Christian Löwbeer

Spontaneous Ischemic Events in the Brain and Heart Adapt the Hearts of Severely Atherosclerotic Mice to Ischemia

To investigate if spontaneous ischemic events in mice with severe multi-organ atherosclerosis could adapt to ischemia, apolipoprotein E/LDL receptor knockout mice were fed an atherogenic diet for 7 to 9 months. Signs of spontaneous ischemia occurred. One to two days later, hearts were excised, Langendorff-perfused with induced global ischemia, and compared with mice without signs of disease. In vivo heart or brain infarctions were verified by heart histology and/or increased serum levels of cardiac troponin T and S100B. Hearts of mice with spontaneous ischemic events had improved function and reduced Langendorff-induced infarctions. To investigate the remote preconditioning effect of brain ischemia, bilateral ligation of the internal carotid arteries was performed in C57BL6 mice. Twenty-four hours later, their isolated hearts were protected against induced global ischemia. A possible role of inducible NO synthase (iNOS) was studied in iNOS knock out mice, who were not preconditioned by induced brain ischemia. Cardiac iNOS was unchanged 24 hours after preconditioning, suggesting that NO is a trigger rather than a mediator of protection. These findings suggest that spontaneous ischemic events in the brain and heart adapt the heart to ischemia. This can be mimicked by induced brain ischemia, with iNOS as a key factor of protection.

Elevated cardiac troponin T in predialysis patients is associated with inflammation and predicts mortality

Abstract.  Löwbeer C, Stenvinkel P, Pecoits‐Filho R, Heimbürger O, Lindholm B, Gustafsson SA, Seeberger A (Karolinska Institutet at Huddinge University Hospital; Capio Diagnostik, St Görans Hospital, Stockholm, Sweden). Elevated cardiac troponin T in predialysis patients is associated with inflammation and predicts mortality. J Intern Med 2003; 253: 153–160.

Serum cardiac troponin T in patients hospitalized with heart failure is associated with left ventricular hypertrophy and systolic dysfunction

Background: Cardiac troponin T (cTnT) is a highly sensitive and specific marker of acute myocardial infarction. Serum cTnT is also slightly elevated in patients with severe heart failure and is associated with left ventricular hypertrophy (LVH) in patients treated with haemodialysis. In this study serum cTnT concentrations and echocardiographic findings were investigated in heart failure patients without acute coronary syndrome. cTnT was also compared with other cardiac markers and plasma levels of brain natriuretic peptide (BNP). Methods: Twenty-six patients hospitalized with heart failure were included in the study. Echocardiographic measurements and blood sampling were carried out 12-36 h after admission. Serum cTnT (3rd generation assay), cardiac troponin I (cTnI), creatine kinase MB (CKMB) and CK were measured. Plasma BNP was analysed using the Shionoria assay. LVH was defined as left ventricular mass index (LVMI)>125 g/m<formula>2</formula> for males and>110 g/m<formula>2</formula> for females. Left ventricular systolic function was estimated from the mitral annulus motion (AV-mean LV). Results: Median cTnT was 0.012 (<0.010-0.032) μg/L. Sixty-two percent of the patients (16 of 26) had elevated serum cTnT≥0.010 μg/L. cTnT was positively correlated with CKMB (ρ=0.40, p=0.04) and BNP (ρ=0.43, p=0.03), but not with cTnI and CK. A negative correlation was found between cTnT and AV-mean LV (ρ=−0.58, p=0.007), and there was a positive correlation between cTnT and LVMI (ρ=0.44, p=0.03). No other analyte was correlated to LVMI. Conclusions: Serum cTnT but not cTnI was associated with left ventricular dysfunction and LVH in patients hospitalized with heart failure. This explains why cTnT tends to be slightly elevated in patients with heart failure without symptoms of acute myocardial ischaemia.

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.

