Delayed miR-199a Administration After Myocardial Infarction Precludes Pro-Regenerative Effects

Figures

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  Figure 1. Experimental Protocols Pigs were randomly assigned to a W1 or W2 protocol and further randomly assigned to administration of adeno-associated virus serotype 6 (AAV6)-control or AAV6-miR-199a. MI = myocardial infarction; MRI = magnetic resonance imaging; PES = programmed electrical stimulation; W1 = week 1; W2 = week 2.

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  Figure 2. miR-199a Delivery Did Not Affect MI Scar Size Infarct mass (A), size (B), infarct core (C), and infarct gray zone (D) (calculated from late gadolinium enhancement [LGE]-positive regions as a percentage of the left ventricle) at 2 days before (baseline) and 28 days after adeno-associated virus (AAV) administration in AAV serotype 6 (AAV6-control (5-6) and AAV6-miR-199a (5-7)), pigs. Data are mean ± SEM. Two-way analysis of variance. (E and F) LGE cardiac magnetic resonance images (from base to apex) are shown from 2 representative pig hearts of the W1 protocol to compare AAV6-control (E) with AAV6-miR-199a (F) at 2 and 28 days after MI. The infarct area is highlighted in red; the corresponding original images without highlight are shown in Supplemental Figure 1. Abbreviations as in Figure 1.

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  Figure 3. Late miR-199a Delivery Does Not Improve Global Cardiac Function (A) Left ventricular (LV) end-diastolic volume (P = 0.079), (B) LV end-systolic volume (P = 0.19), (C) LV ejection fraction (P = 0.062), (D) cardiac output (P = 0.072), (E) LV stroke volume (P = 0.023), and (F) LV mass (P = 0.005) measured by cardiac MRI in noninfarcted controls and infarcted pigs 2 days before (baseline) and 28 days after injection of adeno-associated virus serotype 6 (AAV6-control (5-6) and AAV6-miR-199a (5-7)). Data are mean ± SEM. ∗P < 0.05 vs sham. Two-way analysis of variance with Bonferroni post-hoc correction. See detailed results in Supplemental Table 1 for LV stroke volume and Supplemental Table 2 for LV mass. Abbreviations as in Figure 1.

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  Figure 4. Cardiac Regional Wall Motion Left ventricular (LV) wall thickening long (A) and short-axis (B) cardiac MRI (cMRI) segmentation scheme for regional wall motion evaluation. The red arrow indicates the reference point (ie, the LV inferoseptal segment at the connection of the right ventricle). (C) cMRI tagging segmentation scheme for LV strain evaluation. (D and E) Six-segment curves generated from LV segmental wall thickening of W1 (D) and W2 (E) protocol groups with their corresponding AUCs quantified in arbitrary units (5-6). (F and G) Eight-segment curves generated from LV radial strain in W1 (F) and W2 (G) protocol groups and their corresponding AUCs (4-6). Data are mean ± SEM ∗P < 0.05 vs sham. One-way analysis of variance with Bonferroni post hoc correction. AAV6 = adeno-associated virus serotype 6; AUC = area under curve; circumferential segments: I = inferior; IL = inferolateral; AL = anterolateral; A = anterior; AS = anteroseptal; IS = inferoseptal; other abbreviations as in Figure 1.

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  Figure 5. Electrophysiological Substrate Evaluation (A) Kaplan-Meier curve depicting survival rates in pigs treated 2 weeks and 1 week after MI (AAV6-control (5-6) and AAV6-miR-199a (5-7)); P < 0.05 vs control animals). No differences were found in miR-199a treated and control pigs in terms of ventricular fibrillation induction (B), the time between two successive R-waves of the QRS signal on the electrocardiogram (C), QT interval (D), and QTc (corrected QT) (E) (1 week protocol; (AAV6-control n = 5 and AAV6-miR-199a n = 5). Data are mean ± SEM; unpaired t test. (F) Representative case of ventricular fibrillation induction with the PES protocol in a AAV6-miR-199a pig following the stimulation of the train 600 ms (black arrows), S1-420 ms (green arrow), and S2-350 ms (blue arrow). (G) Representative case of ventricular fibrillation induction with the PES protocol in a AAV6-control following the stimulation with coupling intervals of the train 500 ms (black arrows), S1-320 ms (green arrow), S2-320 ms (blue arrow), S3-240 ms (red arrow). Abbreviations as in Figures 1 and 2.

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  Figure 6. Histologic and Molecular Analyses Systematic assessment of miR-199a-3p expression after AAV6-mediated transduction. (A) Schematic representation of pig heart sectioning for histologic and molecular studies. Four 1-cm-thick transversal slices were cut starting from the base to the apex (1-4). Each slice was subsequently divided into 2 to 9 regions, each 1 labeled with a capital letter, and then into additional subregions (letters plus numbers) for targeted molecular and histologic analyses. Sectors T, I, and D correspond to the infarct border zone, where the vectors were administered, whereas sector L was considered representative of the remote zone. Injection and infarct border segments for each slice were divided into smaller fragments (dashed lines) to accurately assess the levels of expression of the transgene at 56 days after transduction. (B) Real-time polymerase chain reaction quantification of mature miR-199a-3p expressed as fold change over endogenous levels (assessed in AAV6-control pigs) in the indicated regions for a representative animal. (C) Average expression levels of miR-199a-3p in the treated animals. Each data point represents an individual animal, averaging the…