Chronic treatment with the mitochondrial peptide humanin prevents age-related myocardial fibrosis in mice

Abstract

Cardiac fibrosis is a biological process that increases with age and contributes to myocardial dysfunction. Humanin (HN) is an endogenous mitochondria-derived peptide that has cytoprotective effects and reduces oxidative stress. The present study aimed to test the hypothesis that chronic supplementation of exogenous HN in middle-aged mice could prevent and reverse cardiac fibrosis and apoptosis in the aging heart. Female C57BL/6N mice at 18 mo of age received 14-mo intraperitoneal injections of vehicle (old group; n = 6) or HN analog (HNG; 4 mg/kg 2 times/wk, old + HNG group, n = 8) and were euthanized at 32 mo of age. C57BL/6N female mice (young group, n = 5) at 5 mo of age were used as young controls. HNG treatment significantly increased the ratio of cardiomyocytes to fibroblasts in aging hearts, as shown by the percentage of each cell type in randomly chosen fields after immunofluorescence staining. Furthermore, the increased collagen deposition in aged hearts was significantly reduced after HNG treatment, as indicated by picrosirius red staining. HNG treatment also reduced in aging mice cardiac fibroblast proliferation (5′-bromo-2-deoxyuridine staining) and attenuated transforming growth factor-β₁, fibroblast growth factor-2, and matrix metalloproteinase-2 expression (immunohistochemistry or real-time PCR). Myocardial apoptosis was inhibited in HNG-treated aged mice (TUNEL staining). To decipher the pathway involved in the attenuation of the myocardial fibrosis by HNG, Western blot analysis was done and showed that HNG upregulated the Akt/glycogen synthase kinase -3β pathway in aged mice. Exogenous HNG treatment attenuated myocardial fibrosis and apoptosis in aged mice. The results of the present study suggest a role for the mitochondria-derived peptide HN in the cardioprotection associated with aging.

NEW & NOTEWORTHY Cardiac fibrosis is a biological process that increases with age and contributes to myocardial dysfunction. Humanin is an endogenous mitochondria-derived peptide that has cytoprotective effects and reduces oxidative stress. Here, we demonstrate, for the first time, that exogenous humanin treatment attenuated myocardial fibrosis and apoptosis in aging mice. We also detected upregulated Akt/glycogen synthase kinase-3β pathway in humanin analog-treated mice, which might be the mechanism involved in the cardioprotective effect of humanin analog in aging mice.

INTRODUCTION

Humanin (HN) is a 24-amino acid, endogenous, mitochondria-derived peptide that was first identified as a neuroprotective factor that suppressed the neuronal cell death induced by proteins generated in familial Alzheimer’s disease (14). Besides the neuroprotective effect in Alzheimer’s disease, stroke (46), and amyotrophic lateral sclerosis (31), HN and its analogs (such as HN analog HNG) have been demonstrated to show a protective effect in nonneurological diseases, including diabetes (17, 33), atherosclerosis (35, 48), and myocardial infarction (34). Although the exact mechanism of its cytoprotective effect remains to be fully elucidated, it has been demonstrated that HN has protective effects on different cell types under a variety of insults, including oxidative stress (1, 29, 39), serum starvation (22), and hypoxia (32). Oxidative stress is a common mechanism of age-related diseases, which is caused by ROS released from mitochondria. Excessive accumulation of oxidative damage can result in mitochondrial dysfunction, which, in turn, leads to enhanced ROS production and contributes to myocardial cell loss and myocardial fibrosis in aging (8, 26, 36).

Cardiac fibrosis is a biological process that increases with age and contributes to dysfunction of the heart, characterized by diastolic or systolic heart failure (3, 38). Recently, it has been demonstrated that circulating levels of HN decline with age in mice and humans, indicating age-dependent regulation of its expression (33). Based on the cytoprotective effect of HN under oxidative stress, we hypothesized that exogenous HN treatment may be beneficial to aging hearts. The goal of the present study was to examine if replenishing the reduced levels of HN in old mice could reverse cardiac fibrosis and apoptosis in the aging heart.

