mTOR inhibition induces endothelial progenitor cell death.

Immunosuppressants are necessary to prevent graft rejection after solid organ transplantation. However, they are also known to have significant side effects, including endothelial toxicity. Endothelial progenitor cells originate in the bone marrow and are recognized by their angiogenic and endothelial reparative properties. The effects of the immunosuppressants cyclosporine A (CyA), tacrolimus and rapamycin were analyzed on endothelial progenitor-like cells. Rapamycin induced rapid cell death, even at concentrations much lower than those used clinically, in peripheral blood mononuclear cells (PBMC) cultured to favor outgrowth of endothelial progenitors. Cyclosporine A and tacrolimus had no significant effects at clinical concentrations. The effect of rapamycin was specific to endothelial progenitor cells, in particular to the early stages of differentiation, as a lesser effect was observed in late outgrowth endothelial progenitors, mature aortic endothelial cells, and macrophages derived from the same PBMCs. The mechanism of cell death appeared to be apoptosis; however, its induction was probably multifactorial and did not depend on caspase or cathepsin activation. In conclusion, rapamycin induces endothelial progenitor cell death, possibly because it blocks survival signals given by growth factors critically required by these cells.

Optimal myocardial preconditioning in humans.

We developed a model of ischemia and reperfusion (I and R) in human ventricular myocytes (CM). CM injury and metabolics were studied after various interventions: endogenous preconditioning (PC) with anoxia, hypoxia, and anoxic or hypoxic supernatants; endogenous PC with or without SPT or adenosine deaminase; and exogenous adenosine PC before, during, or after I or continuously, with or without SPT. To assess the clinical implications of PC and the possible mediating effects of adenosine, patients undergoing elective coronary bypass surgery (CABG) received either a high or low dose of adenosine. Patients not receiving adenosine served as controls. Adenosine levels, high-energy phosphate levels, the metabolic parameters were evaluated from blood samples and left ventricular biopsy samples. Our cellular model studies indicated that preconditioning conferred protection to human CM via an adenosine-mediated pathway. Adenosine simulated PC without a fall in ATP. Adenosine administered to patients during CABG stimulated myocardial metabolism while preventing the degradation of high energy phosphates. A prospective randomized trial of adenosine administered to high-risk patients for myocardial protection is required.

Optimal myocardial preconditioning in a human model of ischemia and reperfusion.

BACKGROUND: Adenosine (ADE) may mediate the protective effects of preconditioning (PC). However, human data are lacking, and the optimal method of ADE administration and the mechanism of protection remain unresolved. METHODS AND RESULTS: We have developed a model of simulated "ischemia" (I) and "reperfusion" (R) in quiescent human ventricular cardiomyocytes. Cellular injury and metabolic parameters were assessed after various interventions: Cells were preconditioned with anoxia (PC0), hypoxia (PC16), anoxic supernatant (SUP0), or hypoxic supernatant (SUP16) with or without the ADE receptor antagonist (SPT) or ADE deaminase (ADA). ADE was applied before, during, or after I or continuously with and without SPT. Cells were treated with the PKC agonist PMA. PC cells were incubated with the protein kinase-C (PKC) antagonist Calphostin-C (Cal-C). PKC translocation and PKC activity were assessed. PC0 was most protective. Protection was transferable via SUP0, which produced the highest concentrations of ADE. Protection was lost with SPT or ADA. Intracellular ATP fell after PC and prolonged I and R. Exogenous ADE was most protective when administered before I at 50 mumol. ADE during I was partially protective. No additional protection was provided with continuous ADE treatment. ADE prevented ATP degradation but increased lactate immediately after its administration. SPT abolished the protective effects of ADE. PMA conferred protection, which was abolished with Cal-C. ADE stimulated PKC translocation and PKC activity in the absence of SPT. CONCLUSIONS: Maximal I confers maximal PC. The degree of I is reflected in supernatant ADE concentrations. The initial ATP fall with PC may account for a lack of ATP preservation after I and R. ADE reproduces the protective effects of PC, preserves ATP, and increases lactate production, perhaps by stimulating glycolysis. Clinical trials of ADE administered during cardiac surgery are necessary to further define its beneficial effects in humans.

Myocardial aerobic metabolism is impaired in a cell culture model of cyanotic heart disease.

