Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.
Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.
Cardiac disease is the leading cause of morbidity and mortality worldwide1,2. The lack of effective therapeutic strategies for life-threatening heart conditions including myocardial infarction and heart failure attracts intensive exploration of underlying pathomechanisms and identification of novel therapeutic options3. For these scientific explorations, cardiac-specific gene manipulation is widely used4,5. Cardiac gene manipulation can be achieved by genome editing using the powerful transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) tools, or by delivery of ectopic genetic materials (e.g., virus vectors carrying genes encoding proteins of interest)6. Though genome editing allows precise and spatiotemporal genome modifications in living mice, it is still a time-consuming and labor-intensive practice6. Alternatively, cardiac-specific gene manipulation by virus vector or small interfering RNA (siRNA) complex delivery are routinely performed6.
Virus vector delivery to the adult mouse heart is achieved by roughly two strategies: systemic or local injection. Systemic injection of cardiotropic serotype of AAVs such as AAV9 is noninvasive for cardiac specific gene manipulation7. However, this method requires a relatively high amount of vector necessary for efficient transduction and gene expression, and may result in significant transduction of nontarget organs such as the muscle and liver7. Local virus injection is achieved by intramyocardial injection or intracoronary delivery7. Intracoronary delivery leads to a more even distribution of virus within the heart compared to intramyocardial injection. However, the disadvantages of this technique are the rapid wash out of viral vectors to the systemic circulation and transduction in nontarget organs8, and its requirement of devices for pressure measurement during the operation. By contrast, intramyocardial injection enables better virus retention in the myocardium as well as site specific delivery, but it fails to evenly distribute viral vector7. For small animals, intracoronary delivery is technically difficult to perform, while systemic AAV9 injection and intramyocardial injection are more commonly practiced4,5,7. Though systemic injection is easy to perform, conventional intramyocardial injection requires mechanical ventilation and thoracotomy, causes extensive tissue damage, and is time-consuming.
In this report, we described an easy, time-saving, and highly efficient method for intramyocardial injection. Adenovirus encoding luciferase and VDR, or shRNA targeting VDR, was injected to manipulate cardiac gene expression. Once mastered, this method can be used for gene manipulation, as well as injection of cells or other materials in the mouse heart.
All animal experiments were carried out according to the National Institutes of Health Guidelines on the Use of Laboratory Animals, and were approved by the Institute's Animal Ethics Committee. Male C57BL/6J mice (aged 8 – 10 weeks) were used for all the experiments. Mice were housed under pathogen-free conditions at 24 °C ± 4 °C, under a 12-h light/dark cycle, with free access to water and food.
1. Preparation of Adenovirus Solution
2. Anesthesia and Operative Preparation
3. Intramyocardial Injection of Adenovirus in Mouse Heart
4. Postoperative Management
5. In Vivo Imaging for Measuring Cardiac Luciferase Expression
6. Harvesting Tissues
7. Determination of Protein Expression
The experiment protocol and some of the key steps for the reported method are shown in Figure 1. At 5 days after intramyocardial injection of adenovirus encoding luciferase (Adv-luc), in vivo imaging in adv-luc injected mice indicated robust overexpression of luciferase specifically in the heart (Figure 2A, B), which was confirmed by Western blot analysis (Figure 2C), suggesting the absence of nontarget organ transduction. By contrast, no luciferase expression was detected in control mice. Consistent with the successful overexpression of luciferase, Western blot analysis suggested significantly increased VDR expression in the left ventricle of mice injected with adenovirus encoding VDR (adv-VDR) (Figure 3A). Moreover, adv-shVDR injection significantly reduced VDR expression in the left ventricle (Figure 3B). By contrast, VDR expression was not significantly changed in the right ventricles neither in adv-VDR injected mice or adv-shVDR injected mice (Figure 3C, D), because the adenovirus was only injected into the left ventricular myocardium.
Figure 1. Schema for mouse intramyocardial injection and gene expression detection protocol. (A) Illustration showing three injection sites in the myocardium of the left ventricle. (B) Protocol for gene expression detection in mouse heart after injection of indicated viruses. Adv-luciferase: adenovirus encoding luciferase; AdVDR: adenovirus encoding VDR; AdshVDR, adenovirus encoding shRNA targeting VDR. (C) Representative images showing multiple steps of the modified method for mouse intramyocardial injection. a. Removal of the fur by commercially available depilatory cream. b. Sterilization of the surgical site with 3 scrubs of povidone-iodine. c. Covering the surgical site with a sterile drape. d. 0.5-cm skin incision along the line connecting xiphoid and axilla. e. Blunt dissection of the pectoral major and pectoral minor muscles with forceps and a micro-mosquito hemostat. f. Externalization of the heart. g. Injection of 30 µL adenovirus solution into the myocardium of the left ventricle via the Hamilton syringe. h–i. Closure of the skin by a purse-string suturing with a 5-0 silk suture. Please click here to view a larger version of this figure.
