HCR Rats Research Articles

Divergence in aerobic capacity influences hepatic and systemic metabolic adaptations to bile acid sequestrant and short-term high-fat/sucrose feeding in rats.

High versus low aerobic capacity significantly impacts the risk for metabolic diseases. Rats selectively bred for high or low intrinsic aerobic capacity differently modify hepatic bile acid metabolism in response to high-fat diets (HFDs). Here we tested if a bile acid sequestrant would alter hepatic and whole body metabolism differently in rats with high and low aerobic capacity fed a 1-wk HFD. Male rats (8 mo of age) that were artificially selected to be high (HCR) and low-capacity runners (LCR) with divergent intrinsic aerobic capacities were transitioned from a low-fat diet (LFD, 10% fat) to an HFD (45% fat) with or without a bile acid sequestrant (BA-Seq, 2% cholestyramine resin) for 7 days while maintained in an indirect calorimetry system. HFD + BA-Seq increased fecal excretion of lipids and bile acids and prevented weight and fat mass gain in both strains. Interestingly, HCR rats had increased adaptability to enhance fecal bile acid and lipid loss, resulting in more significant energy loss than their LCR counterpart. In addition, BA-Seq induced a greater expression of hepatic CYP7A1 gene expression, the rate-limiting enzyme of bile acid synthesis in HCR rats both on HFD and HFD + BA-Seq diets. HCR displayed a more significant reduction of RQ in response to HFD than LCR, but HFD + BA-Seq lowered RQ in both groups compared with HFD alone, demonstrating a pronounced impact on metabolic flexibility. In conclusion, BA-Seq provides uniform metabolic benefits for metabolic flexibility and adiposity, but rats with higher aerobic capacity display adaptability for hepatic bile acid metabolism.NEW & NOTEWORTHY The administration of bile acid sequestrant (BA-Seq) has uniform metabolic benefits in terms of metabolic flexibility and adiposity in rats with high and low aerobic capacity. However, rats with higher aerobic capacity demonstrate greater adaptability in hepatic bile acid metabolism, resulting in increased fecal bile acid and lipid loss, as well as enhanced fecal energy loss.

Selective Breeding for High Intrinsic Exercise Capacity Slows Pan-Tissue Epigenetic Aging in Rats

Aerobic exercise capacity is strongly associated with longevity and reduction in all-cause mortality. Rats selectively bred for high intrinsic running capacity (HCR) compared with low (LCR), have between a 28-45% increase in median lifespan. It is not known whether the greater HCR survival is associated with slowed epigenetic aging. We hypothesized that HCR rats would express altered rates of pan-tissue DNA methylation corresponding to a slowed epigenetic clock (DNAmAge). Male and female LCR and HCR rats from generation 15-17 (n=22; chronological age=2.34±0.45 yrs (mean±SD)), generation 28 (n=20; 1.09±0.05 yrs) and generation 37 (n=20; 1.04±0.08 yrs) were included. DNA was extracted from skeletal muscle (n=61), cardiac muscle (n=60), adipose (n=61) and liver (n=61). DNAmAge was quantitated using the mammalian methylation array platform (~106k CpGs specific to brown rat). Three epigenetic clocks were used, previously trained on separate rat tissues, including dual species clocks that apply to rats and humans (pan-tissue rat clock; human-rat clocks for relative and chronologic age). Slowing of epigenetic age relative to chronologic age was assessed by multivariable regression adjusted for chronologic age, generation, sex and phenotype (treadmill running distance at 3 months of age). Epigenome wide association with phenotype was performed in Limma, with Benjamini-Hochberg p-value correction and significance set at FDR q<0.05. Gene set enrichment analysis was performed with Gene Ontology Biological Processes. Maximum treadmill running distance was ~8 times greater in HCR than LCR (2,137±500 vs. 246±63 m; p=1.97✕10-20). Across all samples and clocks, epigenetic age was associated with chronological age (r=0.461-0.500; p<3.60✕10-14). Compared with LCR, HCR epigenetic aging was slowed by a mean of -0.18 to -0.24 yrs, which represents 9-42% lower DNAmAge in HCR rats than LCR (p<0.024). Tissue-specific analyses revealed a lower DNAmAge in HCR for adipose, skeletal muscle and cardiac muscle (pan-tissue rat clock; p<0.04), and liver (human-rat clock for relative and chronologic age; p<0.01); effects were strongest at younger chronologic age. Significantly differentially methylated CpGs were found in skeletal muscle (n=11,341), cardiac muscle (n=2,480), adipose (n=3,036) and liver (n=12,595). Of the top 10 differentially methylated pathways, ~58% related to tissue development and ~20% related to cardiac or skeletal muscle. These data show differences in the rate of epigenetic aging manifest in tissues that directly (skeletal and cardiac muscle) and indirectly (adipose, liver) contribute to the energy transfer of aerobic exercise capacity upon which the selective breeding of LCR and HCR rat models were based. These data suggest that a large fraction of the survival benefit associated with high intrinsic exercise capacity is reflected in slowed epigenetic aging across multiple tissues. The Lundquist Institute. Epigenetic Clock Development Foundation. The University of Toledo Department of Physiology & Pharmacology. NIH (R01HL151452, R01HL153460, P50HD098593, P40OD021331). Open Philanthropy (SH). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The acute removal of metformin induces mitochondrial remodeling and increases mitochondrial respiration

