Abstract: Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

Abstract: AIMS: Angiotensin II (Ang II) and tumour necrosis factor alpha (TNFalpha) are involved in the progression from compensated hypertrophy to heart failure. Here, we test their role in the remodelling of ATP-dependent potassium channel (K(ATP)) in heart failure, conferring increased metabolic and diazoxide sensitivity. METHODS AND RESULTS: We observed increased expression of both angiotensinogen and TNFalpha in the failing rat myocardium, with a regional gradient matching that of the K(ATP) subunit Kir6.1 expression. Both angiotensinogen and TNFalpha expression correlated positively with Kir6.1 and negatively with Kir6.2 expression across the post-infarction myocardium. To further identify a causal relationship, cardiomyocytes isolated from normal rat hearts were exposed in vitro to Ang II or TNFalpha. We observed increased Kir6.1 and SUR subunit and reduced Kir6.2 subunit mRNA expression in cardiomyocytes cultured with Ang II or TNFalpha, similar to what was observed in failing hearts. In patch-clamp experiments, cardiomyocytes cultured with Ang II or TNFalpha exhibited responsiveness to diazoxide, in terms of both K(ATP) current and action potential shortening. This was not observed in untreated cardiomyocytes and resembles the diazoxide sensitivity of failing cardiomyocytes that also overexpress Kir6.1. Ang II exerted its effect through induction of TNFalpha expression, because TNFalpha-neutralizing antibody abolished the effect of Ang II, and in failing hearts, regional expression of angiotensinogen matched TNFalpha expression. Finally, Ang II and TNFalpha regulated K(ATP) subunit expression, possibly through differential expression of Forkhead box transcription factors. CONCLUSION: This study identifies Ang II and TNFalpha as mediators of the remodelling of K(ATP) channels in heart failure.

Abstract: AIMS: Advanced heart failure is often associated with reduced myocardial fatty acid oxidation capacity. We have previously observed that failing hearts of mice with overexpression of angiotensinogen in the myocardium exhibit marked reduction of key regulatory proteins of fatty acid oxidation. In the present study, we determined whether exposure of adult rat cardiac (ARC) myocytes to angiotensin II (Ang II) influences expression of fatty acid translocase, muscle-type carnitine palmitoyl transferase-I, and medium-chain acyl-CoA dehydrogenase. METHODS AND RESULTS: Ang II reduced mRNA expression of the three regulatory proteins in ARC myocytes during the entire 14-days culture period. However, protein expression and palmitate oxidation rate remained unaltered for 7 days, but subsequently markedly decreased. The decrease of protein expression and of fatty acid oxidation coincided with the onset of increased protein expression of tumour necrosis factor-alpha (TNF-alpha). The effect of Ang II was completely abolished by either blocking TNF-alpha formation through inhibition of reactive oxygen species-mediated activation of nuclear factor-kappaB or by neutralizing TNF-alpha with a specific antibody. Activation of peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARbeta/delta counteracted Ang II-mediated reduction of the fatty acid oxidation pathway. CONCLUSION: Prolonged exposure of cardiac myocytes to Ang II elicits downregulation of the fatty acid oxidation pathway mediated by enhanced synthesis of TNF-alpha.

Abstract: Insulin resistance is the failure of insulin to stimulate the transport of glucose into its target cells. A highly regulatable supply of glucose is important for cardiomyocytes to cope with situations of metabolic stress. We recently observed that isolated adult rat cardiomyocytes become insulin resistant in vitro. Insulin resistance is combated at the whole body level with agonists of the nuclear receptor complex peroxisome proliferator-activated receptor gamma (PPARgamma)/retinoid X receptor (RXR). We investigated the effects of PPARgamma/RXR agonists on the insulin-stimulated glucose transport and on insulin signaling in insulin-resistant adult rat cardiomyocytes. Treatment of cardiomyocytes with ciglitazone, a PPARgamma agonist, or 9-cis retinoic acid (RA), a RXR agonist, increased insulin- and metabolic stress-stimulated glucose transport, whereas agonists of PPARalpha or PPARbeta/delta had no effect. Stimulation of glucose transport in response to insulin requires the phosphorylation of the signaling intermediate Akt on the residues Thr308 and Ser473 and, downstream of Akt, AS160 on several Thr and Ser residues. Phosphorylation of Akt and AS160 in response to insulin was lower in insulin-resistant cardiomyocytes. However, treatment with 9-cis RA markedly increased phosphorylation of both proteins. Treatment with 9-cis RA also led to better preservation of microtubules in cultured cardiomyocytes. Disruption of microtubules in insulin-responsive cardiomyocytes abolished insulin-stimulated glucose transport and reduced phosphorylation of AS160 but not Akt. Metabolic stress-stimulated glucose transport also involved AS160 phosphorylation in a microtubule-dependent manner. Thus, the stimulation of glucose uptake in response to insulin or metabolic stress is dependent in cardiomyocytes on the presence of intact microtubules.