MATERIAL AND METHODS

Reagents and animals.

HNG was synthesized by Genscript (Piscataway, NJ) (46). SH-SY5Y cells were a gift from Dr. Cohen’s laboratory (Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA). Animal protocols were in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the University of Southern California Institutional Animal Care and Use Committee (no. 11610). Most longevity studies have effects on female animals, and, therefore, we did our first-long term study using female mice. Female C57BL/6N mice (n = 100) obtained from the National Institute on Aging aged mouse colony at 18 mo of age were grouped into old and old + HNG groups (n = 50 each). Mice that survived until the end of the experiment received twice weekly injections of vehicle (water, old group, n = 6) or HNG (4 mg/kg 2 times/wk, old + HNG group, n = 8) for 14 mo. Mice were injected with 5′-bromo-2-deoxyuridine (BrdU; 50 mg/kg) for 8 days before they were euthanized at 32 mo of age. C57BL/6N female mice (young group, n = 5) were purchased at 1 mo of age and treated with intraperitoneal injection of vehicle (water) for 4 mo before euthanasia by CO₂ asphyxiation. Mice were fed normal chow provided by the Department of Animal Research at the University of Southern California. Pellets were replaced weekly; body weight and food consumption were recorded on alternate days, and survival was monitored daily.

Histological examination.

Mouse hearts were excised, fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5-μm sections. After deparaffinized and rehydration in serial changes of xylene and ethanol, representative sections were stained with picrosirius red and examined under polarized light with an EVOS FL auto microscope (ThermoFisher Scientific, Waltham, MA). Images were taken at multiple sites (at least seven) with a ×20 objective and quantified with MetaMorph image-analysis software (Molecular Devices, Sunnyvale, CA). In evaluating interstitial fibrosis, vessels were excluded from the analysis, whereas in evaluating perivascular fibrosis, the extracellular matrix around each vessel was selected and threshold analysis was applied. Interstitial fibrosis was calculated as the ratio between collagen area and the total area of the heart section. The collagen immediately surrounding each intramyocardial vessels was considered to represent perivascular collagen deposition, and perivascular fibrosis was expressed as the ratio between perivascular collagen area and luminal media area (4).

For antigen retrieval, slides were covered with sodium citrate buffer (10 mM, pH 6.0) and heated to 95°C for 20 min. Immunohistochemistry was performed using the Dako Envision horseradish peroxide-diaminobenzidine system (Dako, Carpinteria, CA) with primary antibodies to transforming growth factor (TGF)-β₁ (1:50 dilution, Abcam, Cambridge, MA) and 4-hydroxynonenal (4-HNE; 1:50 dilution, Abcam) and visualized under natural light with an EVOS FL auto microscope. Images were taken at multiple sites (at least seven) with a ×20 or ×4 objective and quantified with MetaMorph image-analysis software (Molecular Devices). Data are expressed as percentages of threshold area representing the ratio of positive (stained) area to total tissue area.

For immunofluorescence, 5-μm sections were stained with TRITC-conjugated wheat germ agglutinin (WGA) and antibodies to cleaved caspase-3 (1:50 dilution, Cell Signaling Technology, Danvers, MA), vimentin (1:50 dilution, Abcam), BrdU (1:100 dilution, Abcam), and DAPI. Primary antibodies were detected by the following Alexa fluorescent dye conjugates (Invitrogen, Carlsbad, CA): Alexa 647 and Alexa 488. Slides were observed with a Zeiss LSM 780 confocal microscope (Carl Zeiss, Jena, Germany), and pictures were taken with ×40 objective. To discriminate cardiomyocytes, tissue sections were stained with WGA and DAPI. WGA stains all cell membranes, allowing for a discrimination of myocytes based on cell size, with myocytes being the largest cells present in the myocardium. For the identification of fibroblasts, serial tissue sections were immunostained with vimentin and DAPI. Nuclei that did not colocalize with either WGA or vimentin were defined as other cell types. The percentage of each cell type was expressed as the ratio of corresponding cell numbers to total cell nuclei (DAPI-positive cells). BrdU staining was quantified as the number of BrdU-positive cells and the total cell number to calculate the proliferation index (BrdU-positive cells/total cell number).

