bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic Interval Exercise Modifies the Myocardial Lipid Profile in a Miniature Swine Model of Heart Failure with Preserved Ejection Fraction
Authors: Emter Craig Allen , Rector R. Scott , Baines Christopher P. , Ford Kaitlin D. , Ford David A.
Published: June 2021

Manipulation of dietary lipid intake and exercise has demonstrated beneficial effects in preventing the progression of heart failure (HF). We have previously shown chronic exercise prevents mitochondrial dysfunction characterized by increased mitochondrial permeability transition. Recent studies examining impaired mitochondrial function have focused on cardiolipin, a mitochondria‐localized phospholipid that decreases during HF. Our purpose was to examine the effect of chronic exercise on myocardial lipid phenotype in aortic‐banded mini‐swine with compensated HF. Shotgun lipidomics was used to examine alterations in ventricular lipids in male control (n=7), sedentary HF (n=4), and exercise trained HF (HF‐TR, n=7) animals. Total cardiolipin was significantly increased in the HF‐TR animals. Specific modifications in the cardiolipin pool were found in the tetralinoleoyl cardiolipin molecular species. Differences in triacylglycerol and ceramide molecular species were also observed. In conclusion, our novel results demonstrate the efficacy of exercise in modulating myocardial lipid profile in a large animal model of HF. We propose that our previous observation of exercise‐dependent preservation of normal mitochondrial function may be mediated via modification of myocardial lipid phenotype.

Research support: NIH HL074214, HL111906 (DAF), HL093982‐01, HL101332 (CAE), and VA‐CDA2 (RSR).
c9,t11‐Conjugated linoleic acid alters HDL functionality
Authors: Nicod Nathalie , Parker Parker Stanley , Giordano Elena , Dávalos Alberto , Visioli Francesco
Published: June 2021

Conjugated linoleic acid (CLA) is known to modulate plasma HDL levels. Here we report the effect of CLA on HDL functionality. Caco‐2 monolayers were grown on transwells, on which they can synthesize HDL and take up HDL‐cholesterol via basal SR‐BI. Cells were apically administered linoleic acid or different isomers of CLA (t9,t11‐CLA; c9,t11‐CLA; t10,c12‐CLA) in mixed micelles. HDL was secreted in the basal chamber for 24h in the absence or presence of an antibody to SR‐BI (aSR‐BI) to inhibit its interaction with HDL. Free (FC) and esterified cholesterol (EC) were determined in the basal chamber. FC in the presence of aSR‐BI was similar for all three isomers of CLA, but in the absence of aSR‐BI, FC accumulated significantly more with c9,t11‐CLA, suggesting a lack of uptake: 13% of FC was taken up via SR‐BI with c9,t11‐CLA vs. 30% with the other isomers. When the formation of pre‐β‐HDL particles was induced, cells treated with c9,t11‐CLA showed no uptake of FC via SR‐BI vs. a 25% uptake of FC with the other isomers. To determine if this effect was due to an alteration in HDL functionality, we added mature HDL to the basal chamber. c9,t11‐CLA induced a greater uptake of FC and EC via SR‐BI compared with the other isomers. Our results show that c9,t11‐CLA treatment induces the formation of a less functional pre‐β‐HDL particle. On the other hand, c9,t11‐CLA stimulates a more efficient uptake of cholesterol from a mature HDL particle.
Garlic (Allium sativum) attenuate glomerular glycation in Streptozotocin‐induced diabetic rats
Authors: Al‐Qattan Khaled K , Thomson Martha , Ali Muslim , Mansour Mohamed H
Published: June 2021

Background

In diabetes, hyperglycemia causes microangiopathy while garlic is effective in keeping euglycemia.

Objective

This study investigated the effect of 8 week‐single daily i.p. garlic extract injection on glomerular glycation in Streptozotocin‐induced diabetic rats.

Method

Urine volume and creatinine and serum insulin, glucose, creatinine and erythrocyte hemoglobin glycation (GHb) were measured at different times in diabetic‐control (D‐C), diabetic‐garlic (D‐G) treated and normal‐control (N‐C) rats. Images, glycation and count of normal and abnormal renal corpuscles (RC) were taken and estimated by light microscopy and Periodic Acid Shiff (PAS) staining.

Results

Compared to N‐C, D‐C showed significant decrease in serum insulin and increase in urine volume and creatinine and serum glucose, creatinine and GHb. D‐C also showed prevalent characteristic RC damage and intense diffused glomerular PAS activity. D‐G showed significant increase in serum insulin and decrease in urine volume and creatinine and serum glucose, creatinine, GHb. In D‐G significantly less damage of RC was obvious. In addition, PAS activity and distribution was close to that in N‐C.

Conclusion

Garlic is effective in inducing euglycemia ‐ one way by insulinemia ‐ thus attenuating glomerular glycation and preserving some renal clearance function.
Effect of Induced Hypothermia on Microvascular Inflammation During Hemorrhagic Shock/Resuscitation
Authors: Coyan Garrett N , Moncure Michael , Thomas James H , Holloway Naomi , Wood John G
Published: June 2021

Introduction

Microvascular inflammation caused by hemorrhagic shock is associated with increased morbidity and mortality in trauma patients. Hypothermia has been shown to be a novel approach in preventing morbidity and mortality in several clinical scenarios.

Objective

Our goal was to determine if induced hypothermia during hemorrhagic shock/resuscitation attenuated leukocyte adherence and mast cell degranulation in the rat mesenteric microcirculation.

Methods

Intravital microscopy was used to visualize the mesentery in real time. Ruthenium red was superfused over the mesentery throughout the experiment to assess mast cell degranulation. After a control period, blood pressure was reduced to 40–45 mm Hg for 1 hour by withdrawal of blood. During 2 hours of resuscitation, temperature was reduced to 32–34°C. The animal was then re‐warmed for 2 hours to 37–38°C.

