Vago-splenic signal transduction of cardioprotection in humans.

BACKGROUND AND AIMS: The spleen serves as an important relay organ that releases cardioprotective factor(s) upon vagal activation during remote ischaemic conditioning (RIC) in rats and pigs. The translation of these findings to humans was attempted. METHODS: Remote ischaemic conditioning or electrical auricular tragus stimulation (ATS) were performed in 10 healthy young volunteers, 10 volunteers with splenectomy, and 20 matched controls. Venous blood samples were taken before and after RIC/ATS or placebo, and a plasma dialysate was infused into isolated perfused rat hearts subjected to global ischaemia/reperfusion. RESULTS: Neither left nor right RIC or ATS altered heart rate and heart rate variability in the study cohorts. With the plasma dialysate prepared before RIC or ATS, respectively, infarct size (% ventricular mass) in the recipient rat heart was 36 ± 6% (left RIC), 34 ± 3% (right RIC) or 31 ± 5% (left ATS), 35 ± 5% (right ATS), and decreased with the plasma dialysate from healthy volunteers after RIC or ATS to 20 ± 4% (left RIC), 23 ± 6% (right RIC) or to 19 ± 4% (left ATS), 26 ± 9% (right ATS); infarct size was still reduced with plasma dialysate 4 days after ATS and 9 days after RIC. In a subgroup of six healthy volunteers, such infarct size reduction was abrogated by intravenous atropine. Infarct size reduction by RIC or ATS was also abrogated in 10 volunteers with splenectomy, but not in their 20 matched controls. CONCLUSIONS: In humans, vagal innervation and the spleen as a relay organ are decisive for the cardioprotective signal transduction of RIC and ATS.

"Expression of concern": publication bias for positive preclinical cardioprotection studies.

The present analysis reports on the robustness of preclinical cardioprotection studies with infarct size as endpoint which were published in Basic Research in Cardiology, Cardiovascular Research, and Circulation Research between January 2013 and December 2023. Only 26 out of 269 papers with technically robust analysis of infarct size by triphenyltetrazolium chloride staining, magnetic resonance imaging or single photon emission tomography applied a prospective power analysis. A retrospective power calculation revealed that only 75% of the reported data sets with statistically significant positive results from all these studies had a statistical power of ≥ 0.9, and an additional 9% had a statistical power ≥ 0.8. The remaining 16% of all significant positive data sets did not even reach the 0.8 threshold. Only 13% of all analyzed data sets were neutral. We conclude that neutral studies are underreported and there is indeed a significant lack of robustness in many of the published preclinical cardioprotection studies which may contribute to the difficulties of translating cardioprotection to patient benefit.

No robust reduction of infarct size and no-reflow by metoprolol pretreatment in adult Göttingen minipigs.

Whereas prior experiments in juvenile pigs had reported infarct size reduction by intravenous metoprolol early during myocardial ischaemia, two major clinical trials in patients with reperfused acute myocardial infarction were equivocal. We, therefore, went back and tested the translational robustness of infarct size reduction by metoprolol in minipigs. Using a power analysis-based prospective design, we pretreated 20 anaesthetised adult Göttingen minipigs with 1 mg kg-1 metoprolol or placebo and subjected them to 60-min coronary occlusion and 180-min reperfusion. Primary endpoint was infarct size (triphenyl tetrazolium chloride staining) as a fraction of area at risk; no-reflow area (thioflavin-S staining) was a secondary endpoint. There was no significant reduction in infarct size (46 ± 8% of area at risk with metoprolol vs. 42 ± 8% with placebo) or area of no-reflow (19 ± 21% of infarct size with metoprolol vs. 15 ± 23% with placebo). However, the inverse relationship between infarct size and ischaemic regional myocardial blood flow was modestly, but significantly shifted downwards with metoprolol, whereas ischaemic blood flow tended to be reduced by metoprolol. With an additional dose of 1 mg kg-1 metoprolol after 30-min ischaemia in 4 additional pigs, infarct size was also not reduced (54 ± 9% vs. 46 ± 8% in 3 contemporary placebo, n.s.), and area of no-reflow tended to be increased (59 ± 20% vs. 29 ± 12%, n.s.).Infarct size reduction by metoprolol in pigs is not robust, and this result reflects the equivocal clinical trials. The lack of infarct size reduction may be the result of opposite effects of reduced infarct size at any given blood flow and reduced blood flow, possibly through unopposed alpha-adrenergic coronary vasoconstriction.

