Jianzhong An

Sevoflurane before or after Ischemia Improves Contractile and Metabolic Function while Reducing Myoplasmic Ca2+Loading in Intact Hearts

Background Ca2+ loading occurs during myocardial reperfusion injury. Volatile anesthetics can reduce reperfusion injury. The authors tested whether sevoflurane administered before index ischemia in isolated hearts reduces myoplasmic diastolic and systolic [Ca2+] and improves function more so than when sevoflurane is administered on reperfusion. Methods Four groups of guinea pig hearts were perfused with crystalloid solution (55 mmHg, 37°C): (1) no treatment before 30 min global ischemia and 60 min reperfusion (CON); (2) 3.5 vol% sevoflurane administered for 10 min before ischemia (SBI); (3) 3.5 vol% sevoflurane administered for 10 min after ischemia (SAI); and (4) 3.5 vol% sevoflurane administered for 10 min before and after ischemia (SBAI). Phasic myoplasmic diastolic and systolic [Ca2+] were measured in the left ventricular free wall with the fluorescence probe indo-1. Results Ischemia increased diastolic [Ca2+] and diastolic left ventricular pressure (LVP). In CON hearts, initial reperfusion greatly increased diastolic [Ca2+] and systolic [Ca2+] and reduced contractility (systolic–diastolic LVP, dLVP/dtmax), relaxation (diastolic LVP, dLVP/dtmin), myocardial oxygen consumption (Mvo2), and cardiac efficiency. SBI, SAI, and SBAI each reduced ventricular fibrillation, attenuated increases in systolic and systolic–diastolic [Ca2+], improved contractile and relaxation indices, and increased coronary flow, percent oxygen extraction, Mvo2, and cardiac efficiency during 60 min reperfusion compared with CON. SBI was more protective than SAI, and SBAI was generally more protective than SAI. Conclusions Sevoflurane improves postischemic cardiac function while reducing Ca2+ loading when it is administered before or after ischemia, but protection is better when it is administered before ischemia. Reduced Ca2+ loading on reperfusion is likely a result of the anesthetic protective effect.

Modulation of myocardial function and [Ca2+] sensitivity by moderate hypothermia in guinea pig isolated hearts

Cardiac hypothermia alters contractility and intracellular Ca2+ concentration ([Ca2+]i) homeostasis. We examined how left ventricular pressure (LVP) is altered as a function of cytosolic [Ca2+]iover a range of extracellular CaCl2 concentration ([CaCl2]e) during perfusion of isolated, paced guinea pig hearts at 37°C, 27°C, and 17°C. Transmural LV phasic [Ca2+] was measured using the Ca2+ indicator indo 1 and calibrated (in nM) after correction was made for autofluorescence, temperature, and noncytosolic Ca2+. Noncytosolic [Ca2+]i, cytosolic diastolic and systolic [Ca2+]i, phasic [Ca2+]i, and systolic Ca2+ released per beat (area Ca2+) were plotted as a function of 0.3-4.5 mM [CaCl2]e, and indexes of contractility [LVP, maximal rates of LVP development (+dLVP/d t) and relaxation (-dLVP/d t), and the integral of the LVP curve per beat (LVParea)] were plotted as a function of [Ca2+]i. Hypothermia increased systolic [Ca2+]iand slightly changed systolic LVP but increased diastolic LVP and [Ca2+]i. The relationship of diastolic and noncytosolic [Ca2+] to [CaCl2]ewas shifted upward at 17°C and 27°C, whereas that of phasic [Ca2+]ito [CaCl2]ewas shifted upward at 17°C but not at 27°C. The relationships of phasic [Ca2+]ito developed LVP, +dLVP/d t, and LVParea were progressively reduced by hypothermia so that maximal Ca2+-activated LVP decreased and hearts were desensitized to Ca2+. Thus mild hypothermia modestly increases diastolic and noncytosolic Ca2+ with little effect on systolic Ca2+ or released (area) Ca2+, whereas moderate hypothermia markedly increases diastolic, noncytosolic, peak systolic, and released Ca2+ and results in reduced maximal Ca2+-activated LVP and myocardial sensitivity to systolic Ca2+.Cardiac hypothermia alters contractility and intracellular Ca2+ concentration ([Ca2+]i) homeostasis. We examined how left ventricular pressure (LVP) is altered as a function of cytosolic [Ca2+]i over a range of extracellular CaCl2 concentration ([CaCl2]e) during perfusion of isolated, paced guinea pig hearts at 37 degrees C, 27 degrees C, and 17 degrees C. Transmural LV phasic [Ca2+] was measured using the Ca2+ indicator indo 1 and calibrated (in nM) after correction was made for autofluorescence, temperature, and noncytosolic Ca2+. Noncytosolic [Ca2+]i, cytosolic diastolic and systolic [Ca2+]i, phasic [Ca2+]i, and systolic Ca2+ released per beat (area Ca2+) were plotted as a function of 0.3-4.5 mM [CaCl2]e, and indexes of contractility [LVP, maximal rates of LVP development (+dLVP/dt) and relaxation (-dLVP/dt), and the integral of the LVP curve per beat (LVParea)] were plotted as a function of [Ca2+]i. Hypothermia increased systolic [Ca2+]i and slightly changed systolic LVP but increased diastolic LVP and [Ca2+]i. The relationship of diastolic and noncytosolic [Ca2+] to [CaCl2]e was shifted upward at 17 degrees C and 27 degrees C, whereas that of phasic [Ca2+]) to [CaCl2]e was shifted upward at 17 degrees C but not at 27 degrees C. The relationships of phasic [Ca2+]i to developed LVP, +dLVP/dt, and LVP(area) were progressively reduced by hypothermia so that maximal Ca2+-activated LVP decreased and hearts were desensitized to Ca2+. Thus mild hypothermia modestly increases diastolic and noncytosolic Ca2+ with little effect on systolic Ca2+ or released (area) Ca2+, whereas moderate hypothermia markedly increases diastolic, noncytosolic, peak systolic, and released Ca2+ and results in reduced maximal Ca2+-activated LVP and myocardial sensitivity to systolic Ca2+.

