Mitochondrial DNA damage and repair during ischemia-reperfusion injury of the heart.

Ischemia-reperfusion (IR) injury of the heart generates reactive oxygen species that oxidize macromolecules including mitochondrial DNA (mtDNA). The 8-oxoguanine DNA glycosylase (OGG1) works synergistically with MutY DNA glycosylase (MYH) to maintain mtDNA integrity. Our objective was to study the functional outcome of lacking the repair enzymes OGG1 and MYH after myocardial IR and we hypothesized that OGG1 and MYH are important enzymes to preserve mtDNA and heart function after IR. Ex vivo global ischemia for 30min followed by 10min of reperfusion induced mtDNA damage that was removed within 60min of reperfusion in wild-type mice. After 60min of reperfusion the ogg1(-/-) mice demonstrated increased mtDNA copy number and decreased mtDNA damage removal suggesting that OGG1 is responsible for removal of IR-induced mtDNA damage and copy number regulation. mtDNA damage was not detected in the ogg1(-/-)/myh(-/-), inferring that adenine opposite 8-oxoguanine is an abundant mtDNA lesion upon IR. The level and integrity of mtDNA were restored in all genotypes after 35min of regional ischemia and six week reperfusion with no change in cardiac function. No consistent upregulation of other mitochondrial base excision repair enzymes in any of our knockout models was found. Thus repair of mtDNA oxidative base lesions may not be important for maintenance of cardiac function during IR injury in vivo. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease."

Adenosine increases LPS-induced nuclear factor kappa B activation in smooth muscle cells via an intracellular mechanism and modulates it via actions on adenosine receptors.

AIM: In inflamed and damaged cardiovascular tissues, local extracellular adenosine concentrations increase coincidentally with activation of the transcription factor nuclear factor kappa B (NFκB). To investigate whether adenosine influences NFκB activation in vascular smooth muscle cells (VSMCs) and, if so, to examine the role of its receptors. METHODS: VSMCs were isolated from NFκB-luciferase reporter mice, cultured and then treated by lipopolysaccharide (LPS) to activate NFκB signalling. Adenosine, adenosine receptor agonists and antagonists, adenosine deaminase and uptake inhibitors were used together with LPS to evaluate the role of adenosine and its receptors on NFκB activation, which was assessed by luciferase activity and NFκB target gene expression. RESULTS: Adenosine potentiated LPS-induced NFκB activation. This was dependent on adenosine uptake and enhanced by an adenosine deaminase inhibitor, suggesting that intracellular adenosine plays an important role. Non-selective adenosine receptor agonists (2Cl-Ado and NECA) inhibited NFκB activation induced by LPS. Selective A1 or A2A antagonist given alone could not completely antagonize the NECA effect, indicating that the inhibitory effect was due to multiple adenosine receptors. The activation of the A3 receptor further increased LPS-induced NFκB activation. CONCLUSIONS: Adenosine increases LPS-induced nuclear factor kappa B activation in smooth muscle cells via an intracellular mechanism and decreases it via actions on A1 and A2A receptors. These results provide novel insights into the role of adenosine as a regulator of inflammation-induced NFκB activation.

Protecting the heart through delivering DNA encoding for heme oxygenase-1 into skeletal muscle.

AIM: To evaluate if remote gene delivery of HMOX-1 prior to myocardial infarction can prevent cardiac remodeling and preserve function, without causing general angiogenesis. MAIN METHODS: Right quadriceps muscles of mice were treated with DNA encoding for HMOX-1 or empty vector (pcDNA) and electroporated to enhance nuclear uptake, while a third group received saline. Transfection efficacy was evaluated by real time PCR and situ hybridization in transfected muscle, contralateral muscle, and heart. Seven days after transfection baseline echocardiography was performed. Myocardial infarction was induced by ligation of the left coronary artery. Six weeks later heart function was reassessed by echocardiography. Hearts were extracted for evaluation of infarct size. Immunoflorescent staining was used to evaluate angiogenesis using the endothelial marker CD31 in cross-sections of the transfected quadriceps muscle, the untreated muscle, and hearts. KEY FINDINGS: Gene delivery of HMOX-1 leads to a local expression of HMOX-1 in the treated muscle, but not in any other organ. HMOX-1 treated mice had reduced infarct size (p=0.03) and improved function evident as higher ejection fraction (p=0.001), improved fractional shortening (p<0.0001) and higher stroke volume (p=0.002). HMOX-1 did not cause angiogenesis in the heart or skeletal muscle. SIGNIFICANCE: Remote delivery of DNA encoding for HMOX-1 was cardioprotective, as evidenced by preserved cardiac structure and function. Angiogenesis was not induced by HMOX-1 treatment.

