c-Jun N-terminal kinases (JNK) antagonize cardiac growth through cross-talk with calcineurin-NFAT signaling

The c-Jun N-terminal kinase (JNK) branch of the mitogen-activated protein kinase (MAPK) signaling pathway regulates cellular differentiation, stress responsiveness and apoptosis in multicellular eukaryotic organisms. Here we investigated the functional importance of JNK signaling in regulating differentiated cellular growth in the post-mitotic myocardium. JNK1/2 gene-targeted mice and transgenic mice expressing dominant negative JNK1/2 were determined to have enhanced myocardial growth following stress stimulation or with normal aging. A mechanism underlying this effect was suggested by the observation that JNK directly regulated nuclear factor of activated T-cell (NFAT) activation in culture and in transgenic mice containing an NFAT-dependent luciferase reporter. Moreover, calcineurin Abeta gene targeting abrogated the pro-growth effects associated with JNK inhibition in the heart, while expression of an MKK7-JNK1 fusion protein in the heart partially reduced calcineurin-mediated cardiac hypertrophy. Collectively, these results indicate that JNK signaling antagonizes the differentiated growth response of the myocardium through direct cross-talk with the calcineurin-NFAT pathway. These results also suggest that myocardial JNK activation is primarily dedicated to modulating calcineurin-NFAT signaling in the regulation of differentiated heart growth.     

A coupled assay for UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc).

UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc) (i.e., core 2 GlcNAc-T) is a developmentally regulated enzyme of the O-linked oligosaccharide biosynthesis pathway. We have developed a coupled-enzyme assay for core 2 GlcNAc-T that is approximately 100 times more sensitive than the standard assay using UDP-[3H]GlcNAc as a sugar donor. Core 2 GlcNAc-T reactions were performed using unlabeled UDP-GlcNAc donor and Gal beta 1-3GalNAc alpha-paranitrophenyl (pNp) as acceptor. The product, Gal beta 1-3(GlcNAc beta 1-6)GalNAc alpha-pNp was then further reacted with purified bovine beta 1-4Gal-T and UDP-[3H]Gal to produce Gal beta 1-3([3H]Gal beta 1-4GlcNAc beta 1-6) GalNAc alpha-pNp, which was separated on an Ultrahydrogel HPLC column. Approximately 10% of the available GlcNAc-terminating acceptor was substituted in the Gal-T reaction, allowing 1 pmol of product to be readily detected. The increased sensitivity of the coupled assay should facilitate studies of core 2 GlcNAc-T activity where material is limiting or specific activity is low.     

Differential regulation of angiotensin II-induced expression of connective tissue growth factor by protein kinase C isoforms in the myocardium

Protein kinase C (PKC) and angiotensin II (AngII) can regulate cardiac function in pathological conditions such as in diabetes or ischemic heart disease. We have reported that expression of connective tissue growth factor (CTGF) is increased in the myocardium of diabetic mice. Now we showed that the increase in CTGF expression in cardiac tissues of streptozotocin-induced diabetic rats was reversed by captopril and islet cell transplantation. Infusion of AngII in rats increased CTGF mRNA expression by 15-fold, which was completely inhibited by co-infusion with AT1 receptor antagonist, candesartan. Similarly, incubation of cultured cardiomyocytes with AngII increased CTGF mRNA expression by 2-fold, which was blocked by candesartan and a general PKC inhibitor, GF109203X. The role of PKC isoform-dependent action was further studied using adenoviral vector-mediated gene transfer of dominant negative (dn) PKC or wild type PKC isoforms. Expression of dnPKCalpha, -epsilon, and -zeta isoforms suppressed AngII-induced CTGF expression in cardiomyocytes. In contrast, expression of dominant negative PKCdelta significantly increased AngII-induced CTGF expression, whereas expression of wild type PKCdelta inhibited this induction. This inhibitory effect was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of PKCdelta (deltaTg mice). Thus, AngII can regulate CTGF expression in cardiomyocytes through a PKC activation-mediated pathway in an isoform-selective manner both in physiological and diabetic states and may contribute to the development of cardiac fibrosis in diabetic cardiomyopathy.     