Remote ischemic conditioning in Ossabaw minipigs induces the release of humoral cardioprotective triggers, but the myocardium does not respond with reduced infarct size.

Ischemic preconditioning (IPC; brief cycles of coronary occlusion/reperfusion) is operative in all species tested so far and reduces infarct size through the release of trigger molecules and activation of signal transducer and activator of transcription (STAT)3 in pigs. We have recently demonstrated that IPC failed to protect Ossabaw minipigs, which had a genetic predisposition to, but not yet established a metabolic syndrome, from infarction and did not activate STAT3. We now subjected Ossabaw minipigs to remote ischemic conditioning (RIC; 4 × 5 min/5 min bilateral hindlimb ischemia-reperfusion) and analyzed the release of cardioprotective triggers into the circulation with the aim to distinguish whether IPC failed to stimulate trigger release or to activate intracellular signaling cascades upstream of STAT3. RIC or a placebo protocol, respectively, was induced in anesthetized pigs before 60 min/180 min coronary occlusion/reperfusion. Plasma, prepared from Ossabaw minipigs after RIC or placebo, was infused into isolated rat hearts subjected to 30 min/120 min global ischemia-reperfusion. In the Ossabaw minipigs, RIC did not reduce infarct size (49.5 ± 12.1 vs. 56.0 ± 11.8% of area at risk with placebo), and STAT3 was not activated. In isolated rat hearts, infusion of RIC plasma reduced infarct size (19.7 ± 6.7 vs. 33.2 ± 5.5% of ventricular mass with placebo) and activated STAT3. Pretreatment of rat hearts with the STAT3 inhibitor stattic abrogated such infarct size reduction and STAT3 activation. In conclusion, Ossabaw minipigs release cardioprotective triggers in response to RIC into the circulation, and lack of cardioprotection is attributed to myocardial nonresponsiveness.NEW & NOTEWORTHY Ischemic conditioning reduces myocardial infarct size in all species tested so far. In the present study, we used Ossabaw minipigs that had a genetic predisposition to, but not yet established a metabolic syndrome. In these pigs, remote ischemic conditioning (RIC) induced the release of cardioprotective triggers but did not reduce infarct size. Transfer of their plasma, however, reduced infarct size in isolated recipient rat hearts, along with signal transducer and activator of transcription (STAT)3 activation.

Non-responsiveness to cardioprotection by ischaemic preconditioning in Ossabaw minipigs with genetic predisposition to, but without the phenotype of the metabolic syndrome.

The translation of successful preclinical and clinical proof-of-concept studies on cardioprotection to the benefit of patients with reperfused acute myocardial infarction has been difficult so far. This difficulty has been attributed to confounders which patients with myocardial infarction typically have but experimental animals usually not have. The metabolic syndrome is a typical confounder. We hypothesised that there may also be a genuine non-responsiveness to cardioprotection and used Ossabaw minipigs which have the genetic predisposition to develop a diet-induced metabolic syndrome, but before they had developed the diseased phenotype. Using a prospective study design, a reperfused acute myocardial infarction was induced in 62 lean Ossabaw minipigs by 60 min coronary occlusion and 180 min reperfusion. Ischaemic preconditioning by 3 cycles of 5 min coronary occlusion and 10 min reperfusion was used as cardioprotective intervention. Ossabaw minipigs were stratified for their single nucleotide polymorphism as homozygous for valine (V/V) or isoleucine (I/I)) in the γ-subunit of adenosine monophosphate-activated protein kinase. Endpoints were infarct size and area of no-reflow. Infarct size (V/V: 54 ± 8, I/I: 54 ± 13% of area at risk, respectively) was not reduced by ischaemic preconditioning (V/V: 55 ± 11, I/I: 46 ± 11%) nor was the area of no-reflow (V/V: 57 ± 18, I/I: 49 ± 21 vs. V/V: 57 ± 21, I/I: 47 ± 21% of infarct size). Bioinformatic comparison of the Ossabaw genome to that of Sus scrofa and Göttingen minipigs identified differences in clusters of genes encoding mitochondrial and inflammatory proteins, including the janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. The phosphorylation of STAT3 at early reperfusion was not increased by ischaemic preconditioning, different from the established STAT3 activation by cardioprotective interventions in other pig strains. Ossabaw pigs have not only the genetic predisposition to develop a metabolic syndrome but also are not amenable to cardioprotection by ischaemic preconditioning.