Abstract: Coordinate adaptation of myocyte metabolism and function is fundamental to survival of the stressed heart, but the mechanisms for this coordination remain unclear. Bioinformatics led us to discover that Foxs are key transcription factors involved. We performed experiments on the mouse atrial cell line HL-1, neonate rat heart myocytes, and an adult rat model of myocardial infarction. In electrophoretic mobility-shift assays, FoxO1 binds to the FoxO concensus site of the KATP channel subunit KIR6.1 promoter. In primary atrial culture, targeting FoxO1 and FoxO3 with siRNA specifically reduces mRNA expression of FoxO1 and -O3 and KIR6.1. Western blots, confocal immunofluorescence, and quantitative RT-PCR was applied for measuring expression of 10 Fox, 6 KATP channel subunits, and 12 metabolic genes. FoxF2, -O1, and -O3 strongly associate with expression of KATP channel subunits (in particular, KIR6.1, SUR1A and SUR2B) in different heart tissues and in the periinfarct zone of the left ventricle. Patch-clamp recordings demonstrate that molecular plasticity of these channels is matched by pharmacological plasticity and increased sensitivity to a metabolic challenge mimicked by the protonophore CCCP. A balance of FoxF2 and FoxO also regulates expression of at least 9 metabolic genes involved in setting the balance of glycolysis and beta-oxidation. Bioinformatics shows that the transcriptional mechanisms are highly conserved among chicken, mouse, rat, and human, and Fox are intimately linked to other metabolic sensors. Thus, FoxF2 and -O are key transcription factors coordinating expression of KATP channels and energy metabolism.

Abstract: Inactivation of peroxisome proliferator-activated receptor (PPARs) isoforms alpha, beta/delta, and gamma mediate distinct facets of hypertrophic transformation of adult cardiac myocytes. PPARs are ligand-activated transcription factors that modulate the transcriptional regulation of fatty acid metabolism and the hypertrophic response in neonatal cardiac myocytes. The purpose of this study was to determine the role of PPAR isoforms in the morphologic and metabolic phenotype transformation of adult cardiac myocytes in culture, which, in medium containing 20% fetal calf serum, undergo hypertrophy-like cell growth associated with downregulation of regulatory proteins of fatty acid metabolism. Expression and DNA-binding activity of PPARalpha, PPARbeta/delta, and PPARgamma rapidly decreased after cell isolation and remained persistently reduced during the 14-day culture period. Cells progressively increased in size and developed both re-expression of atrial natriuretic factor and downregulation of regulatory proteins of fatty acid metabolism. Supplementation of the medium with fatty acid (oleate 0.25 mM/palmitate 0.25 mM) prevented inactivation of PPARs and downregulation of metabolic genes. Furthermore, cell size and markers of hypertrophy were markedly reduced. Selective activation of either PPARalpha or PPARbeta/delta completely restored expression of regulatory genes of fatty acid metabolism but did not influence cardiac myocyte size and markers of hypertrophy. Conversely, activation of PPARgamma prevented cardiomyocyte hypertrophy but had no effect on fatty acid metabolism. The results indicate that PPAR activity markedly influences hypertrophic transformation of adult rat cardiac myocytes. Inactivation of PPARalpha and PPARbeta/delta accounts for downregulation of the fatty acid oxidation pathway, whereas inactivation of PPARgamma enables development of hypertrophy.

Abstract: Myocardial remodeling late after infarction is associated with increased incidence of fatal arrhythmias. Heterogeneous prolongation of the action potential in the surviving myocardium is one of the predominant causes. Sarcolemmal ATP-dependent potassium (K(ATP)) channels are important metabolic sensors regulating electrical activity of cardiomyocytes and are capable of considerably shortening the action potential. We determined whether ATP-dependent potassium channels generate or, on the contrary prevent the heterogeneity in action potential prolongation. Cardiomyocytes were obtained from the infarct border zone, the septum and the right ventricle of rat hearts 20 weeks after coronary occlusion when rats developed signs of heart failure. Expression of the conductance subunit Kir6.1, but not Kir6.2, and of all SUR regulatory subunits was increased up to 3-fold in cardiomyocytes from the infarct border zone. Concomitantly, there was a prominent response of the K(ATP) current to diazoxide that was not detectable in control cardiomyocytes. The action potential was prolonged in cardiomyocytes from the infarct border zone (74 ms) relative to sham (41 ms). However, activation of the K(ATP) channels by diazoxide reduced action potential duration to 42 ms. In myocytes of the septum and right ventricle, expression of channel subunits, duration of action potential, and sensitivity to diazoxide were only slightly increased relative to shams. In conclusion, the myocardium remodeled after infarction displays alterations of K(ATP) expression and function with spatial heterogeneity matching that of the action potential prolongation. Drugs selectively activating diazoxide-sensitive sarcolemmal K(ATP) channels should be considered in the prevention of arrhythmias in post-infarction heart failure.

