XPO1 Gene Therapy Attenuates Cardiac Dysfunction in Rats with Chronic Induced Myocardial Infarction

Transcriptomic signature of XPO1 was highly expressed and inversely related to left ventricular function in ischemic cardiomyopathy patients. We hypothesized that treatment with AAV9-shXPO1 attenuates left ventricular dysfunction and remodeling in a myocardial infarction rat model. We induced myocardial infarction by coronary ligation in Sprague-Dawley rats (n = 10), which received AAV9-shXPO1 (n = 5) or placebo AAV9-scramble (n = 5) treatment. Serial echocardiographic assessment was performed throughout the study. After myocardial infarction, AAV9-shXPO1-treated rats showed partial recovery of left ventricular fractional shortening (16.8 ± 2.8 vs 24.6 ± 4.1%, P < 0.05) and a maintained left ventricular dimension (6.17 ± 0.95 vs 4.70 ± 0.93 mm, P < 0.05), which was not observed in non-treated rats. Furthermore, lower levels of EXP-1 (P < 0.05) and lower collagen fibers and fibrosis in cardiac tissue were observed. However, no differences were found in the IL-6 or TNFR1 plasma levels of the myocardium of AAV9-shXPO1 rats. AAV9-shXPO1 administration attenuates cardiac dysfunction and remodeling in rats after myocardial infarction, producing the gene silencing of XPO1. Electronic supplementary material The online version of this article (10.1007/s12265-019-09932-y) contains supplementary material, which is available to authorized users.


Introduction
Coronary heart disease carries significant morbidity and is the leading cause of death across all diseases of the circulatory system [1]. After myocardial infarction, adverse ventricular remodeling associated with a higher probability of heart failure and mortality occurs [2,3], and numerous cellular and molecular pathways are affected [4][5][6][7], such as the existence of various alterations in the molecular machinery of nuclearcytoplasmic transport [8,9], which precisely regulates the bidirectional selective protein flow between the nucleus and the cytoplasm.
Previous studies have shown that several molecules that participate in nuclear-cytoplasmic transport (Exportin-1 [EXP-1], IMP-β3, Nup160) are intimately related to a reduced left ventricular (LV) function in human ischemic cardiomyopathy. The transcriptomic signature of these alterations has been found and has been identified that changes in gene expression, specifically of XPO1 that encodes EXP-1, were highly related to LV dysfunction in patients with ischemic cardiomyopathy [10].
The short hairpin RNA (shRNA) can be used to silence specific genes and is a powerful tool in studies pertaining to loss of gene function and characterization. The highly cardiotropic adeno-associated virus vector (AAV), with high affinity for the heart and down to other organs, can be introduced simply by intravenous injection [11,12]. In particular, AAV9 has a great potential as a valuable tool for cardiac therapy in cardiovascular disease experimental models for RNA interference and gene therapy [13].
We hypothesize that manipulation of gene deregulation has therapeutic value in myocardial infarction. We aim to investigate whether highly significant relationship between XPO1 and ventricular function is a component of causality. Therefore, we have developed a rodent myocardial infarction experimental model to show whether AAV9-shXPO1 silencing agent induces recovery of myocardial function.

Ethics Statement
The project was approved by the Biomedical Investigation Ethics Committee of Hospital La Fe. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), and the National (RD 53/2013) and European Directive (2010/63/EC). All surgery was performed using accurate anesthesia and analgesia veterinary protocols, to minimize animal suffering. All animal procedures are reported following ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and included as Supplementary Data (Online Resource 1).

Rat Myocardial Infarction Model
Adult male Sprague-Dawley rats weighting 300-400 g were anesthetized and left anterior descending (LAD) coronary artery ligation was performed (n = 10) with polypropylene nonabsorbable sutures (Premilene® Braun) to induce chronic myocardial infarction (Online Resource 1). Tetrazolium staining was performed in transversal slices from infarcted hearts 6 h after LAD coronary artery banding in preliminary myocardial infarction rats to ensure technical procedure [14]. Four months later, a group of infarcted rats showing left ventricular systolic dysfunction received intravenous AAV9-shXPO1 gene therapy (n = 5), and another received intravenous placebo AAV9-scramble (n = 5) (see below). Age-matched non-infarcted sham rats (n = 5) served as healthy non-failing rats, not Fig. 1 Study protocol timeline. Baseline functional echocardiographic parameters were evaluated (stage 0) in rats before the experimental procedure. Functional parameters were studied in sham and LAD (left anterior descending) coronary artery ligation animals after procedure (stage I). Four months later, the animals were administered with either adeno-associated virus vector silencing XPO1 gene (AAV9-shXPO1) or placebo (AAV9-scramble) particles. After gene therapy, changes in functional parameters were evaluated by echocardiography 2 (stage II) and 5 (stage III) months later receiving any treatment. The experimental design of these groups and the experimental procedure ( Fig. 1) were performed based on previous studies focused on this field of research [15][16][17].