A human pediatric cardiomyocyte cell culture model of chronic cyanosis was used to assess the effects of low oxygen tension on mitochondrial enzyme activity to address the postoperative increase in lactate and decreased ATP in the myocardium and the high incidence of low-output failure with restoration of normal oxygen tension, after technically successful corrective cardiac surgery. Chronically hypoxic cells (PO2 = 40 mmHg for 7 days) exhibited significantly reduced activities for pyruvate dehydrogenase, cytochrome-c oxidase, succinate cytochrome c reductase, succinate dehydrogenase, and citrate synthase. The activity of NADH-cytochrome c reductase was unaffected. Lactate production and the lactate-to-pyruvate ratio were significantly greater in hypoxic cardiomyocytes. Western and Northern analysis demonstrated a decrease in the levels of various mRNA and corresponding polypeptides in hypoxic cells. Thus hypoxia influences mitochondrial metabolism through acute and chronic adaptive mechanisms, reflecting allosteric (posttranscriptional) and transcriptional modulation. Transcriptional downregulation of key mitochondrial enzyme systems can explain the insufficient myocardial aerobic metabolism and low-output failure in children with cyanotic heart disease after cardiac surgery.

Insulin stimulates pyruvate dehydrogenase and protects human ventricular cardiomyocytes from simulated ischemia.

UNLABELLED: Impaired myocardial metabolism after cardioplegic arrest results in persistent anaerobic lactate production. Insulin may protect the heart from ischemia and reperfusion by enhancing myocardial metabolic recovery. However, the stimulation of glycolysis during ischemia may be detrimental because of an accumulation of metabolic end-products. We examined the effect of insulin on quiescent human ventricular cardiomyocytes subjected to simulated cardioplegic ischemia and reperfusion. METHODS: Primary cardiomyocyte cultures were established from patients undergoing corrective repair of tetralogy of Fallot. Cells were exposed to varying concentrations of glucose and insulin during 30 minutes of stabilization in 10 mL of phosphate-buffered saline solution. Ischemia was simulated by exposing the cells to a low volume (1.5 mL) of deoxygenated phosphate-buffered saline solution for 90 minutes followed by 30 minutes of simulated reperfusion in 10 mL of normoxic phosphate-buffered saline solution. Cell viability was assessed by trypan blue exclusion. The activity of mitochondrial pyruvate dehydrogenase was measured in 3 states: stabilization, ischemia, and reperfusion. In addition intracellular lactate, adenine nucleotides, extracellular lactate, pyruvate, and acid release were measured. RESULTS: Higher ambient glucose concentrations resulted in greater cellular injury although insulin-treated cells displayed less injury after ischemia and reperfusion. Insulin increased the pyruvate dehydrogenase activity by 31% in cardiomyocytes and reduced extracellular lactate production by 40%. Intracellular adenosine triphosphate was improved by 75% in cells exposed to high glucose concentrations in the presence of insulin. CONCLUSIONS: Insulin protected human ventricular cardiomyocytes from ischemia and reperfusion. This protection may be due to a stimulation of pyruvate dehydrogenase activity which resulted in improved aerobic metabolism.

Preconditioning human cardiomyocytes and endothelial cells.

BACKGROUND: The effects of simulated "ischemia" and "reperfusion" were evaluated in cell cultures of human ventricular cardiomyocytes and human saphenous vein endothelial cells. METHODS: Myocyte and endothelial cell cultures were exposed to a low volume (1.5 ml) of either hypoxic (oxygen tension = 16 mm Hg) or anoxic (oxygen tension = 0 mm Hg) phosphate-buffered saline solution for 90 minutes ("ischemia") followed by 30 minutes of simulated "reperfusion." Cell injury was evaluated by trypan blue exclusion. Next, the effects of a preconditioning stimulus were evaluated by a brief (10 minute) exposure to hypoxic or anoxic ischemia and 10 minutes of reperfusion before prolonged (90 minutes) anoxic ischemia. Finally, the effects of anoxic preconditioning on intracellular lactate accumulation and extracellular lactate and acid release were assessed. RESULTS: "Ischemia" and "reperfusion" resulted in greater injury to endothelial cells than to cardiomyocytes. In both cell types, anoxic ischemia resulted in greater injury than hypoxic ischemia. Preconditioning reduced cell injury in myocytes but not in endothelial cells. Endothelial cells produced more lactate than cardiomyocytes under normoxic conditions. Ischemia increased lactate accumulation and release in cardiomyocytes but not endothelial cells. Preconditioning reduced lactate accumulation and release in cardiomyocytes but not endothelial cells. CONCLUSIONS: Endothelial cells were more susceptible to the same period of simulated ischemia than cardiomyocytes. Preconditioning protected cardiomyocytes but not endothelial cells from a subsequent prolonged period of ischemia and reperfusion.

