Exercise-induced circular RNA circUtrn is required for cardiac physiological hypertrophy and prevents myocardial ischaemia–reperfusion injury

Abstract

Aims

Regular exercise training benefits cardiovascular health and effectively reduces the risk for cardiovascular disease. Circular RNAs (circRNAs) play important roles in cardiac pathophysiology. However, the role of circRNAs in response to exercise training and biological mechanisms responsible for exercise-induced cardiac protection remain largely unknown.

Methods and results

RNA sequencing was used to profile circRNA expression in adult mouse cardiomyocytes that were isolated from mice with or without exercise training. Exercise-induced circRNA circUtrn was significantly increased in swimming-trained adult mouse cardiomyocytes. In vivo, circUtrn was found to be required for exercise-induced physiological cardiac hypertrophy. circUtrn inhibition abolished the protective effects of exercise on myocardial ischaemia–reperfusion remodelling. circUtrn overexpression prevented myocardial ischaemia–reperfusion-induced acute injury and pathological cardiac remodelling. In vitro, overexpression of circUtrn promoted H9 human embryonic stem cell–induced cardiomyocyte growth and survival via protein phosphatase 5 (PP5). Mechanistically, circUtrn directly bound to PP5 and regulated the stability of PP5 in a ubiquitin–proteasome-dependent manner. Hypoxia-inducible factor 1α–dependent splicing factor SF3B1 acted as an upstream regulator of circUtrn in cardiomyocytes.

Conclusion

The circRNA circUtrn is upregulated upon exercise training in the heart. Overexpression of circUtrn can prevent myocardial I/R-induced injury and pathological cardiac remodelling.

Graphical Abstract

Exercise, Circular RNA, Physiological Cardiac Hypertrophy, circUtrn, Protein phosphatase PP5

Time of primary review: 27 days

1. Introduction

Cardiovascular disease is one of the leading causes of death worldwide.¹ Regular exercise training leads to cardiovascular health benefits and effectively reduces the risk of cardiovascular disease, thereby improving cardiovascular function and quality of life, finally leading to reduced morbidity and mortality in patients with cardiovascular diseases.^2,3 Exercise can significantly reduce the impact of risk factors of cardiovascular disease by improving cardiac metabolism, reducing blood pressure, improving chronic inflammation, and increasing cardiovascular resistance to damage.^4–7 Investigating the therapeutic potential of exercise-induced molecular factors provides a new therapeutic approach for heart diseases.^2,8,9

Exercise can induce physiological cardiac hypertrophy.^4,10 Several studies have suggested that some of the best-characterized exercise-induced physiological cardiac hypertrophy regulators, such as C/EBPβ, miR-222, CPhar, lncExACT1, CITED4, and METTL14, can promote cardiomyocyte healthy hypertrophy, renewal, and survival.^11–16 However, the understanding of molecular mechanisms responsible for exercise-induced cardiac hypertrophy is relatively limited. Loss of cardiomyocytes is one of the major causes of heart failure, and hence reducing cardiomyocyte damage is an important approach to improve the therapeutic effect and prognosis of patients with heart diseases.^17,18 Targets identified from exercised hearts can promote the capacity of cardiac renewal in adult mice and resist cardiomyocyte apoptosis to protect the heart from pathological cardiac remodelling.^19,20 Investigating the biological mechanisms underlying exercise-induced cardiac protection would have important implications for both biological fundamental studies and potential drug development.

Circular RNA (circRNA) is a class of newly identified non-coding RNAs and ubiquitously expressed in many mammalian tissues. circRNAs are generated by back-splicing and play pivotal roles in the regulation of biological functions in many human diseases.^21,22 In the heart, abnormal changes of circRNAs have been found to be closely associated with cardiac diseases.^23–32 However, the role of cardiac circRNAs in response to exercise training remains largely unknown. Here, we identified an exercise-induced circRNA circUtrn, which was significantly increased in cardiomyocytes of swim-trained adult mice. In vivo, circUtrn was found to be required for exercise-induced physiological cardiac hypertrophy. circUtrn inhibition abolished the protective effects of exercise on ischaemia–reperfusion (I/R) remodelling. circUtrn overexpression (OE) prevented myocardial I/R-induced acute injury and pathological cardiac remodelling. In H9 human embryonic stem cell–induced cardiomyocytes (hESC-CMs), OE of circUtrn promoted hESC-CM growth and survival via protein phosphatase 5 (PP5). Mechanistically, circUtrn directly bound to PP5 and regulated the stability of PP5 in a ubiquitin–proteasome-dependent manner to activate MAPK/ERK signalling. Hypoxia-inducible factor 1α–dependent splicing factor SF3B1 acted as an upstream regulator of circUtrn in cardiomyocytes. This study provides new data into the regulation of cardiac circRNA in response to exercise, extending our fundamental knowledge and mechanistic insight about the benefits of exercise on cardiovascular health.

2. Methods

Detailed methods are provided in the Supplementary methods online.

2.1 Animal care and use

All animal experiments were conducted in accordance with the guidelines on the use and care of laboratory animals for biomedical research published by the National Institutes of Health (No. 85-23, revised 1996) and approved by the Committee on the Ethics of Animal Experiments of Shanghai University (No. 2019042). Adult male (8–9 weeks) C57BL/6J mice were purchased from Charles River Laboratories (Beijing, China).

To establish swimming exercise-induced physiological cardiac hypertrophy, adult C57BL/6J male mice were swim trained (or sedentary control) twice a day in a water tank for 4 weeks as reported previously.^11,16 Briefly, mice were randomized into two different groups, which were injected with adeno-associated virus serotype 9 (AAV9)–mediated circUtrn shRNA (AAV9-sh-circUtrn) (10¹³ vg/mL, 30 μL) or AAV9-scrambled control (10¹³ vg/mL, 30 μL) via the tail vein a week before swim training. Training began for a duration of 10 min at the first time and was increased to 10 min twice a day interval, until the training maximum of 90 min twice a day had been reached. The mice were closely observed at all times to avoid relative hypoxia during this 4-week exercise training. After the last swim, mice were sacrificed, and left ventricular samples were harvested for further analysis.

To study the role of circUtrn OE in myocardial I/R injury, mice were randomized to two different groups, which underwent tail vein injection of circUtrn OE AAV9 (AAV9-OE-circUtrn) (10¹³ vg/mL, 30 μL) or AAV9-Control (10¹³ vg/mL, 30 μL), before surgery or a sham operation. To establish a myocardial I/R injury mouse model, surgeries were performed under 2.0% isoflurane anaesthesia and using a ventilator to acquire passive respiration. After the left thoracic cavity opened, the left anterior descending (LAD) was ligated with a 7–0 silk for 30 min and was reperfused before the thoracic cavity was closed. Sham-operated mice underwent the procedure without actual LAD ligation. For acute I/R remodelling, after 24-h reperfusion, further analysis was performed. To determine the severity of myocardial I/R injury, 1% Evans blue was injected into the hearts and 2 mm slices of the heart were obtained. This was followed by exposure to 4% paraformaldehyde for 15 min and subsequent dyeing with a 1% triphenyltetrazolium chloride (TTC) solution in a water bath at 37°C. Infarct size (INF) was defined as the area with no staining of TTC or Evans blue while the area at risk (AAR) was defined as the area with staining of TTC but unstaining with Evans blue. INF/AAR ratio was obtained for evaluating the success of surgery. All surgeries and analyses were performed by investigators blinded to the treatment. Mice were sacrificed via intraperitoneal sodium pentobarbital (60 mg/kg) for cardiac tissue collection.

