Recovery Of Left Ventricular Pressure Research Articles

The effect of initial flush for cooling and coronary vascular washout in heart preservation

Initial flush for cooling and coronary vascular washout using University of Wisconsin (UW) solution has presented some problems. We used a separate solution for initial flush and for subsequent storage, and evaluated the effect of initial flush for prolonged heart preservation. Following potassium arrest, the initial flush was performed using UW solution (UW group) or Physiol · 3® solution with additional potassium (PK group). In the PK group, an additional flush with the UW solution was done prior to preservation. The hearts of both groups were stored in the cold UW solution for 12 hours. Myocardial high-energy phosphates were measured using phosphorus 31 nuclear magnetic resonance spectroscopy (UW group: n=7, PK group: n=5). Adenosine triphosphate (ATP) levels in the UW group decreased to 23% of the control after storage. ATP levels in the PK group were well maintained for up to 6 hours, and then decreased to 51% (p<0.01). Orthotopic transplantation was performed in 10 pairs of dogs (UW group: n=7, PK group: n=3). Preserved grafts in the PK group (74%) tended to show better recovery of left ventricular pressure than those in the UW group (52%). These results suggest that the introduction of an initial flush may prevent ATP level reduction during cold storage, and may provide good results after transplantation. An initial flush for cooling and coronary vascular washout is important for prolonged heart preservation.

Adding ROS Quenchers to Cold K+Cardioplegia Reduces Superoxide Emission During 2-Hour Global Cold Cardiac Ischemia

We reported that the combination of reactive oxygen species (ROS) quenchers Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), catalase, and glutathione (MCG) given before 2 hours cold ischemia better protected cardiac mitochondria against cold ischemia and warm reperfusion (IR)-induced damage than MnTBAP alone. Here, we hypothesize that high K(+) cardioplegia (CP) plus MCG would provide added protection of mitochondrial bioenergetics and cardiac function against IR injury. Using fluorescence spectrophotometry, we monitored redox balance, ie reduced nicotinamide adenine dinucleotide and flavin adenine dinucleotide (NADH/FAD), superoxide (O(2) (•-)), and mitochondrial Ca(2+) (m[Ca(2+)]) in the left ventricular free wall. Guinea pig isolated hearts were perfused with either Krebs Ringer's (KR) solution, CP, or CP + MCG, before and during 27°C perfusion followed immediately by 2 hours of global ischemia at 27°C. Drugs were washed out with KR at the onset of 2 hours 37°C reperfusion. After 120 minutes warm reperfusion, myocardial infarction was lowest in the CP + MCG group and highest in the KR group. Developed left ventricular pressure recovery was similar in CP and CP + MCG and was better than in the KR group. O(2) (•-), m[Ca(2+)], and NADH/FAD were significantly different between the treatment and KR groups. O(2) (•-) was lower in CP + MCG than in the CP group. This study suggests that CP and ROS quenchers act in parallel to improve mitochondrial function and to provide protection against IR injury at 27°C.

Reactive Oxygen Species–Induced Stimulation of 5′AMP-Activated Protein Kinase Mediates Sevoflurane-Induced Cardioprotection

5'AMP-activated protein kinase (AMPK), a well-known regulator of cellular energy status, is also implicated in ischemic preconditioning leading to cardioprotection. We hypothesized that AMPK is involved in anesthetic-induced cardioprotection and that this activation is mediated by reactive oxygen species (ROS). Isolated Langendorff-perfused rat hearts were subjected to 35 minutes of global ischemia (I) followed by 120 minutes of reperfusion (I/R). Hearts were assigned to a control group (Con) or a sevoflurane (Sevo) group receiving 3 times 5-minute episodes of sevoflurane (2.5vol%) before I/R. Phosphorylation of both AMPK and endothelial nitric oxide synthase (eNOS) were determined by Western blot analysis. Cardioprotection was assessed after I/R from recovery of left ventricular pressure and from infarct size (triphenyltetrazolium chloride staining). In the control group, ischemia resulted in a 2-fold increase in phosphorylation levels of AMPK (Con 0.13+/-0.01 versus Con-I 0.28+/-0.05, P<0.05), which was sustained after 120 minutes of reperfusion (Con-I/R 0.26+/-0.02, P<0.05). Sevoflurane preconditioning had no affect on AMPK phosphorylation before ischemia (Sevo 0.12+/-0.03, P>0.05), but almost doubled the increase in AMPK phosphorylation relative to control after ischemia (Sevo-I 0.48+/-0.09, P<0.05), an effect that was sustained after reperfusion (Sevo-I/R 0.49+/-0.12, P<0.05). The AMPK-inhibitor compound C (10 micromol/L) reduced the sevoflurane-mediated increase in phosphorylation of AMPK and its target eNOS and abolished cardioprotection. The ROS-scavenger n-(2-mercaptopropionyl)-glycine (1 mmol/L) blunted the sevoflurane-mediated increase in AMPK and eNOS phosphorylation and prevented cardioprotection. Sevoflurane-induced AMPK activation protects the heart against ischemia and reperfusion injury and relies on upstream production of ROS.