Reflection of Cardioprotection by Remote Ischemic Perconditioning in Attenuated ST-Segment Elevation During Ongoing Coronary Occlusion in Pigs: Evidence for Cardioprotection From Ischemic Injury.

RATIONALE: Reduction of infarct size by remote ischemic perconditioning (perRIC) is evident only after several hours reperfusion. OBJECTIVE: To develop a potential real-time estimate of cardioprotection by perRIC, we have analyzed the time course of ST-segment elevation. METHODS AND RESULTS: Anesthetized open-chest pigs were subjected to 60-minute coronary occlusion and 180-minute reperfusion (placebo; n=19). PerRIC (n=18; 4×5 min/5 min hindlimb occlusion/reperfusion) was induced 20 minutes after coronary occlusion. Regional myocardial blood flow was measured with microspheres, areas of no-reflow with thioflavin-S, area at risk with blue dye, and infarct size with triphenyl tetrazolium chloride staining. Phosphorylation of protein kinase B α/ß/γ, extracellular signal-regulated kinase 1/2, and signal transducer and activator of transcription 3 was determined by Western blot. ST-segment elevation was analyzed in a V2-like ECG-lead at baseline, 5- and 55-minute coronary occlusion, and 10-, 30-, 60-, and 120-minute reperfusion. Transmural blood flow at 5-minute coronary occlusion was not different between perRIC (0.029±0.015 mL/min per gram; mean±SD) and placebo (0.024±0.018 mL/min per gram) as was area at risk (perRIC: 24±6% of the left ventricle; placebo: 21±4%). Areas of no-reflow tended to be smaller with perRIC (9±12% of area at risk versus 15±14% with placebo; P=0.13). Infarct size with perRIC was 23±12% of area at risk versus 40±11% with placebo (P<0.001). PerRIC increased phosphorylation of signal transducer and activator of transcription 3 at 120-minute reperfusion by 196±142% versus 109±120% with placebo (P=0.047). The time courses of ST-segment elevation in perRIC and placebo protocols, respectively, were different (P=0.017). With similar ST-segment elevation at 5-minute coronary occlusion (perRIC 282±34 µV; placebo 259±28 µV), partial recovery of ST-segment elevation between 5- and 55-minute coronary occlusion was more pronounced with perRIC than placebo (by 111±84 versus 15±94 µV; P=0.028). CONCLUSION: Infarct size reduction by perRIC is reflected in the ST-segment elevation during coronary occlusion in pigs, supporting the notion of protection from ischemic injury.

Vago-Splenic Axis in Signal Transduction of Remote Ischemic Preconditioning in Pigs and Rats.

RATIONALE: The signal transduction of remote ischemic conditioning is still largely unknown. OBJECTIVE: Characterization of neurohumoral signal transfer and vago-splenic axis in remote ischemic preconditioning (RIPC). METHODS AND RESULTS: Anesthetized pigs were subjected to 60 minutes of coronary occlusion and 180 minutes of reperfusion (placebo+ischemia/reperfusion [PLA+I/R]). RIPC was induced by 4×5/5 minutes of hindlimb I/R 90 minutes before coronary occlusion (RIPC+I/R). Arterial blood samples were taken after placebo or RIPC before I/R. In subgroups of pigs, bilateral cervical vagotomy, splenectomy, or splenic denervation were performed before PLA+I/R or RIPC+I/R, respectively. In pigs with RIPC+I/R, infarct size (percentage of area at risk) was less than in those with PLA+I/R (23±12% versus 45±8%); splenectomy or splenic denervation abrogated (splenectomy+RIPC+I/R: 38±15%; splenic denervation+RIPC+I/R: 43±5%), and vagotomy attenuated (vagotomy+RIPC+I/R: 36±11%) RIPC protection. RIPC increased phosphorylation of STAT3 (signal transducer and activator of transcription 3) in left ventricular biopsies taken at early reperfusion. Splenectomy or splenic denervation, but not vagotomy, abolished this increased phosphorylation. In rats with vagotomy, splenectomy, or splenic denervation, RIPC (3×5/5 minutes of hindlimb occlusion/reperfusion) or placebo was performed, respectively. Hearts were isolated, saline perfused, and subjected to 30/120-minute global I/R. With RIPC, infarct size (percentage of ventricular mass) was less (20±7%) than with placebo (37±6%), and vagotomy, splenectomy, or splenic denervation abrogated RIPC protection (38±12%, 36±9%, and 36±7%), respectively. Rat spleens were isolated, saline perfused, and splenic effluate (SEff) was sampled after infusion with carbachol (SEffcarbachol) or saline (SEffsaline). Pig plasma or SEff was infused into isolated perfused rat hearts subjected to global I/R. Infarct size was less with infusion of RIPC+I/Rplasma+ (24±6%) than with PLA+I/Rplasma (40±8%), vagotomy+PLA+I/Rplasma (39±11%), splenectomy+PLA+I/Rplasma (35±8%), vagotomy+RIPC+I/Rplasma (40±9%), splenectomy+RIPC+I/Rplasma (33±9%), or splenic denervation+RIPC+I/Rplasma (39±8%), respectively. With infusion of SEffcarbachol, infarct size was less than with infusion of SEffsaline (24 [19-27]% versus 35 [32-38]%). CONCLUSIONS: Activation of a vago-splenic axis is causally involved in RIPC cardioprotection.

