Different role of antioxidants in endotoxin-induced late myocardial protection

Previous studies have proposed that endogenous antioxidants play a protective role against cardiac ischemia-reperfusion injury in endotoxin pretreatment. However, the mechanism underlying this effect remains elusive. We therefore evaluated the role of endogenous antioxidants in delayed myocardial protection after different doses of endotoxin administration using cultured rat neonatal cardiomyocytes. Myocytes were treated with normal saline (control) or lipopolysaccharide (Escherichia coli, serotype O111) at doses of 40 and 80 microg/ml (ET40 and ET80). Also, antisense oligodeoxyribonucleotide (1.5 micromol/L) to manganese superoxide dismutase (Mn-SOD) and 3-amino-1,2,4-triazole (25 mg/ml) were added along with a 40 or 80 microg/ml endotoxin pretreatment in the IET40 and IET80 groups. Twenty-four hours later, Cells were subjected to hypoxia (pO2 < 1 kPa, 3 h) and reoxygenation (pO2: 19 kPa, 1 h). Compared with controls, cell viability enhanced significantly (65.3 +/- 5.9, 63.8 +/- 4.6, and 69.7 +/- 5.2% vs 47.2 +/- 4.3%, P < 0.05) and creatine kinase release decreased (7.34 +/- 1.76, 7.11 +/- 1.49, and 6.27 +/- 1.24 U/mg protein vs 11.23 +/- 2.49 U/mg protein, P < 0. 05) in ET40, IET40, and ET80 groups following reoxygenation. No statistically significant difference was found between the control and the IET80 groups. Furthermore, the levels of Mn-SOD (1.12 +/- 0. 31 vs 0.75 +/- 0.15 U/mg. protein, P < 0.05) and catalase activity (1265 +/- 109 vs 996 +/- 85 U/mg. protein, P < 0.05) were higher only in the ET80 group. The results suggest that at a dose of 40 microg/ml, cells were protected by mechanisms other than the augmentation of endogenous antioxidant activity which were more evident at a dose of 80 microg/ml. It seems that different doses of endotoxin pretreatment may induce delayed myocardial protection through various mechanisms.     

Reduced myocardial expression of calcium handling protein in patients with severe chronic mitral regurgitation.

OBJECTIVE: Left ventricle (LV) function was shown to be a principal determinant of morbidity and mortality in both uncorrected and surgically corrected mitral regurgitation (MR). However, the cellular mechanisms that develop in the LV remodeling secondary to volume overload in chronic severe MR is still not well defined. In single ventricular myocyte, a reduced contraction and slowed relaxation have been mainly attributed to defective intracellular Ca2+ currents. Between several Ca2+ handling proteins, sarcoplasmic reticulum Ca2+-ATPase 2 (SERCA2) expression and activity determines not only the extent and rate of relaxation, but also the rate and amplitude of contraction. The aim of the study was to determine whether modifications of SERCA2 gene expression occurs in LV wall remodeling process secondary to chronic severe MR. METHODS: The LV samples were obtained from 12 patients presented LV wall remodeling (LV: diastolic/systolic diameter-70+/-7 mm vs 46+/-10 mm; diastolic/systolic volume-260+/-65 ml vs 102+/-68 ml) due to chronic, severe MR. Expressions of SERCA2 isoforms-SERCA2a and 2b mRNAs were estimated by semiquantitative RT-PCR and normalized to GAPDH. The protein levels of SERCA2 were determined by Western blot after normalization to actin. Results were compared with samples from non-failing human hearts (NFH). RESULTS: On SERCA2 mRNA levels, important reduction on both SERCA isoforms SERCA2a (-40%) and SERCA2b (-49%) compared to NFH, together with significant correlation between isoforms (r = 0.89; p = 0.01) were observed. SERCA2 protein levels were decreased (-38%) in MR compared to NFH. Also significant correlations between SERCA2a/2b and SERCA2 protein expression (r = 0.83, p = 0.017; r = 0.68, p = 0.05, respectively) were observed. Moreover, a negative correlation between protein levels of SERCA2 (r = -0.64, p = 0.053) and left ventricular diastolic diameter was observed. CONCLUSIONS: In chronic volume overload the down-regulation of SERCA2a and 2b at the mRNA and SERCA2 protein levels exist. Moreover, protein levels of SERCA2 tend to correlate to the grade of left ventricular diastolic dilatation and suggest an important role LV remodeling.     