2.2 Echocardiography

Mice were anaesthetized with 2% isoflurane, and the cardiac function parameters of each mouse were detected by Vevo 2100 Echocardiography (VisualSonics Inc, Toronto, Ontario, Canada) with a 30 MHz central frequency scan head. Left ventricular ejection fraction (LVEF) and fractional shortening (FS) were measured. The echocardiographer was blinded to the surgical procedure and group. All the echocardiography data were presented in Supplementary material online, Table S1 and S2.

2.3 H9 hESC-CM differentiation and culture

H9 human embryonic stem cell line (WiCell) was cultured in E8 medium (A1517001, Thermo Fisher, USA) on Matrigel-coated plates (354230, Corning, USA) and differentiated to cardiomyocytes as reported previously.^33,34 Briefly, cardiomyocyte differentiation medium was supplemented with human recombinant albumin (A9731, Sigma, USA) and L-ascorbic acid 2-phosphate (A8960, Sigma, USA) in RPMI1640 medium (72400047, Life Technologies, USA). Briefly, differentiation was induced using 4 μM CHIR99021 (S1263, Selleck, USA) for 2 days. On Day 2, the medium was changed to cardiomyocyte differentiation medium, supplemented with 5 μM IWP-2 (S7085, Selleck, USA) for another 2 days. On Days 4–6, cells were maintained in cardiomyocyte differentiation medium. Spontaneous beating of cardiomyocytes was observed on Day 8. Beating cardiomyocytes were grown on RPMI1640 medium supplied with B27 (17504-044, Thermo Fisher, USA) for further maturation. On Day 14, cardiomyocytes began to undergo metabolic screening and purification and thus were re-plated in RPMI1640 medium lacking glucose and containing Dl-lactate (L4263, Sigma, USA) instead. Finally, cardiomyocytes with high purity were used for in vitro experiments after differentiation and maturation until Day 30. To perform circUtrn gain- and loss-of-function assays, lentivirus was infected with hESC-CMs together with Polybrene. After 48–72 h, immunofluorescence staining and western blotting were performed.

2.4 Statistical analysis

All data are presented as mean ± SD. Analyses were presented using GraphPad Prism 8.0. The independent sample t-test was used for comparison between the two groups by SPSS 20.0 software. Two-way or three-way ANOVA with Tukey post hoc test or one-way was performed to compare multiple groups by GraphPad Prism 8.0. For one-way analysis, The Levene test was used to verify the homogeneity of variance, and the Bonferroni post hoc test or Dunnett T3 post hoc test was performed according to the results. A P-value of <0.05 was considered to indicate a statistically significant difference.

3. Results

3.1 Identification of circRNA expression in adult mouse cardiomyocytes in response to swim exercise training

Mice were trained for 4 weeks to establish exercise-induced physiological cardiac hypertrophy by a swim-training model. Subsequently, adult mouse cardiomyocytes were harvested, and RNA sequencing was performed to profile circRNA expression. A total of 1106 circRNAs were identified in cardiomyocytes from both groups with or without exercise training. To select potential candidates, an initial screen was performed based on two criteria: (i) circRNAs that were differentially expressed by more than 2.0-fold between control and swim samples [FDR (adjusted p-value) < 0.05] and (ii) circRNAs that were detected or non-detected in all biological replicate from the same group samples. As shown in Figure 1A and Supplementary material online, Table S3, 22 of the identified circRNAs were selected for further characterization. Among them, 10 were significantly upregulated while 12 were significantly downregulated. Next, we conducted reverse transcription-quantitative polymerase chain reaction (RT–qPCR) to validate the above RNA sequencing identified circRNAs’ expression in cardiomyocytes from swim and control mice hearts with another set of six independent biological replicates using divergent primers. Among them, as shown in Figure 1B, we successfully validated nine circRNAs (circRNA_1678, circRNA_1647, circRNA_1504, circRNA_0169, circRNA_1283, circRNA_0563, circRNA_0508, circRNA_1884, and circRNA_0191) that were consistently significantly regulated with RNA-sequencing data. To further understand the alteration of circRNA expression in response to other (patho-)physiological stimuli and select specific exercise-induced physiological cardiac hypertrophy-induced circRNAs, we analysed the expression of these nine circRNAs in four pathological cardiac models [transverse aortic constriction surgery (TAC 6 weeks), myocardial infarction (MI 3 weeks), I/R injury (3 weeks), and doxorubicin-induced cardiotoxicity (DOX 5 weeks)]. As demonstrated in Figure 1C and Supplementary material online, Figure S1, among them was circRNA_0191 (circBase ID: mmu_circ_0000144), which was specifically involved in exercise-induced cardiac growth, was derived from the exon 60–64 of Utrn gene at chromosome 10, was downregulated in all stimuli models, capturing our attention, and was thus named as circUtrn to undergo further investigation.

Figure 1

Identification of circRNA expression in adult mice cardiomyocytes in response to swim exercise training. (A) Heat map of differentially expressed circRNAs in adult mouse cardiomyocytes that were isolated from mice with or without exercise training [FDR (adjusted P-value) < 0.05, fold change > 2.0]. (B) RT–qPCR validation of the nine differentially regulated circRNA expressions in adult mouse cardiomyocytes isolated from control mice or swimming training mice (*P < 0.05 and **P < 0.01, n = 6/group). (C) RT–qPCR analysis of circRNA_0191 expression in nine pathological cardiac stimulus models (TAC 6 weeks, transverse aortic constriction surgery 6 weeks of induced pathological cardiac remodelling; MI 3 weeks, myocardial infarction 3 weeks of induced cardiac remodelling; I/R 3 weeks, I/R injury 3 weeks of induced cardiac remodelling; DOX 5 weeks, doxorubicin treatment 5 weeks of induced cardiotoxicity) (*P < 0.05 and **P < 0.01, n = 6/group). (D) PCR analysis of circUtrn using divergent and convergent primers against cDNA and genomic DNA. (E) Confirmation of the back-splicing junction site within circUtrn by Sanger sequence. (F) RT–qPCR analysis of circUtrn and its parent gene Utrn mRNA with or without RNase R treatment (**P < 0.01, n = 6/group). (G) RT–qPCR analysis of circUtrn (hsa_circ_0001648) and its parent gene Utrn mRNA in human AC16 cardiomyocytes treated with or without actinomycin D at the indicated time points (n = 6/group). (H) The relative abundance of circUtrn in adult mouse tissues (**P < 0.01, ns, non-statistically significant; n = 6/group). The asterisks represent significant differences compared with the expression in the heart. (I) RNA FISH of circUtrn in hESC-CMs. Scale bar: 10μm. (J) RT–qPCR analysis of circUtrn in adult mouse fibroblast isolated from control mice or swimming training mice (ns, non-statistically significant; n = 5/group). (K) The serum level of circUtrn in people with regular exercise training (**P < 0.01, ns, non-statistically significant; n = 8/group). Data are presented as means ± SD. (B, C, F, J, and H, t-test; K, one-way ANOVA followed by Bonferroni post hoc test).

