Sodium alterations in isolated rat heart during cardioplegic arrest

Schepkin, V. D., I. O. Choy, and T. F. Budinger. Sodiumalterations in isolated rat heart during cardioplegic arrest. J. Appl. Physiol. 81(6):2696-2702, 1996.Triple-quantum-filtered (TQF) Na nuclearmagnetic resonance (NMR) without chemical shift reagent is used toinvestigate Na derangement in isolated crystalloid perfused rat heartsduring St. Thomas cardioplegic (CP) arrest. Theextracellular Na contribution to the NMR TQF signal of a rat heart isfound to be 73 ± 5%, as determined by wash-out experiments atdifferent moments of ischemia and reperfusion. With the use of thiscontribution factor, the estimated intracellular Na([Na⁺]_i)TQF signal is 222 ± 13% of preischemic level after 40 min of CParrest and 30 min of reperfusion, and the heart rate pressure productrecovery is 71 ± 8%. These parameters aresignificantly better than for stop-flow ischemia: 340 ± 20% and 6 ± 3%, respectively. At 37°C, the initial delay of 15 min in[Na⁺]_igrowth occurs during CP arrest along with reduced growth later (~4.0%/min) in comparison with stop-flow ischemia (~6.7%/min). The hypothermia (21°C, 40 min) for the stop-flow ischemia and CPdramatically decreases the[Na⁺]_igain with the highest heart recovery for CP (~100%). These studiesconfirm the enhanced sensitivity of TQF NMR to[Na⁺]_iand demonstrate the potential of NMR without chemical shift reagent tomonitor[Na⁺]_iderangements.

     

The isolated working heart model in infarcted rat hearts

Congestive heart failure (CHF) is one of the most common causes of death in western countries. The aim of this study was to establish and validate the working heart model in rat hearts with CHF. In the rat model the animals show parameters and symptoms that can be extrapolated to the clinical situation of patients with end-stage heart failure. The focus of attention was the evaluation of cardiodynamics (e.g.contractility) in the isolated 'working heart' model. The geometric properties of the left ventricle were measured by planimetry (stereology). Formulae available in the past for determining certain parameters in the working heart model (e.g.external heart work) have to be fitted to the circumstances of the infarcted rat hearts with its different organ properties.CHF was induced in Wistar Kyoto (WKY/NHsd) and spontaneously hypertensive rats (SHR/NHsd) by creating a permanent (8 week) occlusion of the left coronary artery, 2 mm distal to the origin from the aorta, by a modified technique (Itter et al. 2004). This resulted in a large infarction of the free left ventricular wall.We were able to establish and adapt a new and predictive working heart model in spontaneously hypertensive rat hearts with myocardial infarction (MI) 8-12 weeks after coronary artery ligation. At this stage the WKY rat did not show any symptoms of CHF. The SHR rat represented characteristic parameters and symptoms that could be extrapolated to the clinical situation of patients with end-stage heart failure (NYHA III-IV). Upon inspection, severe clinical symptoms of CHF such as dyspnoea, subcutaneous oedema, palebluish limbs and impaired motion were prominent. On necropsy the SHR showed lung oedema, hydrothorax, large dilated left and right ventricular chambers and hypertrophy of the septum. In the working heart model the infarcted animals showed reduced heart power, diminished contractility and enhanced heart work, much more so in the SHR/NHsd than in the Wistar Kyoto rat (WKY/NHsd).The aim for the future is to find a causal therapy of heart failure treatment. At present, only palliative therapy is possible for patients with heart failure. For this reason the working heart model in CHF rat hearts should provide a valuable method for early testing of new therapeutic approaches for patients with CHF.     