Human pediatric and adult ventricular cardiomyocytes in culture: assessment of phenotypic changes with passaging.

OBJECTIVES: The purpose of this study was to assess morphologically and biochemically the phenotypic changes which occur in vitro with passaging of human pediatric and adult ventricular cardiomyocytes. METHODS: Human ventricular cardiomyocytes from 3 children (1 to 2 years of age) and an adult patient (65 years of age) undergoing open heart surgery and an adult heart transplant patient (55 years of age) were isolated, cultured, purified, and passaged. Growth curves and 3H-thymidine uptake studies were performed. Characterization of the cells was done by light microscopy, transmission electron microscopy, immunofluorescent staining for myoglobin, CK-MB, and cardiac-specific troponin I isoform, human ventricular myosin heavy chain (HVMHC) and light chain 1 (HVMLC1), Northern blot analysis of HVMHC, and CK-MB activity and mass measurements. Passage 3 cardiomyocyte and pediatric myocardial phospholipids were analysed by gas chromatography. RESULTS: Pediatric cells were smaller (P < 0.01) and divided faster (P < 0.001, ANOCOVA) than adult cells. The cardiomyocytes showed phenotypic changes in primary culture with essentially complete loss of sarcomeres by 10 days and a gradual loss of myofilaments with passaging. The cells were identified as cardiomyocytes by immunohistochemistry for myoglobin, CK-MB, cardiac-specific troponin I isoform, HVMHC and HVMLC1, and by Northern blot analysis for the 3'-end of HVMHC mRNA. The composition of phospholipid fatty acids in the cultured pediatric cells was similar to that found in the pediatric myocardium. CK-MB activity and mass could be measured in the cardiomyocytes. The adult cardiomyocytes were more difficult to maintain than the pediatric cells which could be cultured for as long as 6 months. CONCLUSIONS: Primary cultures of human pediatric and adult partially differentiated ventricular cardiomyocytes can be passaged. Although rapid disorganization of the myofibrils occurs, the non-contractile cells can be identified as cardiomyocytes by morphological appearance, immunofluorescent staining, Northern blot analysis for HVMHC, and CK-MB activity.

Adequate distribution of warm cardioplegic solution.

Seventy-five patients undergoing coronary artery bypass grafting were randomized to receive warm antegrade (N = 25), warm retrograde (N = 25), or a combination of warm antegrade and retrograde (N = 25) delivery of blood cardioplegic solution. Myocardial oxygen utilization, lactate and acid metabolism, and adenine nucleotides and their degradation products were measured during the operation and cardiac function was assessed postoperatively. Warm retrograde delivery of cardioplegic solution increased lactate and acid release during cardioplegia and reperfusion, decreased left ventricular adenosine triphosphate concentrations, and reduced the washout of adenine nucleotide degradation products from both left and right ventricles. Warm antegrade delivery of cardioplegic solution resulted in less lactate and acid release during cardioplegia but more lactate accumulated in the territory of the left anterior descending artery during the crossclamp period. Intermittent antegrade delivery of the cardioplegic solution during combination cardioplegia washed out lactate and acid, which suggested inhomogeneous delivery of the cardioplegic solution during continuous retrograde cardioplegia. Combination cardioplegia best preserved adenosine triphosphate in the left ventricle and resulted in the best postoperative left and right ventricular function. A combination of intermittent antegrade and continuous retrograde delivery of cardioplegic solution provided better myocardial protection than either antegrade or retrograde delivery of cardioplegic solution alone.

Cardiac storage with University of Wisconsin solution and a nucleoside-transport blocker.

