Reduction of infarct size by gentle reperfusion without activation of reperfusion injury salvage kinases in pigs.

AIMS: Reperfusion is mandatory to salvage ischaemic myocardium from infarction, but also induces additional reperfusion injury and contributes to infarct size (IS). Gentle reperfusion (GR) has been proposed to attenuate reperfusion injury, but this remains contentious. We now investigated whether (i) GR reduces IS and (ii) GR is associated with the activation of reperfusion injury salvage kinases (RISK). METHODS AND RESULTS: Anaesthetized pigs were subjected to 90 min left anterior descending coronary artery hypoperfusion and 120 min reperfusion. GR was induced by slowly increasing coronary inflow back to baseline over 30 min, using an exponential algorithm [F(t) = F(i)+e(-(0.1)(t)((min)-3)).(F(b)-F(i)); F(b), coronary inflow at baseline; F(i), coronary inflow during ischaemia; n = 12]. Pigs subjected to immediate full reperfusion (IFR; n = 13) served as controls. IS was determined by triphenyl tetrazolium chloride staining. The expression level of phosphorylated RISK proteins was determined by western blot analysis in myocardial biopsies taken at baseline, after 80-85 min ischaemia and at 10, 30, and 120 min reperfusion. In additional experiments with IFR (n = 3) and GR (n = 3), the PI3-AKT and MEK1/2-ERK1/2 pathways were pharmacologically blocked (BL). IS was 37 +/- 2% (mean +/- SEM) of the area at risk with IFR and 29 +/- 1% (P < 0.05) with GR. RISK phosphorylation was similar between GR and IFR at baseline and 85 min ischaemia. At 10 min reperfusion, RISK phosphorylation was increased with IFR, but not with GR. At 30 and 120 min reperfusion, RISK phosphorylation was still greater with IFR than GR. RISK blockade did not abolish the IS reduction by GR (BL-IFR: 27 +/- 4% of the area at risk; BL-GR: 42 +/- 5%; P < 0.05). CONCLUSION: Gentle reperfusion reduces infarct size in pigs, but RISK activation is not causally involved in this infarct size reduction.

Systematic analysis of functional and structural changes after coronary microembolization: a cardiac magnetic resonance imaging study.

OBJECTIVES: Our study aimed to detect the morphological und functional effects of coronary microembolization (ME) in vivo by cardiac magnetic resonance (CMR) imaging in an established experimental animal model. BACKGROUND: Post-mortem morphological alterations of coronary ME include perifocal inflammatory edema and focal microinfarcts. Clinically, the detection of ME after successful coronary interventions identifies a population with a worse long-term prognosis. METHODS: In 18 minipigs, ME was performed by intracoronary infusion of microspheres followed by repetitive in vivo imaging on a 1.5-T MR system from 30 min to 8 h after ME. Additionally, corresponding ex vivo CMR imaging and histomorphology were performed. RESULTS: Cine CMR imaging demonstrated a time-dependent increase of wall motion abnormalities from 9 of 18 animals after 30 min to all animals after 8 h (0.5 h, 50%; 2 h, 78%; 4 h, 75%; 8 h, 100%). Whereas T2 images were negative 30 min after ME, 4 of 18 animals showed myocardial edema at follow-up (0.5 h, 0%; 2 h, 6%; 4 h, 25%; 8 h, 17%). In vivo late gadolinium enhancement (LGE) was observed in none of the animals after 30 min, but in 33%, 50%, and 83% of animals at 2 h, 4 h, and 8 h, respectively, after ME. Ex vivo CMR imaging showed patchy areas of LGE in all but 1 animal (2 h, 83%; 4 h, 100%; 8 h, 100%). A significant correlation was seen between the maximum troponin I level and LGE in vivo (r = 0.63) and the spatial extent of ex vivo LGE (r = 0.76). CONCLUSIONS: Our results show that in vivo contrast-enhanced CMR imaging allows us to detect functional and structural myocardial changes after ME with a high sensitivity. Ex vivo, the pattern of LGE of high-resolution, contrast-enhanced CMR imaging is different from the well-known pattern of LGE in compact myocardial damage. Thus, improvements in spatial resolution are thought to be necessary to improve its ability to visualize ME-induced structural alterations even in vivo.

Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria.

Cardiomyocytes contain subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which differ in their respiratory and calcium retention capacity. Connexin 43 (Cx43) is located at the inner membrane of SSM, and Cx43 is involved in the cardioprotection by ischemic preconditioning (IP). The function of Cx43-formed channels is regulated in part by phosphorylation at residues in the carboxy terminus of Cx43. The aim of the present study was (1) to investigate whether Cx43 is also present in IFM, and (2) to characterize its spatial orientation in the inner mitochondrial membrane (IMM). Confirming previous findings, ADP-stimulated respiration was greater in IFM than in SSM from rat ventricles. In preparations from rats and mice not contaminated with sarcolemmal proteins, Cx43 was exclusively detected in SSM, but not in IFM by Western blot analysis (n = 6). SSM were exposed to different proteinase K concentrations to cleave peptide bonds, and Western blot analysis was performed for ATP synthase alpha (IMM, subunit in the matrix), uncoupling protein 3 (UCP3, IMM, intermembrane space epitope), and manganese superoxide dismutase (MnSOD, matrix). At a proteinase K concentration of 50 microg/ml, immunoreactivities of all the analyzed proteins were completely lost. The use of 5 microg/ml proteinase K resulted in similarly reduced immunoreactivities for Cx43 (19.4 +/- 5.8% of untreated mitochondria, n = 6) and UCP3 (23.0 +/- 4%, n = 7), whereas the immunoreactivities of ATP synthase alpha (49.1 +/- 6.4%, n = 7) and MnSOD (79.9 +/- 17.4%, n = 6) were better preserved, suggesting that the carboxy terminus of Cx43 is directed towards the intermembrane space. The results were confirmed in digitonin-treated mitochondria. Taken together, Cx43 is exclusively localized in SSM, with its carboxy terminus directed towards the intermembrane space. Since loss of mitochondrial Cx43 abolishes IP's cardioprotection, SSM and IFM apparently differ in their function in the signal transduction of IP.

Ischemic postconditioning in pigs: no causal role for RISK activation.

Ischemic postconditioning (IPoC) reduces infarct size following ischemia/reperfusion. Whether or not phosphorylation of RISK (reperfusion injury salvage kinases) (AKT, ERK1/2, P70S6K, GSK3beta) is causal for protection by IPoC is controversial. We therefore studied the impact of RISK on IPoC in anesthetized pigs subjected to 90 minutes of left anterior descending coronary artery hypoperfusion and 120 minutes of reperfusion. In protocol 1, IPoC, by 6 cycles of 20/20 seconds of reperfusion/reocclusion (n=13), was compared with immediate full reperfusion (IFR) (n=15). In protocol 2, IPoC (n=4) or IFR (n=4) was performed with pharmacological RISK blockade by IC coinfusion of Wortmannin and U0126. Infarct size was determined by TTC staining, and the expression of phosphorylated RISK proteins by Western blot analysis in biopsies. In protocol 1, infarct size was 20+/-3% (percentage of area at risk; mean+/-SEM) with IPoC and 33+/-4% (P<0.05) with IFR. RISK phosphorylation increased with reperfusion but was not different between IPoC and IFR. In protocol 2, Wortmannin and U0126 blocked the increases in RISK phosphorylation during reperfusion, but infarct size was still smaller with IPoC (15+/-7%) than with IFR (35+/-6%; P<0.05).

No impact of protein phosphatases on connexin 43 phosphorylation in ischemic preconditioning.