Using the circBank database set and sequence alignment, we observed that circUtrn was highly conserved between humans (circBase ID: hsa_circ_0001648) and mice (see Supplementary material online, Figure S2). The linear host gene Utrn was not regulated in the adult mouse cardiomyocytes between control and swimming mice (see Supplementary material online, Figure S3). The specific PCR product was amplified by divergent primers from cDNA but not from genomic DNA (Figure 1D). Moreover, the back splicing junction site within circUtrn was confirmed by Sanger sequencing (Figure 1E). Furthermore, an RNA stability study revealed that circUtrn was much more resistant to RNase R and actinomycin D treatment than linear Utrn mRNA (Figure 1F and G). The relative expression of circUtrn in adult mouse tissues was further examined by RT–qPCR and suggested that circUtrn was highly abundant in the heart (Figure 1H). Furthermore, as demonstrated by RNA fluorescence in situ hybridization assay, circUtrn was mainly observed in the cytoplasm (Figure 1I). In isolated cardiac mouse fibroblasts deriving from mice that underwent swim exercise, the circUtrn expression level was not changed, indicating that circUtrn might play specificity roles in cardiomyocytes (Figure 1J). To validate the human relevance, we detected circulating circUtrn expression in the serum of basketball athletes during acute and long-term exercise training.³⁵ Stability of serum circUtrn was assessed as previously reported³⁶ as well as RNase R treatment (see Supplementary material online, Figure S4). The serum level of circUtrn in long-term exercise athletes was found to be dramatically increased compared with a control group, indicating a positive correlation between serum circUtrn level and endurance exercise (Figure 1K). Collectively, circUtrn is a conserved circRNAs, which is highly expressed in cardiac tissue, localized in the cytoplasm, and specifically upregulated in cardiomyocytes after exercise training.

3.2 circUtrn regulates hESC-CM hypertrophy and apoptosis

To examine the role of circUtrn in cardiomyocytes, we generated circUtrn OE and knockdown (circUtrn short hairpin RNA, sh-circUtrn) models. As shown in Figure 2A and B and Supplementary material online, Figure S5A, circUtrn OE and sh-circUtrn were able to modulate the expression of circUtrn in human AC16 cardiomyocytes, suggesting that circUtrn OE and sh-circUtrn took effects in human cardiomyocytes. Next, we performed gain- and loss-of-function studies in H9 hESC-CMs.^33,34 Upon circUtrn OE, the cell size of hESC-CMs increased at the basal level, while circUtrn knockdown reduced it (Figure 2C). Besides, circUtrn OE increases the DNA replication (EdU staining) and cell cycle activity (Ki67 staining and pHH3 staining) of hESC-CMs while circUtrn knockdown decreases it (Figure 2C and D and Supplementary material online, Figure S5B). Furthermore, circUtrn OE ameliorated oxygen-glucose deprivation/reperfusion (OGD/R)-induced hESC-CM apoptosis, as suggested by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. Inhibition of circUtrn expression via aggregation of sh-circUtrn OGD/R-treatment led to hESC-CM apoptosis (Figure 2E). Bcl2-associated X protein (Bax) and B-cell leukaemia/lymphoma 2 (Bcl2) proteins have been reported to play important roles in cell apoptosis. Bax is a pro-apoptotic regulator while Bcl2 is an anti-apoptotic regulator.³⁷ Regulatory effects of circUtrn on cardiomyocyte apoptosis was also confirmed by western blot of Bax/Bcl2 in hESC-CMs (Figure 2F). Taken together, these data indicate that circUtrn is sufficient to modulate cardiomyocyte growth and survival.

Figure 2

circUtrn regulates hESC-CM hypertrophy and apoptosis. (A) Verification of human circUtrn (hsa-circUtrn) OE in human AC16 cardiomyocytes by using RT–qPCR (*P < 0.05, n = 6/group). (B) RT–qPCR analysis of human circUtrn shRNA (hsa-sh-circUtrn) knockdown efficiency in human AC16 cardiomyocytes (**P < 0.01, ns, non-statistically significant; n = 6/group). (C) Representative images of immunofluorescence staining and quantification of the relative cell size and Ki67-positive ratio of hESC-CMs transfected with or without hsa-circUtrn or hsa-sh-circUtrn treatment (**P < 0.01, n = 6/group. Scale bar: 100 μm. Magnification scale bar: 20 μm). (D) Representative images of immunofluorescence staining and quantification of the relative 5-ethynyl-2′-deoxyuridine (EdU)-positive ratio of hESC-CMs transfected with hsa-circUtrn OE or hsa-sh-circUtrn treatment (**P < 0.01, n = 6/group. Scale bar: 100 μm. Magnification scale bar: 20 μm). (E) Representative images of immunofluorescence staining and quantification of the relative TUNEL-positive ratio of hESC-CMs transfected with hsa-circUtrn OE or hsa-sh-circUtrn knockdown at indicated group (*P < 0.05 and **P < 0.01, n = 6/group. Scale bar: 100 μm). (F) Western blot analysis of Bax/Bcl2 in hESC-CMs at indicated groups (**P < 0.01, n = 6/group). Hsa, human. pLO5-ciR, control without circUtrn overexpression; circUtrn OE, circUtrn overexpression; shScramble, Scramble short hairpin RNA; sh-circUtrn, short hairpin RNA to knockdown circUtrn. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; OGD/R, oxygen-glucose deprivation/reperfusion. Data are presented as means ± SD. (A, B, C, and D, t-test; E and F, two-way ANOVA with Tukey post hoc test).

