Genetic deletion of IL-6 increases CK-MB, a classic cardiac damage marker, and decreases UPRmt genes after exhaustive exercise.

The intensity, duration, type of contraction, and muscle damage influence interleukin-6 (IL-6) response to acute exercise. However, in response to an exhaustive exercise session, the upregulation of IL-6 in the serum and heart is associated with an inflammatory condition and can inhibit autophagy. This study aimed to investigate the role of IL-6 in autophagy pathway responses and mitochondrial function in the heart of mice submitted to acute exhaustive physical exercise. The mice were allocated into three groups, five animals per group, for the wild type (WT) and the IL-6 knockout (IL-6 KO): Basal (sedentary; Basal), 1 h (after 1 h of the acute exercise; 1 h), and 3 h (after 3 h of the acute exercise; 3 h). After the specific time for each group, the blood was collected, each mouse heart was removed, and the left ventricle (LV) was isolated. In summary, under basal conditions, without the influence of the acute exercise, the IL-6 KO group showed lower number of nuclei in the cardiac tissue, but higher collagen deposition; lower messenger RNA (mRNA) levels of Prkaa1 and Mtco1, but higher mRNA levels of Ulk1; and higher protein levels of the ratio p-AMPK/AMPK in the heart when compared to WT at the same time point. After the acute exercise (1 and 3 h), the IL-6 KO group had lower mRNA levels of Tfam, Mtnd1, Mtco1, and Nampt in the heart when compared to WT after exercise; higher serum levels of creatine kinase (CK), CK-MB, and lactate dehydrogenase for the IL-6 group when compared to the WT group after the exercise. Specifically, the heat-shock protein 60 protein levels in the heart increased 3 h after exhaustive exercise in the WT group, but not in the IL-6 KO group. The study emphasizes that IL-6 may offer cardioprotective effects, including mitochondrial adaptations in response to acute exhaustive exercise.

Interleukin-6 ablation does not alter morphofunctional heart characteristics but modulates physiological and inflammatory markers after strenuous exercise.

Interleukin-6 (IL-6) is associated with pathological cardiac hypertrophy and can be dramatically increased in serum after an acute strenuous exercise session. However, IL-6 is also associated with the increased production and release of anti-inflammatory cytokines and the inhibition of tumor necrosis factor-alpha (TNF-α) after chronic moderate exercise. To elucidate the relevance of IL-6 in inflammatory and hypertrophic signaling in the heart in response to an acute strenuous exercise session, we combined transcriptome analysis using the BXD mice database and exercised IL-6 knockout mice (IL-6KO). Bioinformatic analysis demonstrated that low or high-levels of Il6 mRNA in the heart did not change the inflammation- and hypertrophy-related genes in BXD mice strains. On the other hand, bioinformatic analysis revealed a strong positive correlation between Il6 gene expression in skeletal muscle with inflammation-related genes in cardiac tissue in several BXD mouse strains, suggesting that skeletal muscle-derived IL-6 could alter the heart's intracellular signals, particularly the inflammatory signaling. As expected, an acute strenuous exercise session increased IL-6 levels in wild-type, but not in IL-6KO mice. Despite not showing morphofunctional differences in the heart at rest, the IL-6KO group presented a reduction in physical performance and attenuated IL-6, TNF-α, and IL-1beta kinetics in serum, as well as lower p38MAPK phosphorylation, Ampkalpha expression, and higher Acta1 and Tnf gene expressions in the left ventricle in the basal condition. In response to strenuous exercise, IL-6 ablation was linked to a reduction in the pro-inflammatory response and higher activation of classical physiological cardiac hypertrophy proteins.

Impact of Different Physical Exercises on the Expression of Autophagy Markers in Mice.

Although physical exercise-induced autophagy activation has been considered a therapeutic target to enhance tissue health and extend lifespan, the effects of different exercise models on autophagy in specific metabolic tissues are not completely understood. This descriptive investigation compared the acute effects of endurance (END), exhaustive (ET), strength (ST), and concurrent (CC) physical exercise protocols on markers of autophagy, genes, and proteins in the gastrocnemius muscle, heart, and liver of mice. The animals were euthanized immediately (0 h) and six hours (6 h) after the acute exercise for the measurement of glycogen levels, mRNA expression of Prkaa1, Ppargc1a, Mtor, Ulk1, Becn1, Atg5, Map1lc3b, Sqstm1, and protein levels of Beclin 1 and ATG5. The markers of autophagy were measured by quantifying the protein levels of LC3II and Sqstm1/p62 in response to three consecutive days of intraperitoneal injections of colchicine. In summary, for gastrocnemius muscle samples, the main alterations in mRNA expressions were observed after 6 h and for the ST group, and the markers of autophagy for the CC group were increased (i.e., LC3II and Sqstm1/p62). In the heart, the Beclin 1 and ATG5 levels were downregulated for the ET group. Regarding the markers of autophagy, the Sqstm1/p62 in the heart tissue was upregulated for the END and ST groups, highlighting the beneficial effects of these exercise models. The liver protein levels of ATG5 were downregulated for the ET group. After the colchicine treatment, the liver protein levels of Sqstm1/p62 were decreased for the END and ET groups compared to the CT, ST, and CC groups. These results could be related to diabetes and obesity development or liver dysfunction improvement, demanding further investigations.

