Fibronectin type III domain‐containing 5 improves aging‐related cardiac dysfunction in mice

Abstract Aging is an important risk factor for cardiovascular diseases, and aging‐related cardiac dysfunction serves as a major determinant of morbidity and mortality in elderly populations. Our previous study has identified fibronectin type III domain‐containing 5 (FNDC5) and its cleaved form, irisin, as the cardioprotectant against doxorubicin‐induced cardiomyopathy. Herein, aging or matched young mice were overexpressed with FNDC5 by adeno‐associated virus serotype 9 (AAV9) vectors, or subcutaneously infused with irisin to uncover the role of FNDC5 in aging‐related cardiac dysfunction. To verify the involvement of nucleotide‐binding oligomerization domain‐like receptor with a pyrin domain 3 (NLRP3) and AMP‐activated protein kinase α (AMPKα), Nlrp3 or Ampkα2 global knockout mice were used. Besides, young mice were injected with AAV9‐FNDC5 and maintained for 12 months to determine the preventive effect of FNDC5. Moreover, neonatal rat cardiomyocytes were stimulated with tumor necrosis factor‐α (TNF‐α) to examine the role of FNDC5 in vitro. We found that FNDC5 was downregulated in aging hearts. Cardiac‐specific overexpression of FNDC5 or irisin infusion significantly suppressed NLRP3 inflammasome and cardiac inflammation, thereby attenuating aging‐related cardiac remodeling and dysfunction. In addition, irisin treatment also inhibited cellular senescence in TNF‐α‐stimulated cardiomyocytes in vitro. Mechanistically, FNDC5 activated AMPKα through blocking the lysosomal degradation of glucagon‐like peptide‐1 receptor. More importantly, FNDC5 gene transfer in early life could delay the onset of cardiac dysfunction during aging process. We prove that FNDC5 improves aging‐related cardiac dysfunction by activating AMPKα, and it might be a promising therapeutic target to support cardiovascular health in elderly populations.