Results

Leukocyte adherence and mast cell degranulation were decreased during hypothermic resuscitation compared to normothermic resuscitation. However, both leukocyte adherence and mast cell degranulation increased during the re‐warming period in comparison to the hypothermic period.

Conclusion

We have shown that induced hypothermia during resuscitation attenuates microvascular inflammation, but this response may only be temporary as shown by an increase in leukocyte adherence during the re‐warming period.
Overexpression of Mn superoxide dismutase attenuates age related up‐regulation of TGF‐ß and remodeling in the aging heart
Authors: Lee Yang , Kwak Hyo‐Bum , Kim Jong‐Hee , Van Remmen Holly , Richardson Arlan , Lawler John M
Published: June 2021

Aging results in accumulation of extracellular matrix proteins and impairment of cardiac function. Oxidative stress is a potential mechanism in age‐associated cardiac fibrosis. We have previously show exercise training reduces fibrosis and expression of transforming growth factor‐beta (TGF‐ß1) in the aging heart, concomitant with a reduction in oxidative stress and upregulation of the manganese‐isoform of superoxide dismutase (MnSOD). However, the causal relationship between MnSOD and TGF‐ß1 in the aging heart is unknown. Thus, we tested the hypothesis that overexpression of Mn‐SOD would reduce TGF‐ß1 and markers of fibrosis in the aging heart. Old (27 mo) wild type (OWT) animals had larger cardiac myocyte cross‐sectional areas (CSA) that young (4 mo) wild type mice (YWT), an effect reduced in Old Mn‐SOD transgenic mice animals (Otg). TGF‐β1 and collagen I abundance was higher in OWT vs. YWT, while overexpression of MnSOD attenuated TGF‐β1 and collagen I in hearts of old mice. Matrix metalloproteinase‐9 (MMP‐9) was lower in OWT, with age dependent suppression of MMP‐9 prevented by Mn‐SOD. Results indicate that overexpression of Mn‐SOD overexpression protects against age‐related upregulation of TGF‐ß1 and fibrosis, suggestive that elevation of MnSOD with exercise training may indeed be cardioprotective against fibrosis and remodeling. Supported by AHA 0855158F; NIH AR054084
Long‐term Effects of Aliskiren versus Hydrochlorothiazide on Left Ventricular Diastolic Function in Elderly Hypertensive Patients
Authors: Okada Yoshiyuki , Fujimoto Naoki , Jarvis Sara S. , Shibata Shigeki , Bivens Tiffany B. , Meier Rhonda L. , Levine Benjamin D. , Fu Qi
Published: June 2021

A direct rennin inhibitor, Aliskiren, has been found to improve cardiac function in animals with myocardial infarction. We tested whether Aliskiren would modulate left ventricular (LV) diastolic function in elderly hypertensive patients, and compared it to hydrochlorothiazide (HCTZ). Mild hypertensives [65 ± 4 (SD) yrs] were randomly assigned to Aliskiren (n = 10) or HCTZ (n = 7) for 6 months. Blood pressure (BP), cardiac output (Qc), stroke volume (SV) and echocardiographic variables, such as early (E) and late (A) mitral inflow velocity, tissue Doppler velocities of the early (E’) and late (A’) mitral annulus, and color M‐mode propagation velocity (Vp) were recorded before and after treatment. BP decreased to a similar extent in both groups (p < 0.001). Qc, SV, and LV end‐diastolic volume remained unchanged in both groups. In the HCTZ group, E (77 ± 14 vs 69 ± 13 cm/s, p = 0.044) and Vp significantly decreased with a small decrease in E/A ratio (0.97 ± 0.24 vs. 0.87 ± 0.17) and E’ (7.8 ± 1.4 vs 7.3 ± 2.0 cm/s). However, A’ (10.9 ± 1.4 vs 11.7 ± 1.4 cm/s, p = 0.018) significantly increased, suggesting a compensatory increase in late diastolic filling to maintain stroke volume. In the Aliskiren group, no changes were observed in E, E/A ratio, E’, A’, or Vp. Thus, HCTZ decreased early diastolic filling most likely due to decreased LA driving pressure, while Aliskiren had no effects on early or late diastolic filling in hypertensive seniors.
Aging exacerbates obesity‐induced oxidative stress and inflammation in perivascular adipose tissue in mice: a paracrine mechanism contributing to vascular redox dysregulation and inflammation
Authors: Tucsek Zsuzsanna , Bailey‐Downs Lora C , Toth Peter , Sosnowska Danuta , Gautam Tripti , Sonntag William E , Csiszar Anna , Ungvari Zoltan
Published: June 2021

Obesity in the elderly is increasing at alarming rates and there is evidence suggesting that elderly individuals are more vulnerable to the deleterious cardiovascular effects of obesity than younger individuals. To test the hypothesis that aging exacerbates obesity‐induced inflammation in perivascular adipose tissue we assessed changes in the secretome, ROS production and macrophage infiltration in periaortic adipose tissue of young (7 mo) and aged (24 mo) high fat diet (HFD)‐fed obese mice. In young animals HFD‐induced obesity promoted oxidative stress in the perivascular adipose tissue, which was associated with a marked pro‐inflammatory shift in the profile of secreted cytokines and chemokines. Aging exacerbated obesity‐induced oxidative stress and inflammation and significantly increased macrophage infiltration in periaortic adipose tissue. Inflammatory factors secreted from the perivascular fat tissue of obese aged mice promoted significant pro‐oxidative and pro‐inflammatory phenotypic alterations in cultured arteries isolated from young control mice. Overall, localized perivascular adipose tissue inflammation exacerbates vascular oxidative stress and inflammation in aging, an effect that likely enhances the risk for development of cardiovascular diseases from obesity in the elderly.
Caloric restriction and resveratrol attenuate doxorubicin‐induced vascular dysfunction in old rat mesenteric arteries
Authors: Whidden Melissa Ann , Kirichenko Nataliya , Dutta Debapriya , Xu Jinze , Leeuwenburgh Christiaan , Tumer Nihal
Published: June 2021

