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

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.

Infarct size (IS) is the most robust end point for evaluating the success of preclinical studies on cardioprotection. The gold standard for IS quantification in ischemia/reperfusion (I/R) experiments is triphenyl tetrazolium chloride (TTC) staining, typically done manually. This study aimed to determine if automation through deep learning segmentation is a time-saving and valid alternative to standard IS quantification. High-resolution images from TTC-stained, macroscopic heart slices were retrospectively collected from pig experiments (n = 390) with I/R without/with cardioprotection to cover a wide IS range. Existing IS data from pig experiments, quantified using a standard method of manual and subsequent digital labeling of film-scan annotations, were used as reference. To automate the evaluation process with the aim to be more objective and save time, a deep learning pipeline was implemented; the collected images (n = 3869) were pre-processed by cropping and labeled (image annotations). To ensure their usability as training data for a deep learning segmentation model, IS was quantified from image annotations and compared to IS quantified using the existing film-scan annotations. A supervised deep learning segmentation model based on dynamic U-Net architecture was developed and trained. The evaluation of the trained model was performed by fivefold cross-validation (n = 220 experiments) and testing on an independent test set (n = 170 experiments). Performance metrics (Dice similarity coefficient [DSC], pixel accuracy [ACC], average precision [mAP]) were calculated. IS was then quantified from predictions and compared to IS quantified from image annotations (linear regression, Pearson’s r; analysis of covariance; Bland–Altman plots). Performance metrics near 1 indicated a strong model performance on cross-validated data (DSC: 0.90, ACC: 0.98, mAP: 0.90) and on the test set data (DSC: 0.89, ACC: 0.98, mAP: 0.93). IS quantified from predictions correlated well with IS quantified from image annotations in all data sets (cross-validation: r = 0.98; test data set: r = 0.95) and analysis of covariance identified no significant differences. The model reduced the IS quantification time per experiment from approximately 90 min to 20 s. The model was further tested on a preliminary test set from experiments in isolated, saline-perfused rat hearts with regional I/R without/with cardioprotection (n = 27). There was also no significant difference in IS between image annotations and predictions, but the performance on the test set data from rat hearts was lower (DSC: 0.66, ACC: 0.91, mAP: 0.65). IS quantification using a deep learning segmentation model is a valid and time-efficient alternative to manual and subsequent digital labeling.

Background: Vagal activation during remote ischemic conditioning (RIC) with subsequent release of cardioprotective factors from the spleen into the circulation reduces infarct size. These humoral cardioprotective factors can be transferred with plasma-dialysate from donors undergoing RIC to isolated recipient hearts where they reduce infarct size. Electrical auricular tragus stimulation (ATS) has been demonstrated to reduce the cardiac injury biomarker release and the frequency of ventricular arrhythmias and to improve contractile function in patients with acute myocardial infarction. Aim: To determine whether ATS, as RIC, induces release of cardioprotective factors into the circulation. Methods: Healthy volunteers (3 females, 7 males, 26±5 years) were randomized to receive ATS or RIC, respectively, with an interval of at least two weeks between protocols. ATS was induced by bipolar electrical rectangular pulses of 200μs width at a frequency of 30 Hz (Curamed TEN630, CURAmed, Shenzen Dongdixin Technology, Shenzhen, China). The amplitude (5.5 ± 0.81 mA) was set for each volunteer to not be perceived as uncomfortable over the total stimulation period. A microprocessor-controlled circuit periodically interrupted the stimulation signal after 5 s of stimulation for 5 s. ATS was continued over 30 min. RIC served as a reference to induce the release of cardioprotective factors by 3 x 5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/ 5 min deflation. Venous blood samples were taken before and 60 min after ATS or RIC, respectively, and used to prepare plasma-dialysates (1:10 dialysis against buffer for 24 h, cut-off 12-14 kDa). Male Lewis rats were sacrificed and their hearts isolated and perfused at constant pressure with buffer. Plasma-dialysates from samples taken before and 60 min after ATS or RIC, respectively, were infused for 8 min followed by 2 min washout into rat hearts before global 30 min ischemia/ 120 min reperfusion. Infarct size was demarcated by triphenyl tetrazolium chloride staining and calculated as percent of ventricular mass. Results: With infusion of plasma-dialysate before ATS infarct size was 30.9±4.5% of ventricular mass and not different from that with infusion of plasma-dialysate before RIC (35.8±5.8%). Infusion of plasma-dialysate after ATS reduced infarct size to 18.3±3.9%. This cardioprotective effect was similar to that seen with infusion of plasma-dialysate after RIC (20.1±3.5%). Conclusion: ATS induces release of cardioprotective factors into the circulation of healthy volunteers. Plasma-dialysates after ATS or RIC, respectively, reduce myocardial infarct size by the same magnitude in ex vivo isolated perfused rat hearts with global ischemia/ reperfusion. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

IntroductionDiazoxide 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 resultsIn 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%).ConclusionsCardioprotection 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.

