Negative inotropic effects of high-mobility group box 1 protein in isolated contracting cardiac myocytes - PubMed (original) (raw)

Negative inotropic effects of high-mobility group box 1 protein in isolated contracting cardiac myocytes

Huei-Ping Tzeng et al. Am J Physiol Heart Circ Physiol. 2008 Mar.

Abstract

High-mobility group box 1 (HMGB1) released from necrotic cells or macrophages functions as a late inflammatory mediator and has been shown to induce cardiovascular collapse during sepsis. Thus far, however, the effect(s) of HMGB1 in the heart are not known. We determined the effects of HMGB1 on isolated feline cardiac myocytes by measuring sarcomere shortening in contracting cardiac myocytes, intracellular Ca2+ transients by using fluo-3, and L-type calcium currents by using whole cell perforate configuration of the patch-clamp technique. Treatment of isolated myocytes with HMGB1 (100 ng/ml) resulted in a 70% decrease in sarcomere shortening and a 50% decrease in the height of the peak Ca2+ transient within 5 min (P < 0.01). The immediate negative inotropic effects of HMGB1 on cell contractility and calcium homeostasis were partially reversible upon washout of HMGB1. A significant inhibition of the inward l-type calcium currents was also documented by the patch-clamp technique. HMGB1 induced the PKC-epsilon translocation, and a PKC inhibitor significantly attenuated the negative inotropic effects of HMGB1. These studies show for the first time that HMGB1 impairs sarcomere shortening by decreasing calcium availability in cardiac myocytes through modulating membrane calcium influx and suggest that HMGB1 maybe acts as a novel myocardial depressant factor during cardiac injury.

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Conflict of interest statement

Conflict of Interest

The authors have no conflicts to disclose.

Figures

Figure 1

Figure 1. Effects of HMGB1 on cardiac myocyte contractility and intracellular calcium transients

Recordings of myocyte contractility during the entire period of experimentation in individual cells treated with diluent (A) or HMGB1 (100 ng/mL; B). Vertical axis shows absolute sarcomere length in microns or fluorescence brightness (in arbitrary units). The horizontal axis represents time (in seconds). Representative pacing events recorded under baseline (C) and post treatment with HMGB1 (100 ng/mL; D). Vertical axis represents fluorescence brightness in arbitrary units.

Figure 2

Figure 2. Effects of HMGB1 on cardiac myocyte contractility and peak amplitude of calcium transients

(A) Effect of 100 ng/ml HMGB1 on the sarcomere shortening. (B) Effect of 100 ng/ml HMGB1 on the peak fluorescence brightness. (* p < 0.01 vs. baseline, N = 9 cells from 4 hearts).

Figure 3

Figure 3. Dose-dependent effects of HMGB1 on myocyte contractility and intracellular calcium transients

(A) Effects of increasing concentrations of HMGB1 on sarcomere shortening. (B) Effects of increasing concentrations of HMGB1 on the peak amplitude of calcium transients. (*p < 0.01 vs. diluent; N = 8 cells/condition).

Figure 4

Figure 4. Effect of HMGB1 on L-type calcium currents

(A) Inward L-type calcium currents recorded under baseline conditions, after HMGB1 treatment, and washout. (B) Relative effects of HMGB1 (10 ng/ml) on the L-type calcium currents in isolated myocytes. (C) Time course of the L-type calcium currents in the presence and after washout of HMGB1. (N = 8 cells/condition).

Figure 5

Figure 5. HMGB1 induces PKC-ε translocation in cardiac myoctyes

(A) Representative western blots for cytosolic and membrane PKC-ε. HMGB1 (100 ng/ml: R&D Systems) induced PKC-ε translocation from the cytosol to the membrane fraction. (B) Time course of PKCε translocation in cardiac myocytes after exposure to HMGB1 (100 ng/ml). (*p < 0.01 vs Time 0; n = 4 isolations) (C) Ro-31-8220 (Ro) significantly attenuated the effects of HMGB1 (100 ng/mL; R&D Systems) in a dose dependent manner compared to Diluent (Dil). N= 10–15 cells/condition. (*p < 0.01 vs. Diluent; ‡ p < 0.05 vs. HMGB1 alone). N= 10–15 cells/condition

Figure 6

Figure 6. Effects of anti-RAGE and TLR4 antibodies on HMGB1-induced negative inotropic effects in cardiac myocytes

Cells were pre-incubated (30 min) with each antibody prior to exposure to HMGB1 (100 ng/mL). (A) RAGE blockade improved sarcomere shortening by 32% (B) TLR4 blockade improved sarcomere shortening by 22% (*p < 0.01 vs. Diluent; ‡ p < 0.05 vs. HMGB1 alone). N= 10-15 cells/condition

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