1,12-cyclic apelin-12 ameliorates Ang II and Apelin-13-induced cardiac hypertrophy by reducing mitochondrial oxidative damage

To the Editor: Pathological cardiac hypertrophy is commonly stimulated by stress or induced by cardiovascular diseases, such as hypertension and myocardial infarction. A sustained pathological hypertrophic response can lead to declines in diastolic and systolic functions and eventually develop heart failure. Angiotensin II (Ang II) can mediate cardiac hypertrophy through the angiotensin II type 1 receptor (AT1R).^[1] Angiotensin receptor-like 1 (APJ), a member of the seven-transmembrane G protein-coupled receptor (GPCR) family, has high homology with AT1R in amino acid sequence (identity: 27.56%, similarity: 74.28%) [Supplementary Figure 1A, http://links.lww.com/CM9/B887]. Apelin is the endogenous ligand of APJ. Published studies report that the Apelin/APJ system participates in various physiological processes of myocardial cells. In the previous study, we demonstrated that the Apelin-13/APJ system promotes the development of cardiac hypertrophy.^[2] Therefore, both Ang II/AT1R and Apelin/APJ may contribute to the development of cardiac hypertrophy.

Based on the amino acid sequence of the natural ligand Apelin-13, an artificial linear polypeptide F13A was synthesized by mutating the amino acid site at the end of the N-terminus. Our previous studies found that the artificial linear polypeptide F13A can antagonize the APJ receptor and significantly inhibit Apelin-13-induced cardiomyocyte hypertrophy.^[3] Peptide molecules have many characteristics such as the degradation product of peptide is the amino acid with low biological toxicity.^[4,5] However, in the physiological environment, there are many exopeptidases including aminopeptidase and carboxypeptidase, which can easily degrade linear peptides. Linear peptide cyclization might be the most effective strategy to overcome the unstable defect of linear peptides.^[4,5]

1,12-Cyclic apelin-12 (cApelin-12) is an artificial cyclic peptide that is synthesized by connecting the first and 12th loci of the apelin-12 peptide chain head-to-tail in the form of an amide bond [Supplementary Figure 1B, http://links.lww.com/CM9/B887]. The amino acid sequence of cApelin-12 is cyclic (RPRLSHKGPMPF), where R: arginine, P: proline, L: leucine, S: serine, H: histidine, K: lysine, G: glycine, M: methionine, F: phenylalanine. We used Autodock (Scripps Research, La Jolla, California, USA) and Pymol (Schrodinger, New York, State of New York, USA) software for molecular docking to calculate the binding sites of cApelin-12 with AT1R and APJ receptors. The prediction results showed that cApelin-12 interacts with tyrosine-94, glutarnine-121, threonine-123, glycine-9, and leucine-11 on AT1R [Supplementary Figure 1C, http://links.lww.com/CM9/B887]. cApelin-12 also interacts with lysine-1046 and aspartic acid-1047 on APJ receptor [Supplementary Figure 1D, http://links.lww.com/CM9/B887]. These results imply that cApelin-12 can bind to AT1 and APJ receptors simultaneously.

To verify the role of cApelin-12 in Ang II and Apelin-13-induced cardiac hypertrophy, we constructed a mice model of cardiac hypertrophy by intraperitoneal injection of Ang II (1.44 mg·kg^–1·day^–1) and Apelin-13 (2 mg·kg^–1·day^–1), respectively [Supplementary Methods, http://links.lww.com/CM9/B887]. The protocols of animal experiments in the current study were approved by the Ethics Committee of the University of South China (No.2021[30]). The experiments were performed strictly abiding by international ethical guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Furthermore, we administered injections of cApelin-12 (1 mg·kg^–1·day^–1) and cApelin-12 (10 mg·kg^–1·day^–1) intraperitoneally 14 days after the initial injections [Supplementary Figure 2A, http://links.lww.com/CM9/B887]. During the experiment, we measured the body weight of all mice every four days. We found that Ang II, Apelin-13, and cApelin-12 had no significant effects on the body weight changes of mice [Supplementary Figure 2B, http://links.lww.com/CM9/B887]. Finally, all the experimental mice were sacrificed by cervical dislocation under anesthesia with avertin (tribromoethanol) (0.2 mL/10 g) and the isolated heart tissue was obtained. There was a hypertrophic myocardial area in hematoxylin and eosin (HE) staining in the myocardial tissue treated with Ang II, which was attenuated in the mice treated with Ang II + cApelin-12 [Figure 1A]. As was expected, treatment with cApelin-12 in the Apelin-13-induced mice showed significant improvement in gross morphology, which was observed in HE staining [Figure 1A]. We compared the heart weight and tibia length between groups and calculated the ratios of heart weight to tibia length (HW/TL) and heart weight to body weight (HW/BW) for each group. We found a hypertrophic phenotype in the Ang II treatment group, wherein HW/TL and HW/BW were significantly increased. However, the tissue collected from the cApelin-12 treated mice had attenuated Ang II-induced cardiac hypertrophy [Figure 1B]. Similarly, tissues treated with cApelin-12 had attenuated Apelin-13-induced increases in HW/TL and HW/BW in mice [Figure 1C].

