An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis.

BACKGROUND: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. METHODS: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. RESULTS: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. CONCLUSIONS: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

AIBP Regulates Metabolism of Ketone and Lipids but Not Mitochondrial Respiration.

Accumulating evidence indicates that the APOA1 binding protein (AIBP)-a secreted protein-plays a profound role in lipid metabolism. Interestingly, AIBP also functions as an NAD(P)H-hydrate epimerase to catalyze the interconversion of NAD(P)H hydrate [NAD(P)HX] epimers and is renamed as NAXE. Thus, we call it NAXE hereafter. We investigated its role in NAD(P)H-involved metabolism in murine cardiomyocytes, focusing on the metabolism of hexose, lipids, and amino acids as well as mitochondrial redox function. Unbiased metabolite profiling of cardiac tissue shows that NAXE knockout markedly upregulates the ketone body 3-hydroxybutyric acid (3-HB) and increases or trends increasing lipid-associated metabolites cholesterol, α-linolenic acid and deoxycholic acid. Paralleling greater ketone levels, ChemRICH analysis of the NAXE-regulated metabolites shows reduced abundance of hexose despite similar glucose levels in control and NAXE-deficient blood. NAXE knockout reduces cardiac lactic acid but has no effect on the content of other NAD(P)H-regulated metabolites, including those associated with glucose metabolism, the pentose phosphate pathway, or Krebs cycle flux. Although NAXE is present in mitochondria, it has no apparent effect on mitochondrial oxidative phosphorylation. Instead, we detected more metabolites that can potentially improve cardiac function (3-HB, adenosine, and α-linolenic acid) in the Naxe-/- heart; these mice also perform better in aerobic exercise. Our data reveal a new role of NAXE in cardiac ketone and lipid metabolism.

Enhanced Succinate Oxidation with Mitochondrial Complex II Reactive Oxygen Species Generation in Human Prostate Cancer.

The transformation of prostatic epithelial cells to prostate cancer (PCa) has been characterized as a transition from citrate secretion to citrate oxidation, from which one would anticipate enhanced mitochondrial complex I (CI) respiratory flux. Molecular mechanisms for this transformation are attributed to declining mitochondrial zinc concentrations. The unique metabolic properties of PCa cells have become a hot research area. Several publications have provided indirect evidence based on investigations using pre-clinical models, established cell lines, and fixed or frozen tissue bank samples. However, confirmatory respiratory analysis on fresh human tissue has been hampered by multiple difficulties. Thus, few mitochondrial respiratory assessments of freshly procured human PCa tissue have been published on this question. Our objective is to document relative mitochondrial CI and complex II (CII) convergent electron flow to the Q-junction and to identify electron transport system (ETS) alterations in fresh PCa tissue. The results document a CII succinate: quinone oxidoreductase (SQR) dominant succinate oxidative flux model in the fresh non-malignant prostate tissue, which is enhanced in malignant tissue. CI NADH: ubiquinone oxidoreductase activity is impaired rather than predominant in high-grade malignant fresh prostate tissue. Given these novel findings, succinate and CII are promising targets for treating and preventing PCa.

The Role of Calcium Signaling in Melanoma.

Calcium signaling plays important roles in physiological and pathological conditions, including cutaneous melanoma, the most lethal type of skin cancer. Intracellular calcium concentration ([Ca2+]i), cell membrane calcium channels, calcium related proteins (S100 family, E-cadherin, and calpain), and Wnt/Ca2+ pathways are related to melanogenesis and melanoma tumorigenesis and progression. Calcium signaling influences the melanoma microenvironment, including immune cells, extracellular matrix (ECM), the vascular network, and chemical and physical surroundings. Other ionic channels, such as sodium and potassium channels, are engaged in calcium-mediated pathways in melanoma. Calcium signaling serves as a promising pharmacological target in melanoma treatment, and its dysregulation might serve as a marker for melanoma prediction. We documented calcium-dependent endoplasmic reticulum (ER) stress and mitochondria dysfunction, by targeting calcium channels and influencing [Ca2+]i and calcium homeostasis, and attenuated drug resistance in melanoma management.

