Activated protein C protects against myocardial ischemic/reperfusion injury through AMP‐activated protein kinase signaling

Summary. Background: Activated protein C (APC) is a vitamin K‐dependent plasma serine protease that down‐regulates clotting and inflammatory pathways. It is known that APC exerts a cardioprotective effect by decreasing apoptosis of cardiomyocytes and inhibiting expression of inflammatory mediators after myocardial ischemia. Objectives: The objective of this study was to understand the mechanism of the APC‐mediated cardioprotection against ischemic injury. Methods: Cardioprotective activities of wild‐type APC and two derivatives, having either dramatically reduced anticoagulant activity or lacking signaling activity, were monitored in an acute ischemia/reperfusion injury model in which the left anterior descending coronary artery (LAD) was occluded. Results: APC reduced the myocardial infarct size by a mechanism that was largely independent of its anticoagulant activity. Thus, the non‐anticoagulant APC‐2Cys mutant, but not the non‐signaling APC‐E170A mutant, attenuated myocardial infarct size by EPCR and PAR‐1‐dependent mechanisms. Further studies revealed that APC acts directly on cardiomyocytes to stimulate the AMP‐activated protein kinase (AMPK) signaling pathway. The activation of AMPK by APC ameliorated the post‐ischemic cardiac dysfunction in isolated perfused mouse hearts. Moreover, both APC and APC‐2Cys inhibited production of TNFα and IL‐6 in vivo by attenuating the ischemia/reperfusion‐induced JNK and NF‐κB signaling pathways. Conclusions: APC exerts a cardioprotective function in ischemic/reperfusion injury through modulation of AMPK, NF‐κB and JNK signaling pathways.     

Role of Na+-H+ Exchange on Reperfusion Related Myocardial Injury and Arrhythmias in an Open-Chest Swine Model

The inhibition of Na⁺-H⁺ exchange (NHE) with amiloride analogues in vitro has been shown to prevent reperfusion arrhythmias and additional cell necrosis. Inhibition of intracellular Ca²⁺ overload via NHE inhibition has been suggested as a mechanism of these protective effects. The aim of this study was to examine whether treatment with amiloride analogues reduces the incidence of reperfusion arrhythmias and limits infarct size in vivo. Open-chest swine were exposed to a 30-minute left anterior descending artery (LAD) occlusion and 180 minutes of reperfusion during atrial pacing at 150 ppm. Intravenous 5-(N,N-dimethyI)-amiloride (AML, 5 μg/kg per min) was administered in the treatment group (n = 7) and intravenous saline in the control group (n = 7), starting 10 minutes before coronary occlusion. The infusion was continued during ischemia and reperfusion. The area at risk was defined by monastral blue dye and infarct size by triphenyltetrazolium chloride staining. Limb leads ECG and monophasic action potentials (MAPs) from the epicardium in the ischemic area were recorded. There was no significant difference in the size of the area at risk and hemodynamic parameters between the groups. However, the infarcted area was 0.4%± 1.0% of the area at risk in the treatment group, whereas it was 62%± 29% in the control group (P < 0.05). Pathological examination (Hematoxylin-eosin and mallory s phosphotungstic acid-hematoxylin staining) revealed that all of the infarcted area consisted of contraction band necrosis. MAP duration in both groups was significantly shortened during ischemia. After reperfusion, MAP duration in the treatment group recovered earlier than that of control group. However, there was no significant difference in the incidence of ventricular tachyarrhythmia between the groups. Inhibition of NHE with AML prevented reperfusion related cell necrosis in the in vivo swine model, but did not reduce the incidence of ventricular tachyarrhythmia.     

The infarct size-limiting effect of ischemic postconditioning (IPOC) is suppressed in isolated hyperhomocysteinemic (Hhcy) rat hearts: The reasonable role of PKC-δ

Background

Recently we have demonstrated that the cardioprotective potential of ischemic postconditioning (IPOC) against ischemia and reperfusion (I/R)-induced myocardial injury was markedly suppressed in hyperhomocysteinemic (Hhcy) rat hearts. The present study investigated the possible role of PKC-δ in Hhcy-induced suppression of myocardial infarct size-limiting effect of IPOC.

Methods

Isolated Langendorff's perfused normal and Hhcy rat hearts were subjected to 30-min global ischemia (I), followed by 120-min reperfusion (R). The myocardial damage was assessed by measuring the infarct size, and analyzing the release of LDH and CK-MB in coronary effluent. The oxidative stress in the heart was assessed by measuring lipid peroxidation and superoxide anion generation.