Cardiac connexin 43 (Cx43) is involved in infarct propagation, and the uncoupling of Cx43-formed channels reduces infarct size. Cx43-formed channels open upon Cx43 dephosphorylation, and ischemic preconditioning (IP) prevents the ischemia-induced Cx43 dephosphorylation. In addition to the sarcolemma, Cx43 is also present in the cardiomyocyte mitochondria. We now examined the interaction of Cx43 with protein phosphatases PP1alpha, PP2Aalpha, and PP2Balpha and the role of such interaction for Cx43 phosphorylation in preconditioned myocardium. Infarct size (in %area at risk) in left ventricular anterior myocardium of Göttinger minipigs subjected to 90 min of low-flow ischemia and 120 min of reperfusion was 23.1 +/- 2.7 [n = 7, nonpreconditioned (NIP) group] and was reduced by IP to 10.0 +/- 3.2 (n = 6, P < 0.05). Mitochondrial and gap junctional Cx43 dephosphorylation increased after 85 min of ischemia in NIP myocardium, whereas Cx43 phosphorylation was preserved with IP. PP2Aalpha and PP1alpha, but not PP2Balpha, were detected by Western blot analysis in the left ventricular myocardium. Cx43 coprecipitated with PP2Aalpha but not with PP1alpha. Although the total PP2Aalpha immunoreactivity (confocal laser scan) was increased to 154 +/- 24% and 194 +/- 13% of baseline (P < 0.05) after 85 min of ischemia in NIP and IP myocardium, respectively, the PP2A activities were similar between the groups. The amount of PP2Aalpha coimmunoprecipitated with Cx43 remained unchanged. Only PP2Aalpha coprecipitates with Cx43 in pig myocardium. This interaction is not affected by IP, suggesting that PP2Aalpha is not involved in the prevention of the ischemia-induced Cx43 dephosphorylation by IP.

Inducible nitric oxide synthase expression and cardiomyocyte dysfunction during sustained moderate ischemia in pigs.

In acute myocardial ischemia, regional blood flow and function are proportionally reduced. With prolongation of ischemia, function further declines at unchanged blood flow. We studied the involvement of an inflammatory signal cascade in such progressive dysfunction and whether dysfunction is intrinsic to cardiomyocytes. In 10 pigs, ischemia was induced by adjusting inflow into the cannulated left anterior coronary artery to reduce coronary arterial pressure to 45 mm Hg (ISCH); 4 pigs received the inducible nitric oxide synthase (iNOS) inhibitors aminoguanidine or L-N(6)-(1-iminoethyl)-lysine during ISCH (ISCH+iNOS-Inhib); 6 pigs served as controls (SHAM). Anterior (AW) and posterior (PW) systolic wall thickening (sonomicrometry) were measured. After 6 hours, nitric oxide (NO) synthase (NOS) protein expression, NOS activity, and NO metabolites (nitrite/nitrate/nitroso species) were quantified in biopsies isolated from AW and PW. Cardiomyocyte shortening and intracellular calcium (Indo-1 acetoxymethyl ester) were measured without and with the NOS substrate L-arginine (100 micromol/L). In ISCH, AW wall thickening decreased from 42+/-4% (baseline) to 16+/-3% (6 hours). Wall thickening remained unchanged in ISCH-PW and SHAM-AW/PW. NOS2 (iNOS) protein expression and activity, but not NOS3 (endothelial NO synthase), were increased in ISCH-AW and ISCH-PW. iNOS expression correlated with increased nitrite contents. Cardiomyocyte shortening was reduced in ISCH-AW versus SHAM-AW (4.4+/-0.3% versus 5.6+/-0.3%). L-Arginine reduced cardiomyocyte shortening further in ISCH-AW (to 2.8+/-0.2%) and ISCH-PW (3.4+/-0.4% versus 5.4+/-0.4%) but not in SHAM or in ISCH+iNOS-Inhib; intracellular [Ca(2+)] remained unchanged. With L-arginine, in vitro AW cardiomyocyte shortening correlated with in vivo AW wall thickening (r=0.72). In conclusion, sustained regional ischemia induces myocardial iNOS expression in pigs, which contributes to contractile dysfunction at the cardiomyocyte level.

Improvement of regional myocardial blood flow and function and reduction of infarct size with ivabradine: protection beyond heart rate reduction.

