The role of the large conductance Ca2+-activated K+ channel in adenosine receptor-mediated cytoprotection

The rat embryonic cardiomyoblast-derived H9c2 cell line is increasingly used for studies into cardioprotection, as these cells display similar properties to primary cardiomyocytes. Adenosine receptors are well known mediators of cardioprotection and trigger effectors such as the mitochondrial KATP channel – however, the role of the mitochondrial BKCa channel in adenosine receptor-mediated cardioprotection has not been investigated. GPCR assays provided evidence for functional expression of Gi-coupled adenosine A1 and κ-opioid receptors, Gs-coupled β2 adrenergic receptors and Gq-coupled UTP-binding P2Y purinergic receptors on H9c2 cells. Activation of the adenosine A1 receptor with CPA (N(6)-cyclopentyladenosine) provided significant protection against hypoxia-induced cell death in these cells, as did opening of a BKCa channel with NS1619. The location of this BKCa channel was confirmed to be the mitochondria by the probing of subcellular fractions with BKCa-specific antibodies. Interestingly, CPA-induced protection against hypoxia was blocked by inhibition of the BKCa channel

The role of signal transducer and activator of transcription-3 (STAT-3) in ischaemic and pharmacological preconditioning

Includes bibliographical references (leaves 150-177).Ischemic preconditioning is a protective mechanism whereby repetitive transient ischaemia confer protection against the subsequent prolonged lethal ischaemia that is observed during myocardial infarction. This protective phenomenon that lessens myocardial infarction can be mimicked by the cytokine tumor necrosis factor alpha (TNFα) but the signalling pathways involved still remain unclear

The impact of age on the ability of preconditioning and pharmacological intervention to protect the heart from ischaemic and reperfusion injury.

Introduction: Ischaemic heart disease is the main cause of morbidity and mortality in the United Kingdom, with the greatest proportion of suffers being elderly. Current treatments are not altogether successful, and there is a need for development of improved therapeutic strategies. One area of interest is ischaemic preconditioning as well as the pharmacological prevention of ischaemic and reperfiision injury through upregulation of various reperfusion induced salvage kinases (e.g. p42/p44 MAPK). However, the effect of senescence on the ability of these treatments to influence outcome of ischaemia / reperfusion injury has been overlooked, and the aims of this study has been to address aspects of changed signalling mechanisms and cardioprotection in the aged heart in addition to exploring the role of p42/p44 MAPK in protecting the heart from reperfusion injury. Methods; Whole heart in vitro rat models of myocardial infarction have been used to investigate differences in ischaemic and pharmacological preconditioning in rats of three age groups (young adults, middle age, old). Using similarly aged rats, experiments on isolated mitochondria have investigated age related changes in mitochondrial membrane potential in response to treatment with the mitochondrial KATP channel opener, diazoxide. Further studies in both rat (young and old) and rabbit in vitro and in vivo models of myocardial infarction have determined the role of the agents urocortin and cardiotrophin-1 in protecting the heart from reperfusion injury through activation of the p42/p44 mitogen activated protein kinase (MAPK) pathway. Results; Progressive age has been shown to negatively affect the ability of preconditioning to protect the heart from myocardial infarction, and mitochondrial studies have supported these findings. The importance of the p42/p44 MAPK pathway has been demonstrated in the protection of young adult rats from reperfusion injury, though this effect has been lost in the aged rat heart. Conclusion: Ischaemic and pharmacological preconditioning, as well as pharmacological intervention at reperfusion are both able to reduce infarct size in the adult heart, however the influence of senescence leads to the abrogation of all modalities of cardioprotection investigated in this study

Signalling pathways involved in TNFα-induced cytoprotection : role of reactive oxygen species

Includes bibliographical references (leaves 73-89).Tumour necrosis factor alpha (TNFa) is a pleiotropic cytokine which has both beneficial and deleterious effects. It has previously been shown in our laboratory that TNFa can mimic ischemic preconditioning (IPC). However, the signalling pathways involved in this protection remain incompletely understood. One potential protective pathway involves the generation of reactive oxygen species (ROS), which are known to be activated by TNFa. It was therefore hypothesized that TNFa-induced cytoprotection requires the generation of ROS. In addition, it was postulated that this ROS generation originates in the mitochondria

