Diet-induced obese mouse hearts tolerate an acute high-fatty acid exposure that also increases ischemic tolerance.

An ischemic insult is accompanied by an acute increase in circulating fatty acid (FA) levels, which can induce adverse changes related to cardiac metabolism/energetics. Although chronic hyperlipidemia contributes to the pathogenesis of obesity-/diabetes-related cardiomyopathy, it is unclear how these hearts are affected by an acute high FA-load. We hypothesize that adaptation to chronic FA exposure enhances the obese hearts' ability to handle an acute high FA-load. Diet-induced obese (DIO) and age-matched control (CON) mouse hearts were perfused in the presence of low- or high FA-load (0.4 and 1.8 mM, respectively). Left ventricular (LV) function, FA oxidation rate, myocardial oxygen consumption, and mechanical efficiency were assessed, followed by analysis of myocardial oxidative stress, mitochondrial respiration, protein acetylation, and gene expression. Finally, ischemic tolerance was determined by examining LV functional recovery and infarct size. Under low-FA conditions, DIO hearts showed mild LV dysfunction, oxygen wasting, mechanical inefficiency, and reduced mitochondrial OxPhos. High FA-load increased FA oxidation rates in both groups, but this did not alter any of the above parameters in DIO hearts. In contrast, CON hearts showed FA-induced mechanical inefficiency, oxidative stress, and reduced OxPhos, as well as enhanced acetylation and activation of PPARα-dependent gene expression. While high FA-load did not alter functional recovery and infarct size in CON hearts, it increased ischemic tolerance in DIO hearts. Thus, this study demonstrates that acute FA-load affects normal and obese hearts differently and that chronically elevated circulating FA levels render the DIO heart less vulnerable to the disadvantageous effects of an acute FA-load.NEW & NOTEWORTHY An acute myocardial fat-load leads to oxidative stress, oxygen wasting, mechanical inefficiency, hyperacetylation, and impaired mitochondrial function, which can contribute to reduced ischemic tolerance. Following obesity/insulin resistance, hearts were less affected by a high fat-load, which subsequently also improved ischemic tolerance. This study highlights that an acute fat-load affects normal and obese hearts differently and that obesity renders hearts less vulnerable to the disadvantageous effects of an acute fat-load.

The role of NADPH oxidases in diabetic cardiomyopathy.

Systemic changes during diabetes such as high glucose, dyslipidemia, hormonal changes and low grade inflammation, are believed to induce structural and functional changes in the cardiomyocyte associated with the development of diabetic cardiomyopathy. One of the hallmarks of the diabetic heart is increased oxidative stress. NADPH-oxidases (NOXs) are important ROS-producing enzymes in the cardiomyocyte mediating both adaptive and maladaptive changes in the heart. NOXs have been suggested as a therapeutic target for several diabetic complications, but their role in diabetic cardiomyopathy is far from elucidated. In this review we aim to provide an overview of the current knowledge regarding the understanding of how NOXs influences cardiac adaptive and maladaptive processes in a "diabetic milieu". This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.

Exercise of obese mice induces cardioprotection and oxygen sparing in hearts exposed to high-fat load.

Exercise training is a potent therapeutic approach in obesity and diabetes that exerts protective effects against the development of diabetic cardiomyopathy and ischemic injury. Acute increases in circulating fatty acids (FAs) during an ischemic insult can challenge the heart, since high FA load is considered to have adverse cardiac effects. In the present study, we tested the hypothesis that exercise-induced cardiac effects in diet-induced obese mice are abrogated by an acute high FA load. Diet-induced obese mice were fed a high-fat diet (HFD) for 20 wk. They were exercised using moderate- and/or high-intensity exercise training (MIT and HIT, respectively) for 10 or 3 wk, and isolated perfused hearts from these mice were exposed to a high FA load. Sedentary HFD mice served as controls. Ventricular function and myocardial O2 consumption were assessed after 10 wk of HIT and MIT, and postischemic functional recovery and infarct size were examined after 3 wk of HIT. In addition to improving aerobic capacity and reducing obesity and insulin resistance, long-term exercise ameliorated the development of diet-induced cardiac dysfunction. This was associated with improved mechanical efficiency because of reduced myocardial oxygen consumption. Although to a lesser extent, 3-wk HIT also increased aerobic capacity and decreased obesity and insulin resistance. HIT also improved postischemic functional recovery and reduced infarct size. Event upon the exposure to a high FA load, short-term exercise induced an oxygen-sparing effect. This study therefore shows that exercise-induced cardioprotective effects are present under hyperlipidemic conditions and highlights the important role of myocardial energetics during ischemic stress.NEW & NOTEWORTHY The exercise-induced cardioprotective effects in obese hearts are present under hyperlipidemic conditions, comparable to circulating levels of FA occurring with an ischemic insult. Myocardial oxygen sparing is associated with this effect, despite the general notion that high fat can decrease cardiac efficiency. This highlights the role of myocardial energetics during ischemic stress.