Results

The I/R produced myocardial injury in normal and Hhcy rat hearts by increasing myocardial infarct size, LDH and CK in coronary effluent and oxidative stress. Hhcy rat hearts exhibited enhanced I/R-induced myocardial injury and high oxidative stress as compared to normal rat hearts subjected to I/R. The IPOC (six brief episodes of I/R, 10 s each) afforded cardioprotection against I/R-induced myocardial injury in normal rat hearts; but IPOC-mediated cardioprotection was abolished in Hhcy rat hearts. Treatment with rottlerin (10 μM), a selective inhibitor of PKC-δ, did not affect the cardioprotective effect of IPOC in normal rat hearts; but its treatment significantly restored the myocardial infarct size-limiting effect of IPOC in Hhcy rat hearts.

Conclusion

The high oxidative stress produced in Hhcy rat hearts during reperfusion may activate PKC-δ, which may be responsible for impairing the infarct size-limiting potential of IPOC in Hhcy rat hearts.     

Opioids and cardioprotection

Opioid peptides and exogenous opioids such as morphine are known to exert important cardiovascular effects. However, until recently, it was not appreciated that activation of specific receptors results in a potent cardioprotective effect to reduce infarct size in experimental animals and to reduce cell death in isolated cardiomyocytes. In intact rat and rabbit hearts, nonselective opioid receptor antagonists such as naloxone and a selective delta1-opioid receptor antagonist, 7-benzylidenenaltrexone, have been shown to inhibit the cardioprotective effect of ischemic preconditioning, a phenomenon in which brief periods of ischemia protect the heart against a more prolonged period of ischemia. Selective delta(1) specific agonists such as 2-methyl-4a-alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a-alpha-octahydroquinolino[2,3,3-g]isoquinoline have been shown to exert potent cardioprotective effects in intact animals and cardiac myocytes via activation of Gi/o proteins, protein kinase C, and ultimately, the mitochondrial KATP channel. These protective effects occur immediately following drug administration, and reappear 24-48 hr post treatment. Although further studies are needed to more clearly define the mechanisms by which opioids exert their cardioprotective effects, the data accumulated and summarized in this review suggest that this class of drugs may not only be useful in alleviating the pain associated with a myocardial infarction, but may also be simultaneously reducing the size of the ultimate infarct. Since many of these drugs are already clinically available, a long period of drug development may not be necessary before the use of these drugs reaches the patient with signs of myocardial ischemia.     

Delta2-Specific Opioid Receptor Agonist and Hibernating Woodchuck Plasma Fraction Provide Ischemic Neuroprotection

Objectives: The authors present evidence that the δ opioid receptor agonist Deltorphin‐D_variant (Delt‐D_var) and hibernating woodchuck plasma (HWP), but not summer‐active woodchuck plasma (SAWP), can provide significant neuroprotection from focal ischemia in mice by a mechanism that relies in part on reducing nitric oxide (NO) release in ischemic tissue. Methods: Cerebral ischemia was produced in wild‐type and NO synthase–deficient (NOS^–/–) mice by transient, 1‐hour middle cerebral artery occlusion (MCAO). Behavioral deficits were determined at 22 hours and infarct volume was assessed at 24 hours after MCAO. Mice were treated with saline or Delt‐D_var at 2.0 and 4.0 mg/kg, or 200 μL of HWP or SAWP. NOS^–/– mice were treated with either saline or Delt‐D_var at 4.0 mg/kg. NO release was determined using an N9 microglial cell line pretreated with δ‐ or μ‐specific opioids and HWP or SAWP prior to activation with lipopolysaccharide and interferon‐γ. Nitrate in the medium was measured as an indicator of NO production. Results: Infusion of Delt‐D_var or HWP (but not SAWP) decreased infarct volume and improved behavioral deficits following 1 hour of MCAO and 24 hours of reperfusion. In NOS^–/– mice, endothelial NOS^+/+ is required to provide Delt‐D_var–induced neuroprotection. Delt‐D_var and HWP dose‐dependently decreased NO release in cell culture, while SAWP and other δ‐ and μ‐specific opioids did not. Conclusions: Delt‐D_var and HWP, but not SAWP, are effective neuroprotectant agents in a mouse model of transient MCAO. In cell culture, the mechanism of this ischemic neuroprotection may rely in part on their ability to block NO release.     