Findings from previous investigations conducted at this institution and others have suggested that University of Wisconsin solution (UWS) is preferable for the prolonged hypothermic storage of hearts before transplantation. The benefit seen with UWS may in part be related to the inclusion of adenosine (5 mmol/L) in the UWS. To investigate whether further manipulations of adenosine metabolism might enhance myocardial protection, studies were initially conducted using cultured myocytes, followed by confirmatory experiments using isolated rat hearts. Cultured human ventricular myocytes (7 to 8 dishes/group) were stored for 12 hours at 0 degrees C in unmodified UWS or UWS supplemented with increasing concentrations (1 to 100 mumol/L) of the nucleoside-transport blocker p-nitrobenzylthioinosine. The adenosine triphosphate concentrations were found to be enhanced with nucleoside-transport inhibition, with the best results achieved with the 1- and 3-mumol/L groups (control, 3.37 +/- 0.41 nmol/micrograms DNA; UWS, 2.89 +/- 1.31 nmol/micrograms DNA; 1 mumol/L, 5.91 +/- 3.23 nmol/micrograms DNA; 3 mumol/L, 7.86 +/- 3.45 nmol/micrograms DNA; p < 0.05 versus control or UWS group). Isolated rodent hearts from Sprague-Dawley rats were prepared on a Langendorff apparatus with an intraventricular balloon and subsequently stored for 8 hours at 0 degrees C in unmodified UWS (13 hearts/group) or UWS supplemented with 1 or 3 mumol/L of p-nitrobenzylthioinosine (9 to 10 hearts/group).(ABSTRACT TRUNCATED AT 250 WORDS)

Tepid antegrade and retrograde cardioplegia.

To determine the optimal temperature for the combination of antegrade and retrograde cardioplegia, 42 patients undergoing coronary artery bypass grafting were randomized to receive cold (9 degrees C; n = 14), tepid (29 degrees C; n = 14), or warm (37 degrees C; n = 14) blood cardioplegia delivered continuously retrograde and intermittently antegrade. Myocardial oxygen utilization, lactate and acid metabolism, and coronary vascular resistance were measured during the operation and cardiac function was assessed postoperatively. Myocardial oxygen consumption, lactate release and acid release were greatest with warm, intermediate with tepid, and least with cold cardioplegia (p = 0.0001). However, washout of lactate and acid at the time of cross-clamp release was reduced (p = 0.022) with tepid or cold compared with warm cardioplegia. Early postoperative left ventricular function was best preserved (p = 0.01) after tepid than after cold or warm combination cardioplegia. These results suggest that tepid combination cardioplegia reduced metabolic demands but permitted immediate recovery of cardiac function. This technique may provide better myocardial protection than cold or warm combination cardioplegia.

The optimal cardioplegic temperature.

Seventy-two patients undergoing coronary artery bypass grafting were randomized to receive cold (8 degrees C) antegrade or retrograde, tepid (29 degrees C) antegrade or retrograde, or warm (37 degrees C) antegrade or retrograde blood cardioplegia (n = 12 in each group). Myocardial oxygen utilization as well as lactate and acid metabolism were assessed intraoperatively and cardiac function was assessed postoperatively. Myocardial oxygen consumption and anaerobic lactate release were greatest during warm, intermediate during tepid, and least during cold cardioplegic arrest. Myocardial oxygen consumption and lactate release were underestimated during retrograde cardioplegia because of contamination of aortic root samples. Warm retrograde and tepid retrograde cardioplegia resulted in greater lactate and acid washout with reperfusion. Left ventricular stroke work indices were greater after warm antegrade and tepid antegrade cardioplegia than after cold antegrade cardioplegia, and right ventricular stroke work indices were greatest after warm antegrade cardioplegia. Warm antegrade cardioplegia increased aerobic metabolism during and after cardioplegia and preserved left and right ventricular function. Tepid antegrade cardioplegia reduced anaerobic lactate and acid release during arrest and preserved cardiac function.

Vitamin E for coronary bypass operations. A prospective, double-blind, randomized trial.