Nucleus-mitochondria positive feedback loop formed by ERK5 S496 phosphorylation-mediated poly (ADP-ribose) polymerase activation provokes persistent pro-inflammatory senescent phenotype and accelerates coronary atherosclerosis after chemo-radiation.

The incidence of cardiovascular disease (CVD) is higher in cancer survivors than in the general population. Several cancer treatments are recognized as risk factors for CVD, but specific therapies are unavailable. Many cancer treatments activate shared signaling events, which reprogram myeloid cells (MCs) towards persistent senescence-associated secretory phenotype (SASP) and consequently CVD, but the exact mechanisms remain unclear. This study aimed to provide mechanistic insights and potential treatments by investigating how chemo-radiation can induce persistent SASP. We generated ERK5 S496A knock-in mice and determined SASP in myeloid cells (MCs) by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. Candidate SASP inducers were identified by high-throughput screening, using the ERK5 transcriptional activity reporter cell system. Various chemotherapy agents and ionizing radiation (IR) up-regulated p90RSK-mediated ERK5 S496 phosphorylation. Doxorubicin and IR caused metabolic changes with nicotinamide adenine dinucleotide depletion and ensuing mitochondrial stunning (reversible mitochondria dysfunction without showing any cell death under ATP depletion) via p90RSK-ERK5 modulation and poly (ADP-ribose) polymerase (PARP) activation, which formed a nucleus-mitochondria positive feedback loop. This feedback loop reprogramed MCs to induce a sustained SASP state, and ultimately primed MCs to be more sensitive to reactive oxygen species. This priming was also detected in circulating monocytes from cancer patients after IR. When PARP activity was transiently inhibited at the time of IR, mitochondrial stunning, priming, macrophage infiltration, and coronary atherosclerosis were all eradicated. The p90RSK-ERK5 module plays a crucial role in SASP-mediated mitochondrial stunning via regulating PARP activation. Our data show for the first time that the nucleus-mitochondria positive feedback loop formed by p90RSK-ERK5 S496 phosphorylation-mediated PARP activation plays a crucial role of persistent SASP state, and also provide preclinical evidence supporting that transient inhibition of PARP activation only at the time of radiation therapy can prevent future CVD in cancer survivors.

Plumbagin Elicits Cell-Specific Cytotoxic Effects and Metabolic Responses in Melanoma Cells.

Melanoma is one of the most malignant skin cancers that require comprehensive therapies, including chemotherapy. A plant-derived drug, plumbagin (PLB), exhibits an anticancer property in several cancers. We compared the cytotoxic and metabolic roles of PLB in A375 and SK-MEL-28 cells, each with different aggressiveness. In our results, they were observed to have distinctive mitochondrial respiratory functions. The primary reactive oxygen species (ROS) source of A375 can be robustly attenuated by cell membrane permeabilization. A375 cell viability and proliferation, migration, and apoptosis induction are more sensitive to PLB treatment. PLB induced metabolic alternations in SK-MEL-28 cells, which included increasing mitochondrial oxidative phosphorylation (OXPHOS), mitochondrial ATP production, and mitochondrial mass. Decreasing mitochondrial OXPHOS and total ATP production with elevated mitochondrial membrane potential (MMP) were observed in PLB-induced A375 cells. PLB also induced ROS production and increased proton leak and non-mitochondria respiration in both cells. This study reveals the relationship between metabolism and cytotoxic effects of PLB in melanoma. PLB displays stronger cytotoxic effects on A375 cells, which exhibit lower respiratory function than SK-MEL-28 cells with higher respiratory function, and triggers cell-specific metabolic changes in accordance with its cytotoxic effects. These findings indicate that PLB might serve as a promising anticancer drug, targeting metabolism.

