Biomedical subjects

Christopher J McLeod

Publications and source records attributed to Christopher J McLeod.

6 recordsLinked to original sources

PubMed2006

Diazoxide-induced respiratory inhibition - a putative mitochondrial K(ATP) channel independent mechanism of pharmacological preconditioning.

The ischemic preconditioning biological phenomenon has been explored to identify putative pharmacologic agents to mimic this cytoprotective program against cellular ischemic injury. Diazoxide administration confers this cytoprotection, however, whether this is via direct activation of the putative mitochondrial K(ATP) (mK(ATP)) channel which was originally proposed has been questioned. Here, we present data supporting an alternate hypothesis evoking mitochondrial respiratory inhibition rather than mK(ATP) channel activation, as a mediating event in the diazoxide-activated cytoprotective program. Mitochondrial respiration and reactive oxygen species (ROS) production was measured in digitonin-permeabilized C2C12 myotubes, allowing for the modulation of mK(ATP) conductance by changing the potassium concentration of the medium (0-130 mM). Diazoxide dose-dependently attenuated succinate-supported respiration, an effect that was independent of mK(ATP) channel conductance. Similarly, 5-hydroxydecanoate (5-HD), a putative mK(ATP) channel blocker, released diazoxide-induced respiratory inhibition independently of potassium concentration. Since diazoxide-induced cytoprotection and respiratory inhibition are both integrally linked to ROS generation we repeated above experiments following ROS generation using DCF fluorescence. Cytoprotective doses of diazoxide increased ROS generation independently of potassium concentration and 5-HD inhibited ROS production under the same conditions. Collectively these data support the hypothesis that diazoxide-mediated cytoprotection is independent of the conductance of the mK(ATP) channel and rather implicate mitochondrial respiratory inhibition-triggered ROS signaling.

Adenosine Triphosphate↗

PubMed2005

Uncoupling proteins 2 and 3 function in concert to augment tolerance to cardiac ischemia.

Transient cardiac ischemia activates cell survival signaling, conferring subsequent ischemia tolerance to the heart. This biological phenomenon, termed ischemic preconditioning, results in improved clinical outcome and attenuated infarct size following myocardial infarction. To explore genomic modifications underpinning this ischemia tolerance, we delineated the regulation and function of the cardiac enriched mitochondrial uncoupling proteins 2 and 3 during delayed ischemic preconditioning in the rat. Cardiac transcripts of genes encoding uncoupling proteins 2 and 3 are up-regulated in parallel with infarct size reduction in preconditioned hearts. Mitochondria isolated from preconditioned hearts exhibit an augmented inducible proton leak. In parallel, following anoxia-reoxygenation these mitochondria generate less hydrogen peroxide compared with non-preconditioned mitochondria. Preconditioning in rat cardiac derived myoblasts is abolished following uncoupling protein-2 depletion by RNA-interference. RNAi of uncoupling protein-3 partially attenuates the capacity to precondition these cells. Functional characterization of anoxia and reoxygenation tolerance following uncoupling protein 2 or 3 and combined 2 and 3 RNAi shows the largest reduction in viability follows depletion of both homologues. Uncoupling protein-2 depletion alone significantly attenuates anoxia-reoxygenation tolerance but uncoupling protein-3 depletion does not reduce anoxia tolerance. In parallel combined uncoupling protein depletion and isolated uncoupling protein-2 depletion augments ROS production in viable cardiomyocytes following anoxia-reoxygenation. Concurrent anti-oxidant administration ameliorates the uncoupling protein-depleted anoxia-susceptible phenotype. In conclusion, mitochondrial uncoupling proteins are necessary components of ischemia tolerance and function as components of the cellular antioxidant defense program. In the cytoprotective hierarchy, uncoupling protein-2 appears to play a greater role than uncoupling protein-3 in modulating ischemia/anoxia tolerance in heart-derived cells.

Animals↗

PubMed2005

The mitochondrial biogenesis regulatory program in cardiac adaptation to ischemia--a putative target for therapeutic intervention.

The resurgence of mitochondrial biology research stems from the realization that the distinct regulation of mitochondria to meet diverse homeostatic demands is driven by exquisite biochemical and molecular control mechanisms. This program termed mitochondrial biogenesis is integral to orchestrating mitochondrial function and appears to exhibit adaptive remodeling following biomechanical and oxidative stress. The major bioenergetic function of mitochondria partitions the final utilization of oxygen between oxidative phosphorylation and reactive oxygen species. As disruption in oxidative phosphorylation and excessive reactive oxygen species contribute to cardiac ischemia-reperfusion injury, we hypothesize that the mitochondrial biogenesis regulatory program is an explicit target for cardiac therapeutic interventions. The objectives of this review are to (a) define the advances in understanding the mitochondrial biogenesis regulatory program integrated to its control of mitochondrial bioenergetics and oxygen utilization, (b) reveal how this program is modulated by chronic hypoxia and ischemic preconditioning, and (c) examine the therapeutic potential of modulating the regulation of mitochondrial biogenesis as a strategy to attenuate ischemia-reperfusion injury.

Adaptation, Physiological↗

PubMed2005

PKCepsilon activation augments cardiac mitochondrial respiratory post-anoxic reserve--a putative mechanism in PKCepsilon cardioprotection.