Figure 1.

1,12-cyclic apelin-12 inhibits cardiac hypertrophy induced by Angiotensin II (Ang II) and Apelin-13. (A) Representative transverse sections stained with HE staining (scale bar, 2 mm) (L.D indicates low concentration, H.D indicates high concentration). (B–C) Bar graphs showing quantitative data for HW/BW and HW/TL. Data are presented as mean ± SEM (n = 5 per group. ^*P <0.05, ^†P <0.01, n.s. mean no significant difference). (D–E) Immunofluorescence staining of Phalloidin was performed to evaluate the surface area of HL-1 cells (scale bar, 30 μm). HE: Hematoxylin and Eosin; HW/BW: Ratio of heart weight to body weight; HW/TL: Ratio of heart weight to tibia length; SEM: Standard error of means.

We further explored the potential association of cApelin-12 with cardiomyocyte hypertrophy in vitro. We used Cell Counting Kit-8 (CCK8) cell assay to detect its cytotoxicity. The results showed that cApelin-12 at concentrations of 0.001 µmol/L, 0.01 µmol/L, 0.1 µmol/L, 1 µmol/L, and 10 µmol/L had no significant cytotoxicity after 24 h [Supplementary Figure 3A, http://links.lww.com/CM9/B887]. We labeled the cardiomyocyte cytoskeleton with phalloidin, and determined the cytoskeleton size of cardiomyocytes. Phalloidin is a bicyclic peptide that specifically binds to F-actin [Supplementary Methods, http://links.lww.com/CM9/B887]. Results from immunofluorescence demonstrated that Ang II (10^–8 mol/L, 24 h) promoted cardiomyocyte hypertrophy in HL-1 cells, whereas cApelin-12 (1 µmol/L, 24 h) reversed Ang II-induced cardiomyocyte hypertrophy [Figure 1D]. Likewise, we found that cApelin-12 (1 µmol/L, 24 h) significantly blocked the Apelin-13 (1 µmol/L, 24 h)-induced increase in the cytoskeleton size of cardiomyocytes [Figure 1E]. In addition, we did Western blotting analysis, showing that Ang II (10^–8 mol/L, 24 h) upregulated the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) while pretreatment with cApelin-12 (1 µmol/L, 24 h) significantly suppressed Ang II-induced upregulation of ANP and BNP in HL-1 cells [Supplementary Figure 3B, http://links.lww.com/CM9/B887]. Consistently, cApelin-12 also remarkably repressed Apelin-13-induced (1 µmol/L, 24 h) ANP and BNP expression in HL-1 cells [Supplementary Figure 3C, http://links.lww.com/CM9/B887].

Following the determination of the study of cApelin-12, we determined the diameter and volume of cardiomyocytes using the Scepter^TM Handheld Automated Cell Counter (Millipore, Billerica, Massachusetts, USA) [Supplementary Methods, http://links.lww.com/CM9/B887], and found that both Ang II and Apelin-13 induced the phenotype of cardiomyocyte hypertrophy. We observed that exposure of HL-1 cells to Ang II (10^–8 mol/L, 24 h) was associated with increases of the cell diameter and volume, whereas cApelin-12 (1 µmol/L, 24 h) inhibited the increase in diameter and volume induced by Ang II [Supplementary Table 1, http://links.lww.com/CM9/B887]. In addition, cApelin-12 (1 µmol/L, 24 h) treatment significantly inhibited the Apelin-13-stimulated (1 µmol/L, 24 h) increase in diameter and volume of HL-1 cardiomyocytes [Supplementary Table 2, http://links.lww.com/CM9/B887]. Overall, all these above findings confirmed that both Ang II and Apelin-13 promote cardiomyocyte hypertrophy, but cApelin-12 inhibits cardiomyocyte hypertrophy induced by Ang II and Apelin-13.

