Exploring the effects of lifelong aerobic exercise on early skeletal muscle remodeling between 8 and 14 months in the rat - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice J Physiol Biochem . 2026 Apr 18;82(1):42. doi: 10.1007/s13105-026-01178-y Search in PMC Search in PubMed View in NLM Catalog Add to search Show available content in en fr Exploring the effects of lifelong aerobic exercise on early skeletal muscle remodeling between 8 and 14 months in the rat Alexandra Moreira-Pais Alexandra Moreira-Pais 1 Research Center in Physical Activity, Health and Leisure (CIAFEL), Faculty of Sport, Laboratory for Integrative and Translational Research in Population Health (ITR), University of Porto (FADEUP), Porto, 4200-450 Portugal 2 LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Aveiro, 3810-193 Portugal 3 Centre for Research and Technology of Agro Environmental and Biological Sciences (CITAB), Inov4Agro, University of Trás-os-Montes and Alto Douro (UTAD), Quinta de Prados, Vila Real, 5000-801 Portugal 8 Present Address: Department of Medical Sciences, University of Aveiro, 3810-193 Aveiro, Portugal Find articles by Alexandra Moreira-Pais 1, 2, 3, 8, ✉ , Rita Ferreira Rita Ferreira 2 LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Aveiro, 3810-193 Portugal Find articles by Rita Ferreira 2 , Maria João Neuparth Maria João Neuparth 1 Research Center in Physical Activity, Health and Leisure (CIAFEL), Faculty of Sport, Laboratory for Integrative and Translational Research in Population Health (ITR), University of Porto (FADEUP), Porto, 4200-450 Portugal 4 UCIBIO - Applied Molecular Biosciences Unit, Toxicologic Pathology Research Laboratory, University Institute of Health Sciences (1H-TOXRUN, IUCS-CESPU), 4585-116 Gandra, Portugal Find articles by Maria João Neuparth 1, 4 , Margarida Fardilha Margarida Fardilha 6 Institute of Biomedicine (iBiMED), Department of Medical Sciences, University of Aveiro, Aveiro, 3810-193 Portugal Find articles by Margarida Fardilha 6 , Daniel Moreira-Gonçalves Daniel Moreira-Gonçalves 1 Research Center in Physical Activity, Health and Leisure (CIAFEL), Faculty of Sport, Laboratory for Integrative and Translational Research in Population Health (ITR), University of Porto (FADEUP), Porto, 4200-450 Portugal Find articles by Daniel Moreira-Gonçalves 1 , Paula A Oliveira Paula A Oliveira 3 Centre for Research and Technology of Agro Environmental and Biological Sciences (CITAB), Inov4Agro, University of Trás-os-Montes and Alto Douro (UTAD), Quinta de Prados, Vila Real, 5000-801 Portugal Find articles by Paula A Oliveira 3 , José A Duarte José A Duarte 5 UCIBIO - Applied Molecular Biosciences Unit, Translational Toxicology Research Laboratory, University Institute of Health Sciences (1H-TOXRUN, IUCS-CESPU), Gandra, 4585- 116 Portugal 7 Associate Laboratory i4HB - Institute for Health and Bioeconomy, University Institute of Health Sciences - CESPU, Gandra, 4585-116 Portugal Find articles by José A Duarte 5, 7 Author information Article notes Copyright and License information 1 Research Center in Physical Activity, Health and Leisure (CIAFEL), Faculty of Sport, Laboratory for Integrative and Translational Research in Population Health (ITR), University of Porto (FADEUP), Porto, 4200-450 Portugal 2 LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Aveiro, 3810-193 Portugal 3 Centre for Research and Technology of Agro Environmental and Biological Sciences (CITAB), Inov4Agro, University of Trás-os-Montes and Alto Douro (UTAD), Quinta de Prados, Vila Real, 5000-801 Portugal 4 UCIBIO - Applied Molecular Biosciences Unit, Toxicologic Pathology Research Laboratory, University Institute of Health Sciences (1H-TOXRUN, IUCS-CESPU), 4585-116 Gandra, Portugal 5 UCIBIO - Applied Molecular Biosciences Unit, Translational Toxicology Research Laboratory, University Institute of Health Sciences (1H-TOXRUN, IUCS-CESPU), Gandra, 4585- 116 Portugal 6 Institute of Biomedicine (iBiMED), Department of Medical Sciences, University of Aveiro, Aveiro, 3810-193 Portugal 7 Associate Laboratory i4HB - Institute for Health and Bioeconomy, University Institute of Health Sciences - CESPU, Gandra, 4585-116 Portugal 8 Present Address: Department of Medical Sciences, University of Aveiro, 3810-193 Aveiro, Portugal ✉ Corresponding author. Received 2025 Sep 11; Accepted 2026 Mar 31; Issue date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ . PMC Copyright notice PMCID: PMC13091809 PMID: 41999467 Abstract The insidious onset and progression of sarcopenia make it vital to understand the early skeletal muscle changes and explore therapies to slow its progression. This study explored the gastrocnemius remodeling at an early stage of aging (14 months of age) and the effects of lifelong aerobic exercise. For that, 2-month-old male Wistar rats underwent a 12-month treadmill exercise program. Sedentary age-matched, young sedentary, and young exercised for 6 months rats were considered. The results highlighted an age-related decrease in the relative gastrocnemius muscle mass, suggestive of loss or atrophy of some fibers, which was mitigated by lifelong aerobic exercise. Consequently, an age-related compensatory hypertrophy was suggested to be triggered in the gastrocnemius muscle. Data proposed that aging reduced mitochondrial density, indicated by citrate synthase (CS) activity, which was prevented by lifelong aerobic exercise. The reduced CS activity correlated with increased ATP-dependent 6-phosphofructokinase (PFKM)/ATP synthase subunit beta (ATPB) ratio, suggesting that at an early stage of aging, the skeletal muscle favors the glycolytic metabolism in response to decreased mitochondrial content. The results also pointed to an age-induced AMP-activated protein kinase (AMPK) activation and an AMPK-related apoptosis inhibition, perchance to reduce fiber loss or atrophy. The basal phosphorylated AMPK/AMPK ratio decreased with lifelong aerobic exercise, possibly reflecting the exercise-induced increase in CS activity. This work highlights the importance of studying early skeletal muscle changes in aging for timely disease management and prevention. Keywords: AMPK, Apoptosis, Mitochondria, Muscle wasting, Physical activity, Sarcopenia Key points Compensatory hypertrophy occurs in the gastrocnemius at early aging At early aging, the gastrocnemius relies more on glycolysis for energy production Lifelong aerobic exercise prevents the relative gastrocnemius mass loss with age Lifelong aerobic exercise prevents the age-induced decline in mitochondrial density Introduction Sarcopenia is a progressive and generalized skeletal muscle disease defined by low levels of muscle strength, muscle quantity or quality, and physical performance that can affect 10% to 27% of adults aged 60 years and older [ 1 , 2 ]. Sarcopenia is associated with premature mortality and increased risk of disability, hospitalization, postoperative complications, frailty, and mobility limitations [ 3 ]. In humans, the onset and progression of sarcopenia are insidious [ 4 ]. Skeletal muscle mass and strength typically peak in the mid-20s to 30 years of age, and after this peak, a gradual decline usually begins between ages 30 and 50, progressing at an average rate of about 1% per year until around age 70 [ 5 ]. Thus, sarcopenia appears to develop from processes that are initiated early in life, probably between young adulthood and early middle age [ 6 ]. When these processes clinically manifest, they have been ongoing for decades, making sarcopenia not noticeable until significant dysfunction occurs and the mobility and quality of life of the patient are affected (clinical stage) [ 6 ]. Nonetheless, the European Working Group on Sarcopenia in Older People (EWGSOP2) guidelines recommend that sarcopenia case-finding in clinical practice starts only when a patient reports symptoms or signs of sarcopenia [ 1 ]. On another hand, the EWGSOP2 recognizes that the identification of older individuals at high risk of sarcopenia and the determination of preventive actions for those individuals represent critical gaps in current knowledge [ 1 ]. This is probably a consequence of the use of very young individuals as controls and much older individuals as the sarcopenia group in many preclinical and clinical studies investigating the pathogenesis and possible treatments of this disease [ 7 – 9 ], which may not capture the changes occurring between these age extremes. The existing studies that evaluate the time course of skeletal muscle changes in response to aging and/or to therapeutic approaches like exercise are often focused on specific pathways [ 10 – 12 ], not offering an integrative comprehension of the multiple processes involved in skeletal muscle aging. Therefore, it is critical to obtain a global comprehension of the changes that occur at the early stage of aging (preclinical stage) so that early-stage detection can be made in clinical practice for timely management to slow or prevent the development of sarcopenia. Physical inactivity has been associated with an augmented risk of developing sarcopenia, which aligns with the fact that only 28% to 34% of adults aged 65 years and older enroll in any leisure-time physical activity [ 13 , 14 ]. Therefore, lifelong exercise has been investigated as a potential preventive strategy for sarcopenia [ 15 ]. Still, there is a limited understanding of its impact at the early stage of aging. In old rats, lifelong aerobic exercise seems to prevent the age-induced loss of skeletal muscle mass and quality by modulating protein degradation, autophagy, and mitochondrial function [ 16 – 18 ]. While resistance exercise seems to be more effective in increasing skeletal muscle mass and strength, aerobic exercise effects may range from improvement of skeletal muscle performance and metabolism to skeletal muscle hypertrophy and prevention of future disabilities [ 15 , 19 , 20 ]. Therefore, this study aimed to obtain a global and integrative picture of the gastrocnemius muscle remodeling occurring at an early stage of aging, assessed in rats at 14 months of age, and to evaluate how lifelong aerobic exercise modulates this remodeling. The remodeling of the gastrocnemius muscle was assessed by analyze and integrate anthropometric parameters (body weight, and absolute and relative gastrocnemius muscle mass), the cross-sectional area (CSA) of the gastrocnemius muscle, and markers of anaerobic and aerobic energy metabolism, oxidative stress (protein carbonylation and nitration), Bax-related apoptosis, catabolism (atrogin-1), and neuromuscular junction (NMJ) health. Materials and methods Animal protocol Male Wistar Unilever rats were obtained at the age of 4 weeks from Charles River Laboratories (FR). Before the experiments, the rats were subjected to a 2-week quarantine and then randomly distributed into four experimental groups (maximum of 4–5 rats per cage; 1500U Eurostandard Type IV S cages, Tecniplast, Varese, IT): sedentary and sacrificed at 8 months (SED1, n = 8), exercised and sacrificed at 8 months (EX1, n = 10), sedentary and sacrificed at 14 months (SED2, n = 8), and exercised and sacrificed at 14 months (EX2, n = 10). The ages of 8 (young adulthood) and 14 (middle adulthood) months were chosen to study the skeletal muscle remodeling that occurs at an early stage of the aging process before senescence starts (equivalent to 22 and 35 human years, respectively) [ 21 , 22 ]. The rats were housed in a controlled environment at 18 ± 2 °C and 55 ± 5% of relative humidity with a 12:12 h light-dark cycle with ad libitum food (Mucedola 4RF21, Milan, IT) and water access. The rats from the EX1 and EX2 groups started a treadmill (Treadmill Control LE 8710, Harvard Apparatus, US) exercise program at 2 months of age for, respectively, 6 and 12 months, to mimic continuous exercise training in young humans [ 21 ]. In the first week (habituation period), the rats ran 30 min per day, 5 days per week, at a 0° slope. For the remaining weeks, the rats ran 60 min per day, 5 days per week, at a 0° slope. The speed of the treadmill was set for 70% of the maximal speed capacity of each rat, which was adjusted every 6 weeks. SED1 and SED2 rats were placed on a non-moving treadmill for the same amount of time and in the same conditions to simulate the environmental changes that EX1 and EX2 rats were subjected to. The rats were euthanized 48 h after the last exercise session. At each necropsy, the rats were weighed and euthanized via an intraperitoneal injection of an overdose of ketamine (75 mg.kg − 1 , Imalgene 1000, Merial SAS, Lyon, FR) and xylazine (10 mg.kg − 1 , Rompun 2%, Bayer, Kiel, DE), followed by exsanguination as indicated by the Federation of European Laboratory Animal Science Associations [ 23 ] and the collection of the drawn blood. The two gastrocnemius muscles of each rat were collected, weighed, and treated as follows: one gastrocnemius muscle of each rat was transversely cut in half at mid-belly and fixed in a solution of 4% paraformaldehyde for histological analyses; the remaining skeletal muscles were designated for biochemical analyses and stored at −80 °C until the analyses. The right and left tibiae were collected to assess tibia length, an indicator of animal body size that is independent of changes in skeletal muscle and adipose tissue masses [ 24 ]. All the procedures were approved by the University of Trás-os-Montes and Alto Douro Ethics Review Body ORBEA (Orgão Responsável pelo Bem-Estar e Ética Animal, reference 424-e-DCV-2016) and by the Portuguese Competent Authority DGAV (Direção Geral de Alimentação e Veterinária, license number 021326), according to the European Guidelines, and following the Portuguese law on animal protection for scientific purposes (decree-law number 113/2013). Histological analyses of the gastrocnemius muscle The skeletal muscles were fixed in 4% paraformaldehyde for 24 h at 4 °C and then embedded in paraffin to prepare paraffin blocks. Serial transverse cross-sections (5 μm in thickness) were cut in a manual microtome with the fibers oriented perpendicular to the blade. After deparaffinization with xylol and hydration with decreasing concentrations of alcohol (100%, 90%, and 70%, v/v), the slides were stained with hematoxylin and eosin (H&E) to analyze the CSA of the fibers. Skeletal muscle images were acquired with a microscope (Zeiss Axio Imager Z1, Carl Zeiss, Oberkochen, DE) at 200x magnification and analyzed with ZEN lite software (ZEN v3.2 (blue edition), Carl Zeiss, Oberkochen, DE). Preparation of gastrocnemius muscle homogenates Gastrocnemius muscle samples were homogenized with a Teflon ® pestle in a motor-driven Potter-Elvehjem glass homogenizer at 0–4 °C in a phosphate buffer (0.1 M KH 2 PO 4 , 0.5% Triton X-100, and 0.2 M PMSF) in a proportion of 50 mg of skeletal muscle (transversely cut) to 1 mL of buffer. The protein content of the skeletal muscle homogenates was evaluated with the commercial kit DC™ Protein Assay (Bio-Rad, Hercules, CA, US), according to the manufacturer’s instructions and by utilizing bovine serum albumin as a standard. The obtained skeletal muscle homogenates were preserved at −80 °C for the biochemical analyses described in the following subsections. Citrate synthase activity in gastrocnemius muscle homogenates Citrate synthase (CS) activity was determined in the gastrocnemius muscle homogenates based on the approach described by Coore and collaborators [ 25 ]. Concisely, 5,5’-dithiobis-(2-nitrobenzoic acid) reacted with the thiol groups of coenzyme A (CoA, released by the reaction of acetyl-CoA with oxaloacetate), which was spectrophotometrically measured at 412 nm (molar extinction coefficient of 13.6 mM − 1 .cm − 1 ). The activity of CS was expressed in nmol per minute per milligram of gastrocnemius muscle total protein. Immunoblotting analyses of the gastrocnemius muscle homogenates To determine the content of protein carbonyls in the gastrocnemius muscle, a volume (vol.) corresponding to 40 µg of total protein was mixed with 1vol. of SDS 12% and 2vol. of dinitrophenylhydrazine 10 mM in 10% trifluoroacetic acid, followed by 30 min of dark incubation. Afterward, 1.5vol. of a solution consisting of 18% β-mercaptoethanol and 30% glycerol in Tris 2 M was added. The derivatized skeletal muscle homogenates were electrophoresed on a 12.5% SDS-PAGE gel following Laemmli [ 26 ] and blotted onto a nitrocellulose membrane (Amersham™, Protan ® , GE Healthcare, Chicago, IL, US) for 2 h at 200 mA. The immunodetection was performed by using an anti-dinitrophenol (DNP) primary antibody. For the remaining western blotting analyses, equivalent volumes of 50 µg of total protein of each skeletal muscle homogenate were electrophoresed on a 12.5% SDS-PAGE gel following Laemmli [ 26 ] and blotted onto a nitrocellulose membrane (Amersham™, Protan ® , GE Healthcare, Chicago, IL, US) for 2 h at 200 mA. Protein loading was controlled by Ponceau S staining, as stressful stimuli can influence the normally used housekeeping markers in skeletal muscle [ 27 ]. Nonspecific binding was blocked by incubating the membranes for 1 h in a 5% (w/v) solution of non-fat dry milk in tris-buffered saline with Tween 20 (TBST). Then, the membranes were incubated with the corresponding primary antibody: AMP-activated protein kinase (AMPK, ab80039), autophagy protein 5 (ATG5, ab108327), ATP synthase subunit beta, mitochondrial precursor (ATPB, ab14730), BAX (ab32503), Bcl-2-related protein A1 (BCL2A1, ab33862), brain-derived neurotrophic factor (BDNF, ab226843), electron transfer flavoprotein-ubiquinone oxidoreductase, mitochondrial (ETFDH, ab91508), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ab9485), phosphorylated AMPK (pAMPK, ab23875), ATP-dependent 6-phosphofructokinase, muscle type (PFKM, ab154804) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α, ab191838) from Abcam (Cambridge, UK); atrogin-1 (AP2041) from ECM Biosciences (Aurora, CO, US); NAD-dependent protein deacetylase sirtuin-3 (SIRT3, 2627 S) from Cell Signaling Technology (Danvers, MA, US); calcitonin gene-related peptide (CGRP, PA5-114931) from Invitrogen (Waltham, MA, US); and 3-nitrotyrosine (3-NT, MAB5404) and DNP (MAB2223) from Sigma-Aldrich (St. Louis, MO, US). Afterward, the membranes were washed three times with TBST for 10 min, incubated with the corresponding secondary antibody (anti-rabbit, NA934V, or anti-mouse, NA931V, from GE Healthcare, Chicago, IL, US), and washed again. All the antibodies were diluted 1:1000 in a 5% (w/v) solution of non-fat dry milk in TBST. The incubations with the primary antibody were performed overnight at 4 °C, and with the secondary antibody for 2 h at room temperature and with agitation. The immunoreactive bands were revealed with enhanced chemiluminescence (ECL) reagents (ECL Clarity, Bio-Rad, Hercules, CA, US) following the manufacturer’s procedure. The images were obtained using the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, US) and analyzed with the Image Lab software (v6.0.0., Bio-Rad, Hercules, CA, US). The optical density (OD) values are expressed in arbitrary units. Statistical analyses Data normality was appraised with the Shapiro-Wilk test, except for the fibers’ CSA data normality, which was assessed with the Kolmogorov-Smirnov test. The significant differences among groups were verified with the Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test for fibers' CSA data, and with the one-way analysis of variance test followed by Tukey’s multiple comparison post hoc test for the remaining data, and the values are presented, respectively, as median with interquartile range or mean ± standard deviation. A chi-square test was conducted to evaluate the differences in the distribution of the fibers’ CSA of SED1 and SED2 rats in CSA ranges. Pearson correlations were performed, when considered necessary, for an in-depth interpretation of the results. Results were considered significantly different when p < 0.05 and as a tendency when 0.05 < p < 0.1. The gastrocnemius muscle mass values were normalized to body weight and tibia length. All the statistical analyses were performed with the GraphPad Prism software for Windows (v6.01, Boston, MA, US). Results Anthropometric data and gastrocnemius muscle morphometric remodeling Anthropometric data are presented in Table 1 . Body weight increased with aging, and