Role of interleukin-6 in inhibiting hepatic autophagy markers in exercised mice.

BACKGROUND: Based on the crosstalk of inflammation with apoptosis, autophagy, and endoplasmic reticulum (ER) stress, the main objective of this study was to explore the role of interleukin-6 (IL-6) on genes and proteins related to these phenomena in the livers of mice submitted to acute exhaustive exercise. METHODS: Reverse transcription-quantitative polymerase chain reaction and immunoblotting technique were used to evaluate the livers of wild-type (WT) and IL-6 knockout (KO) mice at baseline (BL) and 3 h after the acute exhaustive physical exercise (EE). RESULTS: Compared to the WT at baseline, the IL-6 KO had lower exhaustion velocity, mRNA levels of Mtor, Ulk1, Map1lc3b, and Mapk14, and protein contents of ATG5 and p-p70S6K/p70S6K. For the WT group, the EE decreased glycemia, mRNA levels of Casp3, Mtor, Ulk1, Foxo1a, Mapk14, and Ppargc1a, and protein contents of ATG5 and p-p70S6K/p70S6K, but increased mRNA levels of Sqstm1. For the IL-6 KO group, the EE decreased glycemia, mRNA levels of Casp3 and Foxo1a, and protein contents of pAkt/Akt and Mature/Pro IL-1beta, but increased mRNA levels of Sqstm1, and protein contents of p-AMPK/AMPK. CONCLUSION: The inhibition of the hepatic autophagy markers induced by the acute EE was attenuated in IL-6 KO mice, highlighting a new function of this cytokine.

Moderate, but Not Excessive, Training Attenuates Autophagy Machinery in Metabolic Tissues.

The protective effects of chronic moderate exercise-mediated autophagy include the prevention and treatment of several diseases and the extension of lifespan. In addition, physical exercise may impair cellular structures, requiring the action of the autophagy mechanism for clearance and renovation of damaged cellular components. For the first time, we investigated the adaptations on basal autophagy flux in vivo in mice's liver, heart, and skeletal muscle tissues submitted to four different chronic exercise models: endurance, resistance, concurrent, and overtraining. Measuring the autophagy flux in vivo is crucial to access the functionality of the autophagy pathway since changes in this pathway can occur in more than five steps. Moreover, the responses of metabolic, performance, and functional parameters, as well as genes and proteins related to the autophagy pathway, were addressed. In summary, the regular exercise models exhibited normal/enhanced adaptations with reduced autophagy-related proteins in all tissues. On the other hand, the overtrained group presented higher expression of Sqstm1 and Bnip3 with negative morphological and physical performance adaptations for the liver and heart, respectively. The groups showed different adaptions in autophagy flux in skeletal muscle, suggesting the activation or inhibition of basal autophagy may not always be related to improvement or impairment of performance.

IL-6 deletion decreased REV-ERBα protein and influenced autophagy and mitochondrial markers in the skeletal muscle after acute exercise.

Interleukin 6 (IL-6) acts as a pro and anti-inflammatory cytokine, has an intense correlation with exercise intensity, and activates various pathways such as autophagy and mitochondrial unfolded protein response. Also, IL-6 is interconnected to circadian clock-related inflammation and can be suppressed by the nuclear receptor subfamily 1, group D, member 1 (Nr1d1, protein product REV-ERBα). Since IL-6 is linked to physical exercise-modulated metabolic pathways such as autophagy and mitochondrial metabolism, we investigated the relationship of IL-6 with REV-ERBα in the adaptations of these molecular pathways in response to acute intense physical exercise in skeletal muscle. The present study was divided into three experiments. In the first one, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were divided into three groups: Basal time (Basal; sacrificed before the acute exercise), 1 hour (1hr post-Ex; sacrificed 1 hour after the acute exercise), and 3 hours (3hr post-Ex; sacrificed 3 hours after the acute exercise). In the second experiment, C2C12 cells received IL-6 physiological concentrations or REV-ERBα agonist, SR9009. In the last experiment, WT mice received SR9009 injections. After the protocols, the gastrocnemius muscle or the cells were collected for reverse transcription-quantitative polymerase chain reaction (RTq-PCR) and immunoblotting techniques. In summary, the downregulation of REV-ERBα, autophagic flux, and most mitochondrial genes was verified in the IL-6 KO mice independent of exercise. The WT and IL-6 KO treated with SR9009 showed an upregulation of autophagic genes. C2C12 cells receiving IL-6 did not modulate the Nr1d1 mRNA levels but upregulated the expression of some mitochondrial genes. However, when treated with SR9009, IL-6 and mitochondrial gene expression were upregulated in C2C12 cells. The autophagic flux in C2C12 suggest the participation of REV-ERBα protein in the IL-6-induced autophagy. In conclusion, the present study verified that the adaptations required through physical exercise (increases in mitochondrial content and improvement of autophagy machinery) might be intermediated by an interaction between IL-6 and REVERBα.

