Masato Tani

Changes in Ischemic Tolerance and Effects of Ischemic Preconditioning in Middle-aged Rat Hearts

BACKGROUND Although both clinical and animal studies have shown that ischemic tolerance is reduced in the senescent myocardium, it has not been clarified when myocardium becomes more vulnerable to ischemia. Preconditioning protects the hearts of young adult animals of various species, but its effects are not identical in human studies. We investigated whether ischemic tolerance and the effect of preconditioning decreased in isolated hearts of middle-aged rats. METHODS AND RESULTS The hearts of young adult rats (12 weeks old: group Y, n = 44) and middle-aged rats (50 weeks old: group M, n = 44) were subjected to global ischemia for 15, 20, or 25 minutes followed by reperfusion. Hearts were also subjected to preconditioning and then to 20 (group Y, n = 22) or 15 (group M, n = 22) minutes of ischemia followed by reperfusion. Left ventricular developed pressure (LVDP) was decreased by 40% to 60%, and the level of ATP was decreased by 60% to 70% in group M compared with group Y. Preconditioning increased LVDP (% LVDP, 40.5% to 72.4%) and levels of high-energy phosphates (ATP, 11.8 to 14.1; creatine phosphate, 17.0 to 23.1 mumol/g dry wt) and reduced left ventricular end-diastolic pressure (LVEDP, 32.8 to 10.3 mm Hg), creatine kinase release (257 to 132 U/g dry wt), and ryanodine-sensitive sarcoplasmic reticulum Ca2+ release after ischemia in group Y. Preconditioning exerted opposite effects in group M (% LVDP, 45.9% to 15.8%; LVEDP, 21.0 to 28.5 mm Hg; ATP, 14.1 to 8.5 mumol/g dry wt; and CK release, 176 to 332 U/g dry wt). Preconditioning was associated with increases in the incidence of reperfusion-induced ventricular fibrillation (0% to 62.5%) and the rate of sarcoplasmic reticulum Ca2+ release in group M. CONCLUSIONS These results indicate that hearts became more vulnerable to ischemia with age and that the beneficial effects of preconditioning were reversed in middle-aged rat hearts.

Na+ accumulation increases Ca2+ overload and impairs function in anoxic rat heart

Maintenance of low coronary flow (1 ml/min) during 40 or 70 min of anoxia maintained function and prevented Ca2+ overload during reoxygenation in isolated rat hearts. In comparison, recovery from 40 min of global ischemia resulted in only 20% of preischemic function and an increase in end-diastolic pressure (LVEDP) to 39 mmHg. Reperfusion Ca2+ uptake rose from 0.6 to 10.2 mumol/g dry tissue. Intracellular Na+ (Nai+) increased from 13 to 61 mumol/g dry tissue after 40 min of global ischemia, but was unchanged in hearts with low flow anoxia. When glucose and pyruvate were omitted from buffer used for anoxic perfusion, recovery was only 15% of preanoxic values, LVEDP rose to 32 mmHg, and reperfusion Ca2+ uptake was 7.2 mumol/g dry. In addition, Nai+ increased (47.4 mumol/g dry tissue) and ATP was depleted (1.0 mumol/g dry tissue) in the absence of substrate. In anoxic hearts supplied substrate, Nai+ stayed low (12 mumol/g dry tissue) and ATP was preserved (11.6 mumol/g dry tissue). Addition of ouabain (100 or 200 microM) and provision of zero-K+ buffer increased Nai+ and resulted in impaired functional recovery, increased LVEDP, and greater reperfusion Ca2+ uptake. These interventions also decreased energy availability in anoxic hearts. To distinguish between effects of Na+ accumulation and ATP depletion, monensin, a Na+ ionophore, was added during low flow anoxia. Monensin increased Nai+, decreased functional recovery and increased reperfusion Ca2+ uptake in a dose-dependent manner (1-10 microM) without changing ATP content. These results suggested that reduction of Nai+ accumulation by maintenance of Na+, K+ pump activity was the major mechanism of the beneficial effects of low coronary flow on reperfusion injury.