| INTRODUC TI ON
Aging is an important risk factor for cardiovascular diseases, and aging-related cardiac dysfunction serves as a major determinant of morbidity and mortality in elderly populations (Marian et al., 2018;Triposkiadis et al., 2019). Despite the poor understanding of all exact molecular and cellular basis, chronic cardiac inflammation has been implicated in the pathogenesis of aging-related cardiac dysfunction through an inflammaging-dependent manner (Liberale et al., 2020). The levels of multiple inflammatory cytokines, including interleukin-6 (IL-6) and tumor necrosis factorα (TNFα), are elevated in serum and heart samples of elderly populations, and closely correlate with their cardiac functional parameters and long-term prognosis. In addition, inhibiting inflammation can provide cardiac benefits to some subjects (Gottdiener et al., 2000;Vasan et al., 2003). Nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome emerges as a critical sensor and regulator of inflammation, and contributes to the progression of aging-related cardiac dysfunction (Marín Aguilar et al., 2019). Upon stimulation, apoptosisassociated speck-like protein (ASC) is recruited to NLRP3 scaffold to promote caspase-1 cleavage and activation, thereby driving the maturation and release of various inflammatory mediators (e.g.,  and orchestrating a pro-inflammatory microenvironment to further amplify the inflammatory response. Therefore, it is reasonable to treat aging-related cardiac dysfunction by inhibiting inflammation. AMP-activated protein kinase α (AMPKα) has pleiotropic biological functions beyond metabolic regulation and plays a critical role in controlling inflammation and cardiac homeostasis. Our previous studies have proven that AMPKα activation could alleviate inflammation and diabetes-or sepsis-induced cardiac dysfunction (Ma et al., 2017;Song et al., 2020). Nuclear factor-κB (NF-κB) is a nodal signaling effector that provokes the transcription of various inflammatory genes, and Chen et al. revealed that AMPKα activation markedly reduced NF-κB phosphorylation and inflammation, and subsequently prevented hypoxia/reoxygenation-mediated cardiomyocyte injury (Chen et al., 2018). In addition, NLRP3 inflammasome pathway in the heart was also impeded by AMPKα activation Yang et al., 2019). AMPKα phosphorylation was found to be dampened in aging hearts, whereas activating AMPKα was sufficient to reduce aging-induced cardiac hypertrophy and interstitial fibrosis in mice (Cieslik et al., 2017). In contrast, Ampkα deficiency resulted in mitochondrial damage, reactive oxygen species (ROS) overproduction, and cardiomyocyte contractile defects in aging mice (Turdi et al., 2010). These findings identify AMPKα as a promising therapeutic candidate for aging-induced cardiac inflammation and dysfunction.
Moderate-to-vigorous physical activity stimulates multiple healthy benefits for elderly populations and protects against the future risk of heart failure . However, elderly individuals, especially those with cardiac dysfunction, are less likely to engage in and tolerate regular exercise. Inappropriate exercise also causes cardiac maladaptation and even acute cardiovascular events in susceptible people (Franklin et al., 2020). Accordingly, effective approaches mimicking the cardioprotective effects of physical exercise are of great significance for the elderly. Fibronectin type III domaincontaining 5 (FNDC5) is a type I transmembrane glycoprotein that can be proteolytically processed at the carboxy-terminal to release irisin, an exercise-responsive myokine conferring cardioprotection in response to different pathological stimulations, such as cardiac remodeling, ischemia/reperfusion injury, and diabetic cardiomyopathy (Bostrom et al., 2012;. Also, our recent study demonstrated that FNDC5 was abundant in the myocardium and that FNDC5 overexpression or irisin infusion significantly attenuated doxorubicin-induced oxidative stress, cardiomyocyte apoptosis, and cardiac dysfunction . In addition, circulating irisin levels are decreased in elderly subjects and positively correlate with the physical conditions and telomere length, suggesting a potential involvement of FNDC5 during aging process (Huh et al., 2014;Planella-Farrugia et al., 2019). Consistently, recent studies have revealed the protective roles of FNDC5 against aging-related memory impairment and cognitive dysfunction (Lourenco et al., 2019). In the present study, we aim to uncover the role of FNDC5 in aging-related cardiac dysfunction and explore the underlying mechanisms.

| Animals and treatments
Male C57BL/6 mice were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and housed in an environment-controlled SPF barrier system with free access to food and water. After 1 week of adaptive feeding, 6-month (M)-old young and 18-M-old aging mice were injected with 1 × 10 11 viral genome AAV9-FNDC5 or AAV9-NC per mouse from the tail vein to specifically overexpress FNDC5 in the myocardium as we previously described (Hu, Zhang, Song et al., 2020;. Eight weeks after AAV9 injection, mice were subjected to cardiac functional measurements and then sacrificed with the heart and serum samples collected for further investigation. To verify the role of NLRP3 and AMPKα, Nlrp3 or Ampkα2 global knockout (KO) mice were used according to our previous studies (Hu et al., 2019;Song et al., 2020). To enhance the clinical impact of our current work, 6-M-old or 18-M-old C57BL/6 mice were subcutaneously infused with irisin (12 nmol/kg/ day) for 2 M as we previously described . For the prevention study, 6-M-old C57BL/6 mice were intravenously injected with either AAV9-FNDC5 or AAV9-NC, and then maintained for an additional 12 M. To ascertain the involvement of GLP-1R, mice were intravenously injected with 1 × 10 11 viral genome shGlp-1r or shRNA carried by AAV9 per mouse at 4 weeks before FNDC5 overexpression.

| Echocardiography and hemodynamics
Echocardiography and hemodynamics were performed as we previously described , Zhang, Hu, Yuan et al., 2021, Zhang, Hu, Zhang et al., 2021. Mice were placed on a preheated pad and anesthetized with 1.5% isoflurane to provide adequate sedation.
Vevo ® 3100 High-Resolution Preclinical Imaging System (FUJIFILM VisualSonics) was used to record functional parameters. Invasive hemodynamic parameters were collected using a 1.4F Millar catheter transducer (SPR-839; Millar Instruments) and analyzed by the PVAN data analysis software.