Abstract: Myocardial metabolism shifts during the perinatal period from predominant utilization of glucose towards oxidation of fatty acids. Expression of enzymes of the fatty acid oxidation (FAO) pathway is under the control of the nuclear receptor/transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha). Insulin-like Growth Factor-I (IGF-I) plays an important role in the post-natal growth and differentiation of the heart. We determined the influence of IGF-I on the maturation of myocardial metabolism. In neonatal rat cardiac myocytes, expression of the FAO enzymes MCAD and M-CPT I was induced by treatment with the specific PPARalpha agonist WY-14643. Concomitant treatment with IGF-I enhanced the expression of both FAO enzymes. By comparison, treatment with FGF-2, which is required for myocyte differentiation of cardiac precursors, did not increase WY-14643-induced expression of FAO enzymes. Despite stimulation of FAO enzyme expression, IGF-I did not further enhance WY-14643-stimulated palmitate oxidation. In contrast, IGF-I relieved WY-14643-mediated inhibition of glucose uptake and promoted storage of fatty acids into cellular neutral lipids. In conclusion, IGF-I promotes a more mature pattern of FAO gene expression but, because of insulin-like metabolic effects, does not concomitantly enhance oxidation of fatty acids.

Abstract: Heart failure is associated with downregulation of the fatty acid oxidation pathway in the ventricular myocardium. Since angiotensin II plays a critical role in myocardial phenotypic changes associated with heart failure, we investigated the effect of chronic angiotensin II stimulation on the fatty acid oxidation pathway in transgenic (TG) mice with targeted overexpression of angiotensinogen in the myocardium (TG1306/1R mice). TG1306/R1 mice progressively developed left ventricular hypertrophy. After 12 months, approximately half of the mice exhibited signs of heart failure including increased lung weight index [>+2 SD of age-matched wild-type (WT) mice] and 5-fold increase of myocardial brain natriuretic peptide expression. Myocardial mRNA and protein expression of peroxisome proliferator-activated receptor alpha (PPARalpha) progressively decreased in both WT and TG1306/R1 mice during the 12 months observation period, but much more pronounced in TG1306/R1 mice. Concomitantly, mRNA expression of enzymes of fatty acid oxidation (medium-chain acyl CoA dehydrogenase, MCAD; carnitine palmitoyl transferase I, CPT-I) was reduced in TG1306/R1 compared with age-matched WT mice. However, protein expression of MCAD and CPT-I was decreased concomitantly only in TG mice with criteria of heart failure. Correspondingly, myocardial oxidation of palmitate, measured during ex vivo working heart perfusion, was reduced by 25% in TG1306/R1 mice with heart failure. These results demonstrate that angiotensin II-induced cardiac hypertrophy is associated with reduction of PPARalpha and of mRNA expression of enzymes of fatty acid metabolism relative to age-matched WT mice. However, both protein expression of fatty acid oxidation enzymes and the rate of fatty acid oxidation remain unchanged unless heart failure occurs, suggesting the involvement of posttranscriptional mechanisms in the metabolic changes associated with heart failure.

Abstract: Sufficient expression of the insulin-sensitive glucose transporter GLUT4 may be crucial for the survival of cardiac myocytes in situations of stress. Expression of GLUT4 in cardiac myocytes correlates with cell differentiation and is reduced in the hypertrophied and failing myocardium. Adult rat cardiomyocytes (ARC) in primary culture undergo dedifferentiation and reduction of GLUT4 expression. Depending on the culture condition partial redifferentiation and/or hypertrophy follows. All-trans (at) and 9-cis retinoic acids (RA) are morphogenetic agents important for cell differentiation. Both atRA and 9-cisRA restored GLUT4 expression in dedifferentiated ARC, while only 9-cisRA could increase GLUT4 expression in hypertrophic ARC. The effects of RA were associated with improved differentiation of the cardiac myocytes, as assessed from the expression of atrial natriuretic factor and the morphology of the contractile apparatus. In neonatal rat cardiomyocytes, 9-cisRA, but not atRA, stimulated transcription from the glut4 promoter. In conclusion, treatment with RA can restore the down-regulated expression of GLUT4 in cardiomyocytes in association with a partial improvement of the differentiated phenotype.

