The double-hit protocol induces HFpEF and impairs myocardial ubiquitin-proteasome system performance in FVB/N mice

Figures

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  FIGURE 1. Changes in body weight (BW) of HFD+L-NAME or chow treated mice. (A), Comparison of the time courses of changes in BW measured immediately before (Week 0) and weekly after the initiation of the HFD or control diet (Chow) treatment. Shown are line graphs superimposed by mean ± SEM of each time point. The p values shown are adjusted p values derived from multiple two-sided unpaired t-tests with correction for multiple comparisons using the Bonferroni-Dunn method; NS, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. The Chow group started with 7 mice but a mouse dropped out at Week 8 and another at Week 14. The HFD+L-NAME group had 6 mice throughout. (B–D), Longitudinal comparison of BW from Week 0 to Week 1 (B), to Week 2 (C), or to Week 18 (D). Each circle represents a mouse. P values are derived from two-sided paired t-tests. (E), BW/TL ratio measured at terminal experiment (Week 18). TL, tibial length. Shown are scatter plots superimposed by mean ± SEM; each circle or dot represents a mouse. The p-value is derived from two-sided unpaired t-test.

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  FIGURE 2. Glucose tolerance tests (GTTs) performed at week 6 and 15. A peritoneal injection of 20% glucose solution (1 g/kg) was administered after 6-h fasting. Blood from the tail was collected immediately before (0 min) and 15, 30, 60, 90, and 120 min after the injection for glucose concentration measurement. (A,B), GTTs at week 6. Shown are the time course of blood glucose levels during GTTs (A) and the area under curve (AUC) comparison (B) between the Chow and the HFD+L-NAME groups. Multiple t-tests reveal the P values for comparisons at 0, 15, 30, 60, 90, and 120 min are respectively 0.791, 0.711, 0.003, 0.002, 0.016, and 0.031; * denotes that the difference at 30- and 60-min is statistically significant after correction for multiple comparisons using the Holm-Sidak method. (C,D), GTTs at week 15 weeks. Shown are the time course of blood glucose levels during GTTs (C) and the area under curve (AUC) comparison (D) between the Chow and the HFD+L-NAME groups. Multiple t-tests reveal the P values for comparisons at 0, 15, 30, 60, 90, and 120 min are respectively 0.955, 0.017, 0.35, 0.008, 0.006, and 0.026; * denotes that the difference at 60 and 90 min is statistically significant after…

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  FIGURE 3. Systolic blood pressure measured at week 17 with the tail-cuff method.

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  FIGURE 4. HFD+L-NAME induced cardiac hypertrophy as revealed by serial echocardiography. (A,B), Representative echographs of the Chow and the HFD+L-NAME groups recorded at week 15. (C), Left ventricular anterior wall thickness at the end of diastole (LVAW; d) at week 7. (D), Left ventricular posterior wall thickness at the end of diastole (LVPW; d) at week 7. (E), LVAW; d at week 15. (F), LVPW; d at week 15.

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  FIGURE 5. Comparisons of LV end-diastolic volume (LVEDV) and end-systolic volume (LVESV) between the Chow and the HFD+L-NMAE groups at week 7 (A,B) and week 15 (C,D).

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  FIGURE 6. Reactivation of the fetal gene program in the ventricles. At the terminal experiment (week 18), LV myocardium was sampled, preserved in the RNA-Later reagent, and stored in −80°C until total RNA extraction. Total RNA was used for reverse transcription to synthesize the first-strand cDNA that was subsequently used for real time PCR to measure the mRNA levels with gene-specific primer sets for Nppa (A), Nppb (B), and Pln (C).

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  FIGURE 7. Comparisons of exercise tolerance between the Chow and the HFD+L-NAME groups at week 7 (A,C) and week 16 (B,D).

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  FIGURE 8. Comparisons of LV ejection fraction (EF) and fractional shortening (FS) between the Chow and the HFD+L-NMAE groups at week 7 (A,B) and week 15 (C, D).

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  FIGURE 9. Myocardial mRNA and protein expression of GFPdgn in HFpEF and control mice. At the terminal experiment (week 18), a segment of left ventricular myocardium of each mouse was collected, snap-frozen in liquid nitrogen, and stored in a −80°C freezer until total protein extraction. The tissue preservation for total RNA extraction was the same as described in Figure 6. (A,B), Representative images (A) and pooled densitometry data (B) of western blot analysis for myocardial GFPdgn. (C), Comparison of GFPdgn mRNA levels as measured with qPCR. (D), Comparison of GFPdgn protein/mRNA level ratios. (E), A summary of overall findings. Dot line denotes a potential causative relationship. The question mark denotes the relationship remains to be determined. EF, ejection fraction; UPS, ubiquitin-proteasome system.