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The goal of this study was to identify the cellular mechanisms responsible for cardiac dysfunction in endotoxemic mice. We aimed to differentiate the roles of cGMP [produced by soluble guanylyl cyclase (sGC)] versus oxidative posttranslational modifications of Ca(2+) transporters. C57BL/6 mice [wild-type (WT) mice] were administered lipopolysaccharide (LPS; 25 μg/g ip) and euthanized 12 h later. Cardiomyocyte sarcomere shortening and Ca(2+) transients (ΔCai) were depressed in LPS-challenged mice versus baseline. The time constant of Ca(2+) decay (τCa) was prolonged, and sarcoplasmic reticulum Ca(2+) load (CaSR) was depressed in LPS-challenged mice (vs. baseline), indicating decreased activity of sarco(endo)plasmic Ca(2+)-ATPase (SERCA). L-type Ca(2+) channel current (ICa,L) was also decreased after LPS challenge, whereas Na(+)/Ca(2+) exchange activity, ryanodine receptors leak flux, or myofilament sensitivity for Ca(2+) were unchanged. All Ca(2+)-handling abnormalities induced by LPS (the decrease in sarcomere shortening, ΔCai, CaSR, ICa,L, and τCa prolongation) were more pronounced in mice deficient in the sGC main isoform (sGCα1(-/-) mice) versus WT mice. LPS did not alter the protein expression of SERCA and phospholamban in either genotype. After LPS, phospholamban phosphorylation at Ser(16) and Thr(17) was unchanged in WT mice and was increased in sGCα1(-/-) mice. LPS caused sulphonylation of SERCA Cys(674) (as measured immunohistochemically and supported by iodoacetamide labeling), which was greater in sGCα1(-/-) versus WT mice. Taken together, these results suggest that cardiac Ca(2+) dysregulation in endotoxemic mice is mediated by a decrease in L-type Ca(2+) channel function and oxidative posttranslational modifications of SERCA Cys(674), with the latter (at least) being opposed by sGC-released cGMP.

The characteristics of nickel (Ni) block of L-type Ca current (I(Ca, L)) were studied in whole cell patch-clamped guinea pig cardiac myocytes at 37 degrees C in the absence and presence of 100 microM cAMP in the pipette solution. Ni block of peak I(Ca,L) had a dissociation constant (K(d)) of 0.33 +/- 0.03 mM in the absence of cAMP, whereas in the presence of cAMP, the K(d) was 0.53 +/- 0.05 mM (P = 0.006). Ni blocked Ca entry via Ca channels (measured as I(Ca, L) integral over 50 ms) with similar kinetics (K(d) of 0.35 +/- 0.03 mM in cAMP-free solution and 0.30 +/- 0.02 mM in solution with cAMP, P = not significant). Under both conditions, 5 mM Ni produced a maximal block that was complete for the first pulse after application. Ni block of I(Ca,L) was largely use independent. Ni (0. 5 mM) induced a positive shift (4 to 6 mV) in the activation curve of I(Ca,L). The block of I(Ca,L) by 0.5 mM Ni was independent of prepulse membrane potential (over the range of -120 to -40 mV). Ni (0.5 mM) also induced a significant shift in I(Ca,L) inactivation: by 6 mV negative in cAMP-free solution and by 4 mV positive in cells dialyzed with 100 microM cAMP. These data suggest that, in addition to blocking channel conductance by binding to a site in the channel pore, Ni may bind to a second site that influences the voltage-dependent gating of the L-type Ca channel. They also suggest that Ca channel phosphorylation causes a conformational change that alters some effects of Ni. The results may be relevant to excitation-contraction coupling studies, which have employed internal cAMP dialysis, and where Ni has been used to block I(Ca,L) and Ca entry into cardiac cells.

We investigated the effect of external Cd2+ on the Na/Ca exchange and the L-type Ca channel current (ICa,L) in whole cell patch-clamped rabbit ventricular myocytes at 36 degrees C. After the interfering ion channels and the Na/K pump were blocked, the exchange current was measured as the membrane current that was inhibited by 5 mM nickel. External Cd2+ inhibited Na/Ca exchange with a dissociation constant (KD) of 320.6 +/- 12.4 microM and a Hill coefficient of 1.5 +/- 0.09 (n = 13 cells) and ICa,L with a KD of 2.14 +/- 0.15 microM and a Hill coefficient of 0.74 +/- 0.03 (n = 11 cells). We observed some overlap in the Cd2+ concentration that blocked each mechanism. Cd2+ (100-500 microM) is used commonly to block ICa,L completely. However, 100 microM Cd2+ also inhibits 20% of the Na/Ca exchange activity, whereas 500 microM Cd2+ inhibits 60%.