MAP Kinase Phosphatase-5 Deficiency Improves Endurance Exercise Capacity

erobic exercise promotes physiological cardiac adaptations, improving cardio- vascular function and endurance exercise capacity. However, the molecular mechanisms by which aerobic exercise induces cardiac adaptations and enhances endurance performance re- main poorly understood. Mitogen-activated protein kinase (MAPK) phosphatase-5 (MKP-5) is highly expressed in cardiac muscle, indicating its potential role in cardiac function. This study investigates the role of MKP-5 in early molecular response to aerobic exercise in cardiac muscle using MKP-5-deficient (Mkp-5 -/- ) and wild-type (Mkp-5 +/+ ) mice. Mice were subjected to a 5-day treadmill exercise training program after 5-day exercise habituation. Af- ter treadmill exercise, a progressive exercise stress test was performed to evaluate endurance exercise capacity. Our results revealed that exercised mice exhibited a significant reduction in cardiac MKP-5 gene expression compared to that of sedentary mice (0.19±5.89-fold; p< 0.0001).Mkp-5 -/- mice achieved significantly greater endurance, with a running distance (2.81±169.8-fold;p< 0.0429) longer thanMkp-5 +/+ mice. Additionally, MKP-5 deficiency enhanced Akt/mTOR signaling

treadmill exercise, a progressive exercise stress test was performed to evaluate endurance exercise capacity. Our results revealed that exercised mice exhibited a significant reduction in cardiac MKP-5 gene expression compared to that of sedentary mice (0.19±5.89-fold; p< 0.0001).Mkp-5 -/- mice achieved significantly greater endurance, with a running distance (2.81±169.8-fold;p< 0.0429) longer thanMkp-5 +/+ mice. Additionally, MKP-5 deficiency enhanced Akt/mTOR signaling (p-Akt/Akt:1.29±0.12-fold ;p= 0.04; p-mTOR/mTOR: 1.59±0.14-fold;p= 0.002) and mitochondrial biogenesis (pgc-1α: 1.56±0.27-fold ;p= 0.03) in cardiac muscle in response to aerobic exercise. Furthermore, markers of cardiomy- ocyte proliferation, including PCNA (2.24±0.31-fold;p< 0.001), GATA4 (1.47±0.10-fold; p< 0.001), and CITED4 (2.03±0.15-fold;p< 0.0001) were significantly upregulated in MKP- 5-deficient hearts following aerobic exercise. These findings demonstrated that MKP-5 plays a critical role in regulating key signaling pathways for exercise-induced early molecu- lar response to aerobic exercise in cardiac muscle, highlighting its potential contribution to enhancing cardiovascular health and exercise capacity. Keywords:aerobic exercise; MKP-5; mitochondrial biogenesis; cardiomyocyte proliferation 1. Introduction The benefits of exercise or physical activity in improving health and treating disease have been well established [1–3]. Extensive studies have consistently shown that levels of physical activity are inversely related to all-cause mortality and cardiovascular disease mortality [4–6]. Specifically, aerobic exercise, also known as endurance exercise, improves Cells2025,14, 410 https://doi.org/10.3390/cells14060410

Cells2025,14, 410 2 of 16 cardiovascular function, reducing the risk of cardiovascular disease [7,8]. Aerobic exercise has been shown to promote physiological cardiac adaptations, including structural, func- tional, and molecular changes that optimize cardiac function and enhance cardiovascular health [9,10]. These adaptations facilitate more efficient transport of blood and oxygen to working muscles, thereby improving overall endurance exercise capacity. Despite the evidence supporting the positive impact of aerobic training on heart function and exercise performance, the mechanisms driving physiological cardiac adaptation to aerobic exercise have not been clearly elucidated. The mitogen-activated protein kinase (MAPK) phosphatase-5 (MKP-5) acts as a neg- ative regulator of MAPK signaling by dephosphorylating phosphothreonine and phos- photyrosine residues on MAPKs [11–13]. Recent studies have explored the involvement of MKP-5-mediated MAPK signaling in various pathological processes [12,14–16]. The studies have revealed that MKP-5 deficiency ameliorates the progression of muscular dys- trophy and that MKP-5-deficient mice are protected from pulmonary fibrosis after lung injury [12,16]. Specifically, we have demonstrated that MKP-5 deficiency confers protection against cardiomyopathy caused by pressure overload, suggesting the importance of MKP-5 in cardiac function [15]. However, the role of MKP-5 in exercise-induced cardiac adaptation has not yet been studied. Therefore, this study aimed to test the effect of MKP-5 on early molecular response to aerobic exercise in cardiac muscle. We hypothesize that MKP-5 is a key regulator of exercise-induced early molecular change in cardiac muscle and that its deficiency enhances endurance exercise capacity. 2. Materials and Methods 2.1. Experimental Animals 8 to 10 week-old-wild type (Mkp-5 +/+ ) and MKP-5 knockout (Mkp-5 -/- ) mice were used in these experiments. MKP-5 knockout mice were engineered as described previously [17]. The mice were maintained under standard conditions, including a 12 h light/dark cycle (lights on at ZT0 and off at ZT12) and ad libitum access to food and water. To minimize vari- ability in feeding-induced signaling responses, tissues were collected at the same Zeitgeber time, and mice were not fasted before tissue collection to reflect physiological conditions. All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Yale University