TUNEL staining.

A TUNEL assay was performed in paraffin-embedded sections with a commercial apoptosis detection kit (In Situ Cell Death Detection Kit, POD, Roche, Indianapolis, IN) according to the manufacturer’s instructions. Hematoxylin was used as a counterstain. To evaluate the apoptosis index (number of apoptotic cells/total number of cells), 10 random heart fields/tissue section were captured at a ×20 objective using an EVOS FL auto microscope and counted using ImageJ software (version 1.43r, National Institutes of Health) (1).

Real-time PCR.

Total RNA was isolated from left ventricular tissues of mice by RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was synthesized from 2 μg RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Quantitative PCR was performed using LightCycler 480 Probes Master (Roche Applied Science, Indianapolis, IN) in the LightCycler 480 multiwell plate at a total volume of 12 μl following the manufacturer’s protocol. Fibroblast growth factor (FGF)-2, matrix metalloproteinase (MMP)-2, and β-actin mRNA was measured with predesigned primers and probe sets (IDT, Coralville, IA) as follows: FGF-2, sense primer 5′-GAAACACTCTTCTGTAACACACACACACTT-3′, antisense primer 5′-GTCAAACTACAACTCCAAGCAG-3′, and probe 5′-/56-FAM/CAGCCGTCC/ZEN/ATCTTCCTTCATAGCA/3IABkFQ/-3′; MMP-2 sense primer 5′-AACTTCACGCTCTTGAGACTT-3′, antisense primer 5′-GAATGCCATCCCTGATAACCT-3′, and probe 5′-/56-FAM/CACCCTTGA/ZEN/AGAAGTAGCTATGACCACC/3IABkFQ/-3′; and β-actin sense primer 5′-GACTCATCGTACTCCTGCTTG-3′, antisense primer 5′-GATTACTGCTCTGGCTCCTAG-3′, and probe 5′-/56-FAM/CTGGCCTCA/ZEN/CTGTCCACCTTCC/3IABkFQ/-3′. Relative levels of mRNA transcripts for FGF-2 and MMP-2 were quantified and normalized to β-actin mRNA. A total of three samples were run for each experimental group, and all PCR samples were in duplicate.

Western blot analysis.

Murine heart tissues and lysates from SH-SY5Y cells were homogenized and sonicated in RIPA buffer (Cell Signaling Technology) with protease inhibitor cocktail (Roche Applied Science). Phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO) were additionally added if needed. After 1 h of extraction at 4°C with rocking, insoluble material was removed by centrifugation. Supernatants were resolved by SDS-PAGE (50 μg protein/lane) and transferred to PVDF membrane (Bio-Rad). Blots were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 and developed with diluted antibodies for Bax (1:1,000 dilution), phospho-Akt (Ser⁴⁷³, 1:1,000 dilution), Akt (1:1,000 dilution), phospho-AMP-activated protein kinase (AMPK)α (Thr¹⁷², 1:1,000 dilution), AMPKα (1:1,000 dilution), phospho-glycogen synthase kinase (GSK)-3β (Ser⁹, 1:1,000 dilution), GSK-3β (1:1,000 dilution) (all Cell Signaling Technology) as well as Bcl-2 (1:500 dilution) and GAPDH (1:2,000 dilution) (all Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. Blots were developed with the ECL chemiluminescence system and captured on autoradiographic films. Films were scanned, and densitometric analysis of the bands was performed with ImageJ software (version 1.43r, National Institutes of Health).

Statistical analysis.

Values are expressed as means ± SD. The statistical significance of differences was assessed with Student’s t-test or ANOVA, as appropriate. Results were considered statistically significant for P < 0.05. Statistical analysis was performed with SPSS 15.0 software (Chicago, IL).

