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We have previously shown that reduction of ischaemic cell swelling via enhanced cell volume regulation is a key mechanism of ischaemic preconditioning (IPC) in cardiomyocytes. We have also shown that pharmacological blockade of Cl(-) channels abolishes cardioprotection achieved by IPC in Langendorff-perfused hearts and freshly isolated cardiomyocytes, thus suggesting that Cl(-) plays a key role in IPC cardioprotection. However, direct evidence of Cl(-) channel activation resulting in transsarcolemmal Cl(-) efflux by IPC had been lacking. To address this issue, 24 h cultured rabbit cardiomyocytes were loaded with 5 mM 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), a specific fluorescence indicator that is quenched by Cl(-) so that cellular efflux of Cl(-) results in an increase in SPQ fluorescence.After stabilization for 10 min, cardiomyocytes were preconditioned either with 10 min simulated ischaemia/10 min simulated reperfusion or with 10 min treatment with 1 microM N(6)-2-(4-aminophenyl)ethyladenosine (APNEA). IPC and APNEA significantly (P0.001) reduced the intracellular Cl(-) concentration ([Cl(-)](i)) to 31.9 +/- 3.2 mM (mean +/- SEM) and 32.5 +/- 2.8 mM, respectively, from an initial [Cl(-)](i) (pooled stabilization 61.5 +/- 7.1 mM). [Cl(-)](i) did not change in control (non-preconditioned) cardiomyocytes (control 58.1 +/- 1.9 mM and control + vehicle 62.6 +/- 4.9 mM, P = 0.98 and 0.99 vs. pooled pre-treatment baseline, respectively). Inhibition of Cl(-) channels with 50 microM indanyloxyacetic acid 94 completely blocked preconditioning-induced Cl(-) efflux. Thus, a net Cl(-) efflux of 29.6 and 29.0 mM was triggered by IPC and APNEA.These findings provide the first direct evidence of activation of sarcolemmal Cl(-) channels by ischaemic and pharmacological preconditioning in cardiomyocytes.

See article by Ikeno et al. [1] (pages 699–709) in this issue. In the paper “Impaired perfusion after myocardial infarction is due to reperfusion-induced δPKC-mediated myocardial damage” by Ikeno et al. [1], Dr. Daria Mochly-Rosen's group at Stanford University have demonstrated a novel molecular interventional strategy, targeted inhibition of the δ isoform of protein kinase C (δPKC), to treat the post-ischemic no-reflow phenomenon in the myocardium using both mouse and pig models. This they achieved by either expressing a δPKC-specific translocation inhibitor protein fragment, δV1-1, only in cardiomyocytes in a transgenic crystalloid perfused mouse ex-vivo acute global myocardial ischemia (30 min)/reperfusion (120 min) model or through intra-coronary delivery of the TAT-conjugated synthetic peptide δV1-1 during the last minute of ischemia in a porcine in-vivo regional (LAD) myocardial ischemia (30 min)/reperfusion (30 min, 24 h, 6 and 12 days) model. Their mouse experiments showed a roughly 70% reduction in both infarction and coronary vascular resistance 5 min after reflow compared to wild-type hearts. Direct coronary delivery of the δV1-1 peptide in wild-type hearts provided similar results and, when added to the δV1-1 transgenic hearts, essentially eliminated the rise in coronary vascular resistance although no further reduction in muscle necrosis was achieved. These results suggest that δPKC inhibition protects both cardiomyocytes and microvasculature, as indicated by these investigators [1]. Their pig model included the contribution of blood cells and energy demand on the in vivo ischemic heart and involved regional ischemia more closely mimicking the clinical setting of reperfusion therapy in acute myocardial infarction. The δV1-1 peptide intra-coronary infusion performed as … *Corresponding author. Division of Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada M5G 1X8. Tel.: +1 416 813 2095; fax: +1 416 813 5965. Email address: gregory.wilson{at}sickkids.ca

