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.

Activation of the nonneuronal cholinergic cardiac system by hypoxic preconditioning protects isolated adult cardiomyocytes from hypoxia/reoxygenation injury.

Activation of the vagus nerve mediates cardioprotection and attenuates myocardial ischemia/reperfusion (I/R) injury. In response to vagal activation, acetylcholine (ACh) is released from the intracardiac nervous system (ICNS) and activates intracellular cardioprotective signaling cascades. Recently, however, a nonneuronal cholinergic cardiac system (NNCCS) in cardiomyocytes has been described as an additional source of ACh. To investigate whether the NNCCS mediates cardioprotection in the absence of vagal and ICNS activation, we used a reductionist approach of isolated adult rat ventricular cardiomyocytes without neuronal cells, using hypoxic preconditioning (HPC) as a protective stimulus. Adult rat ventricular cardiomyocytes were isolated, the absence of neuronal cells was confirmed, and HPC was induced by 10/20 min hypoxia/reoxygenation (H/R) before subjection to 30/5 min H/R to simulate I/R injury. Cardiomyocyte viability was assessed by trypan blue staining at baseline and after HPC+H/R or H/R. Intra- and extracellular ACh was quantified using liquid chromatography-coupled mass spectrometry at baseline, after HPC, after hypoxia, and after reoxygenation, respectively. In a subset of experiments, muscarinic and nicotinic ACh receptor (m- and nAChR) antagonists were added during HPC or during H/R. Cardiomyocyte viability at baseline (69 ± 4%) was reduced by H/R (10 ± 3%). With HPC, cardiomyocyte viability was preserved after H/R (25 ± 6%). Intra- and extracellular ACh increased during hypoxia; HPC further increased both intra- and extracellular ACh (from 0.9 ± 0.7 to 1.5 ± 1.0 nmol/mg; from 0.7 ± 0.6 to 1.1 ± 0.7 nmol/mg, respectively). The addition of mAChR and nAChR antagonists during HPC had no impact on HPC's protection; however, protection was abrogated when antagonists were added during H/R (cardiomyocyte viability after H/R: 23 ± 5%; 13 ± 4%). In conclusion, activation of the NNCCS is involved in cardiomyocyte protection; HPC increases intra- and extracellular ACh during H/R, and m- and nAChRs are causally involved in HPC's cardiomyocyte protection during H/R. The interplay between upstream ICNS activation and NNCCS activation in myocardial cholinergic metabolism and cardioprotection needs to be investigated in future studies.NEW & NOTEWORTHY The intracardiac nervous system is considered to be involved in ischemic conditioning's cardioprotection through the release of acetylcholine (ACh). However, we demonstrate that hypoxic preconditioning (HPC) protects from hypoxia/reoxygenation injury and increases intra- and extracellular ACh during hypoxia in isolated adult ventricular rat cardiomyocytes. HPC's protection involves cardiomyocyte muscarinic and nicotinic ACh receptor activation. Thus, besides the intracardiac nervous system, a nonneuronal cholinergic cardiac system may also be causally involved in cardiomyocyte protection by ischemic conditioning.

"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.

Mitochondrial Kinase Signaling for Cardioprotection.

Myocardial ischemia/reperfusion injury is reduced by cardioprotective adaptations such as local or remote ischemic conditioning. The cardioprotective stimuli activate signaling cascades, which converge on mitochondria and maintain the function of the organelles, which is critical for cell survival. The signaling cascades include not only extracellular molecules that activate sarcolemmal receptor-dependent or -independent protein kinases that signal at the plasma membrane or in the cytosol, but also involve kinases, which are located to or within mitochondria, phosphorylate mitochondrial target proteins, and thereby modify, e.g., respiration, the generation of reactive oxygen species, calcium handling, mitochondrial dynamics, mitophagy, or apoptosis. In the present review, we give a personal and opinionated overview of selected protein kinases, localized to/within myocardial mitochondria, and summarize the available data on their role in myocardial ischemia/reperfusion injury and protection from it. We highlight the regulation of mitochondrial function by these mitochondrial protein kinases.

Is There a Mitochondrial Protection via Remote Ischemic Conditioning in Settings of Anticancer Therapy Cardiotoxicity?