High‐Resolution Optical Mapping of Ventricular Tachycardia in Rats with Chronic Myocardial Infarction

Background: Ventricular tachycardia (VT) is a common cause of mortality in post‐myocardial infarction (MI) patients, even in the current era of coronary revascularization treatment. We report a reproducible VT model in rats with chronic MI induced by ischemia‐reperfusion and describe its electrophysiological characteristics using high‐resolution optical mapping. Methods: An MI was generated by left anterior descending coronary ligation (25 minutes) followed by reperfusion in 20 rats. Electrophysiology study and optical mapping were performed 5 weeks later using a Langendorff‐perfused preparation and compared to normal rats. Results: The conduction velocity of the MI border zone was decreased to 53% of the normal areas remote from the infarct (0.37 ± 0.16 m/sec vs 0.70 ± 0.09 m/sec, P < 0.0001). The rate of VT inducibility in MI rats was significantly greater than in normal control rats (70% vs 0%, P = 0.00002). VT circuits involving the infarct area were identified with optical mapping in 83% MI rats. In addition, fixed and functional conduction block were observed in the infarct border zone. Conclusion: This ischemia‐reperfusion MI rat model is a reliable VT model, which simulates clinical revascularization treatment. High‐resolution optical mapping in this model is useful to study the mechanism of VT and evaluate the effects of therapies. (PACE 2010; 33:687–695)     

Endogenous α2-antiplasmin does not enhance glomerular fibrin deposition or injury in glomerulonephritis

Summary. Background: Fibrin deposition is an important mechanism of glomerular injury in crescentic glomerulonephritis (GN), a severe form of immune renal injury. Both coagulation and fibrinolysis (via the plasminogen–plasmin system) are important in net glomerular fibrin accumulation in GN. α₂‐Antiplasmin (α₂‐AP) is the major circulating inhibitor of plasmin and is expressed in the renal tubulointerstitium. Objective: To determine whether endogenous α₂‐AP contributes to glomerular fibrin accumulation in GN. Methods: Crescentic autologous phase antiglomerular basement membrane GN was induced in mice with intact and deficient endogenous α₂‐AP (α₂‐AP^+/+ and α₂‐AP^?/? mice). Results: In mice with crescentic GN, α₂‐AP was detected in the tubulointerstitium and in segmental deposits within some glomeruli. α₂‐AP^+/+ mice developed crescentic GN (38 ± 9% glomeruli affected) with glomerular fibrin deposition and renal impairment (serum creatinine 30 ± 1 µmol L^?1, normal without GN 11 ± 1 µmol L^?1). Genetic deficiency of α₂‐AP did not result in attenuated glomerular fibrin deposition, crescent formation (39 ± 8% glomeruli affected), glomerular leukocyte infiltration or renal impairment (serum creatinine 33 ± 7 µmol L^?1). α₂‐AP was unmeasurable in kidneys from α₂‐AP^?/? mice, which did not develop compensatory changes in plasminogen, tissue type plasminogen activator (tPA), urokinase type PA (uPA) or plasminogen activator inhibitor‐1 proteins, or changes in tPA or uPA activity. α₂‐AP^?/? mice did have enhanced total renal fibrinolytic capacity as assessed by in situ fibrin overlay (α₂‐AP^+/+ 0.19 ± 0.01, α₂‐AP^?/? 0.36 ± 0.03 lyzed area/total area). Conclusions: α₂‐AP is not important to net glomerular fibrin deposition, crescent formation or renal impairment in crescentic GN.     

Kinetics of 13CO2 elimination after ingestion of 13C bicarbonate: the effects of exercise and acid base balance

Abstract. In order to investigate the effects of muscular work and preceding exercise on the retention of exogenous labelled bicarbonate, we studied the effects of oral administration of [¹³C]bicarbonate (0·1 mg kg^-1) in five subjects at rest before exercise and during and after 1 h of treadmill walking at 73% VO_2max on three separate occasions. Elimination of CO₂ from labelled bicarbonate was 62·6±8·1% at rest, 103·6±11·3% during exercise (P<0·01) and 43·0±4·7% during recovery from exercise (P= 0·01). During exercise mean residence time (MRT) was shorter than at rest (35±7 min vs. 54±9min, P < 0·02) and CO₂ pool size was larger (998±160 ml CO₂kg^-1, vs. 194±28ml CO₂kg^-1, P < 0·001). Compared to values obtained at rest, during recovery from exercise, MRT and CO₂ pool size were reduced (34±5min, P < 0·05; 116±19 ml CO₂kg^-1, P < 0·02, respectively). In an additional five subjects acidosis and alkalosis were induced prior to administration of oral [¹³C]bicarbonate at rest. Elimination of bicarbonate was lower during acidosis (46·1±5·6%, P < 0·01) but was unaltered (50·9±5·6%, NS) during alkalosis, compared to the values obtained at resting pH. During acidosis MRT and CO₂ pool size decreased (37±3min, P<0·01 and 123±10ml CO₂kg^-1, P < 0·01, respectively) whereas in alkalosis MRT was unchanged (65±8 min NS) but CO₂ pool size was increased (230±23ml CO₂kg^-1, P < 0·05). The kinetics of elimination of ¹³CO₂ from administered bicarbonate after exercise are different to those at rest and resemble acidosis. The appropriate correction factor for sequestered ¹³C should be used in metabolic studies of the post-exercise state when using ¹³C tracers.     