Cholesterol Induces CD8+ T Cell Exhaustion in the Tumor Microenvironment.

Tumor-infiltrating T cells often lose their effector function; however, the mechanisms are incompletely understood. We report that cholesterol in the tumor microenvironment induces CD8+ T cell expression of immune checkpoints and exhaustion. Tumor tissues enriched with cholesterol and cholesterol content in tumor-infiltrating CD8+ T cells were positively and progressively associated with upregulated T cell expression of PD-1, 2B4, TIM-3, and LAG-3. Adoptively transferred CD8+ T cells acquired cholesterol, expressed high levels of immune checkpoints, and became exhausted upon entering a tumor. Tumor culture supernatant or cholesterol induced immune checkpoint expression by increasing endoplasmic reticulum (ER) stress in CD8+ T cells. Consequently, the ER stress sensor XBP1 was activated and regulated PD-1 and 2B4 transcription. Inhibiting XBP1 or reducing cholesterol in CD8+ T cells effectively restored antitumor activity. This study reveals a mechanism underlying T cell exhaustion and suggests a new strategy for restoring T cell function by reducing cholesterol to enhance T cell-based immunotherapy.

Improved Cardiovascular Function in Old Mice After N-Acetyl Cysteine and Glycine Supplemented Diet: Inflammation and Mitochondrial Factors.

Metabolic, inflammatory, and functional changes occur in cardiovascular aging which may stem from oxidative stress and be remediable with antioxidants. Glutathione, an intracellular antioxidant, declines with aging, and supplementation with glutathione precursors, N-acetyl cysteine (NAC) and glycine (Gly), increases tissue glutathione. Thirty-month old mice were fed diets supplemented with NAC or NAC+Gly and, after 7 weeks, cardiac function and molecular studies were performed. The NAC+Gly supplementation improved diastolic function, increasing peak early filling velocity, and reducing relaxation time, left atrial volume, and left ventricle end diastolic pressure. By contrast, cardiac function did not improve with NAC alone. Both diet supplementations decreased cardiac levels of inflammatory mediators; only NAC+Gly reduced leukocyte infiltration. Several mitochondrial genes reduced with aging were upregulated in hearts by NAC+Gly diet supplementation. These Krebs cycle and oxidative phosphorylation enzymes, suggesting improved mitochondrial function, and permeabilized cardiac fibers from NAC+Gly-fed mice produced ATP from carbohydrate and fatty acid sources, whereas fibers from control old mice were less able to utilize fatty acids. Our data indicate that NAC+Gly supplementation can improve diastolic function in the old mouse and may have potential to prevent important morbidities for older people.

Polymer Functionalization of Isolated Mitochondria for Cellular Transplantation and Metabolic Phenotype Alteration.

Aberrant mitochondrial energy transfer underlies prevalent chronic health conditions, including cancer, cardiovascular, and neurodegenerative diseases. Mitochondrial transplantation represents an innovative strategy aimed at restoring favorable metabolic phenotypes in cells with dysfunctional energy metabolism. While promising, significant barriers to in vivo translation of this approach abound, including limited cellular uptake and recognition of mitochondria as foreign. The objective is to functionalize isolated mitochondria with a biocompatible polymer to enhance cellular transplantation and eventual in vivo applications. Herein, it is demonstrated that grafting of a polymer conjugate composed of dextran with triphenylphosphonium onto isolated mitochondria protects the organelles and facilitates cellular internalization compared with uncoated mitochondria. Importantly, mitochondrial transplantation into cancer and cardiovascular cells has profound effects on respiration, mediating a shift toward improved oxidative phosphorylation, and reduced glycolysis. These findings represent the first demonstration of polymer functionalization of isolated mitochondria, highlighting a viable strategy for enabling clinical applications of mitochondrial transplantation.

Tead1 is required for maintaining adult cardiomyocyte function, and its loss results in lethal dilated cardiomyopathy.