at ZT0 and off at ZT12) and ad libitum access to food and water. To minimize vari- ability in feeding-induced signaling responses, tissues were collected at the same Zeitgeber time, and mice were not fasted before tissue collection to reflect physiological conditions. All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Yale University and the University of Texas at El Paso and were conducted in strict accordance with their respective guidelines. 2.2. Exercise Training Protocol Prior to the initiation of exercise protocol, mice in the exercise-trained groups under- went a 5-day acclimation period on a treadmill (Columbus Instruments, Columbus, OH, USA). This acclimation involved progressively increasing both the duration and speed of the running sessions. Following a 2-day rest period, the mice ran for 60 min per day for 5 consecutive days at a speed of 15 m/min and a 0% grade. This treadmill speed has been demonstrated for young mice to maintain 70~75% of VO2max[18]. A progressive exercise stress test was performed to assess endurance exercise capacity. The treadmill speed was gradually increased from 2 m/min to 6 m/min every 5 min until the point of exhaustion. Mice were considered exhausted if they failed to move forward from the back of the lane for five consecutive seconds on three separate occasions or for ten consecutive seconds once without attempting to resume running [19,20]. Endurance capacity was evaluated based on the total running distance. Mice in the sedentary group were exposed to the treadmill for the same duration and under the same conditions but without running (speed set to 0 m/min) to control for environmental and handling effects.

Cells2025,14, 410 3 of 16 2.3. Immunoblotting Mouse left ventricles were mechanically homogenized and lysed in lysis buffer composed of 100 mM Tris HCl (pH 7.4) and 25 mM EDTA. Protease and phosphatase inhibitors (1 mM Na3VO4, 10 mM NaF, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1µg/mL pepstain A, 5µg/mL aprotinin, 5µg/mL leupeptin) were added to the lysis buffer. The heart tissues were incubated at 4 ◦ C for 30 min and clarified by centrifugation at 14,000 rpm at 4 ◦ C for 10 min. The protein concentration was measured using the bicinchoninic acid (BCA) reagent according to the manufacturer’s instructions (23225; Thermo Fisher Scientific, Waltham, MA, USA). Total lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked with 1% casein blocking reagent (1610782; Bio-Rad, Hercules, CA, USA) for 1 h at room temperature. Primary antibodies were diluted in 5% BSA in Tris-buffered saline/Tween-20 (TBST). Following overnight incubation with primary antibodies at 4 ◦ C, membranes were washed three times for 10 min each in TBST. The membranes were then incubated with LI-COR secondary antibodies for 1 h at room temperature. The Odyssey CLx Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was used to visualize and quantify antibody binding. 2.4. Antibodies and Reagents All antibodies and reagents were obtained from standard chemical vendors. The anti- bodies used for immunoblotting are as follows. Phospho-p38 MAKP (#9215), phospho-JNK (#4668), phospho-ERK (#9101), phospho-Akt (#9271), phospho-mTOR (#2971), phospho-4E- BP1 (#9459), ERK (#9107), Akt (#2920), mTOR (#4571), p53 (#2524), and GAPDH (#2118) were purchased from Cell Signaling Technology (Danvers, MA, USA). p38 MAPK (sc-535), JNK (sc-571), 4E-BP1 (sc-9977), PCNA (cs-13110), and GATA4 (sc-25310) were sourced from Santa Cruz Biotechnology (Dallas, TX, USA). CITED4 (AV37255) was obtained from Sigma-Aldrich (Louis, MO, USA). OXPHOS antibody cocktail (MS604) was obtained from MitoSciences (Waltham, MA, USA). 2.5. RNA Extraction Followed by Quantitative Real-Time PCR Analysis RNA was isolated from the mouse left ventricles using an RNeasy kit (#4104; QIAGEN, Germantown, MD, USA) following the manufacturer’s instructions. cDNA was generated from 1µg RNA using a reverse transcriptase PCR kit (4368814; Applied Biosystems, Thermo Fisher Scientific,

