Optimizing catecholaminergic polymorphic ventricular tachycardia therapy in calsequestrin-mutant mice.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal arrhythmia provoked by physical or emotional stress and mediated by spontaneous Ca(2+) release and delayed after-depolarizations. Beta-adrenergic blockers are the therapy of choice but fail to control arrhythmia in up to 50% of patients. OBJECTIVE: To optimize antiarrhythmic therapy in recessively inherited CPVT caused by calsequestrin (CASQ2) mutations. METHODS: Murine heart rhythm telemetry was obtained at rest, during treadmill exercise, and after injection of epinephrine. The protocol was repeated after injection of different antiarrhythmic drugs. Results were then validated in human patients. RESULTS: Adult CASQ2 mutant mice had complex ventricular arrhythmia at rest and developed bidirectional and polymorphic ventricular tachycardia on exertion. Class I antiarrhythmic agents (procainamide, lidocaine, flecainide) were ineffective in controlling arrhythmia. Propranolol and sotalol attenuated arrhythmia at rest but failed to prevent VT during sympathetic stimulation. The calcium channel blocker verapamil showed a dose-dependent protection against CPVT. Verapamil was more effective than the dihydropyridine L-type Ca(2+) channel blocker nifedipine, and its activity was markedly enhanced when combined with propranolol. Human patients homozygous for CASQ2(D307H) mutation, remaining symptomatic despite chronic ß-blocker therapy, underwent exercise testing according to the Bruce protocol with continuous electrocardiogram recording. Verapamil was combined with propranolol at maximum tolerated doses. Adding verapamil attenuated ventricular arrhythmia and prolonged exercise duration in five of 11 patients. CONCLUSION: Verapamil is highly effective against catecholamine-induced arrhythmia in mice with CASQ2 mutations and may potentiate the antiarrhythmic activity of ß-blockers in humans with CPVT2.

Dual effect of erythropoietin on liver protection and regeneration after subtotal hepatectomy in rats.

The only currently offered curative option for many patients with primary or secondary liver tumors is the resection of hepatic tumors. The aim of this study was to evaluate the role of recombinant human erythropoietin (rhEPO) in liver protection and regeneration after subtotal hepatectomy in rats. Rats undergoing 70% hepatectomy received an intraperitoneal injection of saline (control) or rhEPO (4 U/g) 30 minutes prior to resection. Liver function was assessed by the measurement of the international normalized ratio (INR) levels, and hepatic injury was assessed by serum alanine aminotransferase and aspartate aminotransferase levels. Hepatic apoptosis was assessed by intrahepatic caspase-3 activity and morphological criteria. The regeneration capacity of remnant livers was assessed over 7 days with the regenerated liver/body weight ratio, immunohistochemistry markers of cell proliferation (Ki-67) and angiogenesis (von Willebrand factor), and phosphorylated extracellular signal-regulated kinase signaling. Two and 4 days after subtotal hepatectomy, the regenerated liver/body weight ratio was significantly higher in animals treated with rhEPO versus the control group (P < 0.005). Serum liver enzymes and INR levels on days 2 and 4 post-hepatectomy were significantly lower in animals pretreated with rhEPO in comparison with the control group (P < 0.005). No statistically significant difference was noted in intrahepatic hepatic caspase-3 activity, immunohistochemistry for caspase-3, or a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay between the hepatectomized groups. In the rhEPO-pretreated group, the mitotic index, Ki-67 and von Willebrand factor expression, and extracellular signal-regulated kinase activity were significantly higher on day 2 post-hepatectomy (P < 0.05) in comparison with the control group. In conclusion, rhEPO treatment may offer a unique beneficial dual-function strategy for hepatic protection and regeneration immediately after subtotal hepatectomy in rats.

Uridine-5'-triphosphate protects against hepatic- ischemic/reperfusion injury in mice.