Sex differences in mouse heart rate and body temperature and in their regulation by adenosine A1 receptors.

AIM: To examine cardiac function, body temperature and locomotor behaviour in the awake adenosine A(1) receptor knock out mouse of both sexes. METHODS: Male and female A(1)R (+/+) and (-/-) mice, instrumented with telemetric devices, were recorded during basal conditions and after drug administration. RESULTS: Female mice had higher heart rate, body temperature and locomotion, both during daytime and during the night. Awake A(1)R (-/-) mice had a slightly elevated heart rate, and this was more clear-cut in males. Heart rate was also higher in Langendorff-perfused denervated A(1)R (-/-) hearts. Body temperature was higher in A(1)R (-/-) males and females; locomotor activity was higher in A(1)R (-/-) females, but not in males. The adenosine receptor agonist R-PIA (0.2 mg kg(-1)) decreased heart rate and body temperature, but less in A(1)R (-/-) animals than in A(1)R (+/+) mice (P < 0.001 in both parameters). The unselective adenosine receptor antagonist caffeine had a minor stimulatory effect on heart rate in lower doses, but depressed it at a dose of 75 mg kg(-1). Body temperature was increased after a low dose (7.5 mg kg(-1)) of caffeine in both sexes and genotypes, and markedly reduced after a high dose (75 mg kg(-1)) of caffeine. An intermediary dose of caffeine 30 mg kg(-1) increased or decreased body temperature depending on genotype and sex. Locomotor responses to caffeine were variable depending both on genotype and sex. CONCLUSION: Thus, the adenosine A(1) receptor is involved in the regulation of heart rate, body temperature and locomotor activity, but the magnitude of the involvement is different in males and females.

Oral pre-treatment with rosuvastatin protects porcine myocardium from ischaemia/reperfusion injury via a mechanism related to nitric oxide but not to serum cholesterol level.

AIMS: The aim of this study was to test whether oral pre-treatment with rosuvastatin at a dosage giving clinically relevant plasma concentrations protects the myocardium against ischaemia/reperfusion injury and to investigate the involvement of nitric oxide (NO) and neutrophil infiltration. METHODS: Pigs were given placebo (n = 7), rosuvastatin (80 mg day(-1), n =7), rosuvastatin (160 mg day(-1), n = 7) or pravastatin (160 mg day(-1), n = 7) orally for 5 days before being subjected to coronary artery ligation and reperfusion. An additional group was given rosuvastatin 160 mg day(-1) and a nitric oxide synthase (NOS) inhibitor. RESULTS: Rosuvastatin 80 and 160 mg day(-1) resulted in plasma concentrations of 2.6 +/- 0.7 and 5.6 +/- 1.0 ng mL(-1), respectively. Serum cholesterol was not affected. Rosuvastatin 160 mg day(-1) and pravastatin limited the infarct size from 82 +/- 3% of the area at risk in the placebo group to 61 +/- 3% (P < 0.05), and to 61 +/- 2% (P < 0.05) respectively. Rosuvastatin 80 mg day(-1) limited the infarct size to 69 +/- 2%, however, this effect was not statistically significant. Rosuvastatin 160 mg day(-1) attenuated neutrophil infiltration in the ischaemic/reperfused myocardium. The protective effect of rosuvastatin 160 mg day(-1) was abolished by NOS inhibition. The expression of NOS2 and NOS3 in the myocardium did not differ between the groups. CONCLUSIONS: Oral pre-treatment with rosuvastatin limited infarct size following ischaemia/reperfusion without affecting cholesterol levels. The cardioprotective effect is suggested to be dependent on maintained bioactivity of NO, without influencing NOS expression.

Adenosine A receptors are necessary for protection of the murine heart by remote, delayed adaptation to ischaemia.

