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.

Diazoxide is a powerful cardioprotectant but is not feasible in a realistic infarct scenario.

Introduction: Diazoxide is a powerful cardioprotective agent that activates mitochondrial ATP-dependent K-channels and stimulates mitochondrial respiration. Diazoxide reduced infarct size in isolated rodent heart preparations and upon pretreatment in juvenile pigs with coronary occlusion/reperfusion. We aimed to study the use of diazoxide in a more realistic adult pig model of reperfused acute myocardial infarction when diazoxide was administered just before reperfusion. Methods and results: In a first approach, we pretreated anaesthetised adult Göttingen minipigs with 7 mg kg-1 diazoxide (n = 5) or placebo (n = 5) intravenously over 10 min and subjected them to 60 min coronary occlusion and 180 min reperfusion; blood pressure was maintained by use of an aortic snare. The primary endpoint was infarct size (triphenyl tetrazolium chloride staining) as a fraction of area at risk; no-reflow area (thioflavin-S staining) was the secondary endpoint. In a second approach, diazoxide (n = 5) was given from 50 to 60 min coronary occlusion, and blood pressure was not maintained. There was a significant reduction in infarct size (22% ± 11% of area at risk with diazoxide pretreatment vs. 47% ± 11% with placebo) and area of no-reflow (14% ± 14% of infarct size with diazoxide pretreatment vs. 46% ± 20% with placebo). With diazoxide from 50 to 60 min coronary occlusion, however, there was marked hypotension, and infarct size (44% ± 7%) and area of no-reflow were not reduced (35% ± 25%). Conclusions: Cardioprotection by diazoxide pretreatment was confirmed in adult pigs with reperfused acute myocardial infarction but is not feasible when diazoxide is administered in a more realistic scenario before reperfusion and causes hypotension.

Attenuation of ST-segment elevation by ischemic preconditioning: Reflection of cardioprotection in Göttingen but not in Ossabaw minipigs.

BACKGROUND: Ischemic preconditioning (IPC; brief cycles of coronary occlusion/ reperfusion) reduces myocardial infarct size. The ST-segment elevation during coronary occlusion is progressively attenuated with increasing number of IPC cycles. Progressive attenuation of ST-segment elevation is considered a result of sarcolemmal KATP channel activation and has been considered to reflect and predict IPC's cardioprotection. We have recently demonstrated that IPC failed to reduce infarct size in minipigs of a particular strain (Ossabaw), which had a genetic predisposition to develop, but not yet established a metabolic syndrome. To determine whether or not Ossabaw minipigs nevertheless had attenuated ST-segment elevation over repetitive IPC cycles, we compared Göttingen vs. Ossabaw minipigs in which IPC reduces infarct size. METHODS AND RESULTS: We analyzed surface chest electrocardiographic (ECG) recordings of anesthetized open-chest contemporary Göttingen (n = 43) and Ossabaw minipigs (n = 53). Both minipig strains were subjected to 60 min coronary occlusion and 180 min reperfusion without or with IPC (3 × 5 min/ 10 min coronary occlusion/ reperfusion). ST-segment elevations during the repetitive coronary occlusions were analyzed. In both minipig strains, IPC attenuated ST-segment elevation with increasing number of coronary occlusions. IPC reduced infarct size in Göttingen minipigs (45 ± 10% without vs. 25 ± 13% of area at risk with IPC), whereas such cardioprotection was absent in Ossabaw minipigs (54 ± 11% vs. 50 ± 11%). CONCLUSION: Apparently, the block of signal transduction of IPC in Ossabaw minipigs occurs distal to the sarcolemma, where KATP channel activation still attenuates ST-segment elevation as it does in Göttingen minipigs.

No sex-related differences in infarct size, no-reflow, and protection by ischaemic pre-conditioning in Göttingen minipigs.