Metformin is a candidate to slow biological aging and extend healthspan, as it reduces the risk of morbidity and mortality in people with type 2 diabetes. However, it is not clear how metformin impacts individuals absent of chronic disease. This unknown is important to answer before prescribing metformin to healthy individuals to prolong the healthspan. It has been shown that metformin creates an inefficiency in energy transfer by accumulating within the mitochondria. Therefore, we hypothesized that the effects of metformin on healthspan are context specific; beneficial in the context of low energy demand/mitochondrial capacity but detrimental in the context of high energy demand/mitochondrial capacity. To test this hypothesis, we used 18-month, male rats selectively bred for low and high treadmill running capacity (LCR/HCR) as models of low and high intrinsic mitochondrial function. For both HCR and LCR, we used high-resolution respirometry (Oroboros O2K) on skeletal muscles from controls (CON), 4 weeks of metformin-treatment (MET), 4 weeks of metformin-treatment with a 48-hour metformin washout (MET-WO), and 48-hour washout with metformin added back to the respirometry chamber (MET-WO-SPIKE). A week prior to sacrifice, we labeled the rats with deuterium oxide to assess protein turnover as an indicator of mitochondrial remodeling. In gastrocnemius muscle, the HCR MET-WO had greater Complex I (CI), CI+Complex II (CII), and maximal respiration (MaxR) (115 ± 23, 164 ± 38, 157 ± 35 pmol/(s✕mg), respectively) compared to the CON (70 ± 24, p = 0.02, 114 ± 32, p = 0.01, 115 ± 40 pmol/(s✕mg), p = 0.04, respectively), and MET (63 ± 54, p = 0.007, 90 ± 50, p < 0.001, 91 ± 48 pmol/(s✕mg), p = 0.002, respectively). The HCR MET-SPIKE group had greater CII (83 ± 33 pmol/(s✕mg)) and MaxR (86 ± 35 pmol/(s✕mg)) compared to the MET (p = 0.007 and p = 0.002, respectively). In the soleus, MET-WO (4.15 ± 0.33%/day) had greater mitochondrial fractional synthesis rates (FSRs) compared to MET (3.12 ± 0.62%/day, p = 0.001) in the HCRs. In the LCRs, MET-WO (4.61 ± 0.42%/day) had greater FSRs compared to CON (p < 0.001, 3.07 ± 0.67%/day) and MET (3.12 ± 0.61%/day, p = 0.001). In the tibialis anterior, MET-WO (3.47 ± 0.80%/day) had greater FSRs compared to the MET (2.41 ± 0.47%/day, p = 0.04) within the HCRs with no differences in the LCR. These data show that metformin reduces mitochondrial respiration in HCR rats but not in LCR rats. The presence of metformin produced a greater subsequent response in HCR compared to LCR as shown by its acute removal revealing metformin-induced mitochondrial remodeling. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Analysis of lipid composition in muscle tissue of obese and lean rats.

Skeletal muscle is a complex component of the body and muscle contraction has significant implications for caloric expenditure. Lipid metabolism is defined as the production and breakdown of different lipids in the body for metabolic demands. Skeletal muscle plays an important role in lipid metabolism. Obesity and other metabolic diseases are characterized by fatty acid accumulation in the form of triglycerides and cholesterol esters. Furthermore, muscle tissue is vulnerable to lipid composition alterations. Here we used tissue from rats bred for high endurance capacity (HCR) and rats bred for low endurance capacity (LCR). It is well known that HCR rats have better metabolic health than LCR rats, who are more prone to metabolicillnesses. To what extent this arises from differences in lipid composition in the two models is unknown. We used these models to investigate the influence of aerobic fitness on global lipid composition in rat skeletal muscle. The lipids were extracted from the HCR and LCR muscle tissue and characterized using thin layer chromatography to separate and differentiate lipid classes in the rat tissue. We hypothesize that there will be a larger content of free fatty acids, triglycerides, and ceramides in LCR compared to HCR models, potentially contributing to a difference in performance and health of the HCR and LCR rats.

Intrinsic cardiorespiratory fitness modulates clinical and molecular response to caloric restriction

ObjectiveCaloric restriction (CR) is one extrinsic intervention that can improve metabolic health, and it shares many phenotypical parallels with intrinsic high cardiorespiratory fitness (CRF), including reduced adiposity, increased cardiometabolic health, and increased longevity. CRF is a highly heritable trait in humans and has been established in a genetic rat model selectively bred for high (HCR) and low (LCR) CRF, in which the HCR live longer and have reduced body weight compared to LCR. This study addresses whether the inherited high CRF phenotype occurs through similar mechanisms by which CR promotes health and longevity. MethodsWe compared HCR and LCR male rats fed ad libitum (AL) or calorically restricted (CR) for multiple physiological, metabolic, and molecular traits, including running capacity at 2, 8, and 12 months; per-hour metabolic cage activity over daily cycles at 6 and 12 months; and plasma lipidomics, liver and muscle transcriptomics, and body composition after 12 months of treatment. ResultsLCR-CR developed a physiological profile that mirrors the high-CRF phenotype in HCR-AL, including reduced adiposity and increased insulin sensitivity. HCR show higher spontaneous activity than LCR. Temporal modeling of hourly energy expenditure (EE) dynamics during the day, adjusted for body weight and hourly activity levels, suggest that CR has an EE-suppressing effect, and high-CRF has an EE-enhancing effect. Pathway analysis of gene transcripts indicates that HCR and LCR both show a response to CR that is similar in the muscle and different in the liver. ConclusionsCR provides LCR a health-associated positive effect on physiological parameters that strongly resemble HCR. Analysis of whole-body EE and transcriptomics suggests that HCR and LCR show line-dependent responses to CR that may be accreditable to difference in genetic makeup. The results do not preclude the possibility that CRF and CR pathways may converge.

