Shiang Y. Lim

Inhibiting Mitochondrial Fission Protects the Heart Against Ischemia/Reperfusion Injury

Background— Whether alterations in mitochondrial morphology affect the susceptibility of the heart to ischemia/reperfusion injury is unknown. We hypothesized that modulating mitochondrial morphology protects the heart against ischemia/reperfusion injury. Methods and Results— In response to ischemia, mitochondria in HL-1 cells (a cardiac-derived cell line) undergo fragmentation, a process that is dependent on the mitochondrial fission protein dynamin-related protein 1 (Drp1). Transfection of HL-1 cells with the mitochondrial fusion proteins mitofusin 1 or 2 or with Drp1K38A, a dominant-negative mutant form of Drp1, increased the percentage of cells containing elongated mitochondria (65±4%, 69±5%, and 63±6%, respectively, versus 46±6% in control: n=80 cells per group; P<0.05), decreased mitochondrial permeability transition pore sensitivity (by 2.4±0.5-, 2.3±0.7-, and 2.4±0.3-fold, respectively; n=80 cells per group; P<0.05), and reduced cell death after simulated ischemia/reperfusion injury (11.6±3.9%, 16.2±3.9%, and 12.1±2.9%, respectively, versus 41.8±4.1% in control: n=320 cells per group; P<0.05). Treatment of HL-1 cells with mitochondrial division inhibitor-1, a pharmacological inhibitor of Drp1, replicated these beneficial effects. Interestingly, elongated interfibrillar mitochondria were identified in the adult rodent heart with confocal and electron microscopy, and in vivo treatment with mitochondrial division inhibitor-1 increased the percentage of elongated mitochondria from 3.6±0.5% to 14.5±2.8% (P=0.023). Finally, treatment of adult murine cardiomyocytes with mitochondrial division inhibitor-1 reduced cell death and inhibited mitochondrial permeability transition pore opening after simulated ischemia/reperfusion injury, and in vivo treatment with mitochondrial division inhibitor-1 reduced myocardial infarct size in mice subject to coronary artery occlusion and reperfusion (21.0±2.2% with mitochondrial division inhibitor-1 versus 48.0±4.5% in control; n=6 animals per group; P<0.05). Conclusion— Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury, suggesting a novel pharmacological strategy for cardioprotection.

Comparative Analysis of Paracrine Factor Expression in Human Adult Mesenchymal Stem Cells Derived from Bone Marrow, Adipose, and Dermal Tissue

Human adult mesenchymal stem cells (MSCs) support the engineering of functional tissue constructs by secreting angiogenic and cytoprotective factors, which act in a paracrine fashion to influence cell survival and vascularization. MSCs have been isolated from many different tissue sources, but little is known about how paracrine factor secretion varies between different MSC populations. We evaluated paracrine factor expression patterns in MSCs isolated from adipose tissue (ASCs), bone marrow (BMSCs), and dermal tissues [dermal sheath cells (DSCs) and dermal papilla cells (DPCs)]. Specifically, mRNA expression analysis identified insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor-D (VEGF-D), and interleukin-8 (IL-8) to be expressed at higher levels in ASCs compared with other MSC populations whereas VEGF-A, angiogenin, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF) were expressed at comparable levels among the MSC populations examined. Analysis of conditioned media (CM) protein confirmed the comparable level of angiogenin and VEGF-A secretion in all MSC populations and showed that DSCs and DPCs produced significantly higher concentrations of leptin. Functional assays examining in vitro angiogenic paracrine activity showed that incubation of endothelial cells in ASC(CM) resulted in increased tubulogenic efficiency compared with that observed in DPC(CM). Using neutralizing antibodies we concluded that VEGF-A and VEGF-D were 2 of the major growth factors secreted by ASCs that supported endothelial tubulogenesis. The variation in paracrine factors of different MSC populations contributes to different levels of angiogenic activity and ASCs maybe preferred over other MSC populations for augmenting therapeutic approaches dependent upon angiogenesis.

Apelin-13 and apelin-36 exhibit direct cardioprotective activity against ischemia-reperfusion injury.

