Carole Lan

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

To assess the influence of isoflurane and pentobarbital anesthesia and the carrier gases on myocardial blood flow (MBF) in the rat heart in vivo.

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.

L-arginine during long-term ischemia: effects on cardiac function, energetic metabolism and endothelial damage

BACKGROUND We have evaluated the addition of L-arginine, a precursor of nitric oxide, to a cardioplegic solution (named CRMBM) designed for long-term heart preservation. METHODS Isolated isovolumic-perfused rat hearts (n = 22) were arrested with the CRMBM solution either with (Arg) or without L-arginine (2 mmol/L) (Arg group, n = 12, vs control group n = 10), submitted to 8 hours of cold storage (4 degrees C) in the solution, and then reperfused for 60 minutes at 37 degrees C. In 11 hearts, we evaluated the quality of cardiac preservation with P-31 magnetic resonance spectroscopy and the measure of function and cellular integrity. Endothelium-dependent and independent vasodilatations were measured in 11 other hearts, using 5-hydroxytryptamine and papaverine to assess endothelial and smooth muscle function. RESULTS Adding L-arginine to the cardioplegic solution improved functional recovery during reflow, as shown by the rate pressure product (31% +/- 3% for control vs 47% +/- 3% for Arg, p = 0.003) together with higher coronary flow and diminished contracture. Purine release in coronary effluents during reperfusion was lower in the Arg group. During ischemia and reflow kinetics of intracellular pH and high-energy phosphates were similar in both groups. Coronary endothelium-dependent vasodilatation was similarly impaired in both groups, but smooth muscle was less altered with L-arginine. CONCLUSIONS As an additive to the CRMBM cardioplegic solution, L-arginine provides a protective effect for long-term heart preservation. Our data do not show coronary endothelial protection as the prominent mechanism.

Optimized cardiac graft preservation: a comparative experimental study using P-31 magnetic resonance spectroscopy and biochemical analyses ☆

BACKGROUND The University of Wisconsin (UW), St. Thomas (ST) and Broussais (B) solutions were compared to the CRMBM solution, that we developed for long term heart preservation. METHODS Isolated isovolumic rat hearts were arrested with each cardioplegic solution (n = 5) to 8 hearts in each group), submitted to 12 hours of cold storage (4 degrees C) in the same solution and then reperfused for 60 minutes at 37 degrees C. Function was measured during control and reflow. High energy phosphates and intracellular pH were monitored by P-31 magnetic resonance spectroscopy. Analyses were performed by biochemical assays and HPLC in coronary effluents (CK, Pi, lactate, purines) and in freeze-clamped hearts (amino acids, nucleotides, CK, LDH) at the end of reperfusion. RESULTS Functional recovery was significantly improved with the new cardioplegic solution (50+/-12% recovery for the rate pressure product at the end of reflow vs 8+/-3% with UW, 0% with B and with ST). This result was correlated with the best metabolic and cellular protection as assessed in particular by higher PCr levels during reflow (30+/-3% vs 10+/-3% with UW, 8+/-4% with B, and 7+/-1% with ST) as well as reduced creatine kinase leakage during reflow (110+/-15 IU/60 minute vs 270 +/- 57 IU/60 minute with UW, 323+/-36 IU/60 minute with Broussais solution and 237+/-18 IU/60 minute with ST). CONCLUSION This new solution is more effective in prolonged myocardial protection than the three most widely used solutions.

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.

Preservation of amino acids during long term ischemia and subsequent reflow with supplementation of L-arginine, the nitric oxide precursor, in the rat heart

Summary. We investigated whether L-arginine, used in heart preservation to limit endothelial damage, may influence the pool of amino acids during long term ischemia and reflow. Isolated isovolumic rat hearts (n = 23) were submitted to 8 h of hypothermic ischemia after cardioplegic arrest with the Centre de Résonance Magnétique Biologique et Médicale (CRMBM) solution with or without L-arginine (Arg and No Arg groups respectively). Hearts were freeze-clamped after ischemia (n = 11) or submitted to 60 min of reflow (n = 12) and freeze-clamped. Eight hearts were perfused aerobically for 20 min and freeze-clamped (No ischemia group). Addition of L-arginine to the CRMBM solution limited aspartate depletion and decreased lysine level at the end of ischemia. After reflow, L-arginine supplementation increased the pool of glutamate and arginine and limited the depletion of serine, asparagine, glycine and taurine. We conclude that adding L-arginine to the CRMBM cardioplegic solution during long term ischemia preserved the amino acids pool.

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 β‐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 (NOx). 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 NOx 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. Copyright

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.

Simultaneous study of metabolism and function following cardioplegic arrest: a novel method of evaluation of the transplanted heart in the rat ☆

BACKGROUND Limitations of the isolated perfused rat heart model for heart preservation studies include short study time due to the lack of stability of the preparation. We aimed to develop a new experimental model based on heterotopic heart transplantation in the rat to achieve simultaneous (31)P magnetic resonance spectroscopy (MRS) and functional study of the transplanted heart during early and late blood reperfusion. METHODS Twenty-five Lewis rats underwent heterotopic abdominal isograft heart transplantation and were randomized in two groups. Hearts were harvested after cardioplegic arrest induced with Centre de Résonance Magnétique Biologique et Médicale (CRMBM) solution and then stored at 4 degrees C for a total ischemic time of 3 hours. Graft contractility measurement and simultaneous (31)P MRS were performed after 1 hour and 24 hours of blood reperfusion, respectively, in groups I (n = 12) and II (n =13). RESULTS Contractility improved during reperfusion. The mean rate pressure product plus or minus standard error of mean increased from 11,373 +/- 1,377 mm Hg/min in group I to 24,363 +/- 3,860 mm Hg/min in group II (P = 0.003), while mean dP/dtmax increased from 1,642 +/- 173 mm Hg/sec to 2,571 +/- 333 mm Hg/sec, respectively (p = 0.03). Simultaneously, both the phosphocreatine/adenosine triphosphate (ATP) and inorganic phosphate/ATP ratios decreased from group I to group II (p = 0.025 and p = 0.015, respectively), suggesting regeneration of the intracellular pool of ATP in group II. CONCLUSIONS Simultaneous functional and metabolic studies of the transplanted heart are feasible in rats. Improvement in contractility during late reperfusion is contemporary with significant changes in energetic metabolism. Our model should be useful for the further improvement of heart preservation, which may result in significant clinical progress.

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.