Plasma from remotely conditioned pigs reduces infarct size when given before or after ischemia to isolated perfused rat hearts.

Short cycles of ischemia/reperfusion in a tissue/organ remote from the heart reduce myocardial ischemia/reperfusion injury. Such remote ischemic conditioning (RIC) can be induced before (pre-), during (per-), or after (post-) the onset of myocardial ischemia. RIC's protection can be transferred with plasma between different individuals, even across species. Infusion of plasma from pigs with remote ischemic per-conditioning(RPERC) reduces infarct size in isolated perfused rat hearts when given before and after the index ischemia. We here determined whether or not infusion of pig plasma is equally protective when given exclusively before or after the index ischemia in isolated perfused rat hearts. Blood was sampled at 10 min reperfusion from Göttingen mini-pigs with 60/180 min coronary occlusion/reperfusion without (placebo, n = 8) or with RPERC (4 × 5 min/5 min hindlimb ischemia/reperfusion, n = 7) starting at 20 min coronary occlusion. Plasma was separated, diluted (1:6), and infused into isolated perfused rat hearts before (plasmabefore) or after (plasmaafter) 30/120 min global zero-flow ischemia/reperfusion. Infarct size (IS) was demarcated and calculated as percent of ventricular mass (means ± standard deviations). The activation of cardioprotective intracellular signaling cascades was analyzed by Western blot. RPERC-plasma reduced IS (placebo-plasmabefore 36 ± 5% and placebo-plasmaafter 36 ± 7% versus RPERC-plasmabefore 19 ± 3% and RPERC-plasmaafter 21 ± 4%; P < 0.001 versus placebo-plasma) and increased the phosphorylation of signal transducer and activator of transcription 3, no matter whether plasma was given before ischemia or during reperfusion. Obviously, the protection, which the released factors exert, is operative during reperfusion. However, pre-ischemic exposure to such cardioprotective factors is remembered throughout ischemia.

Attenuation of ST-segment elevation after ischemic conditioning maneuvers reflects cardioprotection online.

Ischemic conditioning maneuvers, when induced either locally in the heart or remotely from the heart, reduce infarct size. However, infarct size reduction can be assessed no earlier than hours after established reperfusion. ST-segment elevation and its attenuation might reflect cardioprotection by ischemic conditioning online. Pigs were subjected to regional myocardial ischemia/reperfusion (1 h/3 h). Ischemic conditioning was induced prior to ischemia either locally (preconditioning; IPC; n = 15) or remotely (remote preconditioning; RIPC; n = 21), remotely during ischemia (remote perconditioning; RPER; n = 18), or locally at reperfusion (postconditioning; POCO; n = 9). Pigs without conditioning served as controls (PLA; n = 29). Area at risk and infarct size were measured postmortem, and ST-segment elevation was analyzed in a V2-like electrocardiogram lead. Ischemic conditioning reduced infarct size (PLA 42 ± 11% of area at risk; IPC 18 ± 10%; RIPC 22 ± 12%; RPER 23 ± 12%, POCO 22 ± 11%). With PLA, ST-segment elevation was increased at 5 min ischemia, sustained until 55 min ischemia and further increased at 10 min reperfusion. IPC and RIPC did not impact on ST-segment elevation at 5 min ischemia, but attenuated ST-segment elevation at 55 min ischemia. With RPER, ST-segment elevation was not different from that with PLA at 5 min, but attenuated at 55 min ischemia. POCO abolished the further increase of ST-segment elevation with reperfusion. Cardioprotection by ischemic conditioning is robustly reflected by attenuation of ST-segment elevation online.

