MTORC1-Regulated Metabolism Controlled by TSC2 Limits Cardiac Reperfusion Injury.

RATIONALE: The mTORC1 (mechanistic target of rapamycin complex-1) controls metabolism and protein homeostasis and is activated following ischemia reperfusion (IR) injury and by ischemic preconditioning (IPC). However, studies vary as to whether this activation is beneficial or detrimental, and its influence on metabolism after IR is little reported. A limitation of prior investigations is their use of broad gain/loss of mTORC1 function, mostly applied before ischemic stress. This can be circumvented by regulating one serine (S1365) on TSC2 (tuberous sclerosis complex) to achieve bidirectional mTORC1 modulation but only with TCS2-regulated costimulation. OBJECTIVE: We tested the hypothesis that reduced TSC2 S1365 phosphorylation protects the myocardium against IR and is required for IPC by amplifying mTORC1 activity to favor glycolytic metabolism. METHODS AND RESULTS: Mice with either S1365A (TSC2SA; phospho-null) or S1365E (TSC2SE; phosphomimetic) knockin mutations were studied ex vivo and in vivo. In response to IR, hearts from TSC2SA mice had amplified mTORC1 activation and improved heart function compared with wild-type and TSC2SE hearts. The magnitude of protection matched IPC. IPC requited less S1365 phosphorylation, as TSC2SE hearts gained no benefit and failed to activate mTORC1 with IPC. IR metabolism was altered in TSC2SA, with increased mitochondrial oxygen consumption rate and glycolytic capacity (stressed/maximal extracellular acidification) after myocyte hypoxia-reperfusion. In whole heart, lactate increased and long-chain acylcarnitine levels declined during ischemia. The relative IR protection in TSC2SA was lost by lowering glucose in the perfusate by 36%. Adding fatty acid (palmitate) compensated for reduced glucose in wild type and TSC2SE but not TSC2SA which had the worst post-IR function under these conditions. CONCLUSIONS: TSC2-S1365 phosphorylation status regulates myocardial substrate utilization, and its decline activates mTORC1 biasing metabolism away from fatty acid oxidation to glycolysis to confer protection against IR. This pathway is also engaged and reduced TSC2 S1365 phosphorylation required for effective IPC. Graphic Abstract: A graphic abstract is available for this article.

CXCR4-overexpressed exosomes from cardiosphere-derived cells attenuate myocardial ischemia/reperfusion injury by transferring miRNA to macrophages and regulating macrophage polarization.

Cardiosphere-derived cells (CDCs) are emerging as ideal candidates for managing cardiac inflammation, albeit with some limitations. Recent literatures have indicated that exosomes secreted by CDCs with C-X-C motif chemokine receptor 4 (CXCR4) overexpression can promote cardiac function after myocardial infarction and there have been some reports of miRNAs involved in ischemia/reperfusion (I/R) therapy. Therefore, we are interested in the role of CXCR4-overexpressed CDC-derived exosomes in delivering specific miRNA after myocardial I/R injury. In this research, we first constructed CDC-derived exosomes that overexpressed CXCR4 and miR-27a-5p, miR-182, or miR-101a. Then, we co-cultured the engineered exosomes with RAW264.7 cells and injected them intravenously into myocardial I/R model mice. In vitro, results showed that proinflammatory cytokines levels in the culture supernatant were decreased and the expression of M2 phenotypic markers were increased. Administration of engineered exosomes improved cardiac function, reduced infarct size, alleviated macrophage infiltration, and regulated M2 macrophage polarization after myocardial I/R, suggesting their implications in cardiac injury repair.

Magnetic vagus nerve stimulation alleviates myocardial ischemia-reperfusion injury by the inhibition of pyroptosis through the M2AChR/OGDHL/ROS axis in rats.