Direct activation of mitochondrial KATP channels mimics preconditioning but protein kinase C activation is less effective in middle-aged rat hearts

OBJECTIVES This study is aimed to determine whether loss of preconditioning (IP) effects in the middle-aged hearts (MA) is due to the failure of protein kinase C (PKC) activation and, if so, whether direct activation of mitochondrial ATP-sensitive potassium channels (m-K(ATP)) or PKC mimics IP. BACKGROUND PKC is a mediator and m-K(ATP) may be its downstream effector of IP in young adult hearts (YA), but we have demonstrated that IP is not effective in MA. METHODS AND RESULTS Isolated hearts from YA (12-week) and MA (50-week) Fischer 344 rats were preconditioned by three cycles of ischemia and reperfusion (5 min each), and the translocation of PKC isoforms and the effects on reperfusion injury were assessed. In some hearts activation of m-K(ATP) or PKC by diazoxide or 1, 2-dioctanoyl glycerol (DOG) was performed before 25 min of global ischemia/30 min of reperfusion. IP could improve the recovery of LV function and resulted in higher content of ATP after reperfusion in YA but these beneficial effects of IP was not found in MA. The effects of IP in YA were abolished by 5-hydroxydecanoate. In YA but not in MA, immunohistochemical analysis revealed that IP translocated PKC-alpha and delta from the cytosolic or membrane to the perinuclear region but immunoblotting analysis showed translocation of PKC-alpha, delta and epsilon to the membrane fraction. Pretreatment with diazoxide or DOG mimicked IP and decreased the creatine kinase release in YA. Diazoxide was also effective but effects of DOG were less in MA as compared with in YA. CONCLUSIONS IP is not effective in MA hearts partly due to failure of translocation of PKC isoforms. Moreover, less efficacy of PKC activation by DOG as compared with activities of m-K(ATP) by diazoxide in MA may suggest that defect(s) of cell signaling downstream to PKC may also be involved in the loss of IP effects in MA.

Mechanisms of Ca2+ overload induced by extracellular H2O2 in quiescent isolated rat cardiomyocytes.

Abstract Rat cardiomyocytes were exposed to H2O2 (1–100 μmol/L) for 10 min with washout for 10 min. Intracellular Ca2+ concentration ([Ca2+]i) was measured using fluo-3. [Ca2+]i increased with 100 μmol/L H2O2 and further increased during washout, causing irreversible contracture in one-half of the cells. The increase in [Ca2+]i with 10 μmol/L H2O2 was modest with few cells showing irreversible contracture and attenuated by caffeine, and [Ca2+]i gradually decreased during washout and this decrease was accelerated by a calcium-free solution, while 1 μmol/L H2O2 did not have any effects on [Ca2+]i or cell viability. Ca2+ overload caused during exposure to 100 μmol/L H2O2 was attenuated by caffeine with improved cellular viability but not by chelerythrine, KB-R7943 or nifedipine. With 100 μmol/L H2O2 calcium-free solution attenuated the increase during exposure and washout while KB-R7943 or chelerythrine partly attenuated further increase during washout but not improved cell viability, but chelerythrine did not have additional effect on calcium-free treatment. Catalase abolished the effects of H2O2. We concluded that the increased [Ca2+]i during exposure to 100 μmol/L H2O2 was caused both by release of Ca2+ from the intracellular store sites including the sarcoplasmic reticulum and by influx through route(s) other than the voltage-dependent Ca2+ channels or Na+/Ca2+ exchanger, although the Na+/Ca2+ exchanger or protein kinase C-mediated mechanism was partly responsible for a further increase during washout.