USA). OXPHOS antibody cocktail (MS604) was obtained from MitoSciences (Waltham, MA, USA). 2.5. RNA Extraction Followed by Quantitative Real-Time PCR Analysis RNA was isolated from the mouse left ventricles using an RNeasy kit (#4104; QIAGEN, Germantown, MD, USA) following the manufacturer’s instructions. cDNA was generated from 1µg RNA using a reverse transcriptase PCR kit (4368814; Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real-time PCR was performed using the 7500 Fast real-time PCR system (Applied Biosystems, Foster City, CA, USA). Primers for SYBR Green Master Mix (A25742; Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) are listed in Table. All relative gene expression levels were determined using the∆∆CTmethod, with 18S rRNA as the normalization control. Table 1.The list of primer sequences used for quantitative real-time PCR analysis. Primer Name Sequences Mkp-5 5 ′ -ACCGCAGCTAGGAATAATGGA-3 ′ 5 ′ -ACCAAAAGCCTTGACTCCG-3 ′ Pgc-1α 5 ′ -CCCTGCCATTGTTAAGACC-3 ′ 5 ′ -CTTTTGTCCTTGTCGTCGTC-3 ′ Tfam 5 ′ -ATTCCGAAGTGTTTTTCCAGCA-3 ′ 5 ′ -TCTGAAAGTTTTGCATCTGGGT-3 ′ Myh6 5 ′ -GTCCCGGACACTGGACCAGGCC-3 ′ 5 ′ -CTCCTTTTCTTCCAGTTGCCTAGCCAA-3 ′ Myh7 5 ′ -GAGCAAGGCCGAGGAGACGCAGCGT-3 ′ 5 ′ -GAGCCTCCTTCTCGTCCAGCTGCCGG-3 ′ 18S 5 ′ -ACCGCAGCTAGGAATAATGGA-3 ′ 5 ′ -ACCAAAAGCCTTGACTCCG-3 ′

Cells2025,14, 410 4 of 16 2.6. Statistical Analysis All data represent the mean±standard errors of the mean (SEM). The statistical significance between groups was determined using the two-tailed, unpaired Student’s t-test or two-way analysis of variance (ANOVA) followed by a Tukey post-hoc test for multiple group comparisons. Graphing and statistical analyses were performed using Prism 9 software (GraphPad, San Diego, CA, USA). 3. Results 3.1. MKP-5 Deficiency Improves Endurance Exercise Capacity in Response to Aerobic Exercise Training Aerobic exercise improves endurance capacity by inducing various physiological and cellular adaptations in cardiac muscle [21,22]. To examine the effect of 5 consecutive days of aerobic exercise on body weight and cardiac mass, we measured body weight, heart weight, heart weight-to-body weight ratio, and heart weight-to-tibia length ratio following the exercise regimen. The results showed that five consecutive days of aerobic exercise did not lead to significant changes in either body weight or cardiac mass (Figure). To test whether MKP-5 gene expression is regulated in cardiac muscle in response to aerobic exercise, wild-type mice were subjected to treadmill running for five consecutive days after 5 days of exercise habituation. After aerobic exercise training, cardiac MKP-5 gene expression was reduced by 81% in the exercised mice as compared with sedentary mice (FigureA). This finding suggests that MKP-5 plays a role in cardiac muscle in response to aerobic exercise. Based on this result, we anticipated that MKP-5-deficient mice would have an improved ability to endure exercise. To test their exercise tolerance, the maximum exercise capacity of bothMkp-5 +/+ andMkp-5 -/- mice was determined by a progressive exercise stress test. The running distance ofMkp-5 -/- mice was increased by 2.81-fold as compared withMkp-5 +/+ mice (FigureB). Our findings indicate that the absence of MKP-5 enhances endurance capacity in response to aerobic exercise. Figure 1.Body weight, heart weight, and cardiac morphometric ratio. Body weight pre-exercise (A), body weight post-exercise (B), heart weight (C), heart weight-to-body weight ratio (D), and heart weight-to-tibia length ratio (E). All data are presented as mean±SEM. Statistical significance was assessed using 2-way ANOVA, followed by a Tukeypost-hoctest for multiple group comparisons.

capacity in response to aerobic exercise. Figure 1.Body weight, heart weight, and cardiac morphometric ratio. Body weight pre-exercise (A), body weight post-exercise (B), heart weight (C), heart weight-to-body weight ratio (D), and heart weight-to-tibia length ratio (E). All data are presented as mean±SEM. Statistical significance was assessed using 2-way ANOVA, followed by a Tukeypost-hoctest for multiple group comparisons.