Abstract 15560: Mitophagy Dysfunction and Activation of the Inflammasome Cause Aortic Stiffness in Low Aerobic Capacity Aged Rats

Aging and low aerobic capacity are associated with an increased risk of cardiovascular events and mortality. We reported that a polygenic rat model selectively bred for low aerobic capacity (LCR) showed aortic stiffness and dysfunction with age compared to rats bred for a high aerobic capacity (HCR). We observed increased aortic mitochondrial ROS and extracellular mtDNA DAMPs levels, activation of AIM2 inflammasome and its downstream effectors and increased collagen content in old LCR rat aortas. However, the molecular mechanisms linking low aerobic capacity to inflammation in aging have not been elucidated. In order to decipher the changes in genetic networks in low aerobic capacity and aging-associated aortic stiffness, a global transcriptome analysis was performed by RNA-Seq and gene ontology analysis of aortas from young (4 month) and old (27 month) LCR and HCR rats. Down-regulated pathways in old LCR aortas include mitochondrial biogenesis and mitophagy and up-regulated pathways include pro-inflammatory pathways, apoptosis/necroptosis/ferroptosis pathways, in contrast to HCR aortas which have higher levels of pathways regulating longevity, oxidative phosphorylation, and mitophagy and autophagy. In line with that, old LCR rats displayed a 54% reduction in mitophagy (colocalization of mitochondrial marker protein TOM20 with lysosomal marker protein LAMP1) in aortic media and a 76% reduction in cultured VSMCs in comparison to older HCR rats. Mitophagy levels did not differ significantly between young LCR and HCR rats. The autophagic vacuole formation rate was a 12-fold lower in old LCR than in old HCR aortic VSMCs, suggesting that impaired mitophagy may be responsible for accumulation of mtDNA-derived damage associated molecular patterns (mtDNA DAMPs). Treatment with various inducers of mitophagy (rapamycin, deferiprone, and MitoTEMPO) significantly increased mitophagy and decreased mtDNA DAMPs levels in old LCR VSMCs. This was also associated with a reduction in inflammasome activation, mitochondrial dysfunction, and ROS levels. We conclude that impaired mitophagy causes accumulation of DAMPs and inflammasome activation, inducing aortic stiffness and dysfunction in old age LCR rats.

Intrinsic Mitochondrial Function Impacts the Outcomes of Metformin Treatment on Skeletal Muscle Mitochondrial Morphology in Aged Rat

Aging is the main risk factor for many costly, irreversible, and comorbid chronic diseases. The anti‐diabetic drug Metformin is being considered as an anti‐aging treatment although little is known about its effects in healthy individuals. Previous data from our lab suggest that metformin is beneficial to individuals that are relatively insulin resistant and may be detrimental to those who are insulin sensitive. We aimed to understand how intrinsic function of mitochondria affects metformin treatment outcomes in skeletal muscle. We hypothesized that the effects of metformin on skeletal muscle mitochondrial morphology and remodeling will differ based on intrinsic mitochondrial function. To test our hypothesis, we used the High‐Capacity Runner/Low‐Capacity Runner (HCR/LCR) rat model system which comprises two selectively bred lines of genetically heterogeneous rats that diverge for exercise capacity, cardiovascular risk factors, lifespan, and healthspan. In drinking water, we treated six HCR and four LCR male rats (age 18 months) with a dose of 100 mg/kg/day metformin for a week, followed by three weeks of 200 mg/kg/day. We included HCR and LCR control groups (n=5) for a total of four groups. A week prior to sacrifice, we initiated deuterium oxide (D2O) labeling to assess protein turnover and electroporated DNA constructs that encode for mitochondrial matrix‐targeted YFP and tdTomato‐tagged TOM20, a protein located on the mitochondrial outer membrane. We analyzed protein turnover in hindlimb muscles using GC‐MS, and prepared histological sections of the Tibialis Anterior (TA) muscle for further analysis of mitochondrial morphology. Normalized to tibial length, the mass of the gastrocnemius (‐16.6%) and plantaris (‐20.8%) muscles were lower in metformin treated HCR rats compared to untreated, while the normalized mass of the TA muscle was lower in both HCR (‐15%) and LCR (‐20.1%) metformin treated rats compared to control. There were no significant differences in mean fiber cross sectional area (CSA) of the TA muscle. We detected no significant differences in bulk protein synthesis of both the myofibrillar and mitochondrial fractions of the TA, gastrocnemius, and soleus muscles. The number of mitochondrial branches observed in a cross section of TA fibers from metformin treated LCR rats were 30.5% lower compared to control LCR rats. However, mean CSA of each mitochondrial branch in metformin treated LCR rats increased by 82.8% compared to control LCR rats, which, together with the decrease in branch number, resulted in maintenance of the cumulative mitochondrial CSA in each fiber. Analysis based on mitochondrial sub‐populations showed that the subsarcolemmal mitochondrial population was more impacted than intermyofibrillar mitochondria. There were no significant differences in mitochondrial morphology in control vs metformin treated HCR rats. Our data supports our hypothesis that metformin has differential effects in skeletal muscle based on intrinsic mitochondrial function prior to treatment. Additional studies are needed to determine what impact these disparities have on the aging process.