exercise attenuated this increase. No changes between groups were observed in the absolute gastrocnemius muscle mass. To account for the body weight changes, the relative gastrocnemius muscle mass (body weight-normalized) was considered, which in aging contexts is also known as the sarcopenia index [ 28 ]. A decline in the relative gastrocnemius muscle mass in SED2 rats compared to SED1 suggests a failure to maintain the skeletal muscle mass in relation to body weight with aging, whereas exercise mitigated this aging effect, as demonstrated by the increased values in EX2 rats compared to SED2 ones. To account for rat size changes, the gastrocnemius muscle mass values were normalized to tibia length, and no differences between groups were observed. Table 1. Body weight (BW), tibia length (TL), and gastrocnemius (GAS) muscle mass of SED1 ( n = 8), EX1 ( n = 10), SED2 ( n = 8), and EX2 ( n = 10) rats. The gastrocnemius muscle mass values normalized to body weight and tibia length are also depicted SED1 EX1 SED2 EX2 BW (g) 471.73 ± 27.78 404.17 ± 14.45 ### 541.83 ± 44.94 ### 434.81 ± 29.70 **** TL (cm) 4.46 ± 0.13 4.37 ± 0.12 4.51 ± 0.15 4.59 ± 0.17 $$ GAS muscle mass (g) 4.69 ± 0.18 4.45 + 0.25 4.41 + 0.30 4.39 ± 0.33 GAS muscle mass/BW (mg.g − 1 ) 9.97 ± 0.47 11.01 ± 0.57 ## 8.16 ± 0.61 #### 10.09 ± 0.42 ****, $$ GAS muscle mass/TL (g.cm − 1 ) 1.05 ± 0.04 1.02 ± 0.07 0.98 ± 0.06 0.96 ± 0.08 Open in a new tab #### p < 0.0001 vs. SED1, ### p < 0.001 vs. SED1, ## p < 0.01 vs. SED1, $$ p < 0.01 vs. EX1, and **** p < 0.0001 vs. SED2 The gastrocnemius muscle was histologically analyzed to infer the changes in the fibers’ CSA. SED2 rats presented an increased CSA of the gastrocnemius muscle fibers compared to SED1 rats (Fig. 1 a). In addition, SED2 rats presented a high heterogeneity in fiber size compared to SED1 rats (Fig. 1 b). The fibers' CSA of SED2 rats varied between approximately 469–5284 µm 2 , with a higher percentage of fibers with a CSA around 1200 µm 2 , compared to 507–3911 µm 2 in SED1 rats, with a higher percentage of fibers with a CSA around 1600 µm 2 . More specifically, in the SED2 group, approximately 53% of the fibers had a CSA between 400 and 1600 µm 2 , 37% between 1800 and 2800 µm 2 , 9% between 3000 and 4000 µm 2 and 1% between 4200 and 5200 µm 2 , compared to, respectively, 71%, 28%, 1% and 0% in the SED1 group (χ 2 = 63.56, df = 3, p < 0.0001). This concomitant occurrence of large and smaller fibers in the gastrocnemius muscle of SED2 rats compared to SED1 rats (Fig. 1 c) suggests the occurrence of compensatory hypertrophy following the loss or atrophy of surrounding muscle fibers [ 29 ]. Exercise further increased the CSA of gastrocnemius fibers in EX1 and EX2 rats compared to, respectively, SED1 and SED2 ones (Fig. 1 a). Fig. 1. Open in a new tab ( a ) Cross-sectional area (CSA) of the gastrocnemius muscle fibers and ( b ) fiber CSA distribution of the gastrocnemius muscle ( n = 4 and 600 fibers per group). ( c ) Representative photomicrographs of H&E-stained gastrocnemius muscle sections, where in the gastrocnemius muscle of SED2 rats, the co-occurrence of larger fibers (black star) with smaller ones (white star) was observable. The bar scale represents 50 μm. (**** p < 0.0001) Energy metabolism remodeling in the gastrocnemius muscle The levels of general markers of energy metabolism were assessed in the gastrocnemius muscle (Fig. 2 ). No differences between groups were found in the gastrocnemius levels of ATPB, GAPDH, SIRT3, PGC1α, and ETFDH. CS activity, however, decreased with aging (tendency, p = 0.0635). Exercise prevented this decrease in CS activity, with EX2 rats having values similar to those found in SED1 rats. Basal pAMPK gastrocnemius levels increased with aging and decreased in EX2 rats. With no changes in AMPK gastrocnemius levels, the ratio pAMPK/AMPK increased with aging and decreased in EX2 rats (tendency, p = 0.0564). Aging also increased PFKM gastrocnemius levels, suggesting a higher reliance of the gastrocnemius muscle on glycolysis to produce energy as age increases. Despite no differences between groups being observed in the PFKM/ATPB ratio, an increase in this ratio was correlated with the age-induced decrease in the CS gastrocnemius activity and with the age-induced increase in the pAMPK/AMPK ratio (Fig. 2 ). Fig. 2. Open in a new tab Levels of ATP synthase subunit beta (ATPB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), NAD-dependent protein deacetylase sirtuin-3 (SIRT3), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), AMP-activated protein kinase (AMPK), basal phosphorylated AMPK (pAMPK), electron transfer flavoprotein-ubiquinone oxidoreductase (ETFDH) and ATP-dependent 6-phosphofructokinase (PFKM), and GAPDH/ATPB, pAMPK/AMPK, ETFDH/ATPB and PFKM/ATPB ratios in the gastrocnemius muscle evaluated by immunoblotting in SED1 ( n = 4), EX1 ( n = 4–6), SED2 ( n = 4–6), and EX2 ( n = 4–6) rats. Representative immunoblots can be observed. Citrate synthase (CS) activity was spectrophotometrically assessed in the gastrocnemius muscle of SED1 ( n = 4), EX1 ( n = 5), SED2 ( n = 5), and EX2 ( n = 5) rats. Pearson correlations were performed for an in-depth understanding of the results. (** p < 0.01 and * p < 0.05) Markers of oxidative stress, apoptosis, catabolism, and neuromuscular junction remodeling in the gastrocnemius muscle Given the decrease of mitochondrial density with aging in the gastrocnemius muscle (provided by CS activity), oxidative damage and apoptosis were investigated (Fig. 3 ). No changes in DNP or 3-NT gastrocnemius levels were found with aging or exercise, nor differences in stained bands between groups. In contrast, BCL2A1 gastrocnemius levels increased with aging and decreased with exercise, with no changes in BAX gastrocnemius levels being observed. Consequently, the BAX/BCL2A1 ratio decreased with aging, which was correlated with an increased pAMPK/AMPK ratio. No differences were observed in ATG5 gastrocnemius levels. Protein degradation was also studied by the analysis of the E3 ubiquitin ligase atrogin-1 in the gastrocnemius muscle, and no differences between groups were observed (Fig. 3 ). Fig. 3. Open in a new tab Levels of dinitrophenol (DNP), 3-nitrotyrosine (3-NT), Bcl-2-related protein A1 (BCL2A1), BAX, autophagy protein 5 (ATG5), and atrogin-1, and BAX/BCL2A1 ratio in the gastrocnemius muscle evaluated by immunoblotting in SED1 ( n = 4), EX1 ( n = 4–6), SED2 ( n = 4–6), and EX2 ( n = 4–6) rats. Representative immunoblots can be observed along with the molecular weight (MW) of the bands when necessary. Pearson correlations were performed for an in-depth understanding of the results. (* p < 0.05) Since the neuromuscular system has been investigated as a potential contributor to sarcopenia development, the