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Activation of p38 Mitogen-Activated Protein Kinase Contributes to the Early Cardiodepressant Action of Tumor Necrosis Factor

Mohamed Bellahcene, PhD^_*, Sebastien Jacquet, PhD^_*, Xue B. Cao, MD^_*, Masaya Tanno, MD, PhD^_*, Robert S. Haworth, PhD^_*, Joanne Layland, PhD, Alamgir M. Kabir, MB, BS, Matthias Gaestel, PhD, Roger J. Davis, PhD, Richard A. Flavell, PhD^||, Ajay M. Shah, MD, FESC^_*, Metin Avkiran, PhD, DSc^_* and Michael S. Marber, PhD, FACC^_*,_*

^_* Cardiovascular Division, King's College London, The Rayne Institute, St. Thomas' Hospital, London, United Kingdom
Cardiovascular Division, King's College London, King's College Hospital, London, United Kingdom
Institute of Biochemistry, Medical School Hannover, Hannover, Germany
Howard Hughes Medical Institute, University of Massachusetts, Worcester, Massachusetts
^|| Section of Immunobiology, Howard Hughes Medical Institute and Yale University School of Medicine, New Haven, Connecticut

View larger version (28K): [in a new window]   Figure 1 Effect of tumor necrosis factor-alpha (TNF) (10 ng/ml) on mkk3–/– (KO) and mkk3+/+ (WT) hearts during coronary perfusion under conditions of constant pressure. Data represented by mean ± SEM; n = 6 per group. (A) Left ventricular developed pressure (LVDP) before and during 15 min exposure to TNF. (B) Left ventricular volume versus LVDP measured after at least 15 min of TNF. (C) Coronary flow before and during TNF. (D) Upper part shows representative Western blots of total and phosphorylated p38-MAPK and HSP27 in hearts exposed to TNF for 15 min. Activation of p38-MAPK is reflected in the phosphorylation of HSP27. Lower part shows quantitative data expressed as fold increase relative to baseline (mean ± SEM; n = 3). TNF induces a robust activation of p38-MAPK in mkk3+/+ but not in mkk3–/– hearts.

View larger version (25K): [in a new window]   Figure 2 Effect of tumor necrosis factor-alpha (TNF) (10 ng/ml) on mkk3–/– (KO) and mkk3+/+ (WT) hearts during coronary perfusion under conditions of constant flow. Data represented by mean ± SEM; n = 6 per group. (A) Left ventricular developed pressure (LVDP) before and during exposure to TNF. (B) Left ventricular volume versus LVDP measured after at least 15 min of TNF. (C) Perfusion pressure measured before and during TNF. (D) Upper part shows representative Western blots of total and phosphorylated p38-MAPK and HSP27 in hearts exposed to TNF for 15 min. Lower part depicts quantitative data expressed as fold increase relative to baseline (mean ± SEM; n = 3). TNF induces activation of p38-MAPK in mkk3+/+ but not in mkk3–/– hearts under constant flow conditions; however, this effect was less pronounced than under constant pressure (Fig. 1).

View larger version (29K): [in a new window]   Figure 3 Effect of tumor necrosis factor-alpha (TNF) (10 ng/ml) on mk2–/– (KO) and mk2+/+ (WT) hearts during coronary perfusion under conditions of constant pressure. Data represented by mean ± SEM. (A) Left ventricular developed pressure (LVDP) before and during exposure to TNF. (B) Coronary flow before and during TNF. (C) Upper part shows representative Western blots of total and phosphorylated p38-MAPK and MKK3/6 in hearts exposed to TNF for 15 min. Lower part shows quantitative data expressed in arbitrary units as phosphorylation increase related to total amount for a given protein (mean ± SEM; n = 3), because total p38-MAPK is altered by the mk2 genotype. Consequently, p38-MAPK dual phoshorylation is less pronounced in mk2–/– than in mk2+/+. MKK3 is preferentially activated over MKK6 by TNF, with detectable phospho-MKK3/6 signal in both mk2–/– and mk2+/+.

