Interaction of Cardiovascular Nonmodifiable Risk Factors, Comorbidities and Comedications With Ischemia/Reperfusion Injury and Cardioprotection by Pharmacological Treatments and Ischemic Conditioning.

Preconditioning, postconditioning, and remote conditioning of the myocardium enhance the ability of the heart to withstand a prolonged ischemia/reperfusion insult and the potential to provide novel therapeutic paradigms for cardioprotection. While many signaling pathways leading to endogenous cardioprotection have been elucidated in experimental studies over the past 30 years, no cardioprotective drug is on the market yet for that indication. One likely major reason for this failure to translate cardioprotection into patient benefit is the lack of rigorous and systematic preclinical evaluation of promising cardioprotective therapies prior to their clinical evaluation, since ischemic heart disease in humans is a complex disorder caused by or associated with cardiovascular risk factors and comorbidities. These risk factors and comorbidities induce fundamental alterations in cellular signaling cascades that affect the development of ischemia/reperfusion injury and responses to cardioprotective interventions. Moreover, some of the medications used to treat these comorbidities may impact on cardioprotection by again modifying cellular signaling pathways. The aim of this article is to review the recent evidence that cardiovascular risk factors as well as comorbidities and their medications may modify the response to cardioprotective interventions. We emphasize the critical need for taking into account the presence of cardiovascular risk factors as well as comorbidities and their concomitant medications when designing preclinical studies for the identification and validation of cardioprotective drug targets and clinical studies. This will hopefully maximize the success rate of developing rational approaches to effective cardioprotective therapies for the majority of patients with multiple comorbidities. SIGNIFICANCE STATEMENT: Ischemic heart disease is a major cause of mortality; however, there are still no cardioprotective drugs on the market. Most studies on cardioprotection have been undertaken in animal models of ischemia/reperfusion in the absence of comorbidities; however, ischemic heart disease develops with other systemic disorders (e.g., hypertension, hyperlipidemia, diabetes, atherosclerosis). Here we focus on the preclinical and clinical evidence showing how these comorbidities and their routine medications affect ischemia/reperfusion injury and interfere with cardioprotective strategies.

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.

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.

"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 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.

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.

PCSK9 Activity Is Potentiated Through HDL Binding.

Rationale: Proprotein convertase subtilisin/kexin type 9 (PCSK9) circulates in a free and lipoprotein-bound form, yet the functional consequence of the association between PCSK9 and high-density lipoprotein (HDL) remains unexplored. Objective: This study sought to interrogate the novel relationship between PCSK9 and HDL in humans. Methods and Results: Comparing lipoprotein and apolipoprotein profiles by nuclear magnetic resonance and targeted mass spectrometry measurements with PCSK9 levels in the community-based Bruneck (n=656) study revealed a positive association of plasma PCSK9 with small HDL, alongside a highly significant positive correlation between plasma levels of PCSK9 and apolipoprotein-C3, an inhibitor of lipoprotein lipase. The latter association was replicated in an independent cohort, the SAPHIR study (n=270). Thus, PCSK9-HDL association was determined during the postprandial response in two dietary studies (n=20 participants each, 8 times points). Peak triglyceride levels coincided with an attenuation of the PCSK9-HDL association, a loss of apolipoprotein-C3 from HDL and lower levels of small HDL as measured by nuclear magnetic resonance. Crosslinking mass spectrometry (XLMS) upon isolated HDL identified PCSK9 as a potential HDL-binding partner. PCSK9 association with HDL was confirmed through size-exclusion chromatography and immuno-isolation. Quantitative proteomics upon HDL isolated from patients with coronary artery disease (n=172) returned PCSK9 as a core member of the HDL proteome. Combined interrogation of the HDL proteome and lipidome revealed a distinct cluster of PCSK9, phospholipid transfer protein, clusterin and apolipoprotein-E within the HDL proteome, that was altered by sex and positively correlated with sphingomyelin content. Mechanistically, HDL facilitated PCSK9-mediated low-density lipoprotein receptor degradation and reduced low-density lipoprotein uptake through the modulation of PCSK9 internalisation and multimerisation. Conclusions: This study reports HDL as a binder of PCSK9 and regulator of its function. The combination of -omic technologies revealed postprandial lipaemia as a driver of PCSK9 and apolipoprotein-C3 release from HDL.

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.

Mitochondrial Telomerase Reverse Transcriptase Protects From Myocardial Ischemia/Reperfusion Injury by Improving Complex I Composition and Function.

