A New Piece of the Cardioprotection Puzzle?
- myocardial infarction
- cardiac transcription factors cardiomyocytes
- signal transduction
See related article, pages 41–49
The era of research into infarct size modification began in earnest in 1986, when Murry et al unambiguously showed that the heart could be made resistant to myocardial infarction by preconditioning it with brief periods of sublethal ischemia/reperfusion.1 There was an unmet clinical need for an intervention that could protect the ischemic heart from infarction. Sparked by the hope that if its mechanism could be understood, we surely should be able to confer this profound protection to the ischemic patient. Ischemic preconditioning soon became the object of intense investigation. More than 20 years later, we still do not fully understand its mechanism, although much has been learned about it. Nor do we have an approved drug to give to patients to make their hearts resistant to infarction. The fundamental problem with such work is that one simply doesn’t know what one doesn’t know. In 1986, little was known about mitochondrial permeability transition pores or ATP-sensitive potassium channels, structures that play important roles in the preconditioning mechanism. The way science has always worked is that we attack any new phenomenon using the tools that science has provided us up to that moment. It is all we have to work with. Imagine the ancient Greeks trying to do mechanistic studies using just fire, air, earth, and water. The Sigma catalog must have been pretty thin in those days.
By 1986, pharmacology had already provided us with some insight as to how preconditioning might work. In 1905, when John Newport Langley noted that curare and pilocarpine could block the actions of neural transmitters on skeletal muscle, he proposed that various active chemicals in the body exert their actions on cells by attaching to “receptor substances.”2 Between then and 1986, much had been learned about receptors and how they transmit their signals to the inner machinery of the cell. In 1956, Krebs and Fischer3 discovered that inactive phosphorylase b is converted to its active form, phosphorylase a, by adding a single phosphate group to the molecule. The enzyme that performs this posttranslational modification was termed a kinase, and, by 1986, many kinases had been described. Examination of kinase-like sequences in the human genome suggests that there may be more than a thousand kinases present, which should keep this field busy for a long time trying to figure out what all of them do. So, in 1991, an obvious experiment was to test whether receptors to adenosine, a chemical that is quickly made and released by ischemic tissue and that had already been proposed to be a protective substance, might be involved in preconditioning. We were lucky enough to perform this experiment,4 and, as a result, it soon became apparent that preconditioning was receptor-mediated. With another stroke of incredible luck, we were able to establish that a well-known kinase, protein kinase C, was involved in the signal transduction pathway.5 Since that time, most investigators have generally been looking at cardioprotection through kinase-colored glasses. Juhaszova et al,6 using isolated cardiomyocytes, published strong evidence that protection conferred by preconditioning involved prevention of the formation of the mitochondrial permeability transition pore during ischemia/reperfusion and suggested that another kinase, glycogen synthase kinase (GSK)-3β, was the gatekeeper for this action. Indeed, pharmacological inhibition of GSK-3β seems to mimic preconditioning,7 but phosphorylation of GSK-3β is not very well correlated with protection from pharmacological or ischemic preconditioning in whole hearts.8 Thus, how preconditioning prevents pore formation is still controversial.
But then, you don’t know what you don’t know. Posttranslational modifications come in other forms besides just kinase-mediated phosphorylations. Other well-known examples are redox signaling in which reactive oxygen species modify enzymes in a meaningful way, nitric oxide, cyclic nucleotides, calcium, and even carbon monoxide signaling. Another interesting signaling system that is still poorly understood involves the linkage of β-N-acetylglucosamine through an oxygen atom to proteins at their serine and threonine residues. This interesting burgeoning signal transduction cascade appears to be controlled by a single enzyme that attaches the sugar group (uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylglucosaminyltransferase) and another that removes it (β-N-acetylglucosaminidase). Cardiovascular scientists had little interest in this seemingly esoteric system until Jones and colleagues noted that the enhanced O-linked β-N-acetylglucosamine (O-GlcNAc) content of proteins of a cardiomyocyte was correlated with preconditioning-induced resistance to cardiomyocyte death from simulated ischemia.9 Inhibiting β-N-acetylglucosaminidase with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate increased the O-GlcNAc content of mouse hearts and protected them from infarction during regional ischemia/reperfusion by an amount similar to that seen in ischemic preconditioning.9 That got the attention of the community. In this issue of Circulation Research, this group used an isolated cardiomyocyte model of simulated ischemia to further investigate the involvement of this system.10 Genetic manipulation of β-N-acetylglucosaminidase indeed modified both the O-GlcNAc content and the resistance of these cells to simulated ischemia. More interestingly, they showed that the mitochondrial voltage-dependent anion channel (VDAC) is also a target for O-GlcNAc modification. That is important because VDAC, a putative constituent of the elusive permeability transition pore, clearly does not show altered phosphorylation in protected hearts.8 Of course, O-GlcNAc modification of VDAC would fly under the kinase radar, and, as such, O-GlcNAc modification of VDAC may be the real gatekeeper of the permeability transition pore. O-GlcNAc could easily be incorporated into currently proposed signal transduction schemes for preconditioning and may simply serve as a bridge to the transition pore. That, of course, will require much more research to establish, but at least we have somewhere to look. It just shows you don’t know what you don’t know.
Sources of Funding
J.M.D. and M.V.C. are supported by NIH grant HL-20648.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986; 74: 1124–1136.
Liu GS, Thornton J, Van Winkle DM, Stanley AWH, Olsson RA, Downey JM. Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation. 1991; 84: 350–356.
Juhaszova M, Zorov DB, Kim S-H, Pepe S, Fu Q, Fishbein KW, Ziman BD, Wang S, Ytrehus K, Antos CL, Olson EN, Sollott SJ. Glycogen synthase kinase-3β mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest. 2004; 113: 1535–1549.
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Jones SP, Zachara NE, Ngoh GA, Hill BG, Teshima Y, Bhatnagar A, Hart GW, Marbán E. Cardioprotection by N-acetylglucosamine linkage to cellular proteins. Circulation. 2008; 117: 1172–1182.
Ngoh GA, Facundo HT, Hamid T, Dillmann W, Zachara NE, Jones SP. Unique hexosaminidase reduces metabolic survival signal and sensitizes cardiac myocytes to hypoxia/reoxygenation injury. Circ Res. 2009; 104: 41–49.