Heart disease remains the leading cause of death worldwide, highlighting a pressing need to identify novel regulators of cardiomyocyte (CM) function that could be therapeutically targeted. The mammalian Hippo/Tead pathway is critical in embryonic cardiac development and perinatal CM proliferation. However, the requirement of Tead1, the transcriptional effector of this pathway, in the adult heart is unknown. Here, we show that tamoxifen-inducible adult CM-specific Tead1 ablation led to lethal acute-onset dilated cardiomyopathy, associated with impairment in excitation-contraction coupling. Mechanistically, we demonstrate Tead1 is a cell-autonomous, direct transcriptional activator of SERCA2a and SR-associated protein phosphatase 1 regulatory subunit, Inhibitor-1 (I-1). Thus, Tead1 deletion led to a decrease in SERCA2a and I-1 transcripts and protein, with a consequent increase in PP1-activity, resulting in accumulation of dephosphorylated phospholamban (Pln) and decreased SERCA2a activity. Global transcriptomal analysis in Tead1-deleted hearts revealed significant changes in mitochondrial and sarcomere-related pathways. Additional studies demonstrated there was a trend for correlation between protein levels of TEAD1 and I-1, and phosphorylation of PLN, in human nonfailing and failing hearts. Furthermore, TEAD1 activity was required to maintain PLN phosphorylation and expression of SERCA2a and I-1 in human induced pluripotent stem cell-derived (iPS-derived) CMs. To our knowledge, taken together, this demonstrates a nonredundant, novel role of Tead1 in maintaining normal adult heart function.

Mitochondrial Function in Non-ischemic Heart Failure.

Provision for the continuous demand for energy from the beating heart relies heavily on efficient mitochondrial activity. Non-ischemic cardiomyopathy in which oxygen supply is not limiting results from etiologies such as pressure overload. It is associated with progressive development of metabolic stress culminating in energy depletion and heart failure. The mitochondria from the ventricular walls undergoing non-ischemic cardiomyopathy are subjected to long periods of adaptation to support the changing metabolic milieu, which has been described as mal-adaptation since it ultimately results in loss of cardiac contractile function. While the chronicity of exposure to metabolic stressors, co-morbidities and thereby adaptive changes in mitochondria maybe different between ischemic and non-ischemic heart failure, the resulting pathology is very similar, especially in late stage heart failure. Understanding of the mitochondrial changes in early-stage heart failure may guide the development of mitochondrial-targeted therapeutic options to prevent progression of non-ischemic heart failure. This chapter reviews findings of mitochondrial functional changes in animal models and humans with non-ischemic heart failure. While most animal models of non-ischemic heart failure exhibit cardiac mitochondrial dysfunction, studies in humans have been inconsistent despite confirmed reduction in ATP production. This chapter also reviews the possibility of impairment of substrate supply processes upstream of the mitochondria in heart failure, and discusses potential metabolism-targeted therapeutic options.

The Role of Estrogen in Cardiac Metabolism and Diastolic Function.

Heart failure with preserved ejection fraction (HFpEF) has similar prevalence and prognosis as HF with reduced EF, but there is no approved treatment for HFpEF. HFpEF is common in postmenopausal women, which suggests that the absence of estrogen (E2) plays a role in its pathophysiology. With the country's growing elderly population, the prevalence of HFpEF is rapidly increasing. This has triggered a renewed urgency in finding novel approaches to preventing and slowing the progression of HFpEF. In this review, we address the role of E2 in left ventricular diastolic function and how it impacts women with HFpEF as well as animal models. We also discuss the primary potential mechanisms that represent critical nodes in the mechanistic pathways of HFpEF and how new treatments could be developed to target those mechanisms.

Pet Imaging and its Application in Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and represent a great challenge for modern research and medicine. Despite advances in preventing and treating CVD over the decades, there remains an urgent need to develop sensitive and safe methods for early detection and personalized treatment. With refinements of molecular imaging technologies such as positron emission tomography (PET), noninvasive imaging of CVDs is experiencing impressive progress in both preclinical and clinical settings. In this review, we summarize advances in cardiovascular PET imaging, highlight the latest development of CVD imaging probes, and illustrate the potential for individualized therapy based on metabolic phenotype.