Reduced Cytosolic Ca2+ Loading and Improved Cardiac Function After Cardioplegic Cold Storage of Guinea Pig Isolated Hearts

BackgroundHypothermia is cardioprotective, but it causes Ca2+ loading and reduced function on rewarming. The aim was to associate changes in cytosolic Ca2+ with function in intact hearts before, during, and after cold storage with or without cardioplegia (CP). Methods and ResultsGuinea pig hearts were initially perfused at 37°C with Krebs-Ringer’s (KR) solution (in mmol/L: Ca2+ 2.5, K+ 5, Mg2+ 2.4). One group was perfused with CP solution (Ca2+ 2.5, K+ 18, Mg2+ 7.2) during cooling and storage at 3°C for 4 hours; another was perfused with KR. LV pressure (LVP), dP/dt, O2 consumption, and cardiac efficiency were monitored. Cytosolic phasic [Ca2+] was calculated from indo 1 fluorescence signals obtained at the LV free wall. Cooling with KR increased diastolic and phasic [Ca2+], whereas cooling with CP suppressed phasic [Ca2+] and reduced the rise in diastolic [Ca2+]. Reperfusion with warm KR increased phasic [Ca2+] 86% more after CP at 20 minutes and did not increase diastolic [Ca2+] at 60 minutes, compared with a 20% increase in phasic [Ca2+] after KR. During early and later reperfusion after CP, there was a 126% and 50% better return of LVP than after KR; during later reperfusion, O2 consumption was 23% higher and cardiac efficiency was 38% higher after CP than after KR. ConclusionsCP decreases the rise in cardiac diastolic [Ca2+] observed during cold storage in KR. Decreased diastolic [Ca2+] and increased systolic [Ca2+] after CP improves function on reperfusion because of reduced Ca2+ loading during and immediately after cold CP storage.

Inhibition of CDKS by roscovitine suppressed LPS-induced ·NO production through inhibiting NFκB activation and BH4 biosynthesis in macrophages

In inflammatory diseases, tissue damage is critically associated with nitric oxide ((*)NO) and cytokines, which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on (*)NO production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of (*)NO induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IkappaB kinase beta (IKKbeta), IkappaB, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun NH(2)-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1beta, and IL-6 but not tumor necrosis factor (TNF)-alpha. Tetrahydrobiopterin (BH(4)), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (BH(2)). Roscovitine significantly inhibited LPS-induced BH(4) biosynthesis and decreased BH(4)-to-BH(2) ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for BH(4) biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced (*)NO production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited (*)NO production, iNOS, and COX-2 upregulation, activation of NFkappaB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced (*)NO production in macrophages by suppressing nuclear factor-kappaB activation and BH(4) biosynthesis, which might be mediated by CDK1, CDK5, and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation.

