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Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease logoLink to Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease
. 2025 Jul 17;14(15):e040700. doi: 10.1161/JAHA.124.040700

Targeting Mettl14 Using an RNA‐Targeting Clustered Regularly Interspaced Short Palindromic Repeat‐ High‐Fidelity Cas13x System Attenuates Doxorubicin‐Induced Cardiotoxicity

Wensi Wan 1,2,*, Caiyue Cui 1,2,*, Yi Zhou 1,2, Jiaqi Wang 1,2, Xuan Zhao 1,2, Xinxin Cui 1,2, Jiangpeng Sun 1,2, Pujiao Yu 3, Jingyi Feng 1,2, Tianhui Wang 1,2,✉, Lijun Wang 1,2,✉, Jiahong Xu 3,✉
PMCID: PMC12449919  PMID: 40673518

Abstract

Background

Doxorubicin is an effective chemotherapy drug used to treat various types of cancer. However, doxorubicin treatment is associated with cardiotoxicity, which limits its clinical use. Exercise can benefit both cancer and cardiovascular disease. Clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas13 (CRISPR‐associated protein 13) platforms have emerged as effective technologies for targeting the expression of RNA in transcript levels. To develop exercise mimetics that can mimic the beneficial effects of exercise training to attenuate doxorubicin‐induced cardiotoxicity, we are using the CRISPR‐hf (high‐fidelity)Cas13x system.

Methods

Adult male mice were swim‐trained twice a day for 4 weeks to induce exercise‐induced physiological cardiac hypertrophy. Adeno‐associated virus 9‐mediated METTL14 (methyltransferase‐like 14) overexpression under the cardiac‐specific ctnt promoter was used to overexpression METTL14 in vivo. RNA N6‐methyladenosine inhibitor STM2457 was used to modulate global total RNA m6A levels in vivo. CRISPR‐cr3‐4/hfCas13x system was generated by hfCas13x guided crRNA3 and crRNA4 targeting the Mettl14 expressed under ctnt promoter and packaged in an adeno‐associated virus 9.

Results

Swimming exercise alleviated doxorubicin‐induced cardiotoxicity. METTL14 was increased in doxorubicin‐treated hearts but decreased in exercised hearts. METTL14 overexpression inhibited exercise‐induced physiological cardiac hypertrophy. Conversely, STM2457 treatment reversed the suppressive effects of METTL14 overexpression on the physiological cardiac hypertrophy induced by exercise. Treatment with CRISPR‐cr3‐4/hfCas13x effectively inhibiting the expression of METTL14 in the heart, alleviating doxorubicin treatment‐induced cardiac dysfunction and cardiac fibrosis.

Conclusions

Our results suggest that the CRISPR‐hfCas13x system has the potential for generating exercise mimetics. Mimicking exercise by RNA‐targeting Mettl14 suppression could be a therapeutic strategy for doxorubicin‐induced cardiotoxicity.

Keywords: doxorubicin‐induced cardiotoxicity, exercise training, high‐fidelity Cas13, Mettl14

Subject Categories: Animal Models of Human Disease, Basic Science Research

Nonstandard Abbreviations and Acronyms

m6A

N6‐methyladenosine

METTL14

methyltransferase‐like 14

Research Perspective.

What Is New?

  • Swimming exercise alleviates doxorubicin‐induced cardiotoxicity.

  • METTL14 (methyltransferase‐like 14) overexpression prevents exercise‐induced physiological cardiac hypertrophy, and RNA N6‐methyladenosine inhibitor STM2457 treatment can recover the suppression effects of METTL14 overexpression on exercise effects of physiological cardiac hypertrophy.

  • Adeno‐associated virus 9‐packaged high‐fidelity Cas13 (CRISPR‐associated protein) variants (hfCas13x) guided by crRNA3‐4 silence Mettl14 expression in vivo and attenutate doxorubicin‐induced cardiotoxicity.

What Question Should Be Addressed Next?

  • Although this proof‐of‐concept demonstrates a potential alternative therapeutic strategy for doxorubicin‐induced cardiotoxicity by RNA‐targeting CRISPR technology, future work focuses on a more comprehensive comparison of the therapeutic efficiency, safety, and scalability between CRISPR‐hfCas13x and traditional gene‐silencing approaches (eg, shRNA) in the context of doxorubicin‐induced cardiotoxicity holds significant importance to facilitate the translational value of this finding.

With advancements in anticancer therapies, the morbidity and mortality outcomes for cancer patients have improved. Doxorubicin is an effective chemotherapy drug used to treat various types of cancer. However, the administration of doxorubicin is associated with cardiotoxicity, which limits its clinical use. Although several potential mechanisms underlying doxorubicin‐induced cardiotoxicity have been reported, there are currently no effective therapies available for the treatment of this condition. A healthy lifestyle, in particular exercise prescription, can benefit both cancer and cardiovascular disease, and is proposed to be a promising treatment to counteract anticancer treatment side effects. 1 , 2 However, not all patients are suitable for exercise prescription, especially those who require bed rest. Developing exercise mimetics that can mimic the beneficial effects of exercise training paves a promising pathway for both preventive and therapeutic applications.