Acute saline infusion induces extracellular acidification and activation of the Na+/H+ exchanger in man

The effect of acute expansion of the extracellular fluid volume (ECV) with isotonic (0.9%) saline on the activity of the lymphocyte Na⁺/H⁺ antiport (NHE) was studied in a total of 18 healthy volunteers. Saline was infused at a constant rate so that 4 mmol kg^?1 b.w. was administered over 2 h. NHE activity was measured by quantifying cytosolic pH (pH_i) recovery following acidification of the cells with propionic acid and by pH clamping at various pH_i values between 7.2 and 5.8 using nigericin. Both methods demonstrate NHE activation associated with intravenous saline infusion, the kinetic difference being a marked decrease in the Hill coefficient n from 3.28 ± 0.21 (SEM) to 2.22 ± 0.11 in the absence of changes in baseline pH_i (7.14 ± 0.02 vs. 7.08 ± 0.02; P = 0.15), V_max (42.8 ± 2.7 vs. 48.1 ± 2.8 mmol L^?1 min^?1; P = 0.08) and pK (6.32 ± 0.04 vs. 6.35 ± 0.02). NHE activation was associated with significant decreases in serum chloride (P = 0.016), calcium (P = 0.008), total cholesterol (P = 0.008), low-density lipoproteins (P = 0.016) and high-density lipoproteins (P = 0.008). Moreover, saline infusion induced extracellular acidification with a decrease in pH from 7.39 ± 0.01 to 7.37 ± 0.01 (P = 0.016), HCO₃^? from 23.3 ± 0.43 mmol L^?1 to 21.3 ± 0.25 mmol L^?1 (P = 0.008) and base excess from ?1.03 ± 0.38 mmol L^?1 to ?3.00 ± 0.31 mmol L^?1 (P = 0.008). Our results show for the first time that acute ECV expansion with isotonic saline is followed by an activation of the lymphocyte NHE. The underlying mechanism(s) remain to be investigated. However, the demonstration in our study of marked changes in acid–base balance induced by acute saline points to a possible inter-relationship of antiporter activation and extracellular acidification.     

Chronic oral administration of low‐dose combination of fenofibrate and rosuvastatin protects the rat heart against experimentally induced acute myocardial infarction

Fenofibrate and rosuvastatin at low doses might have experimental pleiotropic benefits. This study investigated the combined effect of low doses of fenofibrate and rosuvastatin in isoproterenol‐induced experimental myocardial infarction. Rats administered isoproterenol (85 mg/kg/day, s.c.) for 2 days (day 29 and day 30) of 30 days experimental protocol developed significant myocardial infarction that was accompanied with high myocardial oxidative stress and lipid peroxidation, elevated serum markers of cardiac injury, lipid abnormalities, and elevated circulatory levels of C‐reactive protein. Pretreatment with low doses of fenofibrate (30 mg/kg/day p.o., 30 days) and rosuvastatin (2 mg/kg/day p.o., 30 days) both alone or in combination markedly prevented isoproterenol‐induced myocardial infarction and associated abnormalities while the low‐dose combination of fenofibrate and rosuvastatin was more effective. Histopathological study in isoproterenol control rat heart showed necrosis with edema and acute inflammation at the margins of necrotic area. The rat heart from low‐dose fenofibrate and rosuvastatin pretreated group showed scanty inflammation and no ischemia. In conclusion, fenofibrate and rosuvastatin pretreatment in low doses might have a therapeutic potential to prevent the pathogenesis of myocardial infarction. Moreover, their combined treatment option might offer superior therapeutic benefits via a marked reduction in myocardial infarct size and oxidative stress, suggesting a possibility of their pleiotropic cardioprotective action at low doses.     

Ticagrelor binds to human P2Y12 independently from ADP but antagonizes ADP-induced receptor signaling and platelet aggregation