BACKGROUND: Free radical lipid peroxidation contributes to the abnormal metabolism and ventricular function frequently seen after cardiac operations. Antioxidants may improve metabolic and functional recovery. METHODS: A prospective, randomized, double-blind clinical trial was conducted to determine the effects of vitamin E (alpha-tocopherol) (n = 14) or a corn oil placebo (n = 14) in patients undergoing elective coronary bypass operations. The RRR-alpha-tocopheryl acetate doubled the alpha-tocopherol levels in the heart. Myocardial metabolism and ventricular function were assessed after the operation. RESULTS: Atrial pacing induced myocardial lactate production in the control patients but lactate consumption in the alpha-tocopherol-treated patients on bypass 25 minutes after crossclamp release. Left ventricular stroke work indices were higher, at similar ventricular volumes, in the alpha-tocopherol-treated group, which indicates improved preload recruitable stroke work, and diastolic compliance was greater 4 hours after the operation. The postoperative creatine kinase cardiac isoenzyme levels were lower in the patients who received alpha-tocopherol. CONCLUSIONS: Pretreatment with alpha-tocopherol sufficient to double the myocardial concentrations had a small but significant metabolic and functional effect after elective coronary bypass operations when compared with placebo. These results do not justify pretreatment of low-risk patients, but they do justify an evaluation in high-risk patients.

Preconditioning human ventricular cardiomyocytes with brief periods of simulated ischaemia.

OBJECTIVE: The aim was to test for "ischaemic" preconditioning in monolayer cultures of quiescent human ventricular cardiomyocytes. METHODS: Stabilised cardiomyocytes (n = 8 plates per group) were preconditioned with varying periods of simulated ischaemia and reperfusion, followed in all groups by 90 min of sustained "ischaemia" with or without 30 min of reperfusion. Cellular injury was assessed by trypan blue exclusion and survival was assessed by culturing the cells for 24 h postintervention. In addition, separate groups of cell plates (n = 8 per group) which had first been preconditioned with 20 min ischaemia and 20 min reperfusion were exposed to either 30, 60, or 90 min sustained ischaemia or 90 min sustained ischaemia with 30 min reperfusion. The supernatants and/or cell homogenates were analysed for hydrogen ion, lactate, lactate dehydrogenase (LDH), and adenine nucleotides and degradation products. RESULTS: Preconditioning (PC) decreased trypan blue uptake following subsequent sustained ischaemia, with the 20 min ischaemia/20 min reperfusion (20/20) regimen having the most profound effect [control ischaemia: 37.0(SEM 2.1); 10/10: 23.9(1.5); 20/20: 15.4(1.4); 30/30: 25.8(2.1) percent blue stained cells, p < 0.05 by ANOVA/Duncan]. The 20/20 preconditioning regimen resulted in less hydrogen ion [control: 2.1(0.4); PC: 1.4(0.1) mmol.g-1 protein, p < 0.05] and less LDH release [control: 20.7(3.1); PC: 11.9(4.2) units.g-1 protein, p < 0.05]. At 90 min of sustained ischaemia, the control group had produced significantly greater lactate [intracellular: control 1.55(0.62); PC 0.54(0.23) mol.g-1 DNA, p < 0.05; extracellular: control 0.47(0.09); PC 0.33(0.07) mol.g-1 DNA, p < 0.05]. There were no differences in ATP depletion in the two groups. CONCLUSIONS: Ischaemic preconditioning can be induced in human cardiomyocytes independent of other cell types. The effect can be established in human cell cultures.

Optimal flow rates for retrograde warm cardioplegia.

Retrograde delivery of warm blood cardioplegia may improve nutrient cardioplegic flow beyond coronary obstructions, but may not adequately perfuse the right ventricle and the posterior left ventricle. To determine the optimal flow rate for warm retrograde cardioplegia, we assessed 62 patients undergoing elective coronary artery bypass in two studies. In the low flow study, administration of 50 ml/min (n = 9), 75 ml/min (n = 11), or 100 ml/min (n = 7) was associated with high lactate production and oxygen extraction during cardioplegic administration. At 50 minutes of cardioplegic arrest, the coronary venous effluent pH was low in all groups. In the high flow study, 30 patients all received flow rates of 100, 200, and 300 ml/min in randomized order during the crossclamp period. In addition, five patients received cardioplegia at a rate of 500 ml/min for the duration of the crossclamp period. Administration of 200 ml/min or higher minimized lactate production and maintained coronary venous pH within the physiologic range, but flows of 300 ml/min or higher did not increase oxygen use or reduce lactate or acid production. Patients in the low flow groups had significantly greater myocardial lactate release during cardioplegic infusion and after removal of the crossclamp than the high flow group. Warm retrograde cardioplegia should be delivered at flow rates of at least 200 ml/min during elective coronary artery bypass operations.

Which techniques of cardioplegia prevent ischemia?