beta1,6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T) expression in normal rat tissues and different cell lines: evidence for complex mechanisms of regulation

The distribution of the Golgi enzyme beta1, 6-N- acetylglucosaminyltransferase (core 2 GlcNAc-T for short) has been investigated in several tissue and cell systems by combining the potentials of a polyclonal antibody and a novel, sensitive fluorescent enzyme assay. In normal rat tissues, levels of the protein were found to vary and as a general trend did not correlate with enzyme activities. Additionally, we observed tissue-specific core 2 GlcNAc-T forms of various size: 75 kDa (liver), 70 kDa (spleen), 60 kDA (heart), and 50 kDa (heart and lung). These forms might arise from differential protein modifications; alternatively, the smaller form may be a product of proteolytic cleavage, given the presence of a catalytically inactive 50 kDa species in rat serum. Chinese hamster ovary (CHO), MDAY-D2, PSA- 5E, and PYS-2 cell lines consistently displayed a 70 kDa enzyme. When induced to retrodifferentiate in the presence of butyrate + cholera toxin, CHO cells exhibited a 21-fold increase in enzyme activity, while protein levels remained constant. A similar trend was observed in the embryonal endoderm cell lines PSA-5E and PYS-2, where an approximately 100-fold difference in core 2 GlcNAc-T activity was found notwithstanding unchanged amounts of the protein and identical mRNA levels, as evidenced by RT-PCR. In contrast, levels of core 2 GlcNAc-T activity in MDAY-D2 cells correlated well with protein expression. Taken together, these observations demonstrate that core 2 GlcNAc-T expression may be subjected to multiple mechanisms of regulation and suggest that in at least some instances (i.e., PSA-5E and PYS-2 cells) expression may be regulated exclusively via posttranslational mechanism(s) of control.      

The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice

Members of the mitogen-activated protein kinase (MAPK) cascade such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 are implicated as important regulators of cardiomyocyte hypertrophic growth in culture. However, the role that individual MAPK pathways play in vivo has not been extensively evaluated. Here we generated nine transgenic mouse lines with cardiac-restricted expression of an activated MEK1 cDNA in the heart. MEK1 transgenic mice demonstrated concentric hypertrophy without signs of cardiomyopathy or lethality up to 12 months of age. MEK1 transgenic mice showed a dramatic increase in cardiac function, as measured by echocardiography and isolated working heart preparation, without signs of decompensation over time. MEK1 transgenic mice and MEK1 adenovirus-infected neonatal cardiomyocytes each demonstrated ERK1/2, but not p38 or JNK, activation. MEK1 transgenic mice and MEK1 adenovirus-infected cultured cardiomyocytes were also partially resistant to apoptotic stimuli. The results of the present study indicate that the MEK1-ERK1/2 signaling pathway stimulates a physiologic hypertrophy response associated with augmented cardiac function and partial resistance to apoptotsis.     

Akt activation and augmented fibronectin production in hyperhexosemia

Dysmetabolic state in diabetes may lead to augmented synthesis of extracellular matrix (ECM) proteins. In the endothelial cells, we have previously demonstrated that glucose-induced fibronectin (FN) production and that of its splice variant, EDB(+)FN, is regulated by protein kinase B (PKB, also known as Akt). In this study, we investigated the role of Akt1 in ECM protein production in the organs affected by chronic diabetic complications. We studied Akt1/PKBalpha knockout mice and wild-type control littermates. To avoid confounding effects of systemic insulin, we used 30% galactose feeding to induce hyperhexosemia for 8 wk starting at 6 wk of age. We investigated FN mRNA, EDB(+)FN mRNA, and transforming growth factor (TGF)-beta mRNA expression, Akt phosphorylation, Akt kinase activity, and NF-kappaB and AP-1 activation in the retina, heart, and kidney. Renal and cardiac tissues were histologically examined. Galactose feeding caused significant upregulation of FN, EDB(+)FN, and TGF-beta in all tissues. FN protein levels paralleled mRNA. Such upregulation were prevented in Akt1-deficient galactose-fed mice. Galactose feeding caused ECM protein deposition in the glomeruli and in the myocardium, which was prevented in the Akt knockout mice. NF-kappaB and AP-1 activation was pronounced in galactose-fed wild-type mice and prevented in the galactose-fed Akt1/PKBalpha-deficient group. In the retina and kidney, Ser473 was the predominant site for Akt phosphorylation, whereas in the heart it was Thr308. Parallel experiment in streptozotocin-induced diabetic animals showed similar results. The data from this study indicate that hyperhexosemia-induced Akt/PKB activation may be an important mechanism leading to NF-kappaB and AP-1 activation and increased ECM protein synthesis in the organs affected by chronic diabetic complications.     

