Abstinence Restores Cardiac Function in Mice with Established Alcohol-Induced Cardiomyopathy

Figures

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  Figure 1. Experimental timeline: Acclimation to liquid diet was allowed for 5 days, before animals were switched to ethanol diet. At days 10 and 30, animals were gavaged with 5 g/kg ethanol or isocaloric 9 g/kg maltose–dextrin. Echocardiography was performed at least 12 h after gavage. Terminal LV catheterization was performed the day after echocardiography was completed. There were 4 groups in total: mice receiving alcohol for 30 days with 2 binge doses (30d EtOH), mice on control diet for 30 days (30d Cntl), mice that after 30 days of alcohol exposure (and 2 binges) were transitioned to control diet (30d/30d Abst), and mice that received the control diet for 60 days (60d Cntl).

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  Figure 2. Heart and body weights (A) Body weights. (B–D) Hearts weights were normalized using tibial length. These data were analyzed using one way ANOVA (LV: left ventricle; RV: right ventricle; ns = not significant, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

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  Figure 3. Echocardiography data at 30 and 60 days. (A–D) There were no significant changes in LV wall dimensions at 30 days. (E–H) LV anterior wall (LVAW) in both systole (s) and diastole (d) increased in 30d/30d Abst mice compared to 60d Cntl mice (** p < 0.01) with no significant difference in LV posterior wall (LVPW). Data were analyzed using Student’s t test (ns = not significant).

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  Figure 4. Representative LV pressure-volume loops for each group. Red lines indicate end-systolic pressure-volume relationship (ESPVR), while blue lines indicate end-diastolic pressure-volume relationship (EDPVR). Steady-state pressure-volume loops for: (A) 30d Cntl mice, (B) 30d EtOH mice, (C) 60d Cntl mice, and (D) 30d/30d Abst mice.

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  Figure 5. LV catheterization data at 30 and 60 days. (A–D) 30d EtOH mice had decreased systolic and diastolic function compared to 30d Cntl mice (** p < 0.01). (D) There was a trend for reduction in time relaxation constant, Tau (p < 0.051). (E–H) At 60 days, 30d/30d Abst mice exhibited increased systolic function, and diastolic function increased to match 60d Cntl diastolic function. (E) Stroke work and (F) dP/dt max were significantly increased compared to 60d Cntl mice (* p < 0.05). (G–H) Both dP/dt min and Tau were not significantly different versus 60d Cntl mice. Data were analyzed using Student’s t test (ns = not significant).

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  Figure 6. Load-independent measures of cardiac contractility. (A,B) At the 30-day timepoint, the slope of the end systolic pressure–volume relation, E(es), and preload-recruitable stroke work (PRSW) were significantly decreased in the 30d EtOh mice (* p < 0.05, **** p < 0.0001). (C,D) This reduction was abrogated by abstinence; both E(es) and PRSW in the 30d/30d Abst mice were not significantly different versus control mice at 60 days. Data were analyzed using Student’s t test (ns = not significant).

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  Figure 7. Echocardiography and LV catheterization data in control mice from day 30 to day 60. (A–H) There were no significant differences in LV wall dimensions or load-dependent measures of contractility. (I) E(es) was also not significantly changed. (J) PRSW significantly decreased at day 60 (* p < 0.05). Data were analyzed using Student’s t test (ns = not sinificant).

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  Figure 8. Echocardiography and LV catheterization data in 30d EtOH mice compared to 30d/30d Abst mice. (A,B) LVAW size increased in both systole and diastole with abstinence. (C,D) While LVPW in diastole did not significantly change, LVPW in systole increased. (E,F) Both systolic measures of stroke work and dP/dt significantly increased in 30/30d Abst mice. (G) The diastolic measure dP/dt min also significantly increased. (H) Tau was not significantly changed. (I,J) E(es) significantly increased with abstinence, while PRSW did not. Data were analyzed using Student’s t test (* p < 0.05, ** p < 0.01, **** p < 0.0001; ns = not significant).

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  Figure 9. mRNA assessment using qPCR. (A–C) Relative amounts of inflammatory cytokines. (A) IL-6 was significantly decreased after abstinence (* p < 0.05). (B) IL1β was significantly increased after 30 days of alcohol exposure compared to control (** p < 0.01) and was significantly decreased after abstinence (*** p < 0.001). (C) IL10 was not significantly changed by alcohol exposure or abstinence. (D–G) Relative amounts of fibrotic markers. (D) LARP6 was significantly increased after 30 days of alcohol exposure (* p < 0.05) and returned to control levels with abstinence. (E) COL1A1 was significantly decreased after abstinence relative to 30d EtOH. (F) COL3A1 was decreased with 30 days of alcohol exposure (**** p < 0.0001) and returned to near control levels after abstinence. (G) The COL1A1/COL3A1 ratio was increased after 30 days of alcohol exposure (*** p < 0.001) and restored to control levels by abstinence (*** p < 0.001). Data were analyzed using a one-way ANOVA, and post hoc analysis was performed using Tukey’s multiple comparisons test.

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  Figure 10. (A,B) Representative images of picrosirius red staining of collagen (2× macro; 20× inset). (C,D) Representative images of hematoxylin and eosin staining. (E) Quantification of picrosirius red staining found no significant changes in collagen deposition after 30 days of alcohol. Macro images were edited using Adobe Photoshop to remove cutting artifact and improve color. The 20× images of LV that were used for collagen quantification were processed raw and not modified.