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(Circulation Research. 2006;99:798.)
© 2006 American Heart Association, Inc.

Editorials

Signaling Mechanisms in Ischemic Preconditioning

Interaction of PKC and MitoK_ATP in the Inner Membrane of Mitochondria

Hossein Ardehali

From the Division of Cardiology, Department of Medicine, Northwestern University Medical Center, Chicago, Ill.

Correspondence to: Hossein Ardehali, Tarry 12–725, 303 E Chicago Ave, Chicago, IL 60611. E-mail h-ardehali{at}northwestern.edu

See related article, pages 878–883

Key Words: mitoK_ATP • protein kinase C- • ischemic preconditioning • RACK • protein kinase G

The cardiac "warm up" phenomenon, described more than 50 years ago in patients with coronary artery disease, refers to improvement in cardiac symptoms and physical performance following exposure to short periods of ischemia.¹ Several mechanisms, such as adaptive reduction in oxygen consumption by the ischemic myocardial region, improved oxygen supply via collateral recruitment or dilation of the stenotic vessel, and activation of an intrinsic phenomenon called ischemic preconditioning (IPC) have been proposed to account for this phenomenon. IPC refers to a process in which brief periods of ischemia improves the ability of the heart to tolerate subsequent prolonged ischemic periods.³ It was first identified in the heart in 1986 by Murry et al,² and has since been demonstrated in various experimental and animal models.³

Several triggers have been proposed for IPC, including adenosine, bradykinin, protaglandins, opiod receptors, nitric oxide, and Ca²⁺.⁴ These triggers lead to the activation of several intracellular pathways that ultimately protect myocardial cells against injury. Although the details of these pathways have not been totally characterized, mitochondria have been shown to be key mediators of IPC. Specifically, the opening of a mitochondrial channel, called the mitochondrial ATP-sensitive potassium channel or mitoK_ATP is believed to be critical for the induction of IPC; drugs that activate this channel protect against ischemia and inhibitors of mitoK_ATP reverse these protective effects.⁵

The signaling pathways that lead to the activation of mitoK_ATP are still under investigation.⁵ In a recent article, Oldenburg et al demonstrated that in isolated rabbit adult cardiomyocytes, bradykinin increased the levels of reactive oxygen species (ROS) and this effect was reversed by inhibitors of both mitoK_ATP and protein kinase G (PKG).⁶ Subsequent studies demonstrated that mitoK_ATP can be activated by the addition of exogenous cGMP and PKG, and that this effect is reversed by inhibitors of protein kinase C (PKC).⁷ These results suggest that PKG transmits the cardioprotective signal to mitoK_ATP through a PKC-dependent pathway. It is unclear how this signal is transmitted and which isoforms of PKC are involved in this process.

In this issue of Circulation Research, Jabrek et al, demonstrate in a series of elegant studies that mitoK_ATP and PKC directly interact in the inner mitochondrial membrane, and that PKC is required for the opening of mitoK_ATP.⁸ They first demonstrate the presence of PKC in highly enriched mitochondrial fractions. Subsequently, they show that PKC activators induce the opening of mitoK_ATP, while its inhibitors and a protein phosphatase reverse these effects.

The PKC family of enzymes play a role in several cellular signal transduction pathways and is implicated in numerous physiological and pathological processes.⁹ Thus far, 11 members of the PKC family have been characterized. These proteins are divided into 3 groups based on their responsiveness to diacylglycerol (DAG) or Ca²⁺ for enzyme activity. The , ßI, ßII, and isozymes are both DAG- and Ca²⁺-dependent, while the , , , and are only DAG-dependent and do not need Ca²⁺ for activity. The atypical PKCs ( and ) are neither DAG- nor Ca²⁺-dependent and require lipid-derived molecules for activity.⁹

The potential role of PKC enzymes in cardioprotection has been the subject of many investigations. To evaluate the role of the individual isoforms of PKCs in cardioprotection, recent studies have used isozyme-specific modulators as well as transgenic and knockout mice of specific PKC isozymes. PKC isozymes translocate to distinct cellular locations after activation by binding to their specific anchoring proteins, called receptor for activated C-kinase or RACKs.¹⁰ Peptides against the RACK binding site of each PKC isozymes can inhibit the translocation and activity of the corresponding enzyme and have been used as isozyme-specific inhibitors.¹¹ Peptide activators promote PKC isozymes to translocate to a specific subcellular location by mimicking the function of isozyme-specific RACKs.¹¹