木犀草素改善低温下离体大鼠心脏保存效果的研究

目的:研究木犀草素是否能改善心脏停搏保存液(UW液)对离体大鼠心脏的低温保存效果。方法:将40只成年SD大鼠随机分成4组(n=10):对照组(UW组)、7.5μmol/L木犀草素小剂量组,15μmol/L木犀草素中剂量组及30μmol/L木犀草素大剂量组。利用Langendorff离体心脏灌流法,观察心脏在4℃含或不含木犀草素的UW液中保存12 h复灌60 min后心脏功能及超微结构变化,比较心脏冠脉流量(CF)、心肌含水量及冠脉流出液中磷酸肌酸激酶(CK)的释放量。结果:与对照组比较,添加木犀草素后,复灌期心脏的收缩功能(LVPSP,+dp/dtmax)与心脏舒张功能(-dp/dtmax)、冠脉流量在多个复灌时间点均优于对照组,心率在复灌60 min时也显著优于对照组;复灌过程中磷酸肌酸激酶的漏出量及低温保存后心脏超微结构的损伤也均明显低于对照组;随灌注时间延长木犀草素组心脏结构和功能的改善有剂量依赖性趋势;木犀草素对心肌含水量没有影响。结论:木犀草素能显著改善UW液对离体大鼠心脏的低温保存效果,对心脏有明显的保护作用,以30μmol/L的木犀草素大剂量组作用最显著。     

Endothelin stimulates degradation of phospholipids in isolated rat hearts.

The abilities of endothelin-1 to cause cellular injury and to enhance the levels of inositol-1,4,5-triphosphate and the breakdown of [14C]arachidonate-labeled phospholipids have been examined in the isolated rat heart model. In 10 minutes, endothelin at 1 and 3 nM concentrations significantly increased the myocardial release of creatine kinase, suggesting endothelin-induced cell injury. The enhanced levels of myocardial inositol-1,4,5-triphosphate and diacyl glycerol by endothelin also suggest the increased breakdown of phosphatidylinositol-4,5-bisphosphate. In addition, endothelin also increased the degradation of other membrane phospholipids as observed by (1) a decrease in [14C]arachidonate radiolabel in phospholipids, (2) an increase in [14C]radiolabel in non-esterified fatty acids and triacyl glycerol, and (3) increased levels of non-esterified fatty acids. The potential role of endothelin-1 in myocardial ischemia-reperfusion injury is discussed.     

Arrhythmogenic effect of acoustic cavitation in isolated rat heart perfused with physiological solution

In experiments on isolated rat hearts the effects of focused continuous and impulse ultrasound (543 Hz, with intensity up to 7.8 W/cm2 at a focal region) on a pressure developed by left ventricle and electrograms were studied. In all experiments ultrasound induced extra-excitations of the heart, which appeared when intensity was 1.35 +/- 0.21 W/cm2 (n = 9). Simultaneously with the extra-excitations the cavitation bursts were recorded at intensity of 1.52 +/- 0.18 W/cm2 (n = 6). Acoustic cavitation (after 30 sec of exposure) resulted in a significant decrease of the developed pressure (from 100.8 +/- 3.8 mm Hg to 95.1 +/- 4.3 mm Hg, p 0.001), measured in 2 min after the end of the exposure. In the absence of cavitation the ultrasound was found to have no effects on cardiac performance. Electrograms recorded during acoustic pacing show that a pattern of the heart excitation changed from stimulus to stimulus.     

Relationship between cytosolic calcium and oxygen consumption in isolated rat hearts.

Maximum oxygen consumption was attained in isolated perfused rat hearts using high perfusate calcium and/or isoproterenol, or phenylephrine. The amplitude of calcium transients was directly related to oxygen consumption until oxygen consumed per beat reached maximum. At saturating oxygen consumption the amplitude of [Ca2+]i transients continued to increase, indicative of a calcium overload. In all cases +dP/dt correlated proportionately with +dCa2+/dt. Augmented developed pressure, related to isoproterenol-induced increase in cytosolic cAMP, cannot be attributed totally to elevated levels of [Ca2+]i transients. Adenosine (10(-5) M) added to the medium containing isoproterenol (10(-6) M) negated the isoproterenol-induced increase in cAMP and returned cardiac performance, oxygen consumption, and amplitude of [Ca2+]i transients to control state.     

