R. Arthur Bouwman

Reactive oxygen species-induced stimulation of 5 ' AMP-activated protein kinase mediates sevoflurane-induced cardioprotection

Background— 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). Methods and Results— 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 &mgr;mol/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. Conclusions— Sevoflurane-induced AMPK activation protects the heart against ischemia and reperfusion injury and relies on upstream production of ROS.

Reactive Oxygen Species Precede Protein Kinase C-δ Activation Independent of Adenosine Triphosphate–sensitive Mitochondrial Channel Opening in Sevoflurane-induced Cardioprotection

BackgroundIn the current study, the authors investigated the distinct role and relative order of protein kinase C (PKC)-&dgr;, adenosine triphosphate–sensitive mitochondrial K+ (mito K+ATP) channels, and reactive oxygen species (ROS) in the signal transduction of sevoflurane-induced cardioprotection and specifically addressed their mechanistic link. MethodsIsolated rat trabeculae were preconditioned with 3.8% sevoflurane and subsequently subjected to an ischemic protocol by superfusion of trabeculae with hypoxic, glucose-free buffer (40 min) followed by 60 min of reperfusion. In addition, the acute affect of sevoflurane on PKC-&dgr; and PKC-&egr; translocation and nitrotyrosine formation was established with use of immunofluorescent analysis. The inhibitors chelerythrine (6 &mgr;m), rottlerin (1 &mgr;m), 5-hydroxydecanoic acid sodium (100 &mgr;m), and n-(2-mercaptopropionyl)-glycine (300 &mgr;m) were used to study the particular role of PKC, PKC-&dgr;, mito K+ATP, and ROS in sevoflurane-related intracellular signaling. ResultsPreconditioning of trabeculae with sevoflurane preserved contractile function after ischemia. This contractile preservation was dependent on PKC-&dgr; activation, mito K+ATP channel opening, and ROS production. In addition, on acute stimulation by sevoflurane, PKC-&dgr; but not PKC-&egr; translocated to the sarcolemmal membrane. This translocation was inhibited by PKC inhibitors and ROS scavenging but not by inhibition of mito K+ATP channels. Furthermore, sevoflurane directly induced nitrosylation of sarcolemmal proteins, suggesting the formation of peroxynitrite. ConclusionsIn sevoflurane-induced cardioprotection, ROS release but not mito K+ATP channel opening precedes PKC-&dgr; activation. Sevoflurane induces sarcolemmal nitrotyrosine formation, which might be involved in the recruitment of PKC-&dgr; to the cell membrane.

Comparison of noninvasive continuous arterial waveform analysis (Nexfin) with transthoracic Doppler echocardiography for monitoring of cardiac output

STUDY OBJECTIVES To compare the Nexfin cardiac output (CO) with the CO obtained from transthoracic Doppler echocardiography (TTE) during routine cardiac function screening. DESIGN Observational clinical study. SETTING Echocardiography laboratory. PATIENTS 40 ASA physical status 1 and 2 patients scheduled for routine TTE examination. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS In 40 patients scheduled for routine TTE examination, we obtained simultaneous CO measurements with Doppler ultrasound and derived from Nexfin blood pressure measurements. Correlation and level of agreement between Nexfin and TTE were analyzed using Pearson correlation coefficient and Bland-Altman plots. The Pearson correlation coefficient for Nexfin versus TTE was 0.68 (CI: 0.46 - 0.82, P < 0.0001). Bland-Altman analysis showed a bias of 0.51 ± 1.1 L/min and limits of agreement of -1.6 to 2.6 L/min, with a percentage error of 39%. CONCLUSIONS Considering limits of precision of CO measurements with Doppler echocardiography (± 30%), the agreement between noninvasive CO measurement with the Nexfin and TTE is reasonable.

Cardioprotection Via Activation of Protein Kinase C-δ Depends on Modulation of the Reverse Mode of the Na+/Ca2+ Exchanger

Background— Pretreatment with the volatile anesthetic sevoflurane protects cardiomyocytes against subsequent ischemic episodes caused by a protein kinase C (PKC)-&dgr; mediated preconditioning effect. Sevoflurane directly modulates cardiac Ca2+ handling, and because Ca2+ also serves as a mediator in other cardioprotective signaling pathways, possible involvement of the Na+/Ca2+ exchanger (NCX) in relation with PKC-&dgr; in sevoflurane-induced cardioprotection was investigated. Methods and Results— Isolated right ventricular rat trabeculae were subjected to simulated ischemia and reperfusion (SI/R), consisting of superfusion with hypoxic glucose-free buffer for 40 minutes after rigor development, followed by reperfusion with normoxic glucose containing buffer. Preconditioning with sevoflurane before SI/R improved isometric force development during contractile recovery at 60 minutes after the end of hypoxic superfusion (83±7% [sevo] versus 57±2% [SI/R];n=8; P<0.01). Inhibition of the reverse mode of the NCX by KB-R7943 (10 &mgr;mol/L) or SEA0400 (1 &mgr;mol/L) during preconditioning attenuated the protective effect of sevoflurane. KB-R7943 and SEA0400 did not have intrinsic effects on the contractile recovery. Furthermore, inhibition of the NCX in trabeculae exposed to sevoflurane reduced sevoflurane-induced PKC-&dgr; translocation toward the sarcolemma, as demonstrated by digital imaging fluorescent microscopy. The degree of PKC-&dgr; phosphorylation at serine643 as determined by western blot analysis was not affected by sevoflurane. Conclusions— Sevoflurane-induced cardioprotection depends on the NCX preceding PKC-&dgr; translocation presumably via increased NCX-mediated Ca2+ influx. This may suggest that increased myocardial Ca2+ load triggers the cardioprotective signaling cascade elicited by volatile anesthetic agents similar to other modes of preconditioning.