Life-long exercise training and inherited aerobic endurance capacity produce converging gut microbiome signatures in rodents.

High aerobic endurance capacity can be acquired by training and/or inherited. Aerobic exercise training (AET) and aging are linked to altered gut microbiome composition, but it is unknown if the environmental stress of exercise and host genetics that predispose for higher exercise capacity have similar effects on the gut microbiome during aging. We hypothesized that exercise training and host genetics would have conserved effects on the gut microbiome across different rodents. We studied young sedentary (Y‐SED, 2‐month‐old) mice, old sedentary (O‐SED, 26‐month‐old) mice, old mice with life‐long AET (O‐AET, 26‐month‐old), and aged rats selectively bred for high (HCR [High Capacity Runner], 21‐month‐old) and low (LCR [Low Capacity Runner], 21‐month‐old) aerobic capacity. Our results showed that O‐SED mice had lower running capacity than Y‐SED mice. The fecal microbiota of O‐SED mice had a higher relative abundance of Lachnospiraceae, Ruminococcaceae, Turicibacteriaceae, and Allobaculum, but lower Bacteroidales, Alistipes, Akkermansia, and Anaeroplasma. O‐AET mice had a higher running capacity than O‐SED mice. O‐AET mice had lower fecal levels of Lachnospiraceae, Turicibacteriaceae, and Allobaculum and higher Anaeroplasma than O‐SED mice. Similar to O‐AET mice, but despite no exercise training regime, aged HCR rats had lower Lachnospiraceae and Ruminococcaceae and expansion of certain Bacteroidales in the fecal microbiome compared to LCR rats. Our data show that environmental and genetic modifiers of high aerobic endurance capacity produce convergent gut microbiome signatures across different rodent species during aging. Therefore, we conclude that host genetics and life‐long exercise influence the composition of the gut microbiome and can mitigate gut dysbiosis and functional decline during aging.

Differential weight loss with intermittent fasting or daily calorie restriction in low- and high-fitness phenotypes.

What is the central question of this study? How does intrinsic aerobic capacity impact weight loss with 50% daily caloric restriction and alternate-day fasting? What is the main finding and its importance? Intermittent fasting is effective for weight loss in rats with low fitness, which highlights the importance of how intermittent fasting interacts with aerobic fitness. Recent interest has focused on the benefits of time-restricted feeding strategies, including intermittent fasting, for weight loss. It is not yet known whether intermittent fasting is more effective than daily caloric restriction at stimulating weight loss and how each is subject to individual differences. Here, rat models of leanness and obesity, artificially selected for intrinsically high (HCR) and low (LCR) aerobic capacity, were subjected to intermittent fasting and 50% calorie restrictive diets in two separate experiments using male rats. The lean, high-fitness HCR and obesity-prone, low-fitness LCR rats underwent 50% caloric restriction while body weight and composition were monitored. The low-fitness LCR rats were better able to retain lean mass than the high-fitness HCR rats, without significantly different proportional loss of weight or fat. In a separate experiment using intermittent fasting in male HCR and LCR rats, alternate-day fasting induced significantly greater loss of weight and fat mass in LCR compared with HCR rats, although the HCR rats had a more marked reduction in ad libitum daily food intake. Altogether, this suggests that intermittent fasting is an effective weight-loss strategy for those with low intrinsic aerobic fitness; however, direct comparison of caloric restriction and intermittent fasting is warranted to determine any differential effects on energy expenditure in lean and obesity-prone phenotypes.

Hydrocephalus Following Experimental Subarachnoid Hemorrhage in Rats with Different Aerobic Capacity.

Low aerobic capacity is considered to be a risk factor for stroke, while the mechanisms underlying the phenomenon are still unclear. The current study looked into the impacts of different aerobic capacities on early brain injury in a subarachnoid hemorrhage (SAH) model using rats bred for high and low aerobic capacity (high-capacity runners, HCR; low-capacity runners, LCR). SAH was modeled with endovascular perforation in HCR and LCR rats. Twenty-four hours after SAH, the rats underwent behavioral testing and MRI, and were then euthanized. The brains were used to investigate ventricular wall damage, blood–brain barrier breakdown, oxidative stress, and hemoglobin scavenging. The LCR rats had worse SAH grades (p < 0.01), ventricular dilatation (p < 0.01), ventricular wall damage (p < 0.01), and behavioral scores (p < 0.01). The periventricular expression of HO-1 and CD163 was significantly increased in LCR rats (p < 0.01 each). CD163-positive cells were co-localized with HO-1-positive cells. The LCR rats had greater early brain injuries than HCR rats. The LCR rats had more serious SAH and extensive ventricular wall damage that evolved more frequently into hydrocephalus. This may reflect changes in iron handling and neuroinflammation.