AIMS: Female sex has been proposed to be cardioprotective per se. Studies with myocardial ischaemia/reperfusion and infarct size as endpoint have demonstrated cardioprotection in female, castrated male, and male pigs. These studies are difficult to compare, given the different pig strains, models, durations of ischaemia, and methods of infarct size quantification. The few studies using both female and male pigs reported no differences in infarct size and cardioprotection. We, therefore, prospectively compared infarct size in Göttingen minipigs undergoing ischaemia/reperfusion (I/R) without and with ischaemic pre-conditioning (IPC) between female, castrated male, and male pigs. METHODS AND RESULTS: In a prospective, randomized approach, 28 Göttingen open-chest, anaesthetized minipigs underwent 60 min ischaemia by distal left anterior descending artery (LAD) occlusion and 180 min reperfusion without and with IPC by three cycles of 5 min LAD occlusion/10 min reperfusion. Infarct size with I/R was not different between female, castrated male, and male pigs (45 ± 8 vs. 45 ± 13 vs. 41 ± 9% area at risk), as was the reduction in infarct size with IPC (25 ± 11 vs. 30 ± 8 vs. 19 ± 10% area at risk). In addition, the area of no-reflow was not different between female, castrated male, and male pigs with I/R (57 ± 13 vs. 35 ± 7 vs. 47 ± 26% infarct size) or IPC (4 ± 10 vs.12 ± 20 vs. 0 ± 0% infarct size). Phosphorylation of signal transducer and activator of transcription 3 was increased at 10 min reperfusion by IPC but not by I/R to the same extent in female, castrated male, and male pigs (198 ± 30 vs. 230 ± 165 vs. 179 ± 107% of baseline). CONCLUSION: Our data do not support the notion of sex- or castration-related differences in infarct size, coronary microvascular injury, and cardioprotection by IPC. TRANSLATIONAL PERSPECTIVE: The translation of successful preclinical studies on cardioprotection to the benefit of patients with reperfused myocardial infarction has been difficult. The difficulties have been attributed to confounders such as co-morbidities and co-medications which patients typically have but animals don´t, but also to age and sex. Notably, female sex has been considered as protective per se. We have now, using our established and clinically relevant pig model of reperfused acute myocardial infarction and ischaemic preconditioning as the most robust cardioprotective intervention looked for sex-related differences of infarct size, no-reflow and cardioprotection by ischaemic preconditioning in a prospectively powered approach but found none such difference.

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.

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.

Larger infarct size but equal protection by ischemic conditioning in septum and anterior free wall of pigs with LAD occlusion.

The ischemic area at risk (AAR) is one major determinant of infarct size (IS). In patients, the largest AAR is seen with a proximal occlusion of the left anterior descending (LAD) coronary artery, which serves parts of the septum and of the anterior free wall. It is not clear, whether regional differences in the perfusion territories also impact on IS and the magnitude of cardioprotection by ischemic conditioning. We have retrospectively analyzed 132 experiments in pigs, which have a similar LAD perfusion territory as humans. The LAD was occluded for 60 min with subsequent 180 min reperfusion. Cardioprotection by either local ischemic pre- or postconditioning or remote ischemic pre- or perconditioning was induced in 93 pigs. The AAR was demarcated by blue dye staining, and IS was assessed by triphenyltetrazolium chloride (TTC) staining. Using digital planimetry, the AAR was separated into sections unequivocally located in the septum (AARS ) or the anterior free wall (AARAFW ). Relative IS was calculated for AARS or AARAFW . AARAFW was larger than AARS (51 ± 9% vs. 34 ± 8% of total AAR; mean ± SD, P < 0.001). Regional myocardial blood flow (microspheres) was not different between septum and anterior free wall. IS without ischemic conditioning tended to be larger in AARS than in AARAFW (50 ± 17% vs. 44 ± 19%; % of AARAWF or AARS , respectively; P = 0.075). Also, with robust IS reduction by ischemic conditioning, the difference in relative IS remained (AARS : 27 ± 16%; AARAFW : 21 ± 16%; P = 0.01). There is a somewhat greater susceptibility for infarction in septal than anterior free wall myocardium. However, ischemic conditioning still reduces IS in both septal and anterior free wall myocardium.

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.

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.

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.

Coronary microembolization and microvascular dysfunction.

Plaque erosion, fissuring or rupture occurs spontaneously or during coronary interventions. At some residual blood flow, the atherothrombotic debris is washed into the coronary microcirculation, causing physical obstruction, vasoconstriction, inflammation and ultimately microinfarction. Coronary microembolization also contributes to microvascular obstruction in reperfused acute myocardial infarction. Patients with microvascular obstruction after reperfused myocardial infarction have worse prognosis. Cardioprotective strategies to avoid acute coronary microembolization and rescue myocardium from microvascular obstruction have not yet been established in clinical practice. Subclinical coronary microembolization together with release of thrombogenic, vasoconstrictor and inflammatory substances from a culprit lesion can sensitize the coronary microcirculation and contribute to angina in the absence of major epicardial coronary obstruction. Repetitive coronary microembolization can induce progressive loss of functional cardiomyocytes and induce heart failure in the absence of overt myocardial infarction.

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.

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.

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.