Diabetic cardiomyopathy is characterized by impaired cardiac contractility leading to poor myocardial performance. We investigated the role that the hexosamine pathway, and especially altered nuclear O-GlcNAcylation, plays in the development of diabetic cardiomyopathy. Incubating neonatal rat cardiomyocytes in high glucose (25 mM) resulted in prolonged calcium transients when compared with myocytes incubated in normal glucose (5.5 mM), which is consistent with delayed myocardial relaxation. High glucose-treated myocytes also exhibited reduced sarcoendoplasmic reticulum Ca 2؉ -ATPase 2a (SERCA2a) mRNA and protein expression, decreased SERCA2a promoter activity, and increased O-GlcNAcylation of nuclear proteins compared with myocytes treated with normal glucose. Exposure of myocytes to 8 mM glucosamine or an adenovirus expressing O-GlcNAc-transferase (OGT) resulted in prolonged calcium transient decays and significantly reduced SERCA2a protein levels, whereas treatment with an adenovirus encoding O-GlcNAcase (GCA) resulted in improved calcium transients and SERCA2a protein levels in myocytes exposed to high glucose. Effects of elevated glucose or altered O-GlcNAcylation were also observed on essential transcription factors involved in cardiomyocyte function. High glucosetreated myocytes (with or without OGT adenovirus) exhibited increased levels of O-GlcNAcylated specificity protein 1 compared with control myocytes, whereas infecting high glucose-treated myocytes with GCA adenovirus reduced the degree of specificity protein 1 GlcNAcylation. Treatment of myocytes with 25 mM glucose, 8 mM glucosamine, or OGT adenovirus also significantly reduced levels of myocytes enhancer factor-2A protein compared with control myocytes, whereas infection with GCA adenovirus resulted in improved myocytes enhancer factor-2 expression. Our results suggest that the hexosamine pathway, and O-GlcNAcylation in particular, is important in impaired cardiac myocyte function and the development of diabetic cardiomyopathy.

Three hallmark features of the cardiac hypertrophic growth program are increases in cell size, sarcomeric organization, and the induction of certain cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by α1- adrenergic receptor agonists, such as phenylephrine (PE) and other growth factors that activate mitogen- activated protein kinases (MAPKs). In this study the MAPK family members extracellular signal–regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases possessing gain-of-function mutations. Transfected cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the A- and B-type natriuretic peptides (NPs), as well as the α-skeletal actin (α-SkA) gene. While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and α-SkA promoter activities, neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and α-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE. Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580. These results demonstrate novel and potentially central roles for MKK6 and p38 in the regulation of myocardial cell hypertrophy.

The Ca 2 ϩ ATPase of the sarcoplasmic reticulum (SERCA2) plays a dominant role in lowering cytoplasmic calcium levels during cardiac relaxation and reduction of its activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. To determine the contractile alterations resulting from increased SERCA2 expression, we generated transgenic mice overexpressing a rat SERCA2 transgene. Characterization of a heterozygous transgenic mouse line (CJ5) showed that the amount of SERCA2 mRNA and protein increased 2.6-fold and 1.2-fold, respectively, relative to control mice. Determination of the relative synthesis rate of SERCA2 protein showed an 82% increase. The mRNA levels of some of the other genes involved in calcium handling, such as the ryanodine receptor and calsequestrin, remained unchanged, but the mRNA levels of phospholamban and Na ϩ /Ca 2 ϩ exchanger increased 1.4-fold and 1.8-fold, respectively. The increase in phospholamban or Na ϩ /Ca 2 ϩ exchanger mRNAs did not, however, result in changes in protein levels. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline ( t 1/2 ) and myocyte relengthening ( t 1/2 ) were accelerated by 23 and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster. In isolated papillary muscle from SERCA2 transgenic mice, the time to half maximum postrest potentiation was significantly shorter than in negative littermates. Furthermore, cardiac function measured in vivo, demonstrated significantly accelerated contraction and relaxation in SERCA2 transgenic mice that were further augmented in both groups with isoproterenol administration. Similar results were obtained for the contractile performance of myocytes isolated from a separate line (CJ2) of homozygous SERCA2 transgenic mice.

In cardiac myocytes the stimulation of p38 mitogenactivated protein kinase activates a hypertrophic growth program and the induction of the cardiac-specific genes associated with this program. This study focused on determining whether these novel growth-promoting effects are accompanied by the p38-mediated inhibition of apoptosis, and if so, what signaling pathways might be responsible. Primary neonatal rat ventricular myocytes were driven into apoptosis by treatments known to induce apoptosis in other cell types, e.g. incubation with anisomycin or overexpression constitutively active MEKK-1 (MEKK-1 COOH ), a protein that strongly activates extracellular signal-regulated kinase and N-terminal c-Jun kinase, but not p38. Overexpression of constitutively active MKK6, MKK6 (Glu), which selectively activates p38 in cardiac myocytes, protected cells from either anisomycin-or MEKK-1 COOH -induced apoptosis. This protection was blocked by SB 203580, a selective p38 inhibitor. MKK6 (Glu) also activated transcription mediated by NF-B, a factor which protects other cell types from apoptosis. The activation of NF-B and the protection from apoptosis mediated by MKK6 (Glu) were both blocked by SB 203580. Interestingly, overexpression of a mutant form of I-B␣, which inhibits nuclear translocation of NF-B, completely blocked MKK6 (Glu)-activated NF-B but had little effect on MKK6s anti-apoptotic effects. These findings suggest that, in part, the overexpression of MKK6 (Glu) may foster growth and survival of cardiac myocytes by protecting them from apoptosis in a p38-dependent manner. Additionally, while NF-B is activated in myocardial cells by p38, this does not appear to be the major mechanism by which MKK6 (Glu) exerts its anti-apoptotic effects in this cell type, suggesting a novel pathway for p38-mediated protection from apoptosis.

In cardiac myocytes, the stimulation of p38 MAPK by the MAPKK, MKK6, activates the transcription factor, NF-B, and protects cells from apoptosis. In the present study in primary neonatal rat cardiac myocytes, constitutively active MKK6, MKK6(Glu), bound to IB kinase (IKK)-␤ and stimulated its abilities to phosphorylate IB and to activate NF-B. MKK6(Glu) induced NF-B-dependent interleukin (IL)-6 transcription and IL-6 release in a p38-dependent manner. IL-6 protected myocardial cells against apoptosis. Like IL-6, TNF-␣, which activates both NF-B and p38, also induced p38-dependent IL-6 expression and release and protected myocytes from apoptotis. While TNF-␣ was relatively ineffective, IL-6 activated myocardial cell STAT3 by about 8-fold, indicating a probable role for this transcription factor in IL-6-mediated protection from apoptosis. TNF-␣-mediated IL-6 induction was inhibited by a kinase-inactive form of the MAPKKK, TGF-␤ activated protein kinase (Tak1), which is known to activate p38 and NF-B in other cell types. Thus, by stimulating both p38 and NF-B, Tak1-activating cytokines, like TNF-␣, can induce IL-6 expression and release. Moreover, the myocyte-derived IL-6 may then function in an autocrine and/or paracrine fashion to augment myocardial cell survival during stresses that activate p38.