Dietary Branched‐Chain Amino Acids Modify Postinfarct Cardiac Remodeling and Function in the Murine Heart

Figures

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  Figure 1. Dietary BCAA levels modulate circulating BCAAs. A, Mice were fed a low (1/3×), normal (1×), or high (2×) BCAA diet for 7 days before measurement of (B) plasma BCAA concentrations; (C) body mass; and (D) heart weight (normalized to tibial length). n=4 male and 4 female mice per group. Panel B shows pooled male and female mice. Data are mean±SEM. Normality was assessed via Shapiro–Wilk test, Panel B–D, 1‐way ANOVA with Tukey's post hoc or Kruskal–Wallis with Dunn's post hoc. BCAA indicates branched‐chain amino acid; HW, heart weight, and TL, tibia length.

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  Figure 2. Low dietary BCAA levels prevent cardiac hypertrophy and MI‐induced congestive heart failure. Gravimetric measurements: A, Schematic of study design: mice were fed a low or high BCAA diet for 7 days before nonreperfused MI and continued on the respective diet for 28 days after MI; B, Female and (C) male body mass recorded at baseline and 2 and 4 weeks post MI; D, Heart weight and heart weight normalized to tibia length measured 4 weeks post MI; E, Wet/dry lung mass ratio measured 4 weeks post MI; n=7–10/group. Data are mean±SEM. Normality was assessed via Shapiro–Wilk test Panel B, C, 2‐way repeated measures ANOVA with Šidák post hoc; Panel D, E, Unpaired 2‐tailed t test; BCAA indicates branched‐chain amino acid; HW, heart weight; HW/TL, heart weight normalized to tibia length; MI, myocardial infarction; and TL, tibia length.

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  Figure 3. Low dietary BCAA levels prevent MI‐induced decreases in stroke volume and cardiac output. Echocardiographic measurements for (A) heart rate; (B) end‐systolic volume; (C) end‐diastolic volume; (D) isovolumetric relaxation time; (E) fractional shortening; (F) ejection fraction; (G) stroke volume; (H) cardiac output; (I) cardiac index; n=7 to 10/group. Data are mean±SEM. Two‐way repeated measures ANOVA with Šidák post hoc. BCAA indicates branched‐chain amino acid; CO, cardiac output; EF, ejection fraction; FS, fractional shortening; HR, heart rate; IVRT, isovolumetric relaxation time; LVEDV, left ventricular end‐diastolic volume; LVESV, left ventricular end‐systolic volume; MI, myocardial infarction; and SV, stroke volume.

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  Figure 4. Low dietary BCAA results in smaller LV mass and wall thickness post MI. Echocardiographic measurements of structural features: (A) calculated left ventricular mass (corrected); (B, C) left ventricular internal diameter in systole and diastole; (D, E) left ventricular anterior wall thicknesses in systole and diastole; (F, G) left ventricular posterior wall thicknesses in systole and diastole. 7–10/group. Data are mean±SEM. Two‐way repeated measures ANOVA with Šidák post hoc. BCAA indicates branched‐chain amino acid; LV, left ventricular; LVAW;d, left ventricular anterior wall diastole; LVAW;s, left ventricular anterior wall systole; LVID;d, left ventricular interior diameter diastole; LVID;s, left ventricular interior diameter systole; LVPW;d, left ventricular posterior wall diastole; LVPW;s, left ventricular posterior wall systole; and MI, myocardial infarction.

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  Figure 5. Cardiac fibroblast activation in vitro is dependent on BCAA availability. Isolated murine cardiac fibroblasts were treated with TGF‐β (or vehicle control) for 48 hours in medium containing different concentrations of BCAAs (0, 100, 400 μM) and were assessed for Col1a1, periostin, and α‐smooth muscle Actin abundance by immunoblotting. A, Representative image of immunoblots; B, quantification of Col1a1 abundance; C, quantification of periostin abundance; and D, quantification of α‐SMA abundance. Proteins were normalized to amido black signal from stained membranes. n=8/group, with males and females pooled. Data are shown as mean±SEM. Statistics: Two‐way ANOVA with Tukey's post hoc test. α‐SMA indicates α‐smooth muscle actin; BCAA, branched‐chain amino acid; and TGF‐β, transforming growth factor beta.

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  Figure 6. Modified dietary BCAA does not affect cardiac fibrosis and scar remodeling. Examination of cardiac collagen content and organization: A, Intact midpapillary sections stained with PSR; B, quantification of PSR signal (cardiac fibrosis) and (C) scar thickness (n=6–9 biological replicates); D, Computational assessment of scar collagen architecture in SHG‐imaged infarct segments. Left to right, representative master SHG images from postinfarct scar regions (region of interest: 256×256 pixel), CurveAlign‐generated images displaying the spatial distribution (red dots) and direction (green lines) of collagen fiber segments, corresponding CurveAlign sourced heatmaps depicting the relative alignment of collagen fibers across the region (red denotes regions of high alignment), and, lastly, CT‐FIRE extracted fibers overlaid on master SHG images (individual fibers are separated by varying colored lines). From these computational analyses, macrostructural collagen attributes were enumerated and graphed, including (E) fiber alignment, (F) fiber width, and (G) fiber straightness (n=3 biological replicates per sex per group). Data are mean±SEM. Normality was assessed via Shapiro–Wilk test.…

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  Figure 7. High dietary BCAA consumption is associated with eccentric cardiomyocyte hypertrophy and higher capillary density in male mice. A–C, Cardiomyocyte cross‐sectional area (wheat germ agglutin), and capillary density (Isolectin B4); D, E, Cardiomyocyte length measurements (averaged from 13 to 73 identified cells per section). Data are mean±SEM. n=6 to 9 biological replicates; Normality was assessed via Shapiro–Wilk test. Parametric unpaired Student's t test or nonparametric Mann–Whitney U test as appropriate. BCAA indicates branched‐chain amino acid; and WGA, wheat germ agglutin.