AIMS: Effects of the bradycardic agent ivabradine on regional blood flow, contractile function, and infarct size were studied in a pig model of myocardial ischaemia/reperfusion. Heart rate reduction by beta-blockade is associated with negative inotropism and unmasked alpha-adrenergic coronary vasoconstriction. Ivabradine is the only available bradycardic agent for clinical use. METHODS AND RESULTS: Anaesthetized pigs were subjected to 90 min controlled left anterior descending coronary artery hypoperfusion and 120 min reperfusion. Regional blood flow was measured with microspheres, regional function with sonomicrometry, and infarct size with triphenyl tetrazolium chloride staining. Pigs received placebo or ivabradine (0.6 mg/kg i.v.) before or during ischaemia or before reperfusion, respectively. Pre-treatment with ivabradine reduced infarct size from 35 +/- 4 (SEM) to 19 +/- 4% of area at risk (AAR). Ivabradine 15-20 min after the onset of ischaemia increased regional myocardial blood flow from 2.12 +/- 0.31 to 3.55 +/- 0.56 microL/beat/g and systolic wall thickening from 6.7 +/- 1.0 to 16.3 +/- 3.0%; infarct size was reduced from 12 +/- 4 to 2 +/- 1% of AAR. Ivabradine 5 min before reperfusion still reduced infarct size from 36 +/- 4 to 21 +/- 5% of AAR. The benefit of ivabradine on flow and function was eliminated by atrial pacing, but part of the reduction of infarct size by ivabradine was not. CONCLUSION: Ivabradine's protection goes beyond heart rate reduction.

Reduced calcium responsiveness characterizes contractile dysfunction following coronary microembolization.

AIMS: We addressed calcium responsiveness in microembolized myocardium at 6 h after coronary microembolization (ME). METHODS AND RESULTS: In anesthetized pigs calcium responsiveness was determined as the increase of a myocardial work index (WI; LV pressure development vs. wall thickening) in response to a graded intracoronary infusion of CaCl(2) at baseline and at 6 h after ME or placebo, respectively. At baseline, CaCl(2 )infusion increased WI in both groups (ME: 296 +/- 22 to 468 +/- 47 mmHg*mm; placebo: 324 +/- 24 to 485 +/- 38 mmHg*mm; mean +/- SEM). At 6 h after ME, WI was decreased by 159 +/- 16 mmHg*mm (P < 0.05 vs. baseline) and remained reduced at any calcium concentration, whereas it was unchanged with placebo. The calcium concentration in coronary blood necessary to achieve the half maximal increase in WI remained unchanged from baseline to 6 h and did not differ between placebo and ME. CONCLUSION: The ME-induced myocardial dysfunction is not related to an altered calcium sensitivity, but is characterized by a reduced maximal contractile force.

Mitochondrial respiration and membrane potential after low-flow ischemia are not affected by ischemic preconditioning.

Mitochondrial function following prolonged ischemia and subsequent reperfusion is better preserved by ischemic preconditioning (IP). In the present study, we analyzed whether or not IP has an impact on mitochondrial function at the end of a sustained ischemic period. Göttinger minipigs were subjected to 90-min low-flow ischemia without (n=5) and with (n=5) a preconditioning cycle of 10-min ischemia and 15-min reperfusion. Mitochondria were isolated from the ischemic or preconditioned anterior wall (AW) and the control posterior wall (PW) at the end of ischemia. Basal mitochondrial respiration was not different between AW and PW. The ADP-stimulated (state 3) respiration in AW mitochondria compared to PW mitochondria was equally decreased in non-preconditioned and preconditioned pigs. The uncoupled respiration as well as the membrane potential (rhodamine 123 fluorescence) were not significantly different between groups. However, the recovery of the membrane potential (Delta rhodamine 123 fluorescence/s) after the addition of ADP was delayed in mitochondria obtained from AW compared to PW, both in non-preconditioned and in preconditioned pig hearts. Neither the amount of marker proteins for complexes of the electron transport chain nor the level of reactive oxygen species were affected by ischemia without or with IP. State 3 respiration and recovery of membrane potential were impaired in pig mitochondria after 90 min of low-flow ischemia. IP did not improve mitochondrial function during ischemia. Therefore, the preservation of mitochondrial function by IP may occur during reperfusion rather than during the sustained ischemic period.

Microdialysis-based analysis of interstitial NO in situ: NO synthase-independent NO formation during myocardial ischemia.