Doxorubicin (DOX) is associated with oxidant‐induced damage to the endothelium and vascular dysfunction. Both caloric restriction (CR) and resveratrol (Res) have been shown to lower levels of oxidative stress and slow aging. So, we examined CR and Res alone and in combination on DOX‐induced vascular dysfunction in old mesenteric arteries (MA). 26 month male F‐344xBN rats were divided into: ad libitum (AL), AL+DOX, 20% CR, CR+DOX, Res, Res+DOX, CR+Res, and CR+Res+DOX. Over 6 wks rats consumed a 20% CR diet while Res (5 mg/kg/day) was given in pellets. DOX (10 mg/kg BW) was administered 24 hrs prior to sacrifice. Concentration‐response curves to Ach (10–9–10–5 M) with and without L‐NAME were evaluated in pressurized MAs. Endothelium‐mediated relaxation was significantly reduced in DOX (38 ± 4%) when compared with AL (76 ± 2%) rats (P<0.001). Alone, both 20% CR and Res attenuated the DOX‐induced reduction in dilation; 20% CR (56 ± 3%, P<0.05) and Res (70 ± 4%, P<0.01). There was no additive protection from DOX when treatments were used together. L‐NAME decreased responses to Ach in AL and treatment arteries and inhibited the reduced responses in the DOX MAs. These findings demonstrate that both antioxidant treatments contribute to the attenuation of vascular dysfunction found with DOX, however, Res appears to be more protective to the vascular system.

Supported by NIH AG17994, 1P30AG02874, Department of Veteran Affairs.
Smooth muscle microRNAs regulate serotonin‐induced contraction in pulmonary and systemic arteries
Authors: Dahan Diana , Hellstrand Per , Swärd Karl , Albinsson Sebastian
Published: June 2021

MicroRNA (miRNA) are short, non coding RNA that regulate mRNA expression levels and/or translational efficiency. MiRNAs are important for vascular smooth muscle development and function by regulating phenotypic modulation and contractile responses. Contractility of vascular smooth muscle is essential for regulation of blood pressure and blood flow. A number of contractile agonists can affect vascular reactivity and altered sensitivity to these agonists may play a role in vascular disease.

The aim of this study was to identify the role of miRNAs in vascular responses to various contractile agonists.

Vascular reactivity was assessed in isolated pulmonary arteries and aortic rings from tamoxifen‐inducible and smooth muscle specific Dicer KO mice 5 weeks post tamoxifen and in constitutive miR‐143/145 KO mice.

Our results show a specific increase in the contractile response to serotonin in the pulmonary artery and aorta. This effect was not found in miR‐143/145 KO mice and is therefore likely to be dependent on other smooth muscle miRNAs. The increased contractile response to serotonin in Dicer KO vessels was reduced by Src‐inhibitors but less sensitive to Rho‐kinase inhibition.

The precise mechanisms for augmented serotonin‐induced contraction in Dicer KO vessels are now under investigation and may identify a novel role for specific miRNAs in vascular disease.
New causal relationship between PLA2g6, store‐operated Ca2+ entry, refilling of Ca2+ stores and ER stress in mouse embryonic fibroblasts.
Authors: Oslowski Christine Mary , Zhou Qingde , Schafer Claudia , Kirber Michael , Bolotina Victoria
Published: June 2021

Store‐operated Ca2+ entry (SOCE) is essential for maintenance and timely refilling of Ca2+ stores in the endoplasmic reticulum (ER). Here we used molecular, imaging and genetic approaches to unmask new determinants of this process and their role in ER stress and the unfolded protein response (UPR). Analysis of endogenous SOCE in primary embryonic fibroblasts (MEF) from a new knockout mouse model revealed that constitutive genetic impairment of interaction of specific plasma membrane‐associated phospholipase A2 (PLA2g6) with STIM1 (ER‐resident Ca2+ sensor) leads to impairment of endogenous SOCE. Such deficiency resulted in sustained depletion of intracellular Ca2+ stores, and more than 50% reduction in ionomycin‐induced Ca2+ release from the stores. We found that PLA2g6 and SOCE‐deficient MEF cells experience significant ER stress: qRT‐PCR analysis of ER stress markers showed a 2–4 fold increase in mRNA expression levels of BiP, CHOP, and TXNIP. Discovery of a new causal relationship between PLA2g6, SOCE, filling state of ER Ca2+ stores and the UPR unveiled a novel mechanism of ER stress, and identified new molecular targets that can be used for prevention of ER stress‐related pathologies.
Raptor activation by aldosterone promotes apoptosis resistance in pulmonary artery smooth muscle cells to modulate adverse pulmonary vascular remodeling in pulmonary arterial hypertension
Authors: Aghamohammadzadeh Reza , Heagerty Anthony , Loscalzo Joseph , Maron Bradley A , Leopold Jane A
Published: June 2021