PURPOSE OF REVIEW: To provide an overview of (a) protective effects on mitochondria induced by remote ischemic conditioning (RIC) and (b) mitochondrial damage caused by anticancer therapy. We then discuss the available results of studies on mitochondrial protection via RIC in anticancer therapy-induced cardiotoxicity. RECENT FINDINGS: In three experimental studies in healthy mice and pigs, there was a RIC-mediated protection against anthracycline-induced cardiotoxicity and there was some evidence of improved mitochondrial function with RIC. The RIC-mediated protection was not confirmed in the two available studies in cancer patients. In adult cancer patients, RIC was associated with an adverse outcome. There are no data on mitochondrial function in cancer patients. Studies in tumor-bearing animals are needed to determine whether RIC does not interfere with the anticancer properties of the drugs and whether RIC actually improves mitochondrial function, ultimately resulting in improved cardiac function.

Differences in vasomotor function of mesenteric arteries between Ossabaw minipigs with predisposition to metabolic syndrome and Göttingen minipigs.

Metabolic syndrome predisposes and contributes to the development and progression of atherosclerosis. The minipig strain "Ossabaw" is characterized by a predisposition to develop metabolic syndrome. We compared vasomotor function in Ossabaw minipigs before they developed their diseased phenotype to that of Göttingen minipigs without such genetic predisposition. Mesenteric arteries of adult Ossabaw and Göttingen minipigs were dissected postmortem and mounted on a myograph for isometric force measurements. Maximal vasoconstriction to potassium chloride (KClmax) was induced. Cumulative concentration-response curves were determined in response to norepinephrine. Endothelium-dependent (with carbachol) and endothelium-independent (with nitroprusside) vasodilation were analyzed after preconstriction by norepinephrine. In a bioinformatic analysis, variants/altered base pairs within genes associated with cardiovascular disease were analyzed. KClmax was similar between the minipig strains (15.6 ± 6.7 vs. 14.1 ± 3.4 ΔmN). Vasoconstriction in response to norepinephrine was more pronounced in Ossabaw than in Göttingen minipigs (increase of force to 143 ± 48 vs. 108 ± 38% of KClmax). Endothelium-dependent and endothelium-independent vasodilation were less pronounced in Ossabaw than in Göttingen minipigs (decrease of force to 46.4 ± 29.6 vs. 16.0 ± 18.4% and to 36.7 ± 25.2 vs. 2.3 ± 3.7% of norepinephrine-induced preconstriction). Vasomotor function was not different between the sexes. More altered base pairs/variants were identified in Ossabaw than in Göttingen minipigs for the exon encoding adrenoceptor-α1A. Vasomotor function in lean Ossabaw minipigs is shifted toward vasoconstriction and away from vasodilation in comparison with Göttingen minipigs, suggesting a genetic predisposition for vascular dysfunction and atherosclerosis in Ossabaw minipigs. Thus, Ossabaw minipigs may be a better model for human cardiovascular disease than Göttingen minipigs.NEW & NOTEWORTHY Animal models with a predisposition to metabolic syndrome and atherosclerosis are attracting growing interest for translational research, as they may better mimic the variability of patients with cardiovascular disease. In Ossabaw minipigs, with a polygenic predisposition to metabolic syndrome, but without the diseased phenotype, vasoconstriction is more and vasodilation is less pronounced in mesenteric arteries than in Göttingen minipigs. Ossabaw minipigs may be a more suitable model of human cardiovascular disease.

Perspective: mitochondrial STAT3 in cardioprotection.

Activation of signal transducer and activator of transcription 3 (STAT3) has been identified as a key cardioprotective signal not only in animal studies but also in humans-in animals, STAT3 is causally involved in cardioprotection. In response to late ischemic conditioning, canonical function of STAT3 activation upregulates the expression of cardioprotective and anti-apoptotic proteins. In its non-canonical function, STAT3 is activated during ischemic conditioning and is part of the cardioprotective cytosolic survival activating factor enhancement pathway. Activated STAT3 is imported and localized to the mitochondria. Mitochondrial STAT3 stimulates the activity of mitochondrial electron transport chain complex I, reduces mitochondrial reactive oxygen species production and mitochondrial permeability transition pore opening. Finally, two novel aspects of STAT activation in cardioprotection are discussed: a genetic variance of the STAT encoding region as a potential primordial confounding variable for cardioprotection, and the cardioprotective potential of sodium-glucose cotransporter 2 inhibitors through STAT3 activation.

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.

Mitochondrial respiration analysis in permeabilized porcine left ventricular and human right atrial specimens with ischemia-reperfusion.

