Skeletal Muscle Microvascular Dysfunction Manifests Early in Diabetic Cardiomyopathy

Figures

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  Figure 1. Timeline of diabetes induction and post-diabetes monitoring and assessment. Additional measurements not shown include weekly weight, weekly non-fasting blood glucose, and monthly blood pressure measurements. STZ, streptozotocin; OGTT, oral glucose tolerance test; ITT, insulin tolerance test; IPL, insulin plasma level.

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  Figure 2. Body weight, blood glucose, and blood cholesterol levels. (A) Weekly weight measurements in all control and diabetic male rats. (B) Weekly non-fasting blood glucose measurements in all control and diabetic male rats. For (C–G), data was obtained from n = 3 male control/diabetic at Month 2 post-diabetes; n = 5 male control/diabetic for Month 3–7; and n = 4 male control, n = 6 male diabetic at Month 10. (C) High-density lipoprotein (HDL) cholesterol over time. (D) Low-density lipoprotein (LDL) cholesterol over time. (E) Total cholesterol over time. (F) Triglyceride levels over time. (G) Fasting glucose blood levels over time. Significance at individual timepoints is indicated (*p < 0.05), (**p < 0.01). Data represented as mean ± SEM.

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  Figure 3. Confirmation of type II diabetes and non-invasive blood pressure measurements. (A) Oral glucose tolerance test performed after overnight fasting in control (n = 9) and diabetic (n = 19) rats. (B) Insulin tolerance testing performed after 6-h fasting in control (n = 9) and diabetic (n = 19) rats. (C) Blood insulin levels measured from plasma collected after overnight fasting (μIU/mL) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) calculated for control (n = 7) and diabetic (n = 14) rats. (D) Systolic blood pressure. (E) Diastolic blood pressure. (F) Mean blood pressure. (G) Conscious heart rate [beats per minute (bpm)]. Significance at individual timepoints is indicated (*p < 0.05), (**p < 0.01). Data represented as mean ± SEM.

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  Figure 4. Treadmill exercise tolerance testing. The difference in distance run relative to Month 3 in individual rats is averaged within each cohort (control vs. diabetic). A significant difference between control and diabetic rats is observed at Months 5, 6, and 7. Significance at individual timepoints is indicated (*p < 0.05). Data represented as mean ± SEM.

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  Figure 5. Cardiac LV function and dimensions measured by pulsed wave Doppler and M-mode echocardiography. (A) Mitral valve E/A ratio representing the ratio between peak blood velocity in the left ventricle (LV) during early diastole to peak blood velocity in the LV at late diastole. (B) Isovolumetric relaxation time (IVRT). (C) Isovolumetric contraction time (IVCT). (D) LV ejection time. (E) LV myocardial performance index (MPI). (F) Pulmonary peak diastolic (D) venous wave. (G) Pulmonary peak systolic (S) venous wave. (H) Left ventricle (LV) ejection fraction. (I) Fractional shortening. (J) LV diameter at end-systole. (K) LV diameter at end-diastole. (L) Relative wall thickness (RWT). (M) LV mass index. (N) Pulsed wave Doppler ultrasound images taken at the mitral orifice in the apical four-chamber view at 4 months post-diabetes for male control (top) and diabetic (bottom) rats. (O) Pulsed wave Doppler ultrasound images taken at the entrance of the pulmonary vein into the left atrium at 6 months post-diabetes for male control (top) and diabetic (bottom) rats, highlighting the pulmonary peak systolic (S) and diastolic (D) venous wave. (P) B-mode ultrasound images taken of the LV at…

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  Figure 6. Additional echocardiographic parameters. (A) Unconscious heart rate [beats per minute (bpm)]. (B) LV stroke volume calculated as EDV-ESV. (C) LV cardiac output calculated as heart rate × stroke volume. (D) Mitral valve E/e' ratio representing atrial filling pressure, defined as the ratio between peak blood velocity in the LV and peak mitral annular velocity during early diastole. (E) LV anterior wall thickness at end-systole. (F) LV anterior wall thickness at end-diastole (G) LV posterior wall thickness at end-systole. (H) LV posterior wall thickness at end-diastole. (I) LV volume at end-systole. (J) LV volume at end-diastole. (K) LV Filling time. (L) LV mass. EDV, end-diastolic volume; ESV, end-systolic volume. Significance at individual timepoints is indicated (*p < 0.05), (**p < 0.01). Data represented as mean ± SEM.