RNA N6‐methyladenosine (m6A) modification is the most prevalent reversible modification of RNA in eukaryotes. 3 The installation of RNA m6A is catalyzed by a methyltransferase complex, which consists of METTL3 (methyltransferase‐like 3), METTL14 (methyltransferase‐like 14), and WTAP (Wilms' tumor 1‐associated protein). In contrast, RNA demethylases, including FTO (fat mass and obesity‐associated protein) and ALKBH5 (alkB homolog 5), can remove m6A methylation from modified RNAs. Dysregulation of cardiac RNA m6A has been implicated in the development and progression of myocardial diseases. 4 , 5 , 6 , 7 , 8 Previously, we reported that cardiac METTL14 is downregulated by exercise, and genetic suppression of METTL14 by shRNA target Mettl14 can alleviate myocardial ischemia–reperfusion injury and doxorubicin‐induced cardiotoxicity in murine model. 9 , 10

The clustered regularly Interspaced short palindromic repeat (CRISPR) system has been developed into an effective gene engineering platform. Gene therapies based on RNA‐targeting nucleases are currently under exploration as a safer alternative to DNA editing Cas endonucleases since they are accompanied by a lower risk of introducing permanent genomic alterations. 11 , 12 , 13 Cas13 (CRISPR‐associated protein) is one of the CRISPR‐based technologies that are targeting RNA in transcript levels. 11 Several different Cas13 subtypes have been identified and applied to silent genes in mammalian cells. Among them, Ruminococcus flavefaciens XPD3002 (RfxCas13d, or CasRx) received much attention for higher knockdown efficiency and specificity. 14 The smaller molecular size of RfxCas13d or its mutants made it capable of enveloped into a single adeno‐associated virus (AAV) vector for reversibly manipulating RNA in vivo. 15 , 16 Thus, we sought to determine whether CRISPR‐high‐fidelity(hf)Cas13x could be used to deliver crRNAs to the cardiac to suppress genes.

Here, in this study, we show that swimming exercise alleviates doxorubicin‐induced cardiotoxicity. METTL14 levels were elevated in hearts treated with doxorubicin but inhibited in hearts subjected to exercise. We employed AAV9‐mediated overexpression of METTL14 and the m6A inhibitor STM2457 to investigate the role of METTL14 in exercise‐induced cardiac hypertrophy. Our findings demonstrated that overexpression of METTL14 inhibited exercise‐induced physiological cardiac hypertrophy, and treatment with STM2457 can reverse the suppressive effects of METTL14 overexpression on the physiological adaptations associated with exercise. Treatment with hfCas13x guided crRNA targeting the Mettl14, expressed under the cardiac‐specific ctnt promoter and packaged in an AAV9‐vector, effectively inhibited the expression of METTL14 in the heart, alleviating doxorubicin treatment‐induced cardiac dysfunction and cardiac fibrosis. Our results suggest that the CRISPR‐hfCas13x system has the potential for generating exercise mimetics, mimicking exercise by RNA‐targeting Mettl14 suppression could be a therapeutic strategy for doxorubicin‐induced cardiotoxicity.

METHODS

The authors declare that all supporting data are available within the article and its online supplementary files.

Animal

All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication, 8th edition, updated 2011), 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) and were randomly assigned to different groups. All housed in a barrier facility on a 12‐hour light/dark cycle at 22 °C to 24 °C and 45% to 55% humidity with access to food and water ad libitum. We used the Animal Research: Reporting of In Vivo Experiments checklist when writing our report. 17 Based on prior experiences, to establish doxorubicin‐induced cardiotoxicity mouse model, 23 adult male C57BL/6J mice (8–9 weeks) were intraperitoneally injected with doxorubicin (5 mg/kg) and 19 male mice were intraperitoneally injected with saline (5 mg/kg) once a week and for 5 weeks as previously reported. 9 To establish swimming exercise‐induced physiological cardiac hypertrophy, 45 adult C57BL/6J male mice were swim‐trained (47 sedentary controls) twice a day in a water tank for 4 weeks as reported previously. 10 , 18 To study the role of AAV9‐packaged CRISPR‐cr3‐4/hfCas13x in vivo, AAV9 with 1.5 × 1012 viral genomes/mice carrying CRISPR‐cr3‐4/hfCas13x or its control in a total volume of 100 μL were injected via tail vein a week before being subjected to doxorubicin injection (21 mice) or saline control (20 mice). Health was monitored by body weight weekly. At the end of the time point, the cardiac function was detected by echocardiography. The numbers of mice in each performed experiment were indicated in figure legend. There were no exclusions and mice were euthanized via intraperitoneal sodium pentobarbital (60 mg/kg) for cardiac tissue collection. All of the analyses were performed by investigators blinded to the treatment.

Echocardiography

Mice were anesthetized with 1.5% isoflurane, and 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. M‐mode echocardiograms were obtained from the papillary muscle level. All the echocardiography data are presented in Tables S1 through S3.

Quantitative Real‐Time Polymerase Chain Reaction

Total RNA was isolated using RNAiso Plus (Takara, 9108). Total RNA was reverse transcribed into cDNA by using the RevertAid First Strand cDNA Synthesis Kit (Thermo scientific, K1622) for the subsequent analysis of genes according to the manufacturer's instruction. RNA levels were quantified using Taq's TB Green Premix (Takara, RR001A) by Real‐Time PCR Detection System (Roche LightCycler480). 18S was used as an internal control. Primers are listed in Table S4.

Western Blot

Protein extracts were isolated from heart tissues by using RIPA lysis buffer with protease inhibitor (KenGEN, KGP702). The protein concentration was quantified using a BCA protein assay kit (Takara, T9300A), and the same amount of protein was isolated by SDS‐PAGE and then electro‐blotted onto PVDF membranes. The membranes were blocked and incubated with the appropriate primary antibodies overnight followed by incubation with horseradish peroxidase–conjugated secondary antibodies. Proteins were visualized by a hypersensitive chemiluminescence kit using the ChemiDoc Imaging System (Bio‐Rad, 17 001 402). Band intensities were calculated using Image J. Antibodies are listed in Table S5.

Dot Blot

Total RNA was isolated by miRNeasy Mini Kit (QIAGEN, 217004) with DNase I on column treatment. RNA m6A methylation was detected by m6A antibodies (Abclonal, #A19841) incubation as previously reported. 9 Methylene blue staining was used to confirm the equal amounts of RNA samples.

