STAT subtype specificity and ischemic preconditioning in mice: is STAT-3 enough?

Abstract

The role of other STAT subtypes in conferring ischemic tolerance is unclear. We hypothesized that in STAT-3 deletion alternative STAT subtypes would protect myocardial function against ischemia-reperfusion injury. Wild-type (WT) male C57BL/6 mice or mice with cardiomyocyte STAT-3 knockout (KO) underwent baseline echocardiography. Langendorff-perfused hearts underwent ischemic preconditioning (IPC) or no IPC before ischemia-reperfusion. Following ex vivo perfusion, hearts were analyzed for STAT-5 and -6 phosphorylation by Western blot analysis of nuclear fractions. Echocardiography and postequilibration cardiac performance revealed no differences in cardiac function between WT and KO hearts. Phosphorylated STAT-5 and -6 expression was similar in WT and KO hearts before perfusion. Contractile function in WT and KO hearts was significantly impaired following ischemia-reperfusion in the absence of IPC. In WT hearts, IPC significantly improved the recovery of the maximum first derivative of developed pressure (+dP/dtmax) compared with that in hearts without IPC. IPC more effectively improved end-reperfusion dP/dtmax in WT hearts compared with KO hearts. Preconditioned and nonpreconditioned KO hearts exhibited increased phosphorylated STAT-5 and -6 expression compared with WT hearts. The increased subtype activation did not improve the efficacy of IPC in KO hearts. In conclusion, baseline cardiac performance is preserved in hearts with cardiac-restricted STAT-3 deletion. STAT-3 deletion attenuates preconditioning and is not associated with a compensatory upregulation of STAT-5 and -6 subtypes. The activation of STAT-5 and -6 in KO hearts following ischemic challenge does not provide functional compensation for the loss of STAT-3. JAK-STAT signaling via STAT-3 is essential for effective IPC.

Fig. 1.

Time course of experimental protocols. Wild-type (WT) and knockout (KO) hearts were randomized to undergo ischemia-reperfusion (I/R) with or without preceding preconditioning (n = 6 to 7 for each group). IPC, ischemic preconditioning.

Fig. 2.

Baseline in vivo systolic performance assessed by two-dimensional transthoracic echocardiography. Myocardial performance was similar between WT and KO animals. FS, fractional shortening; EF, ejection fraction.

Fig. 3.

Representative immunoblots of protein expression in nonperfused (NP) WT and KO hearts. There was no significant difference in baseline expression of phosphorylated (p)STAT-5 or -6 in WT and KO hearts.

Fig. 4.

Myocardial function during I/R and IPC protocols, as reflected by the maximum positive first derivative of developed pressure (+dP/dt_max). *P < 0.001 vs. WT I/R; #P < 0.001 vs. KO IPC.

Fig. 5.

Representative immunoblots of protein expression following I/R in the presence or absence of IPC in WT and KO hearts. Expression of pSTAT-5 was significantly increased in KO hearts compared with WT after I/R and IPC. Expression of pSTAT-6 was significantly increased in KO hearts compared with WT after I/R but not after IPC. Following IPC, expression of pSTAT-5 and -6 were significantly increased in WT but not KO hearts compared with hearts subjected to I/R alone. *P < 0.01 vs. WT I/R; **P < 0.01 vs. WT IPC; #P = 0.12 vs. WT IPC; ***P = 0.03 vs. WT I/R.