BACKGROUND: Myocardial ischemia-reperfusion (I/R) injury is accompanied by an imbalance in the cardiac autonomic nervous system, characterized by over-activated sympathetic tone and reduced vagal nerve activity. In our preceding study, we pioneered the development of the magnetic vagus nerve stimulation (mVNS) system. This system showcased precise vagus nerve stimulation, demonstrating remarkable effectiveness and safety in treating myocardial infarction. However, it remains uncertain whether mVNS can mitigate myocardial I/R injury and its specific underlying mechanisms. In this study, we utilized a rat model of myocardial I/R injury to delve into the therapeutic potential of mVNS against this type of injury. RESULTS: Our findings revealed that mVNS treatment led to a reduction in myocardial infarct size, a decrease in ventricular fibrillation (VF) incidence and a curbing of inflammatory cytokine release. Mechanistically, mVNS demonstrated beneficial effects on myocardial I/R injury by inhibiting NLRP3-mediated pyroptosis through the M2AChR/OGDHL/ROS axis. CONCLUSIONS: Collectively, these outcomes highlight the promising potential of mVNS as a treatment strategy for myocardial I/R injury.

Loss of PKA regulatory subunit 1α aggravates cardiomyocyte necrosis and myocardial ischemia/reperfusion injury.

Reperfusion therapy, the standard treatment for acute myocardial infarction, can trigger necrotic death of cardiomyocytes and provoke ischemia/reperfusion (I/R) injury. However, signaling pathways that regulate cardiomyocyte necrosis remain largely unknown. Our recent genome-wide RNAi screen has identified a potential necrosis suppressor gene PRKAR1A, which encodes PKA regulatory subunit 1α (R1α). R1α is primarily known for regulating PKA activity by sequestering PKA catalytic subunits in the absence of cAMP. Here, we showed that depletion of R1α augmented cardiomyocyte necrosis in vitro and in vivo, resulting in exaggerated myocardial I/R injury and contractile dysfunction. Mechanistically, R1α loss downregulated the Nrf2 antioxidant transcription factor and aggravated oxidative stress following I/R. Degradation of the endogenous Nrf2 inhibitor Keap1 through p62-dependent selective autophagy was blocked by R1α depletion. Phosphorylation of p62 at Ser349 by mammalian target of rapamycin complex 1 (mTORC1), a critical step in p62-Keap1 interaction, was induced by I/R, but diminished by R1α loss. Activation of PKA by forskolin or isoproterenol almost completely abolished hydrogen-peroxide-induced p62 phosphorylation. In conclusion, R1α loss induces unrestrained PKA activation and impairs the mTORC1-p62-Keap1-Nrf2 antioxidant defense system, leading to aggravated oxidative stress, necrosis, and myocardial I/R injury. Our findings uncover a novel role of PKA in oxidative stress and necrosis, which may be exploited to develop new cardioprotective therapies.

Oxidation of Protein Kinase A Regulatory Subunit PKARIα Protects Against Myocardial Ischemia-Reperfusion Injury by Inhibiting Lysosomal-Triggered Calcium Release.

BACKGROUND: Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, PKARIα (type-1 protein kinase A) can be reversibly oxidized, forming interprotein disulfide bonds in the holoenzyme complex. However, the effect of PKARIα disulfide formation on downstream signaling in the heart, particularly under states of oxidative stress such as ischemia and reperfusion (I/R), remains unexplored. METHODS: Atrial tissue obtained from patients before and after cardiopulmonary bypass and reperfusion and left ventricular (LV) tissue from mice subjected to I/R or sham surgery were used to assess PKARIα disulfide formation by immunoblot. To determine the effect of disulfide formation on PKARIα catalytic activity and subcellular localization, live-cell fluorescence imaging and stimulated emission depletion super-resolution microscopy were performed in prkar1 knock-out mouse embryonic fibroblasts, neonatal myocytes, or adult LV myocytes isolated from "redox dead" (Cys17Ser) PKARIα knock-in mice and their wild-type littermates. Comparison of intracellular calcium dynamics between genotypes was assessed in fura2-loaded LV myocytes, whereas I/R-injury was assessed ex vivo. RESULTS: In both humans and mice, myocardial PKARIα disulfide formation was found to be significantly increased (2-fold in humans, P=0.023; 2.4-fold in mice, P<0.001) in response to I/R in vivo. In mouse LV cardiomyocytes, disulfide-containing PKARIα was not found to impact catalytic activity, but instead led to enhanced AKAP (A-kinase anchoring protein) binding with preferential localization of the holoenzyme to the lysosome. Redox-dependent regulation of lysosomal two-pore channels by PKARIα was sufficient to prevent global calcium release from the sarcoplasmic reticulum in LV myocytes, without affecting intrinsic ryanodine receptor leak or phosphorylation. Absence of I/R-induced PKARIα disulfide formation in "redox dead" knock-in mouse hearts resulted in larger infarcts (2-fold, P<0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, P<0.001), which was prevented by administering the lysosomal two-pore channel inhibitor Ned-19 at the time of reperfusion. CONCLUSIONS: Disulfide modification targets PKARIα to the lysosome, where it acts as a gatekeeper for two-pore channel-mediated triggering of global calcium release. In the postischemic heart, this regulatory mechanism is critical for protection from extensive injury and offers a novel target for the design of cardioprotective therapeutics.

