Mitochondrial and Contractile Function of Human Right Atrial Tissue in Response to Remote Ischemic Conditioning.
ABSTRACT: Background Remote ischemic preconditioning ( RIPC ) by repeated brief cycles of limb ischemia/reperfusion attenuates myocardial ischemia/reperfusion injury. We aimed to identify a functional parameter reflecting the RIPC -induced protection in human. Therefore, we measured mitochondrial function in right atrial tissue and contractile function of isolated right atrial trabeculae before and during hypoxia/reoxygenation from patients undergoing coronary artery bypass grafting with RIPC or placebo, respectively. Methods and Results One hundred thirty-seven patients under isoflurane anesthesia underwent RIPC (3×5 minutes blood pressure cuff inflation on the left upper arm/5 minutes deflation, n=67) or placebo (cuff uninflated, n=70), and right atrial appendages were harvested before ischemic cardioplegic arrest. Myocardial protection by RIPC was assessed from serum troponin I/T concentrations over 72 hours after surgery. Atrial tissue was obtained for isolation of mitochondria ( RIPC /placebo: n=10/10). Trabeculae were dissected for contractile function measurements at baseline and after hypoxia/reoxygenation (60 min/30 min) and for western blot analysis after hypoxia/reoxygenation ( RIPC /placebo, n=57/60). Associated with cardioprotection by RIPC (26% decrease in the area under the curve of troponin I/T), mitochondrial adenosine diphosphate-stimulated complex I respiration (+10%), adenosine triphosphate production (+46%), and calcium retention capacity (+37%) were greater, whereas reactive oxygen species production (-24%) was less with RIPC than placebo. Contractile function was improved by RIPC (baseline, +7%; reoxygenation, +24%). Expression and phosphorylation of proteins, which have previously been associated with cardioprotection, were not different between RIPC and placebo. Conclusions Cardioprotection by RIPC goes along with improved mitochondrial and contractile function of human right atrial tissue. Clinical Trial Registration URL: https://www.clinicaltrials.gov . Unique identifier: NCT 01406678.
Project description:Erythropoietin (EPO) has been shown to protect against myocardial infarction in animal studies by activating phosphatidylinositol-3 kinase (PI3K)/Akt and ERK1/2. However these pro-survival pathways are impaired in the diabetic heart. We investigated the ability of EPO to protect human atrial trabeculae from non-diabetic and diabetic patients undergoing coronary artery bypass surgery, against hypoxia-reoxygenation injury.Human atrial trabeculae were exposed to 90min hypoxia and 120min reoxygenation. EPO was administered throughout reoxygenation. The developed force of contraction, calculated as a percentage of baseline force of contraction, was continuously monitored. The involvement of PI3K and ERK1/2 and the levels of activated caspase 3(AC3) were assessed.EPO improved the force of contraction in tissue from non-diabetic patients (46.7+/-1.7% vs. 30.2+/-2.2% in control, p<0.001). These beneficial effects were prevented by the PI3K inhibitor, LY294002 and the ERK1/2 inhibitor, U0126. EPO also significantly improved the force of contraction in the diabetic tissue, although to a lesser degree. The levels of activated caspase 3 were significantly reduced in EPO treated trabeculae from both non-diabetic and diabetic patients, relative to their respective untreated controls.EPO administered at reoxygenation protected human myocardial muscle by activating PI3K and ERK1/2 and reducing the level of activated caspase 3. This cardioprotection was also observed in the diabetic group. This data supports the potential of EPO being used as a novel cardioprotective strategy either alone or as an adjunct in the clinical setting alongside existing reperfusion therapies.
Project description:Post-translational modification of proteins by O-linked ?-N-acetylglucosamine (O-GlcNAc) is cardioprotective but its role in cardioprotection by remote ischaemic preconditioning (rIPC) and the reduced efficacy of rIPC in type 2 diabetes mellitus is unknown. In this study we achieved mechanistic insight into the remote stimulus mediating and the target organ response eliciting the cardioprotective effect by rIPC in non-diabetic and diabetic myocardium and the influence of O-GlcNAcylation.The cardioprotective capacity and the influence on myocardial O-GlcNAc levels of plasma dialysate from eight healthy volunteers and eight type 2 diabetic patients drawn before and after subjection to an rIPC stimulus were tested on human isolated atrial trabeculae subjected to ischaemia/reperfusion injury. Dialysate from healthy volunteers exposed to rIPC improved post-ischaemic haemodynamic recovery (40 ± 6 vs. 16 ± 2%; P < 0.01) and increased myocardial O-GlcNAc levels. Similar observations were made with dialysate from diabetic patients before exposure to rIPC (43 ± 3 vs. 16 ± 2%; P < 0.001) but no additional cardioprotection or further increase in O-GlcNAc levels was achieved by perfusion with dialysate after exposure to rIPC (44 ± 4 and 42 ± 5 vs. 43 ± 3%; P = 0.7). The glutamine:fructose-6-phosphate amidotransferase (GFAT) inhibitor azaserine abolished the cardioprotective effects and the increment in myocardial O-GlcNAc levels afforded by plasma from diabetic patients and healthy volunteers treated with rIPC.rIPC and diabetes mellitus per se influence myocardial O-GlcNAc levels through circulating humoral factors. O-GlcNAc signalling participates in mediating rIPC-induced cardioprotection and maintaining a state of inherent chronic activation of cardioprotection in diabetic myocardium, restricting it from further protection by rIPC.