Abstract: OBJECTIVE: In vivo differentiation of cardiac myocytes is associated with downregulation of the glucose transporter isoform GLUT1 and upregulation of the isoform GLUT4. Adult rat cardiomyocytes in primary culture undergo spontaneous dedifferentiation, followed by spreading and partial redifferentiation, which can be influenced by growth factors. We used this model to study the signaling mechanisms modifying the expression of GLUT4 in cardiac myocytes. RESULTS: Adult rat cardiomyocytes in primary culture exhibited spontaneous upregulation of GLUT1 and downregulation of GLUT4, suggesting resumption of a fetal program of GLUT gene expression. Treatment with IGF-1 and, to a minor extent, FGF-2 resulted in restored expression of GLUT4 protein and mRNA. Activation of p38 MAPK mediated the increased expression of GLUT4 in response to IGF-1. Transient transfection experiments in neonatal cardiac myocytes confirmed that p38 MAPK could activate the glut4 promoter. Electrophoretic mobility shift assay in adult rat cardiomyocytes and transient transfection experiments in neonatal cardiac myocytes indicated that MEF2 was the main transcription factor transducing the effect of p38 MAPK activation on the glut4 promoter. CONCLUSION: Spontaneous dedifferentiation of adult rat cardiomyocytes in vitro is associated with downregulation of GLUT4, which can be reversed by treatment with IGF-1. The effect of IGF-1 is mediated by the p38 MAPK/MEF2 axis, which is a strong inducer of GLUT4 expression.

Abstract: Myocardium undergoing remodeling in vivo exhibits insulin resistance that has been attributed to a shift from the insulin-sensitive glucose transporter GLUT4 to the fetal, less insulin-sensitive, isoform GLUT1. To elucidate the role of altered GLUT4 expression in myocardial insulin resistance, glucose uptake and the expression of the glucose transporter isoforms GLUT4 and GLUT1 were measured in adult rat cardiomyocytes (ARC). ARC in culture spontaneously undergo dedifferentiation, hypertrophy-like spreading, and return to a fetal-like gene expression pattern. Insulin stimulation of 2-deoxy-D-glucose uptake was completely abolished on day 2 and 3 of culture and recovered thereafter. Although GLUT4 protein level was reduced, the time-course of unresponsiveness to insulin did not correlate with altered expression of GLUT1 and GLUT4. However, translocation of GLUT4 to the sarcolemma in response to insulin was completely abolished during transient insulin resistance. Insulin-mediated phosphorylation of Akt was not reduced, indicating that activation of phosphatidylinositol 3-kinase (PI3K) was preserved. On the other hand, total and phosphorylated Cbl was reduced during insulin resistance, suggesting that activation of Cbl/CAP is essential for insulin-mediated GLUT4 translocation, in addition to activation of PI3K. Pharmacological inhibition of contraction in insulin-sensitive ARC reduced insulin sensitivity and lowered phosphorylated Cbl. The results suggest that transient insulin resistance in ARC is related to impairment of GLUT4 translocation. A defect in the PI3K-independent insulin signaling pathway involving Cbl seems to contribute to reduced insulin responsiveness and may be related to contractile arrest.

Abstract: OBJECTIVE: To investigate the influence of obesity on the regulation of myocardial glucose metabolism following protein kinase C (PKC) activation in obese (fa/fa) and lean (Fa/?) Zucker rats. DESIGN: Isolated hearts obtained from 17-week-old lean and obese Zucker rats were perfused with 200 nM phorbol 12-myristate 13-acetate (PMA) for different time periods prior to the evaluation of PKC and GLUT-4 translocation. For metabolic studies isolated hearts from 48 h starved Zucker rats were perfused with an erythrocytes-enriched buffer containing increased concentrations (10-100 nM) of PMA. MEASUREMENTS: Immunodetectable PKC isozymes and GLUT-4 were determined by Western blots. Glucose oxidation and glycolysis were evaluated by measuring the myocardial release of 14CO2 and 3H2O from [U-14C]glucose and [5-3H]glucose, respectively. RESULTS: PMA (200 nM) induced maximal translocation of ventricular PKCalpha from the cytosol to the membranes within 10 min. This translocation was 2-fold lower in the heart from obese rats when compared to lean rats. PMA also induced a significant translocation of ventricular GLUT-4 from the microsomal to the sarcolemmal fraction within 60 min in lean but not in obese rats. Rates of basal cardiac glucose oxidation and glycolysis in obese rats were approximately 2-fold lower than those of lean rats. Perfusion with increasing concentrations of PMA (10-100 nM) led to a significant decrease of cardiac glucose oxidation in lean but not in obese rats. CONCLUSION: Our results show that in the heart of the genetically obese Zucker rat, the impairment in PKCalpha activation is in line with a diminished activation of GLUT-4 as well as with the lack of PMA effect on glucose oxidation.

