Concomitant systolic and diastolic alterations during chronic hypertension in pig.

The mechanical and cellular relationships between systole and diastole during left ventricular (LV) dysfunction remain to be established. LV contraction-relaxation coupling was examined during LV hypertrophy induced by chronic hypertension. Chronically instrumented pigs received angiotensin II infusion for4weeks to induce chronic hypertension (133 ±â€¯7 mmHg vs 98 ±â€¯5 mmHg for mean arterial pressure at Day 28 vs 0, respectively) and LV hypertrophy. LV function was investigated with the instrumentation and echocardiography for LV twist-untwist assessment before and after dobutamine infusion. The cellular mechanisms were investigated by exploring the intracellular Ca2+ handling. At Day 28, pigs exhibited LV hypertrophy with LV diastolic dysfunction (impaired LV isovolumic relaxation, increased LV end-diastolic pressure, decreased and delayed LV untwisting rate) and LV systolic dysfunction (impaired LV isovolumic contraction and twist) although LV ejection fraction was preserved. Isolated cardiomyocytes exhibited altered shortening and lengthening. Interestingly, contraction-relaxation coupling remained preserved both in vivo and in vitro during LV hypertrophy. LV systolic and diastolic dysfunctions were associated to post-translational remodeling and dysfunction of the type 2 cardiac ryanodine receptor/Ca2+ release channel (RyR2), i.e., PKA hyperphosphorylation of RyR2, depletion of calstabin 2 (FKBP12.6), RyR2 leak and hypersensitivity of RyR2 to cytosolic Ca2+ during both contraction and relaxation phases. In conclusion, LV contraction-relaxation coupling remained preserved during chronic hypertension despite LV systolic and diastolic dysfunctions. This implies that LV diastolic dysfunction is accompanied by LV systolic dysfunction. At the cellular level, this is linked to sarcoplasmic reticulum Ca2+ leak through PKA-mediated RyR2 hyperphosphorylation and depletion of its stabilizing partner.

Development of original metabolically stable apelin-17 analogs with diuretic and cardiovascular effects.

Apelin, a (neuro)vasoactive peptide, plays a prominent role in controlling cardiovascular functions and water balance. Because the in vivo apelin half-life is in the minute range, we aimed to identify metabolically stable apelin-17 (K17F) analogs. We generated P92 by classic chemical substitutions and LIT01-196 by original addition of a fluorocarbon chain to the N terminus of K17F. Both analogs were much more stable in plasma (half-life >24 h for LIT01-196) than K17F (4.6 min). Analogs displayed a subnanomolar affinity for the apelin receptor and behaved as full agonists with regard to cAMP production, ERK phosphorylation, and apelin receptor internalization. Ex vivo, these compounds induced vasorelaxation of rat aortas and glomerular arterioles, respectively, precontracted with norepinephrine and angiotensin II, and increased cardiac contractility. In vivo, after intracerebroventricular administration in water-deprived mice, P92 and LIT01-196 were 6 and 160 times, respectively, more efficient at inhibiting systemic vasopressin release than K17F. Administered intravenously (nmol/kg range) in normotensive rats, these analogs potently increased urine output and induced a profound and sustained decrease in arterial blood pressure. In summary, these new compounds, which favor diuresis and improve cardiac contractility while reducing vascular resistances, represent promising candidates for the treatment of heart failure and water retention/hyponatremic disorders.-Gerbier, R., Alvear-Perez, R., Margathe, J.-F., Flahault, A., Couvineau, P., Gao, J., De Mota, N., Dabire, H., Li, B., Ceraudo, E., Hus-Citharel, A., Esteoulle, L., Bisoo, C., Hibert, M., Berdeaux, A., Iturrioz, X., Bonnet, D., Llorens-Cortes, C. Development of original metabolically stable apelin-17 analogs with diuretic and cardiovascular effects.

Intra-coronary morphine versus placebo in the treatment of acute ST-segment elevation myocardial infarction: the MIAMI randomized controlled trial.