See article by Kin et al. [1] (pages 74–85) in this issue . In this issue of Cardiovascular Research, Kin et al. [1] have demonstrated in vivo in rats that following a 30-min index regional myocardial ischemia, protection against reperfusion injury assessed 3 h later through necrosis quantification by tetrazolium staining was achieved by a 1-min duration reperfusion modification protocol that consisted of three cycles of 10 s each of unrestricted reperfusion followed by 10 s of re-occlusion of the same coronary artery subjected to the occlusion causing the index ischemia. This protection, while evident, was substantially less than that achieved by one cycle of preconditioning (5 min of ischemia followed by 10 min of reperfusion immediately preceding the index ischemia). Importantly, delaying the three-cycle (10-s ‘on’/10-s ‘off’) reperfusion modification protocol by 1 min (its duration) failed to protect the myocardium at risk against infarction. Furthermore, doubling the reperfusion modification protocol to six cycles (10-s ‘on’/10-s ‘off’) produced no measurable improvement in protection compared to three cycles. These findings focusing on events in the first minute of reperfusion are, in our view, the novel and potentially important contribution of this paper. The same group, led by Dr. Vinten-Johansen, has previously reported in the dog a substantial reduction in infarct size as a percentage of region at risk, producing about the same amount of protection as single-cycle ischemic preconditioning (same duration as in the rats [1]), with a three-cycle, 30-s unrestricted reperfusion/30-s re-occlusion protocol immediately upon reperfusion after a 60-min index regional ischemia [2]. However, when this reperfusion modification protocol was delayed by 5 min, the protection was lost. The present paper by Kin et al. [1], as described above, refines the temporal focus to the first minute of reperfusion. The Vinten-Johansen group has called the reperfusion modification protocols … * Corresponding author. Division of Cardiovascular Research, Research Institute, The Hospital for Sick Children, McMaster Building, Rm. 7019C, 555 University Avenue, Toronto, ON, Canada M5G 1X8. Tel.: +1-416-813-8720; fax: +1-416-813-7480. Email address: diazport{at}sickkids.ca

Objective At 4-year follow-up, paclitaxel-eluting stents (PES, TAXUS®) have demonstrated clinical effectiveness in reducing restenosis without increasing death or myocardial infarction. Concerns remain with all drug-eluting stents, however, regarding potential interference with long-term healing, particularly in zones with adjacent stent overlap due to theoretical doubling in both drug release and tissue contact with coating polymer. Therefore, we evaluated long-term healing of overlapped TAXUS stents in an accepted animal model. Methods Seventy-one non-injured swine underwent coronary artery placement of 138 overlapping stent-pairs (91 PES TAXUS Liberte 1 μg/mm2 slow release formulation, 3.0 or 3.5 mm diameter pairs and 47 control bare metal Liberte pairs) deployed at a 1.1:1 to 1.2:1 target stent-to-artery diameter ratio. Pathological analysis was performed at 30 (9 bare, 10 paclitaxel), 90 (9 bare, 10 paclitaxel), 180 (10 bare, 16 paclitaxel), 360 (10 bare, 21 paclitaxel), and 580 (9 bare, 22 paclitaxel) days. Results At all time intervals overlapped TAXUS stents were consistently endothelialized and free of luminal thrombus or vascular dilatation. Full healing, however, was delayed compared to control, with macrophage processed para-strut fibrin and cellular debris still present, but reduced and sequestered from blood flow by an endothelialized neointima at 360 and 580 days. While neointimal thickness in TAXUS overlap zones was significantly less than control at 30 days, greater neointima formation was observed with TAXUS at ≥90 days, but was stable and did not progress further from 90 to 580 days. Conclusion In this porcine model TAXUS stents demonstrated safety and acceptable healing with prolonged time to resolution of para-strut deposits, and did not produce the sustained neointimal suppression seen clinically.

Isolated cardiomyocytes, obtained by enzymatic digestion of whole hearts, have multiple advantages, most related to their accessibility to microscopic visualization, beyond the obvious elimination of other cell types that exist in the heart. Conversely, they cannot reproduce the mechanical disruption of reperfusion hypercontracture or the vascular phenomena of leukocyte plugging and compression from interstitial edema and contracture that can lead to the no-reflow phenomenon. Nevertheless, ischemic preconditioning has been consistently demonstrated to be a potent protective mechanism in freshly isolated and cultured cardiomyocytes across multiple species, indicating that much of the innate protection of ischemic preconditioning resides in cardiomyocytes. Centrifuging freshly isolated cardiomyocytes into a pellet with only a thin layer of supernatant covered by oil has proven to be an excellent model of simulated ischemia. In culture, cardiomyocytes may be exposed to severe hypoxia only or to various protocols for simulated ischemia in which an acid/lactate-rich, hyperkalemic extracellular environment with substrate deprivation (lacking glucose) is typically added. Reperfusion is simulated by well-oxygenated media of normal ionic composition. Cardiomyocyte injury has been usually evaluated by cell membrane permeability to dyes, often under hypo-osmotic conditions (osmotic fragility) or enzyme release. A survey of the use of cardiomyocyte models to study preconditioning is presented with the emphasis on examples of the innovative measurements, increasingly involving molecular techniques, that point to an increasing future role for these models in preconditioning research and, more generally, in the mechanistic study of myocardial ischemia/reperfusion.