RESULTS

HNG increased the cardiomyocyte percentage in aging hearts.

As shown by immunofluorescence, the young mouse myocardium was comprised primarily of cardiac myocytes (48.0 ± 3.7%), with cardiac fibroblasts making up the largest portion of the nonmyocyte population (37.0 ± 8.1%) and other cells accounting for 17.0 ± 8.6%, in agreement with a previous study (2). In the old mouse heart, the percentage of cardiac myocytes was reduced to 39.8 ± 7.1% (P < 0.01), whereas fibroblasts increased to 43.9 ± 7.3% (P < 0.05) compared with young mice (Fig. 1, A, B, and D). In HNG-treated old mice, the percentage of cardiac myocytes increased to 44.3 ± 6.5% (P < 0.05) and fibroblasts decreased to 38.4 ± 7.6% (P < 0.05) compared with vehicle-treated old mice (Fig. 1, B–D), which were not significantly different from young mice. These findings indicated that HNG treatment increased the relative cardiomyocyte-to-fibroblast ratio in the aging heart. The percentage of other cell types did not change among these groups.

Fig. 1.

Immunostaining for different cell types in the myocardium. Representative photographs of myocardial sections (×40 objective) were stained with DAPI (blue) for nuclei, vimentin (cyan) for fibroblast, and wheat germ agglutinin (red) for cardiomyocytes in young (n = 3; A), old (n = 3; B), and humanin analog (HNG)-treated old mice (n = 5; C). D: quantitative distribution of cell types in the myocardium. All data are expressed as means ± SD. #P < 0.01; *P < 0.05.

HNG inhibited interstitial fibrosis and reduced fibroblast proliferation in the aging myocardium.

Picrosirius red staining revealed that interstitial collagen deposition increased significantly in aged mice compared with young mice (0.24 ± 0.06% vs. 0.02 ± 0.01%, P < 0.01; Fig. 2, A, B, and D). Upon HNG treatment, a reduction in cardiac interstitial collagen deposition was observed in aged mice (to 0.07 ± 0.03%, P < 0.05; Fig. 2, B–D). In perivascular fibrosis, there was no significant difference among groups.

Fig. 2.

Myocardial fibrosis and oxidative stress. A−C: representative images of interstitial myocardial fibrosis from young mice (n = 3; A), old mice (n = 3; B), and humanin analog (HNG)-treated old mice (n = 5; C) as determined by picrosirius red staining with a ×20 objective and examined under a polarized light. D: quantitative analysis of interstitial fibrosis as represented by percentage of threshold area. E−G: representative images of oxidative stress from young mice (n = 3; E), old mice (n = 3; F), and HNG-treated old mice (n = 5; G) as determined by 4-hydroxynonenal (4-HNE) staining with a ×4 objective. H: quantitative analysis of 4-HNE staining as represented by percentage of threshold area. All data are expressed as means ± SD. #P < 0.01; *P < 0.05.

To quantify proliferating cells and discriminate their types, tissue sections of aged mice were triple stained for antibodies against BrdU, vimentin, and TRITC-conjugated WGA (Fig. 3A). We observed no difference in the number of proliferating cells between the two groups of aging mice (Fig. 3B), whereas the percentage of cardiac fibroblast was lower in HNG-treated old mice (74.0 ± 17.1% vs. 60.2 ± 23.0%, P < 0.05; Fig. 3C). A higher percentage of other proliferating cell types was observed in HNG-treated hearts compared with vehicle-treated old mice (36.7 ± 24.5% vs. 23.6 ± 17.7%, P < 0.05; Fig. 3C). The number of proliferating cardiomyocytes is quite small in aging mice, and thus no difference in the percentage of proliferating cardiomyocytes was detected between the two groups.

Fig. 3.

