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Remote ischemic conditioning (RIC) by repetitive brief periods of limb ischemia and reperfusion renders organs more resistant to ischemic injury. The protection is partly through down-regulation of the inflammatory response. Our aim was to investigate the clinical and anti-inflammatory effects of RIC in patients with active ulcerative colitis (UC). We included 22 patients with active UC in this explorative, randomized, sham-controlled clinical trial. The patients were randomly assigned 1:1 to RIC (induced in the arm through four cycles of 5-min inflation and 5-min deflation of a blood-pressure cuff) or sham (incomplete inflation of the blood-pressure cuff) once daily for 10 days. Outcome variables were measured at baseline and on day 11. When compared with sham, RIC did not affect inflammation in the UC patients measured by fecal calprotectin, plasma C-reactive protein, Mayo Score, Mayo Endoscopic Subscore, Nancy Histological Index or inflammatory cytokines involved in UC and RIC. The mRNA and miRNA expression profiles in the UC patients were measured by RNA sequencing and multiplexed hybridization, respectively, but were not significantly affected by RIC. We used the Langendorff heart model to assess activation of the organ protective mechanism induced by RIC, but could not confirm activation of the organ protective mechanism in the UC patients.

Ischemic conditioning and exercise have been suggested for protecting against brain ischemia-reperfusion injury. However, the endogenous protective mechanisms stimulated by these interventions remain unclear. Here, in a comprehensive translational study, we investigated the protective role of extracellular vesicles (EVs) released after remote ischemic conditioning (RIC), blood flow restricted resistance exercise (BFRRE), or high-load resistance exercise (HLRE). Blood samples were collected from human participants before and at serial time points after intervention. RIC and BFRRE plasma EVs released early after stimulation improved viability of endothelial cells subjected to oxygen-glucose deprivation. Furthermore, post-RIC EVs accumulated in the ischemic area of a stroke mouse model, and a mean decrease in infarct volume was observed for post-RIC EVs, although not reaching statistical significance. Thus, circulating EVs induced by RIC and BFRRE can mediate protection, but the in vivo and translational effects of conditioned EVs require further experimental verification.

Purpose: Hyperpolarized [1- 13C]pyruvate MRS can measure cardiac metabolism in vivo. We investigated whether [1- 13C]pyruvate MRS could predict left ventricular remodeling following myocardial infarction (MI), long-term left ventricular effects of heart failure medication, and could identify responders to treatment. Methods: Thirty-five rats were scanned with hyperpolarized [1- 13C]pyruvate MRS 3 days after MI or sham surgery. The animals were re-examined after 30 days of therapy with β-blockers and ACE-inhibitors (active group, n = 12), placebo treatment (placebo group, n = 13) or no treatment (sham group, n = 10). Furthermore, heart tissue mitochondrial respiratory capacity was assessed by high-resolution respirometry. Metabolic results were compared between groups, over time and correlated to functional MR data at each time point. Results: At 30 ± 0.5 days post MI, left ventricular ejection fraction (LVEF) differed between groups (sham, 77% ± 1%; placebo, 52% ± 3%; active, 63% ± 2%, P 13C]pyruvate MRS and respirometry, neither differed between groups nor between baseline and follow-up. Three days post MI, low bicarbonate + CO 2/pyruvate ratio was associated with low LVEF. At follow-up, in the active group, a poor recovery of LVEF was associated with high bicarbonate + CO 2/pyruvate ratio, as measured by hyperpolarized MRS. Conclusion: In a rat model of moderate heart failure, medical treatment improved function, but did not on average influence [1- 13C]pyruvate flux as measured by MRS; however, responders to heart failure medication had reduced capacity for carbohydrate metabolism.

