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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The serine/threonine kinase protein kinase C ⑀ (PKC⑀) has been shown to be a critical component in the heart's resistance to cell death following ischemic insult. Recent studies have indicated that PKC⑀ forms multi-protein signaling complexes to accomplish signal transduction in cardiac protection. Using two-dimensional electrophoresis (2DE), combined with matrix-assisted laser desorption ionization mass spectrometry (MS), the initial analysis of these complexes identified signaling molecules, structural proteins, and stress-activated proteins. The initial analysis, although fruitful, was limited by the number of proteins revealed on the 2D gels. It was also apparent that many known cardiac protective functions of PKC⑀ could not be fully accounted for by the proteins identified in the initial analysis. Here we report the identification of an additional 57 proteins in PKC⑀ complexes using complimentary separation techniques, combined with high sensitivity MS. These techniques include 2DE or large format 1D SDS-PAGE followed by LC/MS/MS and solution trypsin digestion followed by LC/MS/MS, all of which yielded novel data regarding PKC⑀ protein complexes. Nanoscale LC/ MS/MS for the analysis of gel-isolated proteins was performed with sub-femtomole sensitivity. In contrast to 2DE analyses, the identification of proteins from 1D gels was independent of their visualization via staining and allowed for the identification of proteins with high isoelectric points. We found that PKC⑀ complexes contain numerous structural and signaling molecules that had escaped detection by our previous analyses. Most importantly, we identified two new groups of proteins that were previously unrecognized as components of the PKC⑀ complex: metabolism-related proteins and transcription/translation-related proteins. Molecular & Cellular Proteomics 1:421-433, 2002.

Receptors for activated C kinase (RACKs) have been shown to facilitate activation of protein kinase C (PKC). However, it is unknown whether PKC activation modulates RACK protein expression and PKC-RACK interactions. This issue was studied in two PKCepsilon transgenic lines exhibiting dichotomous cardiac phenotypes: one exhibits increased resistance to myocardial ischemia (cardioprotected phenotype) induced by a modest increase in PKCepsilon activity (228 +/- 23% of control), whereas the other exhibits cardiac hypertrophy and failure (hypertrophied phenotype) induced by a marked increase in PKCepsilon activity (452 +/- 28% of control). Our data demonstrate that activation of PKC modulates the expression of RACK isotypes and PKC-RACK interactions in a PKCepsilon activity- and dosage-dependent fashion. We found that, in mice displaying the cardioprotected phenotype, activation of PKCepsilon enhanced RACK2 expression (178 +/- 13% of control) and particulate PKCepsilon-RACK2 protein-protein interactions (178 +/- 18% of control). In contrast, in mice displaying the hypertrophied phenotype, there was not only an increase in RACK2 expression (330 +/- 33% of control) and particulate PKCepsilon-RACK2 interactions (154 +/- 14% of control) but also in RACK1 protein expression (174 +/- 10% of control). Most notably, PKCepsilon-RACK1 interactions were identified in this line. With the use of transgenic mice expressing a dominant negative PKCepsilon, we found that the changes in RACK expression as well as the attending cardiac phenotypes were dependent on PKCepsilon activity. Our observations demonstrate that RACK expression is dynamically regulated by PKCepsilon and suggest that differential patterns of PKCepsilon-RACK interactions may be important determinants of PKCepsilon-dependent cardiac phenotypes.

Although ancillary pathways of glucose metabolism are critical for synthesizing cellular building blocks and modulating stress responses, how they are regulated remains unclear. In the present study, we used radiometric glycolysis assays, [13C6]-glucose isotope tracing, and extracellular flux analysis to understand how phosphofructokinase (PFK)-mediated changes in glycolysis regulate glucose carbon partitioning into catabolic and anabolic pathways. Expression of kinase-deficient or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat neonatal cardiomyocytes co-ordinately regulated glycolytic rate and lactate production. Nevertheless, in all groups, >40% of glucose consumed by the cells was unaccounted for via catabolism to pyruvate, which suggests entry of glucose carbons into ancillary pathways branching from metabolites formed in the preparatory phase of glycolysis. Analysis of 13C fractional enrichment patterns suggests that PFK activity regulates glucose carbon incorporation directly into the ribose and the glycerol moieties of purines and phospholipids, respectively. Pyrimidines, UDP-N-acetylhexosamine, and the fatty acyl chains of phosphatidylinositol and triglycerides showed lower 13C incorporation under conditions of high PFK activity; the isotopologue 13C enrichment pattern of each metabolite indicated limitations in mitochondria-engendered aspartate, acetyl CoA and fatty acids. Consistent with this notion, high glycolytic rate diminished mitochondrial activity and the coupling of glycolysis to glucose oxidation. These findings suggest that a major portion of intracellular glucose in cardiac myocytes is apportioned for ancillary biosynthetic reactions and that PFK co-ordinates the activities of the pentose phosphate, hexosamine biosynthetic, and glycerolipid synthesis pathways by directly modulating glycolytic intermediate entry into auxiliary glucose metabolism pathways and by indirectly regulating mitochondrial cataplerosis.

Acrolein is a ubiquitous component of environmental pollutants such as automobile exhaust, cigarette, wood, and coal smoke. It is also a natural constituent of several foods and is generated endogenously during inflammation or oxidation of unsaturated lipids. Because increased inflammation and episodic exposure to acrolein-rich pollutants such as traffic emissions or cigarette smoke have been linked to acute myocardial infarction, we examined the effects of acrolein on matrix metalloproteinases (MMPs), which destabilize atherosclerotic plaques. Our studies show that exposure to acrolein resulted in the secretion of MMP-9 from differentiated THP-1 macrophages. Acrolein-treatment of macrophages also led to an increase in reactive oxygen species (ROS), free intracellular calcium ([Ca 2+ ] i ), and xanthine oxidase (XO) activity. ROS production was prevented by allopurinol, but not by rotenone or apocynin and by buffering changes in [Ca 2+ ] I with BAPTA-AM. The increase in MMP production was abolished by pre-treatment with the antioxidants Tiron and N-acetyl cysteine (NAC) or with the xanthine oxidase inhibitors allopurinol or oxypurinol. Finally, MMP activity was significantly stimulated in aortic sections from apoE-null mice containing advanced atherosclerotic lesions after exposure to acrolein ex vivo. These observations suggest that acrolein exposure results in MMP secretion from macrophages via a mechanism that involves an increase in [Ca 2+ ] I , leading to xanthine oxidase activation and an increase in ROS production. ROSdependent activation of MMPs by acrolein could destabilize atherosclerotic lesions during brief episodes of inflammation or pollutant exposure.