Pretreatment with methylprednisolone protects the isolated rat heart against ischaemic and oxidative damage

Methylprednisolone (MP), a synthetic glucocorticoid, is widely used clinically and experimentally as acute antiinflammatory treatment. The molecular actions of MP indicate that pretreatment with this drug may be cardioprotective. We investigated if giving rats MP prior to excising their hearts for Langendorff-perfusion protected cardiac function against oxidative stress, and if this was mediated by increasing antioxidant defence or influencing myocardial nitric oxide synthase (NOS). Rats (n=6–11 in each group) were injected with MP (40 mg/kg i.m.) or vehicle 24 and 12 h before Langendorff-perfusion with 30 min global ischaemia and 60 min reperfusion, or 10 min perfusion with 180 μmol/L hydrogen peroxide. Other hearts were exposed to 30 min global ischaemia 5 days after MP-injection. Additional hearts were sampled before, during, and after ischaemia for analyzing tissue activity of antioxidant enzymes. Tissue endothelial and inducible NOS (eNOS and iNOS) were investigated by immunoblotting and semiquantitative RT-PCR in a time-course after MP injection. Pretreatment with MP improved left ventricular function and increased coronary flow during postischaemic reperfusion, and this effect was sustained 5 days afterwards. When exposing hearts to hydrogen peroxide, MP improved coronary flow. Catalase, glutathione peroxidase, and oxidized glutathione were increased during reperfusion of MP-treated hearts compared to vehicle only. MP did not influence eNOS at protein or mRNA level. iNOS could not be detected by immunoblotting, indicating low cardiac enzyme content. Its mRNA initially increased the first hour after injection, thereafter decreased. In conclusions, pretreating rats with MP protects the heart against ischaemia-reperfusion dysfunction. This effect could be due to increase of tissue antioxidant activity during reperfusion. MP did not influence cardiac eNOS. mRNA for iNOS was influenced by MP, but the corresponding protein could not be detected.

PRECONDITIONING WITH HYDROGEN PEROXIDE (H2O2) OR ISCHEMIA IN H2O2-INDUCED CARDIAC DYSFUNCTION

The possible cardioprotective effects of preconditioning by ischaemia (IPC) or a low dose of H2O2 (HPC) prior to a high dose of H2O2 was investigated. Langendorff-perfused rat hearts (n = 10 in each group) were subjected to 10 min of 140 micromol/L H2O2 and 30 min recovery after either (1) control perfusion, (2) 20 micromol/L H2O2 for 10 min, recovery 10 min, or (3) 2 x 2 min global ischaemia and 5 min reperfusion. 140 micromol/L H2O2 increased left ventricular end-diastolic pressure from 0 to 68+/-8 mmHg in controls (mean+/-SEM), which was attenuated by IPC (46+/-9 mmHg, p<0.001) and HPC (18+/-4 mmHg, p < 0.001 compared to controls, p < 0.01 compared to IPC). HPC, but not IPC, improved coronary flow (p < 0.02) and left ventricular developed pressure (p < 0.001) during recovery. Troponin T release was similar in all groups. Tissue thiobarbituric acid reactive substances, antioxidant capacity, catalase, and glutathione peroxidase were not influenced by 140 micromol/L H2O2. H2O2 decreased the level of tissue glutathione. This reduction was augmented by HPC (p <0.02) and attenuated by IPC (p < 0.02). H2O2 increased superoxide dismutase (p < 0.04). The increase was attenuated by IPC (p < 0.05), but not influenced by HPC. HPC efficiently protected cardiac function in H2O2-induced cardiac injury, while IPC had only a small protective effect. The functional protection cannot be explained by reduction of irreversible injury, attenuation of lipid peroxidation, or modification of tissue antioxidant parameters.

Effects of ethanol on platelet function.