Supplemental Digital Content is available in the text., Background: Myocardial utilization of 3-hydroxybutyrate (3-OHB) is increased in patients with heart failure and reduced ejection fraction (HFrEF). However, the cardiovascular effects of increased circulating plasma-3-OHB levels in these patients are unknown. Consequently, the authors’ aim was to modulate circulating 3-OHB levels in HFrEF patients and evaluate: (1) changes in cardiac output (CO); (2) a potential dose-response relationship between 3-OHB levels and CO; (3) the impact on myocardial external energy efficiency (MEE) and oxygen consumption (MVO2); and (4) whether the cardiovascular response differed between HFrEF patients and age-matched volunteers. Methods: Study 1: 16 chronic HFrEF patients (left ventricular ejection fraction: 37±3%) were randomized in a crossover design to 3-hour of 3-OHB or placebo infusion. Patients were monitored invasively with a Swan-Ganz catheter and with echocardiography. Study 2: In a dose-response study, 8 HFrEF patients were examined at increasing 3-OHB infusion rates. Study 3 to 4: 10 HFrEF patients and 10 age-matched volunteers were randomized in a crossover design to 3-hour 3-OHB or placebo infusion. MEE and MVO2 were evaluated using 11C-acetate positron emission tomography. Results: 3-OHB infusion increased circulating levels of plasma 3-OHB from 0.4±0.3 to 3.3±0.4 mM (P

Hyperpolarized [1-Thirty-five rats were scanned with hyperpolarized [1-At 30 ± 0.5 days post MI, left ventricular ejection fraction (LVEF) differed between groups (sham, 77% ± 1%; placebo, 52% ± 3%; active, 63% ± 2%, P.001). Cardiac metabolism, measured by both hyperpolarized [1-In a rat model of moderate heart failure, medical treatment improved function, but did not on average influence [1

Inhibition of succinate dehydrogenase (SDH) by Dimethyl Malonate (DiMal) reduces cardiac ischemia–reperfusion (IR) injury. We investigated the cardioprotective effect of DiMal in a rat model during advancing type 2 diabetes. Zucker Diabetic Fatty rats and lean controls were investigated corresponding to prediabetes, onset and mature diabetes. Hearts were mounted in an isolated perfused model, and subjected to IR for investigation of infarct size (IS) and mitochondrial respiratory control ratio (RCR). DiMal was administered for 10 min before ischemia. Compared with age-matched non-diabetic rats, prediabetic rats had larger IS (49 ± 4% vs. 36 ± 2%, p = 0.007), rats with onset diabetes smaller IS (51 ± 3% vs. 62 ± 3%, p = 0.05) and rats with mature diabetes had larger IS (79 ± 3% vs. 69 ± 2%, p = 0.06). At the prediabetic stage DiMal did not alter IS. At onset of diabetes DiMal 0.6 mM increased IS in diabetic but not in non-diabetic control rats (72 ± 4% vs. 51 ± 3%, p = 0.003). At mature diabetes DiMal 0.1 and 0.6 mM reduced IS (68 ± 3% vs. 79 ± 3% and 64 ± 5% vs. 79 ± 3%, p = 0.1 and p = 0.01), respectively. DiMal 0.1 mM alone reduced IS in age-matched non-diabetic animals (55 ± 3% vs. 69 ± 2% p = 0.01). RCR was reduced at mature diabetes but not modulated by DiMal. Modulation of SDH activity results in variable infarct size reduction depending on presence and the stage of diabetes. Modulation of SDH activity may be an unpredictable cardioprotective approach.

We studied the translational cardioprotective potential of P2Y12 inhibitors against acute myocardial ischemia/reperfusion injury (IRI) in an animal model of acute myocardial infarction and in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). P2Y12 inhibitors may have pleiotropic effects to induce cardioprotection against acute myocardial IRI beyond their inhibitory effects on platelet aggregation. We compared the cardioprotective effects of clopidogrel, prasugrel, and ticagrelor on infarct size in an in vivo rat model of acute myocardial IRI, and investigated the effects of the P2Y12 inhibitors on enzymatic infarct size (48-h area-under-the-curve (AUC) troponin T release) and clinical outcomes in a retrospective study of STEMI patients from the CONDI-2/ERIC-PPCI trial using propensity score analyses. Loading with ticagrelor in rats reduced infarct size after acute myocardial IRI compared to controls (37 ± 11% vs 52 ± 8%, p 0.99 and 49 ± 9%, p > 0.99, respectively). Correspondingly, troponin release was reduced in STEMI patients treated with ticagrelor compared to clopidogrel (adjusted 48-h AUC ratio: 0.67, 95% CI 0.47-0.94). Compared to clopidogrel, the composite endpoint of cardiac death or hospitalization for heart failure within 12 months was reduced in STEMI patients loaded with ticagrelor (HR 0.63; 95% CI 0.42-0.94) but not prasugrel (HR 0.84, 95% CI 0.43-1.63), prior to PPCI. Major adverse cardiovascular events did not differ between clopidogrel, ticagrelor, or prasugrel. The cardioprotective effects of ticagrelor in reducing infarct size may contribute to the clinical benefit observed in STEMI patients undergoing PPCI.