High incidence of pre-excitation syndrome in Japanese families with Leber's hereditary optic neuropathy

Cardiac conduction abnormalities have been reported in families with Lebers hereditary optic neuropathy (LHON). The pre‐excitation syndrome, Wolff‐Parkinson‐White syndrome or Lown‐Ganong‐Levine syndrome, is reportedly common in Finns with LHON, being seen in 14 (9%) of the 163 individuals with mitochondrial DNA (mtDNA) mutations. While this syndrome is thought to be rare in other ethnic groups with LHON, the present study of 35 Japanese LHON families confirmed that it is also relatively common among Japanese families, being seen in 5 (8%) of the 63 individuals with mtDNA mutations. It remains to be determined whether the high incidence of the pre‐excitation syndrome in Finnish and Japanese LHON families is due to a particular genetic composition of ethnic groups such as in Finland and in Japan, or only to a reporting bias.

Roles of the right ventricular free wall and ventricular septum in right ventricular performance and influence of the parietal pericardium during right ventricular failure in dogs

The roles of the right ventricular (RV) free wall and ventricular septum in RV performance were studied in the canine heart. The parietal pericardium was kept intact. Acute ischemia of the RV free wall from right coronary ligation decreased the RV stroke work index more than did that of the ventricular septum from the septal branch of the left coronary artery ligation (41 and 23%, respectively, p less than 0.01). The response of the RV stroke work index to acute volume loading was also decreased. Left ventricular dysfunction was detected only with ventricular septal ischemia. Combined RV free wall and ventricular septal ischemia produced more severe and predominant RV dysfunction with disproportionate elevation of RV end-diastolic pressure. After combined ischemia, pericardiotomy improved the RV stroke work index as well as the left ventricular stroke work index (40 and 27%, respectively, p less than 0.05), although the increase in RV stroke work index was greater than in left ventricular stroke work index (p less than 0.05). The results of this study suggest that (1) the RV free wall has a more important role than the ventricular septum in RV performance, (2) predominant RV failure can be induced experimentally after combined RV free wall and ventricular septal ischemia, and (3) the pericardium has a restrictive effect on the damaged and dilated right ventricle.

Evaluation of combined valvular prolapse syndrome by two-dimensional echocardiography.

The patterns of aortic and tricuspid valve motion in 50 patients with mitral valve prolapse were analyzed by wide-angle, phased-array, two-dimensional echocardiography. Twelve patients (24%) had redundant aortic leaflets bulging into the left ventricular outflow tract during diastole. Eight of 12 patients had aortic regurgitation and seven of 12 had M-mode echocardiographic evidence of aortic valve prolapse. One patient underwent mitral and aortic valve replacement, and the excised valves revealed marked myxomatous degeneration. Eight of 15 patients undergoing contrast echocardiography had tricuspid regurgitation (systolic reflux of contrast material into the inferior vena cava persisting for more than 10 beats), and prolapse in the septal leaflet or the anterior leaflet or both. A similar tricuspid valve pattern was noted in three of seven patients without tricuspid regurgitation. Tricuspid valve prolapse was identified in 20 patients (40%). Nine patients (18%) had combined prolapse of the mitral, aortic and tricuspid valves. In five patients with middiastolic highpitched murmurs recorded along the left sternal border, tricuspid valve prolapse was demonstrated. In one of these patients, the presence of pulmonary regurgitation was confirmed by intracardiac phonocardiography. We conclude that two-dimensional echocardiography is useful for evaluating patients with combined valvular prolapse syndrome.

Loss of protection by hypoxic preconditioning in aging Fischer 344 rat hearts related to myocardial glycogen content and Na+ imbalance

