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

Background Cardiac metabolic abnormalities are present in heart failure. Few studies have followed metabolic changes accompanying diastolic and systolic heart failure in the same model. We examined metabolic changes during the development of diastolic and severe systolic dysfunction in spontaneously hypertensive rats (SHR). Methods and Results We serially measured myocardial glucose uptake rates with dynamic 2-[18F] fluoro-2-deoxy-d-glucose positron emission tomography in vivo in 9-, 12-, and 18-month-old SHR and Wistar Kyoto rats. Cardiac magnetic resonance imaging determined systolic function (ejection fraction) and diastolic function (isovolumetric relaxation time) and left ventricular mass in the same rats. Cardiac metabolomics was performed at 12 and 18 months in separate rats. At 12 months, SHR hearts, compared with Wistar Kyoto hearts, demonstrated increased isovolumetric relaxation time and slightly reduced ejection fraction indicating diastolic and mild systolic dysfunction, respectively, and higher (versus 9-month-old SHR decreasing) 2-[18F] fluoro-2-deoxy-d-glucose uptake rates (Ki). At 18 months, only few SHR hearts maintained similar abnormalities as 12-month-old SHR, while most exhibited severe systolic dysfunction, worsening diastolic function, and markedly reduced 2-[18F] fluoro-2-deoxy-d-glucose uptake rates. Left ventricular mass normalized to body weight was elevated in SHR, more pronounced with severe systolic dysfunction. Cardiac metabolite changes differed between SHR hearts at 12 and 18 months, indicating progressive defects in fatty acid, glucose, branched chain amino acid, and ketone body metabolism. Conclusions Diastolic and severe systolic dysfunction in SHR are associated with decreasing cardiac glucose uptake, and progressive abnormalities in metabolite profiles. Whether and which metabolic changes trigger progressive heart failure needs to be established.

Metformin Improves Cardiac Metabolism and Function, and Prevents Left Ventricular Hypertrophy in Spontaneously Hypertensive Rats.

Background In spontaneously hypertensive rats (SHR) we observed profound myocardial metabolic changes during early hypertension before development of cardiac dysfunction and left ventricular hypertrophy. In this study, we evaluated whether metformin improved myocardial metabolic abnormalities and simultaneously prevented contractile dysfunction and left ventricular hypertrophy in SHR. Methods and Results SHR and control Wistar-Kyoto rats were treated with metformin from 2 to 5 months of age, when SHR hearts exhibit metabolic abnormalities and develop cardiac dysfunction and left ventricular hypertrophy. We evaluated the effect of metformin on myocardial glucose uptake rates with dynamic 2-[18F] fluoro-2-deoxy-D-glucose positron emission tomography. We used cardiac MRI in vivo to assess the effect of metformin on ejection fraction, left ventricular mass, and end-diastolic wall thickness, and also analyzed metabolites, AMP-activated protein kinase and mammalian target-of-rapamycin activities, and mean arterial blood pressure. Metformin-treated SHR had lower mean arterial blood pressure but remained hypertensive. Cardiac glucose uptake rates, left ventricular mass/tibia length, wall thickness, and circulating free fatty acid levels decreased to normal, and ejection fraction improved in treated SHR. Hearts of treated SHR exhibited increased AMP-activated protein kinase phosphorylation and reduced mammalian target-of-rapamycin activity. Cardiac metabolite profiling demonstrated that metformin decreased fatty acyl carnitines and markers of oxidative stress in SHR. Conclusions Metformin reduced blood pressure, normalized myocardial glucose uptake, prevented left ventricular hypertrophy, and improved cardiac function in SHR. Metformin may exert its effects by normalizing myocardial AMPK and mammalian target-of-rapamycin activities, improving fatty acid oxidation, and reducing oxidative stress. Thus, metformin may be a new treatment to prevent or ameliorate chronic hypertension-induced left ventricular hypertrophy.

Metabolic Changes in Spontaneously Hypertensive Rat Hearts Precede Cardiac Dysfunction and Left Ventricular Hypertrophy.