Mitochondrial health is enhanced in rats with higher vs. lower intrinsic exercise capacity and extended lifespan

The intrinsic aerobic capacity of an organism is thought to play a role in aging and longevity. Maximal respiratory rate capacity, a metabolic performance measure, is one of the best predictors of cardiovascular- and all-cause mortality. Rats selectively bred for high-(HCR) vs. low-(LCR) intrinsic running-endurance capacity have up to 31% longer lifespan. We found that positive changes in indices of mitochondrial health in cardiomyocytes (respiratory reserve, maximal respiratory capacity, resistance to mitochondrial permeability transition, autophagy/mitophagy, and higher lipids-over-glucose utilization) are uniformly associated with the extended longevity in HCR vs. LCR female rats. Cross-sectional heart metabolomics revealed pathways from lipid metabolism in the heart, which were significantly enriched by a select group of strain-dependent metabolites, consistent with enhanced lipids utilization by HCR cardiomyocytes. Heart–liver–serum metabolomics further revealed shunting of lipidic substrates between the liver and heart via serum during aging. Thus, mitochondrial health in cardiomyocytes is associated with extended longevity in rats with higher intrinsic exercise capacity and, probably, these findings can be translated to other populations as predictors of outcomes of health and survival.

Intrinsic Exercise Capacity and Mitochondrial DNA Lead to Opposing Vascular-Associated Risks.

Exercise capacity is a strong predictor of all-cause morbidity and mortality in humans. However, the associated hemodynamic traits that link this valuable indicator to its subsequent disease risks are numerable. Additionally, exercise capacity has a substantial heritable component and genome-wide screening indicates a vast amount of nuclear and mitochondrial DNA (mtDNA) markers are significantly associated with traits of physical performance. A long-term selection experiment in rats confirms a divide for cardiovascular risks between low- and high-capacity runners (LCR and HCR, respectively), equipping us with a preclinical animal model to uncover new mechanisms. Here, we evaluated the LCR and HCR rat model system for differences in vascular function at the arterial resistance level. Consistent with the known divide between health and disease, we observed that LCR rats present with resistance artery and perivascular adipose tissue dysfunction compared to HCR rats that mimic qualities important for health, including improved vascular relaxation. Uniquely, we show by generating conplastic strains, which LCR males with mtDNA of female HCR (LCR-mtHCR/Tol) present with improved vascular function. Conversely, HCR-mtLCR/Tol rats displayed indices for cardiac dysfunction. The outcome of this study suggests that the interplay between the nuclear genome and the maternally inherited mitochondrial genome with high intrinsic exercise capacity is a significant factor for improved vascular physiology, and animal models developed on an interaction between nuclear and mtDNA are valuable new tools for probing vascular risk factors in the offspring.

Differences in intrinsic aerobic capacity alters sensitivity to ischemia-reperfusion injury but not cardioprotective capacity by ischemic preconditioning in rats.

IntroductionAerobic capacity is a strong predictor of cardiovascular mortality. Whether aerobic capacity influences myocardial ischemia and reperfusion (IR) injury is unknown.PurposeTo investigate the impact of intrinsic differences in aerobic capacity and the cardioprotective potential on IR injury.MethodsWe studied hearts from rats developed by selective breeding for high (HCR) or low (LCR) capacity for treadmill running. The rats were randomized to: (1) control, (2) local ischemic preconditioning (IPC) or (3) remote ischemic preconditioning (RIC) followed by 30 minutes of ischemia and 120 minutes of reperfusion in an isolated perfused heart model. The primary endpoint was infarct size. Secondary endpoints included uptake of labelled glucose, content of selected mitochondrial proteins in skeletal and cardiac muscle, and activation of AMP-activated kinase (AMPK).ResultsAt baseline, running distance was 203±7 m in LCR vs 1905±51 m in HCR rats (p<0.01). Infarct size was significantly lower in LCR than in HCR controls (49±5% vs 68±5%, p = 0.04). IPC reduced infarct size by 47% in LCR (p<0.01) and by 31% in HCR rats (p = 0.01). RIC did not modulate infarct size (LCR: 52±5, p>0.99; HCR: 69±6%, p>0.99, respectively). Phosphorylaion of AMPK did not differ between LCR and HCR controls. IPC did not modulate cardiac phosphorylation of AMPK. Glucose uptake during reperfusion was similar in LCR and HCR rats. IPC increased glucose uptake during reperfusion in LCR animals (p = 0.02). Mitochondrial protein content in skeletal muscle was lower in LCR than in HCR (0.77±0.10 arbitrary units (AU) vs 1.09±0.07 AU, p = 0.02), but not in cardiac muscle.ConclusionAerobic capacity is associated with altered myocardial sensitivity to IR injury, but the cardioprotective effect of IPC is not. Glucose uptake, AMPK activation immediately prior to ischemia and basal mitochondrial protein content in the heart seem to be of minor importance as underlying mechanisms for the cardioprotective effects.