BACKGROUND: Experimental studies suggest that morphine may protect the myocardium against ischemia-reperfusion injury by activating salvage kinase pathways. The objective of this two-center, randomized, double-blind, controlled trial was to assess potential cardioprotective effects of intra-coronary morphine in patients with ST-segment elevation myocardial infarction (STEMI) referred for primary percutaneous intervention. METHODS: Ninety-one patients with STEMI were randomly assigned to intracoronary morphine (1 mg) or placebo at reperfusion of the culprit coronary artery. The primary endpoint was infarct size/left ventricular mass ratio assessed by magnetic resonance imaging on day 3-5. Secondary endpoints included the areas under the curve (AUC) for troponin T and creatine kinase over three days, left ventricular ejection fraction assessed by echocardiography on days 1 and 6, and clinical outcomes. RESULTS: Infarct size/left ventricular mass ratio was not significantly reduced by intracoronary morphine compared to placebo (27.2% ± 15.0% vs. 30.5% ± 10.6%, respectively, p = 0.28). Troponin T and creatine kinase AUCs were similar in the two groups. Morphine did not improve left ventricular ejection fraction on day 1 (49.7 ± 10.3% vs. 49.3 ± 9.3% with placebo, p = 0.84) or day 6 (48.5 ± 10.2% vs. 49.0 ± 8.5% with placebo, p = 0.86). The number of major adverse cardiac events, including stent thrombosis, during the one-year follow-up was similar in the two groups. CONCLUSIONS: Intracoronary morphine at reperfusion did not significantly reduce infarct size or improve left ventricular systolic function in patients with STEMI. Presence of comorbidities in some patients may contribute to explain these results. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01186445 (date of registration: August 23, 2010).

Improvement of left ventricular filling by ivabradine during chronic hypertension: involvement of contraction-relaxation coupling.

Chronic hypertension is associated with left ventricular (LV) hypertrophy and LV diastolic dysfunction with impaired isovolumic relaxation and abnormal LV filling. Increased heart rate (HR) worsens these alterations. We investigated whether the I f channel blocker ivabradine exerts beneficial effects on LV filling dynamic. In this setting, we also evaluated the relationship between LV filling and isovolumic contraction as a consequence of contraction-relaxation coupling. Therefore, hypertension was induced by a continuous infusion of angiotensin II during 28 days in 10 chronically instrumented pigs. LV function was investigated after stopping angiotensin II infusion to offset the changes in loading conditions. In the normal heart, LV relaxation filling, LV early filling, LV peak early filling rate were positively correlated to HR. In contrast, these parameters were significantly reduced at day 28 vs. day 0 (18, 42, and 26 %, respectively) despite the increase in HR (108 ± 6 beats/min vs. 73 ± 2 beats/min, respectively). These abnormalities were corrected by acute administration of ivabradine (1 mg/kg, iv). Ivabradine still exerted these effects when HR was controlled at 150 beats/min by atrial pacing. Interestingly, LV relaxation filling, LV early filling and LV peak early filling were strongly correlated with both isovolumic contraction and relaxation. In conclusion, ivabradine improves LV filling during chronic hypertension. The mechanism involves LV contraction-relaxation coupling through normalization of isovolumic contraction and relaxation as well as HR-independent mechanisms.

A Brief Period of Hypothermia Induced by Total Liquid Ventilation Decreases End-Organ Damage and Multiorgan Failure Induced by Aortic Cross-Clamping.

BACKGROUND: In animal models, whole-body cooling reduces end-organ injury after cardiac arrest and other hypoperfusion states. The benefits of cooling in humans, however, are uncertain, possibly because detrimental effects of prolonged cooling may offset any potential benefit. Total liquid ventilation (TLV) provides both ultrafast cooling and rewarming. In previous reports, ultrafast cooling with TLV potently reduced neurological injury after experimental cardiac arrest in animals. We hypothesized that a brief period of rapid cooling and rewarming via TLV could also mitigate multiorgan failure (MOF) after ischemia-reperfusion induced by aortic cross-clamping. METHODS: Anesthetized rabbits were submitted to 30 minutes of supraceliac aortic cross-clamping followed by 300 minutes of reperfusion. They were allocated either to a normothermic procedure with conventional ventilation (control group) or to hypothermic TLV (33°C) before, during, and after cross-clamping (pre-clamp, per-clamp, and post-clamp groups, respectively). In all TLV groups, hypothermia was maintained for 75 minutes and switched to a rewarming mode before resumption to conventional mechanical ventilation. End points included cardiovascular, renal, liver, and inflammatory parameters measured 300 minutes after reperfusion. RESULTS: In the normothermic (control) group, ischemia-reperfusion injury produced evidence of MOF including severe vasoplegia, low cardiac output, acute kidney injury, and liver failure. In the TLV group, we observed gradual improvements in cardiac output in post-clamp, per-clamp, and pre-clamp groups versus control (53 ± 8, 64 ± 12, and 90 ± 24 vs 36 ± 23 mL/min/kg after 300 minutes of reperfusion, respectively). Liver biomarker levels were also lower in pre-clamp and per-clamp groups versus control. However, acute kidney injury was prevented in pre-clamp, and to a limited extent in per-clamp groups, but not in the post-clamp group. For instance, creatinine clearance was 4.8 ± 3.1 and 0.5 ± 0.6 mL/kg/min at the end of the follow-up in pre-clamp versus control animals (P = .0004). Histological examinations of the heart, kidney, liver, and jejunum in TLV and control groups also demonstrated reduced injury with TLV. CONCLUSIONS: A brief period of ultrafast cooling with TLV followed by rapid rewarming attenuated biochemical and histological markers of MOF after aortic cross-clamping. Cardiovascular and liver dysfunctions were limited by a brief period of hypothermic TLV, even when started after reperfusion. Conversely, acute kidney injury was limited only when hypothermia was started before reperfusion. Further work is needed to determine the clinical significance of our results and to identify the optimal duration and timing of TLV-induced hypothermia for end-organ protection in hypoperfusion states.