Background Sustained pressure overload leads to changes in cardiac metabolism, function, and structure. Both time course and causal relationships between these changes are not fully understood. Therefore, we studied spontaneously hypertensive rats (SHR) during early hypertension development and compared them to control Wistar Kyoto rats. Methods and Results We serially evaluated myocardial glucose uptake rates (Ki) with dynamic 2-[18F] fluoro-2-deoxy-D-glucose positron emission tomography, and ejection fraction and left ventricular mass to body weight ratios with cardiac magnetic resonance imaging in vivo, determined glucose uptake and oxidation rates in isolated perfused hearts, and analyzed metabolites, mammalian target of rapamycin activity and endoplasmic reticulum stress in dissected hearts. When compared with Wistar Kyoto rats, SHR demonstrated increased glucose uptake rates (Ki) in vivo, and reduced ejection fraction as early as 2 months of age when hypertension was established. Isolated perfused SHR hearts showed increased glucose uptake and oxidation rates starting at 1 month. Cardiac metabolite analysis at 2 months of age revealed elevated pyruvate, fatty acyl- and branched chain amino acid-derived carnitines, oxidative stress, and inflammation. Mammalian target of rapamycin activity increased in SHR beginning at 2 months. Left ventricular mass to body weight ratios and endoplasmic reticulum stress were elevated in 5 month-old SHR. Conclusions Thus, in a genetic hypertension model, chronic cardiac pressure overload promptly leads to increased myocardial glucose uptake and oxidation, and to metabolite abnormalities. These coincide with, or precede, cardiac dysfunction while left ventricular hypertrophy develops only later. Myocardial metabolic changes may thus serve as early diagnostic markers for hypertension-induced left ventricular hypertrophy.

Serial MRI Imaging Reveals Minimal Impact of Ketogenic Diet on Established Liver Tumor Growth.

Rodent models of liver tumorigenesis have reproducibly shown that dietary sugar intake is a powerful driver of liver tumor initiation and growth. In contrast, dietary sugar restriction with ketogenic diets or calorie restriction generally prevents liver tumor formation. Ketogenic diet is viewed positively as a therapeutic adjuvant; however, most ketogenic diet studies described to date have been performed in prevention mode rather than treatment mode. Therefore, it remains unclear whether a ketogenic diet can be administered in late stages of disease to stall or reverse liver tumor growth. To model the clinically relevant treatment mode, we administered a ketogenic diet to mice after liver tumor initiation and monitored tumor growth by magnetic resonance imaging (MRI). Male C57BL/6 mice were injected with diethylnitrosamine (DEN) at 2 weeks of age and fed a chow diet until 39 weeks of age, when they underwent MRI imaging to detect liver tumors. Mice were then randomised into two groups and fed either a chow diet or switched to a ketogenic diet from 40⁻48 weeks of age. Serial MRIs were performed at 44 and 48 weeks of age. All mice had tumors at study completion and there were no differences in total tumor burden between diet groups. Although a ketogenic diet has marked protective effects against DEN-induced liver tumourigenesis in this mouse model, these data demonstrate that ketogenic diet cannot stop the progression of established liver tumors.

A novel chronic advanced stage abdominal aortic aneurysm murine model.

OBJECTIVE: The purpose of this study was to establish a reliable, chronic model of abdominal aortic aneurysm (AAA). METHODS: Wild-type 8-week-old C56BL/6 male mice (n = 120) were equally divided into three groups: (1) BAPN group: 0.2% 3-aminopropionitrile fumarate salt (BAPN) drinking water was provided to mice 2 days before surgery until the end of study. Sham aneurysm induction surgery was performed using 5 µL of heat deactivated elastase. (2) Elastase group: mice were given regular drinking water without BAPN. During aneurysm induction surgery, 5 µL of the active form of elastase (10.3 mg protein/mL, 5.9 U/mg protein) was applied on top of the infrarenal abdominal aorta adventitia for 5 minutes. (3) BAPN+elastase group: mice were given BAPN drinking water and the active form of elastase application, as above. On postoperative days 7, 14, 21, 28, and 100, aortic samples were collected for histology, cytokine array, and gelatin zymography after aortic diameter measurement. RESULTS: Compared with the elastase group, the BAPN+elastase group had a higher AAA formation rate (93% vs 65%; P < .01) with more advanced AAAs (25 of 42 vs 1 of 40 for stage II and III; P < .001). Aneurysms from the BAPN+elastase group demonstrated persistent long-term growth (221.5% ± 36.6%, 285.8% ± 78.6%, and 801% ± 160% on days 21, 28, and 100, respectively; P < .001), with considerable thrombus formation (54%) and rupture (31%) at the advanced stages of AAA development. Cytokine levels (pro-matrix metalloproteinase 9, interleukin-1ß, interleukin-6, chemokine [C-C motif] ligand 5, triggering receptor expressed on myeloid cells 1, monocyte chemotactic protein 1, and tissue inhibitor of metalloproteinase 1) in the BAPN+elastase group were higher than in the elastase group on day 7. After day 7, cytokine levels returned to baseline, with the exception of elevated matrix metalloproteinase 2 activity. By histology, CD3-positive T cells in the BAPN+elastase group were elevated on days 28 and 100. CONCLUSIONS: A combination of oral BAPN administration and periaortic elastase application induced a chronic, advanced-stage AAA with characteristics of persistent aneurysm growth, thrombus formation, and spontaneous rupture. Future studies should use this model, especially for examining tissue remodeling during the late stages of aneurysm development.

Glucose regulation of load-induced mTOR signaling and ER stress in mammalian heart.