On the pivotal role of PPARα in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury.

The role of peroxisome proliferator-activated receptor α (PPARα)-mediated metabolic remodeling in cardiac adaptation to hypoxia has yet to be defined. Here, mice were housed in hypoxia for 3 wk before in vivo contractile function was measured using cine MRI. In isolated, perfused hearts, energetics were measured using (31)P magnetic resonance spectroscopy (MRS), and glycolysis and fatty acid oxidation were measured using [(3)H] labeling. Compared with a normoxic, chow-fed control mouse heart, hypoxia decreased PPARα expression, fatty acid oxidation, and mitochondrial uncoupling protein 3 (UCP3) levels, while increasing glycolysis, all of which served to maintain normal ATP concentrations ([ATP]) and thereby, ejection fractions. A high-fat diet increased cardiac PPARα expression, fatty acid oxidation, and UCP3 levels with decreased glycolysis. Hypoxia was unable to alter the high PPARα expression or reverse the metabolic changes caused by the high-fat diet, with the result that [ATP] and contractile function decreased significantly. The adaptive metabolic changes caused by hypoxia in control mouse hearts were found to have occurred already in PPARα-deficient (PPARα(-/-)) mouse hearts and sustained function in hypoxia despite an inability for further metabolic remodeling. We conclude that decreased cardiac PPARα expression is essential for adaptive metabolic remodeling in hypoxia, but is prevented by dietary fat.-Cole, M. A., Abd Jamil, A. H., Heather, L. C., Murray, A. J., Sutton, E. R., Slingo, M., Sebag-Montefiore, L., Tan, S. C., Aksentijevic, D., Gildea, O. S., Stuckey, D. J., Yeoh, K. K., Carr, C. A., Evans, R. D., Aasum, E., Schofield, C. J., Ratcliffe, P. J., Neubauer, S., Robbins, P. A., Clarke, K. On the pivotal role of PPARα in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury.

Increased oxidative metabolism following hypoxia in the type 2 diabetic heart, despite normal hypoxia signalling and metabolic adaptation.

KEY POINTS: Adaptation to hypoxia makes the heart more oxygen efficient, by metabolising more glucose. In contrast, type 2 diabetes makes the heart metabolise more fatty acids. Diabetes increases the chances of the heart being exposed to hypoxia, but whether the diabetic heart can adapt and respond is unknown. In this study we show that diabetic hearts retain the ability to adapt their metabolism in response to hypoxia, with functional hypoxia signalling pathways. However, the hypoxia-induced changes in metabolism are additive to abnormal baseline metabolism, resulting in hypoxic diabetic hearts metabolising more fat and less glucose than controls. This stops the diabetic heart being able to recover its function when stressed. These results demonstrate that the diabetic heart retains metabolic flexibility to adapt to hypoxia, but is hindered by the baseline effects of the disease. This increases our understanding of how the diabetic heart is affected by hypoxia-associated complications of the disease. ABSTRACT: Hypoxia activates the hypoxia-inducible factor (HIF), promoting glycolysis and suppressing mitochondrial respiration. In the type 2 diabetic heart, glycolysis is suppressed whereas fatty acid metabolism is promoted. The diabetic heart experiences chronic hypoxia as a consequence of increased obstructive sleep apnoea and cardiovascular disease. Given the opposing metabolic effects of hypoxia and diabetes, we questioned whether diabetes affects cardiac metabolic adaptation to hypoxia. Control and type 2 diabetic rats were housed for 3 weeks in normoxia or 11% oxygen. Metabolism and function were measured in the isolated perfused heart using radiolabelled substrates. Following chronic hypoxia, both control and diabetic hearts upregulated glycolysis, lactate efflux and glycogen content and decreased fatty acid oxidation rates, with similar activation of HIF signalling pathways. However, hypoxia-induced changes were superimposed on diabetic hearts that were metabolically abnormal in normoxia, resulting in glycolytic rates 30% lower, and fatty acid oxidation 36% higher, in hypoxic diabetic hearts than hypoxic controls. Peroxisome proliferator-activated receptor α target proteins were suppressed by hypoxia, but activated by diabetes. Mitochondrial respiration in diabetic hearts was divergently activated following hypoxia compared with controls. These differences in metabolism were associated with decreased contractile recovery of the hypoxic diabetic heart following an acute hypoxic insult. In conclusion, type 2 diabetic hearts retain metabolic flexibility to adapt to hypoxia, with normal HIF signalling pathways. However, they are more dependent on oxidative metabolism following hypoxia due to abnormal normoxic metabolism, which was associated with a functional deficit in response to stress.