OBJECTIVES: Nitric oxide (NO) synthesis by NO synthases (NOS) requires oxygen. However, although counterintuitive, NO synthesis is increased in ischemic myocardium. Accordingly, mechanisms independent of the NOS pathway have been suggested to contribute to NO synthesis during ischemia. NO initiates detrimental as well as protective mechanisms in a concentration-dependent manner, thus aggravating or improving the outcome of ischemia. The aim of this study was to measure in situ interstitial NO concentrations in parallel to infarct size in anaesthetized pigs subjected to myocardial ischemia/reperfusion. The contribution of NOS-independent pathways to NO synthesis was studied using NOS blockade. METHODS: Interstitial NO measurements, based on microdialysis combined with the oxyhemoglobin method, were made during 90 min of moderate or severe ischemia and subsequent reperfusion. To examine the effect of NOS inhibition, an initial 30-min ischemic period was followed 60 min later by a second 30-min ischemic period with intracoronary infusion of S-ethyl-isothiourea. RESULTS: During ischemia, the interstitial NO concentration increased for about 30 min and then remained constant at this elevated level. The increase in NO concentration by 253+/-82 nmol/L during moderate and 565+/-169 nmol/L during severe ischemia correlated inversely with subendocardial blood flow (r=-0.76). NOS inhibition increased coronary arterial pressure and decreased the interstitial basal NO concentration and tissue nitrite content. However, it did not diminish the increase in interstitial NO concentration during ischemia. CONCLUSION: NOS-independent pathways are significantly involved in NO synthesis during myocardial ischemia.

Bidirectional role of tumor necrosis factor-alpha in coronary microembolization: progressive contractile dysfunction versus delayed protection against infarction.

In patients with unstable angina, plaque rupture and coronary microembolization (ME) can precede complete coronary artery occlusion and impending infarction. ME-induced microinfarcts initiate an inflammatory reaction with increased tumor necrosis factor-alpha (TNF-alpha) expression, resulting in progressive contractile dysfunction. However, TNF-alpha is not only a negative inotrope but can also protect the myocardium against infarction. In anesthetized pigs, we studied whether ME protects against infarction when TNF-alpha expression is increased. ME (group1; n=7) was induced by intracoronary infusion of microspheres (42 microm; 3000 per mL/min inflow). Controls (group 2; n=8) received saline. Groups 3 and 4 (n=4 each) were pretreated with ovine TNF-alpha antibodies (25 mg/kg body weight) 30 minutes before ME or placebo, respectively. Ischemia (90 minutes) was induced 6 hours after ME when TNF-alpha was increased (66+/-21 pg/g wet weight; mean+/-SEM) or after placebo (TNF-alpha, 21+/-10 pg/g; P<0.05). Infarct size (percentage area at risk) was determined after 2 hours of reperfusion (triphenyl tetrazolium chloride staining). ME decreased systolic wall thickening progressively over 6 hours (group 1 versus group 2, 65+/-4% versus 90+/-1%; percentage of baseline; P<0.05). TNF-alpha antibodies attenuated the progressive decrease in systolic wall thickening following ME (group 3, 77+/-5% of baseline; P<0.05 versus group 1) with no effect in controls (group 4; 90+/-8% of baseline). With ME, infarct size was decreased to 18+/-4% versus 33+/-4% in group 2 (P<0.05). The infarct size reduction was abolished by TNF-alpha antibodies (group 3 versus group 4, 29+/-3% versus 35+/-5%). In ME, TNF-alpha is responsible for both progressive contractile dysfunction and delayed protection against infarction.

Coronary microembolization.

Atherosclerotic plaque rupture is the key event in the pathogenesis of acute coronary syndromes and it also occurs during coronary interventions. Atherosclerotic plaque rupture does not always result in complete thrombotic occlusion of the epicardial coronary artery with subsequent impending myocardial infarction, but may in milder forms result in the embolization of atherosclerotic and thrombotic debris into the coronary microcirculation. This review summarizes the present experimental pathophysiology of coronary microembolization in animal models of acute coronary syndromes and highlights the main consequences of coronary microembolization--reduced coronary reserve, microinfarction, inflammation and oxidative modification of contractile proteins, contractile dysfunction and perfusion-contraction mismatch.Furthermore, the review presents the available clinical evidence for coronary microembolization in patients and compares the clinical observations with observations in the experimental model.

Prevention of the ischemia-induced decrease in mitochondrial Tom20 content by ischemic preconditioning.