3.3 circUtrn is required for exercise-induced physiological cardiac hypertrophy

To further explore the in vivo effects of circUtrn elevation in the heart during exercise-induced physiological cardiac hypertrophy, we generated AAV9-sh-circUtrn or control (AAV9-shScramble), which was subsequently tail vein injected into adult mice. One week after AAV9 administration, mice were randomly assigned to swim training or a sedentary (control) group. Duration of swim training was determined for 4 weeks, in order to induce physiological cardiac hypertrophy (Figure 3A). Expression of circUtrn was inhibited after AAV9-sh-circUtrn treatment in mouse hearts (Figure 3B). circUtrn knockdown suppressed the development of cardiac hypertrophy compared with AAV9-shScramble treatment mice as measured by cardiac morphology, heart weight (HW), and HW/tibia length (HW/TL) (Figure 3C). We next performed wheat germ agglutinin (WGA) staining and found that knockdown of circUtrn reduced the cardiomyocyte cell cross-sectional area in exercised hearts compared with hearts from mice that underwent AAV9-shScramble treatment (Figure 3D). Furthermore, cardiomyocytes of mice that were exercised and treated with AAV9-sh-circUtrn revealed smaller expression levels of proliferation markers as demonstrated by α-actinin co-immunofluorescent staining with EdU, Ki67, and pHH3 (Figure 3E and Supplementary material online, Figure S6). In summary, these findings indicate that circUtrn is required for the development of exercise-induced physiological cardiac hypertrophy.

Figure 3

circUtrn is required for exercise-induced physiological cardiac hypertrophy. (A) The schedule of AAV9 injection and swimming training–induced physiological cardiac hypertrophy model establishment. (B) RT–qPCR analysis of circUtrn expression in mice hearts at indicated treatment group (**P < 0.01, n = 12/group). (C) Cardiac morphology, HW, and HW/TL of mice hearts at indicated treatment group (**P < 0.01, n = 12/group). (D) Representative images of immunofluorescence staining and quantification of WGA staining in mice hearts at indicated treatment (**P < 0.01, n = 6/group. Scale bar: 20 μm). (E) Representative images of immunofluorescence staining and quantification of EdU-positive and Ki67-positive cardiomyocytes in mice hearts at indicated treatment (**P < 0.01, n = 6/group. Scale bar: 20 μm). AAV9-shScramble, Scramble AAV9; AAV9-sh-circUtrn, circUtrn knockdown AAV9. Data are presented as means ± SD. (B, C, D, and E, two-way ANOVA with Tukey post hoc test).

3.4 circUtrn OE protects from acute myocardial I/R injury

To explore if the protective effects of circUtrn elevation on cardiomyocyte apoptosis are also present at the in vivo animal level, RT–qPCR revealed that circUtrn was downregulated in hearts of acute myocardial I/R injury (see Supplementary material online, Figure S7A). We subsequently generated AAV9-OE-circUtrn and administrated these to adult mice via a tail vein injection; 7 days after viral exposure, animals received acute myocardial I/R injury (30-min coronary ligation and 24-h reperfusion) (see Supplementary material online, Figure S7B). OE efficiency of AAV9-OE-circUtrn treatment in mouse hearts was confirmed by RT–qPCR (see Supplementary material online, Figure S7C). Hearts of mice with circUtrn OE demonstrated a significant reduction in infarct size upon acute I/R injury hearts, as evidenced by TTC staining (see Supplementary material online, Figure S7D). In addition, circUtrn OE attenuated acute I/R injury-induced apoptosis as indicated by TUNEL staining and western blot analyses (Bax/Bcl2) (see Supplementary material online, Figure S7E and F). These data show that circUtrn OE decreases acute I/R injury via alleviation of myocardial infarct size and apoptosis.

3.5 circUtrn OE prevents I/R-induced pathological cardiac remodelling

We subsequently investigated the beneficial effects of circUtrn OE in I/R-induced pathological cardiac remodelling. We overexpressed circUtrn in mouse hearts. Seven days after AAV9 injection, I/R and sham surgery were conducted (Figure 4A). RT–qPCR confirmed OE efficiency of circUtrn in mouse hearts (Figure 4B). Cardiac echocardiography, 3 weeks after I/R surgery, suggested higher LVEF and FS in mouse hearts that overexpressed circUtrn compared with control mice (Figure 4C). WGA staining suggested that the appearance of a larger interstitial space among myocardial cells through adverse I/R remodelling was prevented by AAV9-OE-circUtrn administration (Figure 4D). In addition, circUtrn OE also attenuated I/R remodelling-induced cardiac fibrosis as evidenced by Masson trichrome staining (Figure 4E). Furthermore, expression of fibrosis-associated genes (Col1a1, Col3a1, Acta2, and Postn) and hypertrophic genes (Nppa and Nppb) upon I/R-induced remodelling was reduced after circUtrn OE (Figure 4F). Collectively, circUtrn OE improves I/R remodelling-induced cardiac dysfunction and alleviates cardiac fibrosis.

Figure 4

circUtrn OE prevents I/R-induced pathological cardiac remodelling. (A) The schedule of AAV9 injection and I/R remodelling model establishment. (B) RT–qPCR analysis of circUtrn expression in mice hearts treatment with sham operation or I/R surgery (*P < 0.05 and **P < 0.01, n = 10:10:14:13). (C) Representative echocardiographic images and EF and ventricular FS of mice injected with AAV9-circUtrn or AAV9-Control at 3 weeks after I/R surgery (**P < 0.01, n = 10:10:14:13) (scale bars, x: 0.1 s; y: 2 mm). (D) Representative WGA staining images quantification of fibrosis area (%) of mice injected with AAV9-OE-circUtrn or AAV9-Control at 3 weeks after I/R surgery (**P < 0.01, n = 6/group. Scale bar: 20 μm). (E) Representative Masson trichrome staining images quantification of fibrosis area (%) of mice injected with AAV9-OE-circUtrn or AAV9-Control at 3 weeks after I/R surgery (**P < 0.01, n = 6/group. Scale bar = 1 mm). (F) RT–qPCR analysis of fibrotic genes (Acta2, Col1a1, Col3a1, and Postn) and hypertrophic genes (Nppa and Nppb) expression in infarct region of mice hearts treatment with AAV9-OE-circUtrn followed by sham or I/R remodelling (3 weeks) (**P < 0.01, n = 6/group). I/R, ischaemia–reperfusion. AAV9-Control, control AAV9; AAV9-OE-circUtrn, circUtrn OE AAV9. Data are presented as means ± SD. (B, C, D, E, and F, two-way ANOVA with Tukey post hoc test).