Abstract 2321: Reactive Oxygen Species-Induced Activation of 5′AMP-Activated Protein Kinase Mediates Cardioprotection Against Ischemia and Reperfusion Injury

The myocardium possesses intrinsic protective mechanisms against ischemia and reperfusion injury (I/R). 5′AMP-activated protein kinase (AMPK) is known as regulator of cellular energy status and is reduced during diabetes mellitus. Recently, AMPK is also implicated in ischemic preconditioning leading to cardioprotection against I/R. We hypothesize that AMPK is involved in anesthetic-induced cardioprotection and that this AMPK activation is evoked by production of reactive oxygen species (ROS). Isolated Langendorff-perfused rat hearts were subjected to 35 minutes of global ischemia followed by 120 minutes of reperfusion. Hearts were divided into 2 groups: a Control group and a Sevo group receiving three times 5-minute episodes of 2.5 vol% sevoflurane before I/R. AMPK phosphorylation was determined with Western Blot analysis. Cardioprotection was assessed as recovery of left ventricular pressure after I/R and as infarct size using triphenyltetrazolium chloride staining. In the Control group, I/R resulted in a twofold increase in phosphorylation levels of AMPK (Control 1.0 ± 0.1 vs. Control-I/R: 2.3 ± 0.1 a.u., n = 4, p &lt; 0.05). Sevoflurane preconditioning did not immediately, prior to ischemia, affect the AMPK phosphorylation (Sevo 0.9 ± 0.2 vs. Control 1.0 ± 0.2, n = 6), but doubled the increase in AMPK phosphorylation to control after ischemia (Sevo-I: 2.0 ± 0.5 (vs. Control-I), n = 6, p &lt; 0.05), as well as after I/R (Sevo-I/R: 2.1 ± 0.3 vs. (Control-I/R), n = 6, p &lt; 0.05). The AMPK-inhibitor compound C (1 and 10 μM) reduced the increase in AMPK phosphorylation and abolished the cardioprotection derived from functional recovery and infarct size. In addition, the ROS-scavenger n-(2-mercaptopropionyl)-glycine (MPG, 1mM) also reduced the sevoflurane-mediated increase in AMPK phosphorylation and completely prevented cardioprotection. These results demonstrate for the first time a direct link between AMPK activation and the production of ROS in cardioprotection. We conclude that anesthetic-induced AMPK activation protects the heart against I/R and relies on production of ROS, which might be especially important in the context of impaired cardioprotection in the diabetic myocardium.

Granulocyte Colony-Stimulating Factor Prevents Reperfusion Injury After Heart Preservation

Heart transplantation is an accepted method of treatment for selected patients with end-stage heart disease. Making prolonged heart preservation safer will benefit patients awaiting heart transplantation. Granulocyte colony-stimulating factor (G-CSF) exhibited protective effects against myocardial ischemia-reperfusion injury mediated through the Janus kinase (Jak)/(signal transducer and activator of transcription (Stat) pathway. We examined whether pharmacologic preconditioning with G-CSF improves cardiac function after heart preservation. Male rats were divided into four groups: group A, saline injection; group B, G-CSF, 10 microg/kg; group C, G-CSF, 100 microg/kg; and group D, G-CSF, 100 microg/kg plus AG490 (a selective Jak2 inhibitor), 1 mg/kg. The G-CSF and AG490 were given intravenously for 3 consecutive days. Four hours after the final treatment, isolated rat hearts underwent 12 hours of hypothermic (4 degrees C) preservation, followed by 60 minutes of normothermic reperfusion. Stat3 phosphorylation was observed in the heart at 15 minutes after G-CSF treatment in group C, but this was attenuated by additional treatment with AG 490 in group D. Compared with group A, group C exhibited significant recovery of left ventricular pressure, maximum positive rate of left ventricular developed pressure (Max dP/dt), and coronary flow (p < 0.05, respectively), as well as lower creatine phosphokinase leakage during reperfusion (p < 0.05). Group B and group D did not show significant hemodynamic recovery during reperfusion. In group C, increased Bcl-xL and decreased Bax expressions as well as decreased terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL)-positive cardiomyocytes were observed after reperfusion. Immunohistochemical examination showed significantly increased capillary density before hypothermic preservation in group C, but not in other groups. Pharmacologic preconditioning with G-CSF protected hearts from prolonged hypothermic ischemia-reperfusion injury.