Sevoflurane preconditioning before moderate hypothermic ischemia protects against cytosolic [Ca2+] loading and myocardial damage in part via mitochondrial KATP channels

Background Brief sevoflurane exposure and washout (sevoflurane preconditioning [SPC]) before 30-min global ischemia at 37°C is known to improve cardiac function, decrease cytosolic [Ca2+] loading, and reduce infarct size on reperfusion. It is not known if anesthetic preconditioning (APC) applies as well to hypothermic ischemia and reperfusion and if KATP channels are involved. The authors examined in guinea pig isolated hearts the effect of sevoflurane exposure before 4-h global ischemia at 17°C on cardiac function, cytosolic [Ca2+] loading, and infarct size. In addition they tested the potential role of the mitochondrial KATP channel in eliciting the cardioprotection by SPC. Methods Hearts were randomly assigned to (1) a nontreated hypothermic ischemia group (CON), (2) a group given 3.5 vol% sevoflurane for 15 min with a 15-min washout before hypothermic ischemia (SPC), and (3) an SPC group in which anesthetic exposure was bracketed with 200 &mgr;m 5-hydroxydecanoate (5-HD) from 5 min before until 5 min after sevoflurane (SPC + 5-HD). Cytosolic [Ca2+] was measured in the left ventricular (LV) free wall with the intracellularly loaded fluorescence probe indo-1. Results Initial reperfusion in CON hearts markedly increased systolic and diastolic [Ca2+] and reduced contractility (dLVP/dtmax), relaxation (diastolic LVP, dLVP/dtmin), myocardial oxygen consumption (Mvo2), and cardiac efficiency. In SPC hearts, cytosolic [Ca2+] overloading (especially diastolic [Ca2+]) was decreased with increased myocardial [Ca2+] influx (d[Ca2+]/dtmax) and efflux (d[Ca2+]/dtmin), improved contractility, relaxation, coronary flow, Mvo2, cardiac efficiency, and decreased infarct size. In SPC + 5HD hearts, the reduction in infarct size was antagonized by 5-HD, but functional return was less affected by 5-HD. Conclusions Anesthetic preconditioning occurs after long-term hypothermic ischemia, and the infarct size reduction is the result, in part, of mitochondrial KATP channel opening.

Na+/H+ exchange inhibition with cardioplegia reduces cytosolic [Ca2+] and myocardial damage after cold ischemia.

Cold cardioplegia protects against reperfusion damage. Blocking Na+/H+ exchange may be as protective as cardioplegia by improving the left ventricular pressure (LVP)-[Ca2+] relationship after cold ischemia. In guinea pig isolated hearts subjected to cold ischemia (4 h, 17°C) and reperfusion, the cardioprotective effects of a Krebs-Ringer (KR) solution, a cardioplegia solution, a KR solution containing the Na+/H+ exchange inhibitor eniporide (1 &mgr;M), and a cardioplegia solution containing eniporide were compared. Treatments were given before and initially after cold ischemia. Systolic and diastolic [Ca2+] were calculated from indo-1 fluorescence transients recorded at the LV free wall. During ischemia, diastolic [Ca2+] increased in each group but more so in the KR group. Peak systolic and diastolic [Ca2+] on initial reperfusion were highest after KR and smallest after cardioplegia + eniporide. After reperfusion, systolic-diastolic LVP (% of baseline) and infarct size (%), respectively, were KR, 47 ± 3%, 37 ± 4%; cardioplegia, 71 ± 5%*, 20 ± 2.2%*; KR + eniporide, 73 ± 5%*, 11 ± 3%*†; and cardioplegia + eniporide 77 ± 3%*, 10 ± 1.4%*† (* P ≤ 0.05 vs KR; †P ≤ 0.05 vs cardioplegia). Ca2+ overload was reduced in each treated group, and most in the cardioplegia + eniporide group, and was associated with the improved function. Inhibition of Na+/H+ exchange was as effective as cardioplegia in restoring function and better than cardioplegia in reducing infarct size after hypothermic ischemia. The combination of cardioplegia and Na+/H+ exchange inhibition did not produce additive protective effects but caused a larger decrease in Ca2+ loading.

Anesthetic preconditioning enhances Ca2+ handling and mechanical and metabolic function elicited by Na+-Ca2+ exchange inhibition in isolated hearts.