Effect of intravenous TRO40303 as an adjunct to primary percutaneous coronary intervention for acute ST-elevation myocardial infarction: MITOCARE study results.

AIM: The MITOCARE study evaluated the efficacy and safety of TRO40303 for the reduction of reperfusion injury in patients undergoing revascularization for ST-elevation myocardial infarction (STEMI). METHODS: Patients presenting with STEMI within 6 h of the onset of pain randomly received TRO40303 (n = 83) or placebo (n = 80) via i.v. bolus injection prior to balloon inflation during primary percutaneous coronary intervention in a double-blind manner. The primary endpoint was infarct size expressed as area under the curve (AUC) for creatine kinase (CK) and for troponin I (TnI) over 3 days. Secondary endpoints included measures of infarct size using cardiac magnetic resonance (CMR) and safety outcomes. RESULTS: The median pain-to-balloon time was 180 min for both groups, and the median (mean) door-to-balloon time was 60 (38) min for all sites. Infarct size, as measured by CK and TnI AUCs at 3 days, was not significantly different between treatment groups. There were no significant differences in the CMR-assessed myocardial salvage index (1-infarct size/myocardium at risk) (mean 52 vs. 58% with placebo, P = 0.1000), mean CMR-assessed infarct size (21.9 g vs. 20.0 g, or 17 vs. 15% of LV-mass) or left ventricular ejection fraction (LVEF) (46 vs. 48%), or in the mean 30-day echocardiographic LVEF (51.5 vs. 52.2%) between TRO40303 and placebo. A greater number of adjudicated safety events occurred in the TRO40303 group for unexplained reasons. CONCLUSION: This study in STEMI patients treated with contemporary mechanical revascularization principles did not show any effect of TRO40303 in limiting reperfusion injury of the ischaemic myocardium.

CORM-3, a water soluble CO-releasing molecule, uncouples mitochondrial respiration via interaction with the phosphate carrier.

Carbon monoxide is continuously produced in small quantities in tissues and is an important signaling mediator in mammalian cells. We previously demonstrated that CO delivered to isolated rat heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) is able to uncouple mitochondrial respiration. The aim of this study was to explore more in depth the mechanism(s) of this uncoupling effect. We found that acceleration of mitochondrial O2 consumption and decrease in membrane potential induced by CORM-3 were associated with an increase in mitochondrial swelling. This effect was independent of the opening of the mitochondrial transition pore as cyclosporine A was unable to prevent it. Interestingly, removal of phosphate from the incubation medium suppressed the effects mediated by CORM-3. Blockade of the dicarboxylate carrier, which exchanges dicarboxylate for phosphate, decreased the effects induced by CORM-3 while direct inhibition of the phosphate carrier with N-ethylmaleimide completely abolished the effects of CORM-3. In addition, CORM-3 was able to enhance the transport of phosphate into mitochondria as evidenced by changes in mitochondrial phosphate concentration and mitochondrial swelling that evaluates the activity of the phosphate carrier in de-energized conditions. These results indicate that CORM-3 activates the phosphate carrier leading to an increase in phosphate and proton transport inside mitochondria, both of which could contribute to the non-classical uncoupling effect mediated by CORM-3. The dicarboxylate carrier amplifies this effect by increasing intra-mitochondrial phosphate concentration.