Methylene Blue Treatment of Grafts During Cold Ischemia Time Reduces the Risk of Hepatitis C Virus Transmission.

Background: Although organ shortage is a rising problem, organs from hepatitis C virus (HCV) ribonucleic acid (RNA)-positive donors are not routinely transplanted in HCV-negative individuals. Because HCV only infects hepatocytes, other organs such as kidneys are merely contaminated with HCV via the blood. In this study, we established a protocol to reduce HCV virions during the cold ischemic time. Methods: Standard virological assays were used to investigate the effect of antivirals, including methylene blue (MB), in different preservation solutions. Kidneys from mini pigs were contaminated with Jc1 or HCV RNA-positive human serum. Afterwards, organs were flushed with MB. Hypothermic machine perfusion was used to optimize reduction of HCV. Results: Three different antivirals were investigated for their ability to inactivate HCV in vitro. Only MB completely inactivated HCV in the presence of all perfusion solutions. Hepatitis C virus-contaminated kidneys from mini pigs were treated with MB and hypothermic machine perfusion without any negative effect on the graft. Human liver-uPA-SCID mice did not establish HCV infection after inoculation with flow through from these kidneys. Conclusions: This proof-of-concept study is a first step to reduce transmission of infectious HCV particles in the transplant setting and might serve as a model for other relevant pathogens.

Humoral transfer and intramyocardial signal transduction of protection by remote ischemic perconditioning in pigs, rats, and mice.

Remote ischemic perconditioning (RPER) during ongoing myocardial ischemia reduces infarct size. The signal transduction of RPER's cardioprotection is still largely unknown. Anesthetized pigs were therefore subjected to RPER by 4 × 5 min/5 min of hindlimb ischemia-reperfusion during 60 min of coronary occlusion before 3 h of reperfusion. Pigs without RPER served as placebo (PLA). The phosphorylation of Akt and ERK [reperfusion injury salvage kinase (RISK) pathway] and STAT3 [survivor activating factor enhancement (SAFE) pathway] in the area at risk was determined by Western blot analysis. Wortmannin/U0126 or AG490 was used for pharmacological RISK or SAFE blockade, respectively. Pig plasma/plasma dialysate sampled after RPER or PLA, respectively, was transferred to isolated rat and mouse hearts subjected to 30 min/120 min of global ischemia-reperfusion. Mitochondria were isolated from rat hearts at early reperfusion. Isolated mouse cardiomyocytes were subjected to 1 h of hypoxia/5 min of reoxygenation without and with prior plasma dialysate incubation. RPER reduced infarct size in pigs to 21 ± 15% versus 44 ± 9% in PLA (percentage of the area at risk, mean ± SD, P < 0.05) and increased STAT3 phosphorylation at early reperfusion. AG490 but not RISK blockade abolished the protection. RPER plasma/plasma dialysate reduced infarct size in rat (22 ± 3% of ventricular mass vs. 40 ± 11% with PLA plasma, P < 0.05) and mouse (29 ± 4% vs. 63 ± 8% with PLA plasma dialysate, P < 0.05) hearts and improved mitochondrial function (e.g., increased respiration, ATP formation, and calcium retention capacity and decreased reactive oxygen species formation). RPER dialysate also improved the viability of mouse cardiomyocytes after hypoxia/reoxygenation. RISK or SAFE blockade each abrogated these beneficial effects. NEW & NOTEWORTHY Remote ischemic perconditioning salvages the myocardium in patients with acute infarction. We identified a signal transduction with humoral transfer and STAT3 activation in pigs and an involvement of reperfusion injury salvage kinases and STAT3 in rat and mouse hearts, along with better cardiomyocyte viability and mitochondrial function.

STAT3 as a common signal of ischemic conditioning: a lesson on "rigor and reproducibility" in preclinical studies on cardioprotection.

