Sex-Mediated Response to the Beta-Blocker Landiolol in Sepsis: An Experimental, Randomized Study.

OBJECTIVES: To investigate any gender effect of the beta-1 adrenergic blocker, landiolol, on cardiac performance and energy metabolism in septic rats, and to explore the expression of genes and proteins involved in this process. DESIGN: Randomized animal study. SETTING: University research laboratory. SUBJECTS: Male and female Wistar rats. INTERVENTIONS: One hour after cecal ligation and puncture, male and female rats were randomly allocated to the following groups: sham male, cecal ligation and puncture male, cecal ligation and puncture + landiolol male, sham female, cecal ligation and puncture female, and cecal ligation and puncture + landiolol female. Cardiac MRI was carried out 18 hours after cecal ligation and puncture to assess in vivo cardiac function. Ex vivo cardiac function measurement and P magnetic resonance spectroscopy were subsequently performed using an isovolumic isolated heart preparation. Finally, we assessed cardiac gene and protein expression. MEASUREMENTS AND MAIN RESULTS: In males, landiolol increased indexed stroke volume by reversing the indexed end-diastolic volume reduction without affecting left ventricle ejection fraction. In females, landiolol did not increase indexed stroke volume and indexed end-diastolic volume but decreased left ventricle ejection fraction. Landiolol had no effect on ex vivo cardiac function and on high-energy phosphate compounds. The effect of landiolol on the gene expression of natriuretic peptide receptor 3 and on protein expression of phosphorylated-AKT:AKT ratio and endothelial nitric oxide synthase was different in males and females. CONCLUSIONS: Landiolol improved the in vivo cardiac performance of septic male rats while deleterious effects were reported in females. Expression of natriuretic peptide receptor 3, phosphorylated-AKT:AKT, and endothelial nitric oxide synthase are signaling pathways to investigate to better understand the sex differences in sepsis.

Effect of isoproterenol on myocardial perfusion, function, energy metabolism and nitric oxide pathway in the rat heart - a longitudinal MR study.

The chronic administration of the ß-adrenoreceptor agonist isoproterenol (IsoP) is used in animals to study the mechanisms of cardiac hypertrophy and failure associated with a sustained increase in circulating catecholamines. Time-dependent changes in myocardial blood flow (MBF), morphological and functional parameters were assessed in rats in vivo using multimodal cardiac MRI. Energy metabolism, oxidative stress and the nitric oxide (NO) pathway were evaluated in isolated perfused rat hearts following 7 days of treatment. Male Wistar rats were infused for 7 days with IsoP or vehicle using osmotic pumps. Cine-MRI and arterial spin labeling were used to determine left ventricular morphology, function and MBF at days 1, 2 and 7 after pump implantation. Isolated hearts were then perfused, and high-energy phosphate compounds and intracellular pH were followed using ³¹P MRS with simultaneous measurement of contractile function. Total creatine and malondialdehyde (MDA) contents were measured by high-performance liquid chromatography. The NO pathway was evaluated by NO synthase isoform expression and total nitrate concentration (NO(x)). In IsoP-treated rats, left ventricular mass was increased at day 1 and maintained. Wall thickness was increased with a peak at day 2 and a tendency to return to baseline values at day 7. MBF was markedly increased at day 1 and returned to normal values between days 1 and 2. The rate-pressure product and phosphocreatine/adenosine triphosphate ratio in perfused hearts were reduced. MDA, endothelial NO synthase expression and NO(x) were increased. Sustained high cardiac function and normal MBF after 24 h of IsoP infusion indicate imbalance between functional demand and blood flow, leading to morphological changes. After 1 week, cardiac hypertrophy and decreased function were associated with impaired phosphocreatine, increased oxidative stress and up-regulation of the NO pathway. These results provide supplemental information on the evolution of the different contributing factors leading to morphological and functional changes in this model of cardiac hypertrophy and failure.

Male and Female Rats Have Different Physiological Response to High-Fat High-Sucrose Diet but Similar Myocardial Sensitivity to Ischemia-Reperfusion Injury.