Ten-hour preservation of guinea pig isolated hearts perfused at low flow with air-saturated Lifor solution at 26{degrees}C: comparison to ViaSpan solution

There is no suitable solution to preserve hearts for longer than 5 h between donor explant and recipient implant. Lifor is a fully artificial preservation medium containing both a nonprotein oxygen and nutrient carrier (nanoparticles) and cellular nutrients, including amino acids and sugars. We proposed that recirculated Lifor solution would satisfactorily preserve guinea pig isolated hearts perfused at low flow with no added O(2) at room temperature for 10 h. Hearts were isolated from 21 guinea pigs and perfused with Krebs-Ringer (KR) solution (97% O(2) and 3% CO(2)) at 37 degrees C. Heart rate, inflow and outflow O(2) tension, coronary flow, left ventricular pressure (LVP), and maximal and minimal rate of change in LVP (dLVP/dt) were measured. After baseline measurements, hearts were perfused with recirculated Lifor or ViaSpan equilibrated with room air at 15% of control flow at 26 degrees C for 10 h. Hearts were then perfused at 100% flow with KR for 2 h at 37 degrees C. A time control (untreated) group was perfused only with KR solution for 15 h. Lifor arrested and protected hearts against diastolic contracture and maintained a low O(2) extraction. Compared with time controls, Lifor led to a higher developed LVP and coronary flow; %O(2) extraction and cardiac efficiency were similar between these two groups. Hearts similarly treated with ViaSpan exhibited diastolic contracture and lower %O(2) extraction during treatment and, upon reperfusion with KR, exhibited continued diastolic contracture, no return of heart rate or contractility, low coronary flow, low %O(2) extraction, and marked infarction. For long-term cardiac protection, a suitable preservation solution recirculated at low flow and room temperature without supplemental O(2) would reduce the support apparatus required for transport. Lifor was far superior to ViaSpan in meeting these requirements.     

Mechanical factors affecting protein turnover in isolated rat hearts

Induction of cardiac work increased protein synthesis in hearts supplied glucose or a mixture simulating normal plasma levels of glucose, insulin, glucagon, lactate, and beta-hydroxybutyrate. During 2 h of perfusion, cardiac work did not accelerate protein synthesis in hearts supplied a mixture of glucose, lactate, and higher concentrations of insulin. Protein degradation was decreased by work in hearts supplied glucose. Nitrogen balance was negative in Langendorff-perfused hearts provided glucose, but was less so in working preparations. Nitrogen balance was zero or positive in working hearts provided the mixture simulating plasma or the mixture of glucose, lactate, and insulin. In Langendorff preparations, increased aortic pressure accelerated protein synthesis during the second hour of perfusion in hearts supplied glucose, glucose plus insulin, or pyruvate. When ventricular pressure development was prevented by ventricular draining or when drained hearts were arrested with tetrodotoxin, protein synthesis still increased as perfusion pressure was raised from 60 to 120 mm Hg. Oxygen consumption increased as aortic pressure was increased in drained, beating hearts, but was unaffected in arrested, drained hearts. These studies indicated that increased aortic pressure and its attendant stretch of the ventricular wall were the mechanical parameter most closely associated with faster rates of protein synthesis.     

The effects of exogenous histamine in isolated rat hearts

The role of histamine in cardiac physiology and pathophysiology is not clarified, but is dependent on species. The effects of exogenous histamine in Langendorff-perfused rat hearts were investigated. 1 mM, 100, 10, 1 and 0.1 M of histamine (n=7 each) as 15 min infusions were employed in a dose-response study, and compared to control perfused hearts (n=7). In another experimental series, 100 M histamine (n=15) was added during reperfusion after 25 min global ischemia, and compared to control ischemia-reperfusion (n=15). The maximal response to histamine in the dose-response study (100 M) was an increase of left ventricular developed pressure to 126±8% of initial value (mean±SEM, p<0.04), and increase of coronary flow to 152+6% (p<0.02) after 5 min infusion. 100 M histamine did not significantly influence heart rate or rhythm. The lowest concentration (0.1 M) did not have effects cardiac performance. Reperfusion with histamine for 2 min after ischemia reduced left ventricular developed pressure to 68±10% of initial value versus 116+17% in ischemic controls (p<0.05), and increased left ventricular end-diastolic pressure to 24±8 mmHg compared to 6±2 mmHg in controls (p<0.04). Left ventricular pressures were similar in hearts reperfused with histamine and in ischemic controls for the rest of the observation. Coronary flow increased during reperfusion in hearts given histamine. Histamine had a dose-dependent positive inotropic and vasodilatory effect in isolated rat hearts. Exogenous histamine had only minor effects on post-ischemic cardiac function.     

