Yongge Liu

Role of Bradykinin in Protection of Ischemic Preconditioning in Rabbit Hearts

Bradykinin receptor activation has been proposed to be involved in ischemic preconditioning. In the present study, we further investigated the role of this agent in preconditioning in both isolated and in situ rabbit hearts. All hearts were subjected to 30 minutes of regional ischemia followed by reperfusion for 2 hours (in vitro hearts) and 3 hours (in situ hearts). Infarct size was measured by tetrazolium staining and expressed as a percentage of the size of the risk zone. Preconditioning in situ hearts with 5 minutes of ischemia and 10 minutes of reperfusion significantly reduced infarct size to 10.2 +/- 2.2% of the risk region (P < .0005 versus control infarct size of 36.7 +/- 2.6%). Pretreatment with HOE 140 (26 micrograms/kg), a bradykinin B2 receptor blocker, did not alter infarct size in nonpreconditioned hearts (40.6 +/- 5.3% infarction) but abolished protection from ischemic preconditioning (34.1 +/- 1.6% infarction). However, when HOE 140 was administered during the initial reflow period following 5 minutes of ischemia, protection was no longer abolished (15.6 +/- 3.9% infarction versus 13.3 +/- 3.8% without HOE 140, P = NS). Bradykinin infusion in isolated hearts mimicked preconditioning, and protection was not affected by pretreatment with the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester or the prostaglandin synthesis inhibitor indomethacin but could be completely abolished by the protein kinase C (PKC) inhibitors polymyxin B and staurosporine as well as by HOE 140. HOE 140 could not block the protection of ischemic preconditioning in isolated hearts. That failure was apparently due to the absence of blood-borne kininogens rather than autonomic nerves. When the preconditioning stimulus in the in situ model was amplified with four cycles of 5-minute ischemia/10-minute reperfusion, HOE 140 pretreatment could no longer block protection (infarct size was 10.7 +/- 3.5% versus 6.4 +/- 2.0% without HOE 140, P = NS). We propose that bradykinin receptors protect by coupling to PKC as do adenosine receptors, and blockade of either receptor will diminish the total stimulus of PKC below threshold and prevent protection. A more intense preconditioning ischemic stimulus can overcome bradykinin receptor blockade, however, by simply enhancing the amount of adenosine and possibly other agonists released.

Streptozotocin-induced non-insulin-dependent diabetes protects the heart from infarction.

BACKGROUND The vulnerability of the myocardium of a diabetic animal to an ischemic insult is controversial. To address this issue, streptozotocin-induced non-insulin-dependent diabetes (NIDD) was induced in rats, and the effects of regional myocardial ischemia were assessed by measuring infarct size. METHODS AND RESULTS Open-chest rats with NIDD and age-matched control rats underwent 30 or 45 minutes of regional ischemia and 2-hour reperfusion. Infarct size was measured by tetrazolium. Control rats had 32.0 +/- 3.3% infarction of the risk zone after a 30-minute coronary occlusion, whereas NIDD rats had significantly smaller infarcts (11.5 +/- 3.1% of the risk area, P < .005). When ischemic time was extended to 45 minutes, infarct size in control animals averaged 57.9 +/- 6.2%, whereas only 37.3 +/- 5.6% of ischemic myocardium was infarcted in NIDD rats (P < .05). In a subset NIDD group, rats experienced a period of ischemic preconditioning (three cycles of 5-minute ischemia/5-minute reperfusion) before 45-minute ischemia. Infarct size in these rats averaged only 6.9 +/- 3.0% (P < .01 vs nonpreconditioned NIDD rats with 45-minute coronary occlusions). Collateral flow was measured in NIDD rat hearts with radioactive microspheres. Collateral flow was < 1% of normal myocardial blood flow. CONCLUSIONS We conclude that NIDD protects the heart from infarction and that this protection is not related to the development of coronary collaterals. Furthermore, preconditioning can further protect the NIDD heart.