AIMS: Adenosine is involved in classic pre-conditioning (PC) in most species, acting through especially adenosine A1 and A3 receptors. We studied whether the adenosine A1 receptor (A1R) was important for remote, delayed adaptation to ischaemia using a mouse with targeted deletion of the A1R gene. METHODS: Remote, delayed adaptation was evoked by brain ischaemia (BIPC) through bilateral ligation of the internal carotid arteries. Through microdialysis probes placed in the brain and the abdominal aorta, we found that plasma adenosine increased following carotid artery ligation. Twenty-four hours after ligation, hearts were isolated, Langendorff perfused and subjected to 40 min global ischaemia and 60 min reperfusion. Hearts from sham operated and BIPC animals either with (A1R+/+) or without (A1R-/-) the gene for the adenosine A(1)R were compared with each other. RESULTS: In wild types, BIPC reduced infarct size and improved functional recovery during reperfusion, but BIPC did not protect hearts of A1R-/- mice. There were no significant differences between sham-operated A1R+/+ and A1R-/- in recovery of function or infarct size. The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated protein kinase1/2 (ERK1/2), p38 and c-jun N-terminal kinase (JNK) were phosphorylated during reperfusion of sham treated hearts. The increase in ERK1/2 and p38 phosphorylation detected was attenuated in hearts of BIPC or A1R-/- animals. CONCLUSION: During BIPC adenosine acting on the A1R appears necessary for myocardial protection. MAPK signalling may possibly be involved in organ protection during the delayed phase of remote, delayed adaptation.

Effects of sex, gonadectomy, and oestrogen substitution on ischaemic preconditioning and ischaemia-reperfusion injury in mice.

AIM: Ischaemic preconditioning (IPC) has been demonstrated to protect heart function and viability, but has been predominantly studied in male animals. METHODS: We studied a possible influence of sex and oestrogen for protection in IPC. Infarct size and heart function after 40 min global ischaemia and 60 min reperfusion with or without preceding classic IPC was investigated in Langendorff-perfused hearts. Hearts were harvested from 10-week-old male and female C57BL6 mice with or without gonadectomy 6 weeks earlier, or gonadectomy and substitution with 17 beta-oestradiol for 4 weeks (n = 104). RESULTS: Classic IPC reduced depression of left ventricular developed pressure (P < 0.01), attenuated the increase of end-diastolic pressure (P < 0.01), and reduced infarct size (P < 0.01) in hearts of untreated male mice, but failed to protect untreated females which had improved functional recovery and smaller infarctions than untreated males. After gonadectomy of female mice, developed pressure was reduced (P < 0.01) and infarct size increased (P < 0.01) compared with normal females, with no protection of preconditioning. The changes were not reversed by 17 beta-oestradiol substitution. In hearts of gonadectomized males, the post-ischaemic increase of end-diastolic pressure was attenuated (P < 0.01), and enhanced after substitution with 17 beta-oestradiol (P < 0.01). The preconditioning effect disappeared after gonadectomy and gonadectomy with substitution in male mice. CONCLUSION: There is a sex difference in evoking preconditioning in male and female mice which is only partially dependent on sex hormones.

Importance of preanalytical handling of samples for measurement of cardiac troponin T in coronary effluent from isolated rat hearts.

The isolated, buffer-perfused heart is probably the most widely used model in experimental heart research, and the coronary effluent is often analysed for markers of myocardial injury. Adsorption to surrounding materials may be a serious problem of protein measurements in solutions with low protein concentrations. The aims of the present study were to investigate the importance of the preanalytical phase when measuring cardiac troponin T (cTnT) in a buffer perfusate and to investigate whether addition of albumin to the effluent might increase recovery of cTnT and improve the assay. Coronary effluent was collected in tubes of different materials and in tubes with 40 g/L bovine albumin, and then frozen. cTnT was analysed at different time points after withdrawal from the freezer. cTnT was 2.3-119 times higher in effluent with albumin. In effluent without albumin, cTnT concentration declined to 2% of the initial concentration after two episodes of freezing and thawing. The cTnT loss could not be prevented by using polystyrene or siliconized glass, but was partially inhibited in effluent with albumin. Furthermore, creatine kinase and lactate dehydrogenase levels were higher in effluent with albumin. The within-series coefficient of variation for cTnT was markedly improved when using effluent with albumin.

Exposure of rats to hyperoxia enhances relaxation of isolated aortic rings and reduces infarct size of isolated hearts.