Different effect of acute heart failure on stepwise frequency bands of QRS complex in electrocardiogram

We found that high frequency component of QRS complex in EKG decrease in acute heart failure after open‐heart surgery. It became one of the indexes to recognize degree of heart failure electrically. However, which frequency bands of QRS complex are most sensitive to heart failure has not been identified. To clarify this, relationship between voltage of stepwise frequency bands (1–20Hz, 20–40Hz, 40–80Hz, &gt;80Hz) of QRS complex and left ventricular pressure (LVP) was examined in 4‐minopyridine (4‐AP) induced rat heart failure model. LVP was decreased by use of 4‐AP (from 82+/−23 to 37+/−20 mmHg: p&lt;0.005). Although lower frequency bands (1–20Hz, 20–40Hz) were unchanged, higher frequency bands (40–80Hz, &gt;80Hz) were decreased from 4.1+/−2.0 to 1.6+/−1.4 (mV)^2 (p&lt;0.01), from 3.0+/−1.2 to 0.3+/−0.4 (p&lt;0.0001) respectively when LVP decreased. After recovery of LVP, voltage of high frequency both bands returned to pre‐treatment level. Most sensitive frequency bands to heart failure was bands of &gt;80Hz. Since Ca2+ overload to cardiomyocytes decreased high frequency component of QRS complex from our previous study, elevation of intracellular Ca2+ concentration during heart failure may pivotal role in this phenomenon.

Adenosine A 1 receptor mediates delayed cardioprotective effect of sildenafil in mouse

Sildenafil induces powerful cardioprotection against ischemia/reperfusion (I/R) injury. Since adenosine is known to be a major trigger of ischemic preconditioning, we hypothesized that A 1 adenosine receptor (A 1AR) activation plays a role in sildenafil-induced cardioprotective signaling. Adult male C57BL wild-type (WT) mice or their corresponding A 1AR knockout (A 1AR-KO) mice were treated intraperitoneally (i.p.) with either sildenafil (0.71 mg/kg, equivalent to 50 mg dose for a 70 kg patient) or volume-matched saline. The selective A 1AR antagonist 8-cyclopentyl-1,3-dipropyxanthine (DPCPX; 0.1 mg/kg, i.p.) was administered 30 min before sildenafil. The hearts were isolated 24 h later and subjected to 30 min of global ischemia and 1 h of reperfusion in Langendorff mode. Post-ischemic myocardial infarct size (mean ± SEM; % of risk area) was reduced in C57BL-WT mice treated with sildenafil (5.6 ± 0.9) versus saline control group (27.3 ± 2.1; p < 0.05; n = 6/each). However, sildenafil failed to protect the A 1AR-KO hearts (31.6 ± 1.9 vs. 32.3 ± 1.5 with saline, p > 0.05). Additionally, DPCPX treatment abolished the infarct limiting effect of sildenafil (27.3 ± 3.2, p < 0.05). DPCPX alone had no effect on infarct size as compared with the control group. No significant changes in post-ischemic recovery of left ventricular pressure and heart rate were observed in the sildenafil-treated group. We further examined the effect of sildenafil in protection against simulated ischemia and reoxygenation injury in adult cardiomyocytes derived from WT and A 1AR-KO mice. WT myocytes treated with sildenafil (1 μM) demonstrated significantly lower trypan blue-positive necrotic cells. However, cardiomyocytes derived from A 1AR-KO mice or DPCPX-treated WT cells failed to show protection against necrosis with sildenafil. These results suggest that A 1AR activation following treatment with sildenafil plays an integral role in the signaling cascade responsible for delayed protection against global I/R injury.