Background:Anesthetic preconditioning (APC) is well known to protect against myocardial ischemia–reperfusion injury. Studies also show the benefit of Na+–Ca2+ exchange inhibition on ischemia–reperfusion injury. The authors tested whether APC plus Na+–Ca2+ exchange inhibitors given just on reperfusion affords additive protection in intact hearts. Methods:Cytosolic [Ca2+] was measured by fluorescence at the left ventricular wall of guinea pig isolated hearts using indo-1 dye. Sarcoplasmic reticular Ca2+-cycling proteins, i.e., Ca2+ release channel (ryanodine receptor [RyR2]), sarcoplasmic reticular Ca2+-pump adenosine triphosphatase (SERCA2a), and phospholamban were measured by Western blots. Hearts were assigned to seven groups (n = 8 each): (1) time control; (2) ischemia; (3, 4) 10 &mgr;m Na+–Ca2+ exchange inhibitor KB-R7943 (KBR) or 1 &mgr;m SEA0400 (SEA), given during the first 10 min of reperfusion; (5) APC initiated by sevoflurane (2.2%, 0.41 ± 0.03 mm) given for 15 min and washed out for 15 min before ischemia–reperfusion; (6, 7) APC plus KBR or SEA. Results:The authors found that APC reduced the increase in systolic [Ca2+], whereas KBR and SEA both reduced the increase in diastolic [Ca2+] on reperfusion. Each intervention improved recovery of left ventricular function. Moreover, APC plus KBR or SEA afforded better functional recovery than APC, KBR, or SEA alone (P < 0.05). Ischemia-reperfusion–induced degradation of major sarcoplasmic reticular Ca2+-cycling proteins was attenuated by APC, but not by KBR or SEA. Conclusions:APC plus Na+–Ca2+ exchange inhibition exerts additive protection in part by reducing systolic and diastolic Ca2+ overload, respectively, during ischemia–reperfusion. Less degradation of sarcoplasmic reticular Ca2+-cycling proteins may also contribute to cardiac protection.

Inhibition of Na+/H+ isoform-1 exchange protects hearts perfused after 6-hour cardioplegic cold storage

OBJECTIVES Cardiac ischemia-reperfusion activates Na(+)/H(+) exchange; excess Na(+) and the resulting Ca(2+) overload, through reverse Na(+)/Ca(2+) exchange, cause cellular injury and cardiac dysfunction. We postulated that inhibiting the Na(+)/H(+) isoform-1 exchanger would add to the protection of hearts after long-term cold storage in acidic cardioplegic solution. METHODS Guinea pig hearts were isolated and perfused at 37 degrees C with Krebs-Ringers solution (KRS) and then switched to an acidic St. Thomas solution (STS) at 25 degrees C. Perfusion was stopped at 10 degrees C, and hearts were stored for 6 hours in STS at 3.4 degrees C. On reperfusion to 25 degrees C, hearts were perfused with KRS for 60 minutes. Hearts were divided into 4 groups: sham control (SHAM); eniporide (EPR, EMD96785) IV, 1 mg/kg given IV over 15 minutes before heart isolation; EPR intracoronary, 1 micromol/liter in STS given intracoronary after heart isolation; and EPR IV and intracoronary. RESULTS Values at 60 minutes reperfusion (the percentage of control [100%] before cold storage) are given, respectively, for EPR IV, EPR intracoronary, and EPR IV and intracoronary vs drug-free SHAM (SEM, *p < 0.05 vs SHAM): 72% +/- 3%*, 65% +/- 3%*, and 81% +/- 2%* vs 55% +/- 3% for left ventricular pressure; 94% +/- 3%*, 96% +/- 5%*, and 102% +/- 2%* vs 81% +/- 3% for coronary flow; 60% +/- 2%, 58% +/- 3%, and 74%* +/- 3% vs 58% +/- 4% for cardiac efficiency; 106% +/- 2%*, 108% +/- 3%*, and 107% +/- 2%* vs 116% +/- 4% for percentage of O(2) extraction. Infarct size as percentage of ventricular weight was 20% +/- 3%*, 31% +/- 3%, and 6% +/- 2%* vs 35% +/- 3% (SHAM) after 60 minutes of reperfusion. CONCLUSIONS Na(+)/H(+) isoform-1 exchanger inhibition, particularly if given IV before storage and intracoronary during cooling and rewarming, adds to the protection of cardioplegic solutions.

Contribution of reactive oxygen species to isoflurane-induced sensitization of cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel to pinacidil.