Cells2025,14, 410 5 of 16 Figure 2.MKP-5 is downregulated in cardiac muscle in response to aerobic exercise and MKP-5 deficiency improves endurance exercise capacity. The graphs represent the relative mRNA expression ofMkp-5(A) in cardiac muscle from sedentary and exercised wild-type mice and runningdistance (B) from exercisedMkp-5 +/+ andMkp-5 -/- mice. All data are presented as mean±SEM. A two-tailed, unpaired Student’st-test was used for comparisons between the two groups. 3.2. MKP-5 Regulates MAPKs in Cardiac Muscle in Response to Aerobic Exercise Since MKP-5 suppresses the activity of MAPKs through direct dephosphorylation, the phosphorylation of MAPKs, including p38 MAPK, JNK, and ERK, was measured in hearts isolated from sedentary and exercisedMkp-5 +/+ andMkp-5 -/- mice. In sedentary mice, the phosphorylation of p38 MAPK and JNK was significantly increased in the cardiac muscle ofMkp-5 -/- mice as compared withMkp-5 +/+ mice (FigureA,B). In exercised mice, the phosphorylation of p38 MAPK and JNK ofMkp-5 +/+ mice was increased as compared with the sedentary mice (p< 0.0001 andp< 0.0383, respectively). When compared in exercised Mkp-5 +/+ andMkp-5 -/- mice, the phosphorylation of p38 MAPK and JNK was significantly increased inMkp-5 -/- mice (FigureA,B). These results align with our previous observation that MKP-5 dephosphorylates p38 MAPK and JNK, but not ERK [12,15]. These findings indicate that MKP-5-mediated MAPK signaling is activated in cardiac muscle in response to aerobic exercise and that MKP-5 deficiency further enhances p38 MAPK and JNK in cardiac muscle from exercised mice. Figure 3.MKP-5 deficiency enhances MAPK activity in cardiac muscle in response to aerobic exercise. The graphs represent the ratio of pp38 MAPK/p38 MAPK (A), pJNK/JNK (B), and pERK/ERK (C) in cardiac muscle from sedentary and exercisedMkp-5 +/+ and Mkp-5 -/- mice. All data are presented as mean±SEM. Statistical significance was assessed using 2-way ANOVA, followed by a Tukeypost-hoc test for multiple group comparisons.

Cells2025,14, 410 6 of 16 3.3. MKP-5 Deficiency Promotes Protein Synthesis in Cardiac Muscle Following Aerobic Exercise Aerobic exercise has been shown to activate anabolic signaling pathways in the cardiac muscle. Specifically, growing evidence shows that the Akt/mTOR pathway contributes to cardiac physiological remodeling following aerobic exercise [23–25]. To test whether MKP-5 plays a role in anabolic signaling pathways in cardiac muscle in response to aerobic exercise, the activation of the Akt/mTOR pathway was measured in hearts isolated from sedentary and exercisedMkp-5 +/+ andMkp-5 -/- mice. MKP-5-deficient mice exhibited in- creased Akt phosphorylation in cardiac muscle in response to aerobic exercise as compared withMkp-5 +/+ mice (FigureA). The phosphorylation levels of mTOR in cardiac muscle of exercisedMkp-5 -/- mice were significantly increased as compared withMkp-5 +/+ mice (FigureB). The phosphorylation of 4E-BP1, which is one of the downstream targets of mTOR, was significantly increased in cardiac muscle of mice lacking MKP-5 expression as compared withMkp-5 +/+ mice (FigureC). The gene expression of two myosin heavy chain isoforms,Myh 6(myosin heavy chainα) andMyh 7(myosin heavy chainβ), are consid- ered molecular markers of exercise-induced cardiac adaptation (2). Pathological cardiac hypertrophy is characterized by an increase inMyh 7gene expression, whereas exercise- induced physiological cardiac hypertrophy is associated with a decrease inMyh 7gene expression [26–28]. To investigate the effect of MKP-5 deficiency on the alteration ofMyh 6 andMyh 7gene expression in the hearts of exercised mice, qRT-PCR measurement was performed in hearts isolated from sedentary and exercisedMkp-5 +/+ andMkp-5 -/- mice (Figure). In sedentary and exercised mice, relativeMyh 6mRNA expression showed no significant differences betweenMkp-5 +/+ andMkp-5 -/- mice. Similarly,Myh 7mRNA expression did not significantly differ between groups. Although there was a trend toward a reduction inMhy 7mRNA expression and theMhy 7/Mhy 6ratio in response to aerobic exercise inMkp-5 -/- mice, the differences did not reach statistical significance. These findings indicate that MKP-5 deficiency enhances protein synthesis in cardiac muscle following aerobic exercise, potentially contributing to improved endurance exercise capacity. Figure 4.MKP-5 deficiency enhances the activation of the Akt/mTOR pathway in cardiac muscle in response to aerobic exercise. The graphs represent the ratio of pAkt/Akt