BACKGROUND AND AIM: Mitochondrial calcium overload triggers apoptosis and also regulates ATP production. ATP and uridine-5'-triphosphate (UTP) depletion from hepatic tissue after ischemia causes cell death. ATP and UTP binds to cell membranes of the hepatocytes through P2Y receptors. Our aim was to investigate the role of UTP on the hepatic injury induced by ischemia. METHODS: Isolated mouse livers were randomly divided into five groups: (1) control group; (2) ischemic group (90 min); (3) as group 2, but with the administration of UTP; (4) as group 2, but with the administration of suramin, a P2Y antagonist; and (5) as group 3, but with the simultaneous administration of suramin and UTP. RESULTS: There was a postischemic significant reduction in the release of liver enzymes in the animals pretreated with UTP, the intrahepatic caspase-3 activity was significantly decreased, and the intrahepatic ATP content increased compared with group 2 (ischemic untreated). UTP prevented intracellular Ca overload after hypoxia in hepatocyte cultures. In the UTP-treated groups, significantly fewer apoptotic hepatocyte cells were noted by weaker activation of caspase-3 and by the transferase-mediated dUTP nick end labeling assay. The administration of suramin prevented the beneficial effect of endogenous ATP. UTP treatment attenuated the degradation of IkappaBalpha (nuclear factor-kappaB inhibitor) by 80% during reperfusion with no effect on c-Jun N terminal kinase phosphorylation. CONCLUSION: The administration of UTP before induction of ischemia-reperfusion can attenuate hepatic injury. UTP administration decreased cytosolic Ca overload in hypoxic conditions. UTP-mediated protective effects may be regulated through nuclear factor- kappaB inactivation. These findings have important implications for the potential use of UTP in ischemic hepatic injury.

Recombinant human erythropoietin attenuates hepatic injury induced by ischemia/reperfusion in an isolated mouse liver model.

INTRODUCTION: Apoptosis is a central mechanism of cell death following reperfusion of the ischemic liver. Recombinant human erythropoietin (rhEPO) have an important role in the treatment of myocardial ischemia/reperfusion (I/R) injury, by preventing apoptosis. The aim of the study was to investigate the effect of different regimens of rhEPO in preventing apoptosis following I/R-induced hepatic injury. MATERIAL AND METHODS: Isolated mouse livers were randomly divided into five groups: (1) control group, perfused for the whole study period (105 min); (2) 30-min perfusion followed by 90 min of ischemia and 15 min of reperfusion; (3), (4) and (5) like group 2, but with administration of rhEPO 5,000 units/kg i.p. at 30 min, 24 h, or both 30 min and 24 h respectively, before induction of ischemia. Perfusate liver enzyme levels and intrahepatic caspase-3 activity were measured, and apoptotic cells were identified by morphological criteria, TUNEL assay, and immunohistochemistry for caspase-3. Using immunoblot the expression of the proapoptotic JNK and inhibitor of NFkappaB (IkappaBalpha) were also evaluated. von Willebrand factor (vWF) immunohistochemistry was used as a marker of endothelial cells. RESULTS: Compared to the I/R livers, all 3 rhEPO pretreated groups showed: a significant reduction in liver enzyme levels (P < 0.05) and intrahepatic caspase-3 activity (P < 0.05), fewer apoptotic hepatocytes (P < 0.05) and positive vWF staining in numerous endothelial cells lining the sinusoids. EPO decreased JNK phosphorylation and the degradation of the inhibitor of NFkappaB (IkappaBalpha) during I/R. There was no added benefit of the multiple- over the single-dose rhEPO regimen. CONCLUSION: Pretreatment with one dose of rhEPO can attenuate post-I/R hepatocyte apoptotic liver damage. NFkappaB and JNK activation is likely to play a pivotal role in the pathophysiology of I/R hepatic injury and might have a key role in EPO-mediated protective effects. This effect is associated with the increase in sinusoidal vWF immunostaining suggests an additional effect of rhEPO in liver angiogenesis recovery. These findings have important implications for the potential use of rhEPO in I/R injury during liver transplantation.