Myokine Musclin Is Critical for Exercise-Induced Cardiac Conditioning

This study investigates the role and mechanisms by which the myokine musclin promotes exercise-induced cardiac conditioning. Exercise is one of the most powerful triggers of cardiac conditioning with proven benefits for healthy and diseased hearts. There is an emerging understanding that muscles produce and secrete myokines, which mediate local and systemic “crosstalk” to promote exercise tolerance and overall health, including cardiac conditioning. The myokine musclin, highly conserved across animal species, has been shown to be upregulated in response to physical activity. However, musclin effects on exercise-induced cardiac conditioning are not established. Following completion of a treadmill exercise protocol, wild type (WT) mice and mice with disruption of the musclin-encoding gene, Ostn, had their hearts extracted and exposed to an ex vivo ischemia-reperfusion protocol or biochemical studies. Disruption of musclin signaling abolished the ability of exercise to mitigate cardiac ischemic injury. This impaired cardioprotection was associated with reduced mitochondrial content and function linked to blunted cyclic guanosine monophosphate (cGMP) signaling. Genetic deletion of musclin reduced the nuclear abundance of protein kinase G (PKGI) and cyclic adenosine monophosphate (cAMP) response element binding (CREB), resulting in suppression of the master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), and its downstream targets in response to physical activity. Synthetic musclin peptide pharmacokinetic parameters were defined and used to calculate the infusion rate necessary to maintain its plasma level comparable to that observed after exercise. This infusion was found to reproduce the cardioprotective benefits of exercise in sedentary WT and Ostn-KO mice. Musclin is essential for exercise-induced cardiac protection. Boosting musclin signaling might serve as a novel therapeutic strategy for cardioprotection.


Introduction
Significant advances in prevention and mitigation of cardiovascular disease have been made, but related morbidity and mortality remain high [1,2]. These adverse outcomes stem from two main processes: dysrhythmias and derangement of cardiac mechanical function, both related to ongoing cardiac structural and functional deterioration that is independent from the initial insult causing myocardial injury [3,4]. Cardiac conditioning is a therapeutic approach to induce endogenous protective adaptive responses in the heart that counteract myocardial loss or dysfunction [5]. Physical activity is one of the most powerful triggers of cardiac conditioning with proven benefits for both those with healthy and with diseased

Figure 1. Musclin is required for exercise-mediated protection from ischemia-reperfusion injury
Representative transverse sections of individual TTC-stained hearts (Left) (scale bar 2 mm) and summary statistics of infarct size (Right) from sedentary and exercised WT and Ostn-KO mice (n = 7-12). Data are means ± SD; * p < 0.05.

Musclin Promotes Exercise Training-Induced Enhancements in Mitochondrial Biogenesis and Respiratory Capacity
It has been previously demonstrated that exercise-induced cardiac stress resistance is at least in part mediated through mitochondrial adaptation [39][40][41]. Musclin has been shown to govern exercise-induced mitochondrial biogenesis in skeletal muscles [22] However, its effect on physical activity-triggered mitochondrial biogenesis in the heart is yet to be established. To this end, the hearts of sedentary or exercise-trained WT and Ostn-KO mice were assessed for mitochondrial content and oxidative phosphorylation potential.