The biosynthesis of highly branched N-glycans: studies on the sequential pathway and functional role of N-acetylglucosaminyltransferases I, II, III, IV, V and VI

At least 6 N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V and VI) are involved in initiating the synthesis of the various branches found in complex asparagine-linked oligosaccharides (N-glycans), as indicated below: GlcNAc beta 1-6 GlcNAc-T V GlcNAc beta 1-4 GlcNAc-T VI GlcNAc beta 1-2Man alpha 1-6 GlcNAc-T II GlcNAc beta 1-4Man beta 1-4-R GlcNAc T III GlcNAc beta 1-4Man alpha 1-3 GlcNAc-T IV GlcNAc beta 1-2 GlcNAc-T I where R is GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAcAsn-X. HPLC was used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct (I. Brockhausen, J.P. Carver and H. Schachter (1988) Biochem. Cell Biol. 66, 1134-1151). The following sequential rules have been established: GlcNAc-T I must act before GlcNAc-T II, III and IV; GlcNAc-T II, IV and V cannot act after GlcNAc-T III, i.e., on bisected substrates; GlcNAc-T VI can act on both bisected and non-bisected substrates; both Glc-NAc-T I and II must act before GlcNAc-T V and VI; GlcNAc-T V cannot act after GlcNAc-T VI. GlcNAc-T V is the only enzyme among the 6 transferases cited above which can be essayed in the absence of Mn2+. In studies on the possible functional role of N-glycan branching, we have measured GlcNAc-T III in pre-neoplastic rat liver nodules (S. Narasimhan, H. Schachter and S. Rajalakshmi (1988) J. Biol. Chem. 263, 1273-1281). The nodules were initiated by administration of a single dose of carcinogen 1,2-dimethyl-hydrazine.2 HCl 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. The nodules had significant GlcNAc-T III activity (1.2-2.2 nmol/h/mg), whereas the surrounding liver, regenerating liver 24 h after partial hepatectomy and control liver from normal rats had negligible activity (0.02-0.03 nmol/h/mg). These results suggest that GlcNAc-T III is induced at the pre-neoplastic stage in liver carcinogenesis and are consistent with the reported presence of bisecting GlcNAc residues in N-glycans from rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (A. Kobata and K. Yamashita (1984) Pure Appl. Chem. 56, 821-832).     

Nrf2 prevents diabetic cardiomyopathy via antioxidant effect and normalization of glucose and lipid metabolism in the heart

Metabolic disorders and oxidative stress are the main causes of diabetic cardiomyopathy. Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) exerts a powerful antioxidant effect and prevents the progression of diabetic cardiomyopathy. However, the mechanism of its cardiac protection and direct action on cardiomyocytes are not well understood. Here, we investigated in a cardiomyocyte-restricted Nrf2 transgenic mice (Nrf2-TG) the direct effect of Nrf2 on cardiomyocytes in DCM and its mechanism. In this study, cardiomyocyte-restricted Nrf2 transgenic mice (Nrf2-TG) were used to directly observe whether cardiomyocyte-specific overexpression of Nrf2 can prevent diabetic cardiomyopathy and correct glucose and lipid metabolism disorders in the heart. Compared to wild-type mice, Nrf2-TG mice showed resistance to diabetic cardiomyopathy in a streptozotocin-induced type 1 diabetes mouse model. This was primarily manifested as improved echocardiography results as well as reduced myocardial fibrosis, cardiac inflammation, and oxidative stress. These results showed that Nrf2 can directly act on cardiomyocytes to exert a cardioprotective role. Mechanistically, the cardioprotective effects of Nrf2 depend on its antioxidation activity, partially through improving glucose and lipid metabolism by directly targeting lipid metabolic pathway of AMPK/Sirt1/PGC-1α activation via upstream genes of sestrin2 and LKB1, and indirectly enabling AKT/GSK-3β/HK-Ⅱ activity via AMPK mediated p70S6K inhibition.     