Vector Construction

pCBh_NLS_hfCas13X(Cas13X_M17YY)_NLS‐pA‐U6‐DR‐BpiI‐BpiI‐DR‐pSV40‐EGFP‐pA‐pSV40‐mCherry‐pA was a gift from Huawei Tong (Addgene # 190033). 15 AAV‐EFS‐NLS‐CasRx‐U6‐DR‐sgRNA_LacZ‐DR was a gift from Hui Yang (Addgene # 154003). 16 AAV‐CRISPR‐hfCas13x‐U6‐DR‐sgRNA plasmid was generated by amplify hfCas13x gene from Addgene # 190033, then replaced CasRx in AAV‐EFS‐NLS‐CasRx‐U6‐DR‐sgRNA_LacZ‐DR (Addgene#154003). To further ensure the cardiac specificity, we inserted the cTnT (cardiac troponin T) promoter to replace EF1α (elongation factor 1α) promoter to drive hfCas13x expression.

Immunochemistry and Immunofluorescence Staining for Heart Tissue

For TUNEL staining, the DeadEnd Fluorometric Tunel System kit (Promega, G3250) was used for frozen heart sample sections according to the manufacturer's instructions. Cardiomyocyte apoptosis was assessed by calculating TUNEL‐positive together with α‐actinin (Sigma, A7811)‐positive cardiomyocytes. Six slides from 6 mice hearts were analyzed in each group.

For wheat germ agglutinin (WGA) staining, 5 μm frozen heart sections were stained with WGA Alexa Fluor 488 Conjugate (Invitrogen, W11261). Nuclei were counterstained with Hoechst. The images of heart sample sections were captured by a confocal microscope (Olympus). The size of myocardial cells was quantified by Image J. Six slides from 6 mice hearts were analyzed in each group.

For Sirius Red staining, 5 μm heart paraffin sections were fixed and subjected to Sirius Red staining according to the manufacturer's instruction (Sbjbio, BP‐DL030). Images of each section were taken by Leica Microscope (DM3000 LED). Image J software was used to quantify the collagen deposition area (red) and the percentage of fibrosis was measured as collagen deposition areas/total myocardial areas.

Statistical Analysis

All data are presented as mean±SD using GraphPad Prism 8.0. Analyses were performed using SPSS 20.0 software and GraphPad Prism 8.0. An independent‐sample t test (2 tailed) or Mann–Whitney U test was used for comparison between 2 groups, as appropriate and as indicated in the figure legends. Two‐way ANOVA or 3‐way ANOVA was used to compare means among 3 or more independent groups. The P value for interaction is indicated in Data S1. Tukey post hoc tests were used to compare pairs of treatment groups when the interaction value was <0.05. The exact P values in the legends have been indicated in the figures and supplemental figures. The statistical methods used in each performed experiments were indicated in figure legends. Differences were considered statistically significant with P<0.05. No statistical method was performed to predetermine sample size. Instead, sample sizes were determined based on our prior experience with similar in in vitro and in vivo studies. 9 , 10 , 18 , 19

RESULTS

Swimming Exercise Alleviates Doxorubicin‐Induced Cardiotoxicity

To investigate whether exercise prevented doxorubicin‐induced cardiotoxicity, we used the well‐established swimming exercise model followed by doxorubicin‐induced cardiotoxicity mouse model (Figure 1A). After 4 weeks of swimming training (or sedentary control) followed by 5 weeks of doxorubicin administration, mice were evaluated by echocardiography. As shown in Figure 1B, swimming exercise prevented doxorubicin treatment‐induced decline of cardiac function and thinning of ventricular wall thickness as indicated by ejection fraction and left ventricular anterior wall thickness in systole. Also, exercise training preserved the reduction of doxorubicin treatment‐induced cardiac atrophy as demonstrated by cardiac gross morphology (Figure 1C). WGA‐staining revealed that swimming exercise reduced doxorubicin‐induced cardiac cell atrophy (Figure 1D). Further, swimming exercise can prevent doxorubicin treatment‐induced cardiomyocyte apoptosis and cardiac fibrosis as evidenced by TUNEL and Sirius Red staining, respectively (Figure 1E and 1F). Real‐time quantitative polymerase chain reaction (RT‐qPCR) analysis about the hypertrophic‐associated genes (Nppa, Nppb) and fibrotic‐associated genes (Acta2, Col1a1, Col3a1) also suggested the alleviation effects of swimming exercise on doxorubicin treatment‐induced cardiac atrophy and cardiac fibrosis (Figure 1G). Taken together, our data suggest that exercise inhibits the doxorubicin treatment‐induced functional changes of cardiac.

Figure 1. Swimming exercise alleviates doxorubicin‐induced cardiotoxicity.

Figure 1

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A, Schematic diagram showing the experimental strategy. B, Representative images of echocardiography and analysis of LV ejection fraction and LV systolic anterior wall thickness of doxorubicin‐induced cardiotoxicity mice hearts of swimming training or sedentary control (n=10,9,9,14 mice, respectively). C, Cardiac morphology of doxorubicin‐induced cardiotoxicity mice hearts of swimming training or sedentary control. Scale bar: 5 mm. D, Representative images and quantitative analysis of WGA staining of doxorubicin‐induced cardiotoxicity mice hearts of swimming training or sedentary control (n=6 mice/group). Scale bar: 20 μm. E, Representative images and quantitative analysis of TUNEL staining of doxorubicin‐induced cardiotoxicity mice hearts of swimming training or sedentary control (n=6 mice/group). Scale bar: 20 μm. F, Representative Sirius Red staining images and quantification of collagen deposition (%) of doxorubicin‐induced cardiotoxicity mice hearts of swimming training or sedentary control (n=6 mice/group). Scale bar: 50 μm. G, RT‐qPCR analysis of hypertrophic associated genes (Nppa, Nppb) and fibrotic associated genes (Acta2, Col1a1, Col3a1) (n=6 mice/group). Data are presented as means±SD (B, D–G, 2‐way ANOVA with Tukey post hoc test). CM indicates cardiomyocyte; DOX, doxorubicin; LV, left ventricular; RT‐qPCR, real‐time quantitative polymerase chain reaction; and WGA, wheat germ agglutinin.