Project description:Transient episodes of ischemia in a remote organ (remote ischemic preconditioning, RIPC) can attenuate myocardial ischemia/reperfusion injury but the underlying mechanisms of RIPC in the target organ are still poorly understood. Recent animal studies suggested that the small redox protein thioredoxin may be a potential candidate for preconditioning-induced organprotection. Here we employed a human proteome profiler array to investigate the RIPC regulated expression of cell stress proteins and particularly of thioredoxin in heart tissue of cardiosurgical patients with cardiopulmonary bypass (CPB).RIPC was induced by four 5 minute cycles of transient upper limb ischemia/reperfusion using a blood pressure cuff. Right atrial tissue was obtained from patients receiving RIPC (N = 19) and control patients (N = 19) before and after CPB. Cell stress proteome profiler arrays as well as Westernblotting and ELISA experiments for thioredoxin (Thio-1) were performed employing the respective tissue samples.Protein arrays revealed an up-regulation of 26.9% (7/26; CA IX, Cyt C, HSP-60, HSP-70, pJNK, SOD2, Thio-1) of cell stress associated proteins in RIPC tissue obtained before CPB, while 3.8% (1/26; SIRT2) of the proteins were down-regulated. Array results for thioredoxin were verified by semi-quantitative Westernblotting studies which showed a significant up-regulation of thioredoxin protein levels in cardiac tissue samples of RIPC patients taken before CPB (RIPC: 5.36 ± 0.85 a.u.; control: 3.23 ± 0.39 a.u.; P < 0.05). Quantification of thioredoxin levels in tissue of RIPC and control patients by ELISA experiments further confirmed the Westernblotting results (RIPC: 0.30 ± 0.02 ng/mg protein; control: 0.24 ± 0.02 ng/mg protein; P < 0.05).We provide evidence for thioredoxin as a RIPC-induced factor in heart tissue of cardiosurgical patients and identified several cell stress associated proteins that are regulated by RIPC and may play a role in RIPC-mediated cardioprotection.
Project description:Remote ischemic preconditioning (RIPC) by repeated brief limb ischemia/reperfusion reduces myocardial injury in patients undergoing coronary artery bypass grafting (CABG). Activation of signal transducer and activator of transcription 5 (STAT5) in left ventricular (LV) myocardium at early reperfusion is associated with such protection. Autophagy, i.e., removal of dysfunctional cellular components through lysosomes, has been proposed as one mechanism of cardioprotection. Therefore, we analyzed whether or not the protection by RIPC is associated with activated autophagy.CABG patients were randomized to undergo RIPC (3×5 min blood pressure cuff inflation/5 min deflation) or placebo (cuff deflated) before skin incision (n?=?10/10). Transmural myocardial biopsies were taken from the LV before cardioplegia (baseline) and at early (5-10 min) reperfusion. RIPC-induced protection was reflected by decreased serum troponin I concentration area under the curve (194±17 versus 709±129 ng/ml × 72 h, p?=?0.002). Western blotting for beclin-1-phosphorylation and protein expression of autophagy-related gene 5-12 (ATG5-12) complex, light chain 3 (LC3), parkin, and p62 was performed. STAT3-, STAT5- and extracellular signal-regulated protein kinase 1/2 (ERK1/2)-phosphorylation was used as positive control to confirm signal activation by ischemia/reperfusion.Signals of all analyzed autophagy proteins did not differ between baseline and early reperfusion and not between RIPC and placebo. STAT5-phosphorylation was greater at early reperfusion only with RIPC (2.2-fold, p?=?0.02). STAT3- and ERK1/2-phosphorylation were greater at early reperfusion with placebo and RIPC (?2.7-fold versus baseline, p?0.05).Protection through RIPC in patients undergoing CABG surgery does not appear to be associated with enhanced autophagy in LV myocardium at early reperfusion.