Abstract: OBJECTIVES: Increasing evidence suggests that left ventricular remodeling is associated with a shift from fatty acid to glucose metabolism for energy production. The aim of this study was to determine whether left ventricular remodeling with and without late-onset heart failure after myocardial infarction is associated with regional changes in the expression of regulatory proteins of glucose or fatty acid metabolism. METHODS: Myocardial infarction was induced in rats by ligation of the left anterior descending coronary artery (LAD). In infarcted and sham-operated hearts the peri-infarction region (5-mm zone surrounding the region at risk), the interventricular septum and the right ventricular free wall were separated for analysis. RESULTS: At 8 and 20 weeks after LAD ligation, the peri-infarction region and the septum exhibited marked re-expression of atrial natriuretic factor [+252+/-37 and +1093+/-279%, respectively, in the septum (P<0.05)] and of alpha-smooth muscle actin [+34+/-10 and +43+/-14%, respectively, in the septum (P<0.05)]. At 8 weeks, when left ventricular hypertrophy was present without signs of heart failure, myocardial mRNA expression of glucose transporters (GLUT-1 and GLUT-4) was not altered, whereas mRNA expression of medium-chain acyl-CoA dehydrogenase (MCAD) was significantly reduced in the peri-infarction region (-25+/-7%; P<0.05). In hearts exhibiting heart failure 20 weeks after infarct-induction there was a change in all three ventricular regions of both mRNA and protein content of GLUT-1 [+72+/-28 and +121+/-15%, respectively, in the peri-infarction region (P<0.05)] and MCAD [-29+/-9 and -56+/-4%, respectively, in the peri-infarction region (P<0.05)]. CONCLUSION: In rats with large myocardial infarction, progression from compensated remodeling to overt heart failure is associated with upregulation of GLUT-1 and downregulation of MCAD in both the peri-infarction region and the septum.

Abstract: Postischemic recovery of contractile function is better in hearts from fasted rats than in hearts from fed rats. In this study, we examined whether feeding-induced inhibition of palmitate oxidation at the level of carnitine palmitoyl transferase I is involved in the mechanism underlying impaired recovery of contractile function. Hearts isolated from fasted or fed rats were submitted to no-flow ischemia followed by reperfusion with buffer containing 8 mM glucose and either 0.4 mM palmitate or 0.8 mM octanoate. During reperfusion, oxidation of palmitate was higher after fasting than after feeding, whereas oxidation of octanoate was not influenced by the nutritional state. In the presence of palmitate, recovery of left ventricular developed pressure was better in hearts from fasted rats. Substitution of octanoate for palmitate during reperfusion enhanced recovery of left ventricular developed pressure in hearts from fed rats. However, the chain length of the fatty acid did not influence diastolic contracture. The results suggest that nutritional variation of mitochondrial fatty acid transfer may influence postischemic recovery of contractile function.

Abstract: Non-infarcted myocardium after coronary occlusion undergoes progressive morphological and functional changes. The purpose of this study was to determine whether non-infarcted myocardium exhibits (1) alteration of the substrate pattern of myocardial metabolism and (2) concomitant changes in the expression of regulatory proteins of glucose and fatty acid metabolism. Myocardial infarction was induced in rats by ligation of the left coronary artery. One day and eight weeks after coronary occlusion, glucose and palmitate oxidation were measured. Expression of selected proteins of metabolism were determined one day to 12 weeks after infarction. One day after coronary occlusion no difference of glucose and palmitate oxidation was detectable, whereas after eight weeks, glucose oxidation was increased (+84%, P<0.05) and palmitate oxidation did not change significantly (-19%, P=0.07) in infarct-containing hearts, compared with hearts from sham-operated rats. One day after coronary occlusion, myocardial mRNA expression of the glucose transporter GLUT-1 was increased (+86%, P<0.05) and the expression of GLUT-4 was decreased (-28%, P<0.05) in surviving myocardium of infarct-containing hearts. Protein level of GLUT-1 was increased (+81%, P<0.05) and that of GLUT-4 slightly, but not significantly, decreased (-16%, P=NS). mRNA expressions of heart fatty acid binding protein (H-FABP), and of medium chain acyl-CoA dehydrogenase (MCAD), were decreased by 36% (P<0.05) and 35% (P=0. 07), respectively. Eight weeks after acute infarction, the left ventricle was hypertrophied and, at this time-point, there was no difference in the expression of GLUT-1 and GLUT-4 between infarcted and sham-operated hearts. However, myocardial mRNA and protein content of MCAD were decreased by 30% (P<0.01) and 27% (P<0.05), respectively. In summary, in surviving myocardium, glucose oxidation was increased eight weeks after coronary occlusion. Concomitantly, mRNA and protein expression of MCAD were decreased, compatible with a role of altered expression of regulatory proteins of metabolism in post-infarction modification of myocardial metabolism.

Abstract: The relationship between protein kinase C (PKC) activation and Ras function was investigated in cardiac cells. Ras function was required for ERK activation by phorbol esters in cardiac myocytes, but not in cardiac fibroblasts. Accordingly, treatment with phorbol esters resulted in GTP loading of Ras in cardiac myocytes, but not fibroblasts. Ras activation by phorbol esters was abolished by a PKC specific inhibitor, but was insensitive to tyrosine kinase inhibitors. Ras activation was mediated by stimulation of guanine nucleotide exchange. These results suggest the existence of a novel pathway for Ras activation, specific to cardiac myocytes, with implications for myocardial hypertrophy.