METTL14 Contributes to Swimming Exercise‐Induced Cardiac Hypertrophy

METTL14 knockdown could alleviate doxorubicin‐induced cardiotoxicity. 9 We wondered whether METTL14 was also involved in these observed protective effects of exercise on doxorubicin‐induced cardiotoxicity; thus, we examined the expression level of METTL14. As shown in Figure 2A, the inhibition effect of swim exercise on METTL14 has returned to the baseline at saline 5 weeks after the termination of swim exercise in the saline+swim group, whereas the protein level was sustained decreased at doxorubicin 5 weeks in swim‐exercised hearts compared with sedentary control hearts. Further, we determined the expression of METTL14 in isolated adult mouse cardiomyocytes and noncardiomyocytes from saline control and doxorubicin‐treated hearts, our data suggested that doxorubicin treatment markedly increased the expression of METTL14 in adult cardiomyocytes but has a nonsignificant effect in noncardiomyocytes (Figure S1). These data suggest that METTL14 inhibition in cardiomyocytes might participate in the beneficial cardiac effects of exercise on doxorubicin‐induced cardiotoxicity.

Figure 2. RNA m6A methylation modulation contributes to swimming exercise‐induced cardiac hypertrophy.

Figure 2

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A, Representative western blot and statistical data of METTL14 of doxorubicin‐induced cardiotoxicity mice hearts of swimming training or sedentary control (n=6 mice/group). B, Schedule of virus and STM2457 inhibitor injection and swim‐induced mice's physiological cardiac hypertrophy model establishment. C, Cardiac morphology of hearts from exercised mice injected with AAV9 virus and inhibitor as indicated. Scale bar: 5 mm. D, HW, and HW/TL of mice hearts in indicated groups (n=12, 12, 12, 11, 12, 12, 11, 10 mice, respectively). E, Representative western blot and statistical data of METTL14 expression levels of mice hearts in indicated groups (n=6 mice/group). F, Dot blot of total RNA m6A levels in mice hearts as indicated groups. G, Representative images and quantitative analysis of WGA staining of mice hearts in indicated groups (n=6 mice/group). Scale bar: 20 μm. Data are presented as means±SD. (A, 2‐way ANOVA with Tukey post hoc test. E, 2‐way ANOVA, P value for interaction between AAV9‐cTnT‐Mettl14 OE and STM2457>0.05. D and G, 3‐way ANOVA, P value for interaction between STM2457 and control >0.05; P value for interaction between STM2457 and Swim >0.05; others, Tukey post hoc test.) AAV9 indicates adeno‐associated virus 9; cTnT, cardiac troponin T; DOX, doxorubicin; HW, heart weight; m6A, N6‐methyladenosine; MB, methylene blue staining; METTL14, methyltransferase‐like 14; OE, overexpression; TL, tibia length; and WGA, wheat germ agglutinin.

Exercise‐induced cardiac hypertrophy is one of the important physiological adaptations of the heart in response to exercise; physiological cardiac hypertrophy exerts the cardioprotective effects of exercise. Previously, we have found that METTL14 overexpression disrupted the effects of exercise‐induced cardiac hypertrophy. 10 Here, to further examine the role of METTL14 in exercise‐induced cardiac hypertrophy, mice were administrated METTL14 overexpression AAV9 (AAV9‐cTnT‐METTL14 OE) or m6A inhibitor STM2457 simultaneously, and then subjected to swim training (Figure 2B). STM2457 is a potent RNA methyltransferase inhibitor and selectively binds to the METTL3‐METTL14 heterodimer. 20 Swim exercise can induce cardiac hypertrophy, as evidenced by cardiac gross morphology, heart weight, and heart weight/tibia length (Figure 2C and 2D). Whereas METTL14 overexpression prevented exercise‐induced cardiac hypertrophy, conversely, the antihypertrophic effects of METTL14 overexpression were blunted in the presence of STM2457 on swim training (Figure 2C and 2D). The overexpression efficiency of METTL14 overexpression AAV9 was confirmed by western blot (Figure 2E). As previously reported, 20 STM2457 treatment maintained the expression levels of METTL14 in the heart (Figure 2E). The global total RNA m6A levels were decreased after swimming training or STM2457 inhibitor treatment, whereas METTL14 overexpression increased it (Figure 2F). WGA staining further indicated that STM2457 treatment resumed the suppressed effects of exercise‐induced enlargement of the cross‐sectional area of the myocardium by METTL14 overexpression in exercised‐hearts (Figure 2G). There was no significant increase in hypertrophic gene markers (Nppa, Nppb, β‐Mhc) among hearts from mice treated with METTL14 overexpression or STM2457 in swim‐exercised hearts or sedentary control mice hearts, suggesting the observed exercise‐induced cardiac hypertrophy is physiological (Figure S2). In parallel, STM2457 treatment recovered the decreased ratio of EdU‐positive and Ki67‐positive cardiomyocytes in exercised mice hearts with METTL14 overexpression (Figure S3). In summary, our results demonstrate that METTL14 overexpression prevented exercise‐induced physiological cardiac hypertrophy, and STM2457 treatment can recover the suppression effects of METTL14 overexpression on exercise effects of physiological cardiac hypertrophy.