| Western blot and quantitative real-time PCR
Protein extraction and Western blot were performed according to our previous studies (Hu, Zhang, Song et al., 2020;Zhang et al., 2019). Briefly, total proteins were extracted from murine hearts or cells using RIPA lysis buffer, and the concentrations were quantified with a BCA protein assay kit. Then, equal amounts of total proteins were electrophoresed by the SDS-PAGE and transferred onto polyvinylidene fluoride membranes. After being blocked in 5% skimmed milk at room temperature for 1 h, the membranes were probed with primary antibodies at 4 °C overnight, followed by the incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for an additional 1h on the next day. The protein bands were visualized by a ChemiDoc ™ XRS +system and analyzed with the Image Lab software (Bio-Rad Laboratories, Inc.). Nuclear and membrane proteins were extracted by commercial kits according to the manufacturer's instructions, and lamin B1 in nuclear fractions and GAPDH in cell lysates were used as internal controls, respectively. Total RNA was extracted using TRIzol reagent and then reversely transcribed to cDNA with a Maxima First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) (Zhang, Hu, Yuan et al., 2021, Zhang, Hu, Zhang et al., 2021. Gene expression was determined by Roche LightCycler ® 480 detection system using SYBR Green 1 Master Mix (Roche), and Gapdh was selected as an internal control.

| Cell surface biotinylation assay
To detect the level of GLP-1R in cell membrane, NRCMs were incubated with EZ-link ™ Sulfo-NHS-LC-Biotin (0.5 mg/ml) at 4 °C for 30 min, followed by the quenching with 100 nmol/L Tris-Cl (pH = 7.5) for an additional 20 min. Then, cells were lysed and incubated with neutravidin agarose beads at 4 °C overnight. The beads were subsequently washed for 4 times, and the biotinlabeled proteins were subjected to Western blot analysis. The expression of biotin-labeled GLP-1R in cell surface was normalized to GAPDH in cell lysates.

| Histological analysis
Cardiac morphology and fibrotic area were determined by hematoxylin-eosin (HE) or picrosirius red (PSR) staining as we previously described , Zhang, Hu, Yuan et al., 2021, Zhang, Hu, Zhang et al., 2021. Briefly, paraffin-embedded heart sections were dewaxed and rehydrated with dimethylbenzene and ethanol solution, and then subjected to HE or PSR staining using the standard protocols. Cardiomyocyte cross-sectional area and interstitial collagen volume were blindly measured by two independent authors using the Image-Pro Plus 6.0 software. Cell cross-sectional area was averaged from 30 fields per group with at least 5 cardiomyocytes per field analyzed, and interstitial collagen volume was calculated from more than 60 fields per group. For immunohistochemistry staining, the sections were subjected to antigen retrieval with citric acid buffer, followed by the incubation with 3% hydrogen peroxide and 10% goat serum to reduce endogenous peroxidase activity or the nonspecific binding. Subsequently, the samples were probed with anti-CD45 or anti-CD68 at 4 °C overnight, stained with the anti-rabbit/mouse EnVision TM /HRP reagent at 37 °C for 1 h, and then visualized by diaminobenzidine at room temperature (Zhang, Hu, Yuan et al., 2021, Zhang, Hu, Zhang et al., 2021. The images were captured by a light microscopy (Nikon H550L) and analyzed in a blinded manner.

| SA β-gal staining
SA β-gal staining was performed to assess cell senescence in vivo and in vitro using a commercial kit according to the manufacturer's instructions. Briefly, fresh frozen heart sections or cultured NRCMs were fixed with the fixation buffer at room temperature for 15 min and then incubated with the β-gal staining solution at 37°C for 24 h in a dry incubator. The images were captured using the light microscopy, and the percentage of SA β-gal + cells were quantified from at least 5 high-magnification fields.