Ischemic conditioning before (ischemic preconditioning, IPC) or after (ischemic postconditioning, POCO) sustained myocardial ischemia/reperfusion (I/R), induced locally or remotely from the heart (remote IPC, RIPC), reduces infarct size. However, none of the identified signaling steps of ischemic conditioning was robust across models and species to be successfully translated to humans. In prior separate studies in pigs, activation of signal transducer and activator of transcription 3 (STAT3) was causal for infarct size reduction by IPC, POCO, and RIPC but it remains unclear whether or not STAT3 is truly a common denominator of cardioprotective signaling. We therefore, now analyzed the phosphorylation of STAT3 and other signaling proteins in left ventricular biopsies from our prior studies on IPC, POCO and RIPC in one approach. We developed a strategy for the quantification of protein phosphorylation in multiple samples from many experiments on different gels/membranes by Western blot. Along with reduced infarct size, the ratio of STAT3tyr705 phosphorylation/total STAT3 protein at early reperfusion was significantly increased by IPC (IPC 2.0 ± 0.3 vs. I/R 1.2 ± 0.2 arbitrary units), but only trendwise by POCO and RIPC (1.3 ± 0.2; 1.4 ± 0.2 arbitrary units); storage time for IPC samples was shorter than for POCO and RIPC samples. No other signaling protein phosphorylation was associated with reduced infarct size. We confirmed STAT3 phosphorylation with IPC. For POCO and RIPC we could not reproduce the findings from our earlier more focused studies. At this point, we can not distinguish between lack of robustness of the biological signal and methodological issues of our retrospective approach.

Cardioprotection by remote ischemic conditioning and its signal transduction.

Cardioprotective strategies aim to salvage myocardium from ischemia/reperfusion injury and to reduce infarct size and its consequences. Different stimuli, acting at sites remote from the heart (remote conditioning), activate molecular self-defense mechanisms at the target organ heart as well as in other parenchymal organs. Remote conditioning of the heart has been established in many experimental studies and successfully translated to patients. Remote ischemic conditioning by short repetitive cycles of ischemia/reperfusion on an extremity reduces infarct size and improves the prognosis of patients with reperfused myocardial infarction. The present review focuses on three levels of remote conditioning and its resulting cardioprotection: I) at the stimulus level, electrical stimulation, chemical/pharmacological substances, mechanical trauma and cycles of ischemia/reperfusion act at sites remote from the heart, II) at the transfer level, neuronal and humoral mediators transfer the protective signal from the periphery to the heart, and III) at the target level, receptor activation and intracellular signal transduction ultimately affect protection of the myocardium and other organs, as established in different animal models and humans/patients. Remote conditioning is obviously a systemic response. Further mechanistic understanding is mandatory to translate the protection by remote conditioning more successfully to patients with cardiovascular disease.

Impact of electrical defibrillation on infarct size and no-reflow in pigs subjected to myocardial ischemia-reperfusion without and with ischemic conditioning.

Ventricular fibrillation (VF) occurs frequently during myocardial ischemia-reperfusion (I/R) and must then be terminated by electrical defibrillation. We have investigated the impact of VF/defibrillation on infarct size (IS) or area of no reflow (NR) without and with ischemic conditioning interventions. Anesthetized pigs were subjected to 60/180 min of coronary occlusion/reperfusion. VF, as identified from the ECG, was terminated by intrathoracic defibrillation. The area at risk (AAR), IS, and NR were determined by staining techniques (patent blue, triphenyltetrazolium chloride, and thioflavin-S). Four experimental protocols were analyzed: I/R (n = 49), I/R with ischemic preconditioning (IPC; n = 22), I/R with ischemic postconditioning (POCO; n = 22), or I/R with remote IPC (RIPC; n = 34). The incidence of VF was not different between I/R (44%), IPC (45%), POCO (50%), and RIPC (33%). IS was reduced by IPC (23 ± 12% of AAR), POCO (31 ± 16%), and RIPC (22 ± 13%, all P < 0.05 vs. I/R: 41 ± 12%). NR was not different between protocols (I/R: 17 ± 15% of AAR, IPC: 15 ± 18%, POCO: 25 ± 16%, and RIPC: 18 ± 17%). In pigs with defibrillation, IS was 50% larger than in pigs without defibrillation but independent of the number of defibrillations. Analysis of covariance confirmed the established determinants of IS, i.e., AAR, residual blood flow during ischemia (RMBFi), and a conditioning protocol, and revealed VF/defibrillation as a novel covariate. VF/defibrillation in turn was associated with larger AAR and lower RMBFi. Lack of dose-response relation between IS and the number of defibrillations excluded direct electrical injury as the cause of increased IS. Obviously, AAR size and RMBFi account for both IS and the incidence of VF. IS and NR are mechanistically distinct phenomena.NEW & NOTEWORTHY Ventricular fibrillation/defibrillation is associated with increased infarct size. Electrical injury is unlikely the cause of such association, since there is no dose-response relation between infarct size and number of defibrillations. Ventricular fibrillation, in turn, is associated with a larger area at risk and lower residual blood flow.