AAV9‐Packaged High‐Fidelity CAS13 Variants Guided by crRNA3‐4 Silenced METTL14 Expression In Vivo

Consistent with the observed beneficial effects of swimming exercise on doxorubicin‐induced cardiotoxicity, we also have found that Mettl14 knockdown through traditional shRNA gene silencing modality could alleviate doxorubicin‐induced cardiotoxicity. 9 Thus, strategies that are capable of modulating the expression of cardiac m6A methylation suppression by METTL14 modulation may hold potential for exercise‐induced cardiac benefits in doxorubicin‐induced cardiotoxicity. CRISPR‐based platforms have emerged as effective technologies for perturbing the expression of a target gene. Given this, we sought to determine if CRISPR‐based platforms can be used to lower the expression of METTL14 to attenuate cardiac injury. Exercise training is a lifestyle alteration factors that contribute to the expression change of genes in transcription or posttranscription level that does not affect its genome level. CRISPR/Cas13 editing system can specifically and precisely cleave single‐strand RNAs in mammalian cells. 21 To mimic the exercise effects to target Mettl14 without affecting the whole genome expression, we used the previously reported hfCas13x variants that have been reported for targeted RNA degradation with minimal collateral effects 15 (Figure 3A). We designed 4 crRNAs for Mettl14 mRNA by the online computational model design tool 22 (Figure 3B). Then, we transfected C2C12 cells with hfCas13x and 4 crRNAs to detect the knockdown efficiency by RT‐qPCR, respectively (Figure 3C). As shown in Figure 3C, none of the 4 crRNAs can efficiently reduce the expression of Mettl14 >50%, and crRNA 2‐3‐4 exhibited similar reduction efficiency. To improve the knockdown efficiency, we tested a strategy to combine different crRNA individuals (cr2‐3‐4: a combination of crRNA2, crRNA3, and crRNA4. cr3‐4: a combination of crRNA3 and crRNA4.). After being detected by RT‐qPCR to examine the reduction efficiency of Mettl14 mRNA, cr3‐4 achieved the most efficient combination (Figure 3D). Thus, the most efficient Mettl14‐targeting crRNA combination strategy cr3‐4 was selected for further study.

Figure 3. AAV9‐packaged high‐fidelity Cas13 variants guided by crRNA3‐4 silenced Mettl14 expression in vivo.

Figure 3

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A, Overview of the strategy for targeting Mettl14 using hfCas13x. B, Sequences and location of the Mettl14 crRNAs (CRISPR RNAs). C, RT‐qPCR analysis of Mettl14 mRNA expression in C2C12 cells under targeting by different crRNAs (n=6/group). D, RT‐qPCR analysis of Mettl14 mRNA expression in C2C12 cells under targeting by combination treatment of indicated crRNAs (n=6/group). E, Schematic of the AAV vector encoding EF1α promoter‐hfCas13x/crRNA 3–4 and RT‐qPCR analysis of Mettl14 mRNA expression in mice hearts that were injected with AAV9 containing EF1α promoter‐hfCas13x/crRNA 3–4. (n=6/group). F, Schematic of the AAV vector encoding cTnT promoter‐hfCas13x/crRNA 3–4 and RT‐qPCR analysis of Mettl14 mRNA expression in mice hearts that were injected with AAV9 containing cTnT promoter‐hfCas13x/crRNA 3–4. (n=3/group). Data are presented as means±SD (C–E, t test; F, Mann–Whitney U test). AAV9 indicates adeno‐associated virus 9; CRISPR, clustered regularly interspaced short palindromic repeat; cTnT, cardiac troponin T; EF1α, elongation factor 1α; hfCas13x, high‐fidelity CRISPR‐associated protein; ITR, inverted terminal repeat; NT, nontarget; and RT‐qPCR, real‐time quantitative polymerase chain reaction.

Next, we inserted cr3‐4 tandem crRNA/EF1α promoter driven hfCas13x into an all‐in‐one plasmid and packaged into AAV9 capsid to tail vein injected into mice to test the knockdown efficiency. After 2 weeks, mice hearts were collected to quantify the Mettl14 expression. RT‐qPCR data showed that treatment with cr3‐4 led to a 40% knockdown of Mettl14 in the heart tissues (Figure 3E). To further ensure the cardiac specificity, we inserted the cTnT promoter to replace the EF1α promoter to drive hfCas13x expression. cTnT‐AAV9 (1.5 × 1012 viral genomes/mice) carrying cr3‐4/hfCas13x were tail‐vein injected and the Mettl14 expression in cardiac was examined by RT‐qPCR after 2 weeks (Figure 3F). Collectively, these results indicated that AAV9‐delivered cr3‐4/hfCas13x could successfully inhibit Mettl14 transcripts in mice hearts.

CRISPR‐CR3‐4/ hfCas13x Improves Doxorubicin‐Induced Cardiotoxicity In Vivo

We then sought to explore the therapeutic benefit of targeting Mettl14 by hfCas13x in an early‐phase therapeutic model on doxorubicin‐induced cardiotoxicity. Briefly, CRISPR‐cr3‐4/hfCas13x or control (1.5×1012 viral genomes/mice) was tail vein injected on the same day after the first intraperitoneal injection of doxorubicin (Figure 4A). Doxorubicin‐induced cardiotoxicity murine model was established by consecutively intraperitoneal injection of doxorubicin every week for 5 weeks (5 mg/kg per week). Cardiac function was assessed by echocardiography, the therapy effect of CRISPR‐cr3‐4/hfCas13x were demonstrated by preserved cardiac function (ejection fraction) and inhibited the thinning of ventricular wall thickness (left ventricular anterior wall thickness in systole) (Figure 4B). Also, CRISPR‐cr3‐4/hfCas13x preserved the reduction of doxorubicin treatment‐induced cardiac atrophy as demonstrated by cardiac gross morphology (Figure 4C). The targeting effects of CRISPR‐cr3‐4/hfCas13x on Mettl14 were verified by RT‐qPCR and western blot (Figure 4D and 4E). Further, CRISPR‐cr3‐4/hfCas13x treatment can alleviate doxorubicin treatment‐induced cardiomyocytes apoptosis as evidenced by TUNEL staining (Figure 4F). Moreover, doxorubicin‐induced cardiac fibrosis was also attenuated as suggested by Sirius Red staining and qRT‐PCR analysis about fibrotic‐associated genes (Acta2, Col1a1, Col3a1) (Figure 4G and 4H). Additionally, the alleviation effects were also observed by decreased expression of hypertrophic‐associated genes (Nppa, Nppb) in CRISPR‐cr3‐4/hfCas13x treatment mice after doxorubicin administration (Figure 4I). Finally, we detected the cardiac injury markers in serum. As shown in Figure 4J and 4K, the serum levels of cTnI (cardiac troponin I) and CK‐MB (creatine kinase‐myocardial band) were significantly lower in CRISPR‐cr3‐4/hfCas13x treatment mice than in control mice after doxorubicin treatment. Altogether, these data indicate that CRISPR‐cr3‐4/hfCas13x on silencing Mettl14 significantly improves doxorubicin‐induced cardiotoxicity.