Objectives: We have recently proposed that chloride (Cl−) channels contribute to ischemic preconditioning (IPC) in the myocardium. To further evaluate this hypothesis, we investigated the role of Cl− channels in pharmacological preconditioning. Methods: Isolated rabbit cardiomyocytes and isolated buffer-perfused rabbit hearts were initially preconditioned with a 10 min exposure to either an adenosine receptor agonist [2-chloro- N 6-cyclopentyladenosine (CCPA, 200 nM) and/or N 6-2-(4-aminophenyl)ethyladenosine (APNEA, 1 μM)] or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 μM) followed by a 10 or 20 min washout or not preconditioned (control). Cardiomyocytes or whole hearts were then subjected to prolonged ischemic period (45 min simulated ischemia or 40 min of regional myocardial ischemia, respectively) followed by 60 min reperfusion (resuspension in oxygenated medium or release of the transient coronary occlusion, respectively). Results: Indanyloxyacetic acid 94, a selective Cl− channel inhibitor that produced substantial inhibition of the regulatory volume decrease (RVD) when given at 10 μM concentration in cultured cardiomyocytes, was administered before ischemia to block RVD through Cl− channel inhibition. CCPA, APNEA and PMA significantly ( P

Objective: It has recently been suggested that conductance catheter parallel conductance (αVc) is a function of left ventricular volume. To confirm this, αVc was measured in this study over a wide range of steady state volumes. In addition, conductance derived volumes were compared to those obtained by radionuclide angiography to determine if the conductance catheter can be used to measure absolute left ventricular volume accurately in the intact dog heart. Methods: Seven dogs were anaesthetised and instrumented with left ventricular conductance and pressure tip catheters, a flow through rho cuvette to continually measure blood resistance, a thermodilution catheter, and a venous catheter for volume infusion/withdrawal. Conductance and angiographic data were acquired at 8(SD 1) variably loaded states. Parallel conductance was measured twice at each state using a saline dilution technique and a new non-linear algorithm that allows variability in the observations of both maximum and minimum conductance volumes. Results: The mean value of αVc was 89.1(18.0) ml (71.8 to 111.3 ml) with a mean within-animal coefficient of variation of 7.3(3.4)%. Multiple linear regression using dummy variables to account for the large interanimal variability did not reveal any relationship between αVc and either maximum or minimum left ventricular volume. Furthermore, no difference was found when αVc values measured at the lowest and highest loading levels in each dog were compared. Linear regression showed good agreement between conductance and radionuclide derived end diastolic volumes (slope = 0.94, R = 0.9, p

Objective: The aim was to compare the effects of ischaemic preconditioning in the buffer perfused and parabiotic blood perfused Langendorff rabbit heart models. Methods: Isolated hearts were perfused with either Krebs-Henseleit buffer solution or blood from a support rabbit. Hearts were subjected to an initial 30 min stabilisation period followed by 30 min of global ischaemia and 60 min of reperfusion. Ischaemic preconditioned (IP) hearts were also subjected to one cycle of 5 min global ischaemia and 10 min of reperfusion before the 30 min ischaemia. For each experiment, left ventricular function and necrosis were measured. Results: Necrosis, as measured by tetrazolium staining and expressed as a percentage of the left ventricular area, was significantly different between the buffer perfused control [42.5%(SEM 6.9), n=7] and buffer perfused IP group [22.2%(5.4), n=7, p

Heterotopic cardiac transplantation is a useful method for studying allograft rejection. In this study a new technique of cardiac transplantation was carried out, which involved retroperitoneal anastomoses of the donor ascending aorta and main pulmonary artery with the recipient abdominal aorta and inferior vena cava respectively. The procedure was simple and effective and was accomplished with minimal operative mortality and postoperative morbidity. The method allows better access to the allograft for repeated open myocardial biopsies, obviating the limitations of transvenous fluoroscopically directed endomyocardial biopsy. This technique of retroperitoneal heterotopic cardiac transplantation has important advantages compared with similar procedures performed in the neck, abdomen, or thorax.