Protection against myocardial ischemia-reperfusion (I/R) injury involves activation of phosphatidylinositol-3-OH kinase (PI3K)- Akt/protein kinase B and p44/42 mitogen-activated protein kinase (MAPK), components of the reperfusion injury salvage kinase (RISK) pathway. The adipocytokine, apelin, activates PI3K-Akt and p44/42 in various tissues and we, therefore, hypothesised that it might demonstrate cardioprotective activity. Employing both in vivo (open-chest) and in vitro (Langendorff and cardiomyocytes) rodent (mouse and rat) models ofmyocardial I/R injury we investigated if apelin administered at reperfusion at concentrations akin to pharmacological doses possesses cardioprotective activity. Apelin-13 and the physiologically less potent peptide, apelin-36, decreased infarct size in vitro by 39.6% (p<0.01) and 26.1% (p<0.05) respectively. In vivo apelin-13 and apelin-36 reduced infarct size by 43.1% (p<0.01) and 32.7% (p<0.05). LY294002 and UO126, inhibitors of PI3K-Akt and p44/42 phosphorylation respectively, abolished the protective effects of apelin-13 in vitro.Western blot analysis provided further evidence for the involvement of PI3K-Akt and p44/42 in the cardioprotective actions of apelin. In addition,mitochondrial permeability transition pore (MPTP) opening was delayed by both apelin- 13 (127%, p<0.01) and apelin-36 (93%, p<0.01) which, in the case of apelin-13, was inhibited by LY294002 and mitogen-activated protein kinase kinase (MEK) inhibitor 1. This is the first study to yield evidence that the adipocytokine, apelin, produces direct cardioprotective actions involving the RISK pathway and the MPTP.

The novel adipocytokine visfatin exerts direct cardioprotective effects.

Visfatin is an adipocytokine capable of mimicking the glucose‐lowering effects of insulin and activating the pro‐survival kinases phosphatidylinositol‐3‐OH kinase (PI3K)‐protein kinase B (Akt) and mitogen‐activated protein kinase kinase 1 and 2 (MEK1/2)‐extracellular signal‐regulated kinase 1 and 2 (Erk 1/2). Experimental studies have demonstrated that the activation of these kinases confers cardioprotection through the inhibition of the mitochondrial permeability transition pore (mPTP). Whether visfatin is capable of exerting direct cardioprotective effects through these mechanisms is unknown and is the subject of the current study. Anaesthetized C57BL/6 male mice were subjected to in situ 30 min. of regional myocardial ischaemia and 120 min. of reperfusion. The administration of an intravenous bolus of visfatin (5 × 10−6μmol) at the time of myocardial reperfusion reduced the myocardial infarct size from 46.1 ± 4.1% in control hearts to 27.3 ± 4.0% (n≥ 6/group, P < 0.05), an effect that was blocked by the PI3K inhibitor, wortmannin, and the MEK1/2 inhibitor, U0126 (48.8 ± 5.5% and 45.9 ± 8.4%, respectively, versus 27.3 ± 4.0% with visfatin; n≥ 6/group, P < 0.05). In murine ventricular cardiomyocytes subjected to 30 min. of hypoxia followed by 30 min. of reoxygenation, visfatin (100 ng/ml), administered at the time of reoxygenation, reduced the cell death from 65.2 ± 4.6% in control to 49.2 ± 3.7%(n > 200 cells/group, P < 0.05), an effect that was abrogated by wortmannin and U0126 (68.1 ± 5.2% and 59.7 ± 6.2%, respectively; n > 200 cells/group, P > 0.05). Finally, the treatment of murine ventricular cardiomyocytes with visfatin (100 ng/ml) delayed the opening of the mPTP induced by oxidative stress from 81.2 ± 4 sec. in control to 120 ± 7 sec. (n > 20 cells/group, P < 0.05) in a PI3K‐ and MEK1/2‐dependent manner. We report that the adipocytokine, visfatin, is capable of reducing myocardial injury when administered at the time of myocardial reperfusion in both the in situ murine heart and the isolated murine cardiomyocytes. The mechanism appears to involve the PI3K and MEK1/2 pathways and the mPTP.