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 K-ATP 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 Gottingen vs. Ossabaw minipigs in which IPC reduces infarct size. Methods and results: We analyzed surface chest electrocardiographic (ECG) recordings of anesthetized open-chest contemporary Gottingen (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 x 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 Gottingen 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 K-ATP channel activation still attenuates ST-segment elevation as it does in Gottingen 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.

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.

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.

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.

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.

IntroductionBrief cycles of ischemia/reperfusion (I/R) in a tissue/organ remote from the heart (remote ischemic conditioning, RIC) reduce myocardial I/R injury. Although operative in all species tested so far, including humans, the translation of RIC into clinical practice has been largely disappointing. Such failure in translation has in part been attributed to incomplete understanding of RIC’s signal transduction. RIC’s cardioprotection can be transferred with plasma between different individuals, even across species. Prior studies have suggested that the intrinsic nervous system is causally involved in RIC’s protection, since the ganglionic blocker hexamethonium attenuated the infarct size reduction in isolated rat hearts in response to plasma taken from different rats after RIC. However, recently a non‐neuronal cardiomyocyte‐based cholinergic system (NNCS) has been identified, and its involvement in the signal transduction of RIC is entirely unclear.We therefor aimed to study whether the NNCS is activated by humoral factor(s) released by RIC.MethodsAnesthetized open‐chest minipigs were subjected to 60/180 min occlusion/reperfusion of the left anterior descending coronary artery (n=7, placebo, PLA). RIC was induced by 4×5/5 min hindlimb I/R (n=8) 90 min before coronary occlusion. Arterial blood was sampled after PLA/RIC and plasma separated. Rat ventricular cardiomyocytes were isolated and incubated with PLA or RIC plasma‐dialysate (1:10; cut‐off <12–14 kDa) for 30 min ± hexamethonium (1 μmol/L) or ± atropine (100 nmol/L), respectively, subjected to 30/5 min hypoxia/reoxygenation (H/R) and compared to cardiomyocytes exposed to normoxic buffer (time control, TC) for 35 min. Cardiomyocyte viability was quantified before and after H/R or TC, respectively, as the percent fraction of rod‐shaped, unstained (trypan blue) cells over all cells. Diluted (1:10) PLA or RIC plasma was also infused into isolated perfused rat hearts for 8 min before 30/120 min global ischemia/reperfusion (GI/R) ± hexamethonium (50 μmol/L) or ± atropine (100 nmol/L), respectively. Infarct size (IS) was demarcated by triphenyltetrazolium chloride and calculated as percent of ventricular mass.ResultsIncubation with RIC plasma‐dialysate preserved cardiomyocyte viability after H/R in comparison to PLA plasma‐dialysate. This protection was attenuated by hexamethonium and by atropine. Hexamethonium or atropine had no impact on cardiomyocyte viability per se (Figure A). In isolated hearts, IS was less with infusion of plasma from pigs with RIC than with that from pigs with PLA. With infusion of PLA plasma, hexamethonium or atropine did not impact on IS per se. The protective effect of RIC plasma, however, was abrogated by hexamethonium and by atropine (Figure B).ConclusionRIC’s humoral transfer of cardioprotection is mediated via an activation of cholinergic receptors on cardiomyocytes and attenuated to the same extent by hexamethonium or by atropine. Whether or not hexamethonium also and additionally attenuates the cardioprotection of RIC at the ganglionic level therefore remains unclear.Support or Funding InformationSupported by the German Research Foundation (SFB 1116 B8)Figure legend: A) Viability of rat ventricular cardiomyocytes incubated with placebo (PLA)/remote ischemic conditioning (RIC) plasma‐dialysate after hypoxia (H)/reoxygenation(R), or after normoxic time control (TC). Cardiomyocytes with plasma‐dialysate were incubated with saline, ± hexamethonium (hexa), or ± atropine. Data are means ± standard deviations. †p<0.05 vs. PLA plasma‐dialysate; ‡p<0.05 vs. saline RIC plasma‐dialysate; two‐way ANOVA with Fisher’s least significant differences post‐hoc tests. B) Infarct size in isolated perfused rat hearts (± hexa or ± atropine) with infusion of PLA or RIC plasma and after global ischemia/reperfusion. Data are means ± standard deviations. #p<0.05 vs. PLA plasma; *p<0.05 vs. saline RIC plasma; two‐way ANOVA with Fisher’s least significant differences post‐hoc tests.Figure 1