Substantial evidences suggest that mitochondrial function and mitochondrial reactive oxygen species (Mito-ROS) levels may be altered in the process of cardiac hypertrophy.^[6] Previously, we reported that Apelin-13 improved Mito-ROS production, leading to cardiomyocyte hypertrophy.^[3] In that study, mitochondria-targeted superoxide dismutase mimetic with superoxide and alkyl radical scavenging properties (Mito-TEMPO) was used to verify the effects of Ang II and Apelin-13 on Mito-ROS production [Supplementary Methods, http://links.lww.com/CM9/B887]. Nonetheless, we used the Mito-SOX red mitochondrial superoxide indicator (5 µmol/L, 10 min) to detect Mito-ROS levels in the current study since it was validated in our previous studies as well. Immunofluorescence staining showed that Ang II (10^–8 mol/L, 24 h) promoted the level of Mito-ROS in HL-1 cells, and Mito-TEMPO reversed this Ang II-induced effect [Supplementary Figure 4A, http://links.lww.com/CM9/B887]. Moreover, Apelin-13 (1 µmol/L, 24 h) promotes the level of Mito-ROS in cultured HL-1 mouse cardiomyocyte cell line (HL-1) cardiomyocytes. Mito-TEMPO blocked this Apelin-13-induced effect [Supplementary Figure 4B, http://links.lww.com/CM9/B887]. Then we found that cApelin-12 also presented similar effects as Mito-TEMPO. cApelin-12 (1 µmol/L, 24 h) inhibited Ang II-induced (10^–8 mol/L, 24 h) Mito-ROS levels in HL-1 cardiomyocytes [Supplementary Figure 4C, http://links.lww.com/CM9/B887]. In the meantime, cApelin-12 (1 µmol/L, 24 h) inhibited Mito-ROS levels induced by Apelin-13 (1 µmol/L, 24 h) in HL-1 cells [Supplementary Figure 4D, http://links.lww.com/CM9/B887].

A decrease in mitochondria membrane potential is one of the early markers of mitochondrial damage. Mitochondrial membrane potential (MMP) JC-1 staining is widely used to monitor mitochondrial health [Supplementary Methods, http://links.lww.com/CM9/B887]. JC-1 dye can be used as an indicator of MMP in cells. In this study, we found that Ang II (10^–8 mol/L, 24 h) induced the downregulation of MMP, while cApelin-12 (1 µmol/L, 24 h) inhibited the downregulation of MMP induced by Ang II [Supplementary Figure 4E, http://links.lww.com/CM9/B887]. cApelin-12 (1 µmol/L, 24 h) also inhibited the downregulation of MMP induced by Apelin-13 (1 µmol/L, 24 h) [Supplementary Figure 4F, http://links.lww.com/CM9/B887]. Furthermore, representative transmission electron microscope (TEM) images indicated that Ang II (10^–8 mol/L, 24 h) destroyed the mitochondrial structure which appeared as vacuoles (see the red arrows in the Supplementary Figures 4G–H, http://links.lww.com/CM9/B887), and cApelin-12 (1 µmol/L, 24 h) alleviated this destruction stimulated by Ang II [Supplementary Figure 4G, http://links.lww.com/CM9/B887]. TEM results showed that cApelin-12 (1 µmol/L, 24 h) also alleviated Apelin-13-induced (1 µmol/L, 24 h) mitochondrial structural damage [Supplementary Figure 4H, http://links.lww.com/CM9/B887]. In summary, all these results indicated that cApelin-12 can inhibit Mito-ROS production and alleviate mitochondrial damage induced by Ang II and Apelin-13.

Pathological cardiac hypertrophy is one of the main predictors and inducers of heart failure. However, current therapies available for patients with cardiac hypertrophy, such as angiotensin-converting enzyme (ACE) inhibitors and β-blockers, do not fully meet the clinical needs. Therefore, it is invaluable to identify novel protective agents to help develop better preventive and therapeutic strategies. In this study, we demonstrated that cApelin-12 could significantly inhibit the hypertrophy of cardiomyocytes induced by Ang II and Apelin-13 in vivo and in vitro. Moreover, we delineated that the mechanism regarding how cApelin-12 alleviates Ang II and Apelin-13-induced cardiac hypertrophy via modulation of Mito-ROS levels and mitochondrial oxidative damage.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 81970431) and the Hunan Provincial Natural Science Foundation of China (No. 2023JJ50136).

Conflicts of interest

None.