Effects of tocopheryl quinone on the heart: model experiments with xanthine oxidase, heart mitochondria, and isolated perfused rat hearts

It is generally accepted that the protection effect of biological tissues by vitamin E is due to its radical scavenging potency in membranes, thereby being transformed to a vitamin E radical. A deficiency of appropriate reductants, which recycle vitamin E radicals back to its antioxidative active form, causes an irreversible degradation of vitamin E leading to tocopheryl quinone (TQ). TQ-like compounds were shown to result from both vitamin E and corresponding hydrophilic analogues of this antioxidant in vitro. In vivo elevated concentrations of tocopheryl quinones were detected after oxidative stress and TQ supplementation as well. Quinones in general are known to be efficient one-electron donors and acceptors. Therefore the question arises whether TQ-like compounds can undergo redox-cycling in conjunction with redox-active enzymes in the heart, thereby producing harmful oxygen radicals, or whether these compounds exhibit antioxidant properties. In order to elucidate this question we focused our interest on the interaction of TQ and a corresponding short-chain homologue (TQ(0)) with xanthine oxidase and heart mitochondria. Furthermore, we tested the influence of TQ on the recovery of isolated perfused rat hearts after ischemia/reperfusion. Our experiments revealed that hydrophilic TQ(0) was univalently reduced by xanthine oxidase (XOD) yielding semiquinone radicals in the absence of oxygen. However, under aerobic conditions TQ(0) enhanced the O(2)(*)(-) radical output of XOD. In the mitochondrial respiratory chain TQ was shown to interact with high potential cytochrome b in the bc(1) complex specifically. In contrast to the system XOD/TQ(0), lipophilic TQ in submitochondrial particles decreased the O(2)(*)(-) radical release during regular respiration possibly due to its interaction with b-cytochromes in the mitochondrial respiratory chain. In isolated rat hearts perfused with liposomes containing lipophilic TQ, it was efficiently accumulated in the heart tissue. When hearts were subjected to conditions of ischemia/reperfusion, infusion of TQ prior to ischemia significantly improved the recovery of hemodynamic parameters. Our results demonstrate that TQ derivatives may induce pro-oxidative and antioxidative effects depending on the distribution of TQ derivatives in the heart tissue and the interacting redox system.     

Taxol inhibits stretch-induced electrophysiological alterations in isolated rat hearts with acute myocardial infarction

Mechanosensitive channels have been determined to work as transducers of mechanoelectric feedback in the heart, which is associated with the generation of arrhythmias. Recent studies have investigated the role of the cytoskeleton in ion channels control. This study explored the ability of taxol to inhibit stretch-induced electrophysiological alterations in the ischemic myocardium. Thirty-two Wistar rats were randomly divided into four groups: normal control group (n=9), taxol group (n=7), myocardial infarction (MI) group (n=9), and MI+taxol group (n=7). After Langendorff perfusion, the isolated hearts were stretched for 5 s by balloon inflation to 0.2 or 0.3 mL. The effects of stretching on 90% monophasic action potential duration (MAPD₉₀), premature ventricular beats (PVB), and ventricular tachycardia (VT) were observed for 30 s. Stretching increased MAPD₉₀ in both the normal control and MI groups, but MAPD₉₀ increased more in the MI group for the same degree of stretch. Taxol (5 μmol L⁻¹) had no effect on MAPD₉₀ under baseline, unstretched conditions, but MAPD₉₀ in the taxol group was slightly increased after stretching compared with the normal control group (P>0.05). However, taxol reduced MAPD₉₀ in infarcted myocardium (P<0.05 at ΔV=0.3 mL). The incidences of PVB and VT in the MI group were higher than in the normal control group (both P<0.01). Taxol had no effect on the occurrence of arrhythmias in normal myocardium, but it inhibited PVB and VT in infarcted hearts (both P<0.01). Thus changes in MAPD and the occurrence of arrhythmias caused by mechanical stretching of the myocardium could be inhibited by taxol in isolated rat hearts during AMI, indicating the involvement of tubulin in mechanoelectric feedback in AMI.     