3.6 circUtrn suppression inhibits protective effects of exercise on I/R-induced pathological cardiac remodelling

Benefits of exercise on cardiac performance have been well recognized.⁴ We thus aimed to determine whether circUtrn is critical for the protective effects of exercise on I/R-induced pathological cardiac remodelling. Figure 5A shows the experimental design. Briefly, 7 days after AAV9-sh-circUtrn or AAV9-shScramble administration via tail vein injection, adult mice were subjected to 4 weeks of swim training (vs. 4 weeks of sedentary control) to induce physiological cardiac hypertrophy. Then, mice were randomly assigned to I/R vs. sham surgery. Three weeks after I/R remodelling, cardiac function (as evaluated by LVEF and FS) in the swim-trained mice was significantly improved compared with the sedentary control group, while it was decreased in the AAV9-sh-circUtrn-treated mouse hearts (Figure 5B). Interestingly, compared with AAV9-shScramble-treated mice, AAV9-sh-circUtrn-treated mice of the sedentary control group presented a deterioration of cardiac function after 3 weeks of I/R remodelling, indicating an additional protective role for circUtrn in response to stress stimuli (Figure 5B). We then examined the expression of circUtrn and found that circUtrn was effectively knocked down in the AAV9-sh-circUtrn-treated mouse hearts (Figure 5C). The upregulation effect of swim exercise on circUtrn has returned to the baseline at sham 3 weeks after termination of swim exercise in sham group, while the RNA level was sustained at I/R 3 weeks in swim exercised heart compared with sedentary control heart (Figure 5C and Supplementary material online, Figure S8). Next, we assessed the burden of cardiac fibrosis using Masson trichrome staining and found that hearts from mice that underwent exercise training demonstrated decreased cardiac fibrosis, while AAV9-sh-circUtrn attenuated these protective effects (Figure 5D). Consistent with decreased cardiac function, AAV9-sh-circUtrn-treated mice of the sedentary control group developed severe cardiac fibrosis I/R during the remodelling period (Figure 5D). In addition, expression of fibrotic genes (Acta2, Col1a1, and Col3a1) was decreased in exercise-trained mice compared with sedentary control mice, whereas AAV9-sh-circUtrn treatment partially blunted these effects (Figure 5E). Taken together, these data indicate that exercise-induced circUtrn elevation is required for the protective effect of exercise on pathological cardiac remodelling in an in vivo model of I/R injury.

Figure 5

circUtrn suppression inhibits the protective effects of exercise on I/R-induced pathological cardiac remodelling. (A) The schedule of AAV9 injection, swimming-induced physiological cardiac hypertrophy, and I/R remodelling establishment. (B) Representative echocardiographic images and EF and ventricular FS of mice injected with AAV9-sh-circUtrn or AAV9-shScramble treatment at indicated groups (*P < 0.05 and **P < 0.01, n = 5:5:5:5:6:4:4:6) (scale bars, x: 0.1 s; y: 2 mm). (C) RT–qPCR analysis of circUtrn expression in mice hearts injected with AAV9-sh-circUtrn or AAV9-shScramble treatment at indicated groups (**P < 0.01, n = 5:5:5:5:6:4:4:6). (D) Representative Masson trichrome staining images quantification of fibrosis area (%) of mice injected with AAV9-sh-circUtrn or AAV9-shScramble treatment at indicated groups (**P < 0.01, n = 5:5:5:5:6:4:4:6. Scale bar = 1 mm). (E) RT–qPCR analysis of fibrotic genes (Acta2, Col1a1, and Col3a1) expression of mice injected with AAV9-sh-circUtrn or AAV9-shScramble treatment at indicated groups (*P < 0.05 and **P < 0.01, n = 5:5:5:5:6:4:4:6). AAV9-shScramble, Scramble AAV9; AAV9-sh-circUtrn, circUtrn knockdown AAV9. Data are presented as means ± SD. (B, C, D, and E, three-way ANOVA test with Tukey post hoc test).

3.7 circUtrn interacts with PP5 and promotes PP5 degradation in a ubiquitin–proteasome-dependent manner to activate MAPK/ERK signalling

We next investigated the underlying regulatory mechanism by which circUtrn is involved in cardiomyocyte signalling. Biotin-labelled linearized circUtrn sense and antisense strands were used to pull down binding partners from with protein extracts from mouse heart lysates. This was subsequently followed by mass spectrometry identification (Figure 6A). Potential candidate interacting proteins for circUtrn were then verified by western blot (Figure 6B and Supplementary material online, Figure S9A). Among them, serine/threonine PP5 was found to be specifically pulled down by the biotin-labelled linearized circUtrn sense strand, whereas it was not pulled down by the antisense strand. We then conducted endogenous circUtrn pull downs in AC16 cardiomyocytes using a probe designed to be specific to the junction site (antisense to the junction region, AS circUtrn probe) and its sense strand. The presence of enriched endogenous circUtrn was confirmed by RT–qPCR analysis with an AS circUtrn probe (Figure 6C and Supplementary material online, Figure S9B). Also, the specific binding of PP5 after the circUtrn–protein complex was pulled down by an AS circUtrn probe with protein extracts from AC16 cardiomyocytes was verified by immunoblot analysis (Figure 6D). In addition, a PP5 RNA immunoprecipitation (RIP) assay, followed by RT–qPCR, confirmed the endogenous binding of circUtrn to PP5 in AC16 cardiomyocytes (Figure 6E). Thus, PP5 could specifically bind to circUtrn in cardiomyocytes.

Figure 6

circUtrn interacts with PP5 and promotes PP5 degradation in a ubiquitin–proteasome-dependent manner to activate MAPK/ERK signalling. (A) Schematic diagram of RNA pull down and screen strategy. (B) Silver staining and western blot of biotin-labelled linearized circUtrn sense and antisense strands pulled down with protein extracts from mice heart lysates. (C) RT–qPCR analysis of endogenous circUtrn enrichment that was pulled down by circUtrn junction probe (antisense to junction region, AS circUtrn probe) or its sense control (**P < 0.01, n = 6/group). (D) Immunoblot analysis of PP5 after endogenous circUtrn–protein complex pulled down by circUtrn junction probe with protein extracts from AC16 cardiomyocytes. (E) RIP-qPCR verified the binding of circUtrn to PP5 (**P < 0.01, n = 6/group). Normal IgG was used as a negative control for IP. (F) Western blot of PP5 expression in hESC-CMs with or without circUtrn OE (**P < 0.01, n = 6/group). (G) Western blot analysis of the phosphorylation level of MAPK/ERK in hESC-CMs with or without circUtrn OE (**P < 0.01, n = 6/group). (H) Western blot analysis of PP5 protein stability with or without circUtrn OE at indicated time point. CHX, cycloheximide. (I) Co-immunoprecipitation of PP5 and ubiquitin in AC16 cardiomyocytes. (J) Blocking proteasome-mediated protein degradation with MG132 reversed the pro-degradation effect of circUtrn on PP5. (K) Western blot of biotin-labelled circUtrn-Region 1, circUtrn-Region 2, circUtrn-Region 3, and NC pull-down assay in AC16 cardiomyocytes. circUtrn-Region 1, Regions 25–105; circUtrn-Region 2, Regions 272–353; and circUtrn-Region 3, Regions 473–553; and NC, negative control. (L) Flag-tagged PP5 constructs were generated for validation of the direct binding between circUtrn and PP5 by RIP assay in AC16 cardiomyocytes (**P < 0.01, n = 6/group). pLO5-ciR, control without circUtrn overexpression; circUtrn OE, circUtrn overexpression; CHX, cycloheximide. Data are presented as means ± SD. (C, E, F, and L, t-test; G, two-way ANOVA with Tukey post hoc test).