Hypothermic Total Liquid Ventilation Is Highly Protective Through Cerebral Hemodynamic Preservation and Sepsis-Like Mitigation After Asphyxial Cardiac Arrest.

OBJECTIVES: Total liquid ventilation provides ultrafast and potently neuro- and cardioprotective cooling after shockable cardiac arrest and myocardial infarction in animals. Our goal was to decipher the effect of hypothermic total liquid ventilation on the systemic and cerebral response to asphyxial cardiac arrest using an original pressure- and volume-controlled ventilation strategy in rabbits. DESIGN: Randomized animal study. SETTING: Academic research laboratory. SUBJECTS: New Zealand Rabbits. INTERVENTIONS: Thirty-six rabbits were submitted to 13 minutes of asphyxia, leading to cardiac arrest. After resumption of spontaneous circulation, they underwent either normothermic life support (control group, n = 12) or hypothermia induced by either 30 minutes of total liquid ventilation (total liquid ventilation group, n = 12) or IV cold saline (conventional cooling group, n = 12). MEASUREMENTS AND MAIN RESULTS: Ultrafast cooling with total liquid ventilation (32 °C within 5 min in the esophagus) dramatically attenuated the post-cardiac arrest syndrome regarding survival, neurologic dysfunction, and histologic lesions (brain, heart, kidneys, liver, and lungs). Final survival rate achieved 58% versus 0% and 8% in total liquid ventilation, control, and conventional cooling groups (p < 0.05), respectively. This was accompanied by an early preservation of the blood-brain barrier integrity and cerebral hemodynamics as well as reduction in the immediate reactive oxygen species production in the brain, heart, and kidneys after cardiac arrest. Later on, total liquid ventilation also mitigated the systemic inflammatory response through alteration of monocyte chemoattractant protein-1, interleukin-1ß, and interleukin-8 transcripts levels compared with control. In the conventional cooling group, cooling was achieved more slowly (32 °C within 90-120 min in the esophagus), providing none of the above-mentioned systemic or organ protection. CONCLUSIONS: Ultrafast cooling by total liquid ventilation limits the post-cardiac arrest syndrome after asphyxial cardiac arrest in rabbits. This protection involves an early limitation in reactive oxidative species production, blood-brain barrier disruption, and delayed preservation against the systemic inflammatory response.

Translation of TRO40303 from myocardial infarction models to demonstration of safety and tolerance in a randomized Phase I trial.

BACKGROUND: Although reperfusion injury has been shown to be responsible for cardiomyocytes death after an acute myocardial infarction, there is currently no drug on the market that reduces this type of injury. TRO40303 is a new cardioprotective compound that was shown to inhibit the opening of the mitochondrial permeability transition pore and reduce infarct size after ischemia-reperfusion in a rat model of cardiac ischemia-reperfusion injury. METHODS: In the rat model, the therapeutic window and the dose effect relationship were investigated in order to select the proper dose and design for clinical investigations. To evaluate post-ischemic functional recovery, TRO40303 was tested in a model of isolated rat heart. Additionally, TRO40303 was investigated in a Phase I randomized, double-blind, placebo controlled study to assess the safety, tolerability and pharmacokinetics of single intravenous ascending doses of the compound (0.5 to 13 mg/kg) in 72 healthy male, post-menopausal and hysterectomized female subjects at flow rates from 0.04 to 35 mL/min (EudraCT number: 2010-021453-39). This work was supported in part by the French Agence Nationale de la Recherche. RESULTS: In the vivo model, TRO40303 reduced infarct size by 40% at 1 mg/kg and by 50% at 3 and 10 mg/kg given by intravenous bolus and was only active when administered before reperfusion. Additionally, TRO40303 provided functional recovery and reduced oxidative stress in the isolated rat heart model.These results, together with pharmacokinetic based allometry to human and non-clinical toxicology data, were used to design the Phase I trial. All the tested doses and flow rates were well tolerated clinically. There were no serious adverse events reported. No relevant changes in vital signs, electrocardiogram parameters, laboratory tests or physical examinations were observed at any time in any dose group. Pharmacokinetics was linear up to 6 mg/kg and slightly ~1.5-fold, hyper-proportional from 6 to 13 mg/kg. CONCLUSIONS: These data demonstrated that TRO40303 can be safely administered by the intravenous route in humans at doses expected to be pharmacologically active. These results allowed evaluating the expected active dose in human at 6 mg/kg, used in a Phase II proof-of-concept study currently ongoing.

