Ischemic preconditioning exaggerates cardiac damage in PKC-delta null mice

Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-delta isoform. In an accompanying study (26), we described metabolic changes in PKC-delta knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-delta(-/-) and PKC-delta(+/+) mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-delta(-/-) mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-delta(+/+) hearts, no increase in reactive oxygen species was observed in PKC-delta(-/-) hearts. Proteomic analysis of preconditioned PKC-delta(+/+) hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E(2) component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-delta(-/-) hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-delta(+/+) and PKC-delta(-/-) hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-delta(+/+) hearts, whereas lactate concentrations were higher in PKC-delta(-/-) hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-delta(-/-) hearts.     

The nucleotide metabolism in lactate perfused hearts under ischaemic and reperfused conditions

It was examined whether lactate influences postischaemic hemodynamic recovery as a function of the duration of ischaemia and whether changes in high-energy phosphate metabolism under ischaemic and reperfused conditions could be held responsible for impairment of cardiac function. To this end, isolated working rat hearts were perfused with either glucose (11 mM), glucose (11 mM) plus lactate (5 mM) or glucose (11 mM) plus pyruvate (5 mM). The extent of ischaemic injury was varied by changing the intervals of ischaemia, i.e. 15, 30 and 45 min. Perfusion by lactate evoked marked depression of functional recovery after 30 min of ischaemia. Perfusion by pyruvate resulted in marked decline of cardiac function after 45 min of ischaemia, while in glucose perfused hearts hemodynamic performance was still recovered to some extent after 45 min of ischaemia. Hence, lactate accelerates postischaemic hemodynamic impairment compared to glucose and pyruvate. The marked decline in functional recovery of the lactate perfused hearts cannot be ascribed to the extent of degradation of high-energy phosphates during ischaemia as compared to glucose and pyruvate perfused hearts. Glycolytic ATP formation (evaluated by the rate of lactate production) can neither be responsible for loss of cardiac function in the lactate perfused hearts. Moreover, failure of reenergization during reperfusion, the amount of nucleosides and oxypurines lost or the level of high-energy phosphates at the end of reperfusion cannot explain lactate-induced impairment. Alternatively, the accumulation of endogenous lactate may have contributed to ischaemic damage in the lactate perfused hearts after 30 min of ischaemia as it was higher in the lactate than in the glucose or pyruvate perfused hearts. It cannot be excluded that possible beneficial effects of the elevated glycolytic ATP formation during 15 to 30 min of ischaemia in the lactate perfused hearts are counterbalanced by the detrimental effects of lactate accumulation.     

Potential role for pyruvate kinase M2 in the regulation of murine cardiac glycolytic flux during in vivo chronic hypoxia

Carbohydrate metabolism in heart failure shares similarities to that following hypoxic exposure, and is thought to maintain energy homoeostasis in the face of reduced O₂ availability. As part of these in vivo adaptations during sustained hypoxia, the heart up-regulates and maintains a high glycolytic flux, but the underlying mechanism is still elusive. We followed the cardiac glycolytic responses to a chronic hypoxic (CH) intervention using [5-³H]-glucose labelling in combination with detailed and extensive enzymatic and metabolomic approaches to provide evidence of the underlying mechanism that allows heart survivability. Following 3 weeks of in vivo hypoxia (11% oxygen), murine hearts were isolated and perfused in a retrograde mode with function measured via an intraventricular balloon and glycolytic flux quantified using [5-³H]-glucose labelling. At the end of perfusion, hearts were flash-frozen and central carbon intermediates determined via liquid chromatography tandem mass spectrometry (LC-MS/MS). The maximal activity of glycolytic enzymes considered rate-limiting was assessed enzymatically, and protein abundance was determined using Western blotting. Relative to normoxic hearts, CH increased ex vivo cardiac glycolytic flux 1.7-fold with no effect on cardiac function. CH up-regulated cardiac pyruvate kinase (PK) flux 3.1-fold and cardiac pyruvate kinase muscle isoenzyme M2 (PKM2) protein content 1.4-fold compared with normoxic hearts. CH also augmented cardiac pentose phosphate pathway (PPP) flux, reflected by higher ribose-5-phosphate (R5P) content. These findings support an increase in the covalent (protein expression) and allosteric (flux) control of PKM2 as being central to the sustained up-regulation of the glycolytic flux in the chronically hypoxic heart.     