One hundred seven patients undergoing coronary artery bypass grafting were randomized to receive warm antegrade (n = 21), warm retrograde (n = 22), cold antegrade (n = 20), cold retrograde (n = 22), or intermittent cold antegrade (n = 22) blood cardioplegia. Myocardial oxygen consumption and lactate production, adenine nucleotides, and adenine nucleotide degradation products were measured during the operation, and creatine kinase-MB release was assessed postoperatively. Warm cardioplegia resulted in greater myocardial lactate production than cold cardioplegia (p = 0.048). Retrograde cardioplegia was associated with greater lactate production than antegrade cardioplegia (p = 0.015). Adenosine triphosphate depletion was similar among groups. However, poorly diffusible metabolites of adenosine triphosphate accumulated to the greatest extent in the intermittent cold group. Levels of hypoxanthine were highest after warm retrograde cardioplegia. Operative mortality and morbidity were low and were not different among groups. In summary, none of the five techniques of cardioplegia evaluated in this study was able to completely prevent myocardial ischemia. Anaerobic lactate production was minimized with cold cardioplegia and with antegrade cardioplegic delivery. Hypothermia may have impaired regeneration of adenosine triphosphate, however, particularly in association with inadequate or intermittent cardioplegic flow.

[Andrologic therapy in the elderly--therapy seeking, acceptance, risks, satisfaction]. / Terapie andrologiche nel soggetto anziano--richiesta di terapia, accettazione, rischi, soddisfazione.

The paper discusses over questions and hidden requests of andrological and sexological help in the general medical consultation of the elderly; the most frequent specific andrological symptoms and the pertinent sexological implication; the conscious and unconscious ambivalence present in the acceptance of therapy; ongoing risks, in case of inappropriate or inadequate evaluation of the therapeutic request.

Ventricular function after normothermic versus hypothermic cardioplegia.

Warm cardioplegia produced by essentially continuous infusion has been used as an alternative to traditional cold intermittent infusion techniques during cardiac surgery, but its effects on postoperative left ventricular function have not been defined. We performed a randomized clinical trial to assess the effects of warm and cold blood cardioplegia on load-independent indices of ventricular function. Fifty-three patients were randomized to warm (n = 27) or cold (n = 26) cardioplegia. Myocardial oxygen consumption, lactate production, adenine nucleotides, and adenine nucleotide degradation products were measured during cardioplegia and reperfusion. In 13 patients per group, pressure-volume loops were constructed and ventricular function was assessed 3 hours after the operation. Warm cardioplegia resulted in greater myocardial lactate production but improved recovery of oxygen consumption during reperfusion. Depletion of adenosine triphosphate was similar between groups, but total adenine nucleotides (adenosine triphosphate + adenosine diphosphate + adenosine monophosphate) fell further during warm cardioplegia. Cold cardioplegia was associated with an accumulation of adenosine diphosphate and adenosine monophosphate. Creatine kinase MB isoenzyme release was reduced in the warm group. Three hours after the operation, end-systolic elastance and preload-recruitable stroke work index were increased after warm cardioplegia, and early diastolic relaxation was also increased. Increased systolic function after warm cardioplegia may have been related to improved myocardial protection, elevated arterial lactate concentrations, or increased circulating catecholamine levels. Altered diastolic compliance in the warm group may reflect greater active relaxation during early diastolic filling.

Mechanisms of adaptation of glucose transporters to changes in the oxidative chain of muscle and fat cells.

Cells in which glucose uptake is rate limiting respond to hypoxic insults with an increase in glucose transport activity. To understand the underlying cellular mechanisms involved in this adaptive response, the effects of an uncoupler of oxidative phosphorylation, 2,4-dinitrophenol (DNP), and of an inhibitor of the electron transport chain, rotenone, were compared with the effect of hypoxia in L6 muscle cells and 3T3-L1 adipocytes. All three conditions (DNP, rotenone, and 3% oxygen) elevated hexose uptake by approximately twofold in 4 h relative to control cells. All three insults decreased cellular ATP levels rapidly. A subsequent recovery was observed within 1-2 h in the presence of DNP or 3% oxygen, probably as a result of anaerobic production of ATP through increased glucose uptake and glycolysis. DNP and rotenone elevated the content of GLUT-1 protein in isolated plasma membranes and decreased it in intracellular light microsomes, suggestive of translocation of this transporter isoform. No change in GLUT-4 protein distribution was detected. In contrast, 3% oxygen caused a marked specific increase in GLUT-1 protein in both plasma membranes and microsomes. Consistently, cycloheximide had no effect on the hexose transport responses to DNP or rotenone, but prevented the response to hypoxia. However, GLUT-1 mRNA and the total cell content of GLUT-1 protein were elevated by all three treatments. It is proposed that within the time frame studied, reductions in the energy charge may activate the glucose transport system in L6 myotubes and 3T3-L1 adipocytes by GLUT-1 protein biosynthesis and translocation. When both responses exist, the biosynthetic pathway is dispensable, and posttranslational mechanisms, including transporter translocation suffice to sustain the adaptive elevation in glucose transport activity for several hours.