Oxidative metabolism of Polytron versus Nagarse mitochondria in hearts of genetically diabetic mice

We have shown previously that heart mitochondria obtained by the Nagarse method from genetically diabetic mice (C57BL/KsJ db / db) exhibit a defect in oxidizing NAD⁺-linked substrates (Kuo, T.H., Moore, K.H., Giacomelli, F. and Wiener, J. (1983) Diabetes 32, 781–787). In this study, the oxidative phosphorylation characteristics of cardiac mitochondria isolated by the Polytron method were compared with that of Nagarse mitochondria. Evidence is presented here that in the diabetic heart both Nagarse and Polytron mitochondria have defective pyruvate oxidation, whereas only the former exhibit impaired fatty acid oxidation. Assay of two rate-limiting β-oxidation enzymes, namely β-hydroxyacyl-CoA dehydrogenase and β-ketothiolase, indicates no alteration in specific activities from diabetic mice vs. controls. The data suggest that two populations of mitochondria are present in myocardium and that the defective oxidative metabolism in the cardiac mitochondria of db / db mice may be linked to deficiencies in total NAD + NADH content.     

Exercise training attenuated the PKB and GSK-3 dephosphorylation in the myocardium of ZDF rats.

Cardiac dysfunction is a severe secondary effect of Type 2 diabetes. Recruitment of the protein kinase B/glycogen synthase kinase-3 pathway represents an integral event in glucose homeostasis, albeit its regulation in the diabetic heart remains undefined. Thus the following study tested the hypothesis that the regulation of protein kinase B/glycogen synthase kinase-3 was altered in the myocardium of the Zucker diabetic fatty rat. Second, exercise has been shown to improve glucose homeostasis, and, in this regard, the effect of swimming training on the regulation of protein kinase B/glycogen synthase kinase-3 in the diabetic rat heart was examined. In the sedentary Zucker diabetic fatty rats, glucose levels were elevated, and cardiac glycogen content increased, compared with wild type. A 13-wk swimming regimen significantly reduced plasma glucose levels and cardiac glycogen content and partially normalized protein kinase B-serine473, protein kinase B-threonine308, and glycogen synthase kinase-3alpha phosphorylation in Zucker diabetic fatty rats. In conclusion, hyperglycemia and increased cardiac glycogen content in the Zucker diabetic fatty rats were associated with dysregulation of protein kinase B/glycogen synthase kinase-3 phosphorylation. These anomalies in the Zucker diabetic fatty rat were partially normalized with swimming. These data support the premise that exercise training may protect the heart against the deleterious consequences of diabetes.     

Reversible heart failure in G alpha(q) transgenic mice

For many patients with cardiac insufficiency, the disease progresses inexorably to organ dilatation, pump failure, and death. Although there are examples of reversible heart failure in man, our understanding of how the myocardium repairs itself is limited. A well defined animal model of reversible heart failure would allow us to better investigate these restorative processes. Receptors that activate Galpha(q), a signal transduction molecule in the heterotrimeric G protein superfamily, are thought to play a key role in the development of heart failure. We demonstrated previously that mice expressing a recombinant Galpha(q) protein, the activity of which can be turned on or off at will in cardiac myocytes, develop a dilated cardiomyopathy with generalized edema and heart failure following activation of the protein (Fan, G., Jiang, Y.-P., Lu, Z., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (2005) J. Biol. Chem. 280, 40337-40346). Here we report that the contractile dysfunction and pathological structural changes in the myocardium improved significantly after termination of the Galpha(q) signal, even in animals with overt heart failure. Abnormalities in two proteins that regulate Ca(2+) handling in myocytes, phospholamban and the voltage-dependent L-type Ca(2+) channel, were also reversed, as was the increased expression of genes that are associated with heart failure. These results indicate that the heart has a substantial reparative capacity if the molecular signals responsible for the myocardial dysfunction can be identified and blocked.     