Kidney protection by hypothermic total liquid ventilation after cardiac arrest in rabbits.

BACKGROUND: Total liquid ventilation (TLV) with perfluorocarbons has been shown to induce rapid protective cooling in animal models of myocardial ischemia and cardiac arrest, with improved neurological and cardiovascular outcomes after resuscitation. In this study, the authors hypothesized that hypothermic TLV can also limit kidney injury after cardiac arrest. METHODS: Anesthetized rabbits were submitted to 15 min of untreated ventricular fibrillation. After resuscitation, three groups of eight rabbits each were studied such as (1) life support plus hypothermia (32°-33 °C) induced by cold TLV (TLV group), (2) life support without hypothermia (control group), and (3) Sham group (no cardiac arrest). Life support was continued for 6 h before euthanasia and kidney removal. RESULTS: Time to target esophageal temperature was less than 5 min in the TLV group. Hypothermia was accompanied by preserved renal function in the TLV group as compared with control group regarding numerous markers including creatinine blood levels (12 ± 1 vs. 16 ± 2 mg/l, respectively; mean ± SEM), urinary N-acetyl-ß-(D)-glucosaminidase (1.70 ± 0.11 vs. 3.07 ± 0.10 U/mol of creatinine), γ-glutamyltransferase (8.36 ± 0.29 vs. 12.96 ± 0.44 U/mol of creatinine), or ß2-microglobulin (0.44 ± 0.01 vs. 1.12 ± 0.04 U/mol of creatinine). Kidney lesions evaluated by electron microscopy and conventional histology were also attenuated in TLV versus control groups. The renal-protective effect of TLV was not related to differences in delayed inflammatory or immune renal responses because transcriptions of, for example, interferon-γ, tumor necrosis factor-α, interleukin-1ß, monocyte chemoattractant protein-1, toll-like receptor-2, toll-like receptor-4, and vascular endothelial growth factor were similarly altered in TLV and control versus Sham. CONCLUSION: Ultrafast cooling with TLV is renal protective after cardiac arrest and resuscitation, which could increase kidney availability for organ donation.

Regular treadmill exercise restores cardioprotective signaling pathways in obese mice independently from improvement in associated co-morbidities.

Obesity is a major health issue that impedes the ability of preconditioning and postconditioning to protect the myocardium against infarction secondary to dysregulation of kinase signaling pathways. Moreover, exercise decreases cardiovascular mortality in obese patients but the mechanism remains to be established. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise (1h/day, 5 days/7, 4 weeks, 4° slope, 10-30 cm/s) and underwent 30 min of coronary artery occlusion followed by 24h of reperfusion for infarct size measurement. In WT, exercise reduced infarct size by 60% and increased phosphorylation of kinases such as Akt, ERK 1/2, p70S6K, AMPK and GSK3ß. Importantly, the level of corresponding phosphatases PTEN, MKP-3 and PP2C was decreased. Calcium concentration inducing the opening of mitochondrial permeability transition pore (mPTP) was increased by exercise. In ob/ob, regular exercise induced a robust cardioprotection by reducing infarct size (-67%), increasing kinase phosphorylation, decreasing phosphatase levels and improving the resistance to mPTP opening. However exercise did not modify hyperglycemia, hypercholesterolemia, hyperinsulinemia, fat mass and body weight in obese mice. In conclusion, regular exercise induces cardioprotection against myocardial infarction despite obesity and restores pro-survival signaling pathways with simultaneous increase in kinase phosphorylations, decreased levels of phosphatases and increased resistance of mPTP opening, independently from improvement in associated co-morbidities.

Role of hypoxia inducible factor-1α in remote limb ischemic preconditioning.