5′-Bromo-2-deoxyuridine (BrdU) staining in the heart. A: representative confocal images of myocardial sections (×40 objective) stained with DAPI (blue), vimentin (cyan), BrdU (yellow), and wheat germ agglutinin (red) in vehicle-treated (n = 3) and HNG-treated old mice (n = 5). The solid arrow indicates BrdU-positive fibroblasts. The thick arrow indicates a BrdU-positive cardiomyocyte. B: quantification of the percentage of BrdU-positive cells in total cells in the mouse heart. C: quantification of cell types in BrdU-positive cells. All data are expressed as means ± SD. *P < 0.05.

HNG treatment reduced 4-HNE in the myocardium.

Membrane lipids are one of the primary targets of ROS. 4-HNE is one of the major membrane lipid peroxidation products (40). Immunohistological analysis of left ventricular cardiac tissue demonstrated that 4-HNE staining was low in HNG-treated aging hearts compared with young hearts (P < 0.05; Fig. 2, E, G, and H), indicating lower oxidative stress after HNG treatment. There was no significant difference between old mice treated with and without HNG (P = 0.07; Fig. 2, F–H).

HNG downregulated profibrotic cytokines and MMP-2 expression in the aging myocardium.

Immunostaining showed an increased TGF-β₁ expression in old compared with young mice (18.2 ± 9.1% vs. 7.0 ± 5.3%, P < 0.01). Decreased expression was observed in HNG-treated old mice compared with old vehicle-treated mice (3.8 ± 1.9%, P < 0.01; Fig. 4, A–D), which was not different from young mice. A similar significant decrease was detected in mRNA levels of FGF-2 (P < 0.05) and MMP-2 (P < 0.05), which was upregulated in aged compared with young mice and downregulated in HNG-treated old mice compared with vehicle-treated old mice (Fig. 4, E and F).

Fig. 4.

Profibrotic cytokines and matrix metalloproteinase (MMP)-2 expression in aging hearts. A−C: representative images of myocardial transforming growth factor (TGF)-β₁ staining in young (n = 3; A), old (n = 3; B), and humanin analog (HNG)-treated old mice (n = 5; C) (×20 objective). D: quantitative analysis of TGF-β₁ expression in hearts as represented by percentage of threshold area. mRNA was extracted from young (n = 2), old (n = 3), and HNG-treated old hearts (n = 3). E and F: mRNA expression of FGF-2 (E) and MMP-2 (F) was detected by quantitative RT-PCR, and quantitative analysis was done accordingly. All data are expressed as means ± SD or mean as appropriate. Each experiment was independently repeated at least three times. *P < 0.05.

HNG treatment inhibited myocardial apoptosis in aging mice.

TUNEL staining revealed a significant increase in the number of apoptotic cells in old mice with or without HNG treatment compared with young mice (both P < 0.01; Fig. 5, A–D). However, fewer apoptotic cells were observed in HNG-treated old mice compared with vehicle-treated old mice (13.1 ± 5.6% vs. 27.8 ± 7.3%, P < 0.01; Fig. 5D). Moreover, reduced expression of Bax and increased expression of Bcl-2 were observed in HNG-treated old mice compared with vehicle-treated old control mice, as shown by Western blot analysis (Fig. 5E), leading to an elevated ratio of Bcl-2 to Bax in HNG-treated old mice compared with vehicle-treated old control mice (P < 0.01; Fig. 5F). Taken together, all this evidence indicates blunted apoptotic activity in HNG-treated aging hearts. To discriminate apoptotic cell types, we performed triple staining with antibodies against the cleaved form of caspase-3, vimentin, and TRITC-conjugated WGA. Representative images of cardiomyocyte- or fibroblast-expressing cleaved caspase-3 are shown in Fig. 6A. Apoptosis, as assessed by cleaved caspase-3 staining, showed a similar significant trend as data described for TUNEL staining. More cleaved caspase-3-positive cells were detected in vehicle-treated old mice compared with HNG-treated mice (P < 0.05; Fig. 6B). The number of cardiomyocytes in apoptotic cells was lower in HNG-treated old mice (3.6 ± 2.9 vs. 9.3 ± 3.2/sample, P < 0.05), whereas no difference of fibroblast number was detected (Fig. 6C). Together, these results indicate a reduction in apoptotic cardiomyocytes after HNG treatment.