BackgroundMyocardial protection by volatile anesthetics involves activation of cardiac adenosine triphosphate–sensitive potassium (KATP) channels. The authors have previously shown that isoflurane enhances sensitivity of the sarcolemmal KATP channel to the opener, pinacidil. Because reactive oxygen species seem to be mediators in anesthetic preconditioning, the authors investigated whether they contribute to the mechanism of the sensitization effect by isoflurane. MethodsVentricular myocytes were isolated from guinea pig hearts for the whole cell patch clamp recordings of the sarcolemmal KATP channel current (IKATP). Free radical scavengers N-acetyl-l-cysteine, carnosine, superoxide dismutase, and catalase were used to investigate whether reactive oxygen species mediate isoflurane facilitation of the channel opening by pinacidil. A possible role of the mitochondrial KATP channels was tested using a blocker of these channels, 5-hydroxydecanoate. ResultsThe mean density (± SEM) of IKATP elicited by pinacidil (20 &mgr;m) was 18.9 ± 1.8 pA/pF (n = 11). In the presence of isoflurane (0.55 mm), the density of pinacidil-activated IKATP increased to 38.5 ± 2.4 pA/pF (n = 9). Concurrent application of isoflurane and N-acetyl-l-cysteine decreased the sensitization effect by isoflurane in a concentration-dependent manner, whereby the densities of IKATP were 32.6 ± 1.4 (n = 6), 26.2 ± 2.3 (n = 6), and 19.4 ± 2.1 pA/pF (n = 8) at 100, 250, and 500 &mgr;m N-acetyl-l-cysteine, respectively. Concurrent application of isoflurane and carnosine (100 &mgr;m), superoxide dismutase (100 U/ml), or catalase (100 U/ml) attenuated the densities of IKATP to 27.9 ± 2.6, 27.2 ± 2.9, and 25.9 ± 2.2 pA/pF, respectively. None of the scavengers affected activation of IKATP by pinacidil alone. 5-Hydroxydecanoate (100 &mgr;m) did not alter the sensitization effect by isoflurane, and the density of IKATP in this group was 37.1 ± 3.8 pA/pF (n = 6). ConclusionThese results suggest that reactive oxygen species contribute to the mechanism by which isoflurane sensitizes the cardiac sarcolemmal KATP channel to the opener, pinacidil.

Myocardial protection by isoflurane preconditioning preserves Ca2+ cycling proteins independent of sarcolemmal and mitochondrial KATP channels.

INTRODUCTION:Anesthetic preconditioning (APC) with volatile anesthetics improves recovery of contractile function and reduces calcium overload after ischemia/reperfusion (I/R). Mitochondrial and sarcolemmal KATP channel openings have been implicated in APC-induced cardioprotection. In this study, we investigated the effect of APC on major calcium cycling proteins and its relation to KATP channels. METHODS:Isolated perfused rat hearts were divided into seven groups: Time control (n = 10), ischemia control (n = 8), APC (n = 8), Mitochondrial KATP inhibitor 5-hydroxydecanoate (5-HD, 200 &mgr;M, n = 8), Sarcolemmal KATP inhibitor HMR1098 (HMR, 20 &mgr;M, n = 8), and APC plus 5-HD or APC plus HMR1098 (n = 8 each). APC was initiated by administering 1.5% isoflurane for 15 min, followed by a 15 min washout before 30 min of myocardial ischemia and 60 min of reperfusion. Ca2+-release channels (RyR2), Ca2+-adenosine triphosphatase (SERCA2a), phospholamban, plasma membrane Ca2+ ATPase, and sodium–calcium exchanger in the homogenate were determined by Western blot assay. RESULTS:APC improved contractile recovery (left ventricular developed pressure, +dP/dt, −dP/dt) after I/R, which was blocked by 5-HD and HMR. I/R depressed the density of RyR2, SERCA2a, and phospholamban, with no changes in the density of plasma membrane Ca2+ ATPase and sodium–calcium exchanger. APC reversed I/R-induced degradation of RyR2 and SERCA2a in the presence or absence of 5HD and HMR. CONCLUSIONS:I/R-induced depression in cardiac performance is associated with a down-regulation of the major sarcoplasmic reticulum Ca2+-cycling proteins. Anesthesia preconditioning with isoflurane prevents I/R-related degradation of the RyR2 and SERCA2a in the sarcoplasmic reticulum. However, this effect was independent of its activation of KATP channels.