In pulmonary arterial hypertension (PAH), hyperaldosteronism (ALDO) is associated with adverse vascular remodeling typified by pulmonary artery smooth muscle cell (PSMC) proliferation and apoptosis resistance. Raptor activation induces PSMC growth and inhibits apoptosis; however, the role of ALDO‐Raptor signaling in PAH vascular remodeling is unknown. We, therefore, treated human PSMCs with ALDO (10−7 mol/L) or vehicle (V) for 1 h. Compared to V‐treated cells, ALDO increased P‐raptor (Ser792) expression by 197% (p<0.05) without affecting total raptor levels. ALDO exposure promoted apoptosis resistance in PSMCs; in cells treated with ALDO prior to hydrogen peroxide (1 mM) exposure for 24 h, ALDO decreased apoptosis by 60% (p=0.02) as determined by an apoptosis ELISA. Next, we assessed the effect of ALDO‐raptor on vessel remodeling in the monocrotaline (MCT)‐induced PAH rat model. Compared to V‐treated rats, immunohistochemistry of pulmonary arterioles from PAH rats demonstrated an 85% (p<0.05) increase in levels of P‐p70S6K (Thr389), which mediates raptor‐dependent cell proliferation. ALDO antagonism with spironolactone (25 mg/kg/d) in PAH rats decreased P‐p70S6K by 49% (p<0.02) and increased vessel lumen area by 268% (p<0.05) compared to untreated PAH rats. Together, these data suggest that ALDO‐raptor signaling in PSMCs is a novel mechanism to modulate vascular remodeling in PAH. Funding: NIH/BHF.
Americans with Diabetes are Adhering to a Low Glycemic Diet but are Eating a High Fat Southern Diet which is Associated with Cardiovascular Disease
Authors: Pearson Keith , Shikany James , Fernandez Jose , Judd Suzanne
Published: June 2021

Traditional diabetes nutrition education has emphasized consuming a high fiber, low glycemic diet. Healthy diet adherence has previously been analyzed by examining individual foods and nutrients; however, focusing on overall dietary patterns may be more practical for disease monitoring. Through factor analysis on 56 food groups (obtained from the Block98 Food Frequency Questionnaire), using a nationally representative sample of 21,636 black and white participants in the REGARDS study (age >; 45), five dietary patterns emerged. Logistic regression was used to calculate odds ratios (OR) for adherence to each dietary pattern. People with diabetes were more likely (OR=1.18, p<0.001) to adhere to a plant based dietary pattern, less likely to adhere to a high sweets pattern (OR=0.77, p<0.001) and more likely to adhere to a Southern style pattern (OR=1.27, p<0.001) after adjustment for age, race, sex, education and income. Our results suggest that even if people with diabetes are adhering to low glycemic recommendations, they are still consuming fried foods and added fats typical of a Southern dietary pattern. Given our previous work showing a twofold increased risk of cardiovascular disease (CVD) with a Southern diet – and since diabetes is a risk equivalent for CVD ‐‐ future education may need to stress a reduction in Southern dietary patterns to improve cardiovascular health.

Grant Funding Source: NIH‐NINDS and a grant from General Mills
Effects of high‐fat diet on metabolic parameters and exercise performance of young and adult rats
Authors: Li Zhen‐Jie , Chen Chiao‐Nan
Published: June 2021

Background and purpose

High fat diet (HFD) decreases mitochondrial function and impairs metabolic characteristics in animals. Interestingly, it has been shown that the very young animals are more resistant to the HFD‐induced mitochondria impairment than young animals. Since the function of mitochondria and the metabolic characteristics are associated with the exercise performance of animals, it is interesting to know whether the negative influence of HFD on exercise performance and metabolic characteristics is less in young animals compared to adult animals.

Method

Young (4‐month‐old) and adult (9‐month‐old) rats were randomized into regular chow diet (control) and HFD groups and received respective diet intervention for 14 weeks. Body weight (BW), systolic blood pressure (SBP) and exercise performance including grip test, Rota‐rod test, and treadmill running test were determined at baseline, after 7 weeks and after 14 weeks of intervention.

Result

Rats, both young and adult, fed with HFD showed greater increase of BW and SBP and less increase of normalized grip strength than control groups after 7 weeks and 14 weeks of diet intervention. Treadmill running time was found significant decreased in both young and adult rats on HFD for 14 weeks.

Conclusion

HFD has negative effects on BW, SBP and exercise performance not only in adult but also in young animals.
Contractile In/Activity Influence Mitochondrial Morphology and Membrane Interactions in Mouse Skeletal Muscle
Authors: Picard Martin , Azuelos Ilan , White Kathryn , Jung Boris , Petrof Basil J , Turnbull Doug M
Published: June 2021

Dynamic changes in mitochondrial morphology regulate mitochondrial function and contribute to cellular function and adaptation in vitro, particularly in response to fluctuations in the metabolic status (energy supply vs demand). However, in skeletal muscle where the metabolic status fluctuates during exercise and disuse, whether mitochondrial morphology is actively remodeled is unclear. To investigate this question, the metabolic state was manipulated in the mouse by either i) a single 3‐hour bout of voluntary exercise on running wheel, or ii) an acute disuse model consisting of 6 hours of mechanical ventilation to unload the diaphragm. We quantified changes in both subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial morphology and membrane interactions with a new method combining scanning (SEM) and transmission electron microscopy (TEM) in two orthogonal planes. Exercise depleted intramyocellular lipid droplets whereas diaphragm inactivity caused their accumulation (P<0.01). Immediately after exercise, the number of electron‐dense contact sites connecting adjacent mitochondria in the soleus was increased by 110% for SS (P<0.05) and 200% for IMF (P<0.01), and the number of continuous IMF mitochondria across Z‐lines doubled (P<0.01). In contrast, the inactive diaphragm contained more small (P<0.05), rounded (P<0.05) and granular (P<0.05) IMF mitochondria, along with numerous autophagosomes, indicating mitochondrial fragmentation. Thus, consistent with in vitro paradigms, skeletal muscle mitochondrial morphology and membrane interactions are remodeled by the metabolic status during contractile in/activity. Work funded by the Wellcome Trust and CIHR.
Direct observation of phosphate inhibiting the force generating capacity of myosin using a laser trap assay
Authors: Debold Edward P , Woodward Mike , Turner Matthew A
Published: June 2021