lschemic preconditioning in pigs: a causal role for signal transducer and activator of transcription 3.

Ischemic preconditioning (IPC), i.e., brief episodes of nonlethal myocardial ischemia-reperfusion (I/R) before sustained ischemia with subsequent reperfusion, reduces infarct size in all species tested so far, including humans. In rodents, the cardioprotective signal transduction causally involves an activation of Akt, ERK1/2, and STAT3. However, there are apparent species differences in the signal transduction between rodents and larger mammals such as pigs, where data on IPC's signal transduction are inconsistent for Akt and ERK1/2. The role of STAT3 has not yet been analyzed. Pigs were subjected to 60 min of left anterior descending coronary artery occlusion and 180 min of reperfusion without or with IPC (2 cycles of 3-min occlusion separated by 2 min of reperfusion 15 min before sustained I/R). Infarct size was analyzed by triphenyl tetrazolium chloride staining, and Akt, ERK1/2, and STAT3 phosphorylation was quantified in myocardial biopsies taken at baseline and early reperfusion. AG490 was used to block the STAT3 signaling pathway. IPC reduced infarct size (%area at risk; mean ± SE, I/R, 45 ± 3 vs. IPC, 18 ± 3, P < 0.05). Akt and ERK1/2 phosphorylation was increased at early reperfusion without and with IPC. In contrast, STAT3 phosphorylation at early reperfusion was only increased with IPC (%baseline; mean ± SE, I/R, 126 ± 29 vs. IPC, 408 ± 147, P < 0.05). AG490 prevented the IPC-related increase of STAT3 phosphorylation at reperfusion (%baseline; mean ± SE, 82 ± 12) and abolished IPC's cardioprotection (%area at risk; mean ± SE, 35 ± 4). In pigs, increased phosphorylation of STAT3 is causally involved, whereas Akt and ERK1/2 seem to play no role in IPC's cardioprotection.NEW & NOTEWORTHY In pig hearts in situ, ischemic preconditioning (IPC) causally involves increased phosphorylation of STAT3, whereas Akt and ERK1/2 play no role for cardioprotection. The cardioprotective signal transduction of IPC is similar to that of ischemic postconditioning and remote IPC in pigs.

Across-Species Transfer of Protection by Remote Ischemic Preconditioning With Species-Specific Myocardial Signal Transduction by Reperfusion Injury Salvage Kinase and Survival Activating Factor Enhancement Pathways.

RATIONALE: Reduction of myocardial infarct size by remote ischemic preconditioning (RIPC), that is, cycles of ischemia/reperfusion in an organ remote from the heart before sustained myocardial ischemia/reperfusion, was confirmed in all species so far, including humans. OBJECTIVE: To identify myocardial signal transduction of cardioprotection by RIPC. METHODS AND RESULTS: Anesthetized pigs were subjected to RIPC (4×5/5 minutes hindlimb ischemia/reperfusion) or placebo (PLA) before 60/180 minutes coronary occlusion/reperfusion. Phosphorylation of protein kinase B, extracellular signal-regulated kinase 1/2 (reperfusion injury salvage kinase [RISK] pathway), and signal transducer and activator of transcription 3 (survival activating factor enhancement [SAFE] pathway) in the area at risk was determined by Western blot. Wortmannin/U0126 or AG490 was used for pharmacological RISK or SAFE blockade, respectively. Plasma sampled after RIPC or PLA, respectively, was transferred to isolated bioassay rat hearts subjected to 30/120 minutes global ischemia/reperfusion. RIPC reduced infarct size in pigs to 16±11% versus 43±11% in PLA (% area at risk; mean±SD; P<0.05). RIPC increased the phosphorylation of signal transducer and activator of transcription 3 at early reperfusion, and AG490 abolished the protection, whereas RISK blockade did not. Signal transducer and activator of transcription 5 phosphorylation was decreased at early reperfusion in both RIPC and PLA. In isolated rat hearts, pig plasma taken after RIPC reduced infarct size (25±5% of ventricular mass versus 38±5% in PLA; P<0.05) and activated both RISK and SAFE. RISK or SAFE blockade abrogated this protection. CONCLUSIONS: Cardioprotection by RIPC in pigs causally involves activation of signal transducer and activator of transcription 3 but not of RISK. Protection can be transferred with plasma from pigs to isolated rat hearts where activation of both RISK and SAFE is causally involved. The myocardial signal transduction of RIPC is the same as that of ischemic postconditioning.