Remote ischemic preconditioning (RIPC) has emerged as a feasible and attractive therapeutic procedure for heart protection against ischemia/reperfusion (I/R) injury. However, its molecular mechanisms remain poorly understood. Hypoxia inducible factor-1α (HIF-1α) is a transcription factor that plays a key role in the cellular adaptation to hypoxia and ischemia. This study's aim was to test whether RIPC-induced cardioprotection requires HIF-1α upregulation to be effective. In the first study, wild-type mice and mice heterozygous for HIF1a (gene encoding the HIF-1α protein) were subjected to RIPC immediately before myocardial infarction (MI). RIPC resulted in a robust HIF-1α activation in the limb and acute cardioprotection in wild-type mice. RIPC-induced cardioprotection was preserved in heterozygous mice, despite the low HIF-1α expression in their limbs. In the second study, the role of HIF-1α in RIPC was evaluated using cadmium (Cd), a pharmacological HIF-1α inhibitor. Rats were subjected to MI (MI group) or to RIPC immediately prior to MI (R-MI group). Cd was injected 18 0min before RIPC (Cd-R-MI group). RIPC induced robust HIF-1α activation in rat limbs and significantly reduced infarct size (IS). Despite Cd's inhibition of HIF-1α activation, RIPC-induced cardioprotection was preserved in the Cd-R-MI group. RIPC applied immediately prior to MI increased HIF-1α expression and attenuated IS in rats and wild-type mice. However, RIPC-induced cardioprotection was preserved in partially HIF1a-deficient mice and in rats pretreated with Cd. When considered together, these results suggest that HIF-1α upregulation is unnecessary in acute RIPC.

Protection against myocardial infarction and no-reflow through preservation of vascular integrity by angiopoietin-like 4.

BACKGROUND: Increased permeability, predominantly controlled by endothelial junction stability, is an early event in the deterioration of vascular integrity in ischemic disorders. Hemorrhage, edema, and inflammation are the main features of reperfusion injuries, as observed in acute myocardial infarction (AMI). Thus, preservation of vascular integrity is fundamental in ischemic heart disease. Angiopoietins are pivotal modulators of cell-cell junctions and vascular integrity. We hypothesized that hypoxic induction of angiopoietin-like protein 4 (ANGPTL4) might modulate vascular damage, infarct size, and no-reflow during AMI. METHODS AND RESULTS: We showed that vascular permeability, hemorrhage, edema, inflammation, and infarct severity were increased in angptl4-deficient mice. We determined that decrease in vascular endothelial growth factor receptor 2 (VEGFR2) and VE-cadherin expression and increase in Src kinase phosphorylation downstream of VEGFR2 were accentuated after ischemia-reperfusion in the coronary microcirculation of angptl4-deficient mice. Both events led to altered VEGFR2/VE-cadherin complexes and to disrupted adherens junctions in the endothelial cells of angptl4-deficient mice that correlated with increased no-reflow. In vivo injection of recombinant human ANGPTL4 protected VEGF-driven dissociation of the VEGFR2/VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow in mice and rabbits. CONCLUSIONS: These data showed that ANGPTL4 might constitute a relevant target for therapeutic vasculoprotection aimed at counteracting the effects of VEGF, thus being crucial for preventing no-reflow and conferring secondary cardioprotection during AMI.

Hypothermic liquid ventilation prevents early hemodynamic dysfunction and cardiovascular mortality after coronary artery occlusion complicated by cardiac arrest in rabbits.

OBJECTIVES: Ultrafast and whole-body cooling can be induced by total liquid ventilation with temperature-controlled perfluorocarbons. Our goal was to determine whether this can afford maximal cardio- and neuroprotections through cooling rapidity when coronary occlusion is complicated by cardiac arrest. DESIGN: Prospective, randomized animal study. SETTING: Academic research laboratory. SUBJECTS: Male New Zealand rabbits. INTERVENTIONS: Chronically instrumented rabbits were submitted to coronary artery occlusion and ventricular fibrillation. After 8 minutes of cardiac arrest, animals were resuscitated and submitted to a normothermic follow-up (control group) or to 3 hours of mild hypothermia induced by total liquid ventilation (total liquid ventilation group) or by combination of cold saline infusion and cold blankets application (saline group). Coronary reperfusion was permitted 40 minutes after the onset of occlusion. After awakening, rabbits were followed up during 7 days. MEASUREMENTS AND MAIN RESULTS: Ten animals were resuscitated in each group. In the control group, all animals secondarily died of cardiac/respiratory failure (8 of 10) or neurological dysfunction (2 of 10). In the saline group, the target temperature of 32°C was achieved within 30-45 minutes after cooling initiation. This slightly reduced infarct size versus control (41% ± 16% vs 54% ± 8% of risk zone, respectively; p < 0.05) but failed to significantly improve cardiac output, neurological recovery, and survival rate (three survivors, six death from cardiac/respiratory failure, and one from neurological dysfunction). Conversely, the 32°C temperature was achieved within 5-10 minutes in the total liquid ventilation group. This led to a dramatic reduction in infarct size (13% ± 4%; p < 0.05 vs other groups) and improvements in cardiac output, neurological recovery, and survival (eight survivors, two deaths from cardiac/respiratory failure). CONCLUSIONS: Achieving hypothermia rapidly is critical to improve the cardiovascular outcome after cardiac arrest with underlying myocardial infarction.