Ping et al demonstrated that all 11 isoforms of PKC are present in rabbit myocardium and that IPC activates the and isoforms.¹² Subsequent studies have supported a major role of PKC in IPC. Mochly-Rosen’s group has demonstrated that PKC is activated in IPC,¹³ and that treatment with a PKC selective inhibitor during preconditioning reverses the protective effects of IPC.^13,14 Furthermore, overexpression of PKC in the heart of transgenic mice resulted in a lesser degree of ischemic damage,¹⁵ and PKC knockout mice did not retain the protective effects of preconditioning.¹⁶ These results suggest that PKC is required and sufficient for the protective effects of IPC in the heart. Another PKC isozyme, PKC, also plays a role in myocardial cell death. However, unlike PKC, this isozyme is believed to promote damage from an ischemic insult. Activation of PKC causes a higher degree of cell death in response to ischemia and its inhibition leads to protection in isolated cardiomyocytes and intact hearts.¹⁷

Although the signaling mechanisms that link PKC to ischemic preconditioning are not completely characterized, several pathways have been proposed (Figure). These include generation of free radicals, changes in the levels of the pro- and antiapoptotic proteins of Bcl-2 family, and activation of the mitoK_ATP.¹⁸ ROS that are produced during both ischemia and reperfusion have deleterious effects on cardiomyocytes. However, these molecules are also believed to activate multiple signaling pathways, including the activation of PKC enzymes.^19,20 ROS have been shown to lead to PKC activation and translocation.^19,20 However, there is currently no evidence linking ROS formation to PKC mediated cardioprotection. PKC is also shown to enhance the phosphorylation of a proapoptoic Bcl-2 related protein (Bcl-2 associated death domain or BAD), inactivating it and blocking its ability to induce apoptosis.²¹ The expression of Bcl-2 protein may also be regulated by PKC.²² However, a recent study showed that although knockdown of PKC induces apoptosis in glioma cells, it does not affect the expression of Bcl-2 or Bax.²³

View larger version (46K): [in this window] [in a new window]   Schematic diagram outlining current thinking of the molecular mechanism of cardioprotection through PKC and mitoKATP. PKC activation by different mechanisms would lead to its translocation into the mitochondria and interaction with mitoKATP, leading to cardioprotection. It is also shown to phosphorylate and inactivate the proapoptotic protein BAD. PLC, phospholipase C; PLD, phospholipase D; NO, nitric oxide; NOS, nitric oxide synthase.

A number of previous studies provided evidence for a possible link between PKC and mitoK_ATP in IPC. Hassouna et al demonstrated that PKC inhibitors block protection from IPC, and that diazoxide (a mitoK_ATP activator) did not affect the phosphorylation of PKC, suggesting that PKC may act upstream of mitoK_ATP.²⁴ Korge et al has shown that a nonspecific PKC activator can induce cardioprotection and this effect was reversed by mitoK_ATP inhibitors.²⁵ Finally, PKC is shown to interact with several mitochondrial proteins, suggesting that it may translocate into the mitochondria.²⁶ The current manuscript for the first time shows that PKC interacts and activates mitoK_ATP in the inner membrane of the mitochondria.

Although the findings by Jaburek et al are interesting and have addressed an important question, they have also raised many new questions. What is the mechanism for PKC translocation into the mitochondria? Does PKC phosphorylate any protein components of mitoK_ATP? Is there a RACK for PKC in the mitochondria? Besides PKG, what other pathways lead to PKC-mediated activation of mitoK_ATP? The answer to these questions may better delineate the protective roles of PKC and mitoK_ATP.

In summary, the current manuscript by Jaburek et al demonstrates that mitoK_ATP and PKC functionally interact with each other in the inner membrane of the mitochondria. These results improve our understanding of the signals that leads to the opening of mitoK_ATP and the protective effects of IPC (Figure). Although the mechanism of PKC activation of mitoK_ATP is not totally clear, it is tempting to speculate that it directly phosphorylates the protein components of the channel, resulting in the opening of the channel and entrance of K⁺ into the mitochondria. This potential mechanism for the regulation of the mitoK_ATP channel remains to be elucidated.

	Acknowledgments

 
I thank Drs Mike Burke and Kannan Mutharasan for critical reading of the manuscript.

Sources of Funding

H.A. is supported by NIH grant K08 HL079387 and grants from the Schweppe foundation and the Northwestern Memorial Foundation.

Disclosures

None.

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

	References

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