Echocardiographic Assessment
A non-invasive transthoracic echocardiographic method was used to evaluate the morphology and function of the left ventricle. Echocardiography was performed on anesthetized animals with ketamine and valium (26 and 6 mg/kg, respectively) and so their anterior chest was shaved. Heart functional parameters were analyzed with a two-dimensional mode using Philips EnVisor M2540A Ultrasound System before the LAD ligation and AAV9-shXPO1/AAV9-scramble administration as well as 2 and 5 months after gene therapy.

Tissue Sampling
Frozen samples from AVV9-scramble (n = 5) and AAV9-shXPO1 (n = 5) groups (50 mg of heart, brain, skeletal muscle, and liver) were homogenized in a total protein extraction buffer (2% SDS, 10 mM EDTA, 6 mM Tris-HCl, pH 7.4) with protease inhibitors (25 μg/ml aprotinin and 10 μg/ml leupeptin) in a FastPrep-24 homogenizer with specifically designed Lysing Matrix D tubes (MP Biomedicals, USA). The homogenates were centrifuged and the supernatants were aliquoted. The protein content of the aliquots was determined by Peterson's modification [18] of the Lowry method using bovine serum albumin (BSA) as standard. Cardiac samples were obtained from left ventricles, including infarcted area [19].

Western Blot Analysis
Protein samples for detection of EXP-1 and GAPDH were separated using Bis-Tris Midi gel electrophoresis with 4-12% polyacrylamide under reducing conditions. Description of Western blot procedure is extensively described by Ortega et al. [20]. The primary detection antibodies used were anti-Exportin-1 (611833) mouse monoclonal antibody (1:50) from BD Transduction Laboratories™, and anti-GAPDH (ab9484) mouse monoclonal antibody (1:1000) obtained from Abcam and used as a loading control.
The bands were visualized using an acid phosphataseconjugated secondary antibody and nitro blue tetrazolium/5bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Sigma-Aldrich, St. Louis, USA) substrate system. Finally, the bands were digitalized using an image analyzer (DNR Bio-Imagining Systems, Israel) and quantified with the GelQuant Pro (v. 12.2) program. All the experiments were performed in triplicate.

Statistical Methods
The Kolmogorov-Smirnov test was used to analyze the distribution of the variables. All variables were normally distributed. Data are presented as mean value ± standard deviation. Comparisons of variables were analyzed using two-way ANOVA and Student's t test. Significance was accepted at the P < 0.05 level. All statistical analyses were performed using SPSS software v. 20, 2012 for Windows (IBM SPSS Inc., Chicago, IL, USA).

Echocardiographic Assessment
Cardiac function in infarcted and sham rats was measured by non-invasive transthoracic echocardiography to e v a l u a t e v e n t r i c u l a r f u n c t i o n a n d d i a m e t e r. Echocardiographic measurements were taken prior to surgery and treatment of rats (stage 0), after coronary ligation (stage I) and 2 (stage II) and 5 (stage III) months after AAV9 injection.
The rats were under strict supervision of highly qualified personnel, maintaining precise control of the anesthesia of the animal, with low intragroup variation heart rate throughout the follow-up (sham 353 ± 16, AAV9-shXPO1 367 ± 12, and AAV9-scramble 450 ± 25 beats/min).