Modest cardiac-overexpression of constitutively active PKCepsilon (aPKCepsilon) in transgenic mice evokes cardioprotection against ischemia. As aPKCepsilon interacts with mitochondrial respiratory-chain proteins we hypothesized that aPKCepsilon modulates respiration to induce cardioprotection. Using isolated cardiac mitochondria wild-type and aPKCepsilon mice display similar basal mitochondrial respiration, rate of ATP synthesis and adenosine nucleotide translocase (ANT) functional content. Conversely, the aPKCepsilon mitochondria exhibit modest hyperpolarization of their inner mitochondrial membrane potential (DeltaPsi(m)) compared to wild-type mitochondrial by flow cytometry. To assess whether this hyperpolarization engenders resilience to simulated ischemia, anoxia-reoxygenation experiments were performed. Mitochondria were exposed to 45 min anoxia followed by reoxygenation. At reoxygenation, aPKCepsilon mitochondria recovered ADP-dependent respiration to 44 +/- 3% of baseline compared to 28 +/- 2% in WT controls (P = 0.03) in parallel with enhanced ATP synthesis. This preservation in oxidative phosphorylation is coupled to greater ANT functional content [42% > concentration of atractyloside for inhibition in the aPKCepsilon mitochondria vs. WT control (P < 0.0001)], retention of mitochondrial cytochrome c and conservation of DeltaPsi(m). These data demonstrate that mitochondria from PKCepsilon activated mice are intrinsically resilient to anoxia-reoxygenation compared to WT controls. This resilience is in part due to enhanced recovery of oxidative phosphorylation coupled to maintained ANT activity. As maintenance of ATP is a prerequisite for cellular viability we conclude that PKCepsilon activation augmented mitochondrial respiratory capacity in response to anoxia-reoxygenation may contribute to the PKCepsilon cardioprotective program.

Adenosine Triphosphate↗

PubMed2004

Delayed ischemic preconditioning activates nuclear-encoded electron-transfer-chain gene expression in parallel with enhanced postanoxic mitochondrial respiratory recovery.

BACKGROUND: Delayed ischemic preconditioning promotes cardioprotection via genomic reprogramming. We hypothesize that molecular regulation of mitochondrial energetics is integral to this cardioprotective program. METHODS AND RESULTS: Preconditioning was induced by use of 3 episodes of 3-minute coronary artery occlusion separated by 5 minutes of reperfusion. Twenty-four hours later, infarct size was reduced by 58% after preconditioning compared with sham-operated controls (P<0.001). Cardiac mitochondria were isolated from sham and preconditioned rat hearts. Mitochondrial respiration and ATP production were similar between the groups; however, preconditioned mitochondria exhibit modest hyperpolarization of the inner mitochondrial membrane potential (> or =22% versus control, P<0.001). After 35-minute anoxia and reoxygenation, preconditioned mitochondria demonstrated a 191+/-12% improvement in ADP-sensitive respiration (P=0.002) with preservation of electron-transfer-chain (ETC) activity versus controls. This augmented mitochondrial recovery was eradicated when preconditioning was abolished by the antioxidant 2-mercaptopropionyl glycine (2-MPG). These biochemical modulations appear to be regulated at the genomic level in that the expression of genes encoding rate-controlling complexes in the ETC was significantly upregulated in preconditioned myocardium, with a concordant induction of steady-state protein levels of cytochrome oxidase, cytochrome c, and adenine nucleotide translocase-1. 2-MPG abolished preconditioning induction of these transcripts. Moreover, transcripts of nuclear regulatory peptides known to orchestrate mitochondrial biogenesis, nuclear respiratory factor-1 and peroxisome-proliferator-activated receptor gamma coactivator 1alpha, were significantly induced in preconditioned myocardium. CONCLUSIONS: Delayed preconditioned mitochondria display increased tolerance against anoxia-reoxygenation in association with modifications in mitochondrial bioenergetics, with concordant genomic induction of a mitochondrial energetic gene regulatory program. This program appears to be mediated by reactive oxygen species signaling.

Adenosine Triphosphate↗

PubMed2003

Mitochondrial plasticity in classical ischemic preconditioning-moving beyond the mitochondrial KATP channel.

Ischemic preconditioning is a powerful biologic phenomenon that activates innate cell survival programs to protect the heart from ischemic injury. The preponderance of research into classical ischemic preconditioning has focused on signaling pathways orchestrating cardioprotection. Conceptually classified into triggers, mediators and end effectors of preconditioning multiple distinct signaling pathways appear to 'converge' on the mitochondria possibly via activation of the mitochondrial ATP-sensitive potassium (mK(ATP)) channel. The mechanisms by which mK(ATP) channel activation induces preconditioning are incompletely elucidated but include perturbations of mitochondrial architecture and function. Since evidence invoking the mK(ATP) channel has almost exclusively been based on studies using diazoxide and 5-hydroxydecanote the finding that these two compounds have mitochondrial effects independent of the mK(ATP) channel has initiated a controversy regarding the exclusivity of this particular channel in preconditioning. A concerted effort to characterize the mitochondrial phenotype is important to advance our understanding of the mechanistic events that underlie the robust cardioprotective phenotype unmasked by preconditioning. The purpose of this review is to collate the information available on mitochondrial biology associated with classical preconditioning, to delineate the distinct temporal presentation of these mitochondrial perturbations, to reassess the role of the mitochondrial K(ATP) channel and to propose a working model integrating the mitochondrial adaptations into the biology driving this cyto-protective phenotype.

Adaptation, Physiological↗