Therapeutic Inhibition of Acid-Sensing Ion Channel 1a Recovers Heart Function After Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection through the purinergic P2Y13 receptor and nitric oxide signaling.

Red blood cells (RBCs) are suggested to play a role in cardiovascular regulation by exporting nitric oxide (NO) bioactivity and ATP under hypoxia. It remains unknown whether such beneficial effects of RBCs are protective in patients with acute myocardial infarction. We investigated whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia-reperfusion injury and whether such effect involves NO and purinergic signaling in the RBCs. RBCs from patients with STEMI undergoing primary coronary intervention and healthy controls were administered to isolated rat hearts subjected to global ischemia and reperfusion. Compared to RBCs from healthy controls, RBCs from STEMI patients reduced myocardial infarct size (30 ± 12% RBC healthy vs. 11 ± 5% RBC STEMI patients, P < 0.001), improved recovery of left-ventricular developed pressure and dP/dt and reduced left-ventricular end-diastolic pressure in hearts subjected to ischemia-reperfusion. Inhibition of RBC NO synthase with L-NAME or soluble guanylyl cyclase (sGC) with ODQ, and inhibition of cardiac protein kinase G (PKG) abolished the cardioprotective effect. Furthermore, the non-selective purinergic P2 receptor antagonist PPADS but not the P1 receptor antagonist 8PT attenuated the cardioprotection induced by RBCs from STEMI patients. The P2Y13 receptor was expressed in RBCs and the cardioprotection was abolished by the P2Y13 receptor antagonist MRS2211. By contrast, perfusion with PPADS, L-NAME, or ODQ prior to RBCs administration failed to block the cardioprotection induced by RBCs from STEMI patients. Administration of RBCs from healthy subjects following pre-incubation with an ATP analog reduced infarct size from 20 ± 6 to 7 ± 2% (P < 0.001), and this effect was abolished by ODQ and MRS2211. This study demonstrates a novel function of RBCs in STEMI patients providing protection against myocardial ischemia-reperfusion injury through the P2Y13 receptor and the NO-sGC-PKG pathway.

N-Cadherin Overexpression Mobilizes the Protective Effects of Mesenchymal Stromal Cells Against Ischemic Heart Injury Through a ß-Catenin-Dependent Manner.