PP5 is a serine/threonine protein phosphatase that dephosphorylates the phosphorylation site of serine and threonine. In the heart, PP5 has been found to be increased in failing hearts.³⁸ PP5 expression was found to be significantly decreased in mice with cardiac hypertrophy induced by swim training (see Supplementary material online, Figure S10A). Conversely, it was found to be elevated in mice that underwent I/R remodelling (I/R 3 weeks) (see Supplementary material online, Figure S10B). We then tested whether circUtrn can bind to PP5 and thereby regulate its expression. We found that circUtrn negatively regulates the expression of PP5 at the protein level in mouse hearts and in hESC-CMs, while no significant difference was observed at the Pp5 mRNA level (Figure 6F and Supplementary material online, Figure S1C and D). PP5 can dephosphorylate Raf1 and thereby regulate MAPK/ERK signalling, whereas ERK1/2 activation could inhibit cardiomyocyte apoptosis.^38–40 We then detected the phosphorylation level of Raf1 and ERK1/2 in circUtrn-overexpressed hESC-CMs and mouse hearts. Consistent with previously reported studies on the inhibitory effects of PP5, our data here indicated that the OE of circUtrn, which resulted in a decrease in PP5 level, led to a significant increase in the phosphorylation levels of Raf1 and ERK1/2 (Figure 6G and Supplementary material online, Figure S10E and F). Next, we used the protein synthesis inhibitor cycloheximide (CHX) to treat AC16 cardiomyocytes with or without circUtrn OE and determined the time course of PP5 degradation. As shown in Figure 6H, circUtrn OE dramatically reduced the stability of PP5. It is known that in kidney cancer, PP5 can be ubiquitinated by ligase with subsequent proteasomal degradation.⁴¹ We, therefore, examined whether the ubiquitin–proteasome pathway is involved in circUtrn-mediated PP5 degradation in cardiomyocytes. A PP5 co-immunoprecipitation assay suggested that ubiquitinated PP5 was increased in cardiomyocytes overexpressing circUtrn (Figure 6I). In addition, treatment with the proteasome inhibitor MG132 blunted circUtrn OE-mediated PP5 degradation (Figure 6J). Moreover, we performed an in silico analysis predicted PP5-circUtrn interface, as indicated by CatRAPID (see Supplementary material online, Figure S11). The predicted interaction was further confirmed by RNA pull-down assays in AC16 cardiomyocytes via biotin-labelled in vitro transcribed circUtrn fragments (circUtrn-Region 1, Regions 25–105; circUtrn-Region 2, Regions 272–353; and circUtrn-Region 3, Regions 473–553) (Figure 6K). Further, we generated Flag-tagged PP5 constructs and validated of the direct binding between circUtrn and PP5 by RIP assay in AC16 cardiomyocytes. The expression of Flag-tagged PP5 constructs was confirmed by western blot (see Supplementary material online, Figure S12). As shown in Figure 6L, only the intact PP5 construct can sufficiently bind circUtrn while all the other mutant constructs cannot immunoprecipitation circUtrn, suggesting the essential role of intact PP5 to interaction with circUtrn. Taken together, circUtrn binds to PP5 in cardiomyocytes and promotes PP5 degradation in a proteasome-dependent manner to activate the MAPK/ERK signalling pathway.

3.8 Forced expression PP5 reversed the regulatory effects of circUtrn on hESC-CMs

We further explored whether PP5 participates in the regulatory role of circUtrn in hESC-CMs. A PP5 OE construct was generated and the transfection efficiency was confirmed by RT–qPCR (see Supplementary material online, Figure S13A). PP5 OE led to reduced cardiomyocyte size and decreased expression of DNA replication (EdU) and cell cycle activity–related markers (Ki67 and pHH3) at the basal level (Figure 7A and B and Supplementary material online, Figure S13B). Importantly, observed pro-hypertrophy and pro-cell cycle entry effects of circUtrn on hESC-CMs were reversed by PP5 OE (Figure 7A and B and Supplementary material online, Figure S13B). Moreover, PP5 OE aggravated OGD/R-induced hESC-CMs apoptosis and decreased the protective effect of circUtrn OE on cardiomyocyte apoptosis (Figure 7C). These data suggest that PP5 acts downstream of circUtrn mediating its role to regulate hESC-CMs growth and apoptosis.

Figure 7

Forced expression PP5 reversed the regulatory effects of circUtrn on hESC-CMs. (A) Representative images of immunofluorescence staining and quantification of the relative cell size and Ki67-positive ratio of hESC-CMs treatment at indicated groups (*P < 0.05 and **P < 0.01, n = 6/group. Scale bar: 100 μm. Magnification scale bar: 20 μm). (B) Representative images of immunofluorescence staining and quantification of the relative EdU-positive ratio of hESC-CMs treatment at indicated groups (*P < 0.05 and **P < 0.01, n = 6/group. Scale bar: 100 μm. Magnification scale bar: 20 μm). (C) Representative images of immunofluorescence staining and quantification of the relative the relative TUNEL-positive ratio of hESC-CMs at indicated groups (*P < 0.05 and **P < 0.01, n = 6/group. Scale bar: 100 μm). pLO5-ciR, control without circUtrn overexpression; circUtrn OE, circUtrn overexpression; Fugw, control without PP5 overexpression; PP5 OE, PP5 overexpression. Data are presented as means ± SD. (A and B, two-way ANOVA with Tukey post hoc test; C, three-way ANOVA test with Tukey post hoc test).

3.9 SF3B1 regulates circUtrn in cardiomyocytes

We sought to determine the key factor in regulating circUtrn in cardiomyocytes. We used RBPsuite to predict the potential splicing factors that can bind to circUtrn flanking inverted complementary sequences.⁴² Based on the binding predictive value (over 0.90), we identified three RNA splicing factors (SF3B1, SFPQ, and QKI) as circUtrn upstream regulators’ candidate (see Supplementary material online, Figure S14A). circUtrn was functional as an important regulator in exercise-induced physiological cardiac hypertrophy and I/R-induced pathological cardiac remodelling. Therefore, we first examined the expression of SF3B1, SFPQ, and QKI and found that only SF3B1 was both regulated in the murine hearts of exercise training mice and I/R 3 weeks (see Supplementary material online, Figure S14B and C). In the heart, SF3B1 is a HIF1α-dependent splicing factor and can be activated under trans-aortic constriction-induced pathological hypertrophy.⁴³ Oxidative metabolism in the heart is both regulated in the exercise training and I/R surgery. We then explored whether circUtrn was regulated by SF3B1 in cardiomyocytes. As shown in Supplementary material online, Figure S14D–F, knockdown of SF3B1 specifically promoted the expression of circUtrn without affecting its linear cognate. Further, we demonstrated that SF3B1 could bind to the flanking inverted complementary sequences of circUtrn (see Supplementary material online, Figure S14G). Next, we co-transfected siSF3B1 and sh-circUtrn into AC16 cardiomyocytes and found that the downregulation effect of circUtrn was partially rescued by SF3B1 knockdown, without affecting the linear cognate of circUtrn (see Supplementary material online, Figure S14H and I). Taken together, SF3B1 acts as an upstream regulator of circUtrn in cardiomyocytes.