Roles of mitochondrial ATP-sensitive K channels and PKC in anti-infarct tolerance afforded by adenosine A1receptor activation

OBJECTIVES This study intended to assess the role of mitochondrial ATP-sensitive potassium (mitoK ATP) channels and the sequence of signal transduction with protein kinase C (PKC) and adenosine A1 receptors in rabbits. BACKGROUND To our knowledge, the link between trigger receptors of preconditioning, PKC and mitoK ATP channels has not been examined in a whole heart model of infarction. METHODS In the first series of experiments, myocardial infarction was induced in isolated buffer-perfused rabbit hearts by 30-min global ischemia and 2-h reperfusion. Infarct size in the left ventricle was determined by tetrazolium staining and expressed as a percentage of area at risk (i.e., the whole left ventricle) (%IS/AR). In the second series of experiments, mitochondria were isolated from the heart, and their respiratory function was examined using glutamate as a substrate. RESULTS Pretreatment with R-phenylisopropyladenosine (R-PIA, 1 micromol/liter), an A1-receptor agonist, reduced %IS/AR from 49.8 +/- 6.5% to 13.4 +/- 2.9%. This protection was abolished by calphostin C, a PKC inhibitor, and by 5-hydroxydecanoate (5-HD), a selective inhibitor of mitoK ATP channels. A selective mitoK ATP channel opener, diazoxide (100 micromol/liter), mimicked the effect of R-PIA on infarct size (%IS/AR = 11.6 +/- 4.0%), and this protective effect was also abolished by 5-HD. However, calphostin C failed to block the infarct size-limiting effect of diazoxide. Neither calphostin C nor 5-HD alone modified %IS/AR. State III respiration (QO2) and respiratory control index (RCI) were reduced after 30 min of ischemia (QO2 = 147.3 +/- 5.3 vs. 108.5 +/- 12.3, RCI = 22.3 +/- 1.1 vs. 12.1 +/- 1.8, p < 0.05). This mitochondrial dysfunction was persistent after 10 min of reperfusion (QO2 = 96.1 +/- 15.5, RCI = 9.5 +/- 1.9). Diazoxide significantly attenuated the respiratory dysfunction after 30 min of ischemia (QO2 = 142.8 +/- 9.7, RCI = 16.2 +/- 0.8) and subsequent 10-min reperfusion (QO2 = 135.3 +/- 7.2, RCI = 19.1 +/- 0.8). CONCLUSIONS These results suggest that mitoK ATP channels are downstream of PKC in the mechanism of infarct-size limitation by A1-receptor activation and that the anti-infarct tolerance afforded by opening of mitoK ATP channels is associated with preservation of mitochondrial function during ischemia/reperfusion.

Platelet P2Y12 Blockers Confer Direct Postconditioning-Like Protection in Reperfused Rabbit Hearts

Background: Blockade of platelet activation during primary percutaneous intervention for acute myocardial infarction is standard care to minimize stent thrombosis. To determine whether antiplatelet agents offer any direct cardioprotective effect, we tested whether they could modify infarction in a rabbit model of ischemia/reperfusion caused by reversible ligation of a coronary artery. Methods and Results: The P2Y12 (adenosine diphosphate) receptor blocker cangrelor administered shortly before reperfusion in rabbits undergoing 30-minute regional ischemia/3-hour reperfusion reduced infarction from 38% of ischemic zone in control hearts to only 19%. Protection was dose dependent and correlated with the degree of inhibition of platelet aggregation. Protection was comparable to that seen with ischemic postconditioning (IPOC). Cangrelor protection, but not its inhibition of platelet aggregation, was abolished by the same signaling inhibitors that block protection from IPOC suggesting protection resulted from protective signaling rather than anticoagulation. As with IPOC, protection was lost when cangrelor administration was delayed until 10 minutes after reperfusion and no added protection was seen when cangrelor and IPOC were combined. These findings suggest both IPOC and cangrelor may protect by the same mechanism. No protection was seen when cangrelor was used in crystalloid-perfused isolated hearts indicating some component in whole blood is required for protection. Clopidogrel had a very slow onset of action requiring 2 days of treatment before platelets were inhibited, and only then the hearts were protected. Signaling inhibitors given just prior to reperfusion blocked clopidogrel’s protection. Neither aspirin nor heparin was protective. Conclusions: Clopidogrel and cangrelor protected rabbit hearts against infarction. The mechanism appears to involve signal transduction during reperfusion rather than inhibition of intravascular coagulation. We hypothesize that both drugs protect by activating IPOC’s protective signaling to prevent reperfusion injury. If true, patients receiving P2Y12 inhibitors before percutaneous intervention may already be postconditioned thus explaining failure of recent clinical trials of postconditioning drugs.

Mitochondrial ATP-sensitive K+ channels play a role in cardioprotection by Na+-H+ exchange inhibition against ischemia/reperfusion injury.