Musclin Promotes Exercise Training-Induced Enhancements in Mitochondrial Biogenesis and Respiratory Capacity
It has been previously demonstrated that exercise-induced cardiac stress resistance is at least in part mediated through mitochondrial adaptation [39][40][41]. Musclin has been shown to govern exercise-induced mitochondrial biogenesis in skeletal muscles [22]. However, its effect on physical activity-triggered mitochondrial biogenesis in the heart is yet to be established. To this end, the hearts of sedentary or exercise-trained WT and Ostn-KO mice were assessed for mitochondrial content and oxidative phosphorylation potential.
To further ascertain the effect of musclin on exercise-induced cardiac mitochondrial oxidative capacity, citrate synthase activity was measured in isolated mitochondria and maximal mitochondrial respiration in semi-permeabilized cardiac fibers. While both WT and Ostn-KO mice significantly increased their citrate synthase activity with exercise training in mitochondrial extracts (13.59 ± 1.24 vs. 9.59 ± 1.01 AU, n = 6 and p < 0.0001; Next, mitochondrial DNA levels were quantified. Specifically, expression was measured for mitochondrial DNA encoded NADH dehydrogenase 1 (ND1) as a marker of mitochondrial biogenesis, normalized to hexokinase 2 as a marker of nuclear DNA. This showed that exercise training promotes increased ND1 expression in WT but not in Ostn-KO mice (2.46 ± 0.99 vs. 1.00 ± 0.40, n = 6 and p = 0.002; 1.08 ± 0.38 vs. 0.65 ± 0.09 n = 6 and p = 0.56, respectively, in Figure 3B). This physical activity-induced ND1 upregulation resulted in a significantly higher ND1 level in exercised WT mice compared to exercised Ostn-KO mice (1.08 ± 0.38 vs. 2.46 ± 0.99, n = 6 and p = 0.0003 in Figure 3B), while there was no difference in ND1 level between the two groups under sedentary conditions (0.65 ± 0.09 vs. 1.00 ± 0.40, n = 6 and p = 0.70 in Figure 3B).
To further ascertain the effect of musclin on exercise-induced cardiac mitochondrial oxidative capacity, citrate synthase activity was measured in isolated mitochondria and maximal mitochondrial respiration in semi-permeabilized cardiac fibers. While both WT and Ostn-KO mice significantly increased their citrate synthase activity with exercise training in mitochondrial extracts (13.59 ± 1.24 vs. 9.59 ± 1.01 AU, n = 6 and p < 0.0001; 10.12 ± 0.65 vs. 8.26 ± 0.97 AU, n = 6 and p = 0.046, respectively), citrate synthase activity was still significantly lower in exercise-trained Ostn-KO mice in comparison to exercise trained WT mice (10.12 ± 0.65 vs. 13.59 ± 1.24 AU, n = 6 and p = 0.004 in Figure 3C).

Musclin Regulates Cardiac cGMP/Protein Kinase G-Dependent Signaling
NP/cGMP signaling is recognized as a key regulator of mitochondrial biogenesis, and, given that we previously demonstrated the role of musclin in augmenting ANP/cGMP signaling in skeletal muscle [22], and others described this in the heart [23,24,42], we hypothesized that musclin would influence exercise-induced cardiac cGMP signaling. To assess this hypothesis, cGMP levels were first measured in the myocardium of exercised WT and Ostn-KO mice. We confirmed that exercise-trained WT mice had significantly higher cardiac cGMP following 8 days of treadmill exercise compared to Ostn-KO mice (98.51 ± 14.16 vs. 61.94 ± 19.66 pMol/mg, n = 5 and p = 0.01). cGMP is known to activate protein kinase G (PKG) [43], which can translocate into the nucleus (and phosphorylates cAMP response element binding (CREB) in baby hamster kidney (BHK) fibroblasts [44][45][46] promoting its nuclear localization and ability to regulate transcription of peroxisome proliferatoractivated receptor γ coactivator 1α (PGC1α) [47,48]. To investigate if musclin affects this signaling cascade in the heart, the nuclear expressions of PKG1, CREB1, and PGC1α were quantified. In WT hearts collected immediately after last exercise session compared to those of sedentary WT controls, a significant increase was observed in nuclear localization of PKG1 (1.43 ± 0.13 vs. 0.76 ± 0.12 AU, n = 4 and p = 0.0003), CREB1 (1.87 ± 0.44 vs. 0.83 ± 0.01 AU, n = 4 and p = 0.31), and PGC1α (0.57 ± 0.11 vs. 0.28 ± 0.01 AU, n = 4, p = 0.002, Figure 4A Figure S2C). Taken together, these results indicate that musclin enhances physical activity-induced cardiac mitochondrial biogenesis through augmentation of cGMP/PKG/CREB-dependent signaling.