The role of pentoxifylline in endotoxin-induced alterations of red cell deformability and whole blood viscosity in the neonate

Endotoxin induces alterations in the neonatal red cell membrane that result in decreased deformability and an increase in whole blood viscosity. These rheologic alterations are detrimental to flow in the microcirculation. Pentoxifylline (PTX), a methyl xanthine derivation, increases red cell deformability presumably through its effect on intracellular adenosine 5-triphosphate. The purpose of this study was to evaluate the effect of PTX on endotoxin-induced alterations in the neonatal red blood cell. Anticoagulated whole blood specimens obtained from the cord of 12 neonates at birth were used to study the effects of Escherichia coli endotoxin (LPS) with and without PTX (50 micrograms/mL) on red cell deformability and whole blood viscosity. LPS resulted in a significant (P less than .001) decrease in deformability compared with controls. PTX reversed these endotoxin-induced alterations (P less than .01), normalizing deformability to control values (P = NS). LPS resulted in a significant increase (P less than .005) in blood viscosity that was reversed by PTX (P = NS). Pentoxifylline reverses the detrimental rheologic effect of endotoxin in the neonate. This activity may be helpful in sustaining normal microcirculation in neonatal sepsis.     

Impaired fracture healing in the absence of TNF-alpha signaling: the role of TNF-alpha in endochondral cartilage resorption.

TNF-alpha is a major inflammatory factor that is induced in response to injury, and it contributes to the normal regulatory processes of bone resorption. The role of TNF-alpha during fracture healing was examined in wild-type and TNF-alpha receptor (p55(-/-)/p75(-/-))-deficient mice. The results show that TNF-alpha plays an important regulatory role in postnatal endochondral bone formation. INTRODUCTION: TNF-alpha is a major inflammatory factor that is induced as part of the innate immune response to injury, and it contributes to the normal regulatory processes of bone resorption. METHODS: The role of TNF-alpha was examined in a model of simple closed fracture repair in wild-type and TNF-alpha receptor (p55(-/-)/p75(-/-))-deficient mice. Histomorphometric measurements of the cartilage and bone and apoptotic cell counts in hypertrophic cartilage were carried out at multiple time points over 28 days of fracture healing (n = 5 animals per time point). The expression of multiple mRNAs for various cellular functions including extracellular matrix formation, bone resorption, and apoptosis were assessed (triplicate polls of mRNAs). RESULTS AND CONCLUSIONS: In the absence of TNF-alpha signaling, chondrogenic differentiation was delayed by 2-4 days but subsequently proceeded at an elevated rate. Endochondral tissue resorption was delayed 2-3 weeks in the TNF-alpha receptor (p55(-/-)/p75(-/-))-deficient mice compared with the wild-type animals. Functional studies of the mechanisms underlying the delay in endochondral resorption indicated that TNF-alpha mediated both chondrocyte apoptosis and the expression of proresorptive cytokines that control endochondral tissue remodeling by osteoclasts. While the TNF-alpha receptor ablated animals show no overt developmental alterations of their skeletons, the results illustrate the primary roles that TNF-alpha function contributes to in promoting postnatal fracture repair as well as suggest that processes of skeletal tissue development and postnatal repair are controlled in part by differing mechanisms. In summary, these results show that TNF-alpha participates at several functional levels, including the recruitment of mesenchymal stem, apoptosis of hypertrophic chondrocytes, and the recruitment of osteoclasts function during the postnatal endochondral repair of fracture healing.     