The accumulation of phosphate (Pi) is believed to cause much of the loss in the force during fatigue, but the molecular basis of this effect remains poorly understood. To gain insight into this process we determined the effect of a fatiguing level of Pi (30mM) on the force‐generating capacity of a mini‐ensemble of skeletal myosin (~8molecules) interacting with a single actin filament using a three bead laser trap assay, at 100uM ATP. In the absence of Pi the average force generated displayed a bimodal Gaussian distribution with a low force peak centered at 0.30 ± 0.02 pN and second higher peak centered at 1.22 ± 0.08 pN. In the presence of Pi a low force peak centered at 0.28 ± 0.02 pN remained but the high force peak was completely absent, greatly reducing the average force produced. The duration of the low force binding events were statistically similar in both the presence (30 ± 4ms) and absence of Pi (28 ± 3ms) as were the average displacements of these events at 14 ± 1nm and 16 ± 1nm respectively, consistent with a mix of single and double molecule binding events. The high force events generated on average 64 ± 4nm displacements lasting 75 ± 8ms, suggesting these resulted from multiple binding events before detachment from the actin filament. The overall frequency of binding events was similar in the presence (3.0/s) and absence of Pi (2.7s). Taken together these data suggest that Pi reduces the force‐generating capacity of a myosin by eliminating the ability to make the successive and simultaneous actin attachments required to generate higher forces.
Ceramide alters mitochondrial dynamics and reduces mitochondrial respiration in skeletal muscle
Authors: Smith Melissa E , Tucker Braden J , Washburn Trevor D , Brassfield Eric S , Stark Michael R , Kane Daniel A , Bikman Benjamin T
Published: June 2021

Fat gain is associated with ceramide accrual in tissues of high‐metabolic rate, such as liver, brain and skeletal muscle. We predict that ceramide accumulation in skeletal muscle affects mitochondrial morphology and physiology. We found that ceramide treatment on murine myoblasts decreased viable mitochondrial density and induced mitochondrial fission, possibly via dynamin‐related protein 1 (Drp1), which was increased with ceramide accrual. Inhibition of endogenous ceramide synthesis via myriocin and serine palmitoyltransferase 2 (SPT2) siRNA prevented fatty acid‐induced mitochondrial fission in myoblasts. Moreover, inhibition of ceramide biosynthesis prevented the reduction in mitochondrial O2 consumption in myoblasts treated with fatty acids. Altogether, these findings suggest a critical role for ceramide in altering mitochondrial dynamics and function. Ongoing work will determine the extent to which mitochondrial fission mediates the adverse alterations in mitochondrial function. This work was supported by a BYU Graduate Research Fellowship (MES) and a BYU Mentoring Environment Grant (BTB).
Tumor necrosis factor‐α (TNF) effects on mitochondrial metabolism in C2C12 myotubes
Authors: McLean Julie , Smith Jeff , Sullivan Patrick , Reid Michael
Published: June 2021

TNF appears to function as an endocrine stimulus causing muscle dysfunction during chronic inflammatory conditions. TNF‐induced reactive oxygen species (ROS) in skeletal muscle act on myofibrillar proteins to diminish force, yet their source remains elusive. Our data intimate that TNF signals via the TNF receptor 1 subtype to activate neutral sphingomyelinase 3, increasing ceramide and stimulating ROS production. In murine skeletal muscle cells (C2C12s), mitochondrial‐specific ROS inhibitor, SS31, ablates TNF‐induced increase in dichlorfluorescein‐diacetate fluorescence (P<.002). We hypothesize that TNF alters mitochondrial electron transport chain function. Control experiments using an automated extracellular flux analyzer have shown oxygen consumption rate (OCR) in untreated C2C12s to be stable (352±7pmoles/min) over 90 minutes. In initial experiments testing uncoupled, state 3, and state 4 respiration, it appears that TNF may lower maximal respiration (p<.07) and increase extracellular acidification rate (p<.06). When exposed to inhibitors for complexes IV and V, control and TNF OCR were nearly identical, which suggests TNF‐induced proton leak originates upstream of Complex IV. These data suggest TNF alters mitochondrial metabolism, which may contribute to increased ROS production. Supported by NIH grant 5 AR055974–04 (MBR) and NIH/NINDS P30 NS051220 (PGS).
Sex differences in age‐related changes in cerebral vasodilator responses
Authors: Barnes Jill N , Taylor Jennifer L , Nicholson Wayne T , Joyner Michael J
Published: June 2021

Cerebral vasodilator responses to hypercapnia decrease with aging, and recent data suggests sex differences in cerebrovascular regulation exist. Yet, it is unknown how sex and age interact in response to the stress of cyclooxygenase inhibition. We examined cerebral vasodilator responses to hypercapnia in 7 young men (YM), 8 young women (YW), 7 older men (OM), and 6 older women (OW) before (CON) and during cyclooxygenase (COX) inhibition via indomethacin (INDO). There was no age by sex interaction in middle cerebral artery velocity responses to hypercapnia (MCAv Slope). However, when accounting for mean arterial pressure during hypercapnia, there was an age by sex interaction in cerebrovascular conductance index (CVCi Slope). YM demonstrated higher CVCi Slope (1.27 ± 0.21 AU) compared with YW (0.65 ± 0.10 AU) and older adults (0.58 ± 0.10 AU in OM; 0.66 ± 0.06 AU in OW; p<0.05 for all). There was no difference in CVCi Slope between YW and OW, indicating that the age‐associated reduction in CVCi Slope only pertains to men. During INDO, there was an age by sex interaction for the ΔCVCi Slope (from CON to INDO) with YM having a greater slope compared with both YW (0.72 ± 0.15 vs. 0.27 ± 0.13 AU; p<0.01) and OM (0.26 ± 0.18 AU; p<0.01). Taken together, our results suggest that sex differences exist in the cerebrovascular response to hypercapnia with and without COX inhibition and that the effect of age is different in women compared to men.
LPS induced circadian phase shift in macrophages
Authors: Wang Yusi , Chen Feng , Rudic Dan , Fulton David
Published: June 2021

