Progressive Cardiac Metabolic Defects Accompany Diastolic and Severe Systolic Dysfunction in Spontaneously Hypertensive Rat Hearts

Figures

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  Figure 1. Blood pressure, end‐diastolic and end‐systolic volumes, systolic and diastolic function, body weight, left ventricular mass (normalized to body weight), and glucose uptake rates in SHR hearts. A, Blood pressure. Mean arterial pressure (MAP) in spontaneously hypertensive rats (SHR) (n=6) and Wistar Kyoto rats (WKY) (n=6). MAP was recorded every minute for 30 minutes and values were averaged to obtain the reported data. B, End‐diastolic volume (EDV), C, end‐systolic volume (ESV), D, ejection fraction (EF), E, Isovolumetric Relaxation Time (IVRT), F, body weight (BW), and G, left ventricular mass to body weight ratios (LVM/BW) were determined in SHR (n=5‐8) and WKY (n=4–5) using CMR in vivo. At 18 months of age, 3 SHR hearts with severe systolic dysfunction were identified as having ≥20% lower EF (rEF) than control WKY, while 2 SHR hearts had preserved EF (pEF). (H) Heart weight to body weight ratios (HW/BW) ex vivo. Hearts from SHR (n=6) and WKY (n=6) were weighed after dissection and normalized to BWs. I, Myocardial glucose uptake rates in vivo in SHR. Glucose uptake rates (Ki) were computed from serial dynamic FDG PET images of SHR (n=5–8) and WKY (n=4–5) hearts. At 18 months of…

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  Figure 2. mTOR activation and ER stress in SHR. A, SHR (n=6) and WKY (n=6) hearts were immunoblotted for phospho‐p70S6K (p‐p70S6K, a marker for mTOR activity), total p70S6K, and GAPDH. B, Signal intensities for p‐p70S6K and p70S6K were normalized to GAPDH, and p‐p70S6K/p70S6K ratios were determined. Results were normalized within age groups (for which samples were analyzed on the same immunoblots) and fold changes shown. C, SHR (n=6) and WKY (n=6) hearts were immunoblotted for GRP78, a marker for ER stress, and GAPDH. D, Signal intensities for GRP78 normalized to GAPDH are presented. Results were normalized within age groups (for which samples were analyzed on the same immunoblots) and fold changes were graphed. All data are shown as mean±SE. *P≤0.05 SHR vs WKY. Data were compared between SHR and WKY at each age using paired Student t tests. ER indicates endoplasmic reticulum; mTOR, mechanistic target of rapamycin; SHR, spontaneously hypertensive rats; and WKY, Wistar Kyoto rats.

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  Figure 3. Cardiac metabolite changes in 12‐ and 18‐month‐old SHR. Metabolites were analyzed in hearts of SHR and WKY at 12 (n=6) and 18 months (n=6) as described. 10 A, Principal Component Analysis, with light blue cones representing WKY at 12, dark blue cylinders WKY at 18, orange cones SHR at 12, and red cylinders SHR at 18 months (mo). B, Hierarchical Clustering Analysis. C, Random Forest Analysis. D and E, Metabolite profiles. D, Metabolites that were differentially modified in hearts of 12‐ and 18‐month‐old SHR relative to WKY at the same age with most metabolites normal (P>0.05 SHR vs WKY) at 12 months, except valerylcarnitine, (R)‐3‐hydroxybutyrylcarnitine, butyrylcarnitine, and carnitine that were statistically significantly different (P<0.05 SHR vs WKY). Statistically significant (P<0.05 SHR vs WKY) decreases or increases were found for all metabolites shown for 18‐month‐old rats except for (R)‐3‐hydroxybutyrylcarnitine that was normal (P>0.05 SHR vs WKY). E, Metabolites that were changed in the same direction in hearts of 12‐ and 18‐month‐old SHR relative to WKY at the same age. P<0.05 SHR vs WKY at same age, except 0.05≤P≤0.1 for octanoylcarnitine, phosphoenolpyruvate, fumarate,…

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  Figure 4. Circulating metabolite and insulin levels in SHR. A, Branched chain amino acids (BCAA), B, free fatty acids (FFA), C, β‐hydroxybutyrate, D, glucose, and E, insulin were measured in SHR (n=6) and WKY (n=6) at 9, 12, and 18 months of age. Data are shown as mean±SE. *P≤0.05 and **P<0.001 SHR vs WKY. Data between SHR and WKY were compared at each age using Student t tests. SHR indicates spontaneously hypertensive rats; and WKY, Wistar Kyoto rats.

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  Figure 5. Relationship between heart weight and select metabolites. A, Three‐dimensional plot of heart weight and the 2 principal components PC1 and PC2 for SHR and WKY. Linear Principal Component Analysis was performed (on raw area count metabolite levels rescaled to set medians equal to 1) to calculate the proportion of total variability in heart weight (HW; dependent variable) that is explained by a combination of 17 metabolites (independent variables). Data for 12‐ and 18‐month‐old SHR and WKY were combined. The 2 principal components PC1 and PC2 explained 45% and 26% of the variance in the data (total 71%). B, Loadings Plot. The biplot shows the degree of contribution of each metabolite to the principal components PC1 and PC2 with loadings (lengths of arrows [vectors] and extent of angles deviating from 0) illustrating the strengths of the effects on PC1 and PC2. Positive or negative loadings, respectively, indicate that HW and a principal component are positively or negatively correlated. SHR indicates spontaneously hypertensive rats; and WKY, Wistar Kyoto rats.

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  Figure 6. Summary of changes in energy‐providing metabolic pathways in SHR at 12 and 18 months of age. Up‐ and down arrows next to metabolites indicate up‐ and down‐regulation of specific metabolites, with gray arrows indicating changes at both 12 and 18 months, blue arrows at 12 months only, and red arrows at 18 months only. Broken arrows indicate decreases of only some representatives of the group, while solid arrows signify that the majority of metabolites in respective groups are changed. 3‐P‐glycerate indicates 3‐phosphoglycerate; BCAA, branched chain amino acid; BHB, β‐hydroxybutyrate or 3‐hydroxybutyrate; CoA, coenzyme A; DAG, diacylglycerol; G6‐P, glucose 6‐phosphate; MAG, monoacylglycerol; NAD+, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; SHR, spontaneously hypertensive rats; and TAG, triacylglycerol.