| Telomere length measurement
Telomere length was measured based on a real-time PCR method as previously described (Eren et al., 2014). Briefly, genomic DNA was extracted from the heart samples, and then, the ratio of telomere repeat copy number to the copy number of a single-gene, acidic ribosomal phosphoprotein PO forward (36B4) was calculated as the telomere length.

| Immunofluorescence staining
The abundance of GLP-1R in cell surface was determined by immunofluorescence staining Zhang, Hu, Yuan et al., 2021). Briefly, cell coverslips were incubated with anti-GLP-1R at 4 °C overnight and then probed by Alexa Fluor 568-labeled secondary antibody at 37°C for 1 h, followed by the incubation with DAPI for nuclear visualization. The images were visualized and captured with a STELLARIS 5 confocal laser scanning microscope (Leica Microsystems Inc.).

| Oxidative stress detection
DHE staining was performed to detect the level of superoxide anion in heart samples as we previously described (Hu et al., 2019;. Briefly, fresh frozen heart sections were incubated with DHE solution (5 µmol/L) at 37°C for 30 min, and then, the images were captured using a fluorescence microscope (Tokyo, Japan) in a blinded manner. Colorless and non-fluorescent Amplex Red can be oxidized to resorufin by horseradish peroxidase in the presence of hydrogen peroxide (H 2 O 2 ), a highly fluorescent product whose absorbance can be detected at 560 nm.
To measure H 2 O 2 production from the heart, fresh left ventricular blocks were incubated with Amplex Red (100 µmol/L) and horseradish peroxidase (1 U/ml) in Krebs-HEPES buffer protected from light at 37°C for 30 min. Then, the tissue blocks were removed, and the supernatants were transferred to a 96-well plate with the absorbance being measured at 560 nm according to the manufacturer's instructions (Griendling et al., 2016;Matsushima et al., 2013). Lucigenin undergoes one-electron reduction to generate strong fluorescent signals that are related to the level of superoxide anion (O 2 − ) in the sample. To detect O 2 − in the heart, fresh heart samples were homogenized and incubated with 5 µmol/L lucigenin, and then, the lucigenin-enhanced chemiluminescence was continuously measured according to previous studies (Ago et al., 2004;Matsushima et al., 2013). The levels of MDA and 4-HNE in heart samples were detected using commercial kits according to the manufacturer's instructions as we previously described (Hu, Zhang, Song et al., 2020;.

| Biochemical analysis
Fasting blood glucose (FBG) was detected using an automatic glucometer, and the levels of serum irisin, TG, and TC were measured using commercial kits according to the manufacturer's instructions.
The levels of IL-1β, IL-6, IL-18, and TNFα in cardiac extracts were determined by commercially available ELISA kits following the standard protocols. To determine caspase-1 activity, fresh heart samples were homogenized in chilled cell lysis buffer and then incubated with YVAD-AFC substrate (1 mmol/L) at 37 °C for 2 h in the dark, followed by the quantification at an excitation/emission wavelength of 400/505 nm. In addition, fresh heart samples were homogenized in chloroform-methanol (1:20, w:v), and then, the chloroform-rich layer was mixed with the methanol to detect cardiac lipofuscin at an excitation/emission wavelength of 350/485 nm.

| Statistical analysis
All values are expressed as the mean ±standard deviation and analyzed using a SPSS 22.0 software. Unpaired Student's t test was used to compare differences between two groups, while one-way analysis of variance followed by Tukey's post hoc test was performed to compare the differences among multiple groups. p < 0.05 was considered significant.