Exposure of rats to hyperoxia before organ harvesting protected their isolated hearts against global ischaemia-reperfusion injury in a previous study. The present study investigates whether hyperoxia influences vasomotor function and regional ischaemia of the heart. Isolated rings of the thoracic aorta were obtained from rats immediately or 24 h after in vivo exposure to 60 min of hyperoxia (>95% O2), and the in vitro dose-response to phenylephrine (PHE), prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1), acetylcholine (Ach) and sodium nitroprusside (SNP) was assessed. Hyperoxia in vivo increased the relaxation of aortic rings to Ach and SNP, while it delayed contraction to PHE. The effect was more evident when the vessels were harvested immediately rather than 24 h after hyperoxic exposure. In separate experiments rat hearts were isolated immediately after hyperoxia, buffer-perfused, and subjected to 30 min of regional ischaemia and reperfused for 120 min. Infarct size was determined by triphenyl tetrazolium chloride staining. Hyperoxia significantly reduced infarct size. In normoxic controls 23.0 +/- 8.3% of the area at risk was infarcted, while in hyperoxic animals infarct size was 14.8 +/- 5.6% of the area at risk (P = 0.012). Exposure of rats to hyperoxia modifies the vasomotor response of isolated aortic rings, and reduces the infarct size of isolated rat heart. These novel aspects of hyperoxic treatment require further studies to explore the potential of its clinical application.

Cyclic fluctuations in the cardiac performance of the isolated Langendorff-perfused mouse heart: pyruvate abolishes the fluctuations and has an anti-ischaemic effect.

During the development of a Langendorff preparation of isolated mouse hearts, hitherto undescribed cyclic fluctuations in left ventricular pressure and coronary flow were independently observed in three laboratories. Isolated mouse hearts were perfused with crystalloid glucose-containing Krebs-Hensleit buffer in a constant pressure model, and left ventricular pressures were measured via an intraventricular balloon catheter. After acquiring technical skill in preparing the mouse hearts, the perfusionists observed that fluctuations in cardiac performance with a cycle period lasting 5-10 min occurred shortly after initiation of perfusion. Each fluctuation cycle consisted of a phase of increase and a phase of decrease. Synchronized with the fluctuations in left ventricular pressure, increases and decreases in dP/dt max took place. Analogous fluctuations in coronary flow occurred, with onset 1-2 min later than changes in left ventricular systolic pressure. In some preparations a gradual ST-segment elevation was seen on the electrocardiogram during the systolic pressure increase phase. The amplitude of the fluctuations could be augmented by increasing the perfusion pressure, and reduced, but not abolished, by lowering the pressure. Changes in buffer calcium, magnesium, or sodium concentration did not alter the fluctuations, nor did any change of anaesthetics, mouse strain, or left ventricular drainage. Altering the perfusion mode from constant pressure to constant flow did not prevent the occurrence of the cyclic fluctuations. The hearts became stable and the fluctuations disappeared when the buffer was supplemented with 2 mm pyruvate. In the present study, pyruvate given throughout stabilization and reperfusion also markedly attenuated the ischaemic insult, as evidenced by the delayed ischaemic contracture and a reduced magnitude of ischaemic contracture. A cardioprotective effect was only visible at early reperfusion, did not affect the final functional recovery. In conclusion, a phenomenon of cyclic fluctuations in left ventricular pressure followed by fluctuations in coronary flow was observed in isolated mouse hearts. These could be abolished by adding 2 mm pyruvate to the perfusion buffer. Pyruvate in the buffer also markedly attenuated the post-ischaemic deterioration of cardiac performance seen in this mouse model.

Cardioprotection by breathing hyperoxic gas-relation to oxygen concentration and exposure time in rats and mice.

OBJECTIVES: Breathing a hyperoxic gas (> or =95% O(2)) protects against ischaemia-reperfusion injury in rat and mouse hearts. The present study investigated how oxygen concentration and duration of hyperoxic exposure influenced cardioprotection, and whether hyperoxia might induce delayed cardioprotection (after 24 h). METHODS: Animals were kept in normal air or in a hyperoxic environment, and their hearts were isolated and Langendorff-perfused immediately or 24 h thereafter. Global ischaemia was induced for 25 min in rats and 40 min in mice, followed by 60 min of reperfusion. Infarct size was determined by triphenyl tetrazolium chloride staining. RESULTS: In rats exposure to > or =95, 80, and 60%, but not to 40% of oxygen immediately before heart isolation and perfusion improved postischaemic functional recovery. Eighty or more percent of oxygen also reduced infarct size. A preconditioning-like effect could be evoked by 60 or 180 min of hyperoxia, giving both immediate and delayed protection. In the mouse heart protection could be induced by pretreatment for 15 or 30, but not by 60 min with > or =95% oxygen. The protective effect of hyperoxia in mice could be evoked in the immediate model only. CONCLUSIONS: Hyperoxia protects the isolated rat and mouse heart against ischaemia-reperfusion injury, but some species-different responses exist. The protection depends on both oxygen concentration in inspired air, and duration of hyperoxic exposure.