Synthetic Musclin Infusion Mimics Exercise-Mediated Protection against IR Injury
Exercise is one of the most effective ways to improve cardiac function and stress resistance [6][7][8][9][10][11][12][13][15][16][17][18]. However, adherence among those with heart disease to even short exercise programs, such as cardiac rehabilitation, is suboptimal [49]. The reasons are multifactorial, but a major contributor is baseline exercise intolerance, which is common in those with heart disease [7,50]. Synthetic musclin infusion has been demonstrated to be effective for reduction of myocardial injury and recovery of cardiac function when used after coronary artery ligation [23], but the possibility of using musclin infusion as a therapeutic strategy to induce a profile of benefits overlapping with exercise-induced cardiac conditioning in order to prevent myocardial damage and loss of cardiomyocytes is an intriguing possibility. To test this concept, synthetic peptide, corresponding to aa 80-112 of the murine musclin [22,30], was tested. This fragment has been associated with the most prominent modulation of ANP signaling in previous studies [22,30].
First, this synthetic peptide, containing uniformly labeled with 13 C and 15 N alanine and valine at positions 26 and 27, was used to define the volume of distribution (V d ) and plasma elimination half-life based on the calculations described in the Methods section ( Figure 6A-E). These parameters were used to calculate the infusion rate necessary to maintain a musclin plasma level comparable with endogenously produced levels observed after exercise training [22] (Figure 6A-E).
and Tfam (0.94 ± 0.21 vs. 1.01 ± 0.20 fold change, n = 5 and p = 0.60 in Figure S2C) expression of Nrf2 was slightly lower in sedentary Ostn-KO mice (0.82 ± 0.17 vs. 1.09 fold change, n = 5 and p = 0.03 in Figure S2C). Taken together, these results indica musclin enhances physical activity-induced cardiac mitochondrial biogenesis th augmentation of cGMP/PKG/CREB-dependent signaling.  First, this synthetic peptide, containing uniformly labeled with 13 C and 15 N alanine and valine at positions 26 and 27, was used to define the volume of distribution (Vd) and plasma elimination half-life based on the calculations described in the Methods section ( Figure 6A-E). These parameters were used to calculate the infusion rate necessary to maintain a musclin plasma level comparable with endogenously produced levels observed after exercise training [22] (Figure 6A-E).  Musclin treatment for 14 days significantly reduced myocardial infarct size following IR in sedentary WT mice compared to treatment with the control peptide (48.39 ± 2.15 vs. 59.82 ± 1.91%, n = 7-8 and p = 0.002 in Figure 7A,C). While 14 days of musclin infusion did not significantly reduce myocardial infarct size in sedentary Ostn-KO mice ( Figure 7B,C), 28 days of treatment reduced infarct size following IR (48.57 ± 2.14 vs. 59.69 ± 3.30%, n = 7-8 and p = 0.013 in Figure 7B,C). Importantly, enhanced cardiac tolerance to IR injury after musclin infusion was associated with markers of increased mitochondrial biogenesis ( Figure S3). These results demonstrate that exogenous musclin mimics exercise-induced cardiac conditioning and stress resistance. not significantly reduce myocardial infarct size in sedentary Ostn-KO mice ( Figure 7B,C), 28 days of treatment reduced infarct size following IR (48.57 ± 2.14 vs. 59.69 ± 3.30%, n = 7-8 and p = 0.013 in Figure 7B,C). Importantly, enhanced cardiac tolerance to IR injury after musclin infusion was associated with markers of increased mitochondrial biogenesis ( Figure S3). These results demonstrate that exogenous musclin mimics exercise-induced cardiac conditioning and stress resistance.