Rapid increase in fraction of cardiac output to bone in experimental calcium deficiency

The fractional redistribution of cardiac output to bone in dietary calcium deficiency was studied in the immature rat, utilizing the⁸⁶Rb method of Sapirstein. The results of the study indicated that there was a rapid and significant increase in the fraction of cardiac output to the femurs of the calcium-deficient rats relative to the control population. The increase in cardiac output fraction to bone occurred during the same general time period in which significant changes in the density, dry weight and ash content were detected. The increased fraction of the cardiac output to the femur in the calcium-deficient rats returned to normal after nine days, in spite of continuation of the calcium-deficient diet.From the Department of Radiology, Yale University School of Medicine. Supported by USPHS Grants AM-09664, GM-01152, AM-09429 and CA-06519.     

Propofol-induced alterations in myocardial beta-adrenoceptor binding and responsiveness.

Propofol (iv) depresses cardiovascular function in both humans and animals. However, the mechanism underlying this action has not been well described. The present study was designed to test the hypothesis that this effect of propofol results in part from an antagonism of adrenergic control of the heart. Experiments examined effects of propofol on: 1) [3H]CGP12177 (a beta-adrenoceptor antagonist) binding in rat myocardial membranes; and 2) the inotropic and chronotropic actions of isoproterenol in rat left atrial muscle and right atria, respectively. Propofol (25-200 microM) increased the apparent dissociation constant for [3H]CGP12177 without affecting binding site density. Similarly, 200 microM propofol increased the 50% effective concentration values for the dose-dependent positive chronotropic and inotropic actions of isoproterenol in right and left atria, and depressed the maximum increase in spontaneous rate elicited by this beta-adrenoceptor agonist. Other experiments demonstrated that propofol does not alter muscarinic receptor binding as monitored using [3H]quinuclidi-nylbenzilate. In conclusion, these results indicate that propofol can decrease cardiac beta-adrenoceptor responsiveness; however, the concentrations of propofol required suggest that this action contributes to the cardiovascular depression produced by this anesthetic only during large-dose bolus injection. IMPLICATIONS: Experiments in membranes and cardiac preparations isolated from rat heart demonstrate that relatively high concentrations of propofol (25-200 microM) are required to antagonize beta-adrenoceptor binding and tissue responsiveness.     

Altered renal calcium handling in hypercalcemia of malignancy

It has been controversial whether increased renal tubular calcium reabsorption contributes to hypercalcemia in patients with malignancies. Moreover, whether this abnormality is associated with volume depletion, a parathyroid hormone-like effect, or other mechanisms has not been clarified. Eight consecutive patients with hypercalcemia due to a variety of tumor types were studied in detail. The glomerular filtration rate (iothalamate clearance) was reduced in all patients (0.98 +/- 0.10 (mean +/- SE) mL/s.1.73 m2; P less than 0.001) compared with normal controls (N = 9) (1.93 +/- 0.08 mL/s.1.73 m2), but it was similar to that in controls matched for renal insufficiency (N = 6) (1.15 +/- 0.05 mL/s.1.73 m2). During hypercalcemia produced by calcium infusion, urinary calcium excretion (millimoles of calcium per liter of glomerular filtrate) was increased in controls with renal insufficiency compared to those with normal renal function (P = 0.028). In all patients with hypercalcemia of malignancy, urinary calcium excretion was decreased compared with controls with renal insufficiency, but it was low in only five of eight patients compared with normal controls. Extracellular fluid volume (iothalamate volume of distribution) was not decreased in any patient, and urinary cAMP and/or plasma parathyroid hormone-like bioactivity were increased in six of eight patients. After treatment with an inhibitor of bone resorption, aminopropylidene 1,1 diphosphonate, abnormal renal calcium handling was not detected if the serum calcium normalized. It was concluded that increased renal tubular calcium reabsorption was consistently present in patients with hypercalcemia of malignancy compared with controls matched for renal insufficiency, but the proportion with the abnormality was underestimated if normal controls were used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pinacidil improves contractile function and intracellular calcium handling in isolated cardiac myocytes exposed to simulated cardioplegic arrest

Background

We examined the effects of pinacidil on contractile function and intracellular calcium in isolated rat cardiomyocytes exposed to cardioplegic solution.