The role of nitric oxide in ischaemia/reperfusion injury of isolated hearts from severely atherosclerotic mice.

Nitric oxide (NO) may play an essential role for maintenance of cardiac function and perfusion, while endothelial dysfunction of atherosclerotic vessels may aggravate ischaemia/reperfusion injury. This paper investigates the role of nitric oxide in ischaemia/reperfusion injury in hearts with coronary atherosclerosis. Hearts of apolipoprotein E/LDL receptor double knockout (ApoE/LDLr KO) mice fed an atherogenic diet for 7-9 months were isolated and Langendorff-perfused with 40 minutes of global ischaemia and 60 minutes reperfusion, and funtion and infarction compared with hearts of C57BL/6 controls in the prescence or abscence of the NO-donor SNAP or the NOS inhibitor L-NAME. Hearts of animals with atherosclerosis were more susceptible to ischaemia/reperfusion injury than hearts of animals with healthy vessels, evident as more impaired left ventricular performance. SNAP protected function and reduced infarct size in atherosclerotic hearts, but the same concentration of SNAP was detrimental in normal hearts, perhaps due to NO-overproduction and peroxynitrite formation demonstrated immunohistochemically as increased formation of nitrosylated tyrosine. A low concentration of SNAP protected against ischaemia/reperfusion dysfunction in normal hearts. L-NAME decreased left ventricular performance in atherosclerotic hearts. These findings suggest that impaired endothelium dependent function contributes to reperfusion injury in coronary atherosclerosis.

Nuclear factor kappa-B and the heart.

Nuclear factor kappa-B (NFkappaB), a redox-sensitive transcription factor regulating a battery of inflammatory genes, has been indicated to play a role in the development of numerous pathological states. Activation of NFkappaB induces gene programs leading to transcription of factors that promote inflammation, such as leukocyte adhesion molecules, cytokines, and chemokines, although some few substances with possible anti-inflammatory effects are also NFkappaB regulated. The present article reviews basic regulation of NFkappaB and its activation, cell biological effects of NFkappaB activation and the role of NFkappaB in apoptosis. Evidence involving NFkappaB as a key factor in the pathophysiology of ischemia-reperfusion injury and heart failure is discussed. Although activation of NFkappaB induces pro-inflammatory genes, it has lately been indicated that the transcription factor is involved in the signaling of endogenous myocardial protection evoked by ischemic preconditioning. A possible role of NFkappaB in the development of atherosclerosis and unstable coronary syndromes is discussed. Nuclear factor kappa-B may be a new therapeutic target for myocardial protection.

Preconditioning protects the severely atherosclerotic mouse heart.

BACKGROUND: Coronary atherosclerosis has profound effects on vascular and myocardial biology, and it has been speculated that the atherosclerotic heart does not benefit from ischemic preconditioning. METHODS: To investigate if atherosclerosis would influence the preconditioning response, Apolipoprotein E/low density lipoprotein (LDL) receptor double knockout mice (ApoE/LDLr-/-) were fed an atherogenic diet (21% fat, 0.15% cholesterol) for 6 to 8 months. At that time, extensive atherosclerotic lesions throughout the coronary tree were seen in transverse sections stained with Oil Red-O. Hearts of ApoE/LDLr-/- mice were Langendorff-perfused with 40 minutes of global ischemia and 60 minutes reperfusion, and compared with C57BL/6 controls. Preconditioning with two episodes of 2 minutes of ischemia and 5 minutes reperfusion, or exposing the mice to a hyperoxic environment (O2 > 98%) for 60 minutes before heart perfusion, was performed. RESULTS: Hearts of mice with coronary atherosclerosis had worse postischemic function, and increased infarct size and troponin T release compared to hearts of C57BL/6 mice. Ischemic preconditioning improved postischemic ventricular function, and reduced myocardial infarct size and troponin T release in both normal and ApoE/LDLr-/- mice. The effects were most pronounced in ApoE/LDLr-/- hearts. Exposure to hyperoxia exerted a similar protection of function and cell viability of ApoE/LDLr-/- mice hearts. CONCLUSIONS: These findings suggest that the severely atherosclerotic heart may be protected by preconditioning induced by ischemia or hyperoxia.