RATIONALE: Mesenchymal stromal cell-based therapy is promising against ischemic heart failure. However, its efficacy is limited due to low cell retention and poor paracrine function. A transmembrane protein capable of enhancing cell-cell adhesion, N-cadherin garnered attention in the field of stem cell biology only recently. OBJECTIVE: The current study investigates whether and how N-cadherin may regulate mesenchymal stromal cells retention and cardioprotective capability against ischemic heart failure. METHODS AND RESULTS: Adult mice-derived adipose tissue-derived mesenchymal stromal cells (ADSC) were transfected with adenovirus harboring N-cadherin, T-cadherin, or control adenovirus. CM-DiI-labeled ADSC were intramyocardially injected into the infarct border zone at 3 sites immediately after myocardial infarction (MI) or myocardial ischemia/reperfusion. ADSC retention/survival, cardiomyocyte apoptosis/proliferation, capillary density, cardiac fibrosis, and cardiac function were determined. Discovery-driven/cause-effect analysis was used to determine the molecular mechanisms. Compared with ADSC transfected with adenovirus-control, N-cadherin overexpression (but not T-cadherin) markedly increased engrafted ADSC survival/retention up to 7 days post-MI. Histological analysis revealed that ADSC transfected with adenovirus-N-cadherin significantly preserved capillary density and increased cardiomyocyte proliferation and moderately reduced cardiomyocyte apoptosis 3 days post-MI. More importantly, ADSC transfected with adenovirus-N-cadherin (but not ADSC transfected with adenovirus-T-cadherin) significantly increased left ventricular ejection fraction and reduced fibrosis in both MI and myocardial ischemia/reperfusion mice. In vitro experiments demonstrated that N-cadherin overexpression promoted ADSC-cardiomyocyte adhesion and ADSC migration, enhancing their capability to increase angiogenesis and cardiomyocyte proliferation. MMP (matrix metallopeptidases)-10/13 and HGF (hepatocyte growth factor) upregulation is responsible for N-cadherin's effect upon ADSC migration and paracrine angiogenesis. N-cadherin overexpression promotes cardiomyocyte proliferation by HGF release. Mechanistically, N-cadherin overexpression significantly increased N-cadherin/ß-catenin complex formation and active ß-catenin levels in the nucleus. ß-catenin knockdown abolished N-cadherin overexpression-induced MMP-10, MMP-13, and HGF expression and blocked the cellular actions and cardioprotective effects of ADSC overexpressing N-cadherin. CONCLUSIONS: We demonstrate for the first time that N-cadherin overexpression enhances mesenchymal stromal cells-protective effects against ischemic heart failure via ß-catenin-mediated MMP-10/MMP-13/HGF expression and production, promoting ADSC/cardiomyocyte adhesion and ADSC retention.

Role of miRNA-1 and miRNA-21 in Acute Myocardial Ischemia-Reperfusion Injury and Their Potential as Therapeutic Strategy.

Coronary artery disease remains the leading cause of death. Acute myocardial infarction (MI) is characterized by decreased blood flow to the coronary arteries, resulting in cardiomyocytes death. The most effective strategy for treating an MI is early and rapid myocardial reperfusion, but restoring blood flow to the ischemic myocardium can induce further damage, known as ischemia-reperfusion (IR) injury. Novel therapeutic strategies are critical to limit myocardial IR injury and improve patient outcomes following reperfusion intervention. miRNAs are small non-coding RNA molecules that have been implicated in attenuating IR injury pathology in pre-clinical rodent models. In this review, we discuss the role of miR-1 and miR-21 in regulating myocardial apoptosis in ischemia-reperfusion injury in the whole heart as well as in different cardiac cell types with special emphasis on cardiomyocytes, fibroblasts, and immune cells. We also examine therapeutic potential of miR-1 and miR-21 in preclinical studies. More research is necessary to understand the cell-specific molecular principles of miRNAs in cardioprotection and application to acute myocardial IR injury.

M2b macrophages stimulate lymphangiogenesis to reduce myocardial fibrosis after myocardial ischaemia/reperfusion injury.

