Binge Alcohol Exposure in Adolescence Impairs Normal Heart Growth

Figures

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  Figure 1. Adolescent binge alcohol exposure had no effect on whole body growth. A, Binge alcohol exposure paradigm began with gavage on postnatal day (PND) 37, with outbred, male adolescent rats randomized to water, binge, and acute groups. The expected time course of blood alcohol content (BAC) for each group is shown during the protocol, with a y‐axis scale of 0.10 g/dL. B and C, Food and water intake per cage (2 rats from the same treatment group per cage) was not different between the water and binge groups (n=4). D, All 3 groups had similar body growth over the treatment period (n=water: 20; binge: 21; acute: 8). E, BAC was elevated in the binge and acute groups but not the water group (n=water, 16; binge, 15; acute, 8). F, Corticosterone levels were elevated in the acute group (n=water: 8; binge: 8; acute: 8). Data are expressed as mean±SEM.

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  Figure 2. Binge rats exhibited slowed cardiac growth. A, Representative M‐mode echocardiography images from a water and binge rat at baseline (postnatal day [PND] 34) and post‐treatment. The green dashed line represents left ventricular (LV) end‐systolic diameter and the red dotted line represents LV end‐diastolic diameter. B, Normal cardiac growth during adolescence resulted in ≈65% growth in left ventricular end‐diastolic volume in the water group over the protocol (black open circles), but the binge rats (red open circles) had only half this increase in size. C, Perhaps as a compensation for this decrease in diastolic volume, the binge rats exhibited a decrease in LV end‐systolic volume, compared with an increase in the water group from normal growth. D, Water control rats, despite normal growth, exhibited a steady ejection fraction, while binge rats experienced a significant increase. E, The increased systolic function in binge rats was able to compensate for the reduced filling, and cardiac output is unchanged between the groups after treatment. F and G, The binge rats had a larger LV posterior wall diameter (LVPWd) than the water rats (F), while overall LV dimension (G) was…

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  Figure 3. Binge exposure accelerated normal cellular hypertrophy during adolescence. A, Representative hematoxylin and eosin histological images and (B) analysis of the water and the binge rats left ventricular (LV) cardiomyocyte cross‐section areas and quantitation analysis compared with baseline rats from the base, middle, and apex. (n=base: 182–374 cells per group; middle: 239–341 cells per group; apex: 50–68 cells per group from baseline: 6 rats, water: 4 rats, binge: 4 rats). Both water and binge groups were significantly larger (P<0.001) than baseline, from normal cellular hypertrophy, but the myocytes from binge rats grew more in the base and middle. C, Representative hematoxylin and eosin histological images and (D) analysis of the water and the binge rats LV cardiomyocyte cell lengths and the analysis compared with baseline rats at the base, middle, and apex (n=base: 58–326 cells per group; mid: 62–236 cells per group; apex: 41–321 cells per group from baseline: 6 rats, water: 4 rats, binge: 4 rats). Data are expressed as mean±SEM.

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  Figure 4. Myofilament calcium sensitivity is depressed via PKA (protein kinase A) phosphorylation of troponin I. A, The mean force as a function of calcium concentration and fitted curves for skinned myocytes from the water (black) and binge (red) groups. (B) Calculated values for individual myocytes and summary values for calcium sensitivity (EC 50). Binge rats exhibited a significant decrease in calcium sensitivity (n=water: 21 myocytes from 4 animals; binge: 21 myocytes from 4 animals). C, Representative Western blot images for total and phospho‐serine 22/23 cardiac troponin I in the water, binge, and acute groups. D, Quantification showing an increase in cardiac troponin I PKA phosphorylation in the binge group (n=water: 11; binge: 18). Data are expressed as mean±SEM. E, Single skinned myocyte passive tension generated at sarcomere lengths (SLs) between 1.8 and 2.4 μm (with the inset showing a single cell at ×40 magnification). The binge groups showed significantly higher passive tension over all SLs, likely from changes to titin (n=water: 9 cells from 3 rats; binge: 9 cells from 3 rats [**P<0.01, ***P<0.001 by 2‐way repeated measures ANOVA with Sidak multiple comparisons post hoc…

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  Figure 5. Binge rats exhibited a shift in gene expression. A, Extracellular signal‐related (Erk) kinase activation through phosphorylation, a central signaling kinase in physiological hypertrophy, decreased in binge rats (n=water, 10; binge, 9). B, Volcano plot from RNA sequencing data showing genes upregulated (10) or downregulated (58) by binge alcohol exposure in red (P<0.05 and log(2) fold‐change <−1 or >1). A selection number of these genes are labeled.

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  Figure 6. The structural effects of adolescent binge alcohol persist even after the cessation of consumption. A, Rats were left undisturbed for 7 days from gavage treatment after the 8‐day binge alcohol exposure paradigm. All rats had free access to pellet chow and water ad libitum. B, Both groups kept similar body growth post 1 week after treatment. C through G, Echocardiogram data comparisons between the water and the binge groups after 1 week of the cessation of binge alcohol exposure. C, Adolescent heart continued to grow after the 8‐day paradigm, but the binge group remained to have smaller left ventricular end‐diastolic volume compared with the water group. D, The binge group continued to have larger LV posterior wall diameter than the water group. E, The binge group had smaller stroke volume compared with the water group. F, Ejection fraction was no longer different between the binge and water groups after 1 week from binge alcohol exposure. G, The binge group had faster heart rates compared with the water group. n=water: 9; binge: 11. Data are expressed as mean±SEM.

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  Figure 7. Schematic of the impact of adolescent binge alcohol consumption on the heart. Binge alcohol inhibits extracellular signal‐related kinase and atrial natriuretic peptide to switch growth from physiologic to pathologic and activates PKCα (protein kinase C α), leading to titin phosphorylation and increased cellular stiffness. The adolescent heart compensates for the decreased cardiac output from their smaller hearts by increasing systolic function through PKA (protein kinase A) activation, increasing phosphorylation of its targets such as troponin I, leading to myofilament calcium desensitization. The chronic consequences of hypertrophy and impaired relaxation may sensitize the adolescent for heart failure in adulthood.