Figure 2. Detection of luciferase expression in heart. (A) Images collected by the imaging system showing the luminescent signal of the heart in control mice and mice injected with adenovirus encoding luciferase on 5 days after injection. (B) Luminescent signal intensities in different groups (n = 3) are subjected to statistical analysis by t test. **p <0.01 versus control mice. (C) Western blot analysis results showing luciferase protein levels in heart, lung, liver, and spleen in indicated groups at 5 days after adenovirus injection (n = 3). Since luciferase expression was only detected in the heart of Adv-luc injected mice, statistical analysis was not performed. Adv-luc: adenovirus encoding luciferase. Please click here to view a larger version of this figure.
Figure 3. Detection of VDR expression in heart. (A) Top panel: Western blot bands showing VDR levels in the left ventricle (where the adenovirus is injected) at 0 day, 5 days, 7 days, and 14 days after adv-VDR injection. Bottom panel: semi-quantitative analysis of VDR expression levels in different groups (n = 4 per time point). Results were normalized against GAPDH and converted to fold change relative to 0 day. One-way analysis of variance (ANOVA) followed by the Bonferroni post-test (equal variances assumed) or Tamhane post-test (equal variances not assumed) was performed for statistical analysis. *p <0.05 or **p <0.01 versus 0 day. (B) Top panel: Western blot bands showing VDR levels in the left ventricle at 0 day, 3 days, 5 days, and 7 days after adv-shVDR injection. Bottom panel: semi-quantitative analysis of VDR expression levels in different groups (n = 4 per time point). *p <0.05 versus 0 day. (C) Top panel: Western blot bands showing VDR levels in the right ventricle (where the adenovirus is not injected) at 0 day, 5 days, 7 days, and 14 days after adv-VDR injection. Bottom panel: semi-quantitative analysis of VDR expression levels in different groups (n = 4 per time point). (D) Top panel: Western blot bands showing VDR levels in the right ventricle at 0 day, 3 days, 5 days, and 7 days after adv-shVDR injection. Bottom panel: semi-quantitative analysis of VDR expression levels in different groups (n = 4 per time point). Please click here to view a larger version of this figure.
The current report demonstrates a modified technique for intramyocardial injection of viral vectors for cardiac gene manipulation, which was modified from a method for myocardial infarction induction by Gao et al.13 Currently, in vivo characterization of specific gene functions most often involve the generation of knockout or transgenic mice3,14,15,16,17, which is expensive, time-consuming, and labor-intensive. Alternatively, delivery of gene vectors or siRNA by systemic or local injection is also widely practiced for gene manipulation in cardiovascular research4,5,7. In particular, intramyocardial injection cannot be substituted for cardiac gene manipulation under certain circumstances: when site directed injection is required (e.g., border zone injection in the myocardial infarction model)11; when duration-restricted gene manipulation is required (e.g., adenovirus injection). Here, we showed that the modified intramyocardial injection method is simple, time-saving, and highly efficient.
Critical Steps Within the Protocol and Troubleshooting:
For successful operation of this protocol, several critical steps should be noted. Before aspirating virus, the air within the Hamilton syringe and the attached needle must be evacuated, otherwise the air injected into the myocardium may cause topical cardiac injury or even death. To further avoid this issue, the ready-to-use Hamilton syringe filled with an adequate virus volume should not be placed with the plunger end downward, because this may spontaneously aspirate air by the gravity of the metal plunger. Anesthesia should be carefully monitored, as deep anesthesia may delay post-operation recovery, and severely deep anesthesia may cause death. After adequate anesthesia, the heart should not be externalized out of the chest cavity by force, since this may result in severe lung injury. Indeed, proper heart externalization requires only a gentle push of the heart, and any resistance may indicate pushing toward the improper direction.
Limitations of the Technique:
The elimination of intubation in this technique, which reduces the time needed for the procedure, suggests that the intramyocardial injection procedure should be finished within a relatively limited time window to avoid death. According to our experience, the heart should not be externalized for more than 30 s, because this increases the death rate and slows post-operative recovery. Therefore, the use of intubation is recommended for the first attempts of the protocol described here. Another limitation is the uneven distribution of viral vectors delivered by this method, which also exists in conventional methods of intramyocardial injection7. Moreover, the method described here is more useful for cardiac-specific gene manipulation in uninjured hearts, which can be followed by the establishment of different cardiac disease models18,19,20,21,22,23. However, the use of the current method in delivering agents to injured hearts such as infarcted hearts may be limited, because these mice may not tolerate the procedure.