Estrogen receptor alpha activation enhances mitochondrial function and systemic metabolism in high-fat-fed ovariectomized mice.

Estrogen impacts insulin action and cardiac metabolism, and menopause dramatically increases cardiometabolic risk in women. However, the mechanism(s) of cardiometabolic protection by estrogen remain incompletely understood. Here, we tested the effects of selective activation of E2 receptor alpha (ERα) on systemic metabolism, insulin action, and cardiac mitochondrial function in a mouse model of metabolic dysfunction (ovariectomy [OVX], insulin resistance, hyperlipidemia, and advanced age). Middle-aged (12-month-old) female low-density lipoprotein receptor (Ldlr)(-/-) mice were subjected to OVX or sham surgery and fed "western" high-fat diet (WHFD) for 3 months. Selective ERα activation with 4,4',4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl) (PPT), prevented weight gain, improved insulin action, and reduced visceral fat accumulation in WHFD-fed OVX mice. PPT treatment also elevated systemic metabolism, increasing oxygen consumption and core body temperature, induced expression of several metabolic genes such as peroxisome proliferator-activated receptor gamma, coactivator 1 alpha, and nuclear respiratory factor 1 in heart, liver, skeletal muscle, and adipose tissue, and increased cardiac mitochondrial function. Taken together, selective activation of ERα with PPT enhances metabolic effects including insulin resistance, whole body energy metabolism, and mitochondrial function in OVX mice with metabolic syndrome.

Exercise Intolerance In Heart Failure With Preserved Ejection Fraction.

More than 50% of Americans with heart failure have preserved ejection fraction (HFpEF). Exercise intolerance is a hallmark of HFpEF, but the pathophysiology is not well understood. Diverse etiologies and incomplete mechanistic understanding have resulted in ineffective management strategies to improve the outcomes of HFpEF. Traditional therapies that have been beneficial in the treatment of heart failure with reduced ejection fraction (HFrEF), neurohormonal blockade in particular, have not been effective in treating HFpEF. In this review, we address underlying mechanisms of HFpEF and present the rationale supporting exercise as a component of comprehensive management.

Combination of angiotensin II and l-NG-nitroarginine methyl ester exacerbates mitochondrial dysfunction and oxidative stress to cause heart failure.

Mitochondrial dysfunction has been implicated as a cause of energy deprivation in heart failure (HF). Herein, we tested individual and combined effects of two pathogenic factors of nonischemic HF, inhibition of nitric oxide synthesis [with l-N(G)-nitroarginine methyl ester (l-NAME)] and hypertension [with angiotensin II (AngII)], on myocardial mitochondrial function, oxidative stress, and metabolic gene expression. l-NAME and AngII were administered individually and in combination to mice for 5 wk. Although all treatments increased blood pressure and reduced cardiac contractile function, the l-NAME + AngII group was associated with the most severe HF, as characterized by edema, hypertrophy, oxidative stress, increased expression of Nppa and Nppb, and decreased expression of Atp2a2 and Camk2b. l-NAME + AngII-treated mice exhibited robust deterioration of cardiac mitochondrial function, as observed by reduced respiratory control ratios in subsarcolemmal mitochondria and reduced state 3 levels in interfibrillar mitochondria for complex I but not for complex II substrates. Cardiac myofibrils showed reduced ADP-supported and oligomycin-inhibited oxygen consumption. Mitochondrial functional impairment was accompanied by reduced mitochondrial DNA content and activities of pyruvate dehydrogenase and complex I but increased H2O2 production and tissue protein carbonyls in hearts from AngII and l-NAME + AngII groups. Microarray analyses revealed the majority of the gene changes attributed to the l-NAME + AngII group. Pathway analyses indicated significant changes in metabolic pathways, such as oxidative phosphorylation, mitochondrial function, cardiac hypertrophy, and fatty acid metabolism in l-NAME + AngII hearts. We conclude that l-NAME + AngII is associated with impaired mitochondrial respiratory function and increased oxidative stress compared with either l-NAME or AngII alone, resulting in nonischemic HF.

