Abstract P2008: Hypertrophic Cardiomyopathy Treatment Using Ultrasound And Microbubbles

Abstract

Introduction: Hypertrophic cardiomyopathy is a disease characterized by the abnormal growth of cardiomyocytes (CMs), a marker of which is the early downregulation of miR-1. The delivery of miR-1 to hypertrophic CMs presents as a promising treatment strategy, however there are still limitations in the safe and efficient delivery of gene therapeutics.Ultrasound (US) and microbubbles is an emerging approach to site-specific gene delivery. Microbubbles have long been employed in clinical cardiology as a diagnostic contrast agent. Recent work has demonstrated that under specific conditions, they are capable of forming temporary pores on neighboring cell membranes, allowing the delivery of otherwise impermeable macromolecules. This presents an exciting approach to the treatment of cardiovascular disease (CVD) as a targeted and inherently image-guided therapeutic delivery platform. Objective: The goal of this study is to determine the feasibility of viable US and microbubble mediated delivery of miR-1 to hypertrophic CMs with a view towards reversing the disease. Secondly, we aim to show a correlation between bubble echoes and resulting gene delivery to introduce clinical metrics for the translation of this technique. Methods: CMs were harvested from neonatal rat pups (1-3 days old) and hypertrophied using phenylephrine (100μM). Suspension of healthy or diseased CMs, microbubbles and free miR-1 were placed in a chamber within a 37°C degassed water bath. These samples were insonicated at a mechanical index (MI) ranging from 0.063-0.25 for 2 minutes (20 cycles; 5 kHz PRF) and bubble echoes were recorded via a co-aligned transducer to be processed offline. miR-1 delivery was confirmed with RT-qPCR, cell viablity assesed with MTT, protein expression was measured by immunoblotting and cell size via microscopy. All experiments preformed are at least n=2 independent samples. Result and Conclusion: Treated healthy and diseased CMs demonstrate an upregulation in miR-1 with increasing MI, from 0.063 to 0.188. At MI=0.12, a 2.22 and 1.95-fold increase in miR-1 was shown in diseased and healthy CMs respectively ( p= 0.07, p= 0.02) while maintaining viability (86% and 96% respectively). These miR-1 levels begin to show the reversal of hypertrophy as quantified by increases in target protein expression (1.93-fold in TWF1; 1.38-fold in MEF2A; 1.36-fold in CX43) and cell size decrease (16%, p =0.035) back towards heathy levels. Echo data highlights that relative miR-1 expression positively correlates with bubble disruption ( r =0.76, p =0.04), suggesting a means by which treatment efficiency can be monitored in a clinical setting.In conclusion, we demonstrated that US and microbubbles can be used to successfully delivery miR-1 to CMs, with the potential to reverse the disease phenotype. These findings demonstrate the feasibility of US as an image-guided delivery method for molecular therapeutics in CVD.