Wax esters from the marine copepod Calanus finmarchicus reduce diet-induced obesity and obesity-related metabolic disorders in mice.

We showed previously that dietary supplementation with oil from the marine zooplankton Calanus finmarchicus (Calanus oil) attenuates obesity, inflammation, and glucose intolerance in mice. More than 80% of Calanus oil consists of wax esters, i.e., long-chain fatty alcohols linked to long-chain fatty acids. In the present study, we compared the metabolic effects of Calanus oil-derived wax esters (WE) with those of purified eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) ethyl esters (E/D) in a mouse model of diet-induced obesity. C57BL/6J mice received a high-fat diet (HFD; 45% energy from fat). After 7 wk, the diet was supplemented with either 1% (wt:wt) WE or 0.2% (wt:wt) E/D. The amount of EPA + DHA in the E/D diet was matched to the total amount of n-3 (ω-3) polyunsaturated fatty acids (PUFAs) in the WE diet. A third group was given an unsupplemented HFD throughout the entire 27-wk feeding period. WE reduced body weight gain, abdominal fat, and liver triacylglycerol by 21%, 34%, and 52%, respectively, and significantly improved glucose tolerance and aerobic capacity. In abdominal fat depots, WE reduced macrophage infiltration by 74% and downregulated expression of proinflammatory genes (tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1), whereas adiponectin expression was significantly upregulated. By comparison, E/D primarily suppressed the expression of proinflammatory genes but had less influence on glucose tolerance than WE. E/D affected obesity parameters, aerobic capacity, or adiponectin expression by <10%. These results show that the wax ester component of Calanus oil can account for the biologic effects shown previously for the crude oil. However, these effects cannot exclusively be ascribed to the content of n-3 PUFAs in the wax ester fraction.

Oil from the marine zooplankton Calanus finmarchicus improves the cardiometabolic phenotype of diet-induced obese mice.

The aim of the present study was to investigate the effects of oil extracted from the zooplankton Calanus finmarchicus (Calanus oil) on diet-induced obesity and obesity-related disorders in mice. C57BL/6J mice fed a high-fat diet (HFD, 45% energy from fat) exhibited increased body weight and abdominal fat accumulation as well as impaired glucose tolerance compared with mice fed a normal chow diet (10% energy from fat). Supplementing the HFD with 1.5% (w/w) Calanus oil reduced body-weight gain, abdominal fat accumulation and hepatic steatosis by 16, 27 and 41%, respectively, and improved glucose tolerance by 16%. Calanus oil supplementation reduced adipocyte size and increased the mRNA expression of adiponectin in adipose tissue. It also reduced macrophage infiltration by more than 70%, accompanied by reduced mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6 and monocyte chemotactic protein-1). The effects of Calanus oil were not only preventive, but also therapeutic, as the oil proved to be beneficial, regardless of whether the supplementation was started before or after the onset of obesity and glucose intolerance. Although the present study cannot pinpoint the active component(s) of the oil, there is reason to believe that the n-3 fatty acids EPA and DHA and/or antioxidants are responsible for its beneficial effects. It should be noted that the concentration of n-3 fatty acids in the Calanus oil diet was considerably lower than the concentrations used in similar studies reporting beneficial effects on obesity and obesity-related abnormalities.