Project description:In this placebo-controlled randomized controlled trial, we tested whether remote ischemic preconditioning (RIPC) elicited by four 5-minute cycles of 300 mmHg of cuff inflation/deflation of the lower limb would reduce myocardial necrosis in isoflurane-anesthetized patients undergoing on-pump coronary artery bypass graft surgery. Secondary outcomes were the perioperative release of the biomarkers NTproBNP, hsCRP, S100, atrial transcriptional profiles, and short- and long-term clinical outcomes. RIPC with concomitantly applied isoflurane did not affect the release of biomarkers or clinical outcome. NTproBNP release correlated with isoflurane- but not RIPC-induced transcriptional changes. For eleven randomly selected patients from each group (RIPC/CTL=no RIPC) two atrial samples were collected, one at the time of cannulation (T1) and one fifteen min after releasing the cross clamp (T2). The samples were immediately frozen in liquid nitrogen and later used for RNA isolation and subsequent microarray hybridization. Gene-level analysis was performed. the results of exon-level analysis will be published separately (only preliminary results available so far).
Project description:In this placebo-controlled randomized controlled trial, we tested whether remote ischemic preconditioning (RIPC) elicited by four 5-minute cycles of 300 mmHg of cuff inflation/deflation of the lower limb would reduce myocardial necrosis in isoflurane-anesthetized patients undergoing on-pump coronary artery bypass graft surgery. Secondary outcomes were the perioperative release of the biomarkers NTproBNP, hsCRP, S100, atrial transcriptional profiles, and short- and long-term clinical outcomes. RIPC with concomitantly applied isoflurane did not affect the release of biomarkers or clinical outcome. NTproBNP release correlated with isoflurane- but not RIPC-induced transcriptional changes. Overall design: For eleven randomly selected patients from each group (RIPC/CTL=no RIPC) two atrial samples were collected, one at the time of cannulation (T1) and one fifteen min after releasing the cross clamp (T2). The samples were immediately frozen in liquid nitrogen and later used for RNA isolation and subsequent microarray hybridization. Gene-level analysis was performed. the results of exon-level analysis will be published separately (only preliminary results available so far).
Project description:Remote ischaemic preconditioning (RIPC) by inducing brief ischaemia in distant tissues protects the heart against myocardial ischaemia-reperfusion injury (IRI) in children undergoing open-heart surgery, although its effectiveness in adults with comorbidities is controversial. The effectiveness and mechanism of RIPC with respect to myocardial IRI in children with tetralogy of Fallot (ToF), a severe cyanotic congenital cardiac disease, undergoing open heart surgery are unclear. We hypothesized that RIPC can confer cardioprotection in children undergoing ToF repair surgery.Overall, 112 ToF children undergoing radical open cardiac surgery using cardiopulmonary bypass (CPB) were randomized to either a RIPC group (n?=?55) or a control group (n?=?57). The RIPC protocol consisted of three cycles of 5-min lower limb occlusion and 5-min reperfusion using a cuff-inflator. Serum inflammatory cytokines and cardiac injury markers were measured before surgery and after CPB. Right ventricle outflow tract (RVOT) tissues were collected during the surgery to assess hypoxia-inducible factor (Hif)-1? and other signalling proteins. Cardiac mitochondrial injury was assessed by electron microscopy. The primary results showed that the length of stay in the intensive care unit (ICU) was longer in the control group than in the RIPC group (52.30?±?13.43?h vs. 47.55?±?10.34?h, respectively, P?=?0.039). Patients in the control group needed longer post-operative ventilation time compared to the RIPC group (35.02?±?6.56?h vs. 31.96?±?6.60?h, respectively, P?=?0.016). The levels of post-operative serum troponin-T at 12 and 18?h, CK-MB at 24?h, as well as the serum h-FABP levels at 6?h, after CPB were significantly lower, which was coincident with significantly higher protein expression of cardiac Hif-1?, p-Akt, p-STAT3, p-STAT5, and p-eNOS and less vacuolization of mitochondria in the RIPC group compared to the control group.In ToF children undergoing open heart surgery, RIPC attenuates myocardial IRI and improves the short-term prognosis.