CONTEXT: Therapeutic lymphangiogenesis is a new treatment for cardiovascular diseases. Our previous study showed M2b macrophages can alleviate myocardial ischaemia/reperfusion injury (MI/RI). However, the relation between M2b macrophages and lymphangiogenesis is not clear. OBJECTIVE: To investigate the effects of M2b macrophages on lymphangiogenesis after MI/RI. MATERIALS AND METHODS: Forty male Sprague-Dawley (SD) rats were randomized into Sham operation group (control, n = 8), MI/RI group (n = 16) and M2b macrophage transplantation group (n = 16). M2b macrophages (1 × 106) in 100 µL of normal saline or the same volume of vehicle was injected into the cardiac ischaemic zone. Two weeks later, echocardiography and lymphatic counts were performed, and the extent of myocardial fibrosis and the expression of vascular endothelial growth factor C (VEGFC) and VEGF receptor 3 (VEGFR3) were determined. In vitro, lymphatic endothelial cells (LECs) were cultured with M2b macrophages for 6-24 h, and the proliferation, migration and tube formation of the LECs were assessed. RESULTS: In vivo, M2b macrophage transplantation increased the level of lymphangiogenesis 2.11-fold, reduced 4.42% fibrosis, improved 18.65% left ventricular ejection fraction (LVEF) and upregulated the expressions of VEGFC and VEGFR3. In vitro, M2b macrophage increased the proliferation, migration, tube formation and VEGFC expression of LECs. M2b macrophage supernatant upregulated VEGFR3 expression of LECs. DISCUSSION AND CONCLUSIONS: Our study shows that M2b macrophages can promote lymphangiogenesis to reduce myocardial fibrosis and improve heart function, suggesting the possible use of M2b macrophage for myocardial protection therapy.

Mitochondrial transplantation in cardiomyocytes: foundation, methods, and outcomes.

Mitochondrial transplantation is emerging as a novel cellular biotherapy to alleviate mitochondrial damage and dysfunction. Mitochondria play a crucial role in establishing cellular homeostasis and providing cell with the energy necessary to accomplish its function. Owing to its endosymbiotic origin, mitochondria share many features with their bacterial ancestors. Unlike the nuclear DNA, which is packaged into nucleosomes and protected from adverse environmental effects, mitochondrial DNA are more prone to harsh environmental effects, in particular that of the reactive oxygen species. Mitochondrial damage and dysfunction are implicated in many diseases ranging from metabolic diseases to cardiovascular and neurodegenerative diseases, among others. While it was once thought that transplantation of mitochondria would not be possible due to their semiautonomous nature and reliance on the nucleus, recent advances have shown that it is possible to transplant viable functional intact mitochondria from autologous, allogenic, and xenogeneic sources into different cell types. Moreover, current research suggests that the transplantation could positively modulate bioenergetics and improve disease outcome. Mitochondrial transplantation techniques and consequences of transplantation in cardiomyocytes are the theme of this review. We outline the different mitochondrial isolation and transfer techniques. Finally, we detail the consequences of mitochondrial transplantation in the cardiovascular system, more specifically in the context of cardiomyopathies and ischemia.

Exosomes derived from MSC pre-treated with oridonin alleviates myocardial IR injury by suppressing apoptosis via regulating autophagy activation.

This study aimed to investigate the molecular mechanisms underlying the role of bone marrow mesenchymal stem cells (BMMSCs)-derived exosomes in ischaemia/reperfusion (IR)-induced damage, and the role of oridonin in the treatment of IR. Exosomes were isolated from BMMSCs. Western blot analysis was done to examine the expression of proteins including CD63, CD8, apoptotic-linked gene product 2 interacting protein X (AliX), Beclin-1, ATG13, B-cell lymphoma-2 (Bcl-2), apoptotic peptidase activating factor 1 (Apaf1) and Bcl2-associated X (Bax) in different treatment groups. Accordingly, the expression of CD63, CD81 and AliX was higher in BMMSCs-EXOs and IR + BMMSCs-EXOs + ORI groups compared with that in the BMMSCs group. And BMMSCs-derived exosomes inhibited the progression of IR-induced myocardial damage, while this protective effect was boosted by the pre-treatment with oridonin. Moreover, Beclin-1, ATG13 and Bcl-2 were significantly down-regulated while Apaf1 and Bax were significantly up-regulated in IR rats. And the presence of BMMSCs-derived exosomes partly alleviated IR-induced dysregulation of these proteins, while the oridonin pre-treatment boosted the effect of these BMMSCs-derived exosomes. The inhibited proliferation and promoted apoptosis of H9c2 cells induced by hypoxia/reperfusion (HR) were mitigated by the administration of BMMSCs-derived exosomes. Meanwhile, HR also induced down-regulation of Beclin-1, ATG13 and Bcl-2 expression and up-regulation of Apaf1 and Bax, which were mitigated by the administration of BMMSCs-derived exosomes. And oridonin pre-treatment boosted the effect of BMMSCs-derived exosomes. In conclusion, our results validated that BMMSCs-derived exosomes suppressed the IR-induced damages by participating in the autophagy process, while the pre-treatment with oridonin could boost the protective effect of BMMSCs-derived exosomes.