Pretreating rats with hyperoxia attenuates ischemia-reperfusion injury of the heart.

Oxidative stress may precondition the heart. The present study investigated whether hyperoxia elicits a preconditioning-like response. Rats were kept in a hyperoxic (>95% O2) environment for 60 or 180 minutes. Hearts were Langendorff-perfused immediately or 24 hours after hyperoxia, and exposed to 25 minutes of global ischemia and 60 minutes of reperfusion. Whole blood was sampled after 60 and 180 minutes of hyperoxia for oxidative stress markers. Hearts were sampled immediately or 24 hours after hyperoxia for measurement of antioxidants, lipid peroxidation products, heat shock protein 72 and endothelial nitric oxide synthase. At the end of reperfusion after 1 h hyperoxia, infarct size was determined by tetrazolium staining. Hyperoxia increased serum levels of conjugated dienes, reduced serum antioxidative protection, reduced reperfusion arrhythmias in most groups, and improved myocardial function. Infarct size was reduced from 45% of myocardial tissue in controls to 22% in treated animals. The myocardial activity of antioxidant enzymes, content of heat shock protein 72, and endothelial nitric oxide synthase in myocardial tissue were not influenced. In conclusion, hyperoxia induces a low-graded systemic oxidative stress, improves postischemic cardiac function and reduces infarct size. The mediators of protection remain to be determined.

Induction of inflammatory mediators during reperfusion of the human heart.

BACKGROUND: Cardioplegia and reperfusion may induce an inflammatory reaction, which may contribute to postoperative morbidity and mortality. METHODS: Gene expression of cytokines, adhesion molecules, and vasoactive substances was evaluated in left ventricular biopsies taken before cardioplegia (lasting approximately 70 minutes) and after reperfusion (approximately 40 minutes) from 19 patients (5 with valvular or combined disease, 7 with stable angina pectoris, 7 with unstable angina). mRNA was extracted and amplified with a semiquantitative reverse transcription polymerase chain reaction. RESULTS: Cardioplegia-reperfusion increased mRNA for E-selectin by a factor of 17 +/- 5 (p < 0.002) (mean +/- SEM), interleukin-1beta, with 9 +/- 3 (p < 0.007), tumor necrosis factor-alpha with 6 +/- 3 (p < 0.05), interleukin-2 receptor alpha chain CD25 with 2 +/- 0.6 (p < 0.04), and intercellular adhesion molecule-1 with 2 +/- 0.4 (p < 0.005). Before cardioplegia, mRNA for endothelial nitric oxide synthase was predominantly detected in unstable angina patients, and increased by a factor of 11 +/- 6 (p < 0.02) during reperfusion. mRNA for endothelin-1 decreased by a factor of 0.5 +/- 0.1 (p < 0.0005). The changes were more pronounced in unstable patients. The transcription factor nuclear factor kappa B (NFkappaB), which regulates expression of inflammatory mediators, was activated during reperfusion (n = 10, p < 0.0001). CONCLUSIONS: Open heart surgery induces an inflammatory response in the human heart, which is more pronounced in patients with unstable angina. It involves NFkappaB activation and expression of several NFkappaB-regulated genes.

The effect of a low molecular weight inhibitor of lipid peroxidation on ultrastructural alterations to ischemia-reperfusion in the isolated rat heart.

The effects of H290/51, a novel indenoindole derivative inhibitor of lipid peroxidation, on ultrastructural changes during cardiac ischemia-reperfusion injury were investigated. Langendorff-perfused rat hearts were exposed to 30 minutes of global ischemia followed by 20 minutes of reperfusion: Group A: Control hearts with standard buffer perfusion with vehicle added. Group B: H290/51 (10(-6) mol/l) added to buffer throughout stabilisation and reperfusion. In an additional Group C, where hearts were given H290/51, but not subjected to ischemia, the ultrastructure was preserved till the end of reperfusion. Absolute volumes and calculated volume fractions (Vv) of tissue and subcellular components were assessed with quantitative stereologic morphometry. After ischemia the increase in volume of extracellular interstitium was inhibited by H290/51 (247 +/- 80 vs. 159 +/- 50 microl, mean +/- SD, groups A and B, respectively, p<0.05). The Vv (interstitium/myocard) was higher in control hearts (0.318 +/- 0.062 vs. 0.206 +/- 0.067, p<0.05). Vv (cell edema/myocyte) was higher in the control group (0.144 +/- 0.07 vs. 0.083 +/- 0.033, p<0.05). Vv (myocyte/myocard) was higher in group B after ischemia than in the control group (0.622 +/- 0.071 vs. 0.707 +/- 0.052, p<0.05). The decreased Vv (capillary/myocard) after ischemia was inhibited by H290/51. After reperfusion there was no difference between groups. Treatment with H290/51 reduced edema and ensured better preserved sarcolemmal membrane structure during ischemia. The effect was no longer present after reperfusion.

