Deficiency of ataxia-telangiectasia mutated kinase attenuates Western-type diet-induced cardiac dysfunction in female mice

Abstract

Chronic consumption of Western-type diet (WD) induces cardiac structural and functional abnormalities. Previously, we have shown that WD consumption in male ATM (ataxia-telangiectasia mutated kinase) deficient mice associates with accelerated body weight (BW) gain, cardiac systolic dysfunction with increased preload, and exacerbation of hypertrophy, apoptosis, and inflammation. This study investigated the role of ATM deficiency in WD-induced changes in functional and biochemical parameters of the heart in female mice. Six-week-old wild-type (WT) and ATM heterozygous knockout (hKO) female mice were placed on WD or NC (normal chow) for 14 weeks. BW gain, fat accumulation, and cardiac functional and biochemical parameters were measured 14 weeks post-WD. WD-induced subcutaneous and total fat contents normalized to body weight were higher in WT-WD versus hKO-WD. Heart function measured using echocardiography revealed decreased percent fractional shortening and ejection fraction, and increased LV end systolic diameter and volume in WT-WD versus WT-NC. These functional parameters remained unchanged in hKO-WD versus hKO-NC. Myocardial fibrosis, myocyte hypertrophy, and apoptosis were higher in WT-WD versus WT-NC. However, apoptosis was significantly lower and hypertrophy was significantly higher in hKO-WD versus WT-WD. MMP-9 and Bax expression, and Akt activation were higher in WT-WD versus WT-NC. PARP-1 (full-length) expression and mTOR activation were lower in WT-WD versus hKO-WD. Thus, ATM deficiency in female mice attenuates fat weight gain, preserves heart function, and associates with decreased cardiac cell apoptosis in response to WD.

Keywords: ATM; Western-type diet; apoptosis; female; heart.

FIGURE 1

WD‐induced weight gain with time. (a) Visceral (left panel) and subcutaneous (right panel) adipose distribution after 14 weeks in NC or WD groups. (b) Weight gain for normal chow (NC) and Western‐type diet (WD) groups from week 0 to week 14. ^@ p < 0.05 versus WT‐NC, n = 7–11; statistical analysis was performed using two‐tailed Student's t‐test.

FIGURE 2

WD‐induced changes in M‐mode parameters of the heart. Indices of M‐mode parameters: % fractional shortening (%FS), % ejection fraction (%EF), LV end systolic diameter (LVESD), and LV end systolic volume (LVESV) were measured/calculated using M‐mode echocardiographic images following 14 weeks of NC or WD. (a) Representative M‐mode tracings for each group; (b) %FS; (c) %EF; (d) LVESD; (e) LVESV. *p < 0.05 versus WT‐NC, ^# p < 0.05 versus WT‐WD, n = 10. Statistical analysis was performed using two‐way ANOVA followed by Newman–Keuls test.

FIGURE 3

WD exacerbates fibrosis in wild‐type mice. (a) Masson's trichrome‐stained sections of the heart for each group and associated diet. Blue staining indicates fibrosis, while red staining indicates muscle tissue. (b) Quantitative measurements of fibrosis. *p < 0.05 versus WT‐NC, n = 4. Statistical analysis was performed using two‐way ANOVA followed by Newman–Keuls test.

FIGURE 4

ATM deficiency increases myocyte hypertrophy in response to WD. (a) Representative images of wheat germ agglutinin (WGA)‐stained cross‐sections of the heart‐depicting myocytes. (b) Quantitative analysis of myocyte cross‐sectional area. *p < 0.05 versus WT‐NC, ^$ p < 0.05 versus hKO‐NC, ^# p < 0.05 versus WT‐WD, n = 4. Statistical analysis was performed using two‐way ANOVA followed by Newman–Keuls test.

FIGURE 5

ATM deficiency attenuates apoptosis in the heart in response to WD. (a) Representative images of TUNEL (green), WGA (red) and Hoechst (blue) stained hearts. (b) Quantitative analysis of myocyte apoptosis. (c) Quantitative analysis of cardiac cell apoptosis. *p < 0.05 versus WT‐NC, ^$ p < 0.05 versus hKO‐NC, ^# p < 0.05 versus WT‐WD, n = 4. Statistical analysis was performed using two‐way ANOVA followed by Newman–Keuls test.

FIGURE 6

Expression of MMP‐2 and MMP‐9. Heart lysates were analyzed by western blots using anti‐MMP‐2 (a), and MMP‐9 (b) antibodies. Upper panels exhibit immunostaining for MMP‐2 and MMP‐9. Lower panels exhibit quantitative analyses normalized to total protein staining in each lane. *p < 0.05 versus WT‐NC, ^$ p < 0.05 versus hKO‐NC, ^# p < 0.05 versus WT‐WD, n = 6. Statistical analysis was performed using two‐way ANOVA followed by Newman–Keuls test.

FIGURE 7

Expression of Bax and PARP‐1. Heart lysates were analyzed by western blots using anti‐Bax (a) and PARP‐1 (b) antibodies. Upper panels exhibit immunostaining for Bax and PARP‐1. Lower panels exhibit quantitative analyses normalized to total protein staining in each lane. *p < 0.05 versus WT‐NC, ^$ p < 0.05 versus hKO‐NC, ^# p < 0.05 versus WT‐WD, n = 3. Statistical analysis was performed using two‐way ANOVA followed by Newman–Keuls test.

FIGURE 8

Activation of Akt and mTOR. Heart lysates were analyzed by western blots using anti‐p‐Akt (a) and p‐mTOR (b) antibodies. Bar graphs exhibit quantitative analyses normalized to total protein in each lane. *p < 0.05 versus WT‐NC, ^$ p < 0.05 versus hKO‐NC, ^# p < 0.05 versus WT‐WD, n = 3 for p‐Akt; n = 6 for p‐mTOR. Statistical analysis was performed using two‐way ANOVA followed by Newman–Keuls test. The p‐mTOR antibody recognized two proteins on SDS‐PAGE. Predicted molecular weight of mTOR is ~289 kDa. However, it runs ~230 kDa on SDS‐PAGE. Therefore, the upper band below 251 kDa molecular weight marker (std) is used for quantification.