Progressive Decrease in Coronary Vascular Function Associated With Type 2 Diabetic Heart Disease

Figures

• 

  FIGURE 1. A schematic diagram showing the experimental set-up for coronary microangiography in an anesthetized mouse model using monochromatic synchrotron radiation at the SPring-8 facilities in Hyogo, Japan (modified from Pearson et al., 2018).

• 

  FIGURE 2. Representative microangiogram images showing the contrast-enhanced coronary arterial branching network down to the 3rd order in anesthetized db/db non-diabetic (Left) and db/db diabetic (Right) mice at 8, 16, and 24 weeks of age. The tungsten wire in the bottom right corner of both angiogram frames is a reference of 50 μm diameter.

• 

  FIGURE 3. (A) The number of opaque vessels (mean ± SEM) and (B) the range of vessel sizes (box and whisker graph), at each of the first three branching generations of the coronary circulation in anesthetized db/db diabetic and non-diabetic mice at 8 weeks old (n = 8 and n = 7, respectively), 16 weeks old (n = 10 and n = 9, respectively), and 24 weeks old (n = 8 and n = 7, respectively). Quantitative data presented in A show a decrease in the number of 3rd order branches in the 24-week-old diabetic mice compared to the (i) 8 week old diabetic mice (∗P < 0.05) and (ii) age-matched non-diabetic animals (†P < 0.05).

• 

  FIGURE 4. The relationship between coronary vessel size and the magnitude (%) of change in vessel internal diameter (ID) in anesthetized db/db diabetic and non-diabetic mice (mean ± SEM) at 8 weeks (n = 8 and n = 7, respectively), 16 weeks (n = 10 and n = 9, respectively), and 24 weeks of age (n = 8 and n = 7, respectively) in response to (A) acetylcholine (ACh – 10.0 μg/kg/min for 5 min), (B) sodium nitroprusside (SNP – 10.0 μg/kg/min for 5 min), and (C) fasudil (20 mg/kg, i.v.). ∗Significant difference between non-diabetic vs. diabetic (P < 0.05). †Significantly different from 8-week old mice (P < 0.05). ‡Significant reduction/increase in vessel caliber, compared to baseline (P < 0.05).

• 

  FIGURE 5. Magnitude of change (%) in mean arterial blood pressure (MABP) and heart rate (HR) of db/db diabetic and non-diabetic mice (mean ± SEM) at 8 weeks old (n = 8 and n = 7, respectively), 16 weeks old (n = 10 and n = 9, respectively), and 24 weeks old (n = 8 and n = 7, respectively) in response to (A,D) acetylcholine (ACh – 10.0 μg/kg/min for 5 min), (B,E) sodium nitroprusside (SNP – 10.0 μg/kg/min for 5 min), and (C,F) fasudil (20 mg/kg, i.v.). †Significantly different from the response of 8-week-old diabetic mice (P < 0.05).

• 

  FIGURE 6. (A) Microangiogram images showing the effect of fasudil (20 mg/kg, i.v.) in improving coronary blood flow distribution in 24-week-old diabetic mice, via the recruitment of additional 3rd order branches (red arrows). (B) Quantitative data (mean ± SEM) of the coronary angiograms showing an increase in the number of 3rd order branches in the 24-week-old diabetic mice (n = 8) following fasudil administration. ∗Significant difference between diabetic and non-diabetic mice (P < 0.05). †Significantly different from 8 weeks of age (P < 0.05).

• 

  FIGURE 7. Immunohistochemical assessment of Rho-kinase protein (ROCK1) expression within myocardial sections retrieved from db/db (a) non-diabetic mice and, (b) diabetic mice at 8, 16, and 24 weeks of age; (c) the intensity of ROCK staining (brown precipitate) appeared to be progressively enhanced with age in diabetic hearts, compared to non-diabetic hearts, and significantly more so by 24 weeks of age (∗P < 0.05).

• 

  FIGURE 8. Immunohistochemical assessment of eNOS protein expression in myocardial sections retrieved from db/db (a) non-diabetic mice and, (b) diabetic mice at 8, 16, and 24 weeks of age; (c) the intensity of eNOS staining (brown precipitate) appeared to be progressively reduced with age in diabetic hearts, compared to non-diabetic hearts, and significantly more so by 24 weeks of age (∗P < 0.05).