H11 kinase/heat shock protein 22 deletion impairs both nuclear and mitochondrial functions of STAT3 and accelerates the transition into heart failure on cardiac overload.

BACKGROUND: Cardiac overload, a major cause of heart failure, induces the expression of the heat shock protein H11 kinase/Hsp22 (Hsp22). METHODS AND RESULTS: To determine the specific function of Hsp22 in that context, a knockout mouse model of Hsp22 deletion was generated. Although comparable to wild-type mice in basal conditions, knockout mice exposed to pressure overload developed less hypertrophy and showed ventricular dilation, impaired contractile function, increased myocyte length and accumulation of interstitial collagen, faster transition into heart failure, and increased mortality. Microarrays revealed that hearts from knockout mice failed to transactivate genes regulated by the transcription factor STAT3. Accordingly, nuclear STAT3 tyrosine phosphorylation was decreased in knockout mice. Silencing and overexpression experiments in isolated neonatal rat cardiomyocytes showed that Hsp22 activates STAT3 via production of interleukin-6 by the transcription factor nuclear factor-κB. In addition to its transcriptional function, STAT3 translocates to the mitochondria where it increases oxidative phosphorylation. Both mitochondrial STAT3 translocation and respiration were also significantly decreased in knockout mice. CONCLUSIONS: This study found that Hsp22 represents a previously undescribed activator of both nuclear and mitochondrial functions of STAT3, and its deletion in the context of pressure overload in vivo accelerates the transition into heart failure and increases mortality.

Impaired left ventricular function in the presence of preserved ejection in chronic hypertensive conscious pigs.

Systolic function is often evaluated by measuring ejection fraction and its preservation is often assimilated with the lack of impairment of systolic left ventricular (LV) function. Considering the left ventricle as a muscular pump, we explored LV function during chronic hypertension independently of increased afterload conditions. Fourteen conscious and chronically instrumented pigs received continuous infusion of either angiotensin II (n = 8) or saline (n = 6) during 28 days. Hemodynamic recordings were regularly performed in the presence and 1 h after stopping angiotensin II infusion to evaluate intrinsic LV function. Throughout the protocol, the mean arterial pressure steadily increased by 55 ± 4 mmHg in angiotensin II-treated animals. There were no significant changes in stroke volume, LV fractional shortening or LV wall thickening, indicating the lack of alterations in LV ejection. In contrast, we observed maladaptive changes with (1) the lack of reduction in isovolumic contraction and relaxation durations with heart rate increases, (2) abnormally blunted isovolumic contraction and relaxation responses to dobutamine and (3) a linear correlation between isovolumic contraction and relaxation durations. None of these changes were observed in saline-infused animals. In conclusion, we provide evidence of impaired LV function with concomitant isovolumic contraction and relaxation abnormalities during chronic hypertension while ejection remains preserved and no sign of heart failure is present. The evaluation under unloaded conditions shows intrinsic LV abnormalities.

Vascular endothelial dysfunction in Duchenne muscular dystrophy is restored by bradykinin through upregulation of eNOS and nNOS.

Little is known about the vascular function and expression of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) in Duchenne muscular dystrophy (DMD). Bradykinin is involved in the regulation of eNOS expression induced by angiotensin-converting enzyme inhibitors. We characterized the vascular function and eNOS and nNOS expression in a canine model of DMD and evaluated the effects of chronic bradykinin treatment. Vascular function was examined in conscious golden retriever muscular dystrophy (GRMD) dogs with left ventricular dysfunction (measured by echocardiography) and in isolated coronary arteries. eNOS and nNOS proteins in carotid arteries were measured by western blot and cyclic guanosine monophosphate (cGMP) content was analyzed by radioimmunoassay. Compared with controls, GRMD dogs had an impaired vasodilator response to acetylcholine. In isolated coronary artery, acetylcholine-elicited relaxation was nearly absent in placebo-treated GRMD dogs. This was explained by reduced nNOS and eNOS proteins and cGMP content in arterial tissues. Chronic bradykinin infusion (1 µg/min, 4 weeks) restored in vivo and in vitro vascular response to acetylcholine to the level of control dogs. This effect was NO-mediated through upregulation of eNOS and nNOS expression. In conclusion, this study is the first to demonstrate that DMD is associated with NO-mediated vascular endothelial dysfunction linked to an altered expression of eNOS and nNOS, which can be overcome by bradykinin.