Background: Remote ischemic conditioning (RIC) by brief periods of limb ischemia and reperfusion protects against ischemia–reperfusion injury. We studied the cardioprotective role of extracellular vesicles (EV)s released into the circulation after RIC and EV accumulation in injured myocardium. Methods: We used plasma from healthy human volunteers before and after RIC (pre-PLA and post-PLA) to evaluate the transferability of RIC. Pre- and post-RIC plasma samples were separated into an EV enriched fraction (pre-EV + and post-EV +) and an EV poor fraction (pre-EV- and post-EV-) by size exclusion chromatography. Small non-coding RNAs from pre-EV + and post-EV + were purified and profiled by NanoString Technology. Infarct size was compared in Sprague–Dawley rat hearts perfused with isolated plasma and fractions in a Langendorff model. In addition, fluorescently labeled EVs were used to assess homing in an in vivo rat model. (ClinicalTrials.gov, number: NCT03380663) Results: Post-PLA reduced infarct size by 15% points compared with Pre-PLA (55 ± 4% (n = 7) vs 70 ± 6% (n = 8), p = 0.03). Post-EV + reduced infarct size by 16% points compared with pre-EV + (53 ± 15% (n = 13) vs 68 ± 12% (n = 14), p = 0.03). Post-EV- did not affect infarct size compared to pre-EV- (64 ± 3% (n = 15) and 68 ± 10% (n = 16), p > 0.99). Three miRNAs (miR-16-5p, miR-144-3p and miR-451a) that target the mTOR pathway were significantly up-regulated in the post-EV + group. Labelled EVs accumulated more intensely in the infarct area than in sham hearts. Conclusion: Cardioprotection by RIC can be mediated by circulating EVs that accumulate in injured myocardium. The underlying mechanism involves modulation of EV miRNA that may promote cell survival during reperfusion.

Background Remote ischemic conditioning (RIC) by brief periods of limb ischemia and reperfusion protects against ischemia-reperfusion injury. However, the mechanism is unknown. Purpose We studied the role of exosomes for mediating the cardioprotective signal and whether they accumulate in injured myocardium. Methods Blood samples from 12 healthy male volunteers were obtained prior to and one hour after RIC. Plasma obtained before and after RIC (n=4) (P-Pre and P-Post) was used to evaluate the transferability of RIC. Pre- and Post-RIC plasma (n=8) was separated into an exosome rich fraction (Exo-Pre and Exo-Post) and an exosome depleted fraction (Prot-Pre and Prot-Post) by size exclusion chromatography. All studies were carried out in duplicate samples from each volunteer. Infarct size was compared in Sprague-Dawley rat hearts perfused with plasma, exosomes and exosome depleted fractions in a Langendorff model. We investigated changes in the miRNA content of the exosomes after RIC by a human miRNA panel. Additionally, fluorescently labeled exosomes isolated from C2C12 cells were used to assess accumulation in injured myocardium in an in vivo rat model. Rats were divided into an infarct group (n=6) (left anterior descending artery ligation) and a sham group (n=6) (without ligation). Labelled exosomes were injected in the femoral vein prior to reperfusion. Exosome-accumulation in infarcted or sham myocardium was evaluated. Results P-Post reduced infarct size by 15% points compared with P-Pre (55±4% vs 70±6%, p=0.03) (Fig. 1a). Exo-Post reduced infarct size by 16% points compared with Exo-Pre (53±15% vs 68±12%, p=0.03) (Fig. 1b). Prot-Post did not affect infarct size compared to Prot-Pre (64±3% and 68±10%, p>0.99). We found miRNA-16, miRNA-144 and miRNA-451 to be upregulated in exosomes after RIC and the mTOR-pathway as a potential target for these miRNAs. In the in vivo model, labelled exosomes accumulated more intensively in the infarct area than in remote areas and sham hearts (Fig. 1c). Conclusion Cardioprotection by RIC is mediated by exosomes with a changed miRNA profile and exosomes accumulate in injured myocardium. Figure 1 Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Novo synergy