OBJECTIVES The objective of this study was to determine whether hypoxic preconditioning (HP) could lessen the myocardial increase in [Na+]i, thus protecting the aging myocardium against ischemia. BACKGROUND A decrease in ischemic tolerance with aging is associated with an accelerated increase in [Na+]i during ischemia. Ischemic preconditioning fails to protect the middle-aged and senescent myocardium against ischemia. METHODS Isolated hearts of young adult (12-week-old), middle-aged (50-week-old) and senescent (100-week-old) Fischer 344 rats were subjected to 25 min of ischemia with or without HP followed by 30 min of reperfusion. Left ventricular (LV) function, myocardial energy metabolites and [Na+]i were measured. RESULTS In the older groups, the recovery of LV function and high-energy phosphates (HEPs) was lower with an increased release of creatine kinase (CK) during reperfusion than in the young group. The increased [Na+]i at the end of ischemia was greater in the former groups than in the young group. HP decreased myocardial glycogen and lessened the increased [Na+]i in the young group, resulting in an improved recovery of LV function and HEPs, as well as decreased CK release. However, the levels of glycogen before HP in the older groups were higher than in the young group and its levels after HP were similar to that before HP in the young group. HP did not affect the [Na+]i, exacerbated CK release and inhibited the recovery of LV function and HEPs in the older groups. CONCLUSIONS HP failed to lessen the increased [Na+]i or to protect the aging hearts, probably due to the preexistence of increased glycogen level.

Very low dose of the Na+/Ca2+ exchange inhibitor, KB-R7943, protects ischemic reperfused aged Fischer 344 rat hearts: considerable strain difference in the sensitivity to KB-R7943

OBJECTIVE Decreased ischemic tolerance in the aged myocardium is associated with accelerated intracellular Ca(2+) overload. However, few drugs have been shown to attenuate reperfusion injury in aged hearts. Because the Na(+)/Ca(2+) exchanger (NCX1) has been reported to play an important role in Ca(2+) overload during reperfusion, we investigated whether KB-R7943 (KB-R), a novel inhibitor of the reverse mode of Na(+)/Ca(2+) exchange, can protect aged rat hearts against reperfusion injury. METHODS In the pilot study, isolated hearts from young Sprague-Dawley (SD) rats (12 weeks old), and young (12 weeks old) and aged (78 weeks old) Fischer 344 (F) rats were subjected to 25 min of global ischemia followed by 10 min of reperfusion with various concentrations of KB-R (0, 1 nM, 0.1 microM, 10 microM) and additional 20 min of reperfusion without agent. In subsequent studies with the same protocol in aged F rats, we added a protective dose of KB-R (1 nM) during the initial 10 min of reperfusion. In addition, we compared the amount of NCX1 and the sensitivity of the reverse mode of Na(+)/Ca(2+) exchange to KB-R under extracellular Na(+)-free condition in F rats with young SD rats. RESULTS In the pilot study, protective effects were elicited with 1 nM of KB-R in both young and aged F rats, while 10 microM KB-R was needed for SD rats. In subsequent studies using aged F rats, there was better recovery of LV systolic function and high-energy phosphates with reduced creatine kinase release and the duration of reperfusion arrhythmias. 45Ca(2+) uptake via the reverse mode of Na(+)/Ca(2+) exchange was also inhibited with 1 nM of KB-R in young F rats, but not in young SD rats. Although the amount of NCX1 was not different among young SD, young F, and aged F rat hearts. CONCLUSIONS These results demonstrated that KB-R could protect aged F rat hearts as well as young hearts of both strains against ischemia-reperfusion injury. Moreover, the sensitivity to KB-R is very different between these strains, suggesting serious caution when the agent is applied to human beings.

Preconditioning with heat shock further improved functional recovery in young adult but not in middle-aged rat hearts.

Ischemic preconditioning (PC) improves post-ischemic function, and heat shock (HS) mimics delayed PC in young animals. However, PC is not protective and the consequences of HS are not known in the aging hearts. This report examines the efficacy of HS and its synergy with PC in the middle-aged rat hearts. Hearts from 12- or 50-week-old rats were subjected to PC before 25 min ischemia followed by 30 min reperfusion 48 h after HS. HS induced HS proteins (HSP) in both age groups but that PC and HS translocated PKC-alpha and -delta only in young rats. The beneficial effects of HS and PC were additive and enhanced protein kinase C (PKC) translocation in young rats. However, neither HS alone nor in combination with PC conferred any functional advantage or accelerated PKC translocation in old rats. Similarly neither HS alone nor in combination with PC restore PC effects in old rats with impaired PKC activation, despite the induction of HSP, indicating that induction of HSP is insufficient for cytoprotection.