Abstract: A number of observations indicate that myocardial glucose utilization is increased late during post-ischemic reperfusion. The present study was designed to examine whether transient ischemia elicits altered expression of glucose transporters GLUT-1 and GLUT-4. In rats, the left anterior descending coronary artery was occluded for 20 min followed by reperfusion for 1, 3 or 7 days. Regional myocardial uptake and phosphorylation of glucose was determined based on myocardial accumulation of 2-deoxy-D-[2, 6-3H]glucose-6-phosphate. In hearts from fasted rats, after 3 days of reperfusion, myocardial uptake and phosphorylation of glucose was 48% higher in the reperfused region compared to a remote control region. No regional difference in myocardial glucose uptake and phosphorylation was detectable in hearts from fed rats. After 1 day of reperfusion, expression of myocardial glucose transporter GLUT-1 mRNA was increased to 195+/-24% (mean+/-SEM) of the value measured in the remote region and the expression of GLUT-4 mRNA was decreased to 58+/-7%. After 3 days of reperfusion both mRNA and protein of GLUT-1 were higher in the reperfused region, averaging 133+/-23% and 249+/-36%, respectively. The corresponding values for GLUT-4 mRNA and protein were 77+/-7% and 62+/-6%, respectively. The results indicate that a short period of ischemia alters the expression of glucose transporter isoforms GLUT-1 and GLUT-4. Observed changes may be involved in the mechanisms underlying late changes of substrate metabolism during reperfusion.

Abstract: Myocardial hypertrophy is associated with increased basal glucose metabolism. Basal glucose transport into cardiac myocytes is mediated by the GLUT1 isoform of glucose transporters, whereas the GLUT4 isoform is responsible for regulatable glucose transport. Treatment of neonatal cardiac myocytes with the hypertrophic agonist 12-O-tetradecanoylphorbol-13-acetate or phenylephrine increased expression of Glut1 mRNA relative to Glut4 mRNA. To study the transcriptional regulation of GLUT1 expression, myocytes were transfected with luciferase reporter constructs under the control of the Glut1 promoter. Stimulation of the cells with 12-O-tetradecanoylphorbol-13-acetate or phenylephrine induced transcription from the Glut1 promoter, which was inhibited by cotransfection with the mitogen-activated protein kinase phosphatases CL100 and MKP-3. Cotransfection of the myocytes with constitutively active versions of Ras and MEK1 or an estrogen-inducible version of Raf1 also stimulated transcription from the Glut1 promoter. Hypertrophic induction of the Glut1 promoter was also partially sensitive to inhibition of the phosphatidylinositol 3-kinase pathway and was strongly inhibited by cotransfection with dominant-negative Ras. Thus, Ras activation and pathways downstream of Ras mediate induction of the Glut1 promoter during myocardial hypertrophy.

Abstract: Myocardial ischemia elicits translocation of the insulin-sensitive glucose transporter GLUT-4 from intracellular membrane stores to the sarcolemma. Because glucose metabolism is of crucial importance for post-ischemic recovery of the heart, myocardial uptake of [3H]-labeled 2-deoxyglucose and subcellular localization of GLUT-4 were determined during reperfusion in isolated rat hearts perfused with medium containing 0.4 mm palmitate and 8 mm glucose. Hearts were subjected to 20 min of no-flow ischemia, followed by reperfusion for up to 60 min. Subcellular localization of GLUT-4 was determined by cell fractionation followed by immunoblotting. After 15 and 60 min of reperfusion uptake of 2-deoxyglucose was significantly higher (91+/-9 and 96+/-8 nmol/min/g wet weight, respectively) as compared to control values (65+/-1 nmol/min/g wet weight). Ischemia elicited translocation of GLUT-4 to the sarcolemma, which persisted after 15 min of reperfusion. However, after 60 min of reperfusion the subcellular distribution of GLUT-4 was similar to control hearts. In conclusion, reversal of ischemia-induced translocation of GLUT-4 to the sarcolemma is rather slow, possibly facilitating glucose uptake early during reperfusion. However, myocardial uptake and phosphorylation of 2-deoxyglucose remains enhanced late during reperfusion, when pre-ischemic distribution of GLUT-4 is almost completely restored, indicating that additional mechanisms are likely to be involved in post-ischemic stimulation of glucose uptake.Copyright 1998 Academic Press Limited.

Abstract: Left ventricular hypertrophy in patients with hypertensive heart disease is associated with impaired relaxation and myocardial interstitial fibrosis leading to enhanced filling pressure, referred to as left ventricular diastolic dysfunction. Impairment of systolic function, characterized by reduced ejection fraction occurs at a later stage. Activation of the renin-angiotensin-aldosterone system contributes to progression to heart failure by at least two mechanisms: (1) increased left ventricular loading conditions due to vasoconstriction and retention of sodium; (2) direct effects on the myocardium resulting in myocyte hypertrophy and interstitial fibrosis.