Differential Mitochondrial Function in Remodeled Right and Nonremodeled Left Ventricles in Pulmonary Hypertension.

OBJECTIVES: Right ventricular failure is the primary reason for mortality in pulmonary hypertension (PH), but little is understood about the energetics of the failing right myocardium. Our aim was to examine mitochondrial function and proteomic signatures in paired remodeled right (RM-RV) and non-remodeled left (NRM-LV) ventricular tissue samples procured during heart-lung transplantation. METHODS AND RESULTS: Contractile dysfunction in RM-RV and preserved contractile function in NRM-LV were determined clinically and by echocardiography. Mitochondria were isolated from fresh paired RV and LV wall specimens of explanted hearts. Respiratory states in response to 4 substrates and an uncoupler were analyzed. Proteomic analysis on the mitochondrial isolates was performed with the use of liquid chromatography-mass spectrometry. The RM-RV mitochondria exhibited higher succinate state 4 levels with lower respiratory control ratio (RCR) compared with state 4 levels for pyruvate-malate (PM) and glutamate-malate (GM). RM-RV mitochondria also exhibited lower state 3 for palmitoyl-carnitine (PC) and state 4 for all complex I substrates compared with NRM-LV. The mean RCR were greater in RM-RVs than in NRM-LVs for PM and GM, which is consistent with tight coupling (low state 4 rates, higher RCRs); however, low RM-RV state 3 rates suggest concurrent substrate-dependent impairment in respiratory capacity. Mitochondrial proteomics revealed greater levels of mitochondrial ADP-ATP translocase and proteins of ATP synthesis, mitochondrial pyruvate and short branched chain acyl-CoA metabolism in RM-RV. CONCLUSIONS: The mitochondrial respiration and proteomics in RM-RV are different from NRM-LV. These results have important implications in expanding our understanding of RV metabolism and future management of RV failure.

Estrogen: an emerging regulator of insulin action and mitochondrial function.

Clinical trials and animal studies have revealed that loss of circulating estrogen induces rapid changes in whole body metabolism, fat distribution, and insulin action. The metabolic effects of estrogen are mediated primarily by its receptor, estrogen receptor-α; however, the detailed understanding of its mechanisms is incomplete. Recent investigations suggest that estrogen receptor-α elicits the metabolic effects of estrogen by genomic, nongenomic, and mitochondrial mechanisms that regulate insulin signaling, substrate oxidation, and energetics. This paper reviews clinical and experimental studies on the mechanisms of estrogen and the current state of knowledge regarding physiological and pathobiological influences of estrogen on metabolism.

Changes in α7ß1 integrin signaling after eccentric exercise in heat-shocked rat soleus.

INTRODUCTION: α7ß1 integrin links the extracellular matrix to the focal adhesion (FA) in skeletal muscle and serves as a stabilizing and signal relayer. Heat shock (HS) induces expression of proteins that interact with the FA. METHODS: Male Wistar rats were assigned to 1 of 3 groups: control (CON); eccentric exercise (EE); or EE+HS (HS). Soleus muscle was analyzed at 2 h and 48 h post-exercise. RESULTS: The 120-kDa α7 integrin decreased in the EE and HS groups, and the 70-kDa peptide decreased in the EE group at 2 h post-exercise. Total expression of focal adhesion kinase (FAK) and RhoA were decreased in EE and HS at 2 h post-exercise. Expression of phosphorylated FAK(397) decreased in the EE group but not the HS group at 2 h post-exercise. CONCLUSIONS: Long-duration EE may cause alterations in the FA in rat soleus muscle through the α7 integrin subunit and FAK.