4. Discussion

In this study using high-throughput RNA sequencing, we show for the first time that the circRNA circUtrn is specifically increased in cardiomyocytes of adult mice as a response to swim exercise training. Secondly, we show that circUtrn is required for the development of exercise-induced physiological cardiac hypertrophy in vivo. Thirdly, circUtrn OE protects the heart from acute myocardial I/R injury and I/R-induced remodelling; circUtrn is critical for the protective effects of exercise on I/R-induced remodelling. Fourthly, in an in vitro model, circUtrn OE promoted hESC-CMs growth and survival. Lastly, serine/threonine protein phosphatase PP5 was identified to directly bind to circUtrn, and conversely, circUtrn promotes PP5 degradation dependent on the ubiquitin–proteasome system to activate the MAPK/ERK signalling pathway, giving important mechanistic insight.

Exercise-induced cardiac remodelling is involved in multiple cell types of the heart. In this study, we established the swim exercise-induced physiological cardiac hypertrophy model and isolated cardiomyocytes to profile the circRNA expression for in-depth mechanism investigation. Simultaneously regulating cardiomyocytes from multiple avenues (e.g. promote cardiomyocyte healthy hypertrophy, increase the activity of cell cycle, and resist cardiomyocyte apoptosis) to exert cardioprotection effects is one of the important features of key molecules identified from exercised hearts.^12,13,16 Similar to previous studies, here, we observed that circUtrn regulated hESC-CM size, the expression of cell cycle–related markers in hESC-CMs, and OGD/R-induced cardiomyocyte apoptosis. In vivo, circUtrn OE can prevent I/R remodelling-induced cardiac dysfunction and fibrosis. We examined the role of circUtrn in TGF-β1-treated adult mouse cardiac fibroblasts (MCFs). The expression of circUtrn was significantly decreased in TGF-β1-treated MCFs. As shown in Supplementary material online, Figure S15, TGF-β1 treatment led to the trans-differentiation of fibroblast to myofibroblast as well as the increased expression of Col3a1, Col1a1, and Postn; however, circUtrn OE or knockdown does not affect TGF-β1-induced myofibroblast differentiation. Exercise training can reduce cardiac fibrosis in diseased and aging hearts in different ways.^4,7 Firstly, it can directly inhibit fibroblast activation and trans-differentiation, which suppresses fibrosis.⁴⁴ Secondly, exercise can promote cardiomyocyte hypertrophy and survival, which in turn inhibits fibrosis replacement, leading to a reduction in cardiac fibrosis.^13,15,16 In addition, it has been reported that injured cardiomyocytes can produce fibrotic factors to stimulate the activation of fibroblast.⁴⁵ Our observations here indicate that circUtrn cannot be directly involved in regulating fibrosis in the heart. Thus, the observed effects of circUtrn on cardiac fibrosis in this study most probably came from the indirect effects of cardiomyocyte hypertrophy and survival instead of the direct contribution of circUtrn to fibroblast activation. Detailed regulatory mechanism to fully elucidate this beneficial effect of circUtrn on cardiac fibrosis requires further investigation.

Exercise training is pivotal for the prevention of cardiovascular diseases and the maintenance of cardiovascular health.^{2,4,13,46–48} Over the last years, many efforts have been made to deepen the understanding of the underlying mechanisms by which exercise leads to its cardiovascular benefits.^4,5 However, data regarding the underlying mechanism by which exercise protects the heart are scarce. Here, we studied the crucial role of circUtrn, as cardiomyocytes’ key circRNA to regulate exercise-induced physiological cardiac hypertrophy. Exercise-induced cardiac hypertrophy, as opposed to pathological hypertrophy, is physiological and can reverse pathological cardiac remodelling.^{11–13,49–51} In this study, we provide evidence that exogenously forced disruption of exercise-induced circUtrn would deteriorate the cardiac benefits of exercise. In addition to cardiomyocyte hypertrophy, angiogenesis, vascular adaptations, and improved cardiac metabolism also contributed to the physiological cardiac adaptations in response to physical exercise.^4,7,49 Thus, other cell types including endothelial cells may also contribute to the development of physiological cardiac hypertrophy. AAV9-mediated gene delivery in the heart has been widely used.^13,52–54 In this study, we utilized a mammalian ubiquitous promoter CMV-driven promoter AAV9 for OE or a Pol III U6-driven promoter AAV9 for knockdown of circUtrn in vivo. As we did not employ a cardiomyocyte-specific promoter to restrict transgene expression solely to cardiomyocytes, we cannot exclude the possibility that the in vivo OE or knockdown of circUtrn in this study may have also affected other cell types within the heart, and the observed phenotype might be attributed to a combination of all these effects. Interestingly, we evaluated the effect of swim exercise as well as circUtrn on angiogenesis via CD31⁺ staining and VEGF expression. Our data suggest that exercise training significantly enhanced angiogenesis while circUtrn knockdown decreased it (see Supplementary material online, Figure S16). We then examined the direct role of circUtrn in human umbilical vein endothelial cells (HUVECs). Our data suggested that neither circUtrn OE nor knockdown can regulate HUVECs migration or proliferation (see Supplementary material online, Figure S17). These findings indicated that circUtrn cannot directly regulate endothelial cell, though circUtrn participates in regulating angiogenesis in the hearts. However, a detailed mechanism about the indirect role of circUtrn in angiogenesis remains to be uncovered. Moreover, other regulatory mechanisms might also exist in the heart and contribute to the circUtrn-mediated cardioprotection. Future studies focused on other cardiac cell types would offer a great opportunity to understand the regulatory mechanism of physiological cardiac hypertrophy.