Guidelines for in vivo mouse models of myocardial infarction.

Despite significant improvements in reperfusion strategies, acute coronary syndromes all too often culminate in a myocardial infarction (MI). The consequent MI can, in turn, lead to remodeling of the left ventricle (LV), the development of LV dysfunction, and ultimately progression to heart failure (HF). Accordingly, an improved understanding of the underlying mechanisms of MI remodeling and progression to HF is necessary. One common approach to examine MI pathology is with murine models that recapitulate components of the clinical context of acute coronary syndrome and subsequent MI. We evaluated the different approaches used to produce MI in mouse models and identified opportunities to consolidate methods, recognizing that reperfused and nonreperfused MI yield different responses. The overall goal in compiling this consensus statement is to unify best practices regarding mouse MI models to improve interpretation and allow comparative examination across studies and laboratories. These guidelines will help to establish rigor and reproducibility and provide increased potential for clinical translation.

Transplantation of Human Umbilical Cord Blood-Derived Cellular Fraction Improves Left Ventricular Function and Remodeling After Myocardial Ischemia/Reperfusion.

Rationale: Human umbilical cord blood (hUCB) contains diverse populations of stem/progenitor cells. Whether hUCB-derived nonhematopoietic cells would induce cardiac repair remains unknown. Objective: To examine whether intramyocardial transplantation of hUCB-derived CD45-Lin- nonhematopoietic cellular fraction after a reperfused myocardial infarction in nonimmunosuppressed rats would improve cardiac function and ameliorate ventricular remodeling. Methods and Results: Nonhematopoietic CD45-Lin- cells were isolated from hUCB. Flow cytometry and quantitative polymerase chain reaction were used to characterize this subpopulation. Age-matched male Fischer 344 rats underwent a 30-minute coronary occlusion followed by reperfusion and 48 hours later received intramyocardial injection of vehicle or hUCB CD45-Lin- cells. After 35 days, compared with vehicle-treated rats, CD45-Lin- cell-treated rats exhibited improved left ventricular function, blunted left ventricular hypertrophy, greater preservation of viable myocardium in the infarct zone, and superior left ventricular remodeling. Mechanistically, hUCB CD45-Lin- cell injection favorably modulated molecular pathways regulating myocardial fibrosis, cardiomyocyte apoptosis, angiogenesis, and inflammation in postinfarct ventricular myocardium. Rare persistent transplanted human cells could be detected at both 4 and 35 days after myocardial infarction. Conclusions: Transplantation of hUCB-derived CD45-Lin- nonhematopoietic cellular subfraction after a reperfused myocardial infarction in nonimmunosuppressed rats ameliorates left ventricular dysfunction and improves remodeling via favorable paracrine modulation of molecular pathways. These findings with human cells in a clinically relevant model of myocardial ischemia/reperfusion in immunocompetent animals may have significant translational implications.Visual Overview: An online visual overview is available for this article.

microRNA-129 overexpression in endothelial cell-derived extracellular vesicle influences inflammatory response caused by myocardial ischemia/reperfusion injury.