Cardiomyocyte-restricted inhibition of G protein-coupled receptor kinase-3 attenuates cardiac dysfunction after chronic pressure overload.

Transgenic mice with cardiac-specific expression of a peptide inhibitor of G protein-coupled receptor kinase (GRK)3 [transgenic COOH-terminal GRK3 (GRK3ct) mice] display myocardial hypercontractility without hypertrophy and enhanced α(1)-adrenergic receptor signaling. A role for GRK3 in the pathogenesis of heart failure (HF) has not been investigated, but inhibition of its isozyme, GRK2, has been beneficial in several HF models. Here, we tested whether inhibition of GRK3 modulated evolving cardiac hypertrophy and dysfunction after pressure overload. Weight-matched male GRK3ct transgenic and nontransgenic littermate control (NLC) mice subjected to chronic pressure overload by abdominal aortic banding (AB) were compared with sham-operated (SH) mice. At 6 wk after AB, a significant increase of cardiac mass consistent with induction of hypertrophy was found, but no differences between GRK3ct-AB and NLC-AB mice were discerned. Simultaneous left ventricular (LV) pressure-volume analysis of electrically paced, ex vivo perfused working hearts revealed substantially reduced systolic and diastolic function in NLC-AB mice (n = 7), which was completely preserved in GRK3ct-AB mice (n = 7). An additional cohort was subjected to in vivo cardiac catheterization and LV pressure-volume analysis at 12 wk after AB. NLC-AB mice (n = 11) displayed elevated end-diastolic pressure (8.5 ± 3.1 vs. 2.9 ± 1.2 mmHg, P < 0.05), reduced cardiac output (3,448 ± 323 vs. 4,488 ± 342 µl/min, P < 0.05), and reduced dP/dt(max) and dP/dt(min) (both P < 0.05) compared with GRK3ct-AB mice (n = 16), corroborating the preserved cardiac structure and function observed in GRK3ct-AB hearts assessed ex vivo. Increased cardiac mass and myocardial mRNA expression of ß-myosin heavy chain confirmed the similar induction of cardiac hypertrophy in both AB groups, but only NLC-AB hearts displayed significantly elevated mRNA levels of brain natriuretic peptide and myocardial collagen contents as well as reduced ß(1)-adrenergic receptor responsiveness to isoproterenol, indicating increased LV wall stress and the transition to HF. Inhibition of cardiac GRK3 in mice does not alter the hypertrophic response but attenuates cardiac dysfunction and HF after chronic pressure overload.

Overexpression of NOX2 Exacerbates AngII-Mediated Cardiac Dysfunction and Metabolic Remodelling.

The present study aimed to examine the effects of low doses of angiotensin II (AngII) on cardiac function, myocardial substrate utilization, energetics, and mitochondrial function in C57Bl/6J mice and in a transgenic mouse model with cardiomyocyte specific upregulation of NOX2 (csNOX2 TG). Mice were treated with saline (sham), 50 or 400 ng/kg/min of AngII (AngII50 and AngII400) for two weeks. In vivo blood pressure and cardiac function were measured using plethysmography and echocardiography, respectively. Ex vivo cardiac function, mechanical efficiency, and myocardial substrate utilization were assessed in isolated perfused working hearts, and mitochondrial function was measured in left ventricular homogenates. AngII50 caused reduced mechanical efficiency despite having no effect on cardiac hypertrophy, function, or substrate utilization. AngII400 slightly increased systemic blood pressure and induced cardiac hypertrophy with no effect on cardiac function, efficiency, or substrate utilization. In csNOX2 TG mice, AngII400 induced cardiac hypertrophy and in vivo cardiac dysfunction. This was associated with a switch towards increased myocardial glucose oxidation and impaired mitochondrial oxygen consumption rates. Low doses of AngII may transiently impair cardiac efficiency, preceding the development of hypertrophy induced at higher doses. NOX2 overexpression exacerbates the AngII -induced pathology, with cardiac dysfunction and myocardial metabolic remodelling.

Chronic and acute exposure of mouse hearts to fatty acids increases oxygen cost of excitation-contraction coupling.