Summary. Background: P2Y₁₂ plays an important role in regulating platelet aggregation and function. This receptor is the primary target of thienopyridine antiplatelet agents, the active metabolites of which bind irreversibly to the receptor, and of newer agents that can directly and reversibly modulate receptor activity. Objective: To characterize the receptor biology of the first reversibly binding oral P2Y₁₂ antagonist, ticagrelor (AZD6140), a member of the new cyclopentyltriazolopyrimidine (CPTP) class currently in phase III development. Methods: Ticagrelor displayed apparent non‐competitive or insurmountable antagonism of ADP‐induced aggregation in human washed platelets. This was investigated using competition binding against [³H]ADP, [³³P]2MeS‐ADP and the investigational CPTP compound [¹²⁵I]AZ11931285 at recombinant human P2Y₁₂. Functional receptor inhibition studies were performed using a GTPγS‐binding assay, and further binding studies were performed using membranes prepared from washed human platelets. Results: Radioligand‐binding studies demonstrated that ticagrelor binds potently and reversibly to human P2Y₁₂ with K_on and K_off of (1.1 ± 0.2) × 10^?4 nm ^?1 s^?1 and (8.7 ± 1.4) × 10^?4 s^?1, respectively. Ticagrelor does not displace [³H]ADP from the receptor (K_i > 10 μm ) but binds competitively with [³³P]2MeS‐ADP (K_i = 4.3 ± 1.3 nm ) and [¹²⁵I]AZ11931285 (K_i = 0.33 ± 0.04 nm ), and shows apparent non‐competitive inhibition of ADP‐induced signaling but competitive inhibition of 2MeS‐ADP‐induced signaling. Binding studies on membranes prepared from human washed platelets demonstrated similar non‐competitive binding for ADP and ticagrelor. Conclusions: These data indicate that P2Y₁₂ is targeted by ticagrelor via a mechanism that is non‐competitive with ADP, suggesting the existence of an independent receptor‐binding site for CPTPs.     

Delivering stem cells to the healthy heart on biological sutures: effects on regional mechanical function

Current cardiac cell therapies cannot effectively target and retain cells in a specific area of the heart. Cell‐seeded biological sutures were previously developed to overcome this limitation, demonstrating targeted delivery with > 60% cell retention. In this study, both cell‐seeded and non‐seeded fibrin‐based biological sutures were implanted into normal functioning rat hearts to determine the effects on mechanical function and fibrotic response. Human mesenchymal stem cells (hMSCs) were used based on previous work and established cardioprotective effects. Non‐seeded or hMSC‐seeded sutures were implanted into healthy athymic rat hearts. Before cell seeding, hMSCs were passively loaded with quantum dot nanoparticles. One week after implantation, regional stroke work index and systolic area of contraction (SAC) were evaluated on the epicardial surface above the suture. Cell delivery and retention were confirmed by quantum dot tracking, and the fibrotic tissue area was evaluated. Non‐seeded biological sutures decreased SAC near the suture from 0.20 ± 0.01 measured in sham hearts to 0.08 ± 0.02, whereas hMSC‐seeded biological sutures dampened the decrease in SAC (0.15 ± 0.02). Non‐seeded sutures also displayed a small amount of fibrosis around the sutures (1.0 ± 0.1 mm²). Sutures seeded with hMSCs displayed a significant reduction in fibrosis (0.5 ± 0.1 mm², p < 0.001), with quantum dot‐labelled hMSCs found along the suture track. These results show that the addition of hMSCs attenuates the fibrotic response observed with non‐seeded sutures, leading to improved regional mechanics of the implantation region. Copyright © 2014 John Wiley & Sons, Ltd.     

Myocardial protection in reperfusion with postconditioning

Reperfusion is the definitive treatment for coronary occlusive disease. However, reperfusion carries the potential to exacerbate lethal injury, termed ‘reperfusion injury’. Studies have suggested that reperfusion injury events are triggered during the early moments of reflow, and determine, in part, the severity of downstream manifestations of postischemic injury, including endothelial dysfunction, infarction and apoptosis. The application of brief iterative episodes of reflow (reoxygenation) and reocclusion (ischemia, hypoxia) at the immediate onset of reperfusion, which has been termed ‘postconditioning’ by the authors, reduces many manifestations of postischemic injury, notably infarct size, apoptosis, coronary vascular endothelial injury and reperfusion arrhythmias. Cardioprotection with postconditioning has been reported to be comparable with that observed using the gold standard maneuver ischemic preconditioning. In contrast to preconditioning, which exerts its effects primarily during the index ischemia, postconditioning appears to exert its effects during reperfusion alone. Postconditioning modifies the early phase of reperfusion in ways that are just beginning to be understood. It appears to first: reduce the oxidant burden and consequent oxidant-induced injury; secondly, attenuate the local inflammatory response to reperfusion; and thirdly, engage end effectors and signaling pathways implicated in other cardioprotective maneuvers, such as ischemic and pharmacologic preconditioning. Postconditioning seems to trigger the upregulation of survival kinases principally known to attenuate the pathogenesis of apoptosis and possibly necrosis. The postconditioning phenomenon has been reproduced by a number of independent laboratories and has been observed in both large and small animal in vivo models, as well as in ex vivo and cell culture models. In contrast to preconditioning, postconditioning may have widespread clinical application because it can be applied during reperfusion at the point of service for angioplasty, stenting, cardiac surgery and organ transplantation.     