Comparison of two experimental models for assessment of cardiac preservation.

Previous studies from this institution using human cell cultures have suggested that University of Wisconsin solution is preferred for prolonged hypothermic storage for cardiac transplantation. The primary objective of this study was to evaluate the effectiveness of extended cardiac preservation with University of Wisconsin solution by assessing the time-related changes of purine metabolites using two different models of cold storage. Isolated rat hearts (n = 6/group) or human ventricular myocyte cultures (n = 7 dishes/group) were assessed after 0, 6, 12, and 24 hours in University of Wisconsin solution at 0 degrees C using high-performance liquid chromatography. Adenosine triphosphate content decreased from 18.1 +/- 5.4 to 9.6 +/- 2.7 mumol/g dried weight by 12 hours and to 1.0 +/- 0.6 mumol/g by 24 hours (p < 0.0001 by analysis of variance) in the rat model. Adenosine triphosphate content decreased from 0.64 +/- 0.42 to 0.14 +/- 0.11 nmol/micrograms DNA at 6 hours and to 0.04 +/- 0.03 nmol/micrograms DNA by 24 hours (p < 0.00001) in the cardiomyocytes. Inosine monophosphate content increased from 0.1 +/- 0.2 to 10.8 +/- 1.0 by 24 hours (p < 0.0001) in the rat studies. Inosine monophosphate values tended to increase up to 12 hours (p = 0.06) in the cell cultures and then declined. Adenosine concentration increased from 0.3 +/- 0.3 to 2.3 +/- 0.9 mumol/g at 6 hours and declined thereafter (p < 0.0005) in the rodent hearts. Adenosine concentration increased from 0.03 +/- 0.02 to 1.53 +/- 0.72 nmol/micrograms DNA at 6 hours (p < 0.0001) in the cardiomyocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro assessment of the effects of glucose added to the University of Wisconsin solution on myocyte preservation.

BACKGROUND: University of Wisconsin solution (UWS) has been successfully used for liver transplantation and may be beneficial for hypothermic cardiac storage. The addition of glucose may enhance myocardial preservation. METHODS AND RESULTS: Cultured human ventricular myocytes (eight dishes per group) were stored at 0 degree C for 12 hours in either unmodified UWS or UWS with glucose (1, 3, 10, 30, or 100 mmol/l). Cells were assayed for protein by spectrofluorometry and adenine nucleotides by high performance liquid chromatography after storage. Protein recovery, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and total adenine nucleotides (ATP+ADP+AMP) were all depleted after storage (p < 0.0001 by ANOVA). Protein recovery (p < 0.005), ATP (p < 0.05), and ADP (p < 0.05) were increased with glucose administration compared with unmodified UWS. Improvement was maximal using 30 mmol/l (protein, 0 mmol/l = 0.48 +/- 0.14 and 30 mmol/l = 0.65 +/- 0.11 mg per dish; ATP, 0 mmol/l = 3.08 +/- 0.63 and 30 mmol/l = 4.32 +/- 0.90 nmol/mg protein; ADP, 0 mmol/l = 3.76 +/- 0.80 and 30 mmol/l = 4.63 +/- 0.38 nmol/mg protein, mean +/- SD). Total adenine nucleotides tended to increase at any glucose concentration (p = 0.07 by ANOVA) and were significantly better with 30 mmol/l glucose (0 mmol/l = 7.61 +/- 1.58 and 30 mmol/l = 9.62 +/- 1.08 nmol/mg protein). CONCLUSIONS: Increasing the glucose concentration from 0 to 30 mmol/l improved adenine nucleotide and cellular protein preservation in this in vitro assessment.