Methods

Rat myocytes were incubated at 24°C for 2 hours in cardioplegic solution with or without pinacidil (50 μmol/L), then they were perfused with Krebs-Henseleit solution with a gas phase of 95% O₂/5% CO₂ at the same temperature. Contraction and intracellular calcium transients were then measured by video tracking and spectrofluorometry.

Results

During 20 minutes of perfusion after 2 hours in cardioplegic solution with pinacidil, (1) the recovery of contractile function was significantly increased in terms of both amplitude of contraction (98.30% ± 9.90% versus 81.00% ± 11.25%; p < 0.05) and peak velocity of cell shortening (100.90% ± 13.79% versus 76.89% ± 18.14%; p < 0.01) when compared with myocytes in cardioplegic solution without pinacidil; (2) the amplitudes of the intracellular calcium transients evoked by electrical stimulation and caffeine (10 mmol/L) increased by 23.31% to approximately 40.72% and 61.73%, respectively, compared with those in cardioplegic solution without pinacidil; and (3) the decay time of the caffeine-induced intracellular calcium transient decreased by 36.64% ± 15.10% relative to that measured in cardioplegic solution without pinacidil. The effects induced by supplementing the cardioplegic solution with pinacidil were diminished in the presence of glibenclamide (10 μmol/L).

Conclusions

Addition of the adenosine triphosphate-sensitive potassium-channel opener, pinacidil, to a high potassium cardioplegic solution improves recovery of contractile properties and cytosolic calcium in isolated rat cardiac myocytes.     

The role of state-of-the-art echocardiography in the assessment of myocardial injury during and following cardiac surgery.

Use of intraoperative echocardiography during open heart surgery, transesophageal probes, high-frequency transducers, and color Doppler imaging provide important diagnostic information to surgeons and anesthesiologists. Early detection of myocardial ischemia, assessment of valvular disorders, and the ability to monitor for intracardiac air are among the most important roles of intraoperative transesophageal echocardiography. Large prospective studies are necessary to evaluate whether these changes affect the outcome of patients undergoing coronary artery bypass grafting in terms of morbidity, mortality, hospital length of stay, and functional recovery. The application of new techniques such as contrast-enhanced transesophageal echocardiography helps to assess the adequacy of cardioplegia distribution and, thus, myocardial protection during cardiopulmonary bypass, which has a significant influence on outcomes as well.     

Modifying myocardial management in cardiac surgery: a randomized trial.

Among the 160 patients randomly assigned to one of eight protocols of myocardial management, all of which included controlled aortic root reperfusion, no important differences were found between protocols as to the prevalence of death (0 instances), use of an intra-aortic balloon pump (no instances), use of catecholamines, elaboration of CK-MB isoenzymes, new Q-waves, abnormal wall motion scores, or postoperative atrial fibrillation. Ventricular defibrillation was required more often in patients in the protocol with noncardioplegic blood reperfusate. Cardiac index was highest in the operating room in the group receiving hyperkalemic cold cardioplegia and initial hyperkalemic reperfusion. The reperfusion flow at the controlled pressures had initially a low flow rate (pressure 30 mmHg for 2 min; thereafter 50 mmHg), which increased to reach a peak flow rate at about 3 min after the start of reperfusion, followed by a declining flow rate reflecting changes in coronary resistance. Comparison of the overall randomly assigned group with a historical control group of 100 patients operated upon prior to the randomized trial showed no differences except for a higher rate of postoperative atrial fibrillation (25%) in the historical control group than in the randomized protocols with initial cardioplegic reperfusion (14%). The controlled reperfusion technique was found to be easy to use and is now used routinely.     

