Dissecting Cellular Mechanisms of Long-Chain Acylcarnitines-Driven Cardiotoxicity: Disturbance of Calcium Homeostasis, Activation of Ca2+-Dependent Phospholipases, and Mitochondrial Energetics Collapse.

Long-chain acylcarnitines (LCAC) are implicated in ischemia-reperfusion (I/R)-induced myocardial injury and mitochondrial dysfunction. Yet, molecular mechanisms underlying involvement of LCAC in cardiac injury are not sufficiently studied. It is known that in cardiomyocytes, palmitoylcarnitine (PC) can induce cytosolic Ca2+ accumulation, implicating L-type calcium channels, Na+/Ca2+ exchanger, and Ca2+-release from sarcoplasmic reticulum (SR). Alternatively, PC can evoke dissipation of mitochondrial potential (ΔΨm) and mitochondrial permeability transition pore (mPTP). Here, to dissect the complex nature of PC action on Ca2+ homeostasis and oxidative phosphorylation (OXPHOS) in cardiomyocytes and mitochondria, the methods of fluorescent microscopy, perforated path-clamp, and mitochondrial assays were used. We found that LCAC in dose-dependent manner can evoke Ca2+-sparks and oscillations, long-living Ca2+ enriched microdomains, and, finally, Ca2+ overload leading to hypercontracture and cardiomyocyte death. Collectively, PC-driven cardiotoxicity involves: (I) redistribution of Ca2+ from SR to mitochondria with minimal contribution of external calcium influx; (II) irreversible inhibition of Krebs cycle and OXPHOS underlying limited mitochondrial Ca2+ buffering; (III) induction of mPTP reinforced by PC-calcium interplay; (IV) activation of Ca2+-dependent phospholipases cPLA2 and PLC. Based on the inhibitory analysis we may suggest that simultaneous inhibition of both phospholipases could be an effective strategy for protection against PC-mediated toxicity in cardiomyocytes.

NMDA receptor modulation of glutamate release in activated neutrophils.

BACKGROUND: Neutrophil depletion improves neurologic outcomes in experimental sepsis/brain injury. We hypothesized that neutrophils may exacerbate neuronal injury through the release of neurotoxic quantities of the neurotransmitter glutamate. METHODS: Real-time glutamate release by primary human neutrophils was determined using enzymatic biosensors. Bacterial and direct protein-kinase C (Phorbol 12-myristate 13-acetate; PMA) activation of neutrophils in human whole blood, isolated neutrophils or human cell lines were compared in the presence/absence of N-Methyl-d-aspartic acid receptor (NMDAR) antagonists. Bacterial and direct activation of neutrophils from wild-type and transgenic murine neutrophils deficient in NMDAR-scaffolding proteins were compared using flow cytometry (phagocytosis, reactive oxygen species (ROS) generation) and real-time respirometry (oxygen consumption). FINDINGS: Both glutamate and the NMDAR co-agonist d-serine are rapidly released by neutrophils in response to bacterial and PMA-induced activation. Pharmacological NMDAR blockade reduced both the autocrine release of glutamate, d-serine and the respiratory burst by activated primary human neutrophils. A highly specific small-molecule inhibitor ZL006 that limits NMDAR-mediated neuronal injury also reduced ROS by activated neutrophils in a murine model of peritonitis, via uncoupling of the NMDAR GluN2B subunit from its' scaffolding protein, postsynaptic density protein-95 (PSD-95). Genetic ablation of PSD-95 reduced ROS production by activated murine neutrophils. Pharmacological blockade of the NMDAR GluN2B subunit reduced primary human neutrophil activation induced by Pseudomonas fluorescens, a glutamate-secreting Gram-negative bacillus closely related to pathogens that cause hospital-acquired infections. INTERPRETATION: These data suggest that release of glutamate by activated neutrophils augments ROS production in an autocrine manner via actions on NMDAR expressed by these cells. FUND: GLA: Academy Medical Sciences/Health Foundation Clinician Scientist. AVG is a Wellcome Trust Senior Research Fellow.