The aim of the present study was to evaluate the underlying processes involved in the oxygen wasting induced by inotropic drugs and acute and chronic elevation of fatty acid (FA) supply, using unloaded perfused mouse hearts from normal and type 2 diabetic (db/db) mice. We found that an acute elevation of the FA supply in normal hearts, as well as a chronic (in vivo) exposure to elevated FA as in db/db hearts, increased myocardial oxygen consumption (MVo2(unloaded)) due to increased oxygen cost for basal metabolism and for excitation-contraction (EC) coupling. Isoproterenol stimulation, on top of a high FA supply, led to an additive increase in MVo2(unloaded), because of a further increase in oxygen cost for EC coupling. In db/db hearts, the acute elevation of FA did not further increase MVo2. Since the elevation in the FA supply is accompanied by increased rates of myocardial FA oxidation, the present study compared MVo2 following increased FA load versus FA oxidation rate by exposing normal hearts to normal and high FA concentration (NF and HF, respectively) and to compounds that either stimulate (GW-610742) or inhibit [dichloroacetate (DCA)] FA oxidation. While HF and NF + GW-610742 increased FA oxidation to the same extent, only HF increased MVo2(unloaded). Although DCA counteracted the HF-induced increase in FA oxidation, DCA did not reduce MVo2(unloaded). Thus, in normal hearts, acute FA-induced oxygen waste is 1) due to an increase in the oxygen cost for both basal metabolism and EC coupling and 2) not dependent on the myocardial FA oxidation rate per se, but on processes initiated by the presence of FAs. In diabetic hearts, chronic exposure to elevated circulating FAs leads to adaptations that afford protection against the detrimental effect of an acute FA load, suggesting different underlying mechanisms behind the increased MVo2 following acute and chronic FA load.

Cardioprotective effect of the PPAR ligand tetradecylthioacetic acid in type 2 diabetic mice.

Tetradecylthioacetic acid (TTA) is a novel peroxisome proliferator-activated receptor (PPAR) ligand with marked hypolipidemic and insulin-sensitizing effects in obese models. TTA has recently been shown to attenuate dyslipidemia in patients with type 2 diabetes, corroborating the potential for TTA in antidiabetic therapy. In a recent study on normal mice, we showed that TTA increased myocardial fatty acid (FA) oxidation, which was associated with decreased cardiac efficiency and impaired postischemic functional recovery. The aim of the present study was, therefore, to elucidate the effects of TTA treatment (0.5%, 8 days) on cardiac metabolism and function in a hyperlipidemic type 2 diabetic model. We found that TTA treatment increased myocardial FA oxidation, not only in nondiabetic (db/+) mice but also in diabetic (db/db) mice, despite a clear lipid-lowering effect. Although TTA had deleterious effects in hearts from nondiabetic mice (decreased efficiency and impaired mitochondrial respiratory capacity), these effects were not observed in db/db hearts. In db/db hearts, TTA improved ischemic tolerance, an effect that is most likely related to the antioxidant property of TTA. The present study strongly advocates the need for investigation of the cardiac effects of PPAR ligands used in antidiabetic/hypolipidemic therapy, because of their pleiotropic properties.

Interval training normalizes cardiomyocyte function, diastolic Ca2+ control, and SR Ca2+ release synchronicity in a mouse model of diabetic cardiomyopathy.

RATIONALE: In the present study we explored the mechanisms behind excitation-contraction (EC) coupling defects in cardiomyocytes from mice with type-2 diabetes (db/db). OBJECTIVE: We determined whether 13 weeks of aerobic interval training could restore cardiomyocyte Ca(2+) cycling and EC coupling. METHODS AND RESULTS: Reduced contractility in cardiomyocytes isolated from sedentary db/db was associated with increased diastolic sarcoplasmic reticulum (SR)-Ca(2+) leak, reduced synchrony of Ca(2+) release, reduced transverse (T)-tubule density, and lower peak systolic and diastolic Ca(2+) and caffeine-induced Ca(2+) release. Additionally, the rate of SR Ca(2+) ATPase-mediated Ca(2+) uptake during diastole was reduced, whereas a faster recovery from caffeine-induced Ca(2+) release indicated increased Na(+)/Ca(2+)-exchanger activity. The increased SR-Ca(2+) leak was attributed to increased Ca(2+)-calmodulin-dependent protein kinase (CaMKIIdelta) phosphorylation, supported by the normalization of SR-Ca(2+) leak on inhibition of CaMKIIdelta (AIP). Exercise training restored contractile function associated with restored SR Ca(2+) release synchronicity, T-tubule density, twitch Ca(2+) amplitude, SR Ca(2+) ATPase and Na(+)/Ca(2+)-exchanger activities, and SR-Ca(2+) leak. The latter was associated with reduced phosphorylation of cytosolic CaMKIIdelta. Despite normal contractile function and Ca(2+) handling after the training period, phospholamban was hyperphosphorylated at Serine-16. Protein kinase A inhibition (H-89) in cardiomyocytes from the exercised db/db group abolished the differences in SR-Ca(2+) load when compared with the sedentary db/db mice. EC coupling changes were observed without changes in serum insulin or glucose levels, suggesting that the exercise training-induced effects are not via normalization of the diabetic condition. CONCLUSIONS: These data demonstrate that aerobic interval training almost completely restored the contractile function of the diabetic cardiomyocyte to levels close to sedentary wild type.

