At days and hours after MI
At 21 days and 24 hours after MI, the protein expressions of active JNK1 (P < .02 and P < .01, respectively) and NF-κB (P < .02 and P < .05, respectively) were down-regulated in GSTP1-treated rats compared with control (Supplemental Fig. 2A; Fig. 5A). Meanwhile, the protein 10058-F4 of p38 was down-regulated only at 21 days after MI (P < .006; Fig. 5A). Exogenously supplemented GSTP1 enhanced its own binding activity to TRAF2, JNK1, and p38 at 24 hours (P < .02; Supplemental Fig. 2B) and 21 days (P < .001; Fig. 5B) after MI, whereas this treatment effect was absent in control samples. At 24 hours and 21 days after MI, GSTP1 down-regulated the mRNA expression of interleukin (IL) 1β (P < .02 and P < .003, respectively) and IL-2 (P < .03 and P < .04, respectively), suppressed TGF-β (P < .02 and P < .04, respectively), and up-regulated IL-10 (P < .02) mRNA expression (Supplemental Fig. 2C; Fig. 5C) compared with control and sham. IL-17 mRNA expression was up-regulated only at 21 days (P < .005).
Cardiac function at 24 hours was not significantly different between GSTP1-treated and control rats (Fig. 6). Compared with control rats at 21 days after MI, however, GSTP1-treated rats had a significantly higher cardiac index (0.13 ± 0.04 mL min−1 cm−2; P < .02; Fig. 6A), cardiac output (62.44 ± 22 mL/min; P < .04; Fig. 6B), stroke volume (0.261 ± 0.109 mL; P < .03; Fig. 6C), and ejection fraction (40.58 ± 22%; P < .04; Fig. 6D). There was no statistically significant difference for cardiac index, cardiac output, stroke volume, and ejection fraction when GSTP1-treated rats were compared with sham (Fig. 6).
Infract-related wall motion at 3 weeks after MI was significantly decreased in control subjects at 21 days compared with control subjects at 24 hours, GSTP1-treated animals, or sham (P < .0001, Fig. 6E and F). The infarct-related wall thickening at 21 days after MI was significantly decreased in control rats at 21 days compared with both GSTP1-treated and control rats at 24 hours (P = .03) or sham (P = .02, Fig. 6G and H).
Representative MRI images of all animal groups are available online in Supplemental Figs. 3–5.
Discussion This study demonstrates the role of GSTP1 in HF by inhibiting the TRAF2-induced MAPK activation and the potent inhibitory effect of GSTP1 on TNF-α–induced activation of JNK1 and p38 in the in vitro cultures of human failing myocardium. The interaction of GSTP1 with TRAF2 is a central regulator of TNF-α–induced MAPK activation.10, 11, 12, 13 In line with our previous study, our in vitro findings revealed that GSTP1 interacts also directly with JNK1 to inhibit its activation but not with p38. One explanation is that GSTP1-mediated p38 regulation might be mainly due to GSTP1 interaction with TRAF2 rather than physically interaction with p38 itself, resulting in inhibition of TRAF2-induced activation of p38. In agreement with the published data,14, 15, 26 our findings provide evidence that elevated active JNK1 and p38 are implicated in the pathogenesis of HF. Recent studies had used this concept to create treatment strategies by targeting prosurvival MAPK pathway to rescue ischemic cardiomyocytes,27, 28 whereas earlier studies focused on counteracting this pathologic pathway of HF by targeting specific cytokines, such as TNF-α.4, 5 However, owing to the wide spectrum of systems affected by TNF-α, the benefits of using TNF-α blockers in HF remained controversial. Recent reports provide conclusive evidence of why a global blockade of TNF-α was frustrating.29, 30 It became clear that TNF-α receptors (R) 1 and 2 have opposing effects on cardiac remodeling, apoptosis, and inflammation. TNF-α R1 recruits adaptor proteins via its death domain to trigger TRAF2-dependent signaling that activates MAPKs, leading to apoptosis. TNF-α R2, however, ameliorates this effect.29, 30 Based on the above reasoning, targeting the inflammatory pathways activated during post-MI remodeling process downstream of TRAF2-dependent signaling would benefit rescuing cardiomyocytes. Our data indicate an impaired GSTP1-dependent inhibition of TRAF2 signaling in HF associated with up-regulated JNK1 expression and impaired GSTP1/JNK1 complex formation. One explanation for this observation might be that the physiologic levels of GSTP1 are insufficient to suppress proinflammatory signaling under pathologic circumstances. Accordingly, our in vivo studies demonstrate that the replenishment of the endogenous GSTP1 stores with recombinant GSTP1 has a potent cardioprotective effect after MI. Importantly, GSTP1-treated rats exhibited concurrent down-regulation of proinflammatory cytokines, caspases, and MAPKs, and nuclear factor κB, which are downstream of TNF-α R1 signaling, affecting inflammatory and apoptotic responses.