BACKGROUND: The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), has protective functions in the cardiovascular system. TERT is not only present in the nucleus but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection, and the appropriate tools are missing to dissect this. METHODS: We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart, and cellular functions of cardiomyocytes, fibroblasts, and endothelial cells, as well, were determined. RESULTS: All mice were phenotypically normal. Although respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wild-type mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after 1, 2, and 4 weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. In endothelial cells, mitochondrial TERT enhanced migratory capacity and activation of endothelial nitric oxide synthase. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits, explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning. The telomerase activator TA-65 evoked a similar effect in endothelial cells, thereby increasing their migratory capacity, and enhanced myofibroblast differentiation. CONCLUSIONS: Mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. TA-65 has comparable effects ex vivo and improves the migratory capacity of endothelial cells and myofibroblast differentiation. We conclude that mitochondrial TERT is responsible for cardioprotection, and its increase could serve as a therapeutic strategy.

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.

Coronary blood flow in heart failure: cause, consequence and bystander.

Heart failure is a clinical syndrome where cardiac output is not sufficient to sustain adequate perfusion and normal bodily functions, initially during exercise and in more severe forms also at rest. The two most frequent forms are heart failure of ischemic origin and of non-ischemic origin. In heart failure of ischemic origin, reduced coronary blood flow is causal to cardiac contractile dysfunction, and this is true for stunned and hibernating myocardium, coronary microembolization, myocardial infarction and post-infarct remodeling, possibly also for the takotsubo syndrome. The most frequent form of non-ischemic heart failure is dilated cardiomyopathy, caused by genetic mutations, myocarditis, toxic agents or sustained tachyarrhythmias, where alterations in coronary blood flow result from and contribute to cardiac contractile dysfunction. Hypertrophic cardiomyopathy is caused by genetic mutations but can also result from increased pressure and volume overload (hypertension, valve disease). Heart failure with preserved ejection fraction is characterized by pronounced coronary microvascular dysfunction, the causal contribution of which is however not clear. The present review characterizes the alterations of coronary blood flow which are causes or consequences of heart failure in its different manifestations. Apart from any potentially accompanying coronary atherosclerosis, all heart failure entities share common features of impaired coronary blood flow, but to a different extent: enhanced extravascular compression, impaired nitric oxide-mediated, endothelium-dependent vasodilation and enhanced vasoconstriction to mediators of neurohumoral activation. Impaired coronary blood flow contributes to the progression of heart failure and is thus a valid target for established and novel treatment regimens.

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.

Cardioprotection by post-conditioning with exogenous triiodothyronine in isolated perfused rat hearts and isolated adult rat cardiomyocytes.

Ischemic post-conditioning (iPoCo) by coronary re-occlusion/reperfusion during immediate reperfusion after prolonged myocardial ischemia reduces infarct size. Mechanical manipulation of culprit lesions, however, carries the risk of coronary microembolization which may obscure iPoCo's cardioprotection. Pharmacological post-conditioning with exogenous triiodothyronine (T3) could serve as an alternative conditioning strategy. Similar to iPoCo, T3 may activate cardioprotective prosurvival pathways. We aimed to study T3's impact on infarct size and its underlying signal transduction. Hearts were isolated from male Lewis rats (200-380 g), buffer-perfused and subjected to 30 min/120 min global zero-flow ischemia/reperfusion (I/R). In additional hearts, either iPoCo (2 × 30 s/30 s I/R) was performed or T3 (100-500 µg/L) infused at reperfusion. Infarct size was demarcated with triphenyl tetrazolium chloride staining and calculated as percent of ventricular mass. Infarct size was reduced with iPoCo to 16 ± 7% vs. 36 ± 4% with I/R only. The maximum infarct size reduction was observed with 300 µg/L T3 (14 ± 2%). T3 increased the phosphorylation of protein kinase B and mitogen extracellular-regulated-kinase 1/2, both key enzymes of the reperfusion injury salvage kinase (RISK) pathway. Pharmacological RISK blockade (RISK-BL) during reperfusion abrogated T3's cardioprotection (35 ± 10%). Adult ventricular cardiomyocytes were isolated from buffer-perfused rat hearts and exposed to 30 min/5 min hypoxia/reoxygenation (H/R); reoxygenation was initiated without or with T3, respectively, and without or with RISK-BL, respectively. Maximal preservation of viability was observed with 500 µg/L T3 after H/R (27 ± 4% of all cells vs. 5 ± 3% in time-matched controls). Again, RISK-BL abrogated protection (11 ± 3%). Mitochondria were isolated at early reperfusion from buffer-perfused rat hearts without or with iPoCo or 300 µg/L T3, respectively, at reperfusion. T3 improved mitochondrial function (i.e.: increased respiration, adenosine triphosphate production, calcium retention capacity, and decreased reactive oxygen species formation) to a similar extent as iPoCo. T3 at reperfusion reduces infarct size by activation of the RISK pathway. T3's protection is a cardiomyocyte phenomenon and targets mitochondria.