levels of the neurotrophin BDNF and the neuropeptide CGRP in the gastrocnemius muscle were investigated, and no differences were observed between groups (Fig. 4 ). Fig. 4. Open in a new tab Levels of brain-derived neurotrophic factor (BDNF) and calcitonin gene-related peptide (CGRP) in the gastrocnemius muscle evaluated by immunoblotting in SED1 ( n = 4), EX1 ( n = 5), SED2 ( n = 5), and EX2 ( n = 5) rats. Representative immunoblots can be observed Discussion This study demonstrates that at an early stage of aging, compensatory hypertrophy and inhibition of apoptosis were observed, along with a higher reliance on glycolysis for energy production, likely due to the age-induced decrease in mitochondrial content, which was prevented by lifelong aerobic exercise. The gastrocnemius muscle was selected for this study because it is believed to be one of the first skeletal muscles to be adversely affected by aging [ 30 ], and it is modulated by aerobic exercise programs [ 10 , 19 ], and for comparative purposes with other studies [ 31 , 32 ]. Rats were used since rodent models recapitulate the molecular alterations observed in sarcopenia in humans, and because of the invasive nature and ethical concerns regarding skeletal muscle biopsies in healthy humans, and the low adherence rates of humans to long-term exercise protocols [ 32 , 33 ]. Body weight increased with age, but this increase was attenuated by lifelong aerobic exercise. Age decreased the relative gastrocnemius muscle mass, also referred to as the sarcopenia index [ 28 ], which was increased by lifelong aerobic exercise. These data suggest that with aging, the gastrocnemius muscle was not able to maintain its mass in relation to body weight and that lifelong aerobic exercise is capable of mitigating this aging effect. This age-related decreased gastrocnemius muscle mass (body weight-normalized) suggests the loss or atrophy of some fibers with aging, which may trigger the remaining intact fibers to undergo some degree of compensatory hypertrophy, as observed in lesions causing partial denervation, in an attempt to maintain skeletal function [ 34 ]. The histological analysis of the gastrocnemius muscle revealed the simultaneous presence of smaller fibers alongside larger ones, suggesting the occurrence of compensatory hypertrophy. This led to an increased heterogeneity in fiber size and likely contributed to the overall increase in the median CSA of the fibers with age. The similar occurrence of compensatory hypertrophy at an early stage of aging, along with an increase in the median CSA of the fibers, was also noted in the soleus muscle of 12-month-old mice [ 35 ]. This process also aligns with the occurrence of repeating cycles of denervation and reinnervation, which are acknowledged to occur for much of adult life [ 36 ]. The data of the present study suggest that the age-induced remodeling of the skeletal muscle architecture was accompanied by a decrease in mitochondrial density, given the decreased CS activity levels in the gastrocnemius muscle of SED2 rats, proposing that the adverse effects of aging on the skeletal muscle mitochondrion start early in the aging process. It is a fact that literature suggests that mitochondrial density declines gradually with aging, but most of the preclinical studies evaluated much older rodents (ranging from 24- to 36-month-old rats) [ 8 , 37 , 38 ] compared to the present study. Lifelong aerobic exercise, however, prevented this decrease, alluding to its role in protecting and improving the oxidative capacity of the skeletal muscle. The low levels of CS activity were correlated with the high PFKM/ATPB ratio, suggesting that when mitochondrial density decreases with age, the gastrocnemius muscle may favor glycolysis over oxidative phosphorylation (OXPHOS) to meet its energy demands. These results, along with the age-induced increase in PFKM gastrocnemius levels, suggest an attempt at adaptation of the skeletal muscle to the loss of mitochondria at the early stage of aging, making the skeletal muscle improve the glycolytic metabolism for energy production. However, as age and the remodeling of the skeletal muscle fibers and motor units increase, this attempt may not be sufficient or decompensate, eventually leading to a shift from glycolytic to oxidative metabolism, as described in the literature for older ages [ 39 ]. This shift, coupled with the mitochondrial dysfunction observed at older ages, may turn oxidative metabolism and energy production intricate, contributing to the skeletal muscle dysfunction observed in older ages [ 40 ]. In the present study, CS activity did not differ between EX1 and SED1 groups, and in fact, studies have reported either increases [ 41 ] or no changes [ 42 , 43 ] in the gastrocnemius CS activity in rats of similar ages subjected to similar protocols. Previous work has shown that adult rats exhibit limited further increases in CS activity unless exercise intensity is adjusted upward throughout the training period [ 44 ]. Herein, treadmill speed was recalculated to 70% of each rat’s maximal capacity every 6 weeks. The relatively long interval between adjustments might have allowed a mismatch between training intensity and physiological adaptations. These results may also reflect the fiber-type composition of the gastrocnemius muscle, as slow-twitch skeletal muscles such as the soleus typically exhibit more pronounced increases in CS activity in response to chronic exercise training compared to mixed or fast-twitch skeletal muscles [ 45 ]. The proposed decline in mitochondrial density with age, combined with the reliance of the gastrocnemius muscle on glycolysis, may have limited energy production in the skeletal muscle, which likely triggered the activation of the energy sensor AMPK in the gastrocnemius muscle, as suggested by the correlation between increased pAMPK/AMPK ratio and increased PFKM/ATPB ratio. One can also hypothesize that the activation of AMPK may also trigger or contribute to the stimulation of glycolysis through PFKM, as suggested by similar results in the heart [ 46 ]. This AMPK activation at this early stage of aging may constitute an attempt to improve cellular homeostasis, metabolism, and cell survival to mitigate skeletal muscle fiber loss. However, it seems not to be sufficient to restore the mitochondrial density to levels similar to SED1 rats. This attempt may also become insufficient at older ages, where elevated or diminished pAMPK levels or pAMPK/AMPK ratio in skeletal muscle are reported [ 47 , 48 ]. Herein, lifelong aerobic exercise suppressed basal AMPK activation at an early stage of aging, as indicated by the decreased pAMPK/AMPK ratio in EX2 rats compared to SED2 ones, possibly because of the higher oxidative capacity of the