Discussion
This study establishes that the exercise-responsive myokine musclin [22] is vital for exercise-induced cardiac resistance to IR stress. Disruption of musclin signaling in Ostn-KO mice results in reduced oxidative phosphorylation potential and greater vulnerability to ischemic injury. Musclin replacement therapy restored cardiac resistance to stress in Ostn-KO mice. These findings indicate a previously unrecognized pathway for cardiac adaptation to exercise.
The data in the present study also confirm that intact musclin signaling is necessary for exercise-induced activation of PKGI-CREB/PGC1α, mitochondrial biogenesis, and functional adaptations in the myocardium. Notably, the results indicate significantly greater mitochondrial quantity in WT compared to musclin-deficient hearts by multiple methods and demonstrate the functional importance of the observed changes by revealing a significant deficiency in respiration measured in perforated myofibers isolated from Ostn-KO mice. These findings agree with the previously reported musclin interaction with natriuretic peptide receptors [23,24,30,31,42] and the detection of significantly higher levels of cGMP in the myocardium of WT mice, compared with Ostn-KO mice, after an exercise bout. There are accumulating data suggesting cGMP is a critical driver of mitochondrial biogenesis [72][73][74][75][76][77][78]. In fact, cGMP signaling has been linked to PGC1α-dependent mitochondrial biogenesis in many studies in different tissues and organs, including our own prior study indicating musclin-dependent activation of cGMP/PGC1α-driven mitochondrial biogenesis in skeletal muscles [22], while the current data indicate the significance of this musclin-driven signaling in the heart (Figure 8).
to ischemic injury. Musclin replacement therapy restored cardiac resistance to stress in Ostn-KO mice. These findings indicate a previously unrecognized pathway for cardiac adaptation to exercise.
The data in the present study also confirm that intact musclin signaling is necessary for exercise-induced activation of PKGI-CREB/PGC1α, mitochondrial biogenesis, and functional adaptations in the myocardium. Notably, the results indicate significantly greater mitochondrial quantity in WT compared to musclin-deficient hearts by multiple methods and demonstrate the functional importance of the observed changes by revealing a significant deficiency in respiration measured in perforated myofibers isolated from Ostn-KO mice. These findings agree with the previously reported musclin interaction with natriuretic peptide receptors [23,24,30,31,42] and the detection of significantly higher levels of cGMP in the myocardium of WT mice, compared with Ostn-KO mice, after an exercise bout. There are accumulating data suggesting cGMP is a critical driver of mitochondrial biogenesis [72][73][74][75][76][77][78]. In fact, cGMP signaling has been linked to PGC1α-dependent mitochondrial biogenesis in many studies in different tissues and organs, including our own prior study indicating musclin-dependent activation of cGMP/PGC1α-driven mitochondrial biogenesis in skeletal muscles [22], while the current data indicate the significance of this musclin-driven signaling in the heart (Figure 8). In the heart, musclin competitively interferes with binding to NPR c , allowing increased NP/NPR a binding, which in turn increases cGMP production in the heart. cGMP in turn activates PKG1 which leads to a signaling cascade that results in increased CREB1 nuclear localization and transcription of PGC1α which promotes enhancement of mitochondrial biogenesis and respiratory capacity that support cardiac stress resistance.
Importantly, different time points after the last exercise training session were used here to reveal specific features of exercise-induced cardiac conditioning. Hearts were collected immediately after exercise for cGMP measurement and 10-15 min after exercise to evaluate the nuclear presence of PKGI and CREB, while nuclear expression of PGC1α and mitochondrial respiratory complexes were evaluated in hearts collected 2 h after the last exercise session to probe the previously reported dynamics of cGMP/PKGI signaling [44,79,80].
Although treadmill exercise training was used here to stimulate musclin signaling, results indicate a phenotype of lower mitochondrial content and reduced mitochondrial respiration even in untrained Ostn-KO mice compared with WT controls. Importantly, these observed trends and changes in sedentary Ostn-KO became much more obvious and significant after exposure to the exercise protocol. These findings can be interpreted to indicate that musclin production and secretion, although more easily demonstrated in response to vigorous exercise, are physiologically relevant, even for routine daily activities.