| FNDC5 attenuates aging-related cardiac dysfunction
To explore the potential role of FNDC5 in aging-related cardiac dysfunction, we firstly determined FNDC5 expression in the serum and heart of aging mice. As depicted in Figure 1a, serum irisin level was significantly decreased in aging mice. In addition, the protein and mRNA levels of FNDC5 were also reduced in aging hearts (Figure 1b and Figure S1A). Then, we specifically overexpressed FNDC5 in the myocardium with AAV9 vectors to explore the role of FNDC5 in agingrelated cardiac dysfunction. FNDC5 protein level in murine hearts was increased after AAV9 injection, whereas serum irisin abundance was not markedly affected ( Figure 1c and Figure S1B). Intriguingly, FNDC5 overexpression in the heart did not affect the levels of mean arterial pressure (MAP), FBG, serum TG, and TC in aging mice (Figure S1C-F). As shown in Figure 1d-e, aging mice exhibited severe systolic dysfunction and ventricular dilation compared with young mice, as evidenced by the decreased fractional shortening (FS), the peak rates of isovolumic pressure development (+dP/dt) in left ventricles, and increased left ventricular internal dimension at end-diastole (LVIDd) or end-systole (LVIDs), which were attenuated by FNDC5 overexpression. Diastolic dysfunction is a key feature of aging hearts, and our data showed that aging mice with FNDC5 overexpression displayed improved diastolic function, as determined by the increased ratio of the early (E) to late (A) ventricular filling velocities (Figure 1f).
However, no alteration of heart rate was found ( Figure S1G). SA β-gal staining was applied to identify cellular senescence in aging hearts.
As shown in Figure 1g, the numbers of SA β-gal-positive cells in heart samples were significantly increased during aging progression, but to a less extent in those with FNDC5 overexpression. In addition, FNDC5 overexpression also preserved the telomere length in aging hearts ( Figure 1h). Lipofuscin is a senescence-associated pigment and positively correlates with the extent of aging (Marín Aguilar et al., 2019). As shown in Figure 1i, aging-related lipofuscin accumulation in the heart was significantly suppressed by FNDC5 overexpression. Consistently, FNDC5 overexpression also reduced the protein levels of senescent markers in aging hearts, including p16, p19, and p21 (Figure 1j-k). Collectively, our data imply that FNDC5 attenuates aging-related cardiac dysfunction.

| FNDC5 blocks aging-related cardiac remodeling
Cardiac hypertrophy and interstitial fibrosis are key features and de-  (Figure 2f-g). These findings indicate that FNDC5 blocks aging-related cardiac remodeling.

| FNDC5 suppresses aging-associated inflammatory response
Emerging evidences have addressed a direct association between low-grade chronic inflammation and aging-related cardiac dysfunction (Liberale et al., 2020). As shown in Figure 3a and Figure S2A, IL-6 and TNFα levels were increased in aging hearts, but decreased in those with FNDC5 overexpression. Immunohistochemistry staining and PCR data identified an increased infiltration of leukocytes to murine hearts during aging progression, which were largely suppressed by FNDC5 overexpression (Figure 3b and Figure S2B). NF- aging. To further validate the role of FNDC5 against inflammationassociated cardiac aging, TNFα, a critical inflammatory cytokine in aging hearts, was used to treat NRCMs to mimic inflammaging process in vitro according to a previous study (Cong et al., 2016).
As shown in Figure S4A, irisin treatment dramatically reduced SA β-gal-positive cells upon TNFα stimulation. Also, TNFα-induced increases in senescent markers were also decreased by irisin incubation ( Figure S4B-C). Our results validate that FNDC5 suppresses aging-associated inflammatory response.

| FNDC5 protects against aging-related cardiac dysfunction by activating AMPKα
AMPKα is a longevity-related molecule and plays critical roles in controlling inflammation and cardiac homeostasis (Salminen, & Kaarniranta, 2012). Our previous studies have proven that AMPKα activation could alleviate inflammation and diabetes-or sepsisinduced cardiac dysfunction (Ma et al., 2017;Song et al., 2020).
Herein, we found that FNDC5 overexpression significantly elevated AMPKα activity in aging hearts, as determined by the increased phosphorylation of AMPKα and the downstream ACC (Figure 4a-b).
To gain the evidence that AMPKα activation was responsible for the anti-inflammatory and cardioprotective effects of FNDC5 in aging mice, Ampkα2 global KO mice were used. As shown in Figure 4ce, Ampkα deficiency completely abolished FNDC5 overexpressionmediated suppression on NLRP3 inflammasome. In addition, FNDC5 overexpression also failed to inhibit NF-κB activation in Ampkαdeficient aging hearts ( Figure S5A-B). Meanwhile, the decreased IL-6 and TNFα levels seen in FNDC5-overexpressed aging hearts were dramatically blunted in Ampkα2 KO mice ( Figure S5C). As shown in Figure S5D