Augmented Cardiac Mitochondrial Capacity in High Aerobic Capacity “Disease Resistant” Phenotype at Rest is Lost Following Ischemia Reperfusion

RationaleIschemic heart disease is a major cause of morbidity. Regular active exercise is therapeutic, but up to 70% of individual exercise capacity is due to an intrinsic genetic component. Intrinsic capacity can be studied using high (HCR) and low (LCR) aerobic running capacity rat strains. The phenotypes differ by more than 5 fold in sedentary average running distance and time. The HCR rats have been characterized as “disease resistant”, while the LCRs are characterized as “disease prone”. A consistent characteristic of the LCR is reduced metabolic capacity in several tissues, but metabolic capacity in cardiac tissue is not well studied in these phenotypes, particularly following the metabolic stress of ischemia and reperfusion.Methods32 HCR and LCR rats were obtained from the parent colony at the University of Toledo, housed under sedentary conditions, and fed normal chow. LCR and HCR animals were randomly assigned to either control (C, n = 8 each) or ischemia reperfusion (IR, n = 8 each). On each study day, one HCR/LCR pair was anesthetized and hearts were rapidly excised. In IR animals, the hearts were immediately flushed with iced hyperkalemic, hyperosmotic, cardioplegia solution, and subjected to cold global ischemic arrest (80 min). Following arrest, the hearts underwent warm reperfusion (120 min) using a Langendorff style perfusion system. Following reperfusion, the heart was weighed and the LV was isolated. A mid ventricular ring was obtained to estimate infarction size (TTC), and part of the remaining tissue (~150 mg) was transferred to homogenation buffer on ice. Isolated mitochondria (MITO) samples were prepared and used to determine respiratory capacity under different metabolic conditions (OROBOROS). MITO from control animals were obtained and prepared in similar fashion, but immediately following anesthesia and heart removal, and without IR. Citrate synthase activity was measured in each sample.ResultsIn the control rats, HCR MITO showed respiratory rates 32% higher at rest and more than 40% higher under maximally stimulated conditions, compared to LCR MITO (both p &lt; 0.05). After IR, resting MITO respiratory rates were decreased to about 10% in both strains, and the augmented capacity in HCRs was absent. Maximally stimulated rates also were decreased more than 50% from control, and were no longer different between phenotypes. Infarct size was not significantly different between HCR and LCR (49.2% ± 5.6 vs. 53.7% ± 4.9), nor was average coronary flow during reperfusion.ConclusionCardiac mitochondria from HCR were significantly higher in control conditions with each substrate tested. After IR insult, the cardiac mitochondrial respiratory rates were similar between phenotypes, as was infract size. Relatively, the loss of respiratory capacity was actually greater in HCR than LCR. Together, these data could suggest limits in the extent to which the HCR phenotype might be “protective” against acute tissue stressors. The extent to which any of these deficits could be “rescued” by an active exercise component is unknown.

Differential Receptor‐Mediated Inducible Gene Expression in Post Infarction Cardiac Fibroblasts is Altered by Intrinsic Exercise Phenotype

BackgroundIschemic heart disease is a major cause of morbidity. Regular active exercise is therapeutic after myocardial infarction, but up to 70% of individual exercise capacity is due to an intrinsic genetic component. Intrinsic capacity can be studied using high (HCR) and low (LCR) aerobic running capacity rat strains. The phenotypes differ by more than 5 fold in sedentary average running distance and time. The HCR rats have been characterized as “disease resistant”, while the LCRs are characterized as “disease prone”. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction, but little is known about the effect of intrinsic aerobic capacity on the inducible, receptor‐mediated fibroblast gene expression pathways following ischemia reperfusion.MethodsOn each study day, one HCR/LCR pair (n = 8 each) was anesthetized and hearts were rapidly excised. The hearts were immediately flushed with iced hyperkalemic, hyperosmotic, cardioplegia solution, and subjected to cold global ischemic arrest (80 min). Following arrest, the hearts underwent warm reperfusion (120 min) using a Langendorff style perfusion system. Following reperfusion, the heart was weighed and the LV was isolated. A mid ventricular ring was obtained to estimate infarction size (TTC), and part of the remaining tissue (~150 mg) was transferred to homogenation buffer on ice for subsequent isolation of fibroblasts. Cardiac fibroblasts from the LV of HCRs and LCR rats were isolated, and cultured in 6 well plates. Different wells were treated with BMP7, Angiotensin or both, with or without receptor antagonists (BMP2R, noggin; AngR, Losartan). After conditioning, RNA was isolated from the cultured fibroblasts, and RT‐PCR was used to determine baseline gene expression for Collagen I and III, MMP2, TIMP1, BMPR2, ATR1, SMAD5, and Bag3 in each phenotype.ResultsOverall, Collagen I gene expression was significantly higher in LCRs more than HCRs, but the activating pathways were phenotype dependent. Treating with Ang II induced gene expression for collagen I in LCR fibroblasts but not HCR, and was completely blocked with Losartan. By Contrast, treating with BMP7 induced gene expression of collagen I in HCR fibroblasts but not LCRs, and was completely blocked by noggin. Treating the fibroblasts with a combination of receptor agonists confirmed the phenotypic selectivity of the inducible pathways. Inhibiting BMP7 using noggin brought collagen I gene expression back to the control level with no changes in LCRs.ConclusionThe Angiotensin receptor pathway is responsive only in post‐ischemic LCR cardiac fibroblasts, and the BMP receptor pathway is responsive only in post‐ischemic HCR cardiac fibroblasts. How the intrinsic aerobic phenotype governs these pathways is unknown, but could shed new light on individuals who develop post‐infraction fibrosis despite therapeutic dosing with Angiotensin pathway antagonists.