skeletal muscle of EX2 rats, as demonstrated by the lifelong aerobic exercise-induced increase in CS activity. It has also been proposed that AMPK activation can exert pro-survival effects on skeletal muscle by inhibiting apoptosis [ 49 ], which is supported by the present study, given the correlation between the increased pAMPK/AMPK ratio and the decreased BAX/BCL2A1 ratio. This pro-survival effect has been observed in older skeletal muscles, and it is suggested to be a compensatory mechanism to limit skeletal muscle fiber atrophy [ 50 – 52 ]. Herein, the increased gastrocnemius levels of BCL2A1 and decreased BAX/BCL2A1 ratio highlight that this inhibition of apoptotic signaling starts earlier in the aging process. With lifelong aerobic exercise, the gastrocnemius levels of BCL2A1 decreased (without changes in the BAX/BCL2A1 ratio) in EX2 rats. In this case, apoptosis may act as a normal process to remove damaged cells and maintain homeostasis in response to exercise [ 53 , 54 ]. Age-related mitochondrial dysfunction is linked to an increase in oxidative stress, and this time-dependent accumulation of cellular oxidative damage is believed to be involved in skeletal muscle aging [ 55 ]. In the present study, no changes in markers of oxidative damage were observed in the gastrocnemius muscle, indicating that this accumulation may start or be noticeable later in life, as demonstrated in previous studies [ 56 – 58 ]. Another potential driver of sarcopenia may be the impairment of the neuromuscular system with aging. The levels of the neurotrophin BDNF and of the neuropeptide CGRP, which are involved in maintaining skeletal muscle function and modulating skeletal muscle regeneration [ 59 , 60 ], were evaluated in the gastrocnemius muscle, and no changes were observed. These results indicate that neuromuscular system impairment may be more evident later in life, as previously observed (19- and 24-month-old rats) [ 61 , 62 ]. Still, alterations in the neuromuscular system may already be occurring at this early stage of aging. These changes are likely to be specific to certain fibers and localized, potentially becoming obscured in the analyses of total homogenate or more pronounced at the nerve level. Conclusion At an early stage of aging, the relative gastrocnemius muscle mass decreased, indicating the loss or atrophy of some fibers, which was mitigated by lifelong aerobic exercise. This age-related loss of the relative gastrocnemius muscle mass potentially triggered the compensatory hypertrophy observed, where smaller fibers were grouped with larger ones. A diminished mitochondrial density, given by CS activity, was suggested to occur at this early stage of aging, which was prevented by lifelong aerobic exercise. This age-related decrease in CS activity was correlated with an increased PFKM/ATPB ratio, pointing towards a higher reliance of the gastrocnemius muscle on glycolysis for energy production, which was further indicated by the age-induced increase in PFKM levels. This age-induced increase in PFKM/ATPB ratio was correlated with increased pAMPK/AMPK ratio, indicating AMPK activation. This increased pAMPK/AMPK ratio was correlated with the decreased BAX/BCL2A1 ratio observed at this early stage of aging, suggesting an AMPK-related inhibition of apoptosis, which probably acted as a compensatory mechanism to reduce skeletal muscle fiber atrophy and loss. This exploratory study integrates key architectural and metabolic changes that occur in the skeletal muscle at an early stage of aging (14 months of age), laying the groundwork for future research into the remodeling of the skeletal muscle at the underexplored early stage of aging, thereby enabling the identification of potential markers for timely intervention in sarcopenia, particularly in its preclinical stage. Additionally, it highlights the critical need to initiate exercise early in life as a fundamental strategy for preserving skeletal muscle health with age, supporting further research into the preventive effects of lifelong exercise. Acknowledgements This work was supported by CIAFEL (UIDB/00617/2020, https://doi.org/10.54499/UIDB/00617/2020 ; UIDP/00617/2020, https://doi.org/10.54499/UIDP/00617/2020 ), ITR (LA/P/0064/2020), LAQV (UID/50006/2025; https://doi.org/10.54499/UID/50006/2025 ) and CITAB (UIDB/04033/2020, https://doi.org/10.54499/UIDB/04033/2020 ) research units through national founds by the Portuguese Foundation for Science and Technology (FCT)/Ministry of Science, Technology and Higher Education (MCTES). The authors would like to thank Celeste Resende for her assistance in the preparation of the samples for the histological analyses. Author contributions Conceptualization, R.F. and J.A.D.; Methodology, A.M.-P., M.J.N., M.F., P.A.O, and D.M.-G.; Validation, A.M.-P. and J.A.D.; Formal Analysis, A.M.-P.; Investigation, A.M.-P., R.F., and M.J.N.; Writing – Original Draft Preparation, A.M.-P.; Writing – Review & Editing, R.F., J.A.D. and P.A.O.; Visualization, A.M.-P. and R.F.; Supervision, J.A.D., P.A.O. and R.F. Funding Open access funding provided by FCT|FCCN (b-on). A.M.-P. thanks to the Portuguese Foundation for Science and Technology (FCT)/Ministry of Science, Technology and Higher Education (MCTES) and the European Social Fund (ESF) through PT2020 for her grant (SFRH/BD/144396/2019). Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions. Declarations Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Cruz-Jentoft AJ, Bahat G, Bauer J et al (2019) Sarcopenia: revised european consensus on definition and diagnosis. Age Ageing 48:16–31. 10.1093/ageing/afy169 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 2. Petermann-Rocha F, Balntzi V, Gray SR et al (2022) Global prevalence of sarcopenia and severe sarcopenia: a systematic review and meta-analysis. J Cachexia Sarcopenia Muscle 13:86–99. 10.1002/jcsm.12783 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 3. Coletta G, Phillips SM (2023) An elusive consensus definition of sarcopenia impedes research and clinical treatment: a narrative review. Ageing Res Rev 86:101883. 10.1016/j.arr.2023.101883 [ DOI ] [ PubMed ] [ Google Scholar ] 4. Brown WJ, McCarthy MS (2015) Sarcopenia: what every NP needs to know. J Nurse Pract 11:753–760. 10.1016/j.nurpra.2015.05.017 [ Google Scholar ] 5. Leon AS (2017) Attenuation of adverse effects of aging on skeletal muscle by regular exercise and nutritional support. Am J Lifestyle Med 11:4–16. 10.1177/1559827615589319 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 6. GrönholdtKlein M, Gorzi A, Wang L et al (2023) Emergence and progression of behavioral motor deficits and skeletal muscle atrophy across the adult lifespan of the rat. Biology 12:1177. 