Cardioprotective Effects of Eplerenone in the Rat Heart

Mineralocorticoid receptor antagonism with eplerenone reduces mortality in heart failure, possibly because of blockade of the deleterious effects of aldosterone. To investigate these effects, rat Langendorff hearts were exposed to aldosterone and/or eplerenone. Under normal conditions, aldosterone increased left ventricular pressure and decreased coronary flow. Eplerenone did not block these effects. Eplerenone reduced infarct size (from 68+/-2% to 53+/-4%; P<0.05) and increased left ventricular pressure recovery (from 44+/-2% to 60+/-5%; P<0.05) after 45 minutes of coronary artery occlusion and 3 hours of reperfusion, whereas aldosterone did not affect these parameters. To verify the origin of cardiac aldosterone, hearts were perfused with 3 to 30 nmol/L aldosterone and either frozen immediately or exposed to washout. Without washout, cardiac aldosterone was 1.5 times aldosterone in coronary effluent (CE), that is, too high to be explained on the basis of its presence in extracellular fluid. The cardiac levels of aldosterone correlated with its CE levels (r=0.81; P<0.01), and both were unaffected by eplerenone. During washout, tissue aldosterone disappeared monophasically (half life, 9+/-1 minutes), and CE aldosterone disappeared biphasically (half life 1+/-0 and 8+/-1 minutes, respectively). During buffer perfusion, cardiac aldosterone was at or below the detection limit. In conclusion, eplerenone improves the condition of the heart after ischemia and reperfusion. This does not relate to interference with the inotropic and vasoconstrictor effects of aldosterone. The majority of cardiac aldosterone, if not all, is derived from the circulation. The rapid, mineralocorticoid receptor-independent kinetics of aldosterone suggest that its accumulation in the heart involves cell surface binding rather than internalization.

The role of calcitonin gene-related peptide (CGRP) in ischemic preconditioning in isolated rat hearts

Brief coronary artery occlusion can protect the heart against damage during subsequent prolonged coronary artery occlusion; ischemic preconditioning. The role of calcitonin gene-related peptide (CGRP) in ischemic preconditioning is investigated in isolated perfused rat hearts, by measuring CGRP release during ischemic preconditioning and mimicking this by exogenous CGRP infusion, either in the absence or presence of the CGRP antagonist BIBN4096BS. CGRP increased left ventricular pressure and coronary flow in a concentration dependent manner, which was effectively antagonized by BIBN4096BS. Rat hearts ( n = 36) were subjected to 45 min coronary artery occlusion and 180 min reperfusion, which was preceded by: (1) sham pretreatment, (2) BIBN4096BS infusion (1 μM), (3) preconditioning by 15 min coronary artery occlusion and10 min reperfusion, (4) as 3, but with BIBN4096BS, (5) 15 min CGRP infusion (5 nM) and 10 min washout, (6) as 5, but with BIBN4096BS. Cardiac protection was assessed by reactive hyperaemia, creatine kinase release, infarct size related to the area at risk (%), and left ventricular pressure recovery. Preconditioning increased CGRP release into the coronary effluent from 88 ± 13 to 154 ± 32 pg/min/g, and significantly protected the hearts by decreasing reactive hyperaemia (35%), reducing creatine kinase release (53%), limiting infarct size (48%), and improving left ventricular pressure recovery (36%). Exogenous CGRP induced preconditioning-like cardioprotection. BIBN completely abolished the cardioprotection induced by preconditioning as well as by exogenous CGRP. In conclusion, since cardioprotection of preconditioning-induced CGRP release can be mimicked by exogenous CGRP, and both can be blocked by a CGRP antagonist, results indicate an important role for CGRP in ischemic preconditioning.

Bradykinin Preconditioning Improves the Profile of Cell Survival Proteins and Limits Apoptosis After Cardioplegic Arrest

We hypothesized that preconditioning the heart with bradykinin (BK) would improve the profile of antiapoptotic proteins and inhibit myocardial apoptosis. Eighteen rabbit hearts were retrogradely perfused with Krebs-Henseleit buffer (KHB). Six control hearts were perfused with KHB for 90 minutes without cardioplegia ischemia. Six hearts were arrested for 30 minutes (37 degrees C) with crystalloid cardioplegia (CCP). Six BK preconditioning (BKPC) hearts received a 10-minute coronary infusion of 10(-8) M BK-enriched KHB followed by a 5-minute recovery period and were then arrested for 30 minutes with CCP. The hearts were reperfused for 30 minutes with KHB. BKPC significantly improved the recovery of left ventricular pressure (73+/-5 versus 51+/-4 mm Hg; P<0.05) and reduced the percentage of myocardial apoptosis (3.4+/-0.3% versus 1.2+/-0.2%; P<0.05) as compared with CCP. There were no significant differences in total protein levels of caspase 3, Bcl-2, Bad, and Bax, among the groups. Both BKPC and CCP induced phosphorylation of Bad at Ser112, but the BKPC group had higher phosphorylated Bad than CCP (4.4+/-0.5 versus 2.0+/-0.3; P<0.05). Both BKPC and CCP alone increased caspase 3 cleavage and activity as compared with controls (P<0.05 and P<0.01, respectively), but BKPC caused less cleavage and activation of caspase 3 than CCP alone (P<0.05). BKPC increased Bad phosphorylation, inhibited caspase 3 activation, and limited myocardial apoptosis, which were associated with improvement of left-ventricular performance. These results identify novel molecular mechanisms underlying the protective effects of BKPC during cardiac surgery.