Abstract: Myocytes that have survived a period of transient ischemia may present prolonged alterations of cellular function, detectable during several days. The early period of postischemic reperfusion is characterized by restoration of ion homeostasis mediated by rapid resumption of function of ion pumps (Na+/K(+)-ATPase, Ca(2+)-ATPase) and transsarcolemmal ion exchange mechanisms (H+/Na+, Na+/Ca2+ exchangers). There is experimental evidence that cellular injury may be enhanced during the initial seconds or minutes of reperfusion, depending on the conditions of reperfusion. During the late phase of reperfusion mRNA expression of a number of key proteins of myocyte function is altered. The pattern of gene expression during reperfusion exhibits features of cellular adaptation and/or dedifferentiation in addition to cell repair.

Abstract: The pattern of substrate utilization may influence postischemic myocardial injury. To characterize the effect of nutritional state on substrate selection and contractile function during control conditions and postischemic reperfusion, hearts from fed and fasted rats were perfused retrogradely with 0.4 mM palmitate, 8 mM glucose, and 175 mU/l insulin. Under control conditions, hearts from fasted rats exhibited lower glucose oxidation (-59%) and higher palmitate oxidation (+191%) than hearts from fed rats. During reperfusion, postischemic hearts exhibited stimulation of glucose-oxidation, with no difference between hearts from fasted and fed rats. However, oxidation of palmitate remained higher after fasting (+68%). Hearts from fasted rats exhibited lower left ventricular diastolic pressure and higher left ventricular systolic pressure development during reperfusion. The results indicate that 1) substrate selection in myocardium is influenced by the nutritional state independently of substrate availability, 2) during postischemic reperfusion, inhibition of glucose oxidation is removed in hearts from fasted rats, whereas inhibition of fatty acid oxidation in hearts from fed rats is maintained, and 3) myocardial injury is lower after fasting.