Myocardial proteome changes in aortic stenosis rats subjected to long-term aerobic exercise

The effects of exercise training (ET) on the heart of aortic stenosis (AS) rats are controversial and the mechanisms involved in alterations induced by ET have been poorly clarified. In this study, we analyzed the myocardial proteome to identify proteins modulated by moderate-intensity aerobic ET in rats with chronic supravalvular AS. Wistar rats were divided into four groups: sedentary control (C-Sed), exercised control (C-Ex), sedentary aortic stenosis (AS-Sed), and exercised AS (AS-Ex). ET consisted of five treadmill running sessions per week for 16 weeks. Statistical analysis was performed by ANOVA or Kruskal–Wallis and Goodman tests. Results were discussed at a significance level of 5%. At the end of the experiment, AS-Ex rats had higher functional capacity, lower blood lactate concentration, and better cardiac structural and left ventricular (LV) functional parameters than the AS-Sed. Myocardial proteome analysis showed that AS-Sed had higher relative protein abundance related to the glycolytic pathway, oxidative stress, and inflammation, and lower relative protein abundance related to beta-oxidation than C-Sed. AS-Ex had higher abundance of one protein related to mitochondrial biogenesis and lower relative protein abundance associated with oxidative stress and inflammation than AS-Sed. Proteomic data were validated for proteins related to lipid and glycolytic metabolism. Chronic pressure overload changes the abundance of myocardial proteins that are mainly involved in lipid and glycolytic energy metabolism in rats. Moderate-intensity aerobic training attenuates changes in proteins related to oxidative stress and inflammation and increases the COX4I1 protein, related to mitochondrial biogenesis. Protein changes are combined with improved functional capacity, cardiac remodeling, and LV function in AS rats.     

Expression of genes participating in regulation of fatty acid and glucose utilization and energy metabolism in developing rat hearts

The heart is a unique organ that can use several fuels for energy production. During development, the heart undergoes changes in fuel supply, and it must be able to respond to these changes. We have examined changes in the expression of several genes that regulate fuel transport and metabolism in rat hearts during early development. At birth, there was increased expression of fatty acid transporters and enzymes of fatty acid metabolism that allow fatty acids to become the major source of energy for cardiac muscle during the first 2 wk of life. At the same time, expression of genes that control glucose transport and oxidation was downregulated. After 2 wk, expression of genes for glucose uptake and oxidation was increased, and expression of genes for fatty acid uptake and utilization was decreased. Expression of carnitine palmitoyltransferase I (CPT I) isoforms during development was different from published data obtained from rabbit hearts. CPT Ialpha and Ibeta isoforms were both highly expressed in hearts before birth, and both increased further at birth. Only after the second week did CPT Ialpha expression decrease appreciably below the level of CPT Ibeta expression. These results represent another example of different expression patterns of CPT I isoforms among various mammalian species. In rats, changes in gene expression followed nutrient availability during development and may render cardiac fatty acid oxidation less sensitive to factors that influence malonyl-CoA content (e.g., fluctuations in glucose concentration) and thereby favor fatty acid oxidation as an energy source for cardiomyocytes in early development.     

Cardioprotective effects of exercise training on myofilament calcium activation in ovariectomized rats.

The risks associated with hormone replacement therapy, especially cardiac diseases in postmenopausal women, have prompted extensive studies for other preventive or therapeutic alternatives. We investigated the cardioprotective effects of exercise training on the changes in cardiac myofilament Ca2+ activation in 10-wk-old ovariectomized rats. The exercise groups were subjected to a 9-wk running program on a motor-driven treadmill 1 wk after surgery. The relationship between pCa (-log molar free Ca2+ concentration) and myofibrillar MgATPase activity of exercise-sham myofibrils or exercise-ovariectomized myofibrils was the same and could not be distinguished from that of sedentary-sham control hearts. In contrast, a significant suppression in maximum MgATPase activity and a leftward shift of pCa50 (half-maximally activating pCa) in the pCa-ATPase activity relationship were detected in sedentary-ovariectomized rats. Exercise training also prevented the shift in myosin heavy chain (MHC) isoforms toward beta-MHC in ovariectomized hearts. The upregulation of beta1-adrenergic receptors in the left ventricular membranes of ovariectomized rat hearts, as measured by receptor binding and immunoblot analyses, was no longer observed in exercise-ovariectomized hearts. Immunoblot analyses of heat shock protein (HSP) 72, an inducible form of HSP70, demonstrated a significant downregulation in ovariectomized hearts. Exercise training in ovariectomized rats completely reversed the expression of HSP72 to the same level as sham controls. Our results clearly indicate the cardioprotective effects of exercise training on changes in cardiac myofilament Ca2+ activation in ovariectomized rats. Alterations in expression of beta1-adrenergic receptors and HSP72 may, in part, play a mechanistic role in the cardioprotective effects.     