Erythropoietin improves left ventricular function and coronary flow in an experimental model of ischemia‐reperfusion injury

Recent studies show that erythropoietin (EPO) plays a protective role in brain ischemia. In this condition, administration of EPO protects neurons from ischemic damage. Recently, it has been shown that in patients with chronic heart failure (CHF), EPO treatment improved cardiac function. In the present study we assessed the role of EPO and EPO-receptor (EPO-R) in the heart. We studied the presence and functionality of the EPO-R in isolated rat hearts in the Langendorff set-up. Hearts were perfused for 20 min with 10 U/ml EPO or vehicle. Immunohistochemistry revealed the presence of the EPO-R on endothelial cells, fibroblasts and to a lesser extent cardiomyocytes. Furthermore, perfusion with EPO resulted in a 50% increase in the phosphorylated MAP kinases p42/p44. To evaluate the protective role of EPO in cardiac ischemia, we performed low-flow (0.6 ml/min) ischemia/reperfusion experiments in isolated rat hearts. Administration of EPO (10 U/ml) reduced the cellular damage by 56% (P<0.05) during reperfusion, diminished apoptosis by 15% (P<0.05) and resulted in a significantly improved recovery of left ventricular pressure (P=0.02) and coronary flow (P=0.01). The present data suggest that a functional EPO-R is present in rat adult cardiac tissue and that exogenous EPO administration improves cardiac function after ischemia/reperfusion injury.

Isoflurane preconditions myocardium against infarction via release of free radicals.

Isoflurane exerts cardioprotective effects that mimic the ischemic preconditioning phenomenon. Generation of free radicals is implicated in ischemic preconditioning. The authors investigated whether isoflurane-induced preconditioning may involve release of free radicals. Sixty-one alpha-chloralose-anesthetized rabbits were instrumented for measurement of left ventricular (LV) pressure (tip-manometer), cardiac output (ultrasonic flowprobe), and myocardial infarct size (triphenyltetrazolium staining). All rabbits were subjected to 30 min of occlusion of a major coronary artery and 2 h of subsequent reperfusion. Rabbits of all six groups underwent a treatment period consisting of either no intervention for 35 min (control group, n = 11) or 15 min of isoflurane inhalation (1 minimum alveolar concentration end-tidal concentration) followed by a 10-min washout period (isoflurane group, n = 12). Four additional groups received the radical scavenger N-(2-mercaptoproprionyl)glycine (MPG; 1 mg. kg-1.min-1) or Mn(III)tetrakis(4-benzoic acid)porphyrine chloride (MnTBAP; 100 microg.kg-1.min-1) during the treatment period with (isoflurane + MPG; n = 11; isoflurane + MnTBAP, n = 9) or without isoflurane inhalation (MPG, n = 11; MnTBAP, n = 7). Hemodynamic baseline values were not significantly different between groups (LV pressure, 97 +/- 17 mmHg [mean +/- SD]; cardiac output, 228 +/- 61 ml/min). During coronary artery occlusion, LV pressure was reduced to 91 +/- 17% of baseline and cardiac output to 94 +/- 21%. After 2 h of reperfusion, recovery of LV pressure and cardiac output was not significantly different between groups (LV pressure, 83 +/- 20%; cardiac output, 86 +/- 23% of baseline). Infarct size was reduced from 49 +/- 17% of the area at risk in controls to 29 +/- 19% in the isoflurane group (P = 0.04). MPG and MnTBAP themselves had no effect on infarct size (MPG, 50 +/- 14%; MnTBAP, 56 +/- 15%), but both abolished the preconditioning effect of isoflurane (isoflurane + MPG, 50 +/- 24%, P = 0.02; isoflurane + MnTBAP, 55 +/- 10%, P = 0.001). Isoflurane-induced preconditioning depends on the release of free radicals.