Significance with Respect to Existing Methods:
Conventional intramyocardial injection requires intubation and mechanical ventilation24, and makes it difficult to locate the injection site due to the fast mouse heartbeat. These issues significantly prolong the operation time13, thus increasing variations posed by the time delay. The modified technique presented here is quick and allows precise injection site location by manually securing the externalized heart; overall, significantly potentiating subsequent study. Moreover, the elimination of intubation and mechanical ventilation make the modified method accessible to almost any laboratory.
The authors have nothing to disclose.
This work was supported by National Science Fund for Distinguished Young Scholars (81625002), National Natural Science Foundation of China (81470389, 81270282, 81601238), Program of Shanghai Academic Research Leader (18XD1402400), Shanghai Municipal Education Commission Gaofeng Clinical Medicine Grant Support (20152209), Shanghai Shenkang Hospital Development Center (16CR3034A), Shanghai Jiao Tong University (YG2013MS42), Shanghai Jiao Tong University School of Medicine (15ZH1003 and 14XJ10019), Shanghai Sailing Program (18YF1413000), and Postgraduate Innovation Program of Bengbu Medical College (Byycx1722). We thank Dr. Erhe Gao for his previous help in our lab.
Equipments | |||
Laminar flow sterile hood | Fengshi Animal Experimental Equipment Techonology Co., Ltd. (Soochow, China) | FS-CJ-2F | |
Centrifuge | Thermo Scientific (Waltham, USA) | 75005282 | |
Tissue grinding machine | Scientz Biotechnology Co., Ltd. (Ningbo, China) | Scientz-48 | |
High temperature/high pressure sterilizer | Hirayama (Saitama, Japan) | HVE-50 | |
Isoflurane vaporizer | Matrix (Orchard Park, USA) | VIP3000 | |
IVIS Lumina III imaging system | PerkinElmer (Waltham, USA) | CLS136334 | |
Precision balance | Sartorius (Göttingen, Germany) | 28091873 | |
Instruments | |||
Eppendorf pipette (100 µL) | Eppendorf (Westbury, USA) | 4920000059 | |
Eppendorf pipette (10 µL) | Eppendorf (Westbury, USA) | 4920000113 | |
Forceps | Shanghai Medical Instruments (Group) Ltd., Corp. | JD4020 | Curved tip |
Hamilton syringe | Hamilton (Nevada, USA) | 80501 | Volume 50 μL |
Micro-mosquito hemostat | F.S.T (Foster City, USA) | 13011-12 | Curved, tip width 1.3mm |
Needle holder | Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China) | J32110 | |
Surgical scissors | F.S.T (Foster City, USA) | 14002-12 | |
1-mL Syringe | WeiGao Group Medical Polymer Co.,Ltd. (ShangDong, China) | ||
Materials and reagents | |||
Anti-GAPDH antibody | CST (Danvers, USA) | #2118 | |
Anti-Luciferase antibody | Abcam (Cambridge, UK) | ab187340 | |
Anti-rabbit IgG | CST (Danvers, USA) | #7074 | |
Anti-VDR antibody | Abcam (Cambridge, UK) | ab109234 | |
Buprenorphine | Thermo Scientific (Waltham, USA) | PA175056 | |
Chloralic hydras | LingFeng Chemical (ShangHai, China) | ||
Cryogenic Vials | Thermo Scientific (Waltham, USA) | 375418 | 1.8 mL |
Depilatory cream | Veet (Shanghai, China) | ||
Dulbecco's phosphate buffered saline | Gibco (Grand Island, USA) | 14040133 | |
Entoiodine | LiKang (Shanghai, China) | 310132 | |
EP tube | Sarstedt (Newton, USA) | PCR001 | |
Filter | Millipore (Bedford, USA) | Pore size 0.2 µm | |
Isoflurane | Yipin Pharmaceutical Company (Hebei, China) | ||
Luciferin | Promega (Madison, USA) | P1041 | |
Lysis buffer for western blot | Beyotime (Shanghai, China) | P0013J | Without inhibitors |
Ophthalmic cream | Apex Laboratories ( Melbourne, Australia)) | ||
PBS | Gibco (Grand Island, USA) | 10010023 | |
Protease inhibitor cocktail | Thermo Scientific (Waltham, USA) | 78438 | |
5-0 silk suture | Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China) | ||
Steel ball | Scientz Biotechnology Co., Ltd. (Ningbo, China) | Width 1.5 mm | |
Syringe needle | Kindly Medical Devices Co., Ltd. (Zhejiang, China) | 30 gauge | |
Warm mat | Warmtact Electrical Heating Technology Co., Ltd. (Guangdong, China ) | NF-GNCW |