The heart as a working model to explore themes and strategies for anoxic survival in ectothermic vertebrates

Most vertebrates die within minutes when deprived of molecular oxygen (anoxia), in part because of cardiac failure, which can be traced to an inadequate matching of cardiac ATP supply to ATP demand. Cardiac power output (PO; estimated from the product of cardiac output and central arterial pressure and an indirect measure of cardiac ATP demand) is directly related to cardiac ATP supply up to some maximal level during both normoxia (ATP supply estimated from myocardial O(2) consumption) and anoxia (ATP supply estimated from lactate production rates). Thus, steady state PO provides an excellent means to examine anoxia tolerance strategies among ectothermic vertebrates by indicating a matching of cardiac glycolytic ATP supply and demand. Here, we summarize in vitro measurements of PO data from rainbow trout, freshwater turtles and hagfishes to provide a reasonable benchmark PO of 0.7 mW g(-1) for maximum glycolytic potential of ectothermic hearts at 15 degrees C, which corresponds to a glycolytic ATP turnover rate of about 70 nmol ATP g(-1) s(-1). Using this benchmark to evaluate in vivo PO data for hagfishes, carps and turtles, we identify two cardiac survival strategies, which in conjunction with creative waste management techniques to reduce waste accumulation, allow for long-term cardiac survival during anoxia in these anoxia-tolerant species. Hagfish and crucian carp exemplify a strategy of evolving such a low routine PO that routine cardiac ATP demand lies within the range of the maximum cardiac glycolytic potential. Common carp and freshwater turtles exemplify an active strategy of temporarily and substantially decreasing cardiac and whole body metabolism so that PO is below maximum cardiac glycolytic potential during chronic anoxia despite being quite close to this potential under normoxia.     

Intracellular glucose and binding of hexokinase and phosphofructokinase to particulate fractions increase under hypoxia in heart of the amazonian armored catfish (Liposarcus pardalis)

Armored catfish (Liposarcus pardalis), indigenous to the Amazon basin, have hearts that are extremely tolerant of oxygen limitation. Here we test the hypothesis that resistance to hypoxia is associated with increases in binding of selected glycolytic enzymes to subcellular fractions. Preparations of isolated ventricular sheets were subjected to 2 h of either oxygenated or hypoxic (via nitrogen gassing) treatment during which time the muscle was stimulated to contract. The bathing medium contained 5 mM glucose and was maintained at 25 degrees C. Initial experiments revealed increases in anaerobic metabolism. There was no measurable decrease in glycogen level; however, hypoxic treatment led to a twofold increase in heart glucose and a 10-fold increase in lactate content. It is suggested that the increase in heart glucose content is a result of an enhanced rate of facilitated glucose transport that exceeds the rate of phosphorylation of glucose. Further experiments assessed activities of metabolic enzymes in crude homogenates and subsequently tracked the degree of enzyme binding associated with subcellular fractions. Total maximal activities of glycolytic enzymes (hexokinase [HK], phosphofructokinase [PFK], aldolase, pyruvate kinase, lactate dehydrogenase), and a mitochondrial marker, citrate synthase, were not altered with the hypoxic treatment. A substantial portion (>/=50%) of HK is permanently bound to mitochondria, and this level increases under hypoxia. The amount of HK that is bound to the mitochondrial fraction is at least fourfold higher in hearts of L. pardalis than in rat hearts. Hypoxia also resulted in increased binding of PFK to a particulate fraction, and the degree of binding is higher in hypoxia-tolerant fish than in hypoxia-sensitive mammalian hearts. Such binding may be associated with increased glycolytic flux rates through modulation of enzyme-specific kinetics. The binding of HK and PFK occurs before any significant decrease in glycogen level.     

Hexokinase I expression and activity in embryonic mouse heart during early and late organogenesis

 The role of Fas and Fas ligand (Fas-L) in the apoptotic cell death process in cisplatin (CP)-treated human proximal tubular epithelial cells (PTECs) was examined. The human PTECs were treated with various concentrations (20–80 μM) of CP for 24 h, and the incidence of apoptosis in CP-treated cells was assessed by trypan blue staining, propidium iodide staining, in situ end labeling, and electron microscopy. The expression of Fas and Fas-L was detected by immunofluorescence microscopy. The results showed that: (1) CP-treatment resulted in a decreased number of live human PTECs and an increased number of dead cells, (2) CP-treated human PTECs showed an increased rate of apoptosis with its typical morphological features, and (3) expression of both Fas and Fas-L was upregulated in CP-treated human PTECs. These results indicate that CP treatment induces apoptosis in human PTECs and the activation of the Fas/Fas-L system may play an active role in the induction of the apoptotic cell death process. Accepted: 13 January 1999     