Fig. 5.

Humanin analog (HNG) treatment inhibited apoptosis in aging hearts. A−C: representative images of TUNEL staining (×40 objective) of the myocardium from young (n = 3; A), old (n = 3; B), and HNG-treated old mice (n = 5; C). D: quantitative analysis of TUNEL-positive cells in young, old, and HNG-treated old mice. E: total protein was extracted from young (n = 2), old (n = 3), and HNG-treated old hearts (n = 3). Expression of Bax and Bcl-2 were determined by Western blot analysis. F: quantification of the Bcl-2-to-Bax ratio normalized to old mice. All data are expressed as means ± SD or mean as appropriate. The experiment was independently repeated at least three times. #P < 0.01.

Fig. 6.

Immunostaining of cleaved caspase-3 in the myocardium. A: representative images of cleaved caspase-3 (green), wheat germ agglutinin (red), vimentin (cyan), and DAPI (blue) staining in mouse hearts. The solid arrow indicates a fibroblast expressing cleaved caspase-3. The thick arrow indicates a cardiomyocyte expressing cleaved caspase-3. B: quantification of cleaved caspase-3 positive cells in young (n = 3), old (n = 3), and old + humanin analog (HNG; n = 5) groups. C: quantitative distribution of cell types of apoptotic cells. All data are expressed as means ± SD. *P < 0.05.

HNG attenuated myocardial fibrosis with activation of the Akt/GSK-3β pathway.

To determine the molecular mechanism by which HNG inhibited myocardial fibrosis further, we investigated whether HNG influenced the Akt, GSK-3β, or AMPK pathway by Western blot analysis. As shown in Fig. 7, aging tended to upregulate total Akt, GSK-3β, and AMPK protein expression, and HNG further activated phospho-Akt and phospho-GSK-3β in aging hearts but had little effect on phospho-AMPK activation. We also treated SH-SY5Y cells with 100 μM HNG in DMEM supplemented with 10% FBS for the indicated time periods and confirmed that HNG upregulated Akt phosphorylation in a time-dependent manner in vitro (Fig. 7H). Taken together, these results suggest that HNG might exert its beneficial effect on aging heart through activation of the Akt/GSK-3β pathway.

Fig. 7.

Humanin analog (HNG) treatment activated the Akt/glycogen synthase kinase (GSK)-3β pathway in the aging heart. Total protein was extracted from young (n = 2), old (n = 3), and HNG-treated old hearts (n = 3), and phosphorylated (p-)Akt, Akt, p-AMP-activated protein kinase (AMPK)α, AMPKα, p-GSK-3β, GSK-3β, and GAPDH expression were detected by Western blot analysis (A). B−G: quantitative analysis of the p-Akt-to-Akt ratio (B), p-GSK-3β-to-GSK-3β ratio (C), p-AMPK-to-AMPK ratio (D), Akt-to-GAPDH ratio (E), GSK-3β-to-GAPDH ratio (F), and AMPKα-to-GAPDH ratio (G) as normalized to the old mouse group. H: total cell lysates from SH-SY5Y cells after 100 μM HNG treatment in DMEM supplemented with 10% FBS for the indicated time periods were immunoblotted using anti-p-Akt (Ser⁴⁷³) and anti-Akt (H). All data are expressed as mean or individual dots as appropriate. Each experiment was independently repeated at least three times. *P < 0.05.

DISCUSSION

This is the first study to demonstrate that exogenous HNG treatment attenuates myocardial fibrosis and apoptosis in aging mice. Our data further revealed that the cardioprotective effect of HNG is associated with activation of Akt/GSK-3β signaling as well as an alteration in proapoptotic factors. The results of the present study suggest a potential role for the mitochondria-derived peptide in cardioprotection in aging and potential therapeutic implications for aging-related cardiac disease.