circRNA is one of the non-coding RNAs, which has recently been identified and is associated with many biological functions and pathophysiological processes.²² In this study, using a mouse model, we explored the regulation of circRNAs in response to exercise training. By profiling RNA sequencing, we selected 22 differentially expressed circRNAs for further validation with divergent primers by RT–qPCR. Only nine of them had consistent expression with RNA-sequencing data. The differences that led to over 50% of the circRNAs were not validated might be attributed to a totally another different set of samples that were used for the RT–qPCR validation (includes exercise swimming training/control sedentary mice, isolate of adult mouse cardiomyocyte, and RNA isolation) from the samples that were used for RNA-sequencing analysis. Similarly, relatively low validation ratio has also been observed from the independent biological replicate’s validation by other reports.^32,55,56 Besides, the biological replicates in RNA sequencing (three biological replicates) were relatively less than RT–qPCR analysis (six biological replicates); the different number of biological replicates and different methodologies might also contribute to these observed differences. Regardless of the differences, we consistently identified a circRNA circUtrn that is conserved across human and mouse species. circUtrn is one of the exercise-induced circRNAs and is required for the cardiac benefits of exercise in response to I/R injury. Biological functions of circRNAs have been identified as miRNA sponges and protein sponges, and, in addition, circRNAs encode several functional peptides in the heart.^{23,24,26,28–30} The ubiquitin–proteasome system has been reported to regulate important signal transduction pathways such as MAPK, FOXO, and p53 signalling and plays a pertinent role in the pathophysiology of several heart diseases.⁵⁷ Here, we found that circUtrn directly binds to protein phosphatase PP5 to downregulate its expression via enhancement of the ubiquitin–proteasome system, thereby activating the MAPK/ERK signalling pathway to protect the heart from I/R injury. However, it should be noted that the evidence suggesting the promotion of cardiomyocyte growth and survival through degradation of PP5 and subsequent activation of RAF/MAPK/ERK signalling by circUtrn is primarily based on the observed association between circUtrn expression and ERK1/2 phosphorylation. To definitively establish this relationship, further studies involving loss- and gain-of-function experiments targeting components of the RAF/MAPK/ERK signalling pathway are required. In addition, it has been reported that von Hippel-Lindau protein (VHL) E3 ligase can interact with PP5, subsequently ubiquitinate, and degrade PP5 in the proteasome in kidney cancer.⁴¹ Interestingly, we also showed that treatment with the proteasome inhibitor MG132 abolishes circUtrn OE-mediated PP5 degradation in cardiomyocytes. This raises the possibility that circUtrn may regulate the interaction of PP5 with VHL. Further investigation is warranted to determine whether the interaction between PP5 and VHL exists in cardiomyocytes and, if so, what is the exact role of circUtrn in this regulation. Collectively, our data suggest a new mechanism by which the circRNA circUtrn regulates protein phosphorylation via the ubiquitin–proteasome system and modulates signal transduction at the post-translational level.

Protein phosphorylation is one of the most extensively studied post-translational modifications and is involved in regulating many cellular activities and functions. Drugs targeting phosphorylation signalling pathways represent a promising area. Particularly for the treatment of cancers, several inhibitors designed to alternate phosphorylation signalling are currently undergoing clinical trials.⁵⁸ The kinase/phosphatase system controls the reversible phosphorylation/dephosphorylation of phosphoprotein substrates.⁵⁹ Serine/threonine protein phosphatase PP5 belongs to the phosphoprotein phosphatases family and is ubiquitously expressed in nearly all mammalian tissues. It has been reported that PP5 participated in modulating many cellular processes, such as cell proliferation, differentiation, apoptosis, DNA repair, and steroid hormone signalling.⁶⁰ In the heart, PP5 was found to be increased in the failing heart, and alteration of PP5 was associated with cardiac hypertrophy and arrhythmias and in ischaemia/reperfused hearts.^38,61 In this study, we observed in hESC-CMs that circUtrn regulated the cardiomyocyte size, the expression of proliferation markers, and OGD/R-induced cardiomyocyte apoptosis via PP5. circUtrn directly binds to PP5 and leads to reduction of PP5 expression in cardiomyocytes via increasing PP5 ubiquitination. Further, we demonstrated that circUtrn upregulated PP5 downstream MAPK signalling, consistent with negative regulation of PP5 by circUtrn. Limited knowledge is present about the regulatory molecular mechanism of PP5 in cardiomyocytes. In this current study, we were able to show that PP5 activity is impacted by the presence of circUtrn through the ubiquitin–proteasome system in cardiomyocytes. This observation provides insight into the regulation and functional role of PP5 in cardiomyocytes and extends knowledge of the role of protein phosphatases in the cardiovascular system.

Several different RNA-based therapeutic strategies for the diagnosis and treatment of cardiovascular diseases have been proposed.⁶² hESC-CMs have many features of native cardiomyocytes and are used as a unique platform to explore cardiac biology and preclinical drug screening.^63,64 In this current study, we examined the in vitro function of circUtrn in hESC-CMs. circUtrn elevation and its protective role against I/R injury might hold a therapeutic promise in I/R injury patients. Besides, we have shown that serum circUtrn is significantly increased in long-term exercise training adult men, being consistent with elevated expression of circUtrn in exercise training mice. These data indicate that circUtrn may act as a new serum biomarker for prognosis among several cardiac diseases. Therefore, further studies to investigate circUtrn in different patient populations seem warranted.

In conclusion, our study identified a conserved circRNA circUtrn, which was found to be significantly increased as a response to exercise in mouse cardiomyocytes. In vitro, gain- and loss-of-function assays in hESC-CMs suggest that circUtrn OE promotes cardiomyocyte hypertrophy and alleviates OGD/R-induced apoptosis through binding and regulating PP5. In vivo, exercise-induced increase of circUtrn is critical for the protective effects of exercise in the setting of I/R remodelling. Forced OE of circUtrn in mouse hearts decreases acute myocardial I/R injury and prevents I/R remodelling.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Authors’ contributions

J.J.X. designed the study and instructed all experiments. J.J.X. and L.J.W. drafted the manuscript. L.J.W., J.Y.F., X.F., D.N.M., X.Z., J.Q.W., P.J.Y., G.E.X., T.H.W., and M.Y.H. performed the experiments and analysed the data. H.I.L., G.P.L., and J.P.G.S. provided technical assistance and gave input on experimental design and data interpretation in the manuscript.

Funding

This work was supported by the grants from National Key Research and Development Project of China (2018YFE0113500 to J.J.X.), National Natural Science Foundation of China (82020108002 and 82225005 to J.J.X. and 82270291 to L.J.W.), Science and Technology Commission of Shanghai Municipality (23410750100, 20DZ2255400, and 21XD1421300 to J.J.X.), and Natural Science Foundation of Shanghai (23ZR1423000 to L.J.W.).

Data availability

Adult mouse cardiomyocyte RNA sequencing data that support the findings of this study have been deposited in the BioSample database under BioProject ID PRJNA801905. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Translational perspective

Circular RNA (circRNA) circUtrn is required for the development of exercise-induced physiological cardiac hypertrophy. circUtrn inhibition abolished the protective effects of exercise on ischaemia–reperfusion (I/R) remodelling. Moreover, circUtrn overexpression prevented myocardial I/R-induced acute injury and pathological cardiac remodelling. Our findings suggest that circUtrn elevation and its protective role against I/R injury may hold a therapeutic promise in I/R injury and pathological cardiac remodelling.

Author notes

The first four authors contributed equally to the study.

Conflict of interest: None declared.