Renal sodium handling in experimental diabetes: role of NO

Recent studies have suggested that diabetes is a state of increasedrenal nitric oxide (NO) activity as assessed by urinary excretionof nitrites and nitrates (NO_x), and that NO synthase inhibitorsreverse the increased glomerular filtration rate (GFR) observedin experimental diabetes. In addition to being a potent vasodilatorin the renal vasculature, NO also plays a role in modulationof renal sodium excretion. To explore the role of NO in diabetes-associatedalterations in renal excretory function, renal haemodynamicand sodium handling parameters were evaluated in conscious control(C) and streptozotocin diabetic rats (D) and correlated to therenal activity of NO, as assessed by urinary excretion of itsmetabolites NO_x. To further explore this issue, the changesin renal haemodynamics and sodium handling were also assessedafter NO synthase inhibition with a non-pressor dose of L-nitro-arginine-methyl-ester(L-NAME) and after administration of the NO donor, glyceryltrinitrate (GTN). Systolic blood pressure was not differentbetween C and D rats. D rats exhibited marked hyperglycaemia(P<0.001), and increases in GFR (P<0.001), renal plasmaflow, filtration fraction, urinary sodium excretion (U_NaV, P<0.001),filtered load of sodium (FL_Na, P<0.01), and a decrease infractional reabsorption of sodium (FR_Na, P<0.0001). In contrast,total reabsorption of sodium (TR_Na) was increased in D ratscompared to C rats (P<0.0001). The urinary excretion of NOwas markedly increased in D rats (P<0.01). Regression analysesperformed in D rats revealed a close relationship between U_NaVand GFR and a weaker correlation with urinary NO_x. AlthoughFR_Na correlated only with urinary excretion of NO_x, there wasa strong relationship between TR_Na and GFR. In contrast to Drats, control rats demonstrated only a relationship betweenTR_Na and GFR and no other correlations were found, in D rats,NO inhibition with L-NAME (1 mg/kg body weight) resulted ina marked decrease in GFR and urinary NO_x associated with decreasesin FL_Na and TR_Na but did not influence FR_Na. In contrast, inC rats the post-L-NAME decrease in NO_x was not associated withsignificant changes in GFR and renal sodium handling. GTN-treatedC rats exhibited a renal vasodilatory response and an increasein natriuresis and urinary NO_x whereas no renal changes wereobserved in D rats during GTN administration. The present dataindicate that changes in renal sodium handling before and afterNO modulation in experimental diabetes are related to changesin GFR rather than to the renal activity of NO. Therefore, incontrast to the effects on renal haemodynamics, NO does notplay an important role in the altered renal sodium handlingobserved in experimental diabetes.     

Towards an improved definition of periprocedural myocardial infarction: The role of high-sensitivity cardiac troponins

In the past few years, many have disputed the optimal biomarker for confirming or ruling out a diagnosis of periprocedural myocardial infarction (PMI) and the optimal cut-off concentrations to apply. In this issue of the Journal of Cardiac Surgery, Niclauss et al. performed a retrospective analysis of CK-MB and high-sensitivity cardiac troponin T (hs-cTnT) dynamics and peak concentrations following different cardiac surgical interventions in 400 patients during a 2-year period in a single center. The authors found that CK-MB and hs-cTnT predict PMI with a comparable diagnostic accuracy and discriminatory power >95%. They also attempted to propose an improved, more sensitive threshold of hs-cTnT for PMI. Their findings could have implications for clinical practice, but more research is warranted to identify more appropriate cut-offs. This could include hs-cTnT release pattern, slope steepness, and changes. Ultimately, this could results in patient-specific model, able to predict expected and abnormal ranges of hs-cTnT release, enabling an improved and timely diagnosis of PMI.     