Mitochondrial function is critical to myocardial ischemia-reperfusion injury and cardioprotection. The measurement of mitochondrial function in isolated mitochondria requires cardiac specimens of about 300 mg and is therefore only possible at the end of an animal experiment or during cardiosurgical interventions in humans. As an alternative, mitochondrial function can be measured in permeabilized myocardial tissue (PMT) specimens of about 2-5 mg, which are retrieved by sequential biopsies in animal experiments and during cardiac catheterization in humans. We attempted to validate measurements of mitochondrial respiration from PMT by comparison with those from isolated mitochondria of left ventricular myocardium from anesthetized pigs undergoing 60 min coronary occlusion and 180 min reperfusion. Mitochondrial respiration was normalized to the content of mitochondrial marker proteins [cytochrome-c oxidase 4 (COX4), citrate synthase, and manganese-dependent superoxide dismutase]. When normalized to COX4, mitochondrial respiration measurements in PMT and isolated mitochondria agreed well in Bland-Altman plots (bias score, -0.03 nmol/min/COX4; 95% confidence interval: 6.31 nmol/min/COX4 and -6.37 nmol/min/COX4) and correlated well (slope of 0.77 and Pearson's R of 0.87). Mitochondrial dysfunction by ischemia-reperfusion was equally reflected in PMT and isolated mitochondria (44 and 48% reduction of ADP-stimulated complex I respiration). Also in isolated human right atrial trabeculae, simulation of ischemia-reperfusion injury by exposure to 60 min hypoxia and 10 min reoxygenation reduced mitochondrial ADP-stimulated complex I respiration by 37% in PMT. In conclusion, mitochondrial function measurements in permeabilized cardiac tissue can substitute for that in isolated mitochondria to reflect mitochondrial dysfunction following ischemia-reperfusion.NEW & NOTEWORTHY Methods to quantify mitochondrial function in translationally relevant models and in human tissue are needed to improve the translation of cardioprotection to patients. Our present approach, using PMT instead of isolated mitochondria for the quantification of mitochondrial ischemia-reperfusion damage, provides a reference for further studies in translationally relevant large animal models and in human tissue, thus possibly improving the translation of cardioprotection to the benefit of patients with acute myocardial infarction.

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.

Platelet-Mediated Transfer of Cardioprotection by Remote Ischemic Conditioning and Its Abrogation by Aspirin But Not by Ticagrelor.

PURPOSE: The role of platelets during myocardial ischemia/reperfusion (I/R) is ambivalent. They contribute to injury but also to cardioprotection. Repeated blood flow restriction and reperfusion in a tissue/organ remote from the heart (remote ischemic conditioning, RIC) reduce myocardial I/R injury and attenuate platelet activation. Whether or not platelets mediate RIC's cardioprotective signal is currently unclear. METHODS AND RESULTS: Venous blood from healthy volunteers (without or with pretreatment of 500/1000 mg aspirin or 180 mg ticagrelor orally, 2-3 h before the study, n = 18 each) was collected before and after RIC (3 × 5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/5 min deflation). Washed platelets were isolated. Platelet-poor plasma was used to prepare plasma-dialysates. Platelets (25 × 103/µL) or plasma-dialysates (1:10) prepared before and after RIC from untreated versus aspirin- or ticagrelor-pretreated volunteers, respectively, were infused into isolated buffer-perfused rat hearts. Hearts were subjected to global 30 min/120 min I/R. Infarct size was stained. Infarct size was less with infusion of platelets/plasma-dialysate after RIC (18 ± 7%/23 ± 9% of ventricular mass) than with platelets/plasma-dialysate before RIC (34 ± 7%/33 ± 8%). Aspirin pretreatment abrogated the transfer of RIC's cardioprotection by platelets (after/before RIC, 34 ± 7%/33 ± 7%) but only attenuated that by plasma-dialysate (after/before RIC, 26 ± 8%/32 ± 5%). Ticagrelor pretreatment induced an in vivo formation of cardioprotective factor(s) per se (platelets/plasma-dialysate before RIC, 26 ± 7%/26 ± 7%) but did not impact on RIC's cardioprotection by platelets/plasma-dialysate (20 ± 7%/21 ± 5%). CONCLUSION: Platelets serve as carriers for RIC's cardioprotective signal through an aspirin-sensitive and thus cyclooxygenase-dependent mechanism. The P2Y12 inhibitor ticagrelor per se induces a humoral cardioprotective signal.