Relative Profibrotic Gene Expression in Cardiac Fibroblasts from Low Aerobic Capacity “Disease Prone” Rats Following Ischemic Reperfusion

BackgroundIschemic heart disease is a major cause of morbidity. Regular active exercise is therapeutic after myocardial infarction, but up to 70% of individual exercise capacity is due to an intrinsic genetic component. Intrinsic capacity can be studied using high (HCR) and low (LCR) aerobic running capacity rat strains. The phenotypes differ by more than 5 fold in sedentary average running distance and time. The HCR rats have been characterized as “disease resistant”, while the LCRs are characterized as “disease prone”. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction, but little is known about the effect of intrinsic aerobic capacity on early fibroblast gene expression following ischemia reperfusion.MethodsOn each study day, one HCR/LCR pair (n = 8 each) was anesthetized and hearts were rapidly excised. The hearts were immediately flushed with iced hyperkalemic, hyperosmotic, cardioplegia solution, and subjected to cold global ischemic arrest (80 min). Following arrest, the hearts underwent warm reperfusion (120 min) using a Langendorff style perfusion system. Following reperfusion, the heart was weighed and the LV was isolated. A mid ventricular ring was obtained to estimate infarction size (TTC), and part of the remaining tissue (~150 mg) was transferred to homogenation buffer on ice for subsequent isolation of fibroblasts. Cardiac fibroblasts from the LV of HCRs and LCR rats were isolated, cultured and characterized. Immunohistochemistry verified fibroblasts/myofibroblasts markers. RNA was isolated from the cultured fibroblasts, and RT‐PCR was used to determine baseline gene expression for Collagen I and III, MMP2, TIMP1, BMPR2, ATR1, SMAD5, and Bag3 in each phenotype.ResultsImmunocytochemistry successfully confirmed the presence of both fibroblasts and myofibroblasts markers. In these post IR fibroblast/myofibroblasts, LCRs showed significantly higher gene expression (49%) in the collagen I/collagen III ratio but lower expression of extracellular matrix regulatory genes including MMP2 (20%), TIMP1 (17%), and SMAD5 (15%) compared to HCRs counterparts. BMPR2 (50%), Agtr1a (25%), also were both decreased, and the anti‐apoptotic gene, Bag3, was significantly (67%) higher in LCRs.ConclusionThese results indicate that the LCR phenotype is predisposed to expressing a more fibrotic character in fibroblasts harvested immediately following IR, and may indicate poorer long term outcome/earlier onset heart failure following acute ischemic injury to the heart. While the phenotype produced no differences in the acute injury, there may be phenotypic differences that develop later during resolution of the injury.

Rats genetically selected for low and high aerobic capacity exhibit altered soleus muscle myofilament functions.

Aerobic exercise capacity is critical to bodily health. As a model to investigate the mechanisms that determine health and disease, we employed low (LCR) and high (HCR) capacity running rat models selectively bred to concentrate the genes responsible for divergent aerobic running capacity. To investigate the skeletal muscle contribution to this innate difference in running capacity we employed an approach combining examination of the myofilament protein composition and contractile properties of the fast fiber extensor digitorum longus (EDL) and slow fiber soleus (SOL) muscles from LCR and HCR rats. Intact muscle force experiments demonstrate that SOL, but not EDL, muscles from LCR rats exhibit a three times greater decrease in fatigued force. To investigate the mechanism of this increased fatigability in the LCR SOL muscle, we determined the myofilament protein composition and functional properties. Force-Ca2+ measurements demonstrate decreased Ca2+ sensitivity of single skinned SOL muscle fibers from LCR compared with that of HCR rats. Segregating SOL fibers into fast and slow types demonstrates that the decreased Ca2+ sensitivity in LCR SOL results from a specific decrease in slow-type SOL fiber Ca2+ sensitivity such that it was similar to that of fast-type fibers. These results identify that the altered myofilament contractile properties of LCR SOL slow-type fibers result in a fast muscle type Ca2+ sensitivity and the LCR muscle phenotype. Overall our findings demonstrate alterations of the myofilament proteins could contribute to fatigability of the SOL muscle and the decreased innate aerobic running performance of LCR compared with HCR rats.

Animal Modeling for Hologenome: New inbred and conplastic rat exercise models for uncovering crosstalk between nuclear DNA, mitochondrial DNA, and gut microbiota