microRNA expression and its potential role in cardioprotection by ischemic postconditioning in pigs.

Ischemic postconditioning (PoCo) reduces infarct size following myocardial ischemia/reperfusion. To protect, PoCo must be performed early during reperfusion, and causal cardioprotective signaling must occur then. The role of microRNA (miRNA) in PoCo is unclear. Anesthetized pigs were subjected to 60 min left anterior descending coronary artery (LAD) occlusion and 180 min reperfusion. Immediate full reperfusion (IFR, n = 5) was compared to PoCo (four cycles of 60 s/60 s reperfusion/reocclusion, n = 5). Transmural myocardial biopsies from the LAD territory were sampled at baseline, 60 min ischemia, 10 and 180 min reperfusion. RNA was isolated. The expression of 11 miRNAs, including muscle-specific (miRNA-1, -133a, -206, -208b, -214, and -499), fibrosis- (miRNA-21, -24, and -29b), neovascularization- (miRNA-92a), and inflammation-associated (miRNA-146b) candidates, was quantified using real-time PCR (RT-PCR). mRNA expression at baseline and 180 min reperfusion was quantified and validated (microarray and RT-PCR). PoCo reduced infarct size from 44.9 ± 7.7 to 34.8 ± 5.3% of the area at risk. The expression of miRNA-1, -24, -29b, -133a, -146b, -208b, and -499 was increased at 10 min reperfusion with PoCo vs. IFR; however, that of miRNA-1, -24, -208b, and -499 was already increased at 60 min ischemia and probably reflects falsely positive results. Five mRNAs were different with PoCo vs. IFR. In silico analysis identified a tentative connection between three miRNAs and five mRNAs with the biological functions "cell death", "inflammatory response" and/or "glucose metabolism". If at all, only miRNA-29b, -133a, and -146b fulfill the minimal temporal requirements for a potential causal involvement in cardioprotection by PoCo.

Coronary microembolization during early reperfusion: infarct extension, but protection by ischaemic postconditioning.

AIMS: Reperfusion injury following acute myocardial infarction impacts not only on the myocardium but also on the coronary microcirculation, and microembolization from the culprit lesion contributes to microvascular obstruction. Prior experimental studies have not accounted for microembolization in ischaemia/reperfusion injury and not considered microembolization as a confounder and target of protection by ischaemic postconditioning. We therefore investigated the impact of microembolization during reperfusion on infarct size and cardioprotection by postconditioning. METHODS AND RESULTS: Anaesthetized, open-chest pigs were subjected to 90 min low-flow ischaemia. Immediate full reperfusion (n = 8) served as the control. Microembolization was induced by intracoronary infusion of 42 µm microspheres with the onset of reperfusion (n = 8). In a second step, postconditioning was induced by six cycles of 20s reperfusion/20s re-occlusion without (n = 8) and with superimposed microembolization (n = 8). Transmural blood flow and area at risk were determined by radioactive microspheres, infarct size by triphenyl tetrazolium chloride staining. Area at risk and transmural blood flow were not different between groups. Microembolization increased infarct size from 32 ± 3% of the area at risk to 47 ± 3% (P < 0.05). Embolizing particles were re-distributed away from the central infarcted area and accumulated in the infarct border, thus contributing to infarct extension. Postconditioning reduced infarct size without (21 ± 3%; P < 0.05 vs. immediate full reperfusion) and also with additional microembolization (26 ± 5%; P < 0.05 vs. immediate full reperfusion and microembolization); embolizing particles did not accumulate in the infarct border. CONCLUSION: Microembolization at reperfusion augments infarct size, but postconditioning in the presence of microembolization still reduces infarct size and attenuates infarct expansion.