Cardioprotective properties of musclin were demonstrated previously in different murine models of cardiomyopathies [10,23,81,82]. Our work demonstrates the role of musclin in cardiac conditioning and protection that is specifically exercise-induced. This establishes a previously unrecognized link between muscle activity and cardiac conditioning and supports a therapeutic potential in critically ill, injured, or otherwise high cardiac-risk surgical candidates in whom significant loss of muscle mass due to the catabolic stress of immobility and/or inadequate nutrition associated with any critical illness [83] may undermine optimal intrinsic musclin signaling.
In fact, the data from this study indicate that infusion of a synthetic fragment of musclin peptide mimics exercise benefits with respect to protection of the myocardium from IR injury. This is very encouraging as pharmacological options available to mimic exercise-induced cardioprotection have not previously been clinically available. In contrast to ANP or BNP infusion, which can directly stimulate cGMP synthesis, musclin promotes cGMP signaling through inhibition of other NPs via competitive inhibition of NPR C -driven clearance [22,23]. In this way, musclin infusion would enhance the natural dynamics of NP signaling as well as any potential beneficial effects related to local signaling by multiple NPs that are cleared through the NPR C [84], in contrast to systemic infusion of ANP and BNP that would presumably result in non-physiologically high levels in off-targets and possibly insufficient levels in target tissues such as the myocardium. Interestingly, inhibition of another pathway for NP degradation by neutral endopeptidase has been linked to a survival benefit in heart failure (HF) patients [85]. In contrast, ANP or BNP infusion, while leading to improvement in measures of HF, has been linked to an increased risk of side-effects related to hypotension or nephrotoxicity [86][87][88], supporting the concept that off-target effects of direct infusion of natriuretic peptides are less desirable than methods to boost their intrinsic local concentrations and effects.
The present study also includes pharmacokinetic studies that allow for the calculation of the infusion rate necessary to secure a plasma musclin concentration close to that previously linked to exercise training and enhanced cGMP signaling [22]. As such, the findings of this study may help standardize future experiments with synthetic musclin.
Surprisingly, disruption of musclin signaling in Ostn-KO mice practically abolished exercise-induced cardioprotection. Considering the complexity of exercise-induced adaptations, this is somewhat unexpected. However, this finding may occur since musclin is also important for the skeletal muscle response to physical activity and, as such, Ostn-KO mice may not be getting the full benefits of exercise training [22]. Furthermore, this study investigates the role of musclin in augmenting NP/cGMP signaling. However, there is emerging research that indicates the existence of NPR c -specific signaling [89][90][91]. This possible musclin/NPR c interaction should be the subject of future studies.
Finally, this study further verifies the unique interaction of skeletal muscle and heart through the previously reported co-regulation of endocrine signals: cardiac NPs and the myokine musclin [22,42]. Cardiac NPs, particularly ANP, have been increasingly recognized as powerful exercise-responsive regulators of important physiological functions [92][93][94][95]. Uncovering this musclin-dependent effect on exercise-induced cardiac conditioning broadens appreciation of the complexity and physiologic roles of cardiac and skeletal muscles as endocrine organs. In the future it will be interesting to clarify the effect of musclin signaling on other NP-regulated physiologic phenomenon such as blood pressure homeostasis as well as glucose and fat metabolism [84,96,97]. Additional investigation, including direct comparison of different exercise modes (resistance vs. aerobic) as well as forced vs. voluntary exercise, along with different time exposures, are needed to fully understand the range of musclin-induced adaptations that underlie its beneficial cardioprotective effects. Beyond advancing the understanding of musclin's effects and musclin's role in mechanisms of exercise-induced cardiac conditioning, this study has translational implications by extending the concept of musclin-dependent cardioprotection to the preconditioning of hearts to improve their tolerance of future ischemic insults using synthetic musclin infusion. This could represent a new therapeutic strategy to improve outcomes of stressful cardiac events or procedures, especially in exercise-intolerant patients.

