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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.