Background Exercise promotes metabolic remodeling in the heart, which is associated with physiologic cardiac growth; however, it is not known whether or how physical activity-induced changes in cardiac metabolism cause myocardial remodeling. In this study, we tested whether exercise-mediated changes in cardiomyocyte glucose metabolism are important for physiologic cardiac growth. Methods We used radiometric, immunologic, metabolomic, and biochemical assays to measure changes in myocardial glucose metabolism in mice subjected to acute and chronic treadmill exercise. To assess the relevance of changes in glycolytic activity, we determined how cardiac-specific expression of mutant forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) affect cardiac structure, function, metabolism, and gene programs relevant to cardiac remodeling. Metabolomic and transcriptomic screenings were used to identify metabolic pathways and gene sets regulated by glycolytic activity in the heart. Results Exercise acutely decreased glucose utilization via glycolysis by modulating circulating substrates and reducing phosphofructokinase activity; however, upon exercise adaptation and recovery there was an increase in myocardial phosphofructokinase activity and glycolysis. Cardiac-specific expression of a kinase-deficient PFK2 transgene (GlycoLo mice) lowered glycolytic rate and regulated the expression of genes known to promote cardiac growth. Hearts of GlycoLo mice had larger myocytes, enhanced cardiac function, and higher capillary-to-myocyte ratios. Expression of phosphatase-deficient PFK2 in the heart (GlycoHi mice) increased glucose utilization and promoted a more pathological form of hypertrophy devoid of transcriptional activation of the physiologic cardiac growth program. Modulation of phosphofructokinase activity was sufficient to regulate the glucose-fatty acid cycle in the heart; however, metabolic inflexibility caused by invariantly low or high phosphofructokinase activity caused modest mitochondrial damage. Transcriptomic analyses showed that glycolysis regulates the expression of key genes involved in cardiac metabolism and remodeling. Conclusions Exercise-induced decreases in glycolytic activity stimulate physiologic cardiac remodeling, and metabolic flexibility is important for maintaining mitochondrial health in the heart.

Activation of protein kinase C (PKC) ⑀ by nitric oxide (NO) has been implicated in the development of cardioprotection. However, the cellular mechanisms underlying the activation of PKC⑀ by NO remain largely unknown. Nitration of protein tyrosine residues has been shown to alter functions of a variety of proteins, and NO-derived peroxynitrite is known as a strong nitrating agent. In this investigation, we demonstrate that NO donors promote translocation and activation of PKC⑀ in an NO-and peroxynitrite-dependent fashion. NO induces peroxynitrite-mediated tyrosine nitration of PKC⑀ in rabbit cardiomyocytes in vitro, and nitrotyrosine residues were also detected on PKC⑀ in vivo in the rabbit myocardium preconditioned with NO donors. Furthermore, coimmunoprecipitation of PKC⑀ and its receptor for activated C kinase, RACK2, illustrated a peroxynitrite-dependent increase in PKC⑀-RACK2 interactions in NO donor-treated cardiomyocytes. Moreover, using an enzyme-linked immunosorbent assaybased protein-protein interaction assay, PKC⑀ proteins treated with the peroxynitrite donor SIN-1 exhibited enhanced binding to RACK2 in an acellular environment. Our data demonstrate that post-translational modification of PKC⑀ by NO donors, namely nitration of PKC⑀, facilitates its interaction with RACK2 and promotes translocation and activation of PKC⑀. These findings offer a plausible novel mechanism by which NO activates the PKC signaling pathway. Protein kinase C (PKC)1 is a family of serine-threonine kinases that participate in numerous biological processes (1, 2). In the heart, activation of PKC reduces the myocardial ischemic injury, whereas inhibition of PKC abolishes ischemic preconditioning (3-5). Recently, it has been shown that this cardioprotective effect can be fully mimicked by modulating the activity of a single isozyme of this family, the ⑀ isoform of PKC (6 -9). Multiple molecular events have been shown to have an activating effect on this enzyme, among which, of particular interest, is nitric oxide (NO). Although the effects of NO on PKC depend on its biological functions and on the cell types (10 -14), NO-induced activation of PKC is well documented in the heart (15, 16). Several investigations have demonstrated that at doses that produce a cardioprotective effect, exogenous NO (released by NO donors) activates PKC⑀ in an isoformspecific manner (17-20). Furthermore, activation of this isozyme has been demonstrated to play an essential role in orchestrating the signal transduction events during NO-induced cardioprotection against ischemic injury (15, 21). However, the exact molecular mechanism(s) whereby NO activates PKC⑀ in the heart remain largely unknown.As a relatively stable hydrophobic free radical gas, NO can readily diffuse through cell membranes (22). Within cells, NO itself and NO-derived reactive nitrogen species are capable of reacting with various molecular targets that include complex biological molecules, such as proteins, lipids, and DNA, as well as low molecular weight compounds (23). One of the impo...

We have previously shown that protein kinase C (PKC)-epsilon, nuclear factor (NF)-kappaB, and mitogen-activated protein kinases (MAPKs) are essential signaling elements in ischemic preconditioning. In the present study, we examined whether activation of PKCepsilon affects the activation of NF-kappaB in cardiac myocytes and whether MAPKs are mediators of this signaling event. Activation of PKCepsilon (+108% above control) in adult rabbit cardiomyocytes to a degree that has been previously shown to protect myocytes against hypoxic injury increased the DNA-binding activity of NF-kappaB (+164%) and activator protein (AP)-1 (+127%) but not that of Elk-1. Activation of PKCeta did not have an effect on these transcription factors. Activation of PKCepsilon also enhanced the phosphorylation activities of the p44/p42 MAPKs and the p54/p46 c-Jun NH(2)-terminal kinases (JNKs). PKCepsilon-induced activation of NF-kappaB and AP-1 was completely abolished by inhibition of the p44/p42 MAPK pathway with PD98059 and by inhibition of the p54/p46 JNK pathway with a dominant negative mutant of MAPK kinase-4, indicating that both signaling pathways are necessary. Taken together, these data identify NF-kappaB and AP-1 as downstream targets of PKCepsilon, thereby establishing a molecular link between activation of PKCepsilon and activation of NF-kappaB and AP-1 in cardiomyocytes. The results further demonstrate that both the p44/p42 MAPK and the p54/p46 JNK signaling pathways are essential mediators of this event.

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