Animal Experiments
All animal protocols conform to the Guide for the Care and Use of Laboratory Animals generated by the Institute for Laboratory Animal Research, National Research Council of the National Academies. All animal protocols were approved by the University of Iowa Institutional Animal Care and Use Committee. For all experiments, mice were anesthetized with inhaled isoflurane (5% induction, 1-1.5% maintenance; Piramal Healthcare) to maintain a respiratory rate of~50-60 breaths per minute.

Ostn-KO Mouse Model
Ostn-KO mice were generated as described previously [22]. Vector construction and targeted knockout strategy were designed together with genOway: Mice were generated based on deletion of a 2.1-kb sequence flanking Ostn exon 2, resulting in inactivation of the ATG and signal peptide. All mice used in this study were male, 6-8 weeks of age, and on a C57BL6/N background. Control WT C57BL6/N were originally purchased from Charles River (Wilmington, MA, USA). Heterozygous Ostn-KO mice were bred to obtain homozygous Ostn-KO mice and WT controls.

Myocardial Ischemia-Reperfusion Injury
Hearts were extracted from anesthetized mice and retrogradely perfused at 90 mm Hg with Krebs-Henseleit buffer bubbled with 95% O 2 /5% CO 2 , at 37 • C and pH 7.4. Myocardial ischemia-reperfusion (IR) injury was created as follows: after allowing 20 min for stabilization, the hearts were subjected to 20 min of global ischemia and 45 min of reperfusion. At the end of this treatment, the hearts were removed from the Langendorff perfusion apparatus and immediately frozen at −20 • C. The frozen hearts were then cut into transverse slices of approximately equal thickness (∼0.8 mm) from apex to base. The slices were placed into a small cell culture dish and then incubated in 1% triphenyltetrazolium chloride (TTC) in phosphate buffer (Na 2 HPO 4 88 mmol/L, NaH 2 PO 4 1.8 mmol/L, pH 7.8) at 37 • C for 20 min while gently shaking the dish. The development of the red formazan pigment in living tissues relies on the presence of lactate dehydrogenase, or NADH, while failure to stain red indicates a loss of these constituents from necrotic tissue. After staining, the TTC buffer was replaced by 10% formaldehyde. The slices were fixed for the next 4-6 h, and then the areas of infarct tissue were quantified using ImageJ software. The risk area is taken as the total ventricular cross-sectional area of all slices. The infarct size is presented as infarcted percentage of at-risk area.

Treadmill Exercise Training
Three days before exercise, mice were acclimated for 45 min daily on a non-moving treadmill (Columbus Instruments, Columbus, OH, USA) followed by 15 min at a velocity of 3.5 m/min at 15 • incline. For IR studies and assessment of nuclear PKG and PGC1α, mice were then exercised daily for total of 10 days: five consecutive days (Monday to Friday), followed by a 2-day rest, and then exercised daily for three more days (Monday, Tuesday, Wednesday) at a speed of 15 m/min and inclination of 15 • for 45 min. This protocol has been previously shown to increase the production and secretion of musclin [22]. Immediately after the last exercise session, mice were sacrificed, and their hearts were collected for cGMP measurements. To evaluate the nuclear presence of PKGI and CREB, hearts were collected 10-15 min after the last exercise. To test nuclear expression of PGC1α and mitochondrial respiratory complexes, hearts were collected 2 h after the last exercise. Mitochondrial DNA, mRNA, protein expression, and mitochondrial respiration changes were assessed following 15 days of exercise training as described above (5 d/week daily exercise for 3 wks) with hearts collected 2 h after the last exercise.
For infusion, 1.7 mg of the chemically synthesized [ 13 C, 15 N-A26-V27]-labeled musclin peptide was dissolved in 300 µL of PBS. This musclin solution was injected into the jugular vein. A 100 µL blood sample was then drawn from the carotid artery at 1, 3, 5, 7, 10, and 15 min after the peptide infusion. Blood was mixed with 5 µL of 10% NaEDTA and 1 µL of peptidase inhibitor cocktail (Sigma, St. Louis, MI, USA) and kept on ice, then centrifuged at 4 • C for 45 min at 14,000 rpm. Plasma was removed and stored at −80 • C for NMR studies.

