Ultrasound and Microbubble-targeted Delivery of a microRNA Inhibitor to the Heart Suppresses Cardiac Hypertrophy and Preserves Cardiac Function

Figures

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  Figure 1. Microbubbles loaded with antimiR-23a for ultrasound-mediated therapeutic delivery. (A) Fluorescence microscopy image of myosin-stained cells indicating cardiomyocyte purity of >80%. Nuclei were stained with Hoechst (blue). (B) Sequence and structure of LNA-modified antimiR-23a. (C) Schematic rendering of antimiR-loaded cationic lipid-coated microbubbles. (D) Size distribution of microbubbles loaded with antimiR-23a, as measured by Coulter counter.

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  Figure 2. Experimental setup for in vitro and in vivo ultrasound studies. (A) Schematic of experimental setup for in vitro studies of UTMC antimiR delivery to cardiomyocytes. (B) Schematic of experimental setup for in vivo studies of UTMC antimiR delivery to mouse hearts. AntimiR-loaded microbubbles were injected through the jugular vein and insonified in the heart with pulses from a clinical ultrasound imaging system. (C) Representative B-mode ultrasound image of nucleic acid-loaded microbubbles in mouse heart (acquired with Siemens Acuson ultrasound system).

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  Figure 3. Ultrasound delivery of antimiR-23a reduces miR-23a in cardiomyocytes in vitro. UTMC-targeted delivery of antimiR-23a reduced miR-23a levels relative to each negative control condition at various (A) doses (after 24 h phenylephrine stimulation) and (B) duration of phenylephrine exposure (n=5-6/group at 3.5 pmol, n=3-6/group at 7.0 pmol, n=6/group at 6 h, n=9/group at 24 h, n=3/group at 48 h); *p<0.05.

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  Figure 4. Ultrasound delivery of antimiR-23a reduces cardiomyocyte hypertrophy in vitro. (A) UTMC-targeted delivery of antimiR-23a at two different antimiR doses suppressed cardiomyocyte hypertrophy after 24 h of phenylephrine exposure compared to antimiR-NC delivery at the same doses (n=3-4/group); *p<0.05. (B) Representative microscopy images of cardiomyocytes before and after phenylephrine stimulation and UTMC treatment with 3.5 pmol of antimiR-23a or antimiR-NC (scale bar = 10 µm).

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  Figure 5. Ultrasound delivery of antimiR-23a reduces cardiac hypertrophy and improves cardiac function in vivo. Echocardiographic analysis indicating suppression of phenylephrine-induced left ventricular hypertrophy and preservation of systolic function with UTMC targeted delivery of antimiR-23a compared to UTMC delivery of antimiR-NC. The “no phenylephrine/no UTMC” group received neither phenylephrine nor ultrasound. Compared to UTMC + antimiR-NC microbubbles, UTMC + antimiR-23a microbubble treatment (A) blunted development of LV hypertrophy at 1 week (*p=0.02), and (B) preserved fractional shortening at 2 weeks (*p<0.01, ¥ p<0.02 vs baseline, n=5-9 animals/group). (C) Representative end diastolic (left) and end systolic (right) B-mode frames at 2 weeks in two mice, showing preservation of left ventricular systolic function with UTMC + antimiR-23a microbubbles (lower panels) compared to control (upper panels). Scales (right side of each image) are the same for all frames. In these images the measured LV wall thickness at end-diastole was 0.80 mm and 1.00 mm with UTMC + antimiR-23a MB treatment and control treatment, respectively. The internal diameter at end-diastole was 4.29 mm and 4.61…

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  Figure 6. Ultrasound delivery of antimiR-23a reduces miR-23a levels and suppresses cardiomyocyte hypertrophy in vivo. (A) UTMC delivery of antimiR-23a suppressed phenylephrine-induced elevation of miR-23a levels in mouse hearts compared to UTMC delivery of antimiR-NC (*p<0.01, n=5-9/group). (B) Cardiomyocyte size strongly tended to be less following UTMC delivery of antimiR-23a compared to UTMC delivery of antimiR-NC, (p=0.09, n=5-9/group). (C) Representative fluorescence microscopy images of wheat germ agglutinin-stained left ventricular sections, demonstrating that UTMC treatment with antimiR-23a microbubbles suppressed phenylephrine-induced cell growth. Scale bar represents 40 µm.