Figure 4. CRISPR‐cr3‐4/hfCas13x improves doxorubicin‐induced cardiotoxicity in vivo.

Figure 4

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A, Schematic diagram showing the experimental strategy. B, Representative images of echocardiography and analysis of LV ejection fraction and LV systolic anterior wall thickness of doxorubicin‐induced cardiotoxicity mice hearts tail vein injected with or without CRISPR‐cr3‐4/hfCas13x treatment (n=10, 10, 9, 12 mice/group). C, Cardiac morphology of doxorubicin‐induced cardiotoxicity mice hearts tail vein injected with or without CRISPR‐cr3‐4/hfCas13x treatment. Scale bar: 5 mm. D, RT‐qPCR analysis of Mettl14 mRNA expression of doxorubicin‐induced cardiotoxicity mice hearts tail‐vein injected with or without CRISPR‐cr3‐4/hfCas13x treatment (n=6 mice/group). E, Representative western blot and statistical data of METTL14 and Flag‐hfCas13x of doxorubicin‐induced cardiotoxicity mice hearts tail vein injected with or without CRISPR‐cr3‐4/hfCas13x treatment (n=6 mice/group). F, Representative images and quantitative analysis of TUNEL staining of doxorubicin‐induced cardiotoxicity mice hearts tail‐vein injected with or without CRISPR‐cr3‐4/hfCas13x treatment (n=6 mice/group). Scale bar: 20 μm. G, Representative Sirius Red staining images and quantification of collagen deposition (%) of doxorubicin‐induced cardiotoxicity mice hearts tail vein injected with or without CRISPR‐cr3‐4/hfCas13x treatment (n=6 mice/group). Scale bar: 50 μm. H, RT‐qPCR analysis of fibrotic associated genes (Acta2, Col1a1, Col3a1) of doxorubicin‐induced cardiotoxicity mice hearts tail vein injected with or without CRISPR‐cr3‐4/hfCas13x treatment (n=6 mice/group). I, RT‐qPCR analysis of hypertrophic associated genes (Nppa, Nppb) of doxorubicin‐induced cardiotoxicity mice hearts tail‐vein injected with or without CRISPR‐cr3‐4/hfCas13x treatment (n=6 mice/group). J, The serum levels of cTnI of doxorubicin‐induced cardiotoxicity mice hearts tail vein injected with or without CRISPR‐cr3‐4/hfCas13x treatment (n=6 mice/group). K, The serum levels of CK‐MB of doxorubicin‐induced cardiotoxicity mice hearts tail vein injected with or without CRISPR‐cr3‐4/hfCas13x treatment (n=6 mice/group). Data are presented as means±SD (B, D, F–K, 2‐way ANOVA with Tukey post hoc test. E, 2‐way ANOVA, P value for interaction between doxorubicin 5 weeks and cr3‐4 > 0.05). CK‐MB indicates creatine kinase‐myocardial band; CM, cardiomyocyte; CRISPR, clustered regularly interspaced short palindromic repeat; cTnI, cardiac troponin I; DOX, doxorubicin; hfCas13x, high‐fidelity CRISPR‐associated protein; i.p., intraperitoneally; LV, left ventricular; METTL14, methyltransferase‐like 14; NT, nontarget; and RT‐qPCR, real‐time quantitative polymerase chain reaction.

DISCUSSION

In this study, we reported that swimming exercise alleviated doxorubicin‐induced cardiotoxicity. METTL14 was increased in doxorubicin‐treated hearts but decreased in exercised hearts. Subsequently, using AAV9‐mediated METTL14 overexpression and m6A inhibitor STM2457, we demonstrated that METTL14 overexpression inhibited exercise‐induced physiological cardiac hypertrophy. Conversely, STM2457 treatment could reverse the suppressive effects of METTL14 overexpression on the physiological cardiac hypertrophy induced by exercise. Then, we packaged hfCas13x guided by crRNA3‐4 to silence Mettl14 expression in vivo, thereby mimicking the effects of swimming exercise. Our results demonstrated that CRISPR‐cr3‐4/hfCas13x, driven by a cardiac‐specific promoter, attenuated doxorubicin‐induced cardiotoxicity. Taken together, our results provide a potential strategy for generating exercise mimetics and treating doxorubicin‐induced cardiotoxicity.