NMR Spectroscopy
All NMR spectra were acquired at 5 • C on a Bruker Avance II 800 MHz NMR spectrometer equipped with a TCI cryoprobe. 100 µL of plasma (see above) was added 150 µL NMR buffer containing 50 mM sodium phosphate, pH 7.1 with a final volume of 250 µL in 8.5% D 2 O/91.5% H 2 O. This sample was then transferred to a Shigemi NMR tube for NMR studies. One-dimensional (1D) 1 H, two-dimensional (2D) 15 N/ 1 H-heteronuclear single-quantum correlation (HSQC), 2D 13 C/ 1 H-HSQC with constant time experiment (HSQC-CT), and 2D 15 N/ 1 H-slice of the three-dimensional (3D) HNCO experiment [98,99] were acquired for each blood sample drawn at different times after peptide infusion. The collected NMR data were processed using NMRPipe [100] and analyzed using NMRView [101]. The musclin peptide concentration present in these blood samples was calibrated by using a known amount of chemically synthesized [ 13 C, 15 N-A26-V27]-labeled musclin peptide. The peak intensity of these acquired NMR spectra was measured and fitted to the equation for half-life in exponential decay using GraphPad Prism (GraphPad Software version 9.4.1).  [23]. Each amino acid sequence is as follows: musclin SFSGFGSPLDRLSAGSVEHRGKQRK-AVDHSKKR, and control SFSGFGSPLGGLSAGSVEHRGKQRKAVDHSKKR.

Pharmacokinetic Calculations
Peptides dissolved in PBS were loaded into osmotic pumps, based on the pharmacokinetic data obtained with labeled peptide (see above). Pumps were surgically implanted within the peritoneal cavity of mice under sterile conditions and general anesthesia.

Mitochondrial Protein Isolation
Mitochondrial protein fraction was isolated from hearts by using the Mitochondria Isolation Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Mitochondrial protein content was determined as the amount of mitochondrial protein per mg of wet tissue.

RNA Isolation and qRT-PCR
Total RNA from cardiac tissues was isolated by using the RNeasy RNA Isolation Kit (Qiagen). A 1 µg primary myocyte or tissue RNA sample was used to synthesize cDNA in 50 µL reactions using Oligo(dT) as primer and SuperScript III Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was performed on a Mastercycler EP Gradient S (Eppendorf) by using SYBR green-based PCRs. For qRT-PCR, 1 µL of reverse transcription reaction was mixed with 10 pmol of each specific primer and 12.5 µL of SYBR PCR Master Mix (Bio-Rad, Hercules, CA, USA). The reaction was incubated for 40 cycles consisting of denaturation at 95 • C for 10 s and annealing/extension at 59.9 • C for HPRT and Nrf2, and 56 • C for Creb1, ERRA, PGC1α, Tfam, and Nrf1 for 1 min. The quality of the PCR product was routinely checked by a thermal denaturation curve following the qPCR reactions. The threshold cycle (CT) was determined by Realplex2 software (Eppendorf), and quantification of relative mRNA levels was performed by ∆∆CT method or by calculation of the absolute number of copies based on the standard curve. The primers used in this study are as  PGC1α:  PGC1-αF, TGA TGT GAA TGA CTT GGA TAC AGA CA, and PGC1αR, GCT CAT TGT  TGT ACT GGT TGG ATA TG; TfamF, GGA ATG TGG AGC GTG CTA AAA, and TfamR,  TGC TGG AAA AAC ACT TCG GAA TA; Nrf1F, CGC AGC ACC TTT GGA GAA, and  Nrf1R, CCC GAC CTG TGG AAT ACT TG; and Nrf2F, CAG CTC AAG GGC ACA GTG C, and Nrf2R,-GTG GCC CAA GTC TTG CTC C.