View larger version (24K): [in a new window]   Figure 4 Effect of 1 µmol/l SB203580 (SB) on tumor necrosis factor-alpha (TNF)-induced contractile dysfunction. Hearts from outbred C56BL/6 mice were perfused under constant pressure or constant flow conditions. SB was solubilized in dimethyl sulfoxide (DMSO). Hearts were exposed to TNF (10 ng/ml) for 15 min, SB (1 µmol/l), or DMSO (<0.01%) for 20 min. Data represent mean ± SEM; n = 6 per group. (A) Left ventricular developed pressure (LVDP) and (B) coronary flow with constant pressure perfusion. (C) LVDP and (D) perfusion pressure recorded under constant flow conditions. SB reduced TNF-induced contractile dysfunction with constant pressure and to a lesser extent with constant flow. Surprisingly, SB reversed TNF-induced coronary vasoconstriction. (E) Upper part shows representative Western blots of total and phosphorylated p38-MAPK and HSP27 in hearts exposed to TNF, SB, DMSO, and SB + TNF under constant pressure. Lower part depicts quantitative data expressed as phosphorylation increase related to total amount for a given protein (mean ± SEM; n = 3). NA = normalization not appropriate.

View larger version (24K): [in a new window]   Figure 5 Effect of tumor necrosis factor-alpha (TNF) (10 ng/ml) on murine cardiomyocytes. (A) Representative Western blots of total and phosphorylated p38-MAPK from outbred C57BL/6 mouse cardiomyocytes preincubated for 30 min with increasing concentrations of TNF (0, 50, 100 ng/ml). Lower panel shows quantitative data expressed as fold increase of phosphorylation relative to control (mean ± SEM; n = 3). These cells exhibited a substantial basal p38-MAPK dual phosphorylation, and TNF, even at high doses, only induced a 3-fold increase in p38-MAPK dual phosphorylation. (B) Representative Western blots of total and phosphorylated p38-MAPK from C57BL/6 mouse cardiomyocytes and perfused hearts both exposed to TNF (10 ng/ml) for 15 min (n = 3). Cardiomyocytes exhibited considerable basal p38-MAPK dual phosphorylation which was absent in perfused hearts. Furthermore, p38-MAPK dual phosphorylation in perfused hearts was greater than in cardiomyocytes (25-fold vs. 3-fold increase, respectively).

View larger version (59K): [in a new window]   Figure 6 Effects of tumor necrosis factor-alpha (TNF) (10 ng/ml) and SB203580 (SB, 1 µmol/l) on mkk3–/– cardiomyocytes. Freshly isolated cardiomyocytes from outbred C57BL/6, mkk3+/+, and mkk3–/– mice underwent the following exposures: TNF (10 ng/ml; n = 24, 29, and 32, respectively) for 30 min; SB (1 µmol/L; n = 25, 29, and 34, respectively); and SB for 35 min starting 5 min before TNF exposure (n = 20, 28, and 32, respectively). After TNF and SB treatments, some cells were allowed to recover for 45 min in Tyrode's buffer to assess the reversibility of the contractile deficit (n = 16, 13, and 24, respectively). Values are mean ± SEM. (A) Experimental traces showing sarcomere contraction in outbred C57BL/6, mkk3+/+, and mkk3–/– mouse cardiomyocytes preincubated for 30 min with TNF (10 ng/ml). (B) Normalized sarcomere contraction amplitude, maximal rate of sarcomere contraction, and maximal rate of sarcomere relengthening. Continued on next page.(C) Characterization of SB effect on TNF-induced p38-MAPK activation in murine cardiomyocytes. Upper part shows representative Western blots of total and phosphorylated p38-MAPK and HSP27 in mkk3+/+ and mkk3–/– murine cardiomyocytes exposed to TNF (10 ng/ml) for 15 min, SB (1 µmol/l) for 35 min, and SB for 35 min given 5 min before TNF treatment. Lower part shows quantitative data expressed as fold increase of phosphorylation relative to control (mean ± SEM; n = 3). Importantly, TNF treatment induced p38-MAPK activation in mkk3+/+ but not in mkk3–/– myocytes.