| FNDC5 activates AMPKα via blocking the lysosomal degradation of GLP-1R
The members of integrin family, especially ITGAV and ITGB5, are potential receptors of irisin in osteocytes and fat cells, and simultaneous knockdown of ITGAV and ITGB5 also blocked FNDC5-mediated F I G U R E 3 FNDC5 suppresses aging-associated inflammatory response in mice. (a) 6-month (M)-old young and 18-M-old aging mice were injected with AAV9-FNDC5 (1 × 10 11 viral genome per mouse) from the tail vein for 8 weeks to specifically overexpress FNDC5 in the myocardium or AAV9-NC as a control, and then, the myocardial interleukin-6 (IL-6) and tumor necrosis factorα (TNFα) levels were determined by ELISA kits (n = 6). (b) The immunohistochemistry staining of CD45 and CD68 in murine hearts (n = 6). (c-d) Western blot images and the statistical results of p16, p19, and p21 proteins (n = 6). (e) The caspase-1 activity in murine hearts (n = 6). (f) The myocardial IL-1β and IL-18 levels were determined by ELISA kits (n = 6). (g) Representative dihydroethidium (DHE) staining images in murine hearts and the statistical results (n = 6). Values represent the mean ±standard deviation. *p < 0.05 versus the matched group protective effects against diabetic cardiomyopathy (Kim et al., 2019;Lin et al., 2021). Intriguingly, AMPKα activation and antiaging effects by irisin upon TNFα stimulation were not affected by ItgaV/b5 silence ( Figure S7A-E). We previously reported that GLP-1R upregulation was sufficient to activate AMPKα pathway and that Glp-1r silence significantly restrained AMPKα activation in the heart (Ma et al., 2016;Song et al., 2020). Also, Gros et al. determined that Glp-1r-deficient mice exhibited elevated left ventricular end-diastolic pressure and ventricular thickness at baseline, and displayed systolic and diastolic impairment upon insulin administration (Gros et al., 2003). Based on these findings, we tried to explore whether FNDC5 activated AMPKα through GLP-1R. As shown in

| Irisin infusion mitigates aging-related cardiac dysfunction
We next investigated whether infusion of exogenous irisin would mitigate aging-related cardiac dysfunction in mice ( Figure 6a). As shown in Figure 6b-c, irisin infusion for 2 M significantly reduced the numbers of SA β-gal-positive cells in aging hearts. Also, aginginduced cardiac hypertrophy and fibrosis were also suppressed by irisin treatment (Figure 6d-e). Consistent with the phenotypic alterations, irisin infusion dramatically decreased the protein levels of senescent markers, including p16, p19, and p21 ( Figure S10A-B). As expected, aging-related systolic and diastolic impairment was also improved by irisin infusion, as evidenced by the increased FS, +dP/ dt, and E/A ratio (Figure 6f-g). All these data reveal that irisin infusion mitigates aging-related cardiac dysfunction.

| FNDC5 delays the onset of cardiac dysfunction during aging process
Given the cardioprotective role of FNDC5 in aging mice, we sought to determine whether FNDC5 overexpression in early life would delay the onset of cardiac dysfunction during aging process. As shown in Figure S11A-B, FNDC5-overexpressed mice displayed improved cardiac function during aging process. In addition, the protein levels of p16, p19, and p21 were also reduced in aging hearts with FNDC5 overexpression ( Figure S11C-D). Meanwhile, FNDC5 overexpression in early life significantly reduced the numbers of SA β-gal-positive cells and alleviated cardiac hypertrophy and fibrosis in aging hearts ( Figure S11E-H). In conclusion, we verify that FNDC5 delays the onset of cardiac dysfunction during aging process.