OBJECTIVES The possible role of the ATP-sensitive potassium (KATP) channel in cardioprotection by Na+-H+ exchange (NHE) inhibition was examined. BACKGROUND The KATP channel is suggested to be involved not only in ischemic preconditioning but also in some pharmacological cardioprotection. METHODS Infarction was induced by 30-min coronary occlusion in rabbit hearts in situ or by 30-min global ischemia in isolated hearts. Myocardial stunning was induced by five episodes of 5-min ischemia/5-min reperfusion in situ. In these models, the effects of NHE inhibitors (cariporide and ethylisopropyl-amiloride [EIPA]) and the changes caused by KATP channel blockers were assessed. In another series of experiments, the effects of EIPA on mitochondrial KATP (mito-KATP) and sarcolemmal KATP (sarc-KATP) channels were examined in isolated cardiomyocvtes. RESULTS Cariporide (0.6 mg/kg) reduced infarct size in situ by 40%, and this effect was abolished by glibenclamide (0.3 mg/kg), a nonselective KATP channel blocker. In vitro, 1 microM cariporide limited infarct size by 90%, and this effect was blocked by 5-hydroxydecanoate (5-HD), a mito-KATP channel blocker but not by HMR1098, a sarc-KATP channel blocker. Infarct size limitation by 1 microM EIPA was also prevented by 5-HD. Cariporide attenuated regional contractile dysfunction by stunning, and this protection was abolished by glibenclamide and 5-HD. Ethylisopropyl amiloride neither activated the mito-KATP channel nor enhanced activation of this channel by diazoxide, a KATP channel opener. CONCLUSIONS Opening of the mito-KATP channel contributes to cardioprotection by NHE inhibition, though the interaction between NHE and this KATP channel remains unclear.

Transient inhibition of glucose uptake mimics ischemic preconditioning by salvaging ischemic myocardium in the rabbit heart

The aim of this study was to test whether transient inhibition of glucose uptake could precondition the rabbit heart. Rabbit hearts experienced 30 min regional ischemia followed by either 120 min (isolated heart protocol) or 180 min (in situ protocol) reperfusion. Infarct size was determined by tetrazolium staining. In isolated heart experiments, 15 min perfusion with glucose-free Krebs buffer starting 30 min prior to ischemia significantly limited infarct size to 9.9 +/- 2.6% of the risk zone as compared with 29.4 +/- 1.7% infarction in controls. This protection could be blocked (30.8 +/- 3.4%) by polymyxin B (50 microM), a protein kinase C inhibitor, but not by 8-(p-sulfophenyl)theophylline, an adenosine receptor inhibitor, suggesting the mechanism was similar to that of ischemic preconditioning but without involvement of adenosine receptors. Pyruvate and acetate inhibit glucose uptake without incurring a metabolic deficit. When 20 mM pyruvate or 1 mM acetate was added to the glucose-containing buffer for 15 min prior to ischemia, protection was evident (12.0 +/- 3.0% and 10.0 +/- 3.7% infarction, respectively). However, when acetate (1 mM) was present in the perfusate throughout the experiment, neither omission of glucose nor addition of pyruvate caused protection (26.1 +/- 2.2% and 28.9 +/- 4.7% infarction, respectively). Furthermore, when in situ hearts which preferably utilize lipid substrates were treated with pyruvate (2 g/kg i.v. 20 min before ischemia), infarct size was 40.3 +/- 3.0%, which did not differ from that in untreated hearts (38.6 +/- 3.2%). Hence transient inhibition of glucose uptake can precondition the heart, but only if other substrates which are utilized in preference to glucose are absent.

Adenosine and the Antiinfarct Effects of Preconditioning

Preconditioning the myocardium with a brief period of ischemia followed by reperfusion causes it to become very resistant to both infarction [1, 2, 3, 4] and, in some models, arrhythmias [5, 6, 7] from a subsequent more prolonged ischemia. Ischemic preconditioning is the most potent protector against ischemia known for the heart and has received widespread confirmation as to its efficacy. The mechanism of this protection has been elusive, but there is mounting evidence from this and other laboratories that adenosine plays an important role in the antiinfarct effect, at least in some species. During even the briefest coronary occlusion, adenosine is produced and released by the heart [8]. While adenosine has been the focus of cardiovascular studies for many years, adenosine has only recently been proposed to be directly cardioprotective independent of its vascular effects. This concept was first proposed by Ely and colleagues [9], and today the number of papers describing adenosine-mediated protection appears to be increasing geometrically. This chapter will review the evidence that the cellular changes associated with the preconditioning phenomenon are triggered by adenosine released during the preconditioning ischemia.