Effects of sustained low-flow ischemia and reperfusion on Ca2+ transients and contractility in perfused rat hearts.

We investigated changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and in left ventricular contractility during sustained ischemia and reperfusion in isolated beating rat hearts. Hearts from male Sprague-Dawley rats were perfused retrogradely and were loaded with 4 microM fura-2. Low-flow global ischemia was induced by reducing perfusion flow to 10% and by electric pacing. The hearts were exposed to ischemia for 10 min or 30 min and then reperfused. [Ca2+]i was measured by monitoring the ratio of 500 nm fluorescence excited at 340 and 380 nm while simultaneously measuring left ventricular pressure (LVP). To determine diastolic [Ca2+]i, background autofluorescence was subtracted. LVP rapidly decreased from 82.3+/-8.2 to 17.1+/-2.9 mmHg , whereas the amplitude of the Ca2+ transient did not change significantly during the first 1 min of ischemia. After 10 min of ischemia, the amplitude decreased to 60.8+/-10.6% (p < 0.05) and diastolic [Ca2+]i increased by 26.3+/-2.9% (p < 0.001) compared with the pre-ischemic value (n = 8). When the hearts were reperfused after 10 min of ischemia, the amplitude of the Ca2+ transient and LVP recovered to 79.0+/-7.2% and 73.2+/-7.5 mmHg, respectively. Whereas diastolic [Ca2+]i decreased to the preischemic value. In the hearts exposed to 30 min of ischemia (n = 10), diastolic [Ca2+]i increased even further by 32.7+/-5.3% at the end of ischemia and continued increasing during the 10 min of reperfusion by 42.6+/-15.6%. Six of 10 hearts developed ventricular fibrillation (VF) and intracellular Ca2+ overload after reperfusion. Recovery of LVP after reperfusion was significantly smaller in the hearts exposed to 30 min of ischemia than in the hearts exposed to 10 min of ischemia (58.9+/-11.7 vs. 97.2+/-3.0% of pre-ischemic value, p < 0.05). Diastolic [Ca2+]i also increased under hypoxic conditions (N2 bubbling) in this model. These results suggest that increases in diastolic [Ca2+]i might play an important role in myocardial contractile dysfunction and viability in ischemia-reperfusion injury.

Role of tissue factor-mediated coagulation in ischemia/ reperfusion-induced injury of Langendorf-perfused rabbit hearts.

Production of oxygen free radicals, and activation of neutrophils and plasma complement contribute to myocardial reperfusion injury, but the role of coagulation has not been assessed. To characterize tissue-factor-mediated generation of thrombin and its association with tissue injury during reperfusion from normothermic ischemia of isolated, Langendorf-perfused rabbit hearts. Activation of coagulation was assessed by addition of 12% rabbit plasma and human fibrinogen to Krebs-Henseleit-buffer perfusate with measurement of levels of human fibrinopeptide A (hFPA) in the heart effluent as an index of thrombin-mediated formation of fibrin. Concentrations of hFPA in the effluent were minimal during non-ischemic perfusion (5 +/- 5 ng/ml, n=6) and during 50 min of ischemia (13 +/- 3 ng/ml, n=6), but increased markedly during the first 20 min of reperfusion (to 41 +/- 29 ng/ml, P=0.03 versus before reperfusion). Addition to the perfusate of 10 microg/ml recombinant human tissue-factor-pathway inhibitor, the physiologic inhibitor of tissue-factor-mediated coagulation, abolished increases in the level of hFPA after reperfusion. However, indexes of myocardial injury manifested during reperfusion, including decrease in recovery of left ventricular pressure developed, increase in left ventricular end-diastolic pressure, and increase in activity of creatine kinase in the heart effluent, were not improved by anticoagulation with recombinant human tissue-factor-pathway inhibitor. Our results do not support the hypothesis that coagulation plays a major role in ischemia/reperfusion injury of Langendorf-perfused rabbit hearts.