IMproving Preclinical Assessment of Cardioprotective Therapies (IMPACT) criteria: guidelines of the EU-CARDIOPROTECTION COST Action.

Acute myocardial infarction (AMI) and the heart failure (HF) which may follow are among the leading causes of death and disability worldwide. As such, new therapeutic interventions are still needed to protect the heart against acute ischemia/reperfusion injury to reduce myocardial infarct size and prevent the onset of HF in patients presenting with AMI. However, the clinical translation of cardioprotective interventions that have proven to be beneficial in preclinical animal studies, has been challenging. One likely major reason for this failure to translate cardioprotection into patient benefit is the lack of rigorous and systematic in vivo preclinical assessment of the efficacy of promising cardioprotective interventions prior to their clinical evaluation. To address this, we propose an in vivo set of step-by-step criteria for IMproving Preclinical Assessment of Cardioprotective Therapies ('IMPACT'), for investigators to consider adopting before embarking on clinical studies, the aim of which is to improve the likelihood of translating novel cardioprotective interventions into the clinical setting for patient benefit.

Optimized Treatment of ST-Elevation Myocardial Infarction.

Primary percutaneous coronary intervention is nowadays the preferred reperfusion strategy for patients with acute ST-segment-elevation myocardial infarction, aiming at restoring epicardial infarct-related artery patency and achieving microvascular reperfusion as early as possible, thus limiting the extent of irreversibly injured myocardium. Yet, in a sizeable proportion of patients, primary percutaneous coronary intervention does not achieve effective myocardial reperfusion due to the occurrence of coronary microvascular obstruction (MVO). The amount of infarcted myocardium, the so-called infarct size, has long been known to be an independent predictor for major adverse cardiovascular events and adverse left ventricular remodeling after myocardial infarction. Previous cardioprotection studies were mainly aimed at protecting cardiomyocytes and reducing infarct size. However, several clinical and preclinical studies have reported that the presence and extent of MVO represent another important independent predictor of adverse left ventricular remodeling, and recent evidences support the notion that MVO may be more predictive of major adverse cardiovascular events than infarct size itself. Although timely and complete reperfusion is the most effective way of limiting myocardial injury and subsequent ventricular remodeling, the translation of effective therapeutic strategies into improved clinical outcomes has been largely disappointing. Of importance, despite the presence of a large number of studies focused on infarct size, only few cardioprotection studies addressed MVO as a therapeutic target. In this review, we provide a detailed summary of MVO including underlying causes, diagnostic techniques, and current therapeutic approaches. Furthermore, we discuss the hypothesis that simultaneously addressing infarct size and MVO may help to translate cardioprotective strategies into improved clinical outcome following ST-segment-elevation myocardial infarction.

Translational issues for mitoprotective agents as adjunct to reperfusion therapy in patients with ST-segment elevation myocardial infarction.

Pre-clinical studies have indicated that mitoprotective drugs may add cardioprotection beyond rapid revascularization, antiplatelet therapy and risk modification. We review the clinical efficacy of mitoprotective drugs that have progressed to clinical testing comprising cyclosporine A, KAI-9803, MTP131 and TRO 40303. Whereas cyclosporine may reduce infarct size in patients undergoing primary angioplasty as evaluated by release of myocardial ischaemic biomarkers and infarct size imaging, the other drugs were not capable of demonstrating this effect in the clinical setting. The absent effect leaves the role of the mitochondrial permeability transition pore for reperfusion injury in humans unanswered and indicates that targeting one single mechanism to provide mitoprotection may not be efficient. Moreover, the lack of effect may relate to favourable outcome with current optimal therapy, but conditions such as age, sex, diabetes, dyslipidaemia and concurrent medications may also alter mitochondrial function. However, as long as the molecular structure of the pore remains unknown and specific inhibitors of its opening are lacking, the mitochondrial permeability transition pore remains a target for alleviation of reperfusion injury. Nevertheless, taking conditions such as ageing, sex, comorbidities and co-medication into account may be of paramount importance during the design of pre-clinical and clinical studies testing mitoprotective drugs.

