Andreas Skyschally

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, GSK3&bgr;) 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).

Mitochondrial STAT3 Activation and Cardioprotection by Ischemic Postconditioning in Pigs With Regional Myocardial Ischemia/Reperfusion

Rationale: Timely restoration of coronary blood flow is the only way to salvage myocardium from infarction, but reperfusion per se brings on additional injury. Such reperfusion injury and the resulting size of myocardial infarction is attenuated by ischemic postconditioning, ie, the repeated brief interruption of coronary blood flow during early reperfusion. The signal transduction of ischemic postconditioning is under intense investigation, but no signaling step has yet been identified as causal for such protection in larger mammals in situ. Objective: We have now in an in situ pig model of regional myocardial ischemia/reperfusion addressed the role of mitochondrial signal transducer and activator of transcription 3 (STAT3). Methods and Results: We demonstrated reduction of infarct size by ischemic postconditioning (26±3% of area at risk versus 38±2% in controls with immediate full reperfusion) along with more markedly increased tyrosine705 phosphorylation of STAT3 in myocardial biopsies (at 10 minutes reperfusion: 9.2±3.0-fold from baseline versus 6.6±2.9-fold in controls with immediate full reperfusion). Increased tyrosine705 phosphorylation of STAT3 and better preservation of complex 1 respiration and calcium retention capacity were also present in isolated mitochondria from postconditioned myocardium in vitro. Prior janus kinase/STAT inhibition with AG490 in vivo abrogated the infarct size reduction and the better preservation of mitochondrial function, and the STAT3 inhibitor Stattic in vitro also abrogated better preservation of mitochondrial function. Conclusions: Our data support a causal role for mitochondrial STAT3 activation to mediate cardioprotection through better mitochondrial function.

Ischemic postconditioning: experimental models and protocol algorithms

Ischemic postconditioning, a simple mechanical maneuver at the onset of reperfusion, reduces infarct size after ischemia/reperfusion. After its first description in 2003 by Zhao et al. numerous experimental studies have investigated this protective phenomenon. Whereas the underlying mechanisms and signal transduction are not yet understood in detail, infarct size reduction by ischemic postconditioning was confirmed in all species tested so far, including man. We have now reviewed the literature with focus on experimental models and protocols to better understand the determinants of protection by ischemic postconditioning or lack of it. Only studies with infarct size as unequivocal endpoint were considered. In all species and models, the duration of index ischemia and the protective protocol algorithm impact on the outcome of ischemic postconditioning, and gender, age, and myocardial temperature contribute.

Ischemic preconditioning preserves connexin 43 phosphorylation during sustained ischemia in pig hearts in vivo

During myocardial ischemia, connexin 43 (Cx43) is dephosphorylated in vitro, and the subsequent opening of gap junctions formed by two opposing Cx43 hexamers was suggested to propagate ischemia/reperfusion injury. Reduction of infarct size (IS) by ischemic preconditioning (IP) involves activation of protein kinase C (PKC) and p38 mitogen activated protein kinase (MAPK), both of which can phosphorylate Cx43. We now studied in anesthetized pigs whether IP impacts on Cx43 phosphorylation by measuring the density of non‐phosphorylated and total Cx43 (confocal laser) during normoperfusion and 90‐min ischemia in non‐preconditioned and preconditioned hearts. Co‐localization of PKCα, p38MAPKα, and p38MAPKβ with Cx43 and the activity of p38MAPK were assessed. IP by 10 min ischemia and 15 min reperfusion reduced IS. Non‐phosphorylated Cx43 remained unchanged during ischemia in preconditioned hearts, while it increased from 35±3 to 75±8 AU (P<0.05) in non‐preconditioned hearts. Co‐localization of PKCα, p38MAPKα, and p38MAPKβ with Cx43 during ischemia increased only in preconditioned hearts. While the ischemia‐induced increase in p38MAPKα activity was comparable in preconditioned and non‐preconditioned hearts, p38MAPKβ activity was increased only in preconditioned hearts. Blockade of p38MAPK by SB203580 attenuated the IS‐reduction and the increased p38MAPK‐Cx43 co‐localization by IP. We conclude that IP increases co‐localization of protein kinases with Cx43 and preserves phosphorylation of Cx43 during ischemia.

Bidirectional Role of Tumor Necrosis Factor-α 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-&agr; (TNF-&agr;) expression, resulting in progressive contractile dysfunction. However, TNF-&agr; 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-&agr; expression is increased. ME (group1; n=7) was induced by intracoronary infusion of microspheres (42 &mgr;m; 3000 per mL/min inflow). Controls (group 2; n=8) received saline. Groups 3 and 4 (n=4 each) were pretreated with ovine TNF-&agr; antibodies (25 mg/kg body weight) 30 minutes before ME or placebo, respectively. Ischemia (90 minutes) was induced 6 hours after ME when TNF-&agr; was increased (66±21 pg/g wet weight; mean±SEM) or after placebo (TNF-&agr;, 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-&agr; 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-&agr; antibodies (group 3 versus group 4, 29±3% versus 35±5%). In ME, TNF-&agr; is responsible for both progressive contractile dysfunction and delayed protection against infarction.