The protective role of tumour necrosis factor alpha in the heart

The pleiotropic cytokine tumour necrosis factor alpha (TNFα) is produced by the heart in response to the ischaemic preconditioning (PC) stimulus. We hypothesised that this endogenously produced peptide may play a role in activating the ischaemic PC mediated tolerance towards a subsequent ischaemic insult in muscle cells. To test this and to delineate the downstream signalling cascades mediating this programme we developed classic PC protocols in adherent mature murine C2C12 myotubes and in human cardiac derived Girardi cell lines. The C2C12 myotubes were preconditioned using either one hour of simulated ischaemia (SI) or the PC-mimetic adenosine (0.1 mM) or TNFα (0.5 ng/ml) followed by one hour of reoxygenation followed by an eight hour SI insult. Cell viability was assessed by measuring lactate dehydrogenase (LOH) release. Simulated ischaemia (SI), PC, adenosine and TNFα activated the PC programme and increased cell viability by 40±3%, 28±5% and 36±4% respectively compared to the SI controls (p<0.005 in all experiments, n≥4 x 6 well plates in all groups). Cell viability was also evaluated by the measurement of propidium iodide uptake on flow cytometry. Preconditioning and TNFα enhanced cell viability with a reduction in propidium iodide uptake by 28% and 41 % respectively versus the ischaemic controls. To evaluate whether TNFα activation of the nuclear regulatory protein nuclear factor kappa B (NFₖ B) mediates this myocyte protection, the NFₖ B antagonists diethyldithiocarbamate (DDTC 10mM) or sodium salicylate (SA 100μM) were co-administered with TNFα. The myocyte protective effect of TNF a was significantly decrease with both antagonists, although not completely inhibited/blocked (DDTC - attenuated cell viability by 62 ±6% and SA by 45 ±5% compared to the TNFα preconditioned cells (p <0.05 vs SI controls and p<0.05 vs TNFα PC, with either antagonists). To confirm these data, TNFα was used as a PC-mimetic in the isolated Langendorff perfused rat heart (Langendorff) preparation. Infarct size was used as the end point. In parallel with cell culture studies, TNFα again conferred preconditioning induced cardioprotection with partial abrogation of these effects with the pharmacological antagonists of NFₖ B. Thus, TNFα administration mimics the cytoprotective effects of ischaemic PC in cardiac, skeletal myocytes and in the isolated perfused rat heart. Moreover, these data support the role of TNFα production as an endogenous paracrine / autocrine signalling peptide which promotes myocyte cellular survival, in part, through activation of NFₖ B

Autophagy Induced by Ischemic Preconditioning is Essential for Cardioprotection

Based on growing evidence linking autophagy to preconditioning, we tested the hypothesis that autophagy is necessary for cardioprotection conferred by ischemic preconditioning (IPC). We induced IPC with three cycles of 5 min regional ischemia alternating with 5 min reperfusion and assessed the induction of autophagy in mCherry-LC3 transgenic mice by imaging of fluorescent autophagosomes in cryosections. We found a rapid and significant increase in the number of autophagosomes in the risk zone of the preconditioned hearts. In Langendorff-perfused hearts subjected to an IPC protocol of 3 × 5 min ischemia, we also observed an increase in autophagy within 10 min, as assessed by Western blotting for p62 and cadaverine dye binding. To establish the role of autophagy in IPC cardioprotection, we inhibited autophagy with Tat-ATG5K130R, a dominant negative mutation of the autophagy protein Atg5. Cardioprotection by IPC was reduced in rat hearts perfused with recombinant Tat-ATG5K130R. To extend the potential significance of autophagy in cardioprotection, we also assessed three structurally unrelated cardioprotective agents—UTP, diazoxide, and ranolazine—for their ability to induce autophagy in HL-1 cells. We found that all three agents induced autophagy; inhibition of autophagy abolished their protective effect. Taken together, these findings establish autophagy as an end-effector in ischemic and pharmacologic preconditioning

Role of Mitogen-Activated Protein Kinases in Myocardial Ischemia-Reperfusion Injury during Heart Transplantation