Discovery of novel mechanisms and molecular targets for the inhibition of activated thrombin activatable fibrinolysis inhibitor

Summary. Background: Thrombin activatable fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis and an attractive target to develop profibrinolytic drugs. Objective: To analyze the (inhibitory) properties of five monoclonal antibodies (mAbs) directed towards rat TAFI (i.e. MA‐RT13B2, MA‐RT30D8, MA‐RT36A3F5, MA‐RT36B2 and MA‐RT82F12). Methods and results: Direct interference of the mAb with rat activated TAFI (TAFIa) activity was assayed using a chromogenic activity assay. This revealed reductions of 79% ± 1%, 54% ± 4%, and 19% ± 2% in activity in the presence of a 16‐fold molar excess of MA‐RT13B2, MA‐RT36A3F5, and MA‐RT82F12, respectively whereas MA‐RT30D8 and MA‐RT36B2 had no direct inhibitory effect. Additionally, MA‐RT13B2 and MA‐RT36A3F5 reduced rat TAFIa half‐life by 56% ± 2% and 61% ± 3%. Tissue‐type plasminogen activator mediated in vitro clot lysis was determined using rat plasma. Compared to potato tuber carboxypeptidase inhibitor, MA‐RT13B2, MA‐RT30D8, MA‐RT36A3F5, and MA‐RT82F12 reduced clot lysis times by 86% ± 14%, 100% ± 5%, 100% ± 10%, and 100% ± 11%, respectively. During epitope mapping, Arg²²⁷ and Ser²⁵¹ were identified as major residues interacting with MA‐RT13B2. Arg¹⁸⁸ and His¹⁹² contribute to the interaction with MA‐RT36A3F5. Arg^227, Ser²⁴⁹, Ser²⁵¹, and Tyr²⁶⁰ are involved in the binding of MA‐RT30D8 and MA‐RT82F12 with rat TAFI(a). The following mechanisms of inhibition have been deduced: MA‐RT13B2 and MA‐RT36A3F5 have a destabilizing effect on rat TAFIa whereas MA‐RT30D8 and MA‐RT82F12 partially block the access to the active site of TAFIa or interact with the binding of TAFIa to the blood clot. Conclusions: The described inhibitory mAb towards rat TAFIa will facilitate TAFI research in murine models. Additionally, we reveal novel molecular targets for the direct inhibition of TAFIa through different mechanisms.     

7.0T MR T2* mapping与T2 mapping检测急性心肌梗死再灌注模型大鼠心肌内出血

目的 观察7.0T MR T2^* mapping与T2 mapping检测急性心肌梗死(AMI)再灌注模型大鼠心肌内出血(IMH)的图像质量及其价值。方法 以42只SD大鼠制备AMI再灌注模型,于其后2 d及7 d采集左心室7.0T MR T2^* mapping与T2 mapping,获得后处理T2^* map与T2 map图;之后处死大鼠,取心脏进行病理检查,评估IMH。观察IMH大鼠各序列原始图像的信噪比(SNR)、对比噪声比(CNR)、图像质量评分及T2^* map与T2 map图像的信号均一性。以病理结果为标准,分析2个序列图像检出IMH的效能。结果 造模后2 d (9只)和7 d (16只)后,共25只大鼠造模成功并检出IMH (IMH组),10只存在AMI但无IMH,6只无心肌梗死(无心肌梗死组),另1只在扫描过程中死亡;2 d和7 d IMH组心脏T2 mapping原始图像的SNR＞T2^* mapping原始图像(P均=0.001),而CNR差异均无统计学意义(P均＞0.05)。造模后2 d,IMH组心脏T2 mapping原始图像质量评分(3.90±0.30)高于T2^* mapping (3.80±0.40,t=3.67,P＜0.01);造模后7 d,T2 mapping原始图像质量评分(3.60±0.50)与T2^* mapping差异无统计学意义(3.50±0.50,t=1.65,P=0.10)。IMH组T2 map与T2^* map图像的出血心肌及远端心肌的变异系数(COV)差异均无统计学意义(P均＞0.05),提示信号均一性无明显差异。T2 mapping及T2^* mapping检出IMH的敏感度分别为88.00%(22/25)及96.00%(24/25)。结论 7.0T MR T2^* mapping图像信号均一性、SNR及图像质量略低于T2 mapping,二者CNR相当,均可用于检测AMI再灌注大鼠模型IMH。     

Short-term myocardial ischemia induces cardiac modified C-reactive protein expression and proinflammatory gene (cyclo-oxygenase-2, monocyte chemoattractant protein-1, and tissue factor) upregulation in peripheral blood mononuclear cells