One Bout of Aerobic Exercise Can Enhance the Expression of Nr1d1 in Oxidative Skeletal Muscle Samples.

The nuclear receptor subfamily 1, group D member 1 (Nr1d1), plays a role in the skeletal muscle's oxidative capacity, mitochondrial biogenesis, atrophy genes, and muscle fiber size. In light of the effects of physical exercise, the present study investigates the acute response of Nr1d1 and genes related to atrophy and mitochondrial biogenesis on endurance and resistance exercise protocols. In this investigation, we observed, after one bout of endurance exercise, an upregulation of Nr1d1 in soleus muscle, but not in the gastrocnemius, and some genes related to mitochondrial biogenesis and atrophy were enhanced as well. Also, analysis of muscle transcripts from diverse isogenic BXD mice families revealed that the strains with higher Nr1d1 gene expression displayed upregulation of AMPK signaling and mitochondrial-related genes. In summary, a single session of endurance exercise can enhance the Nr1d1 mRNA levels in an oxidative muscle.

TLR4 deletion increases basal energy expenditure and attenuates heart apoptosis and ER stress but mitigates the training-induced cardiac function and performance improvement.

Strategies capable of attenuating TLR4 can attenuate metabolic processes such as inflammation, endoplasmic reticulum (ER) stress, and apoptosis in the body. Physical exercise has been a cornerstone in suppressing inflammation and dysmetabolic outcomes caused by TRL4 activation. Thus, the present study aimed to evaluate the effects of a chronic physical exercise protocol on the TLR4 expression and its repercussion in the inflammation, ER stress, and apoptosis pathways in mice hearts. Echocardiogram, RT-qPCR, immunoblotting, and histological techniques were used to evaluate the left ventricle of wild-type (WT) and Tlr4 knockout (TLR4 KO) mice submitted to a 4-week physical exercise protocol. Moreover, we performed a bioinformatics analysis to expand the relationship of Tlr4 mRNA in the heart with inflammation, ER stress, and apoptosis-related genes of several isogenic strains of BXD mice. The TLR4 KO mice had higher energy expenditure and heart rate in the control state but lower activation of apoptosis and ER stress pathways. The bioinformatics analysis reinforced these data. In the exercised state, the WT mice improved performance and cardiac function. However, these responses were blunted in the KO group. In conclusion, TLR4 has an essential role in the inhibition of apoptosis and ER stress pathways, as well as in the training-induced beneficial adaptations.

Tlr4 participates in the responses of markers of apoptosis, inflammation, and ER stress to different acute exercise intensities in mice hearts.

BACKGROUND: Toll-like receptor 4 (Tlr4) is recognized due to its role in the immune response. Also, this protein can participate in the signaling pathway of events triggered by physical exercise such as apoptosis, inflammation, and endoplasmic reticulum (ER) stress. The main objective of this study was to evaluate the role of Tlr4 in the markers of these events in the myocardium of mice submitted to acute physical exercise (APE) protocols at different intensities. METHODS: Echocardiogram, RT-qPCR, and immunoblotting technique were used to evaluate the left ventricle of wild-type (WT) and Tlr4 knockout (Tlr4 KO) submitted to APE protocols at 45, 60, and 75% of their maximal velocity. Also, we performed the bioinformatics analysis to establish the connection of heart mRNA levels of Tlr4 with heart genes of inflammation and ER stress of several isogenic strains of BXD mice. RESULTS: Under basal conditions, the Tlr4 deletion diminished the performance, and expression of inflammation and ER stress genes in the left ventricle, but increased the serum levels of CK, Il-17, and Tnf-alpha. Under the same exercise conditions, the Tlr4 deletion reduced the glycemia, serum levels of CK, Il-17, and Tnf-alpha, as well as genes and/or proteins related to apoptosis, inflammation and ER stress in the left ventricle, but increased the levels of CK-mb and LDH, as well as other genes related to apoptosis, inflammation, and ER stress in the left ventricle. CONCLUSION: Altogether, the current findings highlighted the effects of different acute exercise intensities were attenuated in the heart of Tlr4 KO mice.