Remote ischemic conditioning: from bench to bedside

Remote ischemic conditioning (RIC) is a therapeutic strategy for protecting organs or tissue against the detrimental effects of acute ischemia-reperfusion injury (IRI). It describes an endogenous phenomenon in which the application of one or more brief cycles of non-lethal ischemia and reperfusion to an organ or tissue protects a remote organ or tissue from a sustained episode of lethal IRI. Although RIC protection was first demonstrated to protect the heart against acute myocardial infarction, its beneficial effects are also seen in other organs (lung, liver, kidney, intestine, brain) and tissues (skeletal muscle) subjected to acute IRI. The recent discovery that RIC can be induced non-invasively by simply inflating and deflating a standard blood pressure cuff placed on the upper arm or leg, has facilitated its translation into the clinical setting, where it has been reported to be beneficial in a variety of cardiac scenarios. In this review article we provide an overview of RIC, the potential underlying mechanisms, and its potential as a novel therapeutic strategy for protecting the heart and other organs from acute IRI.

Mitochondrial cyclophilin-D as a critical mediator of ischaemic preconditioning

Aims It has been suggested that mitochondrial reactive oxygen species (ROS), Akt and Erk1/2 and more recently the mitochondrial permeability transition pore (mPTP) may act as mediators of ischaemic preconditioning (IPC), although the actual interplay between these mediators is unclear. The aim of the present study is to determine whether the cyclophilin-D (CYPD) component of the mPTP is required by IPC to generate mitochondrial ROS and subsequently activate Akt and Erk1/2. Methods and results Mice lacking CYPD (CYPD−/−) and B6Sv129 wild-type (WT) mice were used throughout. We have demonstrated that under basal conditions, non-pathological mPTP opening occurs (indicated by the percent reduction in mitochondrial calcein fluorescence). This effect was greater in WT cardiomyocytes compared with CYPD−/− ones (53 ± 2% WT vs. 17 ± 3% CYPD−/−; P < 0.01) and was augmented by hypoxic preconditioning (HPC) (70 ± 9% WT vs. 56 ± 1% CYPD−/−; P < 0.01). HPC reduced cell death following simulated ischaemia–reperfusion injury in WT (23.2 ± 3.5% HPC vs. 43.7 ± 3.2% WT; P < 0.05) but not CYPD−/− cardiomyocytes (19.6 ± 1.4% HPC vs. 24.4 ± 2.6% control; P > 0.05). HPC generated mitochondrial ROS in WT (four-fold increase; P < 0.05) but not CYPD−/− cardiomyocytes. HPC induced significant Akt phosphorylation in WT cardiomyocytes (two-fold increase; P < 0.05), an effect which was abrogated by ciclosporin-A (a CYPD inhibitor) and N-2-mercaptopropionyl glycine (a ROS scavenger). Finally, in vivo IPC of adult murine hearts resulted in significant phosphorylation of Akt and Erk1/2 in WT but not CYPD−/− hearts. Conclusion The CYPD component of the mPTP is required by IPC to generate mitochondrial ROS and phosphorylate Akt and Erk1/2, major steps in the IPC signalling pathway.

Hypoxic Preconditioning Enhances Survival of Human Adipose-Derived Stem Cells and Conditions Endothelial Cells In Vitro

To grow more robust cardiac tissue for implantation in vivo, strategies to improve survival of implanted stem cells are required. Here we report the protective effects of hypoxic preconditioning (HPC) and identify mechanisms for improving survival of adipose-derived stem cells (ASC) in vitro. Human ASC were preconditioned for 24 h with hypoxia and then exposed to simulated ischemia for a further 24 h. HPC significantly increased ASC viability, and reduced cell injury and apoptosis compared with non-preconditioned cells under ischemic conditions, as shown by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase-release, and caspase activity assays. Preconditioned ASC increased levels of hypoxia-inducible factor-1 alpha and secreted significantly more of the downstream target vascular endothelial growth factor (VEGF-A; 13-fold) compared with control during the 24 h. Exogenous VEGF (50 ng/mL) increased phosphorylation of Akt without affecting ERK1/2, JNK, or p38 MAPK protein levels. Phospho-Akt was also increased in preconditioned ASC compared with non-preconditioned ASC, an effect that may be mediated via VEGF-A. Importantly, the protective effects of HPC were abolished by a neutralizing antibody against VEGF-A and the phosphoinositol 3-kinase inhibitor LY294002, demonstrating the importance of VEGF-A and Akt in hypoxia-induced ASC survival. Importantly, we showed that media derived from hypoxic preconditioned ASC support endothelial cell survival and endothelial tube formation in vitro. Our in vitro findings indicate that HPC may be a promising strategy to improve survival of ASC and promote angiogenesis in ischemic environments.

Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications

Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.

HIF-1 reduces ischaemia–reperfusion injury in the heart by targeting the mitochondrial permeability transition pore

AIMS Hypoxia-inducible factor-1 (HIF-1) has been reported to promote tolerance against acute myocardial ischaemia-reperfusion injury (IRI). However, the mechanism through which HIF-1 stabilization actually confers this cardioprotection is not clear. We investigated whether HIF-1α stabilization protects the heart against acute IRI by preventing the opening of the mitochondrial permeability transition pore (MPTP) and the potential mechanisms involved. METHODS AND RESULTS Stabilization of myocardial HIF-1 was achieved by pharmacological inhibition of prolyl hydroxylase (PHD) domain-containing enzyme using GSK360A or using cardiac-specific ablation of von Hippel-Lindau protein (VHL(fl/fl)) in mice. Treatment of HL-1 cardiac cells with GSK360A stabilized HIF-1, increased the expression of HIF-1 target genes pyruvate dehydrogenase kinase-1 (PDK1) and hexokinase II (HKII), and reprogrammed cell metabolism to aerobic glycolysis, thereby resulting in the production of less mitochondrial oxidative stress during IRI, and less MPTP opening, effects which were shown to be dependent on HKII. These findings were further confirmed when HIF-1 stabilization in the rat and murine heart resulted in smaller myocardial infarct sizes (both in vivo and ex vivo), decreased mitochondrial oxidative stress, and inhibited MPTP opening following IRI, effects which were also found to be dependent on HKII. CONCLUSIONS We have demonstrated that acute HIF-1α stabilization using either a pharmacological or genetic approach protected the heart against acute IRI by promoting aerobic glycolysis, decreasing mitochondrial oxidative stress, activating HKII, and inhibiting MPTP opening.

Mitochondrial cyclophilin-D as a potential therapeutic target for post-myocardial infarction heart failure

The pharmacological inhibition or genetic ablation of cyclophilin‐D (CypD), a critical regulator of the mitochondrial permeability transition pore (mPTP), confers myocardial resistance to acute ischemia‐reperfusion injury, but its role in post‐myocardial infarction (MI) heart failure is unknown. The aim of this study was to determine whether mitochondrial CypD is also a therapeutic target for the treatment of post‐MI heart failure. Wild‐type (WT) and CypD–/– mice were subjected to either sham surgery or permanent ligation of the left main coronary artery to induce MI, and were assessed at either 2 or 28 days to determine the long‐term effects of CypD ablation. After 2 days, myocardial infarct size was smaller and left ventricular (LV) function was better preserved in CypD–/– mice compared to WT mice. After 28 days, when compared to WT mice, in the CypD–/– mice, mortality was halved, myocardial infarct size was reduced, LV systolic function was better preserved, LV dilatation was attenuated and in the remote non‐infarcted myocardium, there was less cardiomyocyte hypertrophy and interstitial fibrosis. Finally, ex vivo fibroblast proliferation was found to be reduced in CypD–/– cardiac fibroblasts, and in WT cardiac fibroblasts treated with the known CypD inhibitors, cyclosporin‐A and sanglifehrin‐A. Following an MI, mice lacking CypD have less mortality, smaller infarct size, better preserved LV systolic function and undergo less adverse LV remodelling. These findings suggest that the inhibition of mitochondrial CypD may be a novel therapeutic treatment strategy for post‐MI heart failure.