Extracellular vesicles (EVs) have the potency to function as modulators in the process of myocardial ischemia/reperfusion (I/R) injury. This investigation was performed to decipher the mechanism of human umbilical vascular endothelial cells (HUVECs)-derived EVs in myocardial I/R injury with the involvement of microRNA-129 (miR-129). HUVECs-secreted EVs were collected and identified. An I/R mouse model was developed, and cardiomyocytes were used for vitro oxygen-glucose deprivation/reperfusion model establishment. Differentially expressed miRNAs in myocardial tissues after EV treatment were assessed using microarray analysis. The target relationship between miR-129 and toll-like receptor 4 (TLR4) was identified using a dual-luciferase assay. Gain- and loss-function studies regarding miR-129 were implemented to figure out its roles in myocardial I/R injury. Meanwhile, the activation of the nuclear factor-kappa-binding (NF-κB) p65 signaling and NOD-like receptor 3 (NLRP3) inflammasome was evaluated. EVs diminished the apoptosis of cardiomyocytes and the secretion of inflammatory factors, and all these trends were reversed by miR-129 reduction. miR-129 bound to the 3'-untranslated region of TLR4 directly. The NF-κB p65 signaling and NLRP3 inflammasome were abnormally activated after I/R injury, whose impairment after EVs was partially restored by miR-129 downregulation. This study illustrated that EVs could carry miR-129 to mitigate myocardial I/R injury via downregulating TLR4 and disrupting the NF-κB signaling and NLRP3 inflammasome.

Carbon Monoxide-Saturated Polymerized Placenta Hemoglobin Optimizes Mitochondrial Function and Protects Heart Against Ischemia-Reperfusion Injury.

ABSTRACT: Ischemia-reperfusion (I-R) injury is detrimental to cardiovascular system. This study was designed to investigate whether carbon monoxide-saturated polymerized human placenta hemoglobin (CO-PolyPHb) attenuates cardiac I-R injury and to elucidate the underlying mechanism(s). Sixty male adult Sprague-Dawley rats were randomly divided into 6 groups: saline + sham group, PolyPHb + sham group, CO-PolyPHb + sham group, saline + I-R group, PolyPHb + I-R group, and CO-PolyPHb + I-R group. Rats were pretreated with injection of PolyPHb, CO-PolyPHb (0.5 g Hb/kg/d), or an equivalent volume of saline via caudal vein for 3 days. After pretreatment, hearts were isolated Langendorff perfused and subjected to 30-minute no-flow ischemia and 120-minute reperfusion. As compared with the saline + I-R group, pretreatment with CO-PolyPHb greatly improved the recovery of cardiac function, reduced infarct size, and suppressed the release of cardiac enzyme. Importantly, CO-PolyPHb showed more prominent cardioprotective effect than PolyPHb, exhibiting a promising therapeutic potential in cardiac I-R injury. Further study demonstrated that CO-PolyPHb activated molecular signaling toward mitophagy and significantly elevated the mitochondrial respiratory function in the heart. In addition, CO-PolyPHb upregulated the phosphorylation of the proteins in insulin signaling pathway and increased the glucose uptake rate in cardiomyocytes. Pharmacological inhibition of this pathway by wortmannin abrogated the anti-I-R effect of CO-PolyPHb. In conclusion, using an isolated rat heart model, we have demonstrated that pretreatment with CO-PolyPHb provided protective effect against cardiac I-R injury, and this protection was mediated by the improvement of mitochondrial function and activation of insulin signaling pathway in the heart.

Effects of inorganic nitrate on ischaemia-reperfusion injury after coronary artery bypass surgery: a randomised controlled trial.

BACKGROUND: Nitric oxide (NO) is an important signalling molecule in the cardiovascular system with protective properties in ischaemia-reperfusion injury. Inorganic nitrate, an oxidation product of endogenous NO production and a constituent in our diet, can be recycled back to bioactive NO. We investigated if preoperative administration of inorganic nitrate could reduce troponin T release and other plasma markers of injury to the heart, liver, kidney, and brain in patients undergoing cardiac surgery. METHODS: This single-centre, randomised, double-blind, placebo-controlled trial included 82 patients undergoing coronary artery bypass surgery with cardiopulmonary bypass. Oral sodium nitrate (700 mg×2) or placebo (NaCl) were administered before surgery. Biomarkers of ischaemia-reperfusion injury and plasma nitrate and nitrite were collected before and up to 72 h after surgery. Troponin T release was our predefined primary endpoint and biomarkers of renal, liver, and brain injury were secondary endpoints. RESULTS: Plasma concentrations of nitrate and nitrite were elevated in nitrate-treated patients compared with placebo. The 72-h release of troponin T did not differ between groups. Other plasma biomarkers of organ injury were also similar between groups. Blood loss was not a predefined outcome parameter, but perioperative bleeding was 18% less in nitrate-treated patients compared with controls. CONCLUSION: Preoperative administration of inorganic nitrate did not influence troponin T release or other plasma biomarkers of organ injury in cardiac surgery. CLINICAL TRIAL REGISTRATION: NCT01348971.