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.

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 (AAR(S)) or the anterior free wall (AAR(AFW)). Relative IS was calculated for AAR(S) or AAR(AFW). AAR(AFW) was larger than AAR(S) (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 AAR(S) than in AAR(AFW) (50 +/- 17% vs. 44 +/- 19%; % of AAR(AWF) or AAR(S), respectively; P = 0.075). Also, with robust IS reduction by ischemic conditioning, the difference in relative IS remained (AAR(S): 27 +/- 16%; AAR(AFW): 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.

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.

Cardioprotection by ischemic conditioning maneuvers, either locally in the heart or remotely from the heart, reduces infarct size. However, infarct size reduction is seen only hours to days after the acute event. To study the potential of ST‐segment elevation (STE) and its attenuation to reflect infarct size reduction already during ongoing coronary occlusion or early during reperfusion, pigs were subjected to 1 h LAD occlusion and 3 h reperfusion. Cardioprotection was recruited prior to the index ischemia by local ischemic preconditioning (IPC; n=14; 2x3 min LAD occlusion, interspersed by 2 min reperfusion, 15 min prior to ischemia) or remote ischemic preconditioning (RIPC; n=20; 4x5 min / 5 min hindlimb ischemia/reperfusion, 90 min prior to ischemia), during the index ischemia by remote ischemic perconditioning1 (RPER; n=18; 4x5 min / 5 min hindlimb ischemia/reperfusion, starting 20 min after the onset of ischemia), or at the onset of reperfusion by ischemic postconditioning (PoCo; n=9; 4x1 min / 1 min coronary reocclusion/reperfusion at 1 min reperfusion). Pigs without ischemic conditioning served as controls (PLA; n=29). Transmural myocardial blood flow (microspheres), area at risk (blue dye), and infarct size (TTC) were measured. STE amplitude was defined as the voltage difference in a V2‐like ECG lead between a point 30 ms before the P‐wave and one 20 ms after the J‐point.Area at risk and transmural myocardial blood flow during ischemia were similar among groups, and the cardioprotective maneuvers robustly reduced infarct size (PLA 42±11% of area at risk; mean±SD; IPC 19±10%; RIPC 23±12%; RPER 23±12%; PoCo 22±11%; all p<0.05 vs. PLA). With PLA, STE was markedly increased at 5 min ischemia and this elevation was sustained throughout ischemia. At 10 min reperfusion, STE increased even further, possibly reflecting additional reperfusion injury, followed by a gradual, but incomplete STE recovery over 120 min reperfusion. RIPC and IPC did not impact on STE at 5 min ischemia, but STE was attenuated at 55 min ischemia. With RPER, STE was attenuated immediately after completion of the maneuver at 55 min ischemia. PoCo abolished the further increase of STE at 10 min reperfusion. (see figure)The infarct size reduction by IPC, RIPC, RPER, or PoCo is robustly reflected in an attenuation of STE. Whereas PoCo and RPER attenuate STE within minutes after completion of the maneuver, the two cardioprotective maneuvers which were performed prior to myocardial ischemia, i.e. IPC and RIPC, did not attenuate STE immediately at 5 min ischemia, but did so at 55 min ischemia. A speculative explanation for such discrepancy is that STE attenuation only reflects protection from irreversible ischemic or reperfusion injury. In any event, STE analysis provides an on‐line estimate of cardioprotection much earlier than a measurement of infarct size.Support or Funding InformationSupported by: DFG (SFB 1116 B08)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Introduction: Short cycles of ischemia/reperfusion (I/R) in a tissue/organ remote from the heart reduce myocardial I/R injury. Such remote ischemic conditioning (RIC) is operative in all species te...

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/β/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.

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.