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Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia
Published on: May 9, 2013
Ischemic preconditioning involves dual cardio-protective axes with p38MAPK as upstream target
Norbert Nagy1, Keisuke Shiroto, Gautam Malik
1FAHA Cardiovascular Research Center, University of Connecticut School of Medicine Farmington, Cardiovascular Research Institute, CT 06030-1110, USA.
Abstract:
The existing literature indicates a crucial role of p38 MAP (mitogen-activated protein) kinase (p38MAPK) and its downstream target MAPKAP kinase 2 (MK2) in ischemic preconditioning (IPC). Accordingly, deletion of MK2 gene should abolish the cardioprotective ability of IPC. Interestingly, we were able to partially precondition the hearts from MK2(-/-) knockout mice suggesting the existence of an as yet unknown alternative downstream target of p38MAPK. A recent study from our laboratory also determined a crucial role of CREB (cyclic AMP response element binding protein) in IPC. Since CREB is a downstream target of MSK-1 (mitogen- and stress-activated protein kinase-1) situated at the crossroad of ERK (extracellular receptor kinase) and p38MAPK signaling pathways, we reasoned that MSK-1 could be a downstream molecular target for p38MAPK and ERK signaling in the IPC hearts. To test this hypothesis, the rat hearts were subjected to IPC by four cyclic episodes of 5 min ischemia and 10 min reperfusion. As expected, IPC induced the activation of ERK1/2, p38MAPK, MK2 and HSP (heat shock protein) 27 as evidenced by their increased phosphorylation; and the inhibition of p38MAPK with SB203580 almost completely, and the inhibition of ERK1/2 with PD098059 partially, abolished cardioprotective effects of IPC. Inhibition of MSK-1 with short hairpin RNA (shRNA) also abolished the IPC-induced cardioprotection. SB203580 partially blocked the effects of MSK-1 suggesting that MSK-1 sits downstream of p38MAPK. shRNA-MSK-1 blocked the contribution of both p38MAPK and ERK1/2 as it is uniquely situated at the downstream crossroad of both of these MAP kinases. Although MSK-1 sits downstream of both ERK1/2 and p38MAPK, ERK1/2 activation appears to play less significant role compared to p38MAPK, since its inhibition blocked MSK activation only partially. Consistent with these results, shRNA-MSK-1 blocked the partial PC in MK2(-/-) hearts, and in combination with SB203580, completely abolished the PC effects in the wild-type hearts. The IPC-induced survival signaling was almost completely inhibited with SB203580, and only partially with PD 098059 as evidenced from the inhibition patterns of IPC induced activation of CREB, Akt and Bcl-2. Again SB203580 alone or in combination with shRNA-MSK-1 inhibited IPC induced survival signal comparatively, suggesting that MSK-1 exists downstream of p38MAPK. Taken together, these results indicate for the first time MSK-1 as an alternative (other than MK2) downstream target for p38MAPK, which also transmits survival signal through the activation of CREB.
Insights
Mitogen-activated protein kinase kinase kinase 1 (MSK-1) acts as an alternative downstream target of p38 MAP kinase (p38MAPK) in ischemic preconditioning (IPC). MSK-1 transmits survival signals through cyclic AMP response element binding protein (CREB) activation, revealing a novel cardioprotective pathway.
Area of Science:
- Cardiovascular Biology
- Molecular Cardiology
- Signal Transduction
Background:
- Ischemic preconditioning (IPC) confers cardioprotection, with p38 MAP kinase (p38MAPK) and MAPK-activated protein kinase 2 (MK2) identified as key mediators.
- Complete deletion of MK2 partially impairs IPC-induced cardioprotection, suggesting the existence of alternative p38MAPK downstream targets.
- Cyclic AMP response element binding protein (CREB) plays a role in IPC, and its upstream regulator, MSK-1, is positioned at the convergence of ERK and p38MAPK signaling pathways.
Purpose of the Study:
- To investigate if MSK-1 serves as an alternative downstream target of p38MAPK and ERK signaling in IPC.
- To elucidate the role of MSK-1 in transmitting IPC-induced survival signals, particularly through CREB activation.
- To determine the contribution of MSK-1 to cardioprotection in the absence of MK2.
Main Methods:
- Rat hearts were subjected to IPC (5 min ischemia/10 min reperfusion cycles).
- Pharmacological inhibitors (SB203580 for p38MAPK, PD098059 for ERK1/2) and short hairpin RNA (shRNA) targeting MSK-1 were used.
- Western blotting assessed phosphorylation of ERK1/2, p38MAPK, MK2, HSP27, CREB, Akt, and Bcl-2.
Main Results:
- IPC activated ERK1/2, p38MAPK, MK2, and HSP27.
- Inhibition of p38MAPK and MSK-1 abolished IPC-induced cardioprotection, while ERK1/2 inhibition partially reduced it.
- MSK-1 inhibition abolished the partial cardioprotection observed in MK2 knockout hearts, and combined inhibition of p38MAPK and MSK-1 completely abolished protection in wild-type hearts.
- MSK-1 activation was partially dependent on p38MAPK and ERK1/2, with p38MAPK playing a more significant role.
- IPC-induced activation of CREB, Akt, and Bcl-2 was significantly inhibited by p38MAPK inhibition and MSK-1 inhibition.
Conclusions:
- MSK-1 is identified as a novel downstream target of p38MAPK in IPC, distinct from MK2.
- MSK-1 mediates IPC-induced cardioprotection by activating survival signaling pathways, including CREB.
- This finding expands our understanding of the molecular mechanisms underlying ischemic preconditioning and offers potential therapeutic targets.