BACKGROUND: Changes in energy substrate metabolism are first responders to hemodynamic stress in the heart. We have previously shown that hexose-6-phosphate levels regulate mammalian target of rapamycin (mTOR) activation in response to insulin. We now tested the hypothesis that inotropic stimulation and increased afterload also regulate mTOR activation via glucose 6-phosphate (G6P) accumulation. METHODS AND RESULTS: We subjected the working rat heart ex vivo to a high workload in the presence of different energy-providing substrates including glucose, glucose analogues, and noncarbohydrate substrates. We observed an association between G6P accumulation, mTOR activation, endoplasmic reticulum (ER) stress, and impaired contractile function, all of which were prevented by pretreating animals with rapamycin (mTOR inhibition) or metformin (AMPK activation). The histone deacetylase inhibitor 4-phenylbutyrate, which relieves ER stress, also improved contractile function. In contrast, adding the glucose analogue 2-deoxy-d-glucose, which is phosphorylated but not further metabolized, to the perfusate resulted in mTOR activation and contractile dysfunction. Next we tested our hypothesis in vivo by transverse aortic constriction in mice. Using a micro-PET system, we observed enhanced glucose tracer analog uptake and contractile dysfunction preceding dilatation of the left ventricle. In contrast, in hearts overexpressing SERCA2a, ER stress was reduced and contractile function was preserved with hypertrophy. Finally, we examined failing human hearts and found that mechanical unloading decreased G6P levels and ER stress markers. CONCLUSIONS: We propose that glucose metabolic changes precede and regulate functional (and possibly also structural) remodeling of the heart. We implicate a critical role for G6P in load-induced mTOR activation and ER stress.

Three-dimensional phase contrast angiography of the mouse aortic arch using spiral MRI.

Atherosclerosis is a complex disease whose spatial distribution is hypothesized to be influenced by the local hemodynamic environment. The use of transgenic mice provides a mechanism to study the relationship between hemodynamic forces, most notably wall shear stress (WSS), and the molecular factors that influence the disease process. Phase contrast MRI using rectilinear trajectories has been used to measure boundary conditions for use in computational fluid dynamic models. However, the unique flow environment of the mouse precludes use of standard imaging techniques in complex, curved flow regions such as the aortic arch. In this article, two-dimensional and three-dimensional spiral cine phase contrast sequences are presented that enable measurement of velocity profiles in curved regions of the mouse vasculature. WSS is calculated directly from the spatial velocity gradient, enabling WSS calculation with a minimal set of assumptions. In contrast to the outer radius of the aortic arch, the inner radius has a lower time-averaged longitudinal WSS (7.06 ± 0.76 dyne/cm(2) vs. 18.86 ± 1.27 dyne/cm(2) ; P < 0.01) and higher oscillatory shear index (0.14 ± 0.01 vs. 0.08 ± 0.01; P < 0.01). This finding is in agreement with humans, where WSS is lower and more oscillatory along the inner radius, an atheroprone region, than the outer radius, an atheroprotective region.

Postinfarction myocardial scarring in mice: molecular MR imaging with use of a collagen-targeting contrast agent.

PURPOSE: To prospectively evaluate a gadolinium-based collagen-targeting contrast agent, EP-3533, for in vivo magnetic resonance (MR) imaging of myocardial fibrosis in a mouse model of healed myocardial infarction (MI). MATERIALS AND METHODS: All procedures were performed in accordance with protocols approved by the animal care and use committee. MI was induced in eight mice by means of occlusion of the left anterior descending coronary artery followed by reperfusion. Four MR examinations were performed in each animal: one examination before, one examination 1 day after, and two examinations 6 weeks after the MI. For the latter two examinations, electrocardiographically gated inversion-recovery gradient-echo MR images were acquired before and serially (every 5 minutes) after the intravenous injection of either gadopentetate dimeglumine or EP-3533. The image enhancement kinetic properties of the postinfarction scar, normal myocardium, and blood were compared. RESULTS: Dynamic T1-weighted MR imaging revealed the washout time constants for EP-3533 to be significantly longer than those for gadopentetate dimeglumine in regions of postinfarction scarring (mean, 194.8 minutes +/-116.8 [standard deviation] vs 25.5 minutes +/- 4.2; P < .05) and in normal myocardium (mean, 45.4 minutes +/- 16.7 vs 25.1 minutes +/- 9.7; P < .05). Findings on postmortem histologic sections stained for collagen correlated well with EP-3533-enhanced areas seen on inversion-recovery MR images. Fifty minutes after EP-3533 injection, the postinfarction scar tissue samples, as compared with the normal myocardium, had a twofold higher concentration of gadolinium. CONCLUSION: Use of the gadolinium-based collagen-targeting contrast agent, EP-3533, enabled in vivo molecular MR imaging of fibrosis in a mouse model of healed postinfarction myocardial scarring.