposure, peripheral monocytes translocate to the brain and have shown to promote anxiety like behavior (Wohleb et al). To look at the affect stress has on peripheral immune cells, phagocytic ability was measured. Trends indicate that norepinephrine inhibits phagocytosis in home cage controlled animals while peripheral immune cells from stressed animals show little response to norepinephrine.

PROBLEM: To date, no studies have evaluated sex-specific differences in response to an acute bout of resistance exercise (RE) on vascular function. Therefore, we sought to determine the sex-specific responses of pulse wave reflection and aortic arterial stiffness after an acute bout of RE. METHODS: Aortic hemodynamics, pulse wave reflection, and aortic arterial stiffness were assessed at rest and 10 minutes after 3 sets of 10 repetitions at 75% 1-repetition maximum on the squat, bench press, and deadlift in resistance-trained men (n=14) and women (n=12). A mixed ANOVA was used to analyze the effects of sex (men, women) across condition (acute resistance exercise, control) and time (rest, recovery). RESULTS: The sexes had similar values at rest and after the acute RE. There was no effect of the RE on brachial or aortic blood pressures. There were significant condition x time interactions for the augmentation index (AIx: rest: 12.1±7.9%; recovery:19.9±10.5%, p=0.003), AIx at 75bpm (rest: 5.3±7.9%; recovery: 24.5±14.3%, p=0.0001), augmentation pressure (rest: 4.9±2.8mmHg; recovery: 8.3±6.0mmHg, p=0.004), and aortic arterial stiffness (rest: 5.3±0.6ms; recovery: 5.9±0.7ms, p=0.02) with significant increases after the acute RE. There was also a significant condition x time for the time of the reflected wave (rest: 150±7ms; recovery: 147±9ms, p=0.02) and the subendocardial variability ratio (rest: 147±17%; recovery: 83±24%, p=0.0001) such that they were reduced after the acute RE. CONCLUSIONS: These data suggest that an acute bout of RE increases pulse wave reflection and aortic arterial stiffness similarly between the sexes without significantly altering aortic hemodynamics.

Oxidative stress has been shown to be an early predictor and key pathological feature of neurodegenerative diseases including Alzheimer’s disease (AD). Previous work in our laboratory has demonstrated that administration of the peptide hormone amylin can reduce levels of oxidative stress in a transgenic mouse model of AD (APP/PS1) as well as Neuroscreen-1 cultures (neuronal cell model). Although it is apparent that amylin has antioxidant activity, its mechanism of action remains unclear. Because mitochondria are the main producers of reactive oxygen species (ROS) that account for oxidative stress in a cell, they have been a target of our investigation. Specifically, we determined whether amylin treatment regulates mitochondrial dynamics-associated proteins and mobility deficits in the APP/PS1 AD mouse model. Our data show that amylin treatment regulates mitochondrial dynamics-associated proteins as well as proteins associated with mitochondrial biogenesis. Taken together, our data suggest that some of the effects of amylin on oxidative stress regulation may stem from improving mitochondrial function.

Abstract

The reduced metabolic cost of isolated eccentric contractions compared to concentric contractions is well established. However, the net impact of this difference across an entire session of resistance exercise has yet to be determined. Thus, the purpose of this investigation was to determine the extent to which metabolic and cardiovascular stress varied between training sessions comprised only of eccentric, concentric or traditional resistance exercises. Twelve subjects completed a traditional (TRAD), concentric (CON), and eccentric (ECC) full-body resistance workout comprised of 6 upper and lower body exercises. Resistance was set at 65% of their 1 repetition max and mechanical work was matched across conditions. Oxygen consumption (VO₂), respiratory exchange ratio (RER), heart rate (HR), mean arterial pressure (MAP), and blood lactate were recorded throughout the exercise sessions. Both the TRAD (9.26±1.83ml/kg/min) and CON (10.03±1.63ml/kg/min) conditions resulted in greater VO₂ values when compared to the ECC (6.67±1.25ml/kg/min) condition. HR and MAP values were 46% and 4% greater in CON compared to that of ECC conditions. Furthermore, RER during CON (1.00±0.03) and TRAD (1.03±0.04) were greater than during ECC (0.88±0.09). Blood lactate was also greater in CON (8.9±1.7mM) and TRAD (9.0±3.0mM) compared to ECC (2.5±1.9mM) (p

Luteinizing hormone (LH) plays an important role in learning and memory as well as Alzheimer’s Disease (AD). Previously, our laboratory has demonstrated that there is an inverse relationship between blood and brain levels of LH and that when levels are normalized post-ovariectomy, both cognition and spine density losses are reversed. Because these differences appear to depend on the reproductive status of the mouse, we determined whether normal cyclic fluctuations in LH during the estrous cycle correlate with neuronal markers of neuroplasticity that are associated with cognition. To address this, we measured serum LH levels using sandwich RIA ELISAs and determined changes in synaptophysin and spinophilin in cognition-associated brain regions. As expected, preliminary data suggests that LH levels fluctuate during the estrous cycle and may correlate with synaptic markers associated with cognitive function. Given that others have found that cognition function fluctuates during the estrous cycle, our data demonstrates that these fluctuations may be at least, in part, dependent on changes in serum and brain LH.

Obesity is a leading health problem in the United States, despite the abundance of weight-loss programs available. There is an increasing interest in weight-loss methods that use time-restricted feeding, including intermittent fasting, where days of limited food access are alternated with days of normal food intake. To determine the benefits of intermittent fasting to obesity, we employed a rat model of inherent leanness and obesity, with the lean rats being more physically active (high-capacity runners) than the obesity-prone (low-capacity runners). Our hypothesis stated that intermittent fasting would induce loss of fat and lean mass in both rat types with the obesity-prone rats losing more body weight. Body weight and composition were measured before intermittent fasting in lean and obesity-prone male rats (n=8/group). Rats were allowed free access to food on fed days, alternating with fasting days (no calories consumed). Rats were weighed daily and the body composition was analyzed weekly using magnetic resonance spectroscopy (EchoMRI). All rats lost weight over eight weeks of intermittent fasting. Obesity-prone rats lost significantly more fat mass and body weight compared to the lean rats, whereas loss of lean mass differed by only a few grams. Results were similar in female rats but contrasted with previous findings when weight loss was induced using 50% calorie restriction. Compared to standard calorie restriction, intermittent fasting decreased the loss of lean mass while enhancing the loss of fat mass. Intermittent fasting was thus found to be successful in inducing weight loss in obese rats.