In solid organ transplantation, ischemia/reperfusion (IR) injury during organ procurement, storage and reperfusion is an unavoidable detrimental event for the graft, as it amplifies graft inflammation and rejection. Intracellular mitogen-activated protein kinase (MAPK) signaling pathways regulate inflammation and cell survival during IR injury. The four best-characterized MAPK subfamilies are the c-Jun NH2-terminal kinase (JNK), extracellular signal- regulated kinase-1/2 (ERK1/2), p38 MAPK, and big MAPK-1 (BMK1/ERK5). Here, we review the role of MAPK activation during myocardial IR injury as it occurs during heart transplantation. Most of our current knowledge regarding MAPK activation and cardioprotection comes from studies of preconditioning and postconditioning in nontransplanted hearts. JNK and p38 MAPK activation contributes to myocardial IR injury after prolonged hypothermic storage. p38 MAPK inhibition improves cardiac function after cold storage, rewarming and reperfusion. Small-molecule p38 MAPK inhibitors have been tested clinically in patients with chronic inflammatory diseases, but not in transplanted patients, so far. Organ transplantation offers the opportunity of starting a preconditioning treatment before organ procurement or during cold storage, thus modulating early events in IR injury. Future studies will need to evaluate combined strategies including p38 MAPK and/or JNK inhibition, ERK1/2 activation, pre- or postconditioning protocols, new storage solutions, and gentle reperfusion

An in-vitro assessment of myocardial ischaemia

Ischaemic heart disease is the most common causes of death in the United Kingdom. In addition to the mortality associated with this disease there are the human and economic costs associated with chronic illness. Many strategies for the treatment and prevention of heart disease are in use and under investigation. The consequences of myocardial infarction have been well described but the causes and mechanisms underlying the disease and its sequeli remain largely unknown. In order to investigate the mechanisms of ischaemic injury and the interventions that might lead to new or improved therapeutic strategies a number of model systems have been devised. The work described in this thesis concerns the construction and validation of a new model system. In order to investigate the mechanisms of ischaemic injury at the cellular level a model has been constructed based on the adult cardiac myocyte in culture. In the first series of experiments described in this thesis the characteristics of adult cardiomyocytes in culture were investigated and a model of ischaemia / reperfusion injury was devised. The model was tested to determine the effects of the individual components of the ischaemic milieu, in living tissues, on cells in culture. Combinations of these components that would cause reproducible lethal and sub-lethal stimuli analogous to those observed in other models and in living tissues were also investigated. It was also determined that cells in culture retained the responses to injury found in the in-vivo heart and that these responses could be modified by similar interventions. The second series of experiments described in this study dealt with the potential for gene-transfer to myocardial cells as a strategy for modifying the response of these cells to ischaemic injury. This work was divided into three parts. The first was concerned with finding the optimal gene delivery vehicle for use in cells in culture that would also have utility in other model systems such as the in-vitro or in-vivo heart. In this section of the work, both viral and non-viral gene delivery systems were investigated. The second part of the work involved an assessment of the modification of response to injury of gene transfer techniques per se. The final part of the work was to have looked at the action of the transfer of specific genes on the response to ischaemic injury in the in-vitro model

Role of Mitogen-Activated Protein Kinases in Myocardial Ischemia-Reperfusion Injury during Heart Transplantation

In solid organ transplantation, ischemia/reperfusion (IR) injury during organ procurement, storage and reperfusion is an unavoidable detrimental event for the graft, as it amplifies graft inflammation and rejection. Intracellular mitogen-activated protein kinase (MAPK) signaling pathways regulate inflammation and cell survival during IR injury. The four best-characterized MAPK subfamilies are the c-Jun NH2-terminal kinase (JNK), extracellular signal- regulated kinase-1/2 (ERK1/2), p38 MAPK, and big MAPK-1 (BMK1/ERK5). Here, we review the role of MAPK activation during myocardial IR injury as it occurs during heart transplantation. Most of our current knowledge regarding MAPK activation and cardioprotection comes from studies of preconditioning and postconditioning in nontransplanted hearts. JNK and p38 MAPK activation contributes to myocardial IR injury after prolonged hypothermic storage. p38 MAPK inhibition improves cardiac function after cold storage, rewarming and reperfusion. Small-molecule p38 MAPK inhibitors have been tested clinically in patients with chronic inflammatory diseases, but not in transplanted patients, so far. Organ transplantation offers the opportunity of starting a preconditioning treatment before organ procurement or during cold storage, thus modulating early events in IR injury. Future studies will need to evaluate combined strategies including p38 MAPK and/or JNK inhibition, ERK1/2 activation, pre- or postconditioning protocols, new storage solutions, and gentle reperfusion