Remuscularization with triiodothyronine and β1-blocker therapy reverses post-ischemic left ventricular dysfunction and adverse remodeling

Figures

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  Figure 1. T3-induced ERK1/2 phosphorylation and cyclin expression in adult ventricular CMs is potentiated by β1-AR siRNA. (A) Schematic showing the protocol for β1-AR siRNA and T3 administration and analysis times. (B) Representative immunoblots show expression of indicated proteins in lysates obtained from CMs at 40 h after T3 administration. (C–M) Quantitative data obtained from analysis of immunoblots for β1-AR (C), cyclin D1 (D), cyclin A2 (E), cyclin B1 (F), ECT2 (G), p-MEK1/2 (H), MEK1/2 (I), p-ERK1 (J), ERK1 (K), p-ERK2 (L) and ERK2 (M). The data were normalized with loading control (GAPDH) and data were expressed as relative value for scrambled control siRNA without T3 therapy. Immunoblots are representative of 4 biologically independent replicates. Data are mean ± SEM. ***P < 0.001.

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  Figure 2. M + T3 therapy muscularizes the adult heart and increases LV contractile function. (A) Decay of T3 in the circulation after the end of T3 therapy (2 ng/g body weight, daily for 5 days; i.p.) or vehicle (V) control. Comparisons at each time point were made using an unpaired, 2-sided t-test. n = 5 mice/group. ***P < 0.001. (B) Ventricular CM numbers at 14- and 42-days post-therapy (dpt) with vehicle, T3, metoprolol (M) or M + T3. Comparisons were made using 2-way ANOVA, followed by Sidak’s multiple comparison test. n = 7–9 mice/group. ***P < 0.001 for within group (i.e., at 14 or 42 dpt) comparison. (C–E) Changes in body weight (C), LV mass (D) and LVEF (E) at 0 and 14 dpt with V, T3, M or M + T3. In each case, comparisons between values at 0 and 14 dpt were made using a paired, 2-sided t-test. n = 6 mice/group. ***P < 0.001.

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  Figure 3. Sustained reversal of systolic heart failure after short-term M + T3 therapy. (A) Schematic of the MI experiment in adult mice. The timing and dosage of metoprolol and T3 treatment are indicated (red and blue boxes), as are the timing of MI (red arrow) and duration of posttherapy follow-up. Green arrows indicate when serial echocardiography measurements were taken. (B,C) LV ejection fraction (B) and end-systolic volumes (C) of untreated MI-injured mice or those given T3 or metoprolol monotherapy or M + T3 therapy. Data from uninjured age-matched mice are also shown. n = 4 mice per group. Individual data points and mean values ± SEM are shown. **P < 0.01, ***P < 0.001 comparing the therapy group to all other post-MI groups at each assessment period.

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  Figure 4. Newly formed CMs in M + T3-treated post-MI hearts are derived from preexisting CMs. (A) Schematic showing the Confetti construct and the protocol for pre-MI labeling of CMs by 4-hydroxytamoxifen and subsequent post-MI therapy with M + T3. (B) Cre-induced recombination could either induce the expression of green fluorescent protein (GFP), yellow (YFP), red (RFP) or cyan (CFP) in CMs following 4-hydroxytamoxifen administration. (C) Pie charts showing RFP- and YFP-expressing monochromatic CM cluster sizes. The number of individual RFP or YFP CMs evaluated for each cluster analysis is indicated in the middle of the Pie charts. Representative images (right) of monochromatic CM clusters with cell outlines delineated using wheat germ agglutinin (WGA)-staining (white). Dense WGA staining (bottom right panel) identifies regions of fibrosis. Numbers indicate contiguous CMs in a cluster. (D) Bar graph shows frequency of monochromatic CM clusters at 15 dpt. n = 4 mice per group. Individual data points and mean values ± SEM are shown.

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  Figure 5. Early regrowth of the post-infarcted ventricular myocardium after M + T3 therapy. (A,B) Longitudinally cut hearts (both halves) of a 30 days post-MI injury (dpi) (A) and a 15 days post-therapy (dpt) (B) heart. The white arrowheads show the site of coronary artery ligation, the green arrows show the infarct and the red arrowheads show extension of the intraventricular septum (IVS) toward the ligature site. Insets show trichrome-stained sections from indicated hearts. (C) An uninjured age-matched control heart. Each image set is representative of 2–4 hearts at each time point.

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  Figure 6. M + T3 therapy remuscularizes the infarcted mid-apical LV PW. (A,B) Gross anatomy (A) and trichrome staining (B) of hearts from mice that were either uninjured (left), left untreated after an MI (middle) or treated with M + T3 therapy post-MI (right) at 150 days post-therapy (dpt). The images are representative of 3–4 hearts per group. The insets in (B) show a magnified view of the boxed region. Supplementary Fig. S4A–E, show other examples of gross anatomy of post-MI heart, including those shown in “A”, that were untreated or given T3 monotherapy, metoprolol monotherapy or M + T3 therapy. (C) Quantitative analysis of viable myocardium in the LV mid-apical PW at 150 dpt. Data represent values from 3 independent hearts per group. ***P < 0.001. (D,E) Representative longitudinal tissue section (7 µm thick) images, representing 0.2 mm-long views of the LV posterior wall at the mid-apical region, from an untreated post-MI mouse (A) and from a post-MI mouse after M + T3 therapy (B). These hearts are from mice followed for 150 dpt. CMs are visualized using cardiac troponin T (cTnT) staining (red) and cell boundaries are outlined by wheat-germ agglutinin (WGA) staining (white).…

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  Figure 7. M + T3 therapy restores motion to a post-infraction segment of LV mid-apical PW. (A) Schematic of a post-MI heart showing the site of echocardiographic measurements. (B, C) Quantitative analysis of LV mid-apical fractional shortening ((LV end-diastolic dimension − end-systolic dimension) × 100/LV end diastolic dimension) (B) and absolute PW thickening (mid-apical LV PW thickness at end-systole – end-diastole) (C) at 30 dpi and 150 dpt. n = 4 mice per group. ***P < 0.001. Individual data points and mean values ± SEM are shown. See also Supplementary Movies S1–S3.

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  Figure 8. M + T3 therapy reverse-remodels the post-MI LV. (A) Illustration showing the LV end-diastolic long- and short-axis dimensions, which were determined using echocardiography. (B) Bar graph showing long-to-short axis ratio before MI surgery (baseline, BL), at 30-days post-MI injury (30 dpi) and at 30-, 90- and 150-days after cessation of M + T3 therapy (days post-therapy, dpt). n = 4 mice per group. Data are shown as individual values and the mean ± SEM. Comparisons were made by two-way ANOVA followed by Sidak's multiple comparisons test. **P < 0.01, ***P < 0.001. (C) LV volumes at end-diastole of untreated MI-injured mice or those given T3 or metoprolol monotherapy or M + T3 therapy. Data from uninjured age-matched control hearts are also shown. Two-way analysis of variance (ANOVA), followed by Sidak's multiple comparisons test. **P < 0.01, ***P < 0.001 comparing the combination therapy group to all other post-MI groups at each assessment period. n = 4 mice per group. Data are shown as individual values and the mean ± SEM.