Renal handling of calcium in the early newborn period

Urinary calcium excretion was studied in two matched groups of 28 clinically-well preterm and fullterm infants between 24 and 48 hours of life. In the developmental period from 28 to 40 weeks gestation, urinary calcium excretion was positively correlated to the gestational age (r = 0.583, P less than 0.01), serum calcium levels (r = 0.512, P less than 0.01), the rate of glomerular filtration (r = 0.715, P less than 0.001), and urinary cyclic AMP excretion (r = 0.717, P less than 0.001). Urinary calcium excretion was independent of sodium and calcium intake, and urinary sodium and phosphate excretion. The mean fractional total calcium excretion and fractional ionized calcium excretion in babies less than or equal to 32 weeks was less than 1.0%, compared to greater than 2.5% for sodium. Serum calcium levels were positively correlated with gestational age (r = 0.803, P less than 0.001), and serum phosphate levels (r = 0.85, P less than 0.001). Whole blood ionized calcium levels were positively correlated to gestational age (r = 0.625, P less than 0.001), and serum total calcium levels (r = 0.440, P less than 0.05). The newborn kidney in babies less than or equal to 32 weeks gestation, did not have impaired conservation of calcium as for sodium; and neither sodium nor calcium intake appeared to affect urinary calcium excretion in the well newborn infant. Probably neither excess excretion of calcium nor serum phosphate levels contribute to early neonatal hypocalcemia.     

Increased myocardial calcium cycling and reduced myofilament calcium sensitivity in early endotoxemia.

BACKGROUND: Mechanisms of cardiac dysfunction during endotoxemia are multiple and their targets uncertain. This study tested the hypothesis that endotoxin (LPS) induces abnormal calcium-activated contractile force in the heart. METHODS: Adult rabbits were given LPS intravenously; 2 hours later hearts were studied in the Langendorff mode. Measurements included peak developed pressure (PDP), myocardial oxygen consumption (MVO2), high-energy phosphates by 31P-NMR, and beat-to-beat intracellular calcium (Cai) by fluorescence spectroscopy. Myofibrillar calcium sensitivity was assessed from the relationship of PDP to Cai and the rate of diastolic Cai removal (tau Ca) was quantified. RESULTS: Force-calcium relationships were markedly depressed in LPS hearts despite increased Cai. MVO2 was increased in parallel with increased Cai. Taken together, these data denote myofilament calcium insensitivity and mechanical inefficiency. tau Ca was markedly prolonged in LPS hearts, indicating impaired calcium reuptake and/or extrusion. High-energy phosphates and intracellular pH were unaffected by LPS; however, inorganic phosphate (Pi) was significantly increased. Dobutamine further increased Cai and MVO2 in LPS hearts without significantly improving calcium-activated force. Pyruvate, an inotrope that reduces Pi, significantly improved contractility in LPS hearts. CONCLUSIONS: Endotoxemia rapidly induced futile calcium cycling and reduced myofibrillar calcium sensitivity. This state was resistant to beta-agonist inotropic stimulation; inotropes that normalize the calcium-force relationship may be more effective.     

The role of a defined protocol for cardiac risk assessment in decreasing perioperative myocardial infarction in vascular surgery.

Major elective peripheral vascular surgery has historically carried a significant risk of perioperative myocardial infarction; this risk has been quantified further by its association with proved reduction in cardiac reserve/presence of coronary artery disease by stress testing or invasive monitoring. Recognition of this risk logically should lead to protocols that delineate coronary artery disease/cardiac reserve before surgery and correct for observed abnormalities during surgery. This study sought to show that a coherent algorithm of preoperative cardiac assessment combined with aggressive perioperative management could indeed reduce perioperative myocardial infarction rates. Six hundred thirty consecutive elective vascular operations were performed by the author during 6 years. All patients were entered into a prospective protocol for preoperative cardiac risk assessment, which then determined the choice of operation, type of anesthesia, and level of hemodynamic monitoring. Sixty-eight percent of the patients demonstrated clinical coronary artery disease, 15% had previously undergone coronary catheterization or surgery, and 9% had ejection fractions less than 35%. All patients underwent baseline detailed cardiac histories, radionuclide cardioangiography, and electrocardiograms. Patients with significant historic coronary artery disease or ejection fraction less than 50% underwent stress thallium testing; patients with positive fixed or redistribution defects then underwent catheterization, constituting 7% of the series. Risk stratification by age and cardiac assessment then dictated the perioperative care. The overall perioperative myocardial infarction rate was 0.7% (5/628), ranging from 0% for 156 aortic operations and 114 carotid endarterectomies to 0.6% for 159 femoropopliteal and 3.3% for 90 femorotibial revascularizations.(ABSTRACT TRUNCATED AT 250 WORDS)