10.3390/biology12091177 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 7. Zhang F-M, Wu H-F, Wang K-F et al (2024) Transcriptome profiling of fast/glycolytic and slow/oxidative muscle fibers in aging and obesity. Cell Death Dis 15:459. 10.1038/s41419-024-06851-y [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 8. Rossiter HB, Howlett RA, Holcombe HH et al (2005) Age is no barrier to muscle structural, biochemical and angiogenic adaptations to training up to 24 months in female rats. J Physiol 565:993–1005. 10.1113/jphysiol.2004.080663 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 9. Sonjak V, Jacob KJ, Spendiff S et al (2019) Reduced mitochondrial content, elevated ROS, and modulation by denervation in skeletal muscle of pre-frail/frail elderly women. Journals Gerontol Ser A 74:1887–1895. 10.1093/gerona/glz066 [ DOI ] [ PubMed ] [ Google Scholar ] 10. Bao F, Zhao X, You J et al (2024) Aerobic exercise alleviates skeletal muscle aging in male rats by inhibiting apoptosis via regulation of the Trx system. Exp Gerontol 194:112523. 10.1016/j.exger.2024.112523 [ DOI ] [ PubMed ] [ Google Scholar ] 11. Michel JM, Godwin JS, Kerr NR et al (2025) Skeletal muscle atrophy induced by aging and disuse atrophy are strongly associated with the upregulation of the endoplasmic stress protein CHOP in rats. Mol Biol Rep 52:322. 10.1007/s11033-025-10415-4 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 12. Cefis M, Marcangeli V, Hammad R et al (2025) Impact of physical activity on physical function, mitochondrial energetics, ROS production, and Ca2 + handling across the adult lifespan in men. Cell Reports Med 6:101968. 10.1016/j.xcrm.2025.101968 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 13. Yuan S, Larsson SC (2023) Epidemiology of sarcopenia: prevalence, risk factors, and consequences. Metabolism 144:155533. 10.1016/j.metabol.2023.155533 [ DOI ] [ PubMed ] [ Google Scholar ] 14. Nascimento CM, Ingles M, Salvador-Pascual A et al (2019) Sarcopenia, frailty and their prevention by exercise. Free Radic Biol Med 132:42–49. 10.1016/j.freeradbiomed.2018.08.035 [ DOI ] [ PubMed ] [ Google Scholar ] 15. Wiedmer P, Jung T, Castro JP et al (2021) Sarcopenia – molecular mechanisms and open questions. Ageing Res Rev 65:101200. 10.1016/j.arr.2020.101200 [ DOI ] [ PubMed ] [ Google Scholar ] 16. Baek K-W, Kim S-J, Kim B-G et al (2022) Effects of lifelong spontaneous exercise on skeletal muscle and angiogenesis in super-aged mice. PLoS ONE 17:e0263457. 10.1371/journal.pone.0263457 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 17. Liang J, Zhang H, Zeng Z et al (2021) Lifelong aerobic exercise alleviates sarcopenia by activating autophagy and inhibiting protein degradation via the AMPK/PGC-1α signaling pathway. Metabolites 11:323. 10.3390/metabo11050323 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 18. Gao H-E, Li F-H, Xie T et al (2021) Lifelong exercise in age rats improves skeletal muscle function and microRNA profile. Med Sci Sports Exerc 53:1873–1882. 10.1249/MSS.0000000000002661 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 19. Konopka AR, Harber MP (2014) Skeletal muscle hypertrophy after aerobic exercise training. Exerc Sport Sci Rev 42:53–61. 10.1249/JES.0000000000000007 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 20. Distefano G, Goodpaster BH (2018) Effects of exercise and aging on skeletal muscle. Cold Spring Harb Perspect Med 8:a029785. 10.1101/cshperspect.a029785 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 21. Sengupta P (2013) The laboratory rat: relating its age with human’s. Int J Prev Med 4:624–630 [ PMC free article ] [ PubMed ] [ Google Scholar ] 22. Ghasemi A, Jeddi S, Kashfi K (2021) The laboratory rat: age and body weight matter. EXCLI J 20:1431–1445. 10.17179/excli2021-4072 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 23. Forbes D, Blom H, Kostomitsopoulos N et al (2007) Euroguide on the accommodation and care of animals used for experimental and other scientific purposes. London 24. Yin FC, Spurgeon HA, Rakusan K et al (1982) Use of tibial length to quantify cardiac hypertrophy: application in the aging rat. Am J Physiol 243:H941–H947. 10.1152/ajpheart.1982.243.6.h941 [ DOI ] [ PubMed ] [ Google Scholar ] 25. Coore HG, Denton RM, Martin BR, Randle PJ (1971) Regulation of adipose tissue pyruvate dehydrogenase by insulin and other hormones. Biochem J 125:115–127. 10.1042/bj1250115 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 26. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. 10.1038/227680a0 [ DOI ] [ PubMed ] [ Google Scholar ] 27. Vigelsø A, Dybboe R, Hansen CN et al (2015) GAPDH and β-actin protein decreases with aging, making stain-free technology a superior loading control in western blotting of human skeletal muscle. J Appl Physiol 118:386–394. 10.1152/japplphysiol.00840.2014 [ DOI ] [ PubMed ] [ Google Scholar ] 28. Shu H, Huang Y, Zhang W et al (2023) An integrated study of hormone-related sarcopenia for modeling and comparative transcriptome in rats. Front Endocrinol (Lausanne) 14:1073587. 10.3389/fendo.2023.1073587 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 29. Greaves P, Chouinard L, Ernst H et al (2013) Proliferative and non-proliferative lesions of the rat and mouse soft tissue, skeletal muscle and mesothelium. J Toxicol Pathol 26:1S-26S. 10.1293/tox.26.1S [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 30. Shavlakadze T, Xiong K, Mishra S et al (2023) Age-related gene expression signatures from limb skeletal muscles and the diaphragm in mice and rats reveal common and species-specific changes. Skelet Muscle 13:11. 10.1186/s13395-023-00321-3 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 31. Xie W, He M, Yu D et al (2021) Mouse models of sarcopenia: classification and evaluation. J Cachexia Sarcopenia Muscle 12:538–554. 10.1002/jcsm.12709 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 32. Börsch A, Ham DJ, Mittal N et al (2021) Molecular and phenotypic analysis of rodent models reveals conserved and species-specific modulators of human sarcopenia. Commun Biol 4:194. 10.1038/s42003-021-01723-z [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 33. Mahmood A, Nayak P, Deshmukh A et al (2023) Measurement, determinants, barriers, and interventions for exercise adherence: a scoping review. J Bodyw Mov Ther 33:95–105. 10.1016/j.jbmt.2022.09.014 [ DOI ] [ PubMed ] [ Google Scholar ] 34. Dumitru D, Amato AA (2002) Introduction to myopathies and muscle tissue’s reaction to injury. In: Dumitru D, Amato AA, Zwarts M (eds) Electrodiagnostic medicine, 2nd ed. Hanley & Belfus, Philadelphia, pp 1229–1264 35. Sayed RKA, de Leonardis EC, Guerrero-Martínez JA et al (2016) Identification of morphological markers of sarcopenia at early stage of aging in skeletal muscle of mice. Exp Gerontol 83:22–30. 