Prediabetes is a strong predictor of type 2 diabetes and its associated cardiovascular complications, but few studies explore sexual dimorphism in this context. Here, we aim to determine whether sex influences physiological response to high-fat high-sucrose diet (HFS) and myocardial tolerance to ischemia-reperfusion injury. Male and female Wistar rats were subjected to standard (CTRL) or HFS diet for 5 months. Then, ex-vivo experiments on isolated perfused heart model were performed to evaluate tolerance to ischemia-reperfusion injury. HFS diet induced fasting hyperglycemia and increased body fat percent to a similar level in both sexes. However, glucose intolerance was more pronounced in female HFS. Cholesterol was increased only in female while male displayed higher level of plasmatic leptin. We observed increased heart weight to tibia length ratio only in males, but we showed a similar decrease in tolerance to ischemia-reperfusion injury in female and male HFS compared with respective controls, characterized by impaired cardiac function, energy metabolism and coronary flow during reperfusion. In conclusion, as soon as glucose intolerance and hyperglycemia develop, we observe higher sensitivity of hearts to ischemia-reperfusion injury without difference between males and females.

Upregulation of eNOS and unchanged energy metabolism in increased susceptibility of the aging type 2 diabetic GK rat heart to ischemic injury.

We investigated the tolerance of the insulin-resistant diabetic heart to ischemic injury in the male Goto-Kakizaki (GK) rat, a model of type 2 diabetes. Changes in energy metabolism, nitric oxide (NO) pathway, and cardiac function were assessed in the presence of physiological substrates. Age-matched control Wistar (n = 19) and GK (n = 18) isolated rat hearts were perfused with 0.4 mM palmitate, 3% albumin, 11 mM glucose, 3 U/l insulin, 0.2 mM pyruvate, and 0.8 mM lactate for 24 min before switching to 1.2 mM palmitate (11 rats/group) during 32 min low-flow (0.5 ml·min(-1)·g wet wt(-1)) ischemia. Next, flow was restored with 0.4 mM palmitate buffer for 32 min. A subset of hearts from each group (n = 8 for control and n = 7 for GK groups) were freeze-clamped for determining baseline values after the initial perfusion of 24 min. ATP, phosphocreatine (PCr), and intracellular pH (pH(i)) were followed using (31)P magnetic resonance spectroscopy with simultaneous measurement of contractile function. The NO pathway was determined by nitric oxide synthase (NOS) isoform expression and total nitrate concentration (NOx) in hearts. We found that coronary flow was 26% lower (P < 0.05) during baseline conditions and 61% lower (P < 0.05) during reperfusion in GK vs. control rat hearts. Rate pressure product was lower during reperfusion in GK vs. control rat hearts (P < 0.05). ATP, PCr, and pH(i) during ischemia-reperfusion were similar in both groups. Endothelial NOS expression was increased in GK rat hearts during baseline conditions (P < 0.05). NOx was increased during baseline conditions (P < 0.05) and after reperfusion (P < 0.05) in GK rat hearts. We report increased susceptibility of type 2 diabetic GK rat heart to ischemic injury that is not associated with impaired energy metabolism. Reduced coronary flow, upregulation of eNOS expression, and increased total NOx levels confirm NO pathway modifications in this model, presumably related to increased oxidative stress. Modifications in the NO pathway may play a major role in ischemia-reperfusion injury of the type 2 diabetic GK rat heart.

Protective Effect of Resveratrol against Ischemia-Reperfusion Injury via Enhanced High Energy Compounds and eNOS-SIRT1 Expression in Type 2 Diabetic Female Rat Heart.