Blocking Na+–H+Exchange by Cariporide Reduces Na+-overload in Ischemia and is Cardioprotective

In myocardial ischemia, rapid inactivation of Na+–K+-ATPase and continuing influx of sodium induce Na+-overload which is the basis of Ca2+-overload and irreversible tissue injury following reperfusion. The Na+–H+-exchanger of subtype 1 (NHE-1) is assumed to play a major role in this process, but previously available inhibitors were non-specific and did not allow to verify this hypothesis. Cariporide (HOE 642) is a recently synthesized NHE-1 inhibitor. We have investigated its effects on Na+homeostasis (23Na NMR spectroscopy), cardiac function and energy metabolism (31P NMR) in ischemia and reperfusion. In the well-oxygenated, isolated guinea-pig heart, cariporide (10 μ m) had no effect on intracellular Na+, pH or cardiac function. NHE-1 inhibition by cariporide was demonstrated using the NH4Cl prepulse technique. When hearts were subjected to 15 min of ischemia, cariporide markedly inhibited intracellular Na+-accumulation (1.3±0.1 vs 2.1±0.1-fold rise) but had no effect on the decline in pHi. In reperfusion, NHE-1-blockade significantly delayed pH recovery. With longer periods of ischemia (36 min), cariporide delayed the onset of contracture, reduced ATP depletion, Na+-overload and again had no effect on pH. In reperfusion, hearts treated with cariporide showed an improved recovery of left ventricular pressure (60±1 vs 16±8 mmHg); end-diastolic pressure was normalized and phosphocreatine fully recovered, while there was only a partial recovery in controls. The data demonstrate that Na+–H+-exchange is an important port of Na+-entry in ischemia and contributes to H+-extrusion in reperfusion. By reducing Na+-overload in ischemia and prolonging acidosis in reperfusion, NHE-blockade represents a promising cardioprotective principle.

Intracellular calcium dynamics in mouse model of myocardial stunning.

Intracellular calcium (Cai2+) and left ventricular (LV) function were determined in the coronary-perfused mouse heart to study Cai2+-related mechanisms of injury from myocardial ischemia and reperfusion. Specifics for loading of the photoprotein aequorin into isovolumically contracting mouse hearts under constant-flow conditions are provided. The method allows detection of changes in Cai2+ on a beat-to-beat basis in a model of myocardial stunning and permits correlation of interventions that regulate Ca2+ exchange with functional alterations. Twenty-three coronary-perfused mouse hearts were subjected to 15 min of ischemia followed by 20 min of reperfusion. In 13 hearts, the perfusate included the calmodulin antagonist W7 (10 microM) to inhibit Ca(2+)-calmodulin-regulated mechanisms. Peak Cai2+ was 0.77 +/- 0.03 microM in the control group and was unaffected by W7 at baseline. Ischemia was characterized by a rapid decline in LV function, followed by ischemic contracture, accompanied by a gradual rise in Cai2+. Reperfusion was characterized by an initial burst of Cai2+ and a gradual recovery to nearly normal systolic Cai2+ while LV pressure recovered to 55% after 20 min of reperfusion (stunned myocardium). These results in the mouse heart confirm that stunning does not result from deficiency of Cai2+ but rather from a decreased myofilament responsiveness to Cai2+ due to changes in the myofilaments themselves. In hearts perfused with W7, the rise in Cai2+ during ischemia was significantly attenuated, as was the magnitude of mean Cai2+ during early reflow. Ischemic contracture was abolished or delayed. Hearts perfused with W7 showed significantly improved recovery of LV pressure, rate of contraction, and rate of relaxation. Diastolic Cai2+ was increased in control hearts during stunning but returned to baseline in hearts perfused with W7. Simultaneous assessment of Cai2+ and LV function demonstrates that calmodulin-regulated mechanisms may contribute to the pathogenesis of myocardial stunning in the mouse heart.

The effect of initial flush for cooling and coronary vascular washout in heart preservation

Initial flush for cooling and coronary vascular washout using University of Wisconsin (UW) solution has presented some problems. We used a separate solution for initial flush and for subsequent storage, and evaluated the effect of initial flush for prolonged heart preservation. Following potassium arrest, the initial flush was performed using UW solution (UW group) or Physiol · 3® solution with additional potassium (PK group). In the PK group, an additional flush with the UW solution was done prior to preservation. The hearts of both groups were stored in the cold UW solution for 12 hours. Myocardial high-energy phosphates were measured using phosphorus 31 nuclear magnetic resonance spectroscopy (UW group: n = 7, PK group: n = 5). Adenosine triphosphate (ATP) levels in the UW group decreased to 23% of the control after storage. ATP levels in the PK group were well maintained for up to 6 hours, and then decreased to 51% (p < 0.01). Orthotopic transplantation was performed in 10 pairs of dogs (UW group: n = 7, PK group: n = 3). Preserved grafts in the PK group (74%) tended to show better recovery of left ventricular pressure than those in the UW group (52%). These results suggest that the introduction of an initial flush may prevent ATP level reduction during cold storage, and may provide good results after transplantation. An initial flush for cooling and coronary vascular washout is important for prolonged heart preservation.