The circadian clock regulates the expression of a subset of genes important in cellular function. Recent studies have revealed that the recognition of pathogens and subsequent initiation of inflammatory response is strongly regulated by a macrophage‐intrinsic clock. We hypothesized that the circadian pattern of gene expression can be influenced by inflammatory stimuli and that changes in circadian function in inflammatory cells can influence cardiovascular disease. To investigate circadian rhythms in inflammatory cells, peritoneal macrophage were isolated from Per2‐luciferase transgenic mice. Luminescence was measured following exposure of Per2‐Luc macrophages to various stimuli. Surprisingly we found that the LPS significantly lengthened the circadian period in macrophages. Real‐time PCR data suggests that the expressions of core circadian genes (Bmal1 and Per2) are suppressed following LPS treatment. To investigate the mechanism by which LPS impacts circadian oscillation, we targeted TLR4 signaling. The TLR4 antagonist (LPS‐RS) which directs under‐acylated LPS competitively binding on MD‐2 reversed the phase‐shift effect caused by LPS, while CLI‐095, which blocks the intracellular domain of TLR4 showed no effect on circadian gene oscillation. To confirm the role of TLR4 in circadian phase‐shift by LPS, we plan to genetically manipulate TLR4 signaling in macrophages and in other cell lines.
Genetic and Dietary Affects on Oxygen Deprivation Response and Survival in C. elegans
Authors: Padilla Pamela , Garcia Anastasia , Goy Jo
Published: June 2021

Oxygen deprivation, which is central to many human‐health related issues (Ex: stroke, cardiac and pulmonary disorders) occurs frequently in the elderly and can have a more detrimental affect on individuals who are diabetic or obese. We are using Caenorhabditis elegans to examine how environment, diet, age and genotype influences oxygen deprivation response and survival. C. elegans fed the standard OP50 diet and grown at 20C will survive severe oxygen deprivation (hypoxia and anoxia). However, survival rate is severely decreased if anoxia exposure time is increased or if genetic mutations lead to sensitivity to hypoxia. We are examining the genetic and dietary affects on oxygen deprivation response and survival. Factors associated with long‐term anoxia tolerance include age (3–5 day old adults hermaphrodites survive long‐term anoxia), a decrease in ovulation rate, development at 25C instead of 20C, a diet of HT115 E. coli or pretreatment with the diabetic drug metformin. Long‐term anoxia survival rate is associated with an increase in carbohydrate stores (glycogen, treholose) in the intestine however a glucose‐supplemented diet leads to sensitivity to anoxia exposure, suggesting that a homeostatic balance of carbohydrate stores is important for oxygen deprivation survival. Analysis of mutants identified the following signaling pathways as influencing anoxia survival: insulin‐like signaling (daf‐2/daf‐16), developmental pathways that alter germline function (glp‐1), carbohydrate metabolism (aak‐2), hexosamine signaling (oga‐1) and methyglycoxal protein modifications. Insulin‐signaling, carbohydrate homeostasis, oxidative stress and hexosamine signaling impact diabetes in humans suggesting that C. elegans can be used as a cellular and genetic model to understand the relationship between diet, metabolism, oxygen deprivation stress and disease states such as type II diabetes.
Combined aminophylline and ambrisentan is a potentially safe treatment of health complications after rapid ascent to high altitudes
Authors: Schroeder Thies , Irwin David , Hamilton Karyn , Boico Alina , Blueschke Gert
Published: June 2021

Combined theophylline and ambrisentan improve exercise tolerance in rats after rapid ascent to high altitude, by accelerating blood flow and oxygen delivery to peripheral tissues. We hypothesized that this combination is also safe in humans, and that the single drugs do not interact.

Methods

Healthy human subjects were randomized to one of two sequences. Sequence A was a single 400 mg dose of aminophylline (soluble form of theophylline) followed by 48 h washout, then 5 mg of ambrisentan followed by 48h washout, then 400mg aminophylline and 5mg ambrisentan combined. Sequence B was the same, except subjects were dosed first with ambrisentan and then aminophylline. Adverse event (AE) and pharmacokinetic (PK) profiles were assessed.

Results

Of 47 subjects recruited, 18 completed all procedures. Total 70 transient AEs of mild (71%), moderate (26%) or severe (3%) intensity occurred, most in the first cohort of subjects [64 (91%)] and following aminophylline given alone (38%) or combined with ambrisentan (47%). As expected, PK curves showed no drug interaction.

