Ultrasound-Targeted Microbubble Destruction Enhances Gene Expression Of Microrna-21 In Swine Heart

Objective Ultrasound-targeted microbubble destruction (UTMD) has proved to be a promising method for gene delivery. However, the feasibility and efficacy of UTMD-mediated gene delivery to the heart of large animals remain unclear. The present study was to explore the probability of increasing the transfection of microRNA-21 (miR-21) into swine heart by UTMD and to search for the most suitable transfection conditions.Methods Eukaryotic expression plasmid pGV262-EGFP-premiR-21 was constructed and transfected into SUVECs to identify its expressing activity. We first optimized ultrasound intensity for successful miR-21 delivery. After intravenous injection of miR-21/microbubble mixture (miR-21/MB), transthoracic ultrasound irradiation (US) was applied from the left anterior chest using different intensities (1 W/cm,2W/cm2, and 3W/cm2). Then the efficacy of UTMD-mediated miR-21 delivery into myocardium via intracoronary injection was explored. Solution of miR-21/MB was infused intravenously or intracoronarily with US over the heart. Swine undergoing PBS injection, miR-21/MB injection via ear vein or coronary artery without US served as the control. Cardiac function was measured by echocardiography; serum troponin Ⅰ (cTnI) was detected by electrochemiluminescence (ECL). Then left ventricular myocardium was harvested 4 days later. Frozen sections were made and fluorescence microscope was used to observe the expression of EGFP; morphological changes of myocardium were explored with HE staining. The expression levels of miR-21 and PDCD4 were detected by FQ-PCR and Western blot, respectively.Results Eukaryotic expression plasmid pGV262-EGFP-premiR-21 was constructed successfully and could express in vitro. Results from echocardiography showed that there was no statistical difference in cardiac function over time regardless of different intensities of pulse ultrasound (P>0.05). None of the animals receiving 1W/cm2 or 2W/cm2 US had an increase in serum cTnI at any time point (P>0.05); however, in animals that received 3W/cm2 US, serum cTnI was increased at 3h, peaked at 12h, decreased gradually thereafter, and returned to normal at 4 days (P<0.05). Fortunately, structures of the myocardium in the three groups were all well preserved, no microscopic evidence of myocardial injury, hemorrhage or inflammation were found as shown by HE staining. The expression level of miR-21 in 2W/cm2 US group was significantly higher than that in 1W/cm2 and 3 W/cm2 US group (P<0.05); as a result, PDCD4 was significantly inhibited and was lowest in 2W/cm2 US group as shown by Western blot (P<0.05).As revealed by fluorescent microscope, no EGFP could be observed in blank control group; simply intravenous or intracoronary injection of miR-21/MB, EGFP could be seen and was mainly located at subendocardium or around the vessels; while UTMD could significantly enhance EGFP expression. Results from FQ-PCR showed that simply intravenous injection of miR-21/MB could not increase miR-21 expression (P>0.05), and intracoronary injection could somehow improve miR-21 expression, but it was still too low (P<0.05). Compared with miR-21/MB alone, UTMD could significantly enhance miR-21 expression regardless of the injection routes (P<0.05). Interestingly, miR-21 expression level was found to be a little bit higher with intracoronary injection than that with intravenous injection, though the dose for intracoronary injection was half that for intravenous injection (P<0.05). Again, PDCD4 was dramatically inhibited by UTMD-mediated miR-21 delivery as compared to that of simple miR-21/MB injection, and the decrease in PDCD4 expression was significantly related to UTMD with miR-21/MB given via coronary artery. However, the difference in transfection efficiency between intracoronary injection and intravenous injection in UTMD-mediated gene delivery was rather small.Conclusion Under pulse ultrasound at an intensity of 2W/cm2, a frequency of 1MHz and a 50% duty ratio for 20 minutes, UTMD can efficiently enhance gene expression in swine heart regardless of the delivery routes. Intravenous injection might be superior to intracoronary injection with less invasiveness and lower requirement of technique. However, for those undergoing percutanesous coronary intervention, intracoronary injection seems to be another alternative.