NADPH Oxidase 2 Mediates Myocardial Oxygen Wasting in Obesity.

Obesity and diabetes are independent risk factors for cardiovascular diseases, and they are associated with the development of a specific cardiomyopathy with elevated myocardial oxygen consumption (MVO2) and impaired cardiac efficiency. Although the pathophysiology of this cardiomyopathy is multifactorial and complex, reactive oxygen species (ROS) may play an important role. One of the major ROS-generating enzymes in the cardiomyocytes is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), and many potential systemic activators of NOX2 are elevated in obesity and diabetes. We hypothesized that NOX2 activity would influence cardiac energetics and/or the progression of ventricular dysfunction following obesity. Myocardial ROS content and mechanoenergetics were measured in the hearts from diet-induced-obese wild type (DIOWT) and global NOK2 knock-out mice (DIOKO) and in diet-induced obese C57BL/6J mice given normal water (DIO) or water supplemented with the NOX2-inhibitor apocynin (DIOAPO). Mitochondrial function and ROS production were also assessed in DIO and DIOAPO mice. This study demonstrated that ablation and pharmacological inhibition of NOX2 both improved mechanical efficiency and reduced MVO2 for non-mechanical cardiac work. Mitochondrial ROS production was also reduced following NOX2 inhibition, while cardiac mitochondrial function was not markedly altered by apocynin-treatment. Therefore, these results indicate a link between obesity-induced myocardial oxygen wasting, NOX2 activation, and mitochondrial ROS.

3-Weeks of Exercise Training Increases Ischemic-Tolerance in Hearts From High-Fat Diet Fed Mice.

Physical activity is an efficient strategy to delay development of obesity and insulin resistance, and thus the progression of obesity/diabetes-related cardiomyopathy. In support of this, experimental studies using animal models of obesity show that chronic exercise prevents the development of obesity-induced cardiac dysfunction (cardiomyopathy). Whether exercise also improves the tolerance to ischemia-reperfusion in these models is less clear, and may depend on the type of exercise procedure as well as time of initiation. We have previously shown a reduction in ischemic-injury in diet-induced obese mice, when the exercise was started prior to the development of cardiac dysfunction in this model. In the present study, we aimed to explore the effect of exercise on ischemic-tolerance when exercise was initiated after the development obesity-mediated. Male C57BL/6J mice were fed a high-fat diet (HFD) for 20-22 weeks, where they were subjected to high-intensity interval training (HIT) during the last 3 weeks of the feeding period. Sedentary HFD fed and chow fed mice served as controls. Left-ventricular (LV) post-ischemic functional recovery and infarct size were measured in isolated perfused hearts. We also assessed the effect of 3-week HIT on mitochondrial function and myocardial oxygen consumption (MVO2). Sedentary HFD fed mice developed marked obesity and insulin resistance, and demonstrated reduced post-ischemic cardiac functional recovery and increased infarct size. Three weeks of HIT did not induce cardiac hypertrophy and only had a mild effect on obesity and insulin resistance. Despite this, HIT improved post-ischemic LV functional recovery and reduced infarct size. This increase in ischemic-tolerance was accompanied by an improved mitochondrial function as well as reduced MVO2. The present study highlights the beneficial effects of exercise training with regard to improving the ischemic-tolerance in hearts with cardiomyopathy following obesity and insulin resistance. This study also emphasizes the exercise-induced improvement of cardiac energetics and mitochondrial function in obesity/diabetes.