Low-frequency Spectral Power of Heart Rate Variability Is Not a Specific Marker of Cardiac Sympathetic Modulation

Background Heart rate variability in the frequency domain has been proposed to reflect cardiac autonomic control. Therefore, measurement of heart rate variability may be useful to assess the effect of epidural anesthesia on cardiac autonomic tone. Accordingly, the effects of preganglionic cardiac sympathetic blockade by segmental epidural anesthesia were evaluated in humans on spectral power of heart rate variability. Specifically, the hypothesis that cardiac sympathetic blockade attenuates low‐frequency spectral power, assumed to reflect cardiac sympathetic modulation, was tested. Methods Ten subjects were studied while supine and during a 15‐min 40‐ degrees head‐up tilt both before and after cardiac sympathetic blockade by segmental thoracic epidural anesthesia (sensory block: C6‐T6). ECG, arterial pressure, and respiratory excursion (Whitney gauge) were recorded, and a fast‐Fourier‐transformation was applied to 512‐s data segments of heart rate derived from the digitized ECG at the end of each intervention. Results With cardiac sympathetic blockade alone and the subjects supine, both low‐frequency (LF, 0.06–0.15 Hz) and high‐frequency (HF, 0.15–0.80 Hz) spectral power remained unchanged. During tilt, epidural anesthesia attenuated the evoked increase in heart rate (+11 *symbol* min sup ‐1 + 7 SD vs. +6 + 7, P ‐ 0.024). However, while during tilt cardiac sympathetic blockade significantly decreased the LF/HF ratio (3.68 plus/minus 2.52 vs. 2.83 plus/minus 2.15, P = 0.041 vs. tilt before sympathetic blockade), a presumed marker of sympathovagal interaction, absolute and fractional LF and HF power did not change. Conclusions Although preganglionic cardiac sympathetic blockade reduced the LF/HF ratio during tilt, it did not alter spectral power in the LF band during rest or tilt. Accordingly, low‐frequency spectral power is unlikely to specifically reflect cardiac sympathetic modulation in humans.

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 Ivabradines protection goes beyond heart rate reduction.

Calcium responsiveness in regional myocardial short-term hibernation and stunning in the in situ porcine heart : Inotropic responses to postextrasystolic potentiation and intracoronary calcium

BACKGROUND We tested the hypothesis that decreased calcium responsiveness is responsible for the reduction in contractile function in regional hibernating and stunned myocardium in situ. METHODS AND RESULTS In 19 anesthetized swine, the left anterior descending coronary artery flow was reduced to decrease anterior myocardial work index (sonomicrometry) by approximately 60%. During 90 minutes of hypoperfusion, creatine phosphate recovered (as determined by biopsy specimens and bioluminescence) and no necrosis developed (as determined by staining with triphenyl tetrazolium chloride). In 10 swine, changes in the intracellular calcium concentration were induced by systematic variation of the postextrasystolic time interval at a constant prematurity. In 9 additional swine, a graded IC calcium infusion was performed. Under control conditions, anterior myocardial work increased with a fully compensated postextrasystolic time interval from 380+/-93 (mean+/-SD) to 523+/-98 mm Hg . mm. IC calcium infusion increased anterior myocardial work under control conditions from 356+/-85 to a maximum of 428+/-93 mm Hg . mm. Although the maximal responses were decreased during postextrasystolic potentiation (222+/-68 versus 523+/-98 mm Hg . mm) and calcium infusion (176+/-32 versus 428+/-93 mm Hg . mm) after 90 minutes of ischemia, the relationships between increases in anterior myocardial work and, respectively, postextrasystolic time interval and IC calcium were not different. The same was true after 30 minutes of reperfusion. CONCLUSIONS Both regional hibernating myocardium and stunned myocardium in situ are characterized by a decrease in overall myocardial calcium responsiveness; however, there appears to be no significant myocardial desensitization to calcium.

Pathophysiology of myocardial infarction: protection by ischemic pre- and postconditioning.

One or several short cycles of ischemia/reperfusion before (preconditioning) or after (postconditioning) a sustained coronary occlusion with subsequent reperfusion reduce the ultimate infarct size. The protection is potent, but limited to a narrow time frame. In animal experiments, a complex signal transduction cascade was identified which results specifically in a reduction of reperfusion injury. There is evidence for both ischemic pre- and postconditioning in patients with coronary artery disease.ZusammenfassungEin oder mehrere kurze Zyklen von Ischämie/Reperfusion kurz vor (Präkonditionierung) oder nach (Postkonditionierung) einem länger dauernden Koronarverschluss mit anschließender Reperfusion reduzieren die Infarktgröße. Die protektive Wirkung ist potent, aber auf einen engen zeitlichen Rahmen beschränkt. Im Experiment wurde eine komplexe Signaltransduktionskaskade identifiziert, die spezifisch den Reperfusionsschaden reduziert. Belege für ischämische Prä- und Postkonditionierung gibt es auch bei Patienten mit koronarer Herzkrankheit.

Pathophysiology of Myocardial Infarction

One or several short cycles of ischemia/reperfusion before (preconditioning) or after (postconditioning) a sustained coronary occlusion with subsequent reperfusion reduce the ultimate infarct size. The protection is potent, but limited to a narrow time frame. In animal experiments, a complex signal transduction cascade was identified which results specifically in a reduction of reperfusion injury. There is evidence for both ischemic pre- and postconditioning in patients with coronary artery disease.ZusammenfassungEin oder mehrere kurze Zyklen von Ischämie/Reperfusion kurz vor (Präkonditionierung) oder nach (Postkonditionierung) einem länger dauernden Koronarverschluss mit anschließender Reperfusion reduzieren die Infarktgröße. Die protektive Wirkung ist potent, aber auf einen engen zeitlichen Rahmen beschränkt. Im Experiment wurde eine komplexe Signaltransduktionskaskade identifiziert, die spezifisch den Reperfusionsschaden reduziert. Belege für ischämische Prä- und Postkonditionierung gibt es auch bei Patienten mit koronarer Herzkrankheit.