| DISCUSS ION
Epidemiological studies indicate that cardiovascular diseases are leading causes of death in the elderly and that aging-related cardiac dysfunction imposes a major financial burden on the whole soci- Therefore, our present study identifies FNDC5 as a promising therapeutic target to support cardiovascular health in elderly populations.
Aging-related cardiac dysfunction exhibits distinctive epidemiological, histological, and biological features after the long-term exposure to various risk factors and intrinsic aging disabilities  As a central metabolic biosensor during aging process, AMPKα is increasingly recognized as a potential longevity-related molecule with anti-inflammatory properties (Salminen, & Kaarniranta, 2012).
In this study, we also validate that FNDC5 overexpression improves aging-related inflammation and cardiac dysfunction by activating AMPKα. Despite the verification of a relationship between FNDC5 and AMPKα in many studies, relatively little is known about how FNDC5 activates AMPKα. GLP-1R is a member of the G proteincoupled receptors that transduces extracellular signals to intracellular molecular network through AC/cAMP axis. Emerging studies have indicated that GLP-1R is expressed in multiple tissues, including the heart (Helmstadter et al., 2021). Also, Gros et al. also determined the necessity of GLP-1R in aging-related cardiac remodeling and dysfunction. They showed that Glp-1r-deficient mice exhibited elevated left ventricular end-diastolic pressure and ventricular thickness at baseline, and displayed systolic and diastolic impairment upon insulin administration (Gros et al., 2003). In addition, our previous studies demonstrated that GLP-1R activation was sufficient to attenuate pressure overload-, obesity-, and sepsis-induced cardiac dysfunction. Currently, various GLP-1R agonists approved for the treatment of diabetes also stimulate cardiovascular benefits in some populations (Helmstadter et al., 2021). However, agonist-mediated endocytosis can provoke the degradation of GLP-1R in some cases, which extremely impedes the clinical application of GLP-1R agonists . In the present study, we proved that FNDC5 significantly suppressed the lysosomal degradation of GLP-1R, thereby elevating GLP-1R expression and membrane localization. The degradation of GLP-1R commonly involves clathrin-or caveolin-dependent endocytic machinery, spatiotemporal sorting, intracellular degradation, and recycling back to the plasma membrane Sonoda et al., 2008;Wootten et al., 2016). Yet, the exact mechanisms through which FNDC5 regulates the lysosomal degradation and net surface expression of GLP-1R remain elusive in this study.
GLP-1R degradation in most cases depends on the recruitment of clathrin or caveolin adaptors to promote endocytosis and desensitization; however, GLP-1R internalization is also required for signal transduction of some GLP-1R agonists (e.g., exendin-4). Therefore, completely blocking GLP-1R endocytosis may suppress downstream signaling pathways (Widmann et al., 1995). After internalization, GLP-1R is differentially sorted into proteasomes or lysosomes with the help of sorting nexins (SNX), where it is degraded by ubiquitinproteasome proteolytic pathway or lysosomal enzymes. In addition, Buenaventura et al. previously determined that SNX27 and SNX1 were essential for controlling the balance between GLP-1R lysosomal degradation and plasma membrane recycling . Lysosomes are well accepted as a major post-endocytic GLP-1R destination, and our present data consistently reveal that FNDC5 downregulation upon inflammaging stimulation accelerates the lysosomal degradation of GLP-1R and decreases its membrane localization, thereby inhibiting downstream AMPKα signaling and cardioprotection. In addition, the net surface GLP-1R expression also depends on GLP-1R membrane recycling (Widmann et al., 1995).
Although we prove that FNDC5 suppresses GLP-1R lysosomal degradation and subsequently activates downstream AMPKα pathway, whether other mechanisms exist to increase its net surface abundance demands further investigation.
In summary, we prove that FNDC5 improves aging-related cardiac dysfunction by activating AMPKα, and it might be a promising therapeutic target to support cardiovascular health in elderly populations.