Fenofibrate modulates cardiac and hepatic metabolism and increases ischemic tolerance in diet-induced obese mice.

Peroxisome proliferator-activated receptors (PPARs) play an important role in the transcriptional regulation of lipid utilization and storage in several organs, including liver and heart. Our working hypothesis is that treatment of obesity/hyperlipedemia with the PPARalpha ligand fenofibrate leads to drainage of plasma lipids by the liver, resulting in reduced myocardial lipid supply, reduced myocardial fatty acid oxidation and improved myocardial tolerance to ischemic stress. Thus, we investigated changes in substrate utilization in heart and liver, as well as post-ischemic functional recovery in hearts from diet-induced obese (DIO) mice following long-term (11-12 weeks) treatment with fenofibrate. The present study shows that DIO mice express increased plasma lipids and glucose, as well as increased myocardial fatty acid oxidation and a concomitant decrease in glucose oxidation. The lipid-lowering effect of fenofibrate was associated with increased hepatic mitochondrial and peroxisomal fatty acid oxidation, as indicated by a more than 30% increase in hepatic palmiotyl-CoA oxidation and more than a 10-fold increase in acyl-CoA oxidase (ACO) activity. In line with an adaptation to the reduced myocardial lipid supply, isolated hearts from fenofibrate-treated DIO mice showed increased glucose oxidation and decreased fatty acid oxidation, as well as reduced ACO activity. Fenofibrate treatment also prevented the diet-induced decrease in cardiac function and improved post-ischemic functional recovery. We also found that, while fenofibrate treatment markedly increased the expression of PPARalpha target genes in the liver, there were no such changes in the heart. These data demonstrate that fenofibrate results in a direct activation of PPARalpha in the liver with increased hepatic drainage of plasma lipids, while the cardiac effect of the compound most likely is secondary to its lipid-lowering effect.

Novel MRI method to detect altered left ventricular ejection and filling patterns in rodent models of disease.

The aim of this study was to determine whether high-temporal-resolution (HTR) cardiac cine-MRI could be used to identify subtle alterations in contractility and diastolic function in rodent models of disease. Following standard 45-min in vivo MRI measurements of left ventricular (LV) volumes, a single mid-ventricular slice was selected for 3-min HTR imaging. Cavity volume was measured every 2.4 ms, yielding approximately 60 images through the cardiac cycle. From these images, peak ejection and filling rates were calculated and two separate filling phases (comparable with the early (E) and late (A) phases of a Doppler echocardiogram) were identified during diastole. Repeated HTR imaging of the same animals on sequential days indicated reproducibility of E'/A' ratios of 11%. In chronically infarcted rat hearts, HTR imaging revealed lower peak ejection rates (PERs), peak early filling rates (E') and E'/A' ratios, and higher peak late filling rates (A') than in sham-operated rats. Diabetic db/db mouse hearts had the same function as controls when using standard cine-MRI, yet HTR imaging identified significantly lower PERs, early filling rates and E'/A' ratios in diabetic mouse hearts. In conclusion, the HTR MRI technique revealed changes in function that were below the limits of detection of standard cine-MRI.

Myocardial mechanical dysfunction and calcium overload following rewarming from experimental hypothermia in vivo.

Rewarming patients from accidental hypothermia are regularly complicated with cardiovascular instability ranging from minor depression of cardiac output to fatal circulatory collapse also termed "rewarming shock". Since altered Ca2+ handling may play a role in hypothermia-induced heart failure, we studied changes in Ca2+ homeostasis in in situ hearts following hypothermia and rewarming. A rat model designed for studies of the intact heart in a non-arrested state during hypothermia and rewarming was used. Rats were core cooled to 15 degrees C, maintained at 15 degrees C for 4h and thereafter rewarmed. As time-matched controls, one group of animals was kept at 37 degrees C for 5h. Total intracellular myocardial Ca2+ content ([Ca2+]i) was measured using 45Ca2+. Following rewarming we found a significant reduction of stroke volume and cardiac output compared to prehypothermic control values as well as to time-matched controls. Likewise, we found that hypothermia and rewarming resulted in a more than six-fold increase in [Ca2+]i to 3.01+/-0.43 micromol/g dry weight compared to 0.44+/-0.05 micromol/g dry weight in normothemia control. These findings indicate that hypothermia-induced alterations in the Ca2+-handling result in Ca2+ overload during hypothermia, which may contribute to myocardial failure during and after rewarming.