In our study, we observed by picrosirius red staining a decreased interstitial collagen content in HNG-treated compared with vehicle-treated old mice, implying that HNG might have a beneficial effect on cardiac function in aging hearts. Consistent with previous findings (5, 27), we found that old mice expressed higher levels of TGF-β₁, a potent stimulator of collagen synthesis via fibroblasts, which played an important role in age-related fibrosis. The expression of FGF-2 and MMP-2, which had profibrotic effect in hearts, tend to increase in the aging heart, as confirmed by our study (19, 47). Similarly, we observed a downregulated expression of FGF-2 and MMP-2 mRNA in HNG-treated aging hearts, which further confirmed the inhibitory role of HNG in cardiac fibrosis.

Progressive loss of myocytes with age through the apoptotic or necrotic pathway is a major element of age-associated changes in cardiac physiology. Myocyte death and augmented afterload lead to hypertrophy of the remaining myocytes, proliferation of cardiac fibroblasts, and interstitial fibrosis (18). In the present study, we observed increased apoptotic cell numbers with a larger proportion of cardiomyocyte in aging mice, which was reversed after HNG treatment. Therefore, our results demonstrated that HNG treatment has the potential to rescue cardiomyocytes from apoptosis during aging. Previous studies have demonstrated that HN is endogenously expressed in the heart, with the greatest levels found in cardiomyocytes (34). Both in vitro and in vivo studies have confirmed its protective effect on cardiomyocytes from ROS-induced injury. HNG pretreatment has been shown to exert cardioprotection against ischemia-reperfusion injury by reducing myocardial infarct size and attenuating cardiac dysfunction by two research groups. Muzumdar et al. (34) revealed decreased cardiomyocyte apoptosis with HNG treatment under ROS injury in vitro, whereas Thummasorn et al. (41) reported reduced cardiac mitochondrial ROS levels, mitochondrial depolarization, and mitochondrial swelling in vivo with HNG pretreatment before ischemia-reperfusion injury. The protective effect of HNG on mitochondrial function was further confirmed in H9C2 cells stimulated by H₂O₂, in which HNG treatment reduced intracellular ROS and preserved mitochondrial membrane potential, ATP levels, and mitochondrial structure (25). ROS increase with aging and is a common mechanism in age-related disease. Although we did not find increased ROS in aging mice, as shown by 4-HNE staining, probably because of the small sample size, we demonstrated downregulation of ROS in HNG-treated old mice compared with young mice. Taken together, the protective effect of HNG under ROS injury may explain the benefit of HNG treatment observed in our study.

HN mediates its antiapoptotic effect through extracellular and/or intracellular pathways. HN activates the extracellular pathway by interacting with specific receptors such as G protein-coupled formylpeptide receptor like-1 or trimeric complex receptors (10, 12) and initiating different downstream signaling cascades, including phosphatidylinositol 3-kinase/Akt, JAK/STAT3, or ERK1/2 signaling pathways (24, 45). In our study, we found that administration of HNG activated the Akt pathway both in vivo and in vitro, in agreement with the antiapoptotic activity of HN. Activated Akt inactivates proapoptotic proteins, including Bax, Bad, Bim, Bid, and caspase-9 (6). Moreover, activated Akt directly phosphorylates GSK-3β at Ser⁹, which negatively regulates its kinase activity, inhibits the opening of mitochondrial permeability transition pores (21), and inhibits myocardial apoptosis and fibrosis in heart failure (16). As enhanced phosphorylation of GSK-3β was observed in HNG-treated old mice, GSK-3β might be the downstream target of Akt in our study. Previously, Wang et al. (44) reported increased phospho-Akt and decreased phospho-GSK-3β in a mouse model of intracerebral hemorrhage after HNG treatment, which is not consistent with our study, which showed enhanced phosphorylation of GSK-3β. This might be due to various signaling pathways that HN may activate on different organs and under different pathophysiological conditions. The intracellular pathway is determined by the binding of HN to proapoptotic proteins (Bax, Bid, and Bim), inactivating their function and thus attenuating apoptosis (7, 11, 30). The observed downregulation of Bax in HNG-treated old mice may be the combined result of activation of the Akt/GSK-3β pathway by HN and inactivation of Bax function through direct binding with HN. We also found increased Bcl-2 expression in HNG-treated old mice, which has also been demonstrated in the neuroprotective effects of HN (44). Moreover, we examined if the AMPK pathway was activated after HNG, as activation of this pathway was associated with longevity and Muzumdar et al. reported activation of this pathway after HNG treatment in myocardial ischemia and reperfusion model (9, 34). However, there was no significant change in phospho-AMPK between vehicle- and HNG-treated old mouse groups. The potential mechanism of the effect of HN on cardioprotection in cardiomyocytes is shown in Fig. 8.