Summary. Background: Prompt coronary thrombus resolution, reducing time of ischemia, improves cardiac recovery. The factors triggered by ischemia that contribute to the clinical outcome are not fully known. We hypothesize that unabated inflammation due to cardiac ischemia may be a contributing factor. Aims: As a proof‐of‐concept, we evaluated the effect of short‐term myocardial ischemia on the local and systemic inflammatory response. Methods: Pigs underwent either 90‐min mid‐left anterior descending (LAD) coronary artery balloon occlusion (infarct size 25% ± 1% left ventricle; 29% heart function deterioration) or a sham‐operation procedure. Peri‐infarcted and non‐ischemic cardiac tissue was obtained for histopathologic, molecular and immunohistochemical analysis of inflammatory markers [interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), modified C‐reactive protein (mCRP), and human alveolar macrophage‐56 (HAM‐56)]. Blood (femoral vein) was withdrawn prior to myocardial infarction (MI) induction (t = 0) and at 30 and 90 min to evaluate: (i) systemic cytokine levels (IL‐6, TNF‐α, CRP); (ii) proinflammatory gene and protein expression in peripheral blood mononuclear cells (PBMCs) of tissue factor (TF), cyclo‐oxygenase‐2 (Cox‐2), monocyte chemoattractant protein‐1 (MCP‐1), and CRP; and (iii) platelet activation (assessed by perfusion studies and RhoA activation). Results: Short‐term ischemia triggered cardiac IL‐6 and TNF‐α expression, recruitment of inflammatory cells, and mCRP expression in infiltrated macrophages (P < 0.05 vs. t = 0 and sham). PBMC mRNA and protein expression of MCP‐1, Cox‐2 and TF was significantly increased by ischemia, whereas no differences were detected in CRP. Ischemia increased cardiac troponin‐I, IL‐6 and TNF‐α systemic levels, and was associated with higher platelet deposition and RhoA activation (P < 0.001 vs. t = 0 and sham). Conclusion: Short‐term myocardial ischemia, even without atherosclerosis, induces an inflammatory phenotype by inducing local recruitment of macrophages and systemic activation of mononuclear cells, and renders platelets more susceptible to activation.     

PAR-1-dependent pp60src activation is dependent on protein kinase C and increased [Ca2+]i: evidence that pp60src does not regulate PAR-1-dependent Ca2+ entry in human platelets

Summary. Background: The role of the tyrosine kinase pp60^src in PAR‐1‐dependent Ca²⁺ entry was investigated in human platelets. pp60^src plays a role in thapsigargin (TG)‐evoked store‐operated Ca²⁺ entry (SOCE), which is thought to be a major component of thrombin‐evoked Ca²⁺ entry. Methods: pp60^src tyr⁴¹⁶ phosphorylation was used to assess pp60^src activation. Fura‐2‐loaded platelets were used to monitor intracellular Ca²⁺ concentration ([Ca²⁺]_i). Results: Activation of PAR‐1 with the specific agonist SFLLRN increased pp60^src activation within 10 s. This required phospholipase C (PLC) activity, Ca²⁺ release and a rise in intracellular Ca²⁺. PP2, an inhibitor of Src‐family tyrosine kinases, inhibited SFLLRN‐evoked Ca²⁺ entry, but also inhibited Ca²⁺ release and the extrusion of Ca²⁺ by the plasma membrane Ca²⁺ ATPase. Actin polymerization and conventional protein kinase C (cPKC) activity were required for TG‐ and SFLLRN‐evoked pp60^src activation. Although Gö6976, an inhibitor of cPKCs, inhibited TG‐evoked SOCE, it had little effect on SFLLRN‐ or thrombin‐evoked Ca²⁺ entry. Conclusions: These data indicate that stimulation of PAR‐1 leads to activation of pp60^src in human platelets, through PLC and cPKC activation, Ca²⁺ release and actin polymerization. However, as PKC and actin polymerization are not needed for SFLLRN‐evoked Ca²⁺ entry, we suggest that pp60^src is also not required. The apparent inhibition of SFLLRN‐evoked Ca²⁺ entry by PP2 is likely to be secondary to reduced Ca²⁺ release. These data argue against a contribution of this SOCE pathway to PAR‐1‐dependent Ca²⁺ entry.     

Does remote ischemic conditioning salvage left ventricular function after successful primary PCI?

Evaluation of: Munk K, Andersen NH, Schmidt MR et al. Remote ischemic conditioning in myocardial infarct patients treated with primary angioplasty: impact on left ventricular function assessed by comprehensive echocardiography and gated SPECT. Circ. Cardiovasc. Imaging 3(6), 656–662 (2010).

The translation of ischemic preconditioning to a viable therapy that benefits patients has been slow. This has been largely due to the difficultly in preempting when ischemia will occur. Recent advances in the field have demonstrated that cardioprotection from brief episodes of ischemia is possible when applied immediately after reperfusion (ischemic postconditioning) or remotely in another tissue during myocardial ischemia, prior to reperfusion (remote ischemic conditioning). This has facilitated the therapeutic application to patients presenting with acute myocardial infarction. In this article, we will discuss the results of a recent study published by Munk et al., concerning the application of remote ischemic conditioning during primary percutaneous coronary intervention to salvage myocardial function following ST-elevation myocardial infarction.     