Conclusions

Combined dosing with the xanthine aminophylline and the endothelin receptor antagonist ambrisentan is safe in healthy humans, and these drugs do not interact. AE profiles were as expected and mostly due to the high dose of aminophylline. An upcoming trial will test this drug combination for safety and efficacy in hypoxic exercising human subjects.
Phosphorylation of rat skeletal muscle pyruvate dehydrogenase phosphatase in response to insulin stimulation
Authors: Choptiany Jonathan , MacPherson Rebecca , LeBlanc Paul , Peters Sandra
Published: June 2021

Pyruvate dehydrogenase phosphatase (PDP) regulates carbohydrate oxidation through the mitochondrial pyruvate dehydrogenase (PDH) complex. PDP dephosphorylates and activates PDH, enabling increased carbohydrate flux towards oxidative energy production. In cell culture, both PDP1 and 2 appear to undergo covalent activation in direct response to insulin–stimulation by PKCδ. Our objective was to examine the effect of insulin on PDP phosphorylation and PDH activation. Intact rat extensor digitorum longus muscles were incubated (oxygenated at 25°C, fixed to 1g of resting tension) tendon–to–tendon for 30min in basal or insulin–stimulated (10mU/mL) media. PDH activity increased 58% following insulin stimulation, (p=0.057, n=11). Serine phosphorylation of PDP1 (p=0.037) and PDP2 (p=0.027) increased by 44% and 54% respectively following stimulation (n=4), and PKCδ protein content was enriched in the mitochondrial fraction by 45% in response to insulin stimulation (p=0.0009, n=8). These data suggest that the insulin–stimulated increase in PDH activity in whole tissue is mediated through mitochondrial migration of PKCδ and subsequent PDP phosphorylation.

Research supported by NSERC, Canada.
The role of suppressor enhancer lin‐12 1 like (SEL1L) in cholesterol metabolism
Authors: Krumm Christopher , Francisco Adam , Chang Wei‐Hua , Long Qiaoming
Published: June 2021

Cholesterol is a physiologically essential sterol synthesized in the liver. Cholesterol homeostasis in the liver is regulated by a network of genes including those encoding catalytic enzymes and transcription factors. Suppressor Enhancer Lin‐12 1 Like (SEL1L) encodes an endoplasmic reticulum (ER) membrane protein that is highly expressed in the liver. To investigate the possible role of SEL1L in the regulation of cholesterol metabolism, we have developed a liver specific mouse model (SEL1L LKO). SEL1L LKO mice show comparable body and liver growth to wild‐type mice on normal diet. When fed a high cholesterol/fat diet however, the body mass of the SEL1L LKO mice were significantly smaller than the controls, despite exhibiting a larger liver and gallbladder. Immunostaining analysis revealed an inflamed and damaged liver, which indicate the development of non‐alcoholic fatty liver disease and fibrosis. Furthermore, serum analysis revealed abnormally increased cholesterol and triglyceride levels. Immunoblotting analysis had shown down regulation of the previously mentioned rate limiting enzymes in the SEL1L LKO mice, which demonstrate the challenges in maintaining normal intracellular distributions. Therefore, our findings reveal implications that a liver‐specific knockout of SEL1L can lead to alterations in cholesterol metabolism, liver damage and disease.
Validation of method using 2H2O to measure rates of protein breakdown in myoblasts
Authors: Watkins Molly , Ehrlicher Sarah , Khademi Shadi , Reuland Danielle J. , Miller Benjamin F. , Hamilton Karyn L.
Published: June 2021

Rates of protein synthesis and breakdown are thought to relate to longevity in several organisms. Since assessing protein breakdown is important for research aimed at identifying interventions to prolong healthspan, an effective, practicable method for determining rates of protein breakdown is needed. We sought to validate a method for measuring protein breakdown in vitro using deuterium oxide (2H2O) labeling of proteins and the subsequent loss of the label. C2C12 myoblasts were grown in medium supplemented with 10% 2H2O. After 5 passages, the 2H2O solution was replaced with unlabeled media. Cycloheximide (CHX, 50μg/ml) was included to prevent reintegration of 2H liberated from cell breakdown. In the treated cells, Rapamycin (Rap, 5nM) was utilized to stimulate protein breakdown. At 4hr, 8hr, 12hr, and 18hr, duplicate samples of each treatment group were collected. The samples were fractionated into mixed, cytoplasmic, and mitochondrial fractions and analyzed by gas chromatography‐mass spectroscopy. Initial analysis shows protein concentrations decreasing from the 0–18 hr time points before plateauing/slightly increasing. This suggests the 2H2O labeling method could be used to examine how rates of protein degradation differ in long‐lived vs. non‐long‐lived organisms, or how rates differ for conditions related to lengthened healthspan. Supported by the APS Undergraduate Summer Research Fellowship.
Cardiac interfibrillar mitoKATP channels are sensitive to diazoxide and have a regulatory subunit profile distinct from subsarcolemmal mitoKATP
Authors: Fancher Ibra S. , Croston Tara , Shepherd Danielle , Dick Gregory M. , Hollander John M.
Published: June 2021

Two distinct populations of mitochondria exist in cardiac muscle, subsarcolemmal and interfibrillar (SSM and IFM). SSM and IFM differ in location, respiratory capacity, and susceptibility to disease. Mitochondrial KATP channels (mitoKATP) play an important role in cardioprotective signaling; however, characterization of differences in mitoKATP between IFM and SSM is absent. The rationale that cellular demand during insult could affect SSM and IFM differently makes it plausible to hypothesize that differences exist in mitoKATP subunit composition and pharmacology. We tested isolated cardiac SSM and IFM of adult male FVB mice using Rhodamine123 fluorometry and JC‐1 flow cytometry. We measured membrane potential (ΔΨm) in response to the mitoKATP agonist diazoxide. We also compared immunoblots of putative mitoKATP subunits. Our results indicate that IFM mitoKATP are more sensitive to diazoxide, as ΔΨm depolarized more so than did SSM ΔΨm. Immunoblots showed differential expression of SUR subunits, with SSM containing SUR2A and SUR2B, while IFM appear to have a novel splice variant of SUR1. These data suggest that mitoKATP subunits are expressed differently in cardiac mitochondrial subpopulations and that IFM are more sensitive to diazoxide. These findings have implications for cardioprotective mechanisms, metabolic disease, and individual roles that IFM and SSM may have. T32‐HL090610
Impact of dietary induced fatty liver on mitochondrial oxidative phosphorylation
Authors: Newell Christopher , Hughey Curtis , Nyamandi Vongai , Johnsen Virginia , Shearer Jane
Published: June 2021