Fig. 8.

Possible mechanism of the cardioprotective effect of humanin (HN) on the aging heart. In cardiomyocytes, HN mediates an antiapoptotic effect through the extracellular pathway [HN interacts with specific receptors and upregulates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, thus inactivating Bax and activating Bcl-2 and glycogen synthase kinase (GSK)-3β] and/or intracellular pathway (binding to Bax to inactivate its function). HN also reduces ROS production in cardiomyoytes and thus inhibits apoptosis. mPTP, mitochondrial permeability transition pore.

In the present study, we observed upregulated expression of Akt, GSK-3β, and AMPKα in aged mouse hearts compared with young mouse hearts. Increased Akt expression in aging hearts might lead to increased cardiomyocyte size, promotion of cell survival, and prevention of apoptosis (20, 28), and it might be the feedback of the body to the aging process to avoid degenerative processes. As a downstream target of Akt and an endogenous negative regulator of cardiac hypertrophy (13), GSK-3β may be upregulated by Akt as a part of feedback loops in aging. AMPK serves as a cellular energy sensor and maintains energy homeostasis in the heart (15), and its expression and its activity are upregulated in myocardial hypertrophy (23, 42). The increased expression of AMPKα might be related to an imbalance of energy metabolism and myocardial hypertrophy in aging heart.

The correlation between increasing collagen content in the myocardium and decreased diastolic function with aging has been confirmed (37). The beneficial effect of HN on inhibiting myocardial fibrosis in aging heart implicated its potential in treating age-related myocardial dysfunction. Additional studies are needed to prove the protective effect of HN on cardiac function during aging.

Study limitations.

We were not able to assess in vivo cardiac or perfusion function in this study. Therefore, we cannot make direct conclusion that attenuation of fibrosis after HNG treatment will lead to improvements in cardiac function. Moreover, it has been reported that the extent of fibrosis in hearts from aged C57BL/6N mice (the mice used in our experiment) is less than aged C57BL/6J mice (43), and this might lead to <1% fibrosis in aged mouse hearts in our experiment. Third, although the immunofluorescence pictures were taken by confocal microscope, it is possible that nuclei might be in a different frame than vimentin-positive extension. We think the possibility is small and should be similar between the groups.

Conclusion.

In summary, we demonstrated that treatment with a humanin analog attenuated myocardial fibrosis and apoptosis in aging heart. The mechanism involved was activation of the Akt/GSK-3β pathway. This study indicates that HNG may play a role in myocardial remodeling and may be a potential treatment for myocardial remodeling in aging hearts. Further studies are needed to confirm the cardiac functional benefits related to HNG treatment.

GRANTS

This work was supported by National Natural Science Foundation of China Grant 81570315 (to Q. Qin).

DISCLOSURES

Drs. Cohen, Yen, and Lerman are consultants for CohBar.

AUTHOR CONTRIBUTIONS

P.C. and A.L. conceived and designed research; H.M., K.Y., G.N., and S.B. performed experiments; Q.Q. and J.W. analyzed data; S.D. interpreted results of experiments; Q.Q. drafted manuscript; X.Z. revised manuscript; L.O.L., P.C., and A.L. edited and revised manuscript; A.L. approved final version of manuscript.