The Effect of Initial Flush for Cooling and Coronary Vascular Washout in Heart Preservation

Initial flush for cooling and coronary vascular washout using University of Wisconsin (UW) solution has presented some problems. We used a separate solution for initial flush and for subsequent storage, and evaluated the effect of initial flush for prolonged heart preservation. Following potassium arrest, the initial flush was performed using UW solution (UW group) or Physiol · 3® solution with additional potassium (PK group). In the PK group, an additional flush with the UW solution was done prior to preservation. The hearts of both groups were stored in the cold UW solution for 12 hours. Myocardial high-energy phosphates were measured using phosphorus 31 nuclear magnetic resonance spectroscopy (UW group: n = 7, PK group: n = 5). Adenosine triphosphate (ATP) levels in the UW group decreased to 23% of the control after storage. ATP levels in the PK group were well maintained for up to 6 hours, and then decreased to 51% (p < 0.01). Orthotopic transplantation was performed in 10 pairs of dogs (UW group: n = 7, PK group: n = 3). Preserved grafts in the PK group (74%) tended to show better recovery of left ventricular pressure than those in the UW group (52%). These results suggest that the introduction of an initial flush may prevent ATP level reduction during cold storage, and may provide good results after transplantation. An initial flush for cooling and coronary vascular washout is important for prolonged heart preservation.

Effects of myocardial ischemia on the release of cardiac troponin I in isolated rat hearts

Background: The twofold aim of this experimental study was (1) to verify the correlation between the duration of ischemia and concentration of cardiac troponin I and (2) to compare the release of cardiac troponin I with histologic findings. Methods: Experiments were done on 18 rat hearts, which were perfused according to the Langendorff method, immediately after excision in group I (control group) and after immersion for 3 hours (group II) and 6 hours (group III) in St. Thomas' Hospital solution at 4º C. During reperfusion, the release of cardiac troponin I, creatine kinase isoenzyme MB, and lactate dehydrogenase, the recovery of left ventricular pressure, and heart rates were compared among the three groups. After the experiment, three samples of myocardium (left ventricle, right ventricle, and septum) were taken for histologic examination. Results: Cardiac troponin I concentration was significantly higher in group III than in groups I and II and in group II compared with group I. Cardiac troponin I concentration increased as the ischemic period increased. The relation between cardiac troponin I release and ischemic duration tended to be linear. Creatine kinase MB and lactate dehydrogenase concentrations did not differ from one group to the other. Left ventricular pressure was not significantly different among the groups. In the control group, no heart had more than 10% of the myocytes affected. One of six hearts in group II and three of six in group III had more than 10% of myocytes affected. Conclusion: This experimental study showed (1) that cardiac troponin I is an early marker of ischemic injury and (2) that cardiac troponin I concentration increases as the ischemic period increases. Early cardiac troponin I release appears to correlate with the extent of ischemic injury in rats undergoing buffer perfusion. (J T horac C ardiovasc S urg 1996;112:508-13)

DIMINISHED HEAT SHOCK RESPONSE IN THE AGED MYOCARDIUM 845

Exercise or heat induction of heat shock proteins (HSPs), particularly HSP 72, has been associated with an enhanced post-ischemic myocardial recovery. To determine if hearts from aged animals demonstrate an altered heat shock response and subsequent myocardial protection, adult (6 months old) and aged(22 months old) Fisher 344 rats were heat stressed by raising their rectal temperatures to 41°C for 10 min. At selected times following heat stress,(0-24 hours) hearts were examined for heat shock transcription factor trimerization and DNA binding activity (HSF activation), HSP 72 and HSF content as well as protection from ischemia using the Langendorff isolated heart model. Following heat stress, compared to hearts from adults, hearts from aged animals demonstrated a 47% reduction in HSF activation, and a 35% reduction in HSP 72 protein content. Interestingly, HSF protein content was similar between aged and adult. Hearts from heat stressed adult animals (24 h prior) demonstrated an enhanced post-ischemic recovery as indicated by a greater recovery of left ventricular pressure (p<0.05), while hearts from heat stressed aged animals failed to demonstrate an enhanced post-ischemic recovery. These results suggest that hearts from aged animals exhibit an impaired ability to produce the protective HSPs and thus may explain, at least in part, the increased susceptibility of aged hearts to episodes of stress.