To obtain evidence for an ethanol mediated disruption of hemostasis we compared the effects of acute and chronic ethanol ingestion on platelet reactivity. Since ADP may be important for hemostasis in vivo it was used to induce platelet aggregation in platelet-rich plasma. Thromboxane B2 (TXB2) formed during the aggregation was measured by radioimmunoassay. Ethanol (1.5 g/kg) given to 8 healthy non-alcoholic male volunteers increased platelet reactivity to ADP and the associated TXB2 formation rose from 289 +/- 60 (mean +/- SE) to 984 +/- 263 fmol/10(7) platelets (p less than 0.025). The effects lasted for as long as ethanol was present in blood. In 13 non-cirrhotic male alcoholics the withdrawal of ethanol caused a 4-fold increase in TXB2 formation within one week but the basal levels before ethanol withdrawal were the same as in controls. These findings are discussed in relation to the higher risk of brain infarction seen in alcoholics and even associated with binge drinking. Further studies are needed to establish the effects of ethanol on prostacyclin formation.

Does Hypothermia or Hyperkalemia Influence the Preconditioning Response

Ischemic preconditioning (IPC) is a potent mode of myocardial protection, but not in all models of cold cardioplegia. The present study investigates possible effects of hypothermia and hyperkalemia on the preconditioning response. Langendorff-perfused rat hearts were preconditioned (2 min global ischemia and 5 min reperfusion) or control-perfused prior to 35 min normothermic, global ischemia (series 1, n = 17 in each group); 50 min normothermic cardioplegia (St. Thomass II) (series 2, n = 10 in each); 75 min 23 degrees C, global ischemia (series 3, n = 7 in each); or 5 h 6-8 degrees C, global ischemia (series 4, n = 9 in each). Left ventricular developed (LVDP) and end-diastolic (LVEDP) pressures, coronary flow (CF), heart rate, incidence of severe reperfusion arrhythmias, and release of troponin T (TnT) were measured. IPC attenuated reduction of LVDP and CF, and increase of LVEDP during reperfusion in series 1-3. TnT release was reduced by IPC in series 3 only. IPC did not attenuate dysfunction after hypothermic ischemia (series 4). Neither hyperkalemia nor moderate hypothermia alone inhibited the preconditioning response, but IPC was not protective in deep hypothermia.

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.

Mechanical conversion of post-ischaemic ventricular fibrillation : effects on function and myocyte injury in isolated rat hearts

Ventricular fibrillation (VF) and ventricular tachycardia (VT) are common phenomena during reperfusion. In experimental research many hearts have to be excluded from haemodynamic evaluation because of severe arrhythmias. Theoretically, electroconversion or mechanical conversion (MC) might be used to convert VF or VT. MC induces a physical shock analogous to a chest thump. The aim of this study was to investigate the efficacy of MC in isolated, perfused rat hearts, and to see whether MC itself induced myocardial cell injury and functional impairment. Langendorff-perfused rat hearts (n = 89) from several experimental series subjected to 30 min of global ischaemia and 60 min of reperfusion were retrospectively analysed. Left ventricular systolic (LVSP), end-diastolic (LVEDP), and developed (LVDP) pressures, coronary flow (CF), and heart rate (HR) were measured. If VF or VT continued for 1 min during reperfusion, MC was attempted by a flick of the forefinger to the right ventricle. If VF or VT still occurred, MC was repeated. Hearts that did not have regular beating after 20 min of reperfusion were excluded. Release of cardiac troponin T (cTnT) was measured before ischaemia and after 20 min of reperfusion. Forty-four out of 89 hearts had VF or VT during reperfusion. Thirty-five hearts were converted, 18 of which were converted by one or two MCs only. The higher the total number of MCs employed, the more cTnT was released. After 20 min of reperfusion, LVEDP, LVDP and CF were better in hearts with a higher number of MCs and with increased release of cTnT. After 60 min of reperfusion, LVEDP was still improved in hearts with more cTnT release, whereas LVSP was lower, and LVDP and CF were independent of the number of MCs. There was no consistent correlation between release of cTnT and heart dysfunction. In conclusion, MC effectively converted VF or VT. MCs increased post-ischaemic myocardial cell damage, as judged from increased cTnT release. Post-ischaemic dysfunction was partly attenuated in hearts with multiple MCs, and did not correlate with release of cTnT. We feel that MCs should not be used in isolated, perfused hearts.