Phosphodiesterases as Targets for Intermittent Claudication

Intermittent claudication (IC) is one of the most frequent forms of lower extremity peripheral arterial disease (PAD) and is most commonly caused by arterial atherosclerosis. Its clinical manifestation includes fatigue, discomfort, or pain occurring in limb muscles due to exercise-induced ischemia, thus limiting the ability of IC patients to walk and exercise. In addition to lifestyle changes (diet, exercise, and smoking cessation), pharmacological treatments are needed. Pathologically, atherosclerotic lesions cause a mismatch in oxygen supply and metabolic demand in the leg muscles during walking/exercise. This subjects the muscles to repeated ischemia and reperfusion injury that can alter structure and oxidative metabolism, resulting in insufficient utilization of oxygen supply. Despite extensive research efforts, cilostazol and pentoxifylline are the only drugs indicated for relieving the symptoms of IC, with cilostazol demonstrating significant improvement in walking distance and quality of life in these patients. Originally developed as a PDE3 inhibitor, cilostazol was later found to have several other pharmacological actions, and its success has been attributed to its multifactorial actions on platelets, endothelium, smooth muscle, and lipid profiles. Using cilostazol as an example, we discuss the rationales and pitfalls of targeting PDEs in IC, and potential strategies for the development of new and more effective pharmacological treatments.

Activation of Protein Kinase C is Critical to the Protection of Preconditioning

The phenomenon of cardiac preconditioning was first clearly described in 1986 by Murry et al1 when they announced that brief ischemia could actually make the heart more tolerant to further ischemia. In dogs infarct size following a 40 minute coronary occlusion was reduced by 75% if four cycles of 5 minute occlusion/5 minute reperfusion preceded the 40 minute ischemia. Perhaps not unpredictably these data received little attention until a series of reports appeared in 1990-1991 confirming and extending Murry’s observation.2–4 In the succeeding 3–4 years the preconditioning phenomenon has been well characterized and many laboratories have been attempting to determine the mechanism. The latter has not yet been fully characterized, but efforts to date have strongly suggested the participation of protein kinase C (PKC).5 In this review the evidence for involvement of this important cell enzyme will be outlined, and our current hypothesis of the mechanism of preconditioning will be detailed.

Delamanid: From discovery to its use for pulmonary multidrug-resistant tuberculosis (MDR-TB)

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is the leading cause of death from an infectious disease globally. The widespread and ever-increasing resistance to TB drugs is reducing the effectiveness of treatment and jeopardizing TB control. New effective drugs with acceptable safety profiles are needed to turn the tide. Since the early 1990s, Otsuka Pharmaceutical Co., Ltd. has had a TB drug development program that resulted in the selection and development of delamanid (OPC-67683, Deltyba®), a first-in-class bicyclic nitroimidazole. Delamanid was initially approved by the European Medicines Agency (EMA) in 2014 for the treatment of adult pulmonary multi-drug resistant (MDR)-TB when an effective treatment regimen cannot otherwise be composed for reasons of resistance or tolerability. It has since been approved by several other countries/regions. In this review, we describe the history of delamanids development, including the screening process, in vitro and in vivo characterization, as well as various clinical studies. Delamanid possesses potent activity against replicating, dormant, and intracellular MTB bacilli, and is bactericidal in mouse and guinea pig TB models. Delamanid resistance mechanisms have been attributed to genes in the F420-dependent deazaflavin nitroreductase bio-activation pathway, found in mycobacterium species but not in common bacterial or mammalian cells. Published susceptibility testing results from 744 clinical isolates from delamanid-naïve patients indicate that the natural resistance rate to delamanid is very low (1.3%). Delamanid is largely metabolized by albumin in serum, and to a much less extent by cytochrome P450 enzymes. Furthermore, it neither inhibits nor induces P450 enzymes. In terms of efficacy, delamanid demonstrated activity in an early bactericidal activity trial in drug susceptible pulmonary TB patients and increased 2-month sputum culture conversion rates when added to an optimized background regimen in MDR-TB patients in a phase 2b global clinical trial. In addition, recent results outside clinical studies show favourable responses in highly resistant TB patients including extensively drug resistant (XDR)-TB when treated with delamanid-containing regimens in routine programmatic settings. The primary safety concern with delamanid is QTcF interval prolongation, although this observation has thus far not been associated with any clinical cardiac events. Overall, delamanid appears to be a well-tolerated and safe anti-TB drug when compared to other drugs used to treat MDR-TB.