Identification and pharmacological properties of E339-3D6, the first nonpeptidic apelin receptor agonist.

Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To explore further upstream the role played by this peptide, nonpeptidic agonists and antagonists of the apelin receptor are required. To identify such compounds that do not exist to date, we used an original fluorescence resonance energy transfer-based assay to screen a G-protein-coupled receptor-focused library of fluorescent compounds on the human EGFP-tagged apelin receptor. This led to isolated E339-3D6 that displayed a 90 nM affinity and behaved as a partial agonist with regard to cAMP production and as a full agonist with regard to apelin receptor internalization. Finally, E339-3D6 induced vasorelaxation of rat aorta precontracted with noradrenaline and potently inhibited systemic vasopressin release in water-deprived mice when intracerebroventricularly injected. This compound represents the first nonpeptidic agonist of the apelin receptor, the optimization of which will allow development of a new generation of vasodilator and aquaretic agents.

Chronic heart rate reduction with ivabradine improves systolic function of the reperfused heart through a dual mechanism involving a direct mechanical effect and a long-term increase in FKBP12/12.6 expression.

AIMS: To investigate the adaptations of left ventricular function and calcium handling to chronic heart rate reduction with ivabradine in the reperfused heart. METHODS AND RESULTS: Rabbits underwent 20 min coronary artery occlusion followed by 3 weeks of reperfusion. Throughout reperfusion, rabbits received ivabradine (10 mg/kg/day) or vehicle (control). Ivabradine reduced heart rate by about 20% and improved both ejection fraction (+35%) and systolic displacement (+26%) after 3 weeks of treatment. Interestingly, this was associated with a two-fold increase expression of FKBP12/12.6. There was no difference in the expressions of phospholamban, SERCA2a, calsequestrin, ryanodine, phospho-ryanodine, and Na(2+)/Ca(2+) exchanger in the two groups. Infarct scar and vascular density were similar in both groups. Administration of a single intravenous bolus of ivabradine (1 mg/kg) in control rabbits at 3 weeks of reperfusion also significantly improved acutely ejection fraction and systolic displacement. CONCLUSION: Chronic heart rate reduction protects the myocardium against ventricular dysfunction induced by myocardial ischaemia followed by 3 weeks of reperfusion. Beyond pure heart rate reduction, ivabradine improves global and regional systolic function of the reperfused heart through a dual mechanism involving a direct mechanical effect and a long-term adaptation in calcium handling, as supported by the increase in FKBP12/12.6 expression.

TRO40303, a new cardioprotective compound, inhibits mitochondrial permeability transition.

3,5-Seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) is a new cardioprotective compound coming from a chemical series identified initially for neuroprotective properties. TRO40303 binds specifically to the mitochondrial translocator protein 18 kDa (TSPO) at the cholesterol site. After intravenous administration, TRO40303 tissue distribution was comparable to that of TSPO, and, in particular, the drug accumulated rapidly in the heart. In a model of 35 min of myocardial ischemia/24 h of reperfusion in rats, TRO40303 (2.5 mg/kg) reduced infarct size by 38% (p < 0.01 versus control), when administered 10 min before reperfusion, which was correlated with reduced release of apoptosis-inducing factor from mitochondria to the cytoplasm in the ischemic area at risk. Although TRO40303 had no effect on the calcium retention capacity of isolated mitochondria, unlike cyclosporine A, the drug delayed mitochondrial permeability transition pore (mPTP) opening and cell death in isolated adult rat cardiomyocytes subjected to 2 h of hypoxia followed by 2 h of reoxygenation and inhibited mPTP opening in neonatal rat cardiomyocytes treated with hydrogen peroxide. The effects of TRO40303 on mPTP in cell models of oxidative stress are correlated with a significant reduction in reactive oxygen species production and subsequent calcium overload. TRO40303 is a new mitochondrial-targeted drug and inhibits mPTP triggered by oxidative stress. Its mode of action differs from that of other mPTP inhibitors such as cyclosporine A, thus providing a new pharmacological approach to study mPTP regulation. Its efficacy in an animal model of myocardial infarctions makes TRO40303 a promising new drug for the reduction of cardiac ischemia-reperfusion injury.