Exercise training is a lifestyle alteration factor that contributes to the expression change of genes in transcription or posttranscription level that does not affect its genome level. The beneficial effects of exercise training on human disorders have been widely recognized. 23 , 24 Mimicking exercise's beneficial effects to develop new therapeutic strategies has been attempted in many preclinical studies. 18 , 25 , 26 Gene therapy is one of the most prevalent strategies developed to mimic the beneficial effects of exercise to mitigate many human diseases. 10 , 27 , 28 , 29 , 30 , 31 Though different heart diseases such as dilated cardiomyopathy, pathological cardiac remodeling, and ischemic heart disease result in different cardiac maladaptation, exercise‐induced cardioprotection against diseased hearts from multiple aspects. 32 , 33 In addition, targets identified from exercised hearts protect against cardiac diseases by multiple avenues, including cardiomyocytes hypertrophy, reducing oxidative stress, improving metabolic adaptations, and so on. 34 For example, CITED4, which is upregulated by exercise training and is essential for physiological cardiac hypertrophy, have been shown to promote functional recovery after ischemic injury and pressure overload induced cardiac remodeling. 26 , 27 , 35 Recently, CITED4 gene therapy has been explored and exhibited protective effects on postischemic cardiac remodeling in murine models. 36 Although many issues remain to be resolved before the clinical application of CITED4 gene therapy, this study highlights the potential therapeutic application of exercise‐derived factors. In addition to exercise‐upregulated factors, many molecules that have been suppressed by exercise training in the heart that have been found to exhibit beneficial effects. 10 , 37 The cardiac protective effects of METTL14 suppression have been observed in exercised hearts, where it serves to prevent myocardial ischemia–reperfusion injury and is required for exercise‐induced cardiac hypertrophy. 10 Consistent with our previously study, we observed that RNA m6A methylation level was increased in doxorubicin‐induced cardiotoxicity compared with saline control hearts, whereas swimming exercise or METTL14 inhibition reduced the total RNA m6A methylation level in doxorubicin‐induced cardiotoxicity (Figure S4). 9 Strategies that are capable of modulating the expression of cardiac m6A methylation suppression by METTL14 modulation might hold potential for doxorubicin‐induced cardiotoxicity. Therefore, we explored the proof‐of‐concept CRISPR‐hfCas13x ‐based RNA‐targeting therapy to knockdown Mettl14 of potential relevance for therapeutic directions addressing doxorubicin‐induced cardiotoxicity. We observed that CRISPR‐hfCas13x targeting Mettl14 decreased the upregulated total RNA m6A methylation in doxorubicin‐induced cardiotoxicity (Figure S5). Doxorubicin treatment is known to induce dilated cardiomyopathy with cardiac atrophy and apoptosis being hallmarks of doxorubicin‐induced cardiotoxicity. METTL14 knockdown can attenuate doxorubicin‐induced cardiomyocytes apoptosis and oxidative stress by inhibiting circular RNA circ‐ZNF609. 9 In contrast, METTL14 inhibition would provide a protective effect against cardiac atrophy, as evidenced by the partial preservation of heart weight, heart weight/tibia length, and WGA staining (Figure S6). 9 Therefore, our observations regarding the function of METTL14 may be attributed to the combined effects of METTL14 inhibition, which reduces cardiac atrophy, apoptosis, and oxidative stress. Further investigations to explore the roles of METTL14 in exercised hearts in response to doxorubicin challenge simultaneously would contribute to a deep understanding of the cardiac adaptation to exercise and METTL14 regulatory mechanisms in the heart. In this study, we used CRISPR‐cr3‐4/hfCas13x technology to silence Mettl14 expression in vivo to mimic swimming exercise effects, further supporting the potential therapeutic use of RNA‐targeting therapy to knockdown Mettl14 in mimicking the beneficial effects of exercise training.

CRISPR/Cas13 editing system can specifically and precisely cleave single‐strand RNAs in mammalian cells. 21 Currently, to in vivo mimic the exercise‐induced suppression effects through AAV‐based gene therapy, shRNA technologies are the most commonly employed knockdown strategy. In contrast to shRNA‐based knockdown technologies, Cas13‐mediated knockdown demonstrates comparable RNA interference efficacy while exhibiting reduced off‐target effects, making it a suitable platform for therapeutic applications. 11 , 38 In addition, RNA‐targeting CRISPR system Cas13 could avoid the risk associated with permanent DNA alteration caused by CRISPR‐Cas9. 13 High‐fidelity Cas13 variant hfCas13x is a recently developed Cas13 system that has shown similar RNA knockdown activity to wild‐type Cas13 with no detectable collateral damage in vivo. 15 , 36 , 39 In this study, we modified this CRISPR‐cr3‐4/hfCas13x system to restrict hfCas13x expression in cardiomyocytes by replacing the EF1α promoter with cardiac‐specific cTnT promoter, our results demonstrated that cardiac‐specific promoter‐driven CRISPR‐cr3‐4/hfCas13x improved doxorubicin‐induced cardiotoxicity, providing a new approach for therapeutic application. Notably, none of the 4 individual crRNAs selected in the first batch could reduce the expression of Mettl14 >50%, a knockdown efficiency similar to that of shRNA shMETTL14. This observation, where target transcript knockdown levels are comparable under both shRNA and Cas13 conditions, has also been reported in previous studies. 11 To improve the knockdown efficiency of Mettl14, we adopted a strategy of combining different crRNA individuals as previously reported. 16 After being detected by RT‐qPCR, the combination of crRNA3 and crRNA4 (cr3‐4) was selected because it achieved the highest reduction efficiency for Mettl14 mRNA (~50%). Additionally, we compared the efficiency of CRISPR‐cr3‐4/hfCas13x in mice with AAV9‐cTnT‐shMETTL14 for the knockdown of METTL14. As shown in Figure S7, shMETTL14 resulted in approximately a 40% reduction and CRISPR‐cr3‐4/hfCas13x achieved >50% reduction of Mettl14. This indicates that the knockdown efficiency of the CRISPR‐hfCas13x‐based combination knockdown strategy is more effective than that of a shRNA in targeting Mettl14. Previously, circ‐ZNF609 has been identified as a downstream factor of METTL14 in mouse hearts. 9 METTL14 knockdown inhibited the expression of circ‐ZNF609. 9 Consistently, in this study, we observed a significant reduction in circ‐ZNF609 expression following the successful knockdown of Mettl14 (Figure S8). In particular, compared with shMETTL14, circ‐ZNF609 exhibited a more significant inhibition when using the CRISPR‐cr3‐4/hfCas13x combination knockdown strategy (Figure S8). This observation further confirmed the specificity knockdown of Mettl14. Unwanted off‐target silencing is one of the limitations for the applications of RNA interference. 38 In this study, we performed a BLAST analysis of the Top10 potential off‐target transcripts for shMETTL14 and detected the expression of them by RT‐qPCR (Figure S9). Our data suggested that no significant off‐target effects were observed for those 10 transcripts in either shMETTL14‐treated or CRISPR‐cr3‐4/hfCas13x‐treated hearts, though CRISPR‐cr3‐4/hfCas13x exhibited a superior knockdown efficiency on targeting Mettl14 (Figure S9). However, it is worth noting that the knockdown strategy differs between the 2‐crRNA (crRNA3 and crRNA4) in CRISPR‐cr3‐4/hfCas13x system and the single shRNA‐based target RNA interference approach in shMETTL14. Additionally, shRNA‐targeting strategy have been reported to induce widespread off‐target effects, additional changed off‐target transcripts in whole transcriptome should be existed. 11 , 16 , 38 Therefore, it would be important to further comprehensively explore the global off‐target transcripts of CRISPR/hfCas13x system compared with a position/sequence‐matched shRNA construct specifically designed for silencing Mettl14 in cardiac tissues using transcriptome‐wide mRNA sequencing. Although this proof‐of‐concept demonstrates a potential alternative therapeutic strategy for doxorubicin‐induced cardiotoxicity by RNA‐targeting CRISPR technology, future work focused on a more comprehensive comparison of the therapeutic efficiency, safety, and scalability between CRISPR‐hfCas13x and traditional gene‐silencing approaches (eg, shRNA) in the context of doxorubicin‐induced cardiotoxicity holds importance to facilitate the translational value of this finding.