High fat (HF) diet has been linked to mitochondrial dysfunction. Alteration of HF diet duration has shown that although mitochondrial impairments are present initially (2–4 weeks), these impairments are abolished or exceed control level function after prolonged HF feeding (>; 8 weeks). Male C‐57 mice (n=14) were randomly assigned to one of two dietary groups; chow (CH, 10% kcal fat) and high fat (HF, 60% kcal fat). HF mice were obese with elevated liver triglycerides. Isolated mitochondria from mouse liver tissue were measured using high‐resolution respirometry. Animals within the HF group possessed higher state 3 & 4 respiration (p<0.05), as well as higher respiratory control ratios (p<0.05). Mitochondria are termed to be in state 3 respiration when actively respiring, meaning the rate where they are capable of converting adenosine diphosphate (ADP) to adenosine triphosphate (ATP) through oxygen consumption. State 4 refers to the rate of oxygen depletion after all ADP has been phosphorylated, also termed leak respiration. This indicates that mitochondria in HF animals are able to adapt to metabolic stressors such as a high fat diet by elevating state 3 respiratory rates above normal levels. Moreover, mitochondria can increase their active respiratory rate within fatty liver tissues as a direct result of excess lipid availability and accumulation.
Dynamic expression and localization of Protein Kinase A regulatory subunit RIα in cardiac mitochondria controls response to oxidative stress
Authors: Haushalter Kristofer , Patel Hemal , Taylor Susan
Published: June 2021

Recent studies have implicated many possible roles for protein kinase A (PKA) activity localized to mitochondria, especially in organs such as the heart with high metabolic activity and/or increased susceptibility to oxidative stress. Because mitochondria serve as a critical control point for cellular adaption to environmental stress, we hypothesize that regulation of mitochondrial‐localized PKA (mt‐PKA) modifies mitochondrial integrity and cellular homeostasis. In particular, we propose that PKA associated proteins are actively regulated in response to oxidative stress in order to modulate mt‐PKA activity. Using biochemical and proteomic analysis of differing mitochondrial subpopulations within cardiac tissue, we found a striking difference between the relative localization and expression of PKA and its associated proteins. By combining acute oxidative stress models, biochemical, microscopic and pharmacological assessment, as well as physiological and metabolic functional analysis methods, our data suggests that a stress‐induced loss of mitochondrial localized PKA RIα (mt‐RIα) is directly correlated to deterioration of mitochondria integrity, decrease in respiratory activity, increase in ROS production, and an overall reduction in cell viability and heart function. Therefore, we conclude that mt‐RIα is a necessary component for regulating mitochondrial response to oxidative stress. Support for this work was provided by HHMI and NIH funding.
Overexpression of Bnip3 in the Myocardium Leads to Mitochondrial Dysfunction and Enhanced Mitochondrial Autophagy
Authors: Lee Youngil , Thomas Robert L , Kubli Dieter A , Zhang Xiaoxue , Dorn Gerald W , Gustafsson Åsa B
Published: June 2021

A pro‐apoptotic BH3‐only protein Bnip3 in cells leads to mitochondrial dysfunction and cell death, and induction of mitochondrial autophagy. To date, most studies on Bnip3 have been performed in vitro. Using cardiac specific Bnip3 transgenic (Tg), we have investigated the consequences of Bnip3 overexpression in the heart. We found that mitochondrial respiratory function was significantly impaired in 10 weeks old Bnip3 Tg hearts compared to WT. Moreover, number of mitochondria (decreased mtDNA copy number) and yield of isolated mitochondria were significantly reduced compared to WT. Bnip3 overexpression also caused increased induction of autophagy in the heart. We also found increased mitochondrial translocation of the fission protein Drp‐1 and the E3 ubiquitin ligase Parkin which correlated with increased ubiquitination of mitochondrial proteins, and that PINK1 and histone deacetylase 6 (HDAC6), known markers of mitochondrial autophagy, were increased in the heart of Bnip3 Tg mice. Interestingly, echocardiography revealed that Bnip3 Tg cardiac function was not significantly different from WT mice. Taken together, these data suggest that Bnip3‐mediated mitochondrial damage and increased clearance do not lead to heart failure, indicating that the damage does not exceed the mitochondrial reserve capacity in the heart under non‐stressed conditions.

Supported by NIH R01 HL087023
A school feeding program based on the Eco‐Health approach: A case study from rural Lebanon
Authors: Batal Malek , Ghattas Hala , Abou‐Ezze Rheam
Published: June 2021

Using the Eco‐Health approach, a local women's cooperative was paired with a rural school to provide traditional foods to children through a school feeding and nutrition education program. We investigated the effect of this program versus nutrition education alone on nutritional status, breakfast consumption and attitude towards traditional diets. Two elementary schools from the village of Aarsal, Lebanon participated in the study. At baseline and endpoint, height, weight, hemoglobin and breakfast recall were taken. All children received four nutrition education sessions and children from the intervention school received a daily school meal for 4 days/week for 5 months. Qualitative data were collected from meetings with parents. At baseline, stunting, overweight and anemia prevalence were at 8%, 20% and 6%, respectively. There was a significant decrease in anemia prevalence among schoolchildren taking part in the school feeding intervention (p<0.0001). Breakfast consumption habits improved at both schools with a significant decrease in number of children not consuming breakfast (p<0.001). Schoolchildren in the intervention school demonstrated improved attitudes towards traditional diet and healthy lifestyle practices. This school feeding program provides a potentially sustainable local model for school feeding programs in similar rural contexts in countries undergoing a nutrition transition.

This research has been funded by Canada's International Development Research Center (IDRC).