The contribution of reactive oxygen species and p38 mitogen-activated protein kinase to myofilament oxidation and progression of heart failure in rabbits

Abstract

Background and purpose: The formation of reactive oxygen species (ROS) is increased in heart failure (HF). However, the causal and mechanistic relationship of ROS formation with contractile dysfunction is not clear in detail. Therefore, ROS formation, myofibrillar protein oxidation and p38 MAP kinase activation were related to contractile function in failing rabbit hearts.

Experimental approach and key results: Three weeks of rapid left ventricular (LV) pacing reduced LV shortening fraction (SF, echocardiography) from 32 +/- 1% to 13 +/- 1%. ROS formation, as assessed by dihydroethidine staining, increased by 36 +/- 8% and was associated with increased tropomyosin oxidation, as reflected by dimer formation (dimer to monomer ratio increased 2.28 +/- 0.66-fold in HF vs. sham, P < 0.05). Apoptosis (TdT-mediated dUTP nick end labelling staining) increased more than 12-fold after 3 weeks of pacing when a significant increase in the phosphorylation of p38 MAP kinase and HSP27 was detected (Western blotting). Vitamins C and E abolished the increases in ROS formation and tropomyosin oxidation along with an improvement of LVSF (19 +/- 1%, P < 0.05 vs. untreated HF) and prevention of apoptosis, but without modifying p38 MAP kinase activation. Inhibition of p38 MAP kinase by SB281832 counteracted ROS formation, tropomyosin oxidation and contractile failure, without affecting apoptosis.

Conclusions and implications: Thus, p38 MAP kinase activation appears to be upstream rather than downstream of ROS, which impacts on LV function through myofibrillar oxidation. p38 MAP kinase inhibition is a potential target to prevent or treat HF.

Figure 1

The myocardial ROS concentration (DHE signal) increased following 3 weeks of LV pacing (n = 8) compared with sham rabbits (n = 7). Such increase in the myocardial ROS concentration was prevented by both, pretreatment with vitamins C and E (n = 6) and p38 MAP kinase blockade with SB281832 (n = 6). Data are expressed as mean ± SEM. DHE, dihydroethidine; HF3, heart failure rabbits after 3 weeks of pacing; LV, left ventricular; ROS, reactive oxygen species.

Figure 2

Upper panel: representative Western blots of tropomyosin oxidation. Lane 1 (L1) illustrates the highest degree of oxidation obtained by adding 1 mM H₂O₂ for 15 min to isolated tropomyosin. Lanes 3 and 5 illustrate typical examples of Western blots from failing rabbit hearts after 3 weeks of LV pacing as compared with control hearts from sham operated rabbits (lanes 2, L2 and 4, L4). Electrophoreses of lanes 2 and 3 were carried out under non-reducing conditions as opposed to conventional reducing conditions used for lanes 4 and 5. The comparison between reducing and non-reducing electrophoresis permits the attribution of tropomyosin dimers in lanes 2 and 3 (L2, L3) to disulfide cross-bridge formation. Lower panel: tropomyosin oxidation increased following 3 weeks of LV pacing (n = 8) compared with sham rabbits (n = 7). Such increases in tropomyosin oxidation were prevented by both, pretreatment with vitamins C and E (n = 6) and p38 MAP kinase blockade with SB281832 (n = 6). Data are expressed as mean ± SEM. HF3, heart failure rabbits after 3 weeks of pacing; LV, left ventricular.

Figure 3

(A) Representative Western blots of phosphorylated p38 MAP kinase, total p38 MAP kinase, phosphorylated HSP27 and total HSP27 in Sham and HF3 rabbits. (B) Representative Western blots of phosphorylated p38 MAP kinase, total p38 MAP kinase, phosphorylated HSP27 and total HSP27 in Sham and HF3 rabbits, treated with vitamins C and E. (C) Representative Western blots of phosphorylated p38 MAP kinase, total p38 MAP Kinase, phosphorylated HSP27 and total HSP27 in Sham and HF3 rabbits, blocked with SB281832. HF3, heart failure rabbits after 3 weeks of pacing.

Figure 4

Relationship between ROS concentration and LV shortening fraction at the end of the 3 week study protocol. There is a close inverse relationship between ROS concentration and LV shortening fraction, and the beneficial effects of vitamins C and E or SB281832, respectively, fall along the relationship. DHE, dihydroethidine; HF3, heart failure rabbits after 3 weeks of pacing; LV, left ventricular; ROS, reactive oxygen species.