Remote ischemic conditioning (RIC) by brief periods of limb ischemia and reperfusion protects against ischemia-reperfusion injury. We studied the cardioprotective role of extracellular vesicles (EV)s released into the circulation after RIC and EV accumulation in injured myocardium.We used plasma from healthy human volunteers before and after RIC (pre-PLA and post-PLA) to evaluate the transferability of RIC. Pre- and post-RIC plasma samples were separated into an EV enriched fraction (pre-EV + and post-EV +) and an EV poor fraction (pre-EV- and post-EV-) by size exclusion chromatography. Small non-coding RNAs from pre-EV + and post-EV + were purified and profiled by NanoString Technology. Infarct size was compared in Sprague-Dawley rat hearts perfused with isolated plasma and fractions in a Langendorff model. In addition, fluorescently labeled EVs were used to assess homing in an in vivo rat model. (ClinicalTrials.gov, number: NCT03380663) RESULTS: Post-PLA reduced infarct size by 15% points compared with Pre-PLA (55 ± 4% (n = 7) vs 70 ± 6% (n = 8), p = 0.03). Post-EV + reduced infarct size by 16% points compared with pre-EV + (53 ± 15% (n = 13) vs 68 ± 12% (n = 14), p = 0.03). Post-EV- did not affect infarct size compared to pre-EV- (64 ± 3% (n = 15) and 68 ± 10% (n = 16), p 0.99). Three miRNAs (miR-16-5p, miR-144-3p and miR-451a) that target the mTOR pathway were significantly up-regulated in the post-EV + group. Labelled EVs accumulated more intensely in the infarct area than in sham hearts.Cardioprotection by RIC can be mediated by circulating EVs that accumulate in injured myocardium. The underlying mechanism involves modulation of EV miRNA that may promote cell survival during reperfusion.

A multitargeted strategy to treat the consequences of ischemia and reperfusion (IR) injury in acute myocardial infarction may add cardioprotection beyond reperfusion therapy alone. We investigated the cardioprotective effect of mild hypothermia combined with local ischemic preconditioning (IPC) or remote ischemic conditioning (RIC) on IR injury in isolated rat hearts. Moreover, we aimed to define the optimum timing of initiating hypothermia and evaluate underlying cardioprotective mechanisms. Compared to infarct size in normothermic controls (56 ± 4%), mild hypothermia during the entire or final 20 min of the ischemic period reduced infarct size (34 ± 2%, p

Background Ischemia-reperfusion (IR) injury can be attenuated through modulation of mitochondrial metabolism with succinate dehydrogenase (SDH) inhibition by dimethyl malonate (DiMAL). However, it is unknown whether SDH inhibition yields protection in aged, diabetic individuals and whether protection translate into human cardiac tissue. Purpose We wished to evaluate if SDH inhibition can be protective in aged, diabetic rat hearts and could be translated to the human myocardium. Methods We studied infarct size in aged non-diabetic and diabetic rat hearts subjected to isolated, retrograde perfusion and global ischemia and reperfusion. The hearts were randomized to: Sham hearts, IR injured hearts and IR injured hearts co-perfused with 0.1 mM or 0.6 mM DiMAL. Infarct size and mitochondrial respiratory capacity were evaluated post-ischemic. To translate our findings into human cardiac tissue, we tested the efficacy of DiMAL treatment in human atrial trabeculae from patients with and without diabetes mellitus undergoing cardiac surgery. We randomized atrial trabeculae to: IR injury, IR injury treated with ischemic preconditioning (IPC) and IR injury treated with 5 mM DiMAL. Contractile force recovery and mitochondrial respiratory capacity were evaluated post-ischemic. Results In non-diabetic rat hearts, DiMAL 0.1 mM reduced infarct size/area-at-risk (IS/AAR) compared to IR hearts (53±7% vs. 69±6% of IS/AAR; p0.99). Mitochondrial complex I linked respiration capacity did not improve by IPC or DiMAL compared to IR in non-diabetic trabeculae (p>0.99). Conclusion Inhibition of the SDH by DiMAL protects the aged non-diabetic and diabetic heart from IR injury but the threshold for cardioprotection is increased in diabetic rat hearts. DiMAL provides cardioprotection similar to IPC in non-diabetic trabeculae but does not protect the human diabetic myocardium when given in identical concentrations. SDH inhibition can provide protection in both animal and human tissue and may provide a new target for pharmacologic conditioning in patients.