IntroductionThe hologenome is an ensemble of nuclear DNA (nDNA), mitochondrial DNA (mtDNA), and the microbiome that forms a unit of genetic selection that can propagate from one generation to the next. There is increasing evidence for inherited mitochondria‐gut microbiota involvement in cardiovascular and metabolic diseases. Here, we expand on the selectively‐bred Low Capacity Runner/High Capacity Runner (LCR/HCR) rat exercise model system into a genetically defined rat model framework for studying holobionts in controlled laboratory settings.MethodsAt Generation 23 of selection, we identified extreme representatives of female and male LCR and HCR rats and conducted a series of brother‐sister mating to produce inbred strains‐ LCR/Tol and HCR/Tol. We also developed two conplastic strains, one with nDNA from HCR and mtDNA from LCR, and the other with nDNA from LCR and mtDNA from HCR ‐LCR.HCRmt/Tol and HCR.LCRmt/Tol. mtDNA from LCR/Tol and HCR/Tol strains were compared with publicly available mtDNA from common inbred strains. Rats were phenotyped for cardiovascular risks, which included treadmill exercise capacity, VO2max, echocardiography, and blood pressure (BP) telemetry. Fecal microbial communities were profiled using 16S rRNA sequencing and metatranscriptomics.ResultsAs expected, HCR/Tol had significantly higher exercise capacity compared to LCR/Tol but little effect by mismatched mtDNA suggesting that the nDNA is dominant for regulating aerobic exercise capacity. VO2max measurement confirmed high exercise capacity in HCR/Tol and low in LCR/Tol. LCR mtDNA was identical to mtDNA from Wistar Kyoto inbred strain, while HCR mtDNA was identical to mtDNA from Fischer 344 × Brown Norway F1‐hybrid strain. Consistent with reports from selected LCR HCR rats, LCR/Tol showed significantly increased mean arterial pressure (MAP), systolic BP (SBP), and diastolic BP (DBP) compared to the HCR/Tol. Transfer of mtDNA from HCR to LCR nDNA background decreased MAP, SBP, DBP, and preserved diastolic cardiac function. In contrast, there was no difference in BP or cardiac parameters between HCR/Tol and HCR.LCRmt/Tol. Microbiome analysis indicated that host genomes co‐inhabit with differential commensal microbiota. Metatranscriptomics showed HCR/Tol had enhanced function in carbohydrate and amino acid metabolism, microbial metabolites production, and stress management.ConclusionWe successfully generated and systematically characterized genotypes and phenotypes of inbred and conplastic LCR/Tol and HCR/Tol animal models for holobiont studies. The inbred and conplastic strains retained key features of LCR HCR selectively bred models ‐‐ some influenced by nDNA‐mtDNA interaction. These models provide for a new pre‐clinical method to explore gut microbiota as therapeutic targets for complex diseases such as hypertension.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Skeletal muscle metabolism in rats with low and high intrinsic aerobic capacity: Effect of aging and exercise training.

PurposeExercise training increases aerobic capacity and is beneficial for health, whereas low aerobic exercise capacity is a strong independent predictor of cardiovascular disease and premature death. The purpose of the present study was to determine the metabolic profiles in a rat model of inborn low versus high capacity runners (LCR/HCR) and to determine the effect of inborn aerobic capacity, aging, and exercise training on skeletal muscle metabolic profile.MethodsLCR/HCR rats were randomized to high intensity low volume interval treadmill training twice a week or sedentary control for 3 or 11 months before they were sacrificed, at 9 and 18 months of age, respectively. Magnetic resonance spectra were acquired from soleus muscle extracts, and partial least square discriminative analysis was used to determine the differences in metabolic profile.ResultsSedentary HCR rats had 54% and 30% higher VO2max compared to sedentary LCR rats at 9 months and 18 months, respectively. In HCR, exercise increased running speed significantly, and VO2max was higher at age of 9 months, compared to sedentary counterparts. In LCR, changes were small and did not reach the level of significance. The metabolic profile was significantly different in the LCR sedentary group compared to the HCR sedentary group at the age of 9 and 18 months, with higher glutamine and glutamate levels (9 months) and lower lactate level (18 months) in HCR. Irrespective of fitness level, aging was associated with increased soleus muscle concentrations of glycerophosphocholine and glucose. Interval training did not influence metabolic profiles in LCR or HCR rats at any age.ConclusionDifferences in inborn aerobic capacity gave the most marked contrasts in metabolic profile, there were also some changes with ageing. Low volume high intensity interval training twice a week had no detectable effect on metabolic profile.

Differential regulation of cysteine oxidative post-translational modifications in high and low aerobic capacity

Given the association between high aerobic capacity and the prevention of metabolic diseases, elucidating the mechanisms by which high aerobic capacity regulates whole-body metabolic homeostasis is a major research challenge. Oxidative post-translational modifications (Ox-PTMs) of proteins can regulate cellular homeostasis in skeletal and cardiac muscles, but the relationship between Ox-PTMs and intrinsic components of oxidative energy metabolism is still unclear. Here, we evaluated the Ox-PTM profile in cardiac and skeletal muscles of rats bred for low (LCR) and high (HCR) intrinsic aerobic capacity. Redox proteomics screening revealed different cysteine (Cys) Ox-PTM profile between HCR and LCR rats. HCR showed a higher number of oxidized Cys residues in skeletal muscle compared to LCR, while the opposite was observed in the heart. Most proteins with differentially oxidized Cys residues in the skeletal muscle are important regulators of oxidative metabolism. The most oxidized protein in the skeletal muscle of HCR rats was malate dehydrogenase (MDH1). HCR showed higher MDH1 activity compared to LCR in skeletal, but not cardiac muscle. These novel findings indicate a clear association between Cys Ox-PTMs and aerobic capacity, leading to novel insights into the role of Ox-PTMs as an essential signal to maintain metabolic homeostasis.