Abstract: Extracellular matrix vesicles (MV) are the loci of initial mineralization in several calcifying tissues. We recently reported that MV isolated from chicken epiphyseal cartilage are equipped with a Na-dependent P(i) transport (NaPiT) system. The activity of the NaPiT system appeared to be crucial for the development of MV-mediated calcification. In the present study we investigated the expression of NaPiT activity in MV produced by the osteoblast-like cells MC3T3-E1. The relationship between changes in NaPiT activity in the intact cells and in the released MV was also examined. NaPiT activity in MV harvested from cultured MC3T3-E1 cells was transiently expressed. It was markedly increased between Days 8 and 10 (5- to 6-fold), and then gradually decreased. NaPiT activity was enriched in MV as compared with the parent osteoblast-like cells, while the Na-dependent transport system for alanine (NaAlaT) was not. When NaPiT activity was enhanced in osteoblast-like cells by fetal calf serum (FCS) or P(i) depletion, P(i) transport stimulation was observed in the derived MV as well. Alkaline phosphatase (AP) was differentially expressed and regulated in MV from MC3T3-E1 cell cultures, as compared with NaPiT. In contrast to the transient expression of NaPiT, AP activity in MV increased continuously with time in culture. It was stimulated by FCS treatment of the parent cells, but decreased in MV obtained from P(i)-depleted cultures. These results suggest that the presence in osteogenic cells of selective regulatory mechanisms for the insertion and enrichment of P(i) transport activity in released MV.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Inorganic phosphate (Pi) is a key element for the growth and mineralization of the epiphyseal cartilage. In this study, the characteristics of the transport of Pi in growth plate chondrocytes have been determined using primary cultures of chicken growth plate cartilage cells. The uptake of Pi was significantly increased in the presence of extracellular sodium. The kinetic parameters of the saturable sodium-dependent Pi transport (NaPiT) were determined. The Michaelis constant for Pi was 0.443 +/- 0.095 mM, and the concentration of sodium with which half-maximal Pi transport was observed was 48.0 +/- 8.7 mM. Stoichiometric analysis suggested that more than one sodium ion was cotransported with each Pi molecule. NaPiT was sensitive to inhibition by Pi analogues such as phosphonoformic acid and arsenate. These data strongly suggest that Pi uptake by chicken growth plate chondrocytes is a carrier-mediated process driven by the transmembrane electrochemical gradient of sodium. Two important regulators of biosynthetic activities of growth plate chondrocytes, insulin-like growth factor I (IGF-I) and parathyroid hormone (PTH), selectively regulated Pi transport. With IGF-I, maximal stimulation (117 +/- 7% above control) was observed at doses > 5 nM, with an half-maximal effective concentration of 0.46 +/- 0.18 nM. A significant effect was observed after 1 h of exposure and was maintained for up to 24 h. PTH increased Pi transport with a biphasic dose-response curve. The change in NaPiT was transient, being maximally observed after 8 h (58 +/- 8%) and unexpressed after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The mechanisms by which calcium (Ca2+) and inorganic phosphate (Pi) accumulate into matrix vesicles (MV) have not been elucidated. In the present study the characteristics of Pi uptake into MV isolated from mildly rachitic chicken growth plate cartilage have been investigated. The results indicate that Pi accumulates into MV mainly via a Na(+)-dependent Pi transport system. In the absence of NaCl in the extravesicular medium, Pi uptake was a nonsaturable process. In the presence of 150 mM NaCl, the initial rate of Pi uptake was 4.38 +/- 1.02-fold higher than with 150 mM choline chloride (mean +/- S.E., n = 8, p less than 0.005). Other cations showed partial activity to drive Pi into MV as compared to Na+:Li+ (64.4%) greater than K+ (39.8%) greater than choline (39.0%) greater than tetramethylammonium (30.0%) greater than N-methylglucamine (26.3%). Na(+)-dependent Pi transport activity displayed saturability towards increasing extra-vesicular concentrations of Na+ and Pi. The apparent Km for Pi was 0.68 +/- 0.16 mM. The Na+ concentration producing half-maximum Pi transport activity was 106.2 +/- 11.0 mM. Kinetic analysis suggests that Na+ interacts with the Pi carrier with a stoichiometry of more than one Na+ ion with one Pi molecule. In MV isolated from normal chicken growth plate cartilage, this Na(+)-dependent Pi transport system was barely expressed. In contrast to the effect on Pi uptake by MV, the activity of alkaline phosphatase was not changed when NaCl was substituted for choline chloride in the assay medium. In addition to this observation which suggests that this enzyme is not related to the Pi transport activity described in this study, levamisole, which inhibited alkaline phosphatase activity did not affect the Na(+)-dependent uptake of Pi. Both arsenate and phosphonoformic acid, two inhibitors of the epithelial Na(+)-dependent Pi transport systems, were active inhibitors of the Na(+)-dependent Pi uptake by MV with a higher potency for phosphonoformic acid. Associated with the expression of a facilitated Na(+)-coupled Pi transport in MV, in vitro calcification assessed by 45Ca2+ uptake also showed a marked dependence on extravesicular sodium. This relationship was markedly attenuated in MV isolated from normal chicken growth plate cartilage expressing a weak Na(+)-facilitated Pi transport activity. In conclusion, a saturable Na(+)-dependent Pi carrier has been characterized which facilitates Pi transport in MV. Its potential role for Ca-Pi accumulation into MV and subsequent development of vesicular calcification followed by mineralization of the osteogenic matrix is proposed and remains to be further investigated.

Abstract: Administration of GH increases both the tubular reabsorption of inorganic phosphate (Pi) and the plasma level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. These two effects could be induced by a common mediator, possibly the GH-generated insulin-like growth factor 1 (IGF-1). In the present work, the influence of recombinant human IGF-1 on renal Pi transport and plasma 1,25-(OH)2D3 was examined in hypophysectomized (HPX) rats. IGF-1, infused by miniosmotic pump at the dose of 10 micrograms/h for 6 days, significantly increased the maximal tubular reabsorption of Pi per unit volume of glomerular filtrate (max TRPi/m1GFR): IGF-1 3.50 +/- 0.16; vehicle: 2.78 +/- 0.14 mumol/m1GFR, P less than 0.005. The response was associated with a marked stimulation of plasma 1,25-(OH)2D3 (IGF-1; 409 +/- 23; vehicle: 208 +/- 22 pmol/liter, P less than 0.001). As previously reported for GH, IGF-1 also increased GFR and reduced urinary sodium excretion. In brush border membrane vesicles isolated from renal cortex of HPX rats, the Na-dependent Pi transport was stimulated by IGF-1. Neither the Na-dependent glucose transport nor that of alanine was affected by the growth factor. The stimulatory effect of IGF-1 on maxTRPi/m1GFR was also expressed in thyroparathyroidectomized (TPTX) HPX rats (IGF-1: 5.20 +/- 0.29; vehicle: 3.88 +/- 0.37 mumol/m1GFR, P less than 0.025). In conclusion, administration of IGF-1 in HPX rats mimics the stimulatory effects of GH on maxTRPi/m1GFR and on plasma 1,25-(OH)2D3. As described for GH the change in maxTRPi/m1GFR is mediated by a PTH independent mechanism and is expressed at the level of the luminal membrane of proximal tubules. These results suggest that IGF-1 could be an important factor in the control of Pi metabolism, particularly during growth, and might play a significant role in mediating the effect of GH on the renal handling of Pi and production of 1,25-(OH)2D3.