Modification of ischemia/reperfusion induced infarct size by ischemic preconditioning in hypertrophied hearts.

This study examined the effects of ischemic preconditioning (IP) on the ischemia/reperfusion (I/R) induced injury in normal and hypertrophied hearts. Cardiac hypertrophy in rabbits was induced by L-thyroxine (0.5 mg/kg/day for 16 days). Hearts with or without IP (3 cycles of 5 min ischemia and 10 min reperfusion) were subjected to I/R (60 min ischemia followed by 60 min reperfusion). IP reduced the I/R-induced infarct size from 68% to 24% and 57% to 33% in the normal and hypertrophied hearts, respectively. Leakage of creatine phosphokinase in the perfusate from the hypertrophied hearts due to I/R was markedly less than that form the normal hearts; IP prevented these changes. Although IP augmented the increase in phosphorylated p38-mitogen-activated protein kinase (p38-MAPK) content due to I/R, this effect was less in the hypertrophied than in the normal heart. These results suggest that reduced cardioprotection by IP of the I/R-induced injury in hypertrophied hearts may be due to reduced activation of p38-MAPK in comparison with normal hearts.

miR-19a mitigates hypoxia/reoxygenation-induced injury by depressing CCL20 and inactivating MAPK pathway in human embryonic cardiomyocytes.

OBJECTIVE: Myocardial infarction (MI) is a prevalent cardiovascular puzzle and a mainspring of disease-induced mortality. We performed this investigation to detect the role of putative important miRNAs or genes in MI. RESULTS: CCL20 may be a potential therapeutic target, which was directly targeted and negatively regulated by miR-19a. CCL20 expression was significantly increased in MI tissue samples, but miR-19a was expressed at lower levels in MI. H/R treatment inhibited cell viability and induced an increase of apoptotic rate compared with Sham group. However, miR-19a mimic relieved the H/R-stimulated injury to cardiomyocytes. Protective effect of miR-19a against H/R in cardiomyocytes was reversed by CCL20 enhancement, and MAPK pathway was inactivated during this progression. CONCLUSIONS: miR-19a eliminates the H/R-induced injury in cardiomyocytes through directly targeting CCL20 and attenuating the activity of MAPK signaling pathway. These observations highlighted the therapeutic roles of miR-19a and CCL20 for MI treatment.

Treatment of Oxidative Stress with Exosomes in Myocardial Ischemia.

A thrombus in a coronary artery causes ischemia, which eventually leads to myocardial infarction (MI) if not removed. However, removal generates reactive oxygen species (ROS), which causes ischemia-reperfusion (I/R) injury that damages the tissue and exacerbates the resulting MI. The mechanism of I/R injury is currently extensively understood. However, supplementation of exogenous antioxidants is ineffective against oxidative stress (OS). Enhancing the ability of endogenous antioxidants may be a more effective way to treat OS, and exosomes may play a role as targeted carriers. Exosomes are nanosized vesicles wrapped in biofilms which contain various complex RNAs and proteins. They are important intermediate carriers of intercellular communication and material exchange. In recent years, diagnosis and treatment with exosomes in cardiovascular diseases have gained considerable attention. Herein, we review the new findings of exosomes in the regulation of OS in coronary heart disease, discuss the possibility of exosomes as carriers for the targeted regulation of endogenous ROS generation, and compare the advantages of exosome therapy with those of stem-cell therapy. Finally, we explore several miRNAs found in exosomes against OS.