Decreasing Tropomyosin Phosphorylation Rescues Tropomyosin-induced Familial Hypertrophic Cardiomyopathy

Studies indicate that tropomyosin (Tm) phosphorylation status varies in different mouse models of cardiac disease. Investigation of basal and acute cardiac function utilizing a mouse model expressing an α-Tm protein that cannot be phosphorylated (S283A) shows a compensated hypertrophic phenotype with significant increases in SERCA2a expression and phosphorylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N., Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G., Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., and Wieczorek, D. F. (2012) J. Biol. Chem. 287, 44478–44489). With these results, we hypothesized that decreasing α-Tm phosphorylation may be beneficial in the context of a chronic, intrinsic stressor. To test this hypothesis, we utilized the familial hypertrophic cardiomyopathy (FHC) α-Tm E180G model (Prabhakar, R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J., and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815–1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca²⁺ sensitivity, and contractile defects. The FHC mice die by 6–8 months of age. We generated mice expressing both the E180G and S283A mutations and found that the hypertrophic phenotype was rescued in the α-Tm E180G/S283A double mutant transgenic animals; these mice exhibited no signs of cardiac hypertrophy and displayed improved cardiac function. These double mutant transgenic hearts showed increased phosphorylation of phospholamban Ser-16 and Thr-17 compared with the α-Tm E180G mice. This is the first study to demonstrate that decreasing phosphorylation of tropomyosin can rescue a hypertrophic cardiomyopathic phenotype.     

N—乙酰氨基葡萄糖转移酶Ⅲ,Ⅳ及Ⅴ在7721人肝癌细胞周期中的变化

In order to investigate the changes of N-acetylglucosaminyl transferase (GlcNAc-T) III, IV and V in cell cycle, the synchronization of 7721 human hepatocellular carcinoma cells was performed using serum hunger method. The percentages of cells in different phases during cell cycle were measured by flow cytometry (FCM) and the cell cycle was checked by determining the activity of cellular p34cdc2 kinase. It was found that the activities of GlcNAc-T III increased in G0/G1 cell peak phase and had correlation with the cell percentage of G0/G1 phase (r = 0.760, P < 0.05), while GlcNAc-T V showed the highest activity when G2/M cells were most abundant and had an apparent correlation with the cell percentage of G2/M phase (r = 0.868, P < 0.001). The changes of GlcNAc-T IV activity seemed not related to the cell cycle. The changes in opposite directions of relative activities (percentage of total GlcNAc-T III, IV, V) of GlcNAc-T III and GlcNAc-T V were observed during cell cycle (r = -0.951, P < 0.001), suggesting that these two enzymes might be regulated differently and functioned oppositely in the cells: GlcNAc-T V may be related to the proliferation of 7721 cells, while GlcNAc-T III may be related to the non-mitotic silence phase of the cells, or, it may be a factor against proliferation. Immunohistochemical results showed that the protein content of GlcNAc-T V was not significantly changed during cell cycle, and had no correlation with the activity of GlcNAc-T V, suggesting that the changes of GlcNAc-T V activity in cell cycle might not be resulted from the alteration of enzyme protein synthesis. The correlation between the activities of GlcNAc-T V and p34cdc2 kinase (r = 0.752, P < 0.05) was observed in cell cycle, implicating that GlcNAc-T V might possibly be regulated by p34cdc2 kinase.     