Type 2 diabetic women have a high risk of mortality via myocardial infarction even with anti-diabetic treatments. Resveratrol (RSV) is a natural polyphenol, well-known for its antioxidant property, which has also shown interesting positive effects on mitochondrial function. Therefore, we aim to investigate the potential protective effect of 1 mg/kg/day of RSV on high energy compounds, during myocardial ischemia-reperfusion in type 2 diabetic female Goto-Kakizaki (GK) rats. For this purpose, we used 31P magnetic resonance spectroscopy in isolated perfused heart experiments, with a simultaneous measurement of myocardial function and coronary flow. RSV enhanced adenosine triphosphate (ATP) and phosphocreatine (PCr) contents in type 2 diabetic hearts during reperfusion, in combination with better functional recovery. Complementary biochemical analyses showed that RSV increased creatine, total adenine nucleotide heart contents and citrate synthase activity, which could be involved in better mitochondrial functioning. Moreover, improved coronary flow during reperfusion by RSV was associated with increased eNOS, SIRT1, and P-Akt protein expression in GK rat hearts. In conclusion, RSV induced cardioprotection against ischemia-reperfusion injury in type 2 diabetic female rats via increased high energy compound contents and expression of protein involved in NO pathway. Thus, RSV presents high potential to protect the heart of type 2 diabetic women from myocardial infarction.

Cardiac remodeling and higher sensitivity to ischemia-reperfusion injury in female rats submitted to high-fat high-sucrose diet: An in vivo/ex vivo longitudinal follow-up.

Prediabetes is an important risk factor for Type 2 diabetes and cardiovascular complications, such as myocardial infarction. However, few studies explore female sex in this context. Here, we aim to investigate the effects of high-fat high-sucrose diet on cardiac parameters and sensitivity to ischemia-reperfusion injury of female Wistar rats. Female Wistar rats received for 5 months normal diet (CTRL group) or high-fat high-sucrose diet (HFS group). Every month, MRI was performed to follow myocardial morphology, function and perfusion; cardiac and hepatic triglyceride content; and amount of sub-cutaneous and visceral adipose tissues. Then, ex vivo experiments were performed on isolated perfused hearts to evaluate tolerance to ischemia-reperfusion, with simultaneous measurement of energy metabolism by 31P MRS and contractile function. Coronary flow was measured before and after ischemia. At the end of the experiments, hearts were freeze-clamped for biochemical assays. Five months of high-fat high-sucrose diet induced a prediabetic condition in female Wistar rats, associated with an increase in myocardial perfusion, systolic and diastolic wall thickness. HFS rats also exhibited higher sensitivity to ischemia-reperfusion injury in comparison to controls, characterized by impaired cardiac function, energy metabolism and endothelial function. Biochemical analyses in hearts highlighted eNOS uncoupling, higher malondialdehyde level and lower S-Glutathionylation of proteins in HFS rats, indicating higher oxidative stress. Prediabetes induced by energy-dense diet was associated with modification of cardiac morphology and higher myocardial sensitivity to ischemia-reperfusion injury. These results may be related to the high risk of cardiovascular complications among Type 2 diabetic women.

Exercise training impacts exercise tolerance and bioenergetics in gastrocnemius muscle of non-obese type-2 diabetic Goto-Kakizaki rat in vivo.

The functional and bioenergetics impact of regular physical activity upon type-2 diabetic skeletal muscle independently of confounding factors of overweight remains undocumented. Here, gastrocnemius muscle energy fluxes, mitochondrial capacity and mechanical performance were assessed noninvasively and longitudinally in non-obese diabetic Goto-Kakizaki rats using magnetic resonance (MR) imaging and dynamic 31-phosphorus MR spectroscopy (31P-MRS) throughout a 6-min fatiguing bout of exercise performed before, in the middle (4-week) and at the end of an 8-week training protocol consisting in 60-min daily run on a treadmill. The training protocol reduced plasmatic insulin level (-61%) whereas blood glucose and non-esterified fatty acids levels remained unaffected, thereby indicating an improvement of insulin sensitivity. It also increased muscle mitochondrial citrate synthase activity (+45%) but this increase did not enhance oxidative ATP synthesis capacity in working muscle in vivo while glycolytic ATP production was increased (+33%). On the other hand, the training protocol impaired maximal force-generating capacity (-9%), total amount of force produced (-12%) and increased ATP cost of contraction (+32%) during the fatiguing exercise. Importantly, these deleterious effects were transiently worsened in the middle of the 8-week period, in association with reduced oxidative capacity and increased basal [Pi]/[PCr] ratio (an in vivo biomarker of muscle damage). These data demonstrate that the beneficial effect of regular training on insulin sensitivity in non-obese diabetic rat occurs separately from any improvement in muscle mitochondrial function and might be linked to an increased capacity for metabolizing glucose through anaerobic process in exercising muscle.