Discussion
This study may provide a new therapeutic strategy based on gene therapy to restore ventricular function in patients with ischemic cardiomyopathy, which is the leading cause of death worldwide and lacking effective treatment [21]. In this study, AAV9-scramble (i), and AAV9-shXPO1 (j) rats. ***P < 0.001 sham vs AAV9-scramble and AAV9-shXPO1; # P < 0.05 AAV9-scramble vs AAV9-shXPO1 we report a successful delivery of an AAV-based gene therapy in a long-term chronic myocardial infarction rat model, which simulates the clinical features seen in patients with coronary heart disease after myocardial infarction. We have expanded the follow-up until 5 months after gene delivery, proving the long-term efficacy of the treatment and showing the safety of this procedure, since there was no evidence of side-effects in the animals.
EXP-1 mediates the nuclear export of proteins, rRNA, snRNA, and some mRNAs. Previous studies in patients with ischemic cardiomyopathy showed elevated expression levels of both EXP-1 mRNA and protein, and interestingly, these levels were inversely related with ejection fraction and positively correlated with LVESD and LVEDD [8,9], i.e., higher EXP-1 expression is linked with LV function impairment. Hence, we intended to demonstrate the cause-effect of this relationship in this study through gene therapy, which may result in a useful approach for the treatment of heart disease [22][23][24]. Cardiac gene therapy uses vectors that can robustly, specifically, and persistently deliver therapeutic genetic materials to the heart without generating local and/or systemic toxicity. The adenoviral vectors represent an efficient but unstable gene delivery vector for the heart. Nonetheless, longterm myocardial transduction in adult animals has been accomplished with the development of AAV [25]. Research has established AAV9 as a cardiotropic vector superior to all the other serotypes in rodents, making it the most appropriate vector for gene delivery to the heart [11,12]. Our results support this property of AAV9, since we observed that the cardiac tissue was the only sample analyzed where EXP-1 levels decreased in infarcted AAV9-shXPO1 rats compared with those in AAV9-scramble rats. Furthermore, we show the effectiveness and stability of the vector AAV9-shXPO1 as EXP-1 levels decreased in heart tissue 5 months after transduction. Regarding the magnitude of the effect, systematic or local, our results do not show differences in the inflammatory parameters studied in rats treated with AAV9-shXPO or AVV9-scramble.
LV function parameters are directly related to ventricular remodeling that occurs after injury of the heart muscle. Ventricular function of infarcted rats appeared to be in partial recovery following XPO1 silencing. At 2 months after injection (stage II), cardiac function in the rats was similar to that immediately after coronary ligation (stage I). Nevertheless, at 5 months after injection, the differences in these parameters were significant and resulted in improvements in the state of cardiac function in infarcted rats with XPO1 silencing. Furthermore, we have observed a decrease in fibrosis after treatment. This silencing could have similar effects at revascularization, recovering the hibernating myocardium and thereby improving ventricular function in infarcted rats. Although this partial recovery would be slower than that achieved by performing a bypass procedure, it is less invasive and harmful. We did not observe an improvement in the cardiac function of AAV9-scramble infarcted rats; these rats maintained similar parameters during the follow-up after coronary ligation.  5 Echocardiographic parameters of infarcted AAV9-shXPO1 and AAV9-scramble rats measured before surgery and injection (stage 0), before injection (stage I), and 2 (stage II) and 5 months (stage III) after vector injection. a Fractional shortening. b Left ventricular end-systolic diameter. c Left ventricular end-diastolic diameter. ### P < 0.001 stage 0 vs stages 1, 2, and 3 in AAV9-scramble; ***P < 0.001 stage 0 vs stages 1 and 2 in AAV9-shXPO1; *P < 0.05 and **P < 0.01 stage 3 vs stages 1 and 2 in AAV9-shXPO1

Study Limitations
In order to rationalize funding resources and minimize animal testing, we decided to compare only gene therapy responses in rats with chronic infarction, not studying AAV9-shXPO1 administration in healthy control rats [11]. For the development of the myocardial infarction model, a standardized protocol of veterinary pharmacology and surgery was followed; still, the manual procedure of coronary ligation may introduce some variability between infarcted rats.

Conclusions
In conclusion, AAV9-shXPO1 administration attenuates cardiac dysfunction in rats after myocardial infarction, producing the gene silencing of XPO1. This study provides a new therapeutic strategy based on gene therapy to restore ventricular function in patients with ischemic cardiomyopathy.

Clinical Relevance
This study offers a new way to restore cardiac function in patients who have suffered from myocardial infarction, by gene therapy through the silencing of XPO1.
Acknowledgments The authors thank the Animal Experimentation Platform of the IIS La Fe, for the technical support in this work.

Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of interest.
Ethical Approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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