Proteomic changes in the heart of diet-induced pre-diabetic mice

The development of type 2 diabetes (T2D) is strongly associated with obesity. In humans, T2D increases the risk for end organ complications. Among these, heart disease has been ranked as the leading cause of death. We used a proteomic methodology to test the hypothesis that a pre-diabetic state generated by high-fat diet leads to changes in proteins related to heart function and structure. Over 300 protein spots were resolved by two-dimensional gel electrophoresis (2-DE). Fifteen protein spots were found to be altered (7 decreased and 8 increased) in pre-diabetic hearts. The protein spots were then identified by mass spectrometry and immunoblots. Among the decreased proteins, 3 are involved in heart structure (one isoform of desmin, troponin T2 and α-cardiac actin), 3 are involved in energy metabolism (mitochondrial ATP synthase β subunit, adenylate kinase and creatine kinase) and one is a component of the citric acid cycle (isocitrate dehydrogenase 3). In contrast, proteins involved in fatty acid oxidation (two isoforms of peroxisomal enoyl-CoA hydratase) and the citric acid cycle (three isoforms of malate dehydrogenase) were increased in pre-diabetic hearts. The results suggest that changes in the levels of several heart proteins may have implications in the development of the cardiac phenotype associated to T2D.     

Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.     

Rapid purification of mammalian cardiac troponin T and its isoform switching in rat hearts during development

A rapid purification of troponin T from adult hearts of various species has been developed. The purification procedure included 60 degrees C treatment of the high salt extract, ammonium sulfate fractionation, and DEAE-cellulose column chromatography. The troponin T purified from the bovine left ventricle contained two isoforms, which differed in both apparent molecular mass and isoelectric point. Both isoforms were able to bind to F-actin filaments only in the presence of tropomyosin. Monoclonal antibody JLT12 against rabbit skeletal troponin T cross-reacted with both isoforms of bovine cardiac troponin T. There was no detectable difference in the relative amount of these two isoforms among different portions (atria, right and left ventricles) of the bovine heart. The purified protein was used as an antigen to immunize mice, and a mouse antiserum with high titer and specificity to both isoforms was subsequently obtained. This antiserum also cross-reacted with cardiac troponin T from chicken, rabbit, and rat. The antibodies were further used to probe cardiac development in rats by Western blotting and immunoprecipitation. The results clearly showed that there was a switch of troponin T isoforms between hearts from 20-day-old rat embryos and hearts from 14-day-old rats. Immunoprecipitation of the in vitro translation products of poly(A)+ RNA isolated from day 5 rat hearts revealed the presence of two isoforms of troponin T, suggesting that two mRNAs coding for these two isoforms existed in the heart cells. It is of interest to not that some profound changes in the morphology and function of cardiac muscle have also been detected at this time of development. Troponin T isoform switching thus may well represent an important marker for cardiac development and function.     

Preservation of glucose metabolism in hypertrophic GLUT4-null hearts

GLUT4-null mice lacking the insulin-sensitive glucose transporter are not diabetic but do exhibit abnormalities in glucose and lipid metabolism. The most striking morphological consequence of ablating GLUT4 is cardiac hypertrophy. GLUT4-null hearts display characteristics of hypertrophy caused by hypertension. However, GLUT4-null mice have normal blood pressure and maintain a normal cardiac contractile protein profile. Unexpectedly, although they lack the predominant glucose transporter in the heart, GLUT4-null hearts transport glucose and synthesize glycogen at normal levels, but gene expression of rate-limiting enzymes involved in fatty acid oxidation is decreased. The GLUT4-null heart represents a unique model of hypertrophy that may be used to study the consequences of altered substrate utilization in normal and pathophysiological conditions.     

Glucose is a pH-Dependent Motor for Sperm Beat Frequency during Early Activation

To reach the egg in the ampulla, sperm have to travel along the female genital tract, thereby being dependent on external energy sources and substances to maintain and raise the flagellar beat. The vaginal fluid is rich in lactate, whereas in the uterine fluid glucose is the predominant substrate. This evokes changes in the lactate content of sperm as well as in the intracellular pH (pH(i)) since sperm possess lactate/proton co-transporters. It is well documented that glycolysis yields ATP and that HCO(3)- is a potent factor in the increase of beat frequency. We here show for the first time a pathway that connects both parts. We demonstrate a doubling of beat frequency in the mere presence of glucose. This effect can reversibly be blocked by 2-deoxy-D-glucose, dichloroacetate and aminooxyacetate, strongly suggesting that it requires both glycolysis and mitochondrial oxidation of glycolytic end products. We show that the glucose-mediated acceleration of flagellar beat and ATP production are hastened by a pH(i) ≥7.1, whereas a pH(i) ≤7.1 leaves both parameters unchanged. Since we observed a diminished rise in beat frequency in the presence of specific inhibitors against carbonic anhydrases, soluble adenylyl cyclase and protein kinase, we suggest that the glucose-mediated effect is linked to CO(2) hydration and thus the production of HCO(3)- by intracellular CA isoforms. In summary, we propose that, in sperm, glycolysis is an additional pH(i)-dependent way to produce HCO(3)-(,) thus enhancing sperm beat frequency and contributing to fertility.