Reduced up-regulation of the nitric oxide pathway and impaired endothelial and smooth muscle functions in the female type 2 diabetic goto-kakizaki rat heart.

BACKGROUND: Type 2 diabetes is associated with greater relative risk of cardiovascular diseases in women than in men, which is not well understood. Consequently, we have investigated if male and female displayed differences in cardiac function, energy metabolism, and endothelial function which could contribute to increased cardiovascular complications in type 2 diabetic female. METHODS: Male and female Control and type 2 diabetic Goto-Kakizaki (GK) isolated rat hearts were perfused during 28 min with a physiological buffer before freeze-clamping for biochemical assays. High energy phosphate compounds and intracellular pH were followed using 31P magnetic resonance spectroscopy with simultaneous measurement of contractile function. Nitric oxide (NO) pathway and endothelium-dependent and independent vasodilatations were measured as indexes of endothelial function. Results were analyzed via two-way ANOVA, p < 0.05 was considered as statistically significant. RESULTS: Myocardial function was impaired in male and female diabetic versus Control groups (p < 0.05) without modification of energy metabolism. Coronary flow was decreased in both diabetic versus Control groups but to a higher extent in female GK versus male GK rat hearts (p < 0.05). NO production was up-regulated in diabetic groups but to a less extent in female GK rat hearts (p < 0.05). Endothelium-dependent and independent vasodilatations were impaired in female GK rat compared with male GK (p < 0.05) and female Control (p < 0.05) rat hearts. CONCLUSIONS: We reported here an endothelial damage characterized by a reduced up-regulation of the NO pathway and impaired endothelial and smooth muscle functions, and coronary flow rates in the female GK rat hearts while energy metabolism was normal. Whether these results are related to the higher risk of cardiovascular complications among type 2 diabetic female needs to be further elicited in the future.

Dietary docosahexaenoic acid-enriched glycerophospholipids exert cardioprotective effects in ouabain-treated rats via physiological and metabolic changes.

Docosahexaenoic acid (DHA) might prevent heart failure or optimise drug treatments by improving cardiac contraction. We investigated whether DHA-enriched avian glycerophospholipids (GPL-DHA) exert cardioprotection in ouabain-treated rats after 4 weeks of dietary supplementation with 10, 35 or 60 mg DHA per kg body weight versus none (DHA10, DHA35, DHA60 and control groups, respectively). The contractile responsiveness to different doses of ouabain (10(-7) to 10(-4) M), ouabain intoxication (at 3 × 10(-4) M), and relative variations in cardiac energy metabolism were determined using (31)P NMR in isolated perfused rat hearts. The fatty acid composition of cardiac membranes was analysed by gas chromatography. DHA accretion in the heart was dose-dependent (+8%, +30% and +45% for DHA10, DHA35 and DHA60, respectively). The cardiac phosphocreatine content significantly increased at the baseline in DHA35 (+45%) and DHA60 groups (+85%), and at the different doses of ouabain in the DHA60 group (+73% to 98%). The maximum positive inotropy achieved at 10(-4) M ouabain was significantly increased in all DHA groups versus control (+150%, +122.5% and +135% for DHA10, DHA35 and DHA60, respectively), and ouabain intoxication was delayed. The increase in myocardial phosphocreatine content and the improved efficacy of ouabain on myocardial contraction without toxicity suggest the potential of GPL-DHA as a dietary supplement or ingredient for functional food, and possibly as a co-treatment with digitalis drugs in humans.