Background: The Sodium Glucose Transporter 2 (SGLT2)-inhibitor, empagliflozin, reduces death fromcardiovascular causes. We hypothesized that the mechanism involved direct protection against Ischemia-Reperfusion (IR) injury and improved post-ischemic mitochondrial function.Methods: We examined infarct size (series I) and mitochondrial respiration (series II) in four groups of isolatedperfused hearts from male Wistar rats: Sham-operated hearts (Sham group), IR-injured hearts (IR group), heartstreated with ischemic preconditioning (IPC) by 2 × 5 min. cycles of IR prior to sustained ischemia (IPC group), andhearts co-perfused with 2.14 mg/l of empagliflozin 10 min. prior to sustained ischemia (EMPA group).Results: In contrast to IPC, empagliflozin did not reduce infarct size compared to the IR group, when given 10min prior to the acute myocardial infarction. Empagliflozin improved post-ischemic complex I+II respiration comparedto the IR group. This improvement was similar to IPC. In contrast to the improved complex I respiration by IPC,empagliflozin mainly improved complex II respiration. Empagliflozin hearts had significantly higher respiration inoligomycin induced state 4 than the sham and IR group, indicating that empagliflozin modulates the innermitochondrial membrane.Conclusion: In conclusion, empagliflozin yielded no acute cardioprotection in the isolated perfused non-diabeticrat heart. Empagliflozin mainly improved complex II respiration and increased permeability of the inner membrane,providing a potential explanation for the positive long-term effects observed in post infarction myocardial dysfunction.

Left ventricular (LV) remodeling following a myocardial infarction (MI) involves formation of reactive oxygen species (ROS). Paroxetine, a selective serotonin reuptake inhibitor, has an antioxidant effect in the vascular wall. We investigated whether paroxetine reduces myocardial ROS formation and LV remodeling following a MI. In a total of 32 Wistar rats, MI was induced by a 30-min ligation of the left anterior descending artery followed by 7- or 28-day reperfusion. During the 28 days of reperfusion, LV remodeling was evaluated by magnetic resonance imaging (MRI) and echocardiography (n = 20). After 28 days of reperfusion, the susceptibility to ventricular tachycardia was evaluated prior to sacrifice and histological assessment of myocyte cross-sectional area, fibrosis, and presence of myofibroblasts. Myocardial ROS formation was measured with dihydroethidium after 7 days of reperfusion in separate groups (n = 12). Diastolic LV volume, evaluated by MRI (417 ± 60 vs. 511 ± 64 µL, p < 0.05), and echocardiography (515 ± 80 vs. 596 ± 83 µL, p < 0.05) as well as diastolic LV internal diameter evaluated with echocardiography (7.2 ± 0.6 vs. 8.1 ± 0.7 mm, p < 0.05) were lower in the paroxetine group than in controls. Furthermore, myocyte cross-sectional area was reduced in the paroxetine group compared with controls (277 ± 26 vs. 354 ± 23 mm(3), p < 0.05) and ROS formation was reduced in the remote myocardium (0.415 ± 0.19 normalized to controls, p < 0.05). However, no differences in the presence of fibrosis or myofibroblasts were observed. Finally, paroxetine reduced the susceptibility to ventricular tachycardia (induced in 2/11 vs. 6/8 rats, p < 0.05). Paroxetine treatment following MI decreases LV remodeling and susceptibility to arrhythmias, probably by reducing ROS formation.

Treatment with the connexin43 modulator, rotigaptide (ZP123) has been shown to protect against myocardial ischemia-reperfusion (I-R) injury in experimental studies. Our aim was to assess whether ZP123 exerts cardioprotection in isolated rabbit hearts subjected to I-R, and whether this effect