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  Figure 7. Flow-mediated dilation measurements of femoral artery by Doppler ultrasound. Blood velocity measured via color Doppler with a cuff on the ankle at Month 5, 6, and 10 post-diabetes. (A) Femoral artery Doppler ultrasound images at Month 6 - baseline for male control (top) and diabetic (bottom) rats. (B) Representative femoral artery color Doppler ultrasound image at Month 6 – baseline. (C) Peak systolic velocity and (D) end diastolic velocity at Month 6 post-diabetes measured in control (n = 5) and diabetic (n = 5) rats. Significance was evaluated between pre-cuffing (5 min) and cuff release (6 min), and difference was observed for both control and diabetic cohorts. Relative change in (E) peak systolic velocity and (F) end diastolic velocity post-cuff release, measured at Month 5 (n = 5 control, n = 5 diabetic), Month 6 (n = 5 control, n = 5 diabetic), and Month 10 (n = 4 control, n = 6 diabetic) post-diabetes. Line graphs represented as mean ± SEM. Box plot mean value is represented with the symbol “X.” Significance at individual timepoints is indicated (*p < 0.05).

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  Figure 8. Photoacoustic imaging in leg skeletal muscle and myocardium. (A) Photoacoustic ultrasound images of male control (top) and diabetic (bottom) skeletal muscle at 10 months post-diabetes. Greyscale images are B-Mode images, and colormaps are the photoacoustic blood oxygenation saturation overlay. (B) Photoacoustic ultrasound images of male control (top) and diabetic (bottom) cardiac muscle at 10 months post-diabetes. (C) Blood oxygen saturation average in skeletal muscle at 4 months (n = 3 control and n = 4 diabetic), 8 months (n = 3 control and n = 8 diabetic), and 10 months (n = 4 control and n = 6 diabetic) post-diabetes. (D) Blood oxygen saturation average in the myocardium at 8 months (n = 3 control and n = 5 diabetic) and 10 months (n = 4 control and n = 6 diabetic) post-diabetes. Box plot mean value is represented with the symbol “X.” Significance at individual timepoints is indicated (**p < 0.01).

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  Figure 9. Histology analysis on rodent hearts. Timepoints refer to time post-diabetes at 3 months (n = 2 control, n = 8 diabetic), 6 months (n = 2 control, n = 3 diabetic), and 10 months (n = 5 control, n = 9 diabetic). (A) Schematic representing the three sections of the heart where histology was performed (atria, ventricles, and apex). (B) Heart weight measured immediately post-sacrifice and heart weight-to-tibial length ratio. (C) Interstitial fibrosis quantified as percentage of collagen surface area coverage on stained heart sections; shown on the right are slide scans of the LV at 10 months post-diabetes in control (top) and diabetic (bottom) rats. (D) Myocardial capillary density quantified as the number of vessels per mm2 of tissue on stained heart sections; shown on the right are slide scans of the LV at 10 months post-diabetes in control (top) and diabetic (bottom) rats. Arrow shows a small area of reduced capillary density. (E) Cardiomyocyte cross-sectional area quantified at 6 months (n = 2 control, n = 3 diabetic) and 10 months post-diabetes (n = 2 control, n = 3 diabetic); shown on the right are H&E slide scans at 10 months in control (top) and diabetic (bottom) rodent…

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  Figure 10. Histology assessment of skeletal muscle capillary density. Timepoints refer to time post-diabetes at 6 months (n = 2 control, n = 3 diabetic) and 10 months (n = 3 control, n = 3 diabetic). (A) Gastrocnemius muscle capillary density quantified as the number of vessels per mm2 of tissue on CD31 stained sections. (B) Gastrocnemius sections stained with CD31 of control (left) and diabetic (right) rats at 10 months post-diabetes. (C) Gracilis muscle capillary density quantified as the number of vessels per mm2 of tissue on CD31 stained sections. (D) Gracilis sections stained with CD31 of control (left) and diabetic (right) rats at 10 months post-diabetes. Box plot mean value is represented with the symbol “X”.