心脏特异表达肾素（原）受体转基因小鼠的建立

目的建立心脏特异表达（p）RR的转基因小鼠,研究（p）RR在心肌病发病过程中的调节作用。方法将（p）RR基因插入到心脏特异表达启动子α-MHC下游,构建转基因表达载体,通过显微注射的方法建立（p）RR转基因小鼠,PCR法鉴定转基因小鼠的基因型;Western blot和免疫组化的方法检测（p）RR在心脏组织中的表达;利用小动物超声仪检测转基因小鼠的心脏结构和功能;双色免疫荧光共聚焦进行（p）RR蛋白在转基因小鼠中的亚细胞定位,Western blot方法检测了钙泵（ATP2A2）、钠-钙交换体1（NCX 1）以及肌钙蛋白T（cTnT）在转基因小鼠心脏组织中表达量的变化情况。结果建立了心脏高表达（p）RR的转基因小鼠品系;内源性和转基因高表达的（p）RR蛋白均定位在细胞内高尔基体和内质网上;心脏特异高表达（p）RR蛋白使小鼠心脏收缩功能增强,主要表现在与同窝阴性对照小鼠相比,转基因小鼠收缩期左室内径（LVID,systolic）降低9%（P〈0.05,n=18）,收缩期容积（LVESV,systolic）减少了23%（P〈0.05,n=18）,射血分数EF（ejection fraction）升高14%（P〈0.01,n=18）,短轴缩短率FS（fraction shortening）升高20%（P〈0.01,n=18）;和心脏收缩功能和钙离子相关的ATP2A2和NCX 1表达均下调,而cTnT表达量上调。结论在心脏中过表达（p）RR能够引起心脏收缩功能明显增强,同时直接或间接调节ATP2A2、NCX 1和cTnT表达。提示（p）RR可能是调节心脏中的钙离子而参与影响心肌功能。     

FGF21 deletion exacerbates diabetic cardiomyopathy by aggravating cardiac lipid accumulation

Fibroblast growth factor 21 (FGF21) plays an important role in energy homoeostasis. The unaddressed question of FGF21's effect on the development and progression of diabetic cardiomyopathy (DCM) is investigated here with FGF21 knockout (FGF21KO) diabetic mice. Type 1 diabetes was induced in both FGF21KO and C57BL/6J wild‐type (WT) mice via streptozotocin. At 1, 2 and 4 months after diabetes onset, the plasma FGF21 levels were significantly decreased in WT diabetic mice compared to controls. There was no significant difference between FGF21KO and WT diabetic mice in blood glucose and triglyceride levels. FGF21KO diabetic mice showed earlier and more severe cardiac dysfunction, remodelling and oxidative stress, as well as greater increase in cardiac lipid accumulation than WT diabetic mice. Western blots showed that increased cardiac lipid accumulation was accompanied by further increases in the expression of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and its target protein CD36, along with decreases in the phosphorylation of AMP‐activated protein kinase and the expression of hexokinase II and peroxisome proliferator‐activated receptor gamma co‐activator 1α in the heart of FGF21KO diabetic mice compared to WT diabetic mice. Our results demonstrate that FGF21 deletion‐aggravated cardiac lipid accumulation is likely mediated by cardiac Nrf2‐driven CD36 up‐regulation, which may contribute to the increased cardiac oxidative stress and remodelling, and the eventual development of DCM. These findings suggest that FGF21 may be a therapeutic target for the treatment of DCM.     

Increased inhibition of SERCA2 by phospholamban in the type I diabetic heart

The sarcoplasmic reticulum (SR) plays a critical role in mediating cardiac contractility and its function is abnormal in the diabetic heart. However, the mechanisms underlying SR dysfunction in the diabetic heart are not clear. Because protein phosphorylation regulates SR function, this study examined the phosphorylation state of phospholamban, a key SR protein that regulates SR calcium (Ca2+) uptake in the heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (STZ; 65 mg kg(-1) i.v.), and the animals were humanely killed after 6 weeks and cardiac SR function was examined. Depressed cardiac performance was associated with reduced SR Ca2+-uptake activity in diabetic animals. The reduction in SR Ca2+-uptake was consistent with a significant decrease in the level of SR Ca2+-pump ATPase (SERCA2a) protein. The level of phospholamban (PLB) protein was also decreased, however, the ratio of PLB to SERCA2a was increased in the diabetic heart. Depressed SR Ca2+-uptake was also due to a reduction in the phosphorylation of PLB by the Ca2+-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA). Although the activities of the SR-associated Ca2+-calmodulin-dependent protein kinase (CaMK), cAMP-dependent protein kinase (PKA) were increased in the diabetic heart, depressed phosphorylation of PLB could partly be attributed to an increase in the SR-associated protein phosphatase activities. These results suggest that there is increased inhibition of SERCA2a by PLB and this appears to be a major defect underlying SR dysfunction in the diabetic heart.