10.1016/j.exger.2016.07.007 [ DOI ] [ PubMed ] [ Google Scholar ] 36. Hepple RT, Rice CL (2016) Innervation and neuromuscular control in ageing skeletal muscle. J Physiol 594:1965–1978. 10.1113/JP270561 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 37. Lyons CN, Mathieu-Costello O, Moyes CD (2006) Regulation of skeletal muscle mitochondrial content during aging. Journals Gerontol - Ser Biol Sci Med Sci 61A:3–13. 10.1093/gerona/61.1.3 [ DOI ] [ PubMed ] [ Google Scholar ] 38. Chistiakov DA, Sobenin IA, Revin VV et al (2014) Mitochondrial aging and age-related dysfunction of mitochondria. Biomed Res Int 2014:238463. 10.1155/2014/238463 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 39. Peterson CM, Johannsen DL, Ravussin E (2012) Skeletal muscle mitochondria and aging: a review. J Aging Res 2012:194821. 10.1155/2012/194821 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 40. Daily JW, Park S (2022) Sarcopenia is a cause and consequence of metabolic dysregulation in aging humans: effects of gut dysbiosis, glucose dysregulation, diet and lifestyle. Cells 11:338. 10.3390/cells11030338 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 41. Naito H, Powers SK, Demirel HA, Aoki J (2001) Exercise training increases heat shock protein in skeletal muscles of old rats. Med Sci Sports Exerc 33:729–734. 10.1097/00005768-200105000-00008 [ DOI ] [ PubMed ] [ Google Scholar ] 42. Farhat F, Dupas J, Amérand A et al (2015) Effect of exercise training on oxidative stress and mitochondrial function in rat heart and gastrocnemius muscle. Redox Rep 20:60–68. 10.1179/1351000214Y.0000000105 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 43. Pinho RA, Andrades ME, Oliveira MR et al (2006) Imbalance in SOD / CAT activities in rat skeletal muscles submitted to treadmill training exercise. Cell Biol Int 30:848–853. 10.1016/j.cellbi.2006.03.011 [ DOI ] [ PubMed ] [ Google Scholar ] 44. Schenk S, Sagendorf TJ, Many GM et al (2024) Physiological adaptations to progressive endurance exercise training in adult and aged rats: insights from the Molecular Transducers of Physical Activity Consortium (MoTrPAC). Function 5:zqae014. 10.1093/function/zqae014 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 45. Zonderland M, Bär P, Reijneveld J et al (1999) Different metabolic adaptation of heart and skeletal muscles to moderate-intensity treadmill training in the rat. Eur J Appl Physiol 79:391–396. 10.1007/s004210050527 [ DOI ] [ PubMed ] [ Google Scholar ] 46. Marsin A-S, Bertrand L, Rider MH et al (2000) Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia. Curr Biol 10:1247–1255. 10.1016/S0960-9822(00)00742-9 [ DOI ] [ PubMed ] [ Google Scholar ] 47. Qiang W, Weiqiang K, Qing Z et al (2007) Aging impairs insulin-stimulated glucose uptake in rat skeletal muscle via suppressing AMPKα. Exp Mol Med 39:535–543. 10.1038/emm.2007.59 [ DOI ] [ PubMed ] [ Google Scholar ] 48. Thomson DM, Gordon SE (2005) Diminished overload-induced hypertrophy in aged fast-twitch skeletal muscle is associated with AMPK hyperphosphorylation. J Appl Physiol 98:557–564. 10.1152/japplphysiol.00811.2004 [ DOI ] [ PubMed ] [ Google Scholar ] 49. Yan Y, Li M, Lin J et al (2022) Adenosine monophosphate activated protein kinase contributes to skeletal muscle health through the control of mitochondrial function. Front Pharmacol 13:947387. 10.3389/fphar.2022.947387 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 50. Baker DJ, Hepple RT (2006) Elevated caspase and AIF gene expression correlate with progression of sarcopenia during aging in male F344BN rats. Exp Gerontol 41:1149–1156. 10.1016/j.exger.2006.08.007 [ DOI ] [ PubMed ] [ Google Scholar ] 51. Siu PM, Pistilli EE, Alway SE (2005) Apoptotic responses to hindlimb suspension in gastrocnemius muscles from young adult and aged rats. Am J Physiol Regul Integr Comp Physiol 289:R1015–R1026. 10.1152/ajpregu.00198.2005 [ DOI ] [ PubMed ] [ Google Scholar ] 52. Marzetti E, Privitera G, Simili V et al (2010) Multiple pathways to the same end: mechanisms of myonuclear apoptosis in sarcopenia of aging. The Scientific World JOURNAL 10:340–349. 10.1100/tsw.2010.27 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 53. Azad M, Khaledi N, Hedayati M, Karbalaie M (2021) Apoptotic response to acute and chronic exercises in rat skeletal muscle: eccentric & sprint interval. Life Sci 270:119002. 10.1016/j.lfs.2020.119002 [ DOI ] [ PubMed ] [ Google Scholar ] 54. Phaneuf S, Leeuwenburgh C (2001) Apoptosis and exercise. Med Sci Sports Exerc 33:393–396. 10.1097/00005768-200103000-00010 [ DOI ] [ PubMed ] [ Google Scholar ] 55. Chen M, Wang Y, Deng S et al (2022) Skeletal muscle oxidative stress and inflammation in aging: focus on antioxidant and anti-inflammatory therapy. Frontiers in Cell and Developmental Biology 10:964130. 10.3389/fcell.2022.964130 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 56. Tohma H, El-Shafey AF, Croft K et al (2014) Protein thiol oxidation does not change in skeletal muscles of aging female mice. Biogerontology 15:87–98. 10.1007/s10522-013-9483-y [ DOI ] [ PubMed ] [ Google Scholar ] 57. Pandya CD, Lee B, Toque HA et al (2019) Age-dependent oxidative stress elevates arginase 1 and uncoupled nitric oxide synthesis in skeletal muscle of aged mice. Oxid Med Cell Longev 2019:1704650. 10.1155/2019/1704650 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 58. Pearson T, McArdle A, Jackson MJ (2015) Nitric oxide availability is increased in contracting skeletal muscle from aged mice, but does not differentially decrease muscle superoxide. Free Radic Biol Med 78:82–88. 10.1016/j.freeradbiomed.2014.10.505 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 59. Machado J, Silveira WA, Gonçalves DA et al (2019) α − calcitonin gene-related peptide inhibits autophagy and calpain systems and maintains the stability of neuromuscular junction in denervated muscles. Mol Metab 28:91–106. 10.1016/j.molmet.2019.06.024 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 60. Rentería I, García-Suárez PC, Fry AC et al (2022) The molecular effects of BDNF synthesis on skeletal muscle: a mini-review. Front Physiol 13:934714. 10.3389/fphys.2022.934714 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 61. Moreira-Pais A, Ferreira R, Aiires I et al (2025) Age, cancer, and the dual burden of cancer and doxorubicin in skeletal muscle wasting in female rats: which one to blame? Biogerontology 26:47. 10.1007/s10522-024-10182-y [ DOI ] [ PubMed ] [ Google Scholar ] 62. Liang J, Zhang H, Zeng Z et al (2023) MicroRNA profiling of different exercise interventions for alleviating skeletal muscle atrophy in naturally aging rats. J Cachexia Sarcopenia Muscle 14:356–368. 10.1002/jcsm.13137 [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions. 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