Sonographic features of dialysis-related amyloidosis of the shoulder.

This study evaluated the diagnostic role of ultrasonography in dialysis-related amyloidosis in shoulders of chronically hemodialyzed patients. Fourteen shoulders of 12 long-term hemodialysis patients were examined. All patients had been on dialysis for at least 10 years. All patients had varying degrees of pain and limitations of movement in the studied shoulders. Dialysis-related amyloidosis was the presumed diagnosis in all patients. Any patient with a history of any disease, other than dialysis-related amyloidosis, capable of producing a pathologic shoulder condition was excluded. The following parameters were studied: supraspinatus and biceps tendon thickness, tendon tears, synovial thickening, and the presence of hypoechoic material around tendons and within bursae. All shoulders had a nonhomogeneous thickening, greater than 7 mm, of the supraspinatus tendon. Seven shoulders (50%) had abnormal thickening of the biceps tendon (4 mm or greater), and two shoulders had abnormal thickening of the subscapularis tendon. Hypoechoic deposits were seen in the subdeltoid bursae and biceps sheaths in five and six shoulders, respectively. Three shoulders showed partial tears of the supraspinatus tendon, one shoulder showed a tear in the biceps tendon, and one shoulder had a tear in the subscapularis tendon. Ultrasonography is an excellent imaging modality in diagnosing the presence of dialysis-related amyloidosis in symptomatic shoulders of long-term hemodialysis patients, without having to resort to invasive procedures. The results of previous studies have been confirmed and new ultrasonographic findings described. Of particular interest is the involvement of the subscapularis tendon in dialysis-related amyloidosis. Repeat ultrasonography can become an important way to follow-up progression of shoulder dialysis-related amyloidosis in hemodialyzed patients.

Endothelial dysfunction in atherosclerotic mice: improved relaxation by combined supplementation with L-arginine-tetrahydrobiopterin and enhanced vasoconstriction by endothelin.

1. Mice lacking the apolipoprotein E and low density lipoprotein receptor genes (E degrees xLDLR degrees ) develop atherosclerosis and endothelial dysfunction. The aim of this study was to characterize the roles of L-arginine and tetrahydrobiopterin (BH(4)) for endothelium-dependent relaxation and the changes in the vasoconstrictor response to endothelin-1 (ET-1) in thoracic aortic rings of E degrees xLDLR degrees mice. 2. Histological examination revealed severe atherosclerosis of the thoracic aorta of E degrees xLDLR degrees mice. Relaxations induced by acetylcholine (Ach), but not that to sodium nitroprusside, were significantly impaired in E degrees xLDLR degrees mice compared to control mice indicating attenuated endothelium-dependent relaxations. 3. Preincubation with the nitric oxide (NO) substrate L-arginine did not affect, whereas the co-factor for NO synthase, BH(4), slightly improved the relaxations induced by Ach. Combined preincubation with L-arginine and BH(4) induced a pronounced enhancement of Ach-induced relaxations in E degrees xLDLR degrees mice. The relaxations induced by Ach in E degrees xLDLR degrees mice in the presence of L-arginine and BH(4) were not different from those observed in control mice. 4. Preincubation with superoxide dismutase did not affect Ach-induced relaxations in aorta from E degrees xLDLR degrees mice. 5. The contractile response to ET-1 was enhanced in E degrees xLDLR degrees mouse aorta. The contractions were abolished by the ET(A) receptor antagonist LU 135252. The ET(B) receptor agonist sarafotoxin 6c did not induce contractions or relaxations. 6. It is concluded that endothelial dysfunction of E degrees xLDLR degrees mouse aorta is reversed by combined administration of L-arginine and BH(4). In addition, the ET(A) receptor-mediated vasoconstriction by ET-1 is enhanced in E degrees xLDLR degrees mice.