Lipopolysaccharides directly decrease Ca2+ oscillations and the hyperpolarization-activated nonselective cation current If in immortalized HL-1 cardiomyocytes.
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 microg/ml), derived either from Escherichia coli or from Salmonella enteritidis, reversibly abolished Ca2+ oscillations and decreased basal [Ca2+]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 [Ca2+]i, but it reduced the rate of Ca2+ oscillations. Interestingly, Pam3CSK4, a TLR-2 ligand, affected neither Ca2+ 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 (I(f)), which is expressed in HL-1 cells, we utilized whole cell voltage-clamp techniques to demonstrate that LPS (1 microg/ml) reduced I(f) 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 I(f). 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 I(f), whereas Pam3CSK4 had no effect on I(f). We conclude that LPS inhibits activation of I(f), enhances its deactivation, and impairs regulation of [Ca2+]i in HL-1 cardiomyocytes via TLR-4 and other mechanisms.
Wondergem
Robert
R
Dept. of Physiology, James H. Quillen College of Medicine, East Tennessee State Univ., P. O. Box 70576, Johnson City, TN 37614-1708, USA. wonderge@etsu.edu
Graves
Bridget M
BM
Ozment-Skelton
Tammy R
TR
Li
Chuanfu
C
Williams
David L
DL
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000502:physiology; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D004926:Escherichia coli; D007473:Ion Channels / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008564:Membrane Potentials; D051379:Mice; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012477:Salmonella enteritidis; D051197:Toll-Like Receptor 4 / Q000096:biosynthesis
D020746:Calcium Channels, L-Type; D007473:Ion Channels; D008070:Lipopolysaccharides; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D002118:Calcium