to aerobic exercise inMkp-5 -/- mice, the differences did not reach statistical significance. These findings indicate that MKP-5 deficiency enhances protein synthesis in cardiac muscle following aerobic exercise, potentially contributing to improved endurance exercise capacity. Figure 4.MKP-5 deficiency enhances the activation of the Akt/mTOR pathway in cardiac muscle in response to aerobic exercise. The graphs represent the ratio of pAkt/Akt (A), pmTOR/mTOR (B), and p4E-BP1/4E-BP1 (C) in cardiac muscle from sedentary and exercisedMkp-5 +/+ and Mkp-5 -/- mice. All data are presented as mean±SEM. Statistical significance was assessed using 2-way ANOVA, followed by a Tukeypost-hoctest for multiple group comparisons.

Cells2025,14, 410 7 of 16 Figure 5.MKP-5 deficiency tends to shift myosin heavy chain isoform in cardiac muscle in response to aerobic exercise. The graphs represent the relative mRNA expression ofMyh 6(A),Myh 7(B), and the ratio ofMyh 7andMyh 6(C) in cardiac muscle from sedentary and exercisedMkp-5 +/+ and Mkp-5 -/- mice. All data are presented as mean±SEM. Statistical significance was assessed using 2-way ANOVA, followed by a Tukeypost-hoctest for multiple group comparisons. 3.4. MKP-5 Deficiency Promotes Aerobic Exercise-Induced Mitochondrial Biogenesis Mitochondrial function is necessary for the performance of enduranceexercise [29,30] . It is well-established that aerobic exercise enhances mitochondrial biogenesis and respi- ratory function [31,32]. The process of mitochondrial biogenesis is regulated by several key factors, including the peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC-1α), which is activated during aerobic exercise [33,34]. The activated PGC-1α promotes the expression of genes such as transcription factor A (Tfam), which is involved in mitochondrial replication and function [35,36]. In order to investigate the role of MKP-5 in cardiac mitochondrial biogenesis, the mRNA expression ofPgc-1αandTfamwas mea- sured in cardiac muscle derived from sedentary and exercised mice (Figure). The cardiac muscle ofMkp-5 -/- mice showed increasedPgc-1αandTfammRNA expression in response to aerobic exercise as compared withMkp-5 +/+ mice (FigureA,B). Tfam is known to en- hance mitochondrial biogenesis through its interaction with p53, which positively regulates mitochondrial biogenesis [37,38]. Thus, we assessed the expression of p53 in the cardiac muscle of sedentary and exercised mice. The protein expression of p53 in the cardiac muscle ofMkp-5 -/- mice was significantly increased in response to aerobic exercise as compared withMkp-5 +/+ mice (FigureC). These observations imply that MKP-5 deficiency facilitates cardiac mitochondrial biogenesis following aerobic exercise. The increased mitochondrial biogenesis triggered by aerobic exercise activates mito- chondrial oxidative phosphorylation, leading to improved ATP production, which in turn enhances endurance exercise performance [39,40]. Mitochondrial oxidative phosphoryla- tion occurs through a series of protein complexes in the inner mitochondrial membrane [41]. To determine whether MKP-5 deficiency enhances mitochondrial protein complex expres- sion in response to aerobic exercise, the levels of these complexes were assessed in cardiac muscle of sedentary and exercisedMkp-5 +/+

This study investigates MKP-5's role in cardiac response to aerobic exercise.