Anaemia is associated with severe RBC dysfunction and a reduced circulating NO pool: vascular and cardiac eNOS are crucial for the adaptation to anaemia.

Anaemia is frequently present in patients with acute myocardial infarction (AMI) and contributes to an adverse prognosis. We hypothesised that, besides reduced oxygen carrying capacity, anaemia is associated with (1) red blood cell (RBC) dysfunction and a reduced circulating nitric oxide (NO) pool, (2) compensatory enhancement of vascular and cardiac endothelial nitric oxide synthase (eNOS) activity, and (3) contribution of both, RBC dysfunction and reduced circulatory NO pool to left ventricular (LV) dysfunction and fatal outcome in AMI. In mouse models of subacute and chronic anaemia from repeated mild blood loss the circulating NO pool, RBC, cardiac and vascular function were analysed at baseline and in reperfused AMI. In anaemia, RBC function resulted in profound changes in membrane properties, enhanced turnover, haemolysis, dysregulation of intra-erythrocytotic redox state, and RBC-eNOS. RBC from anaemic mice and from anaemic patients with acute coronary syndrome impaired the recovery of contractile function of isolated mouse hearts following ischaemia/reperfusion. In anaemia, the circulating NO pool was reduced. The cardiac and vascular adaptation to anaemia was characterised by increased arterial eNOS expression and activity and an eNOS-dependent increase of end-diastolic left ventricular volume. Endothelial dysfunction induced through genetic or pharmacologic reduction of eNOS-activity abrogated the anaemia-induced cardio-circulatory compensation. Superimposed AMI was associated with decreased survival. In summary, moderate blood loss anaemia is associated with severe RBC dysfunction and reduced circulating NO pool. Vascular and cardiac eNOS are crucial for the cardio-circulatory adaptation to anaemia. RBC dysfunction together with eNOS dysfunction may contribute to adverse outcomes in AMI.

Potent anti-inflammatory properties of HDL in vascular smooth muscle cells mediated by HDL-S1P and their impairment in coronary artery disease due to lower HDL-S1P: a new aspect of HDL dysfunction and its therapy.

Dysfunctional HDL is associated with coronary artery disease (CAD), but its effect on inflammation in vascular smooth muscle cells (VSMCs) in atherosclerosis is unknown. We investigated the effect of healthy human HDL and CAD-HDL on TNF-α-driven inflammation in VSMCs and examined whether HDL-associated sphingosine-1-phosphate (HDL-S1P) could modulate inflammation with the aim of designing novel HDL-based anti-inflammatory strategies. Healthy human HDL, human CAD-HDL, and mouse HDL were isolated by ultracentrifugation, S1P was measured by liquid chromatography-tandem mass spectrometry, and TNF-α-induced inflammation was characterized by gene expression and analysis of NF-κB-dependent signaling. Mechanisms of S1P interference with TNF-α were assessed by S1P receptor antagonists, mouse knockouts, and short interfering RNA. We observed that healthy HDL potently inhibited the induction of TNF-α-stimulated inflammatory genes, such as iNOS (inducible NO synthase) and MMP9 (matrix metalloproteinase 9), a process that was entirely dependent on HDL-S1P, as evidenced by loss-of-function using S1P-less HDL and mimicked by genuine S1P. Inhibition was based on suppression of TNF-α-activated Akt signaling resulting in reduced IkBαSer32 and p65Ser534 NF-κB phosphorylation based on a persistent phosphatase and tensin homolog activation by S1P through the S1P receptor 2. Intriguingly, S1P suppressed inflammation even hours after initial TNF-α stimulation. The anti-inflammatory effect of healthy HDL correlated with HDL-S1P content and was superior to that of CAD-HDL featuring lower HDL-S1P. Nevertheless, therapeutic loading of HDL with S1P completely restored the anti-inflammatory capacity of CAD-HDL and greatly boosted that of both healthy and CAD-HDL. Suppression of inflammation by HDL-S1P defines a novel pathophysiologic characteristic that distinguishes functional from dysfunctional HDL. The anti-inflammatory HDL function can be boosted by S1P-loading and exploited by S1P receptor-targeting to prevent and even turn off ongoing inflammation.-Keul, P., Polzin, A., Kaiser, K., Gräler, M., Dannenberg, L., Daum, G., Heusch, G., Levkau, B. Potent anti-inflammatory properties of HDL in vascular smooth muscle cells mediated by HDL-S1P and their impairment in coronary artery disease due to lower HDL-S1P: a new aspect of HDL dysfunction and its therapy.

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

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

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

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