Isolated perfused working hearts provide valuable additional information during phenotypic assessment of the diabetic mouse heart.

Although murine models for studying the development of cardiac dysfunction in diabetes mellitus are well established, their reported cardiac phenotypes vary. These reported divergences may, in addition to the severity of different models, also be linked to the methods used for cardiac functional assessment. In the present study, we examined the functional changes using conventional transthoracic echocardiography (in vivo) and isolated heart perfusion techniques (ex vivo), in hearts from two mouse models; one with an overt type 2 diabetes (the db/db mouse) and one with a prediabetic state, where obesity was induced by a high-fat diet (HFD). Analysis of left ventricular function in the isolated working hearts from HFD-fed mice, suggested that these hearts develop diastolic dysfunction with preserved systolic function. Accordingly, in vivo examination demonstrated maintained systolic function, but we did not find parameters of diastolic function to be altered. In db/db mice, ex vivo working hearts showed both diastolic and systolic dysfunction. Although in vivo functional assessment revealed signs of diastolic dysfunction, the hearts did not display reduced systolic function. The contrasting results between ex vivo and in vivo function could be due to systemic changes that may sustain in vivo function, or a lack of sensitivity using conventional transthoracic echocardiography. Thus, this study demonstrates that the isolated perfused working heart preparation provides unique additional information related to the development of cardiomyopathy, which might otherwise go unnoticed when only using conventional echocardiographic assessment.

Increased myocardial oxygen consumption reduces cardiac efficiency in diabetic mice.

Altered cardiac metabolism and function (diabetic cardiomyopathy) has been observed in diabetes. We hypothesize that cardiac efficiency, the ratio of cardiac work (pressure-volume area [PVA]) and myocardial oxygen consumption (MVo(2)), is reduced in diabetic hearts. Experiments used ex vivo working hearts from control db/+, db/db (type 2 diabetes), and db/+ mice given streptozotocin (STZ; type 1 diabetes). PVA and ventricular function were assessed with a 1.4-F pressure-volume catheter at low (0.3 mmol/l) and high (1.4 mmol/l) fatty acid concentrations with simultaneous measurements of MVo(2). Substrate oxidation and mitochondrial respiration were measured in separate experiments. Diabetic hearts showed decreased cardiac efficiency, revealed as an 86 and 57% increase in unloaded MVo(2) in db/db and STZ-administered hearts, respectively. The slope of the PVA-MVo(2) regression line was increased for db/db hearts after elevation of fatty acids, suggesting that contractile inefficiency could also contribute to the overall reduction in cardiac efficiency. The end-diastolic and end-systolic pressure-volume relationships in db/db hearts were shifted to the left with elevated end-diastolic pressure, suggesting left ventricular remodeling and/or myocardial stiffness. Thus, by means of pressure-volume technology, we have for the first time documented decreased cardiac efficiency in diabetic hearts caused by oxygen waste for noncontractile purposes.

Nox4 reprograms cardiac substrate metabolism via protein O-GlcNAcylation to enhance stress adaptation.

Cardiac hypertrophic remodeling during chronic hemodynamic stress is associated with a switch in preferred energy substrate from fatty acids to glucose, usually considered to be energetically favorable. The mechanistic interrelationship between altered energy metabolism, remodeling, and function remains unclear. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated in the overloaded heart, where it ameliorates adverse remodeling. Here, we show that Nox4 redirects glucose metabolism away from oxidation but increases fatty acid oxidation, thereby maintaining cardiac energetics during acute or chronic stresses. The changes in glucose and fatty acid metabolism are interlinked via a Nox4-ATF4-dependent increase in the hexosamine biosynthetic pathway, which mediates the attachment of O-linked N-acetylglucosamine (O-GlcNAcylation) to the fatty acid transporter CD36 and enhances fatty acid utilization. These data uncover a potentially novel redox pathway that regulates protein O-GlcNAcylation and reprograms cardiac substrate metabolism to favorably modify adaptation to chronic stress. Our results also suggest that increased fatty acid oxidation in the chronically stressed heart may be beneficial.