Preserved mitochondrial function (respiration, calcium handling) and integrity (cytochrome c release) is central for cell survival following ischemia/reperfusion. Mitochondrial function also requires import of proteins from the cytosol via the translocase of the outer and inner membrane (TOM and TIM complexes). Since mitochondrial function following ischemia/reperfusion is better preserved by ischemic preconditioning (IP), we now investigated whether expression of parts of the import machinery is affected by ischemia/reperfusion without or with IP in vivo. We analyzed the mitochondrial content of the presequence receptor Tom20, the pore forming unit Tom40 and Tim23. Goettinger minipigs were subjected to 90 min of low-flow ischemia without or with preconditioning by 10 min ischemia and 15 min reperfusion. Mitochondria were isolated from the ischemic or preconditioned anterior wall of the left ventricle and from the control posterior wall. Infarct size was significantly reduced by IP (20.1 +/- 1.6% of area at risk (non-preconditioned) vs. 6.5 +/- 2.5% of area at risk (IP)). Using Western blot analysis, the ratio of Tom20 (normalized to Ponceau S) between mitochondria isolated from the anterior ischemic and posterior control wall was reduced (0.72 +/- 0.11, a.u., n = 8), whereas the mitochondrial Tom20 content was preserved by IP (1.17 +/- 0.16 a.u., n = 7, P < 0.05). The mitochondrial Tom40, Tim23 and adenine nucleotide transporter (ANT) contents were not significantly different between non-preconditioned and preconditioned myocardium. The preservation of the mitochondrial Tom20 protein level may contribute to the improved mitochondrial function after IP.

Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.

We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.

Oxidative modification of tropomyosin and myocardial dysfunction following coronary microembolization.

AIMS: We addressed a potential mechanism of myocardial dysfunction following coronary microembolization at the level of myofibrillar proteins. METHODS AND RESULTS: Anaesthetized pigs underwent intracoronary infusion of microspheres. After 6 h, the microembolized areas (MEA) had decreased systolic wall thickening to 38 +/- 7% of baseline and a 2.62 +/- 0.40-fold increase in the formation of disulphide cross-bridges (DCB) in tropomyosin relative to that in remote areas. The impairment in contractile function correlated inversely with DCB formation (r = -0.68; P = 0.015) and was associated with increased TNF-alpha content. DCB formation was reflected by increased tropomyosin immunoreactivity and abolished in vitro by dithiothreitol. Ascorbic acid prevented contractile dysfunction as well as increased DCB and TNF-alpha. In anaesthetized dogs, 8 h after intracoronary microspheres infusion, contractile function was reduced to 8+/-10% of baseline and DCB in MEA was 1.48+/-0.12 higher than that in remote areas. In conscious dogs, 6 days after intracoronary microspheres infusion, myocardial function had returned to baseline and DCB was no longer different between remote and MEA. Again contractile function correlated inversely with DCB formation (r = -0.83; P = 0.005). CONCLUSION: Myofibrillar protein oxidation may represent a mechanistic link between inflammation and contractile dysfunction following coronary microembolization.

Preinfarction angina: no interference of coronary microembolization with acute ischemic preconditioning.

Transient episodes of angina preceding acute myocardial infarction may both, protect the myocardium by ischemic preconditioning or damage it when associated with coronary microembolization. We now studied the potential loss of ischemic preconditioning with coronary microembolization. Anesthetized pigs (group 1; n=8) were subjected to 90 min sustained low-flow ischemia. Group 2 (n=8) was subjected to coronary microembolization (i.e. microspheres; 42 microm slashed circle; 3000 per ml min-1 inflow) 35 min before sustained ischemia. In group 3, coronary microembolization was followed 10 min later by one cycle of ischemic preconditioning (10 min ischemia/15 min reperfusion) before subsequent sustained ischemia. Infarct size was determined after 2 h reperfusion by triphenyl tetrazolium chloride staining. Infarct size after sustained ischemia alone (group 1) was 19.4+/-3.4% of the area at risk (mean+/-S.E.M.). With coronary microembolization before sustained ischemia (group 2) infarct size was only slightly larger (23.6+/-4.6%, ns). In group 3 with microembolization followed by ischemic preconditioning, infarct size was reduced to 12.7+/-3.0% (P<0.05 vs. group 2). The relationships between infarct size and transmural blood flow in groups 1 and 3 were not different, giving the impression that ischemic preconditioning failed to protect microembolized myocardium. However, additional coronary microembolization shifted the relationship between infarct size and blood flow upwards to a larger infarct size at any given blood flow. Thus when comparing the relationship of group 3 to its true control (group 2), it was shifted downwards (P<0.05; analysis of covariance (ANCOVA)) indicating persistent protection of microembolized myocardium by ischemic preconditioning. Coronary microembolization induces additional infarction when superimposed on sustained ischemia but does not interfere with the endogenous protection by ischemic preconditioning.

Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning.

OBJECTIVE: Connexin 43 (Cx43) is involved in infarct size reduction by ischemic preconditioning (IP); the underlying mechanism of protection, however, is unknown. Since mitochondria have been proposed to be involved in IP's protection, the present study analyzed whether Cx43 is localized at mitochondria of cardiomyocytes and whether such localization is affected by IP. METHODS AND RESULTS: Western blot analysis on mitochondrial preparations isolated from rat, mouse, pig, and human hearts showed the presence of Cx43. The preparations were not contaminated with markers for other cell compartments. The localization of Cx43 to mitochondria was also confirmed by FACS sorting (double staining with MitoTracker Red and Cx43) and immuno-electron and confocal microscopy. To study the role of Cx43 in IP, mitochondria were isolated from the ischemic anterior wall (AW) and the control posterior wall (PW) of pig myocardium at the end of 90 min low-flow ischemia without (n=13) or with (n=13) a preceding preconditioning cycle of 10 min ischemia and 15 min reperfusion. With IP, the mitochondrial Cx43/adenine nucleotide transporter ratio was 3.4+/-0.7 fold greater in AW than in PW, whereas the ratio remained unchanged in non-preconditioned myocardium (1.1+/-0.2, p<0.05). The enhancement of the mitochondrial Cx43 protein level occurred rapidly, since an increase of mitochondrial Cx43 was already detected with two cycles of 5 min ischemia/reperfusion in isolated rat hearts to 262+/-63% of baseline. CONCLUSION: These data demonstrate that Cx43 is localized at cardiomyocyte mitochondria and that IP enhances such mitochondrial localization.

Regional differences of myocardial infarct development and ischemic preconditioning.

UNLABELLED: The spatial and temporal development of myocardial infarction depends on the area at risk (AAR), the severity and duration of blood flow reduction (energy supply) as well as on heart rate and regional wall function (energy demand). Both supply and demand can vary within the AAR of a given heart, potentially resulting in differences in infarct development. We therefore retrospectively analyzed infarct size (IS, %AAR, TTC) in 24 anesthetized pigs in vivo following 90 min hypoperfusion and 120 min reperfusion of the LAD coronary artery, which supplies parts of the LV septum (LVS) and anterior free wall (LVAFW). The total LAD perfusion territory averaged 49.8 +/- 14.2 (SD) g (49.2 +/- 8.4% of LV); 61.4 +/- 8.1% of the AAR was LVAFW. IS within the LVS was 25.3 +/- 15.1%, while IS within the LVAFW was 16.6 +/-10.1% (p<0.05). While ischemic blood flow (radiolabeled microspheres) did not differ between LVS (0.05 +/- 0.02 ml/min/g) and LVAFW (0.05 +/- 0.03 ml/min/g), perivascular connective tissue (56 +/- 9 vs. 38+/-7 microm(2), p < 0.05) and the capillary-to-myocyte distance (1.65 +/- 0.23 vs. 1.18 +/- 0.23 mm, p < 0.05) were larger in LVS than in LVAFW. Interestingly, IS in LVS (9.3 +/- 9.6%, n = 24) and LVAFW (9.2 +/- 9.1%) were reduced to the same absolute extent by ischemic preconditioning with one cycle of 10 min ischemia and 15 min reperfusion, suggesting that a similar regional difference exists also in the protection afforded by ischemic preconditioning. The mechanism(s) for that remain(s) to be established. CONCLUSION: In pigs, regional differences in infarct development and protection from it exist in the LAD perfusion territory, which are independent of ischemic blood flow but apparently related to pre-existing structural differences.

Coronary microembolization does not induce acute preconditioning against infarction in pigs-the role of adenosine.