Western Blotting
Whole-cell protein extracts were prepared by homogenizing cardiac tissue in RIPA Buffer (Sigma), supplemented with protease and phosphatase inhibitors (Roche Inc. Basel, Switzerland). Nuclear extracts were obtained by using the Subcellular Protein Fractionation Kit for Tissues (Thermo Scientific). Electrophoresis was performed on 3-8% gradient Nu-Page Tris-Acetate or 10% Nu-Page Bis-Tris gels and transferred to 0.2 µm Sequi-Blot PVDF membranes (Bio-Rad). The membranes were blotted with total Creb1 (Cell Signaling, Danvers, MA, USA), PKG1 (Cell Signaling), total OXPHOS (Abcam, Cambridge, UK), PGC1α (Abcam), TBP, Lamin B1, and GAPDH (Cell Signaling) antibodies. Densitometric analysis of Western blots was performed using Adobe Photoshop (Adobe Systems, San Jose, CA, USA). For representative individual gel/blots, the reference proteins (laminB1 and TBP) are shown immediately below the proteins of interest.

CGMP Measurement
cGMP levels were measured using the cyclic GMP enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI). On the day of the experiment, mice were exercised at 12 m/min for 20 min while sedated with isoflurane, and heart tissue was collected immediately at the end of exercise. Cyclic nucleotides were extracted by using 5% (wt/vol) trichloroacetic acid. Samples were acetylated and used for cGMP level measurements according to the manufacturer's protocol.

Mitochondrial Respiration in Isolated Myofibers
Mitochondria respiratory function was assessed using an Oroboros Instruments Oxygraph 2K (Innsbruck, Austria) as described previously [102]. Cardiac fibers from a small piece of the endocardium (~15 mg) were dissected and separated into bundles in ice-cold BIOPS buffer (7.23  Following incubation, 1-1.5 mg of tissue was added into the O2K chamber, which was filled with 2 mL of respiration buffer. Carbohydrate (CHO)-supported respiration was assessed with pyruvate (5 mM) and malate (2 mM) with subsequent addition of ADP (5 mM) and glutamate (5 mM). Maximal CHO-supported respiratory capacity was determined in the presence of these substrates with the subsequent addition of succinate (5 mM).

Citrate Synthase Activity
Citrate synthase activity was determined using a colorimetric assay based on the reaction between 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) and CoA-SH to form 5-thio-2nitrobenzoic acid (TNB), which exhibits maximum absorbance at 412 nm, according to the manufacturer's instructions (Sigma). A mitochondrial pellet fraction was isolated from the heart by using the Mitochondria Isolation Kit (Thermo Scientific) and resuspended in CS Assay Buffer.

Statistics
Data are presented as individual data points with means ± the standard deviation. Statistical significance was evaluated using two-way ANOVA with Tukey's testing for multiple comparisons or a two-sided Student's t-test with tests for normality/lognormality as appropriate (GraphPad Prism version 9.4).

•
The exercise-responsive myokine musclin, which has high homology to natriuretic peptides (NPs), is critical for exercise-induced myocardial protection. • Normal musclin signaling is critical for exercise-induced cardiac mitochondrial biogenesis. • Musclin-dependent cardioprotective remodeling is driven by activation of cGMP/PKGI/ CREB/PGC1α signaling.
• Uncovering a musclin-dependent effect on exercise-induced cardiac conditioning broadens the appreciation of the complexity and physiologic roles of cardiac and skeletal muscles as endocrine organs. • Synthetic musclin infusion reproduces the exercise-induced cardioprotective response to ischemia-reperfusion.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in the article.

Conflicts of Interest:
The authors declare no conflict of interest.