Defective myocardial blood flow and altered function of the left ventricle in type 2 diabetic rats: a noninvasive in vivo study using perfusion and cine magnetic resonance imaging.

OBJECTIVE: In type 2 diabetes mellitus, cardiovascular complications are related to microvascular abnormalities. In this work, we aimed at characterizing in vivo myocardial blood flow and left ventricular function of the Goto-Kakizaki (GK) rat as a nonobese model of type 2 diabetes. MATERIALS AND METHODS: We performed arterial spin labeling magnetic resonance imaging (MRI) for myocardial blood flow quantification and cine MRI for functional evaluation in free-breathing isoflurane-anesthetized animals. RESULTS: Myocardial blood flow was altered in adult female GK rats compared with age-matched female Wistar rats (4.7 +/- 1.6 vs. 7.1 +/- 1.2 mL/g/min respectively, P = 0.0022). Ejection fraction was decreased in GK compared with Wistar rats (64 +/- 7 vs. 78 +/- 8% respectively, P <0.005), mainly as a result of a loss in left ventricular longitudinal contraction. CONCLUSIONS: Adult female GK rats have defective myocardial blood flow associated with altered left ventricular function. This multiparametric MRI approach in the GK rat is of particular interest for the study of type 2 diabetic cardiomyopathy.

Insulin Resistance Is Not Associated with an Impaired Mitochondrial Function in Contracting Gastrocnemius Muscle of Goto-Kakizaki Diabetic Rats In Vivo.

Insulin resistance, altered lipid metabolism and mitochondrial dysfunction in skeletal muscle would play a major role in type 2 diabetes mellitus (T2DM) development, but the causal relationships between these events remain conflicting. To clarify this issue, gastrocnemius muscle function and energetics were investigated throughout a multidisciplinary approach combining in vivo and in vitro measurements in Goto-Kakizaki (GK) rats, a non-obese T2DM model developing peripheral insulin resistant without abnormal level of plasma non-esterified fatty acids (NEFA). Wistar rats were used as controls. Mechanical performance and energy metabolism were assessed strictly non-invasively using magnetic resonance (MR) imaging and 31-phosphorus MR spectroscopy (31P-MRS). Compared with control group, plasma insulin and glucose were respectively lower and higher in GK rats, but plasma NEFA level was normal. In resting GK muscle, phosphocreatine content was reduced whereas glucose content and intracellular pH were both higher. However, there were not differences between both groups for basal oxidative ATP synthesis rate, citrate synthase activity, and intramyocellular contents for lipids, glycogen, ATP and ADP (an important in vivo mitochondrial regulator). During a standardized fatiguing protocol (6 min of maximal repeated isometric contractions electrically induced at a frequency of 1.7 Hz), mechanical performance and glycolytic ATP production rate were reduced in diabetic animals whereas oxidative ATP production rate, maximal mitochondrial capacity and ATP cost of contraction were not changed. These findings provide in vivo evidence that insulin resistance is not caused by an impairment of mitochondrial function in this diabetic model.

NOS substrate during cardioplegic arrest and cold storage decreases stunning after heart transplantation in a rat model.

BACKGROUND: In this study, we evaluated how adding L-arginine to Centre de Résonance Magnétique Biologique et Médicale (CRMBM) solution affected myocardial performance during post-ischemic in vivo reperfusion. METHODS: Experiments were conducted using a modified Lewis-Lewis heterotopic heart transplantation model, with a total ischemic time of 3 hours followed by 1 or 24 hours of blood reperfusion. Heart grafts were arrested using intra-aortic injection of CRMBM solution, either supplemented or not supplemented with 2 mmol/liter L-arginine (n = 12 in each group). We measured systolic indexes and simultaneously performed phosphorus magnetic resonance spectroscopy ((31)P MRS). We quantified total endothelial nitric oxide synthase (eNOS) protein using the Western blot test of freeze-clamped hearts. RESULTS: Contractility during early reperfusion was significantly better in grafts arrested with CRMBM solution enriched with L-arginine: mean rate pressure product, 11249 +/- 1548 vs 5637 +/- 1118 mm Hg/min (p = 0.05), and maximal first derivative of the pressure signal (dP/dt(max)), 1721 +/- 177 vs 1214 +/- 321 mm Hg/sec (p = 0.013). Conversely, during late reperfusion, contractility did not relate to the nature of the preservation solution. The presence of L-arginine in the CRMBM solution did not alter time-related variations of high-energy phosphate ratios measured using in vivo (31)P MRS. The eNOS protein level decreased significantly during early compared with late reperfusion, with no effect caused by L-arginine. CONCLUSIONS: During early reperfusion, the limited myocardial stunning observed with CRMBM solution containing L-arginine does not relate to energy metabolism but to better preservation of the NO pathway.