OBJECTIVE: After coronary microembolization (ME) adenosine is released from ischemic areas of the microembolized myocardium. This adenosine dilates vessels in adjacent nonembolized myocardium and increases coronary blood flow. For ischemic preconditioning (IP) to protect the myocardium against infarction, an increase in the interstitial adenosine concentration (iADO) prior to the subsequent ischemia/reperfusion is necessary. We hypothesized that the adenosine release after ME is sufficient to increase iADO and protect the myocardium against infarction from subsequent ischemia/reperfusion. We have therefore compared myocardial protection by either coronary microembolization or ischemic preconditioning prior to ischemia/reperfusion. METHODS: In anesthetized pigs, the left anterior descending (LAD) was cannulated and perfused from an extracorporeal circuit. In 11 pigs, sustained ischemia was induced by 85% inflow reduction for 90 min (controls). Two other groups of pigs were subjected either to IP (n = 8; 10-min ischemia/15-min reperfusion) or coronary ME (n = 9; i.c. microspheres; 42 microm Ø; 3000 x ml(-1) x min inflow) prior to sustained ischemia. Coronary venous adenosine concentration (vADO) and iADO (microdialysis) were measured. Infarct size was determined after 2-h reperfusion by triphenyl tetrazolium chloride staining. RESULTS: In pigs subjected to IP, infarct size was reduced to 2.6 +/- 1.1% (mean +/- S.E.M.) vs. 17.0 +/- 3.2% in controls. iADO was increased from 2.4 +/- 1.3 to 13.1 +/- 5.8 micromol x l(-1) during the reperfusion following IP. In pigs subjected to ME, at 10 min after ME, coronary blood flow (38.6 +/- 3.6 to 53.6 +/- 4.3 ml x min(-1)) and vADO (0.25 +/- 0.04 to 0.48 +/- 0.07 micromol x l(-1)) were increased. However, iADO (2.0 +/- 0.5 at baseline vs. 2.3 +/- 0.6 micromol x l(-1) at 10 min after ME) did not increase. Infarct size induced by sustained ischemia following ME (22.5 +/- 5.2%) was above that of controls for any given subendocardial blood flow. CONCLUSION: ME released adenosine into the vasculature and increased coronary blood flow. The failure of iADO to increase with ME possibly explains the lack of protection against infarction after ME.

Activation of ATP-dependent potassium channels is a trigger but not a mediator of ischaemic preconditioning in pigs.

1. Activation of ATP-dependent potassium channels (K(ATP)) is involved in ischaemic preconditioning (IP). In isolated buffer-perfused rabbit hearts, activation of mitochondrial K(ATP)--through a generation of free radicals--acted as a trigger rather than a mediator of IP; the isolated buffer-perfused heart preparation, however, favours free radical generation. In contrast, in vivo studies in rats and dogs suggested that activation of K(ATP) acts as a mediator of IP's protection. A detailed analysis on the role of K(ATP) in IP's protection in vivo by varying the time and dose of K(ATP) blocker administration is, however, lacking. 2. In 54 enflurane-anaesthetized pigs, the left anterior descending coronary artery was perfused by an extracorporeal circuit. Infarct size (IS, %, TTC) following 90 min sustained low-flow ischaemia and 120 min reperfusion was 26.6+/-3.5 (s.e.m.) (n=8). IP with one cycle of 10 min ischaemia and 15 min reperfusion reduced IS to 6.5+/-2.1 (n=7, P<0.05). Blockade of K(ATP) with glibenclamide (0.5 mg kg(-1) i.v., 50 microg min(-1) continuous infusion) starting 10 min before or immediately following the preconditioning ischaemia abolished IS reduction by IP (20.7+/-2.7, n=7 and 21.9+/-6.6, n=6, respectively) while having no effect on IS per se (22.2+/-5.2, n=7), supporting a trigger role of K(ATP) in IP. In contrast, starting glibenclamide following the preconditioning ischaemia 10 min prior to the sustained ischaemia did not prevent IS reduction by IP (3.7+/-2.3, n=6), even when its bolus dose was increased to 1.5 mg kg(-1) (26.6+/-3.8 with IP vs 37.5+/-2.9 without IP; n=7 and 6 respectively, P<0.05), thereby refuting a mediator role of K(ATP) in IP. 3. In conclusion, activation of K(ATP) in the immediate reperfusion following the preconditioning ischaemia is pivotal for triggering IP.