Lipopolysaccharide (LPS) has been implicated in sepsis-mediated heart failure and chronic cardiac myopathies. We determined that LPS directly and reversibly affects cardiac myocyte function by altering regulation of intracellular Ca2+ concentration ([Ca2+]i) in immortalized cardiomyocytes, HL-1 cells. [Ca2+]i oscillated (<0.4 Hz), displaying slow and transient components. LPS (1 μg/ml), derived either from Escherichia coli or from Salmonella enteritidis, reversibly abolished Ca2+ oscillations and decreased basal [Ca 2+]i by 30-40 nM. HL-1 cells expressed Toll-like receptors, i.e., TLR-2 and TLR-4. Thus, we differentiated effects of LPS on [Ca2+]i and Ca2+ oscillations by addition of utlrapure LPS, a TLR-4 ligand. Ultrapure LPS had no effect on basal [Ca 2+]i, but it reduced the rate of Ca2+ oscillations. Interestingly, Pam3CSK4, a TLR-2 ligand, affected neither Ca 2+ parameter, and the effect of ultrapure LPS and Pam3CSK4 combined was similar to that of utlrapure LPS alone. Thus, unpurified LPS directly inhibits HL-1 calcium metabolism via TLR-4 and non-TLR-4-dependent mechanisms. Since others have shown that endotoxin impairs the hyperpolarization-activated, nonselective cationic pacemaker current (If), which is expressed in HL-1 cells, we utilized whole cell voltage-clamp techniques to demonstrate that LPS (1 μg/ml) reduced If in HL-1 cells. This inhibition was marginal at physiologic membrane potentials and significant at very negative potentials (P < 0.05 at -140, -150, and -160 mV). So, we also evaluated effects of LPS on tail currents of fully activated If. LPS reduced the slope conductance of the tail currents from 498 ± 140 pS/pF to 223 ± 65 pS/pF (P < 0.05) without affecting reversal potential of -11 mV. Ultrapure LPS had similar effect on If, whereas Pam3CSK4 had no effect on If. We conclude that LPS inhibits activation of I f, enhances its deactivation, and impairs regulation of [Ca 2+]i in HL-1 cardiomyocytes via TLR-4 and other mechanisms.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

>Magister Scientiae - MSc / This study focused on the synthesis of a series of novel tricycloundecane derivatives and evaluation of these compounds for neuroprotection using the fluorescent ratiometric calcium assay that indicates the ability of the test compounds to inhibit NMDA receptors and VGCC. The cycloaddition reaction between p-benzoquinone and monomerised dicyclopentadiene yielded tricycloundeca- 4,9-diene-3,6-dione which was used as the base structure and further derivatised. These derivatives were conjugated with benzylamine to form a series of imines and amines. A total of 10 compounds were synthesised for evaluation of inhibition of calcium influx through NMDA receptor channels and voltage-gated calcium channels. The structures were confirmed using NMR, IR and MS. On the proton NMR, the characteristic AB-quartet system was observed in the region of 1-2 ppm for all the compounds and the aromatic moiety was observed between 6.5-7.5 ppm for the novel polycyclic amines. These, with other functional groups, were used to confirm the individual structures