Limitation of myocardial and endothelial injury of the rat heart graft after preservation with Centre de Résonance Magnétique Biologique et Médicale (CRMB) solution.

Myocardial injury caused by prolonged storage compromises post-transplantation contractile performance and induces endothelial injury. The aim of this study was to compare a solution developed in our laboratory [Centre de Résonance Magnétique Biologique et Médicale (CRMBM) solution] with a widely used solution (Celsior, Genzyme, Saint Germain en Laye, France). Metabolic and contractile parameters as well as indexes of endothelial injury were measured in a heterotopic rat heart transplantation model with a 3-h ischaemia and a 1-h reperfusion. The two solutions were randomly used for cardioplegia and graft preservation in six experiments each. During reperfusion, developed pressure and rate pressure product were higher with CRMBM compared with Celsior (P = 0.0002 and P = 0.0135, respectively). Phosphocreatine and adenosine triphosphate (ATP) concentrations after reperfusion were significantly higher with CRMBM (P = 0.0069 and P = 0.0053, respectively). Endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) protein expression were decreased to the same extent after reperfusion compared with baseline with CRMBM (P = 0.0001 and P < 0.0001, respectively) and Celsior (P = 0.0007 and P < 0.0001, respectively). Total nitrate concentration (NOx) was significantly increased after reperfusion with CRMBM (P < 0.0001 versus baseline and P < 0.0001 versus Celsior). Na,K-ATPase activity was decreased in both groups versus baseline after reperfusion (P < 0.0001 for CRMBM and P < 0.0001 for Celsior). We showed limitation of both myocardial and endothelial damage with CRMBM compared with Celsior during heterotopic rat heart transplantation in vivo.

In vivo assessment of myocardial blood flow in rat heart using magnetic resonance imaging: effect of anesthesia.

PURPOSE: To assess the influence of isoflurane and pentobarbital anesthesia and the carrier gases on myocardial blood flow (MBF) in the rat heart in vivo. MATERIALS AND METHODS: MBF was quantified in vivo using arterial spin-labeling (ASL) magnetic resonance imaging (MRI). Left ventricular (LV) function was estimated during the same experiment using cine-MRI. Thirty-four male Wistar-Kyoto rats were divided in four groups, one anesthetized with isoflurane in oxygen:nitrous oxide mix (ISO), the three others with intraperitoneal pentobarbital, and breathing either room air (PB), oxygen:nitrous oxide (PB + N(2)O), or oxygen:nitrogen (PB + N(2)). RESULTS: MBF was significantly higher in the ISO and PB + N(2)O groups vs. PB and in ISO vs. PB + N(2), with the following respective MBF values: ISO, 5.9 +/- 1.1; PB, 4.0 +/- 0.8; PB + N(2)O, 5.1 +/- 1.4; and PB + N(2), 4.6 +/- 0.8 mL/g/minute, mean +/- SD. Ejection fractions were reduced by 10% in PB and PB + N(2)O rats vs. ISO rats. Cardiac output (CO) and index (CI) were 25 to 30% lower in all rats anesthetized with pentobarbital than with isoflurane. CONCLUSION: Isoflurane and nitrous oxide induce a higher MBF than pentobarbital. Isoflurane also induces a higher ejection fraction in healthy rats.