Norepinephrine-induced expression of cytokines in isolated biventricular working rat hearts

Whereas ATP consumption increases with neural activity and is buffered by phosphocreatine (PCr), it is not known whether PCr synthesis by ubiquitous mitochondrial creatine kinase (uMtCK) supports energy metabolism in all neurons. To explore the possibility that uMtCK expression in neurons is modulated by activity and during development, we used immunocytochemistry to detect uMtCK-containing mitochondria. In the adult brain, subsets of neurons including layer Va pyramidal cells, most thalamic nuclei, cerebellar Purkinje cells, olfactory mitral cells and hippocampal interneurons strongly express uMtCK. uMtCK is transiently expressed by a larger group of neurons at birth. Neurons in all cortical layers express uMtCK at birth (P0), but uMtCK is restricted to layer Va by P12. uMtCK is detected in cerebellar Purkinje cells at birth, but localization to dendrites is only observed after P5 and is maximal on P14. Hippocampal CA1 and CA3 pyramidal neurons contain uMtCK-positive mitochondria at birth, but this pattern becomes progressively restricted to interneurons. Seizures induced uMtCK expression in cortical layers II–III and CA1 pyramidal neurons. In the cortex, but not in CA1, blockade of seizures prevented the induction of uMtCK. These findings support the concept that uMtCK expression in neurons is (1) developmentally regulated in post-natal life, (2) constitutively restricted in the adult brain, and (3) regulated by activity in the cortex and hippocampus. This implies that mitochondrial synthesis of PCr is restricted to those neurons that express uMtCK and may contribute to protect these cells during periods of increased energy demands.     

Biochemical consequences of electrical pacing in ischemic-reperfused isolated rat hearts

It is still unclear if performance recovery in postischemic hearts is related to their tissue level of high-energy phosphates before reflow. To test the existence of this link, we monitored performance, metabolism and histological damage in isolated, crystalloid-perfused rat hearts during 20 min of low-flow ischemia (90% coronary flow reduction) and reflow. To prevent interference from different ischemia times and perfusing media compositions, the ischemic ATP level was varied by changing energy demand (electrical pacing at 330 min–1). Under full coronary flow conditions, work output, as well as ATP and phosphocreatine contents were the same in control, spontaneously contracting (n = 23) and paced (n = 21) hearts. During low-flow ischemia, the higher work output (p < 0.0001) in paced hearts decreased their tissue content of ATP, phosphocreatine and total adenylates and purines (p < 0.05), as opposed to maintained values in control hearts. During reflow, the recovery of mechanical performance and O2 uptake was 94 ± 5% and 110 ± 9% (p = NS vs. baseline) in controls, vs. 71 ± 5% and 74 ± 6% in paced hearts (p < 0.004 vs. baseline). The levels of ATP and total adenylates and purines remained constant in control, but were markedly depressed (p < 0.05 vs. baseline) in paced hearts. Phosphocreatine+creatine was the same in both groups. These data, together with the observed lack of creatine kinase leakage and of structural damage, indicate that myocardial recovery during reflow reflects the tissue level of ATP, phosphocreatine and total adenylates and purines during ischemia, regardless of physical cell damage.     

Synthesis of stress-induced protein in isolated and perfused rat hearts

Isolated and perfused rat hearts were examined by two-dimensional gel electrophoresis and liquid scintillation counting for alterations in protein synthesis following incubation with L-[3H]leucine at 0.5-2.5, 2.5-4.5, or 4.5-6.5 h of perfusion. When 35-mL volumes of three different buffers were recycled for a 2-h period from 0.5 to 2.5 h, by fluorography little effect was seen on the normal patterns of protein synthesis and there was a moderate synthesis of a stress-induced protein (heat-shock protein) with a molecular mass of 71 X 10(3) daltons (SP71). However, hearts perfused with Krebs-improved Ringer 1 bicarbonate had the highest incorporation of L-[3H]leucine. When buffers were recycled for 30-min periods from 0.5 to 2.5 h, SP71 was synthesized in hearts perfused with Krebs-Henseleit original Ringer bicarbonate. Hearts perfused in a similar fashion with Krebs-improved Ringer 1 bicarbonate had the lowest incorporation of label into SP71 and in fact SP71 was undetectable on fluorograms. Overall protein synthesis was decreased and the ratio of SP71 to the total synthesis was increased at 4.5-6.5 h of perfusion when 35-mL volumes of Krebs-improved Ringer 1 bicarbonate was recycled for 2-h periods. A similar result was observed at 2.5-4.5 h of perfusion when this buffer was recycled for either the duration of the experiment or 30-min periods.