Possible involvement of PKC-δ in the abrogated cardioprotective potential of ischemic preconditioning in hyperhomocysteinemic rat hearts

The present study has been designed to investigate the possible role of protein kinase C-delta (PKC-δ) in hyperhomocysteinemia-induced attenuation of cardioprotective potential of ischemic preconditioning (IPC). Rats were administered l-methionine (1.7 g/kg/day, p.o.) for 4 weeks to produce hyperhomocysteinemia. Isolated Langendorff perfused normal and hyperhomocysteinemic rat hearts were subjected to global ischemia for 30 min followed by reperfusion for 120 min. Myocardial infarct size was assessed macroscopically using triphenyltetrazolium chloride (TTC) staining. Coronary effluent was analyzed for lactate dehydrogenase (LDH) and creatine kinase (CK) release to assess the degree of cardiac injury. Moreover, the oxidative stress in heart was assessed by measuring lipid peroxidation and superoxide anion generation. The ischemia-reperfusion (I/R) was noted to produce myocardial injury as assessed in terms of increase in myocardial infarct size, LDH and CK in coronary effluent and oxidative stress in normal and hyperhomocysteinemic rat hearts. In addition, the hyperhomocysteinemic rat hearts showed enhanced I/R-induced myocardial injury with high degree of oxidative stress as compared with normal rat hearts subjected to I/R. Four episodes of IPC (5 min each) afforded cardioprotection against I/R-induced myocardial injury in normal rat hearts as assessed in terms of reduction in myocardial infarct size, LDH, CK and oxidative stress. On the other hand, IPC mediated myocardial protection against I/R-injury was abolished in hyperhomocysteinemic rat hearts. Treatment with rottlerin (10 μM), a selective inhibitor of PKC-δ did not affect the cardioprotective effects of IPC in normal rat hearts; but its treatment significantly restored the cardioprotective potentials of IPC in hyperhomocysteinemic rat hearts. The high degree of oxidative stress produced in hyperhomocysteinemic rat hearts during reperfusion may activate PKC-δ, which may be implicated in the observed paradoxically abrogated cardioprotective potentials of IPC in hyperhomocysteinemic rat hearts.     

MR imaging assessment of the kinetics of P846, a new gadolinium‐based MR contrast medium,in ischemically injured myocardium

The objectives of the study were: (1) to compare the kinetics of a new gadolinium‐based low‐diffusibility magnetic resonance (MR) contrast medium, P846 and Gd‐DOTA in left ventricular (LV) blood and in normal and ischemically injured myocardium using inversion recovery echo‐planar imaging (IR‐EPI) and (2) to compare the enhancement pattern after injection of P846 with Gd‐DOTA, using T₁‐weighted spin‐echo imaging (T₁‐SE). Sixteen rats were subjected to left descending artery (LAD) occlusion for 30 min, followed by 2.5 h reperfusion. MR imaging was performed before and after administration of the contrast medium in two different groups of animals: one group (n = 8) received 0.05 mmol kg^?1 P846 and the other (n = 8) 0.1 mmol kg^?1 Gd‐DOTA. A blipped IR‐EPI and a multislice T₁‐SE were performed before injection and for 90 min after injection. T₁‐values were derived by fitting regional signal intensity on the IR‐EPI images, the R₁, ΔR₁ (R_{1postcontrast} – R_1precontrast) and ΔR₁ ratios were calculated in LV blood, normal and injured myocardium. On SE‐T₁, the signal intensity ratio (SI) and extent of injury were measured. True infarct size was measured using histochemical staining. Changes in ΔR₁ were 4.8 times greater with 0.05 mmol kg^?1 P846 than with 0.1 mmol kg^?1 Gd‐DOTA in LV blood (6.3 ± 0.9 vs 0.9 ± 0.1 s^?1, p < 0.0001), normal (1.7 ± 0.2 vs 0.34 ± 0.03 s^?1, p < 0.0001) and ischemically injured myocardium (5.4 ± 0.4 vs 1.6 ± 0.1 s^?1, p < 0.0001). MR imaging experiments showed that the signal enhancement with P846 is longer (90 min), which might be explained by a lower diffusion of P846 compared with Gd‐DOTA (30 min). P846 differentiates viable and nonviable myocardium. Despite lower gadolinium dose, P846 permits differentiation of viable and nonviable myocardium owing to a high contrast and a long imaging window with conventional t₁‐weighted SE sequence. Copyright © 2008 John Wiley & Sons, Ltd.