Project description:Remote ischemic preconditioning (RIPC) by repeated brief cycles of limb ischemia/reperfusion may reduce myocardial ischemia/reperfusion injury and improve patients' prognosis after elective coronary artery bypass graft (CABG) surgery. The signal transducer and activator of transcription (STAT)5 activation in left ventricular myocardium is associated with RIPC´s cardioprotection. Cytokines and growth hormones typically activate STATs and could therefore act as humoral transfer factors of RIPC´s cardioprotection. We here determined arterial plasma concentrations of 25 different cytokines, growth hormones, and other factors which have previously been associated with cardioprotection, before (baseline)/after RIPC or placebo (n?=?23/23), respectively, and before/after ischemic cardioplegic arrest in CABG patients. RIPC-induced protection was reflected by a 35% reduction of serum troponin I release. With the exception of interleukin-1?, none of the humoral factors changed in their concentrations after RIPC or placebo, respectively. Interleukin-1?, when normalized to baseline, increased after RIPC (280?±?56%) but not with placebo (97?±?15%). The interleukin-1? concentration remained increased until after ischemic cardioplegic arrest and was also higher than with placebo in absolute concentrations (25?±?6 versus 16?±?3?pg/mL). Only interleukin-1? possibly fulfills the criteria which would be expected from a substance to be released in response to RIPC and to protect the myocardium during ischemic cardioplegic arrest.
Project description:Background. Nitric oxide can successfully compete with oxygen for sites of electron-transport chain in conditions of myocardial hypoxia. These features may prevent excessive oxidative stress occurring in cardiomyocytes during sudden hypoxia-reoxygenation. Aim. To study the action of the potent stable NO donor dinitrosyl iron complex with glutathione (Oxacom®) on the recovery of myocardial contractile function and Ca2+ transients in cardiomyocytes during hypoxia-reoxygenation. Results. The isolated rat hearts were subjected to 30?min hypoxia followed by 30?min reoxygenation. The presence of 30?nM Oxacom in hypoxic perfusate reduced myocardial contracture and improved recovery of left ventricular developed pressure partly due to elimination of cardiac arrhythmias. The same Oxacom concentration limited reactive oxygen species generation in hypoxic cardiomyocytes and increased the viability of isolated cardiomyocytes during hypoxia from 12 to 52% and after reoxygenation from 0 to 40%. Oxacom prevented hypoxia-induced elevation of diastolic Ca2+ level and eliminated Ca2+ transport alterations manifested by slow Ca2+ removal from the sarcoplasm and delay in cardiomyocyte relaxation. Conclusion. The potent stable NO donor preserved cardiomyocyte integrity and improved functional recovery at hypoxia-reoxygenation both in the isolated heart and in cardiomyocytes mainly due to preservation of Ca2+ transport. Oxacom demonstrates potential for cardioprotection during hypoxia-reoxygenation.
Project description:Remote ischemic preconditioning (RIPC) can evoke cardioprotection following ischemia/reperfusion and this may depend on the anesthetic used. We tested whether 1) extracellular vesicles (EVs) isolated from humans undergoing RIPC protect cardiomyoblasts against hypoxia-induced apoptosis and 2) this effect is altered by cardiomyoblast exposure to isoflurane or propofol. EVs were isolated before and 60 min after RIPC or Sham from ten patients undergoing coronary artery bypass graft surgery with isoflurane anesthesia and quantified by Nanoparticle Tracking Analysis. Following EV-treatment for 6 hours under exposure of isoflurane or propofol, rat H9c2 cardiomyoblasts were cultured for 18 hours in normoxic or hypoxic atmospheres. Apoptosis was detected by flow cytometry. Serum nanoparticle concentrations in patients had increased sixty minutes after RIPC compared to Sham (2.5x1011±4.9x1010 nanoparticles/ml; Sham: 1.2x1011±2.0x1010; p = 0.04). Hypoxia increased apoptosis of H9c2 cells (hypoxia: 8.4%±0.6; normoxia: 2.5%±0.1; p<0.0001). RIPC-EVs decreased H9c2 cell apoptosis compared to control (apoptotic ratio: 0.83; p = 0.0429) while Sham-EVs showed no protection (apoptotic ratio: 0.97). Prior isoflurane exposure in vitro even increased protection (RIPC-EVs/control, apoptotic ratio: 0.79; p = 0.0035; Sham-EVs/control, apoptotic ratio:1.04) while propofol (50?M) abrogated protection by RIPC-EVs (RIPC-EVs/control, Apoptotic ratio: 1.01; Sham-EVs/control, apoptotic ratio: 0.94; p = 0.602). Thus, EVs isolated from patients undergoing RIPC under isoflurane anesthesia protect H9c2 cardiomyoblasts against hypoxia-evoked apoptosis and this effect is abrogated by propofol. This supports a role of human RIPC-generated EVs in cardioprotection and underlines propofol as a possible confounder in RIPC-signaling mediated by EVs.