In parallel with increased cardiomyocytes size, exercise also stimulates the increase in endogenous cardiomyocyte proliferation. 40 , 41 In this study, we demonstrated that METTL14 overexpression blocked exercise‐induced cardiac hypertrophy, whereas RNA m6A methyltransferase inhibitor STM2457 treatment reversed the antihypertrophy effects of METTL14 overexpression on exercised hearts. Consistently, METTL14 overexpression inhibited the DNA replication (EdU staining) and cell cycle activity (Ki67 staining) of cardiomyocytes compared with AAV9‐cTnT‐Ctrl administration in exercised hearts, and STM2457 treatment rescued these effects (Figure S3). Measurements of cardiomyocytes cell‐cycle activity based on different markers ranging from ~0.2% to ~2% in exercised hearts. 10 , 42 , 43 Swim exercise induces ~2% EdU+ cardiomyocytes (compared with 0.5% at basal level), which is comparable to the ~4.6‐fold (based on incorporation of 15 N‐thymidine by multi‐isotope imaging mass spectrometry). 40 Usually, swim exercise induces ~2% EdU+ adult cardiomyocytes, which is higher than Ki67+ ratio (~1%) due to the presence of DNA synthesis, which does not necessarily indicate the generation of new cardiomyocytes. 10 , 42 STM2457 is a commercial compound developed to inhibit RNA m6A as a potential anticancer therapeutic by increasing cell apoptosis. 20 However, STM2457 could reduce alveolar epithelial cell apoptosis in acute lung injury. 44 Here we found that treatment of STM2457 during METTL14 overexpression increases cardiomyocytes proliferation‐related markers in exercise‐trained mice. The observed opposite effects (proapoptosis and prosurvival) of STM2457 treatment might be due to the difference in cell types and physiopathological conditions. Besides, STM2457 treatment can inhibit cardiomyocyte apoptosis and reduce cardiomyocytes oxidative stress in doxorubicin‐induced cardiotoxicity. 9 , 45 However, it remains unclear whether STM2457 can facilitate cardiomyocyte proliferation in the myocardium after doxorubicin treatment. Given that developing strategies to promote cardiomyocytes proliferation following injury represents an attractive approach to treat cardiovascular disease, 46 further investigation into the role of STM2457 in regulating cardiomyocytes proliferation in damaged myocardium is warranted.

Prior studies have reported gender differences in doxorubicin‐induced cardiac dysfunction in mice. 47 , 48 To avoid the potential interference of hormonal fluctuations, the present study focused solely on male mice. Thus, the expression changes and protective effects of Mettl14 inhibition against doxorubicin‐induced cardiotoxicity are examined limited in male mice. It remains to be determined whether similar outcomes would be observed in female animals. Further investigation is warranted to elucidate its role and function in female animals and to ascertain whether sexual dimorphism exists in this context.

CONCLUSIONS

In conclusion, our study demonstrates that swimming exercise alleviates doxorubicin‐induced cardiotoxicity. RNA m6A methyltransferase METTL14, which is suppressed expression in exercised hearts, is critical for the effects of exercise‐induced physiological cardiac hypertrophy. Mimicking aerobic exercise by targeting Mettl14 using an RNA‐targeting CRISPR‐hfCas13x system attenuates doxorubicin‐induced cardiotoxicity. Taken together, our work demonstrates that the RNA‐targeting CRISPR system has the potential for generating exercise mimetics and treating doxorubicin‐induced cardiotoxicity.

Sources of Funding

This work was supported by the grants from National Natural Science Foundation of China (82070411 and 82370370 to Jiahong Xu, 82470369 and 82270291 to Lijun Wang). Shanghai Rising‐Star Program (24QA2703000 to Lijun Wang). Natural Science Foundation of Shanghai Municipality (23ZR1423000 to Lijun Wang). Medical Discipline Construction Program of Shanghai Pudong New Area Health Commission (PWZxk2022‐15 to Jiahong Xu). Key Discipline Construction Program of Shanghai Municipal Health System (2024ZDXK0024 to Jiahong Xu).

Disclosures

None.

Supporting information

Data S1

Tables S1–S5

Figures S1–S9

Unedited bots

JAH3-14-e040700-s001.zip (2.3MB, zip)

This article was sent to Sakima Ahmad Smith, MD, MPH, Associate Editor, for review by expert referees, editorial decision, and final disposition.

For Sources of Funding and Disclosures, see page 12.

Contributor Information

Tianhui Wang, Email: wangth@shu.edu.cn.

Lijun Wang, Email: lijunwang@shu.edu.cn.

Jiahong Xu, Email: jiahongxushu@shu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1

Tables S1–S5

Figures S1–S9

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