Diaphragm fiber strength is reduced in critically ill patients and restored by a troponin activator

To the Editor: Diaphragm weakness in the intensive care unit (ICU) plays an important role in difficult weaning from mechanical ventilation. Diaphragm strength in mechanically ventilated (MV) critically ill patients has been assessed indirectly using phrenic nerve stimulation, which demonstrated that the pressure-generating capacity of the diaphragm was reduced in these patients (1–3). However, this technique cannot distinguish between impaired phrenic nerve function, abnormal neuromuscular transmission, and intrinsic abnormalities in the diaphragm muscle itself. Consequently, it is unknown whether intrinsic contractile weakness of diaphragm muscle fibers occurs in MV critically ill patients. If so, targeted treatment strategies that enhance contractility may improve the success of weaning. Such treatment strategies may include the administration of a novel class of small-molecule drugs, named fast skeletal troponin activators, which improve the contractile strength of skeletal muscle fibers (4). In this study, we obtained diaphragm biopsy specimens from critically ill patients (n = 10; MV for 28–603 h) undergoing laparotomy or thoracotomy, and compared them with control patients undergoing elective lung surgery (n = 10; MV 1–2 h, see Table E1 in the online supplement). The size and the contractile performance of isolated diaphragm muscle fibers were determined. In addition, we tested the ability of the fast skeletal troponin activator, CK-2066260, to improve contractile strength. Diaphragm fiber cross-sectional area (CSA) was determined by means of immunohistochemical analyses with myosin heavy chain antibodies performed on cryosections of the biopsy specimens (5, 6). Figure 1A demonstrates atrophy of slow- and fast-twitch diaphragm fibers in critically ill patients (CSA slow-twitch fibers: control patients, 3,284 ± 793 μm2 vs. critically ill patients, 2,328 ± 763 μm2, P = 0.004; fast-twitch fibers: control patients, 2,766 ± 606 μm2 vs. critically ill patients, 1,819 ± 527 μm2, P < 0.0001). Figure 1. (A) Severe diaphragm muscle fiber atrophy in mechanically ventilated (MV) critically ill patients. Typical examples of serial diaphragm cross-sections stained with antibodies against slow-twitch myosin heavy chain (green). Wheat germ agglutinin (WGA) ... We measured the contractile performance of permeabilized single diaphragm fibers isolated from the biopsy specimens. Fibers were mounted between a force transducer and a length motor, and exposed to activating calcium solutions. Maximal contractile strength was markedly lower in critically ill patients (absolute force slow-twitch fibers: control patients, 0.44 ± 0.16 mN vs. critically ill patients, 0.19 ± 0·07 mN, P < 0.0001; fast-twitch fibers: control patients, 0.49 ± 0.21 mN vs. critically ill patients, 0.24 ± 0.09 mN, P = 0.0002; Figure 1B). After normalization of force to the CSA of these fibers (i.e., specific force), a deficit remained in diaphragm fibers of critically ill patients (see Figure E1). This suggests that, in these critically ill patients, there is not only a loss of contractile proteins, but also dysfunction of the remaining ones. In addition, we measured the sensitivity of force to calcium. The negative logarithm of the calcium concentration needed to obtain 50% of maximal force (pCa50) was unaffected in slow-twitch fibers (control patients, 5.64 ± 0.03 vs. critically ill patients, 5.61 ± 0.08, P = 0.30), whereas, in fast-twitch fibers, the pCa50 was significantly lower in critically ill patients (control patients, 5.76 ± 0.07 vs. critically ill patients, 5.70 ± 0.06, P = 0.036) (Figure 1B). Thus, fast-twitch diaphragm fibers from critically ill patients not only have reduced maximal force, but also require more calcium to generate force. We exposed diaphragm fibers of a representative subset of control patients (nos. I, IV, VI) and critically ill patients (nos. 1, 3, 4, 5) to the fast skeletal troponin activator, CK-2066260, which improves the sensitivity of the calcium sensor in the muscle sarcomere. Compared with vehicle, 5 μM of CK-2066260 significantly increased the calcium sensitivity of diaphragm fibers both in control patients (pCa50: 5.75 ± 0.04 vs. 6.18 ± 0.1, respectively; P < 0.001) and in critically ill patients (5.70 ± 0.07 vs. 6.00 ± 0.13, respectively; P < 0.01) (Figure 1C). Importantly, at physiological calcium concentrations, CK-2066260 restored the contractile force of fast-twitch diaphragm fibers of critically ill patients back to levels observed in untreated fibers from control patients (force at pCa 5.8: untreated control patients, 0.22 ± 0.05 vs. treated critically ill patients, 0.22 ± 0.07 mN; P = 0.954). See the online supplement for details. The current study is the first to show that atrophy and contractile weakness of diaphragm muscle fibers develop in a clinically relevant group of MV critically ill patients. Interestingly, the reduction in the contractile force of diaphragm fibers of these critically ill patients is comparable to the reduction in diaphragm strength estimated previously by phrenic nerve pacing (1, 2), indicating that the reduction in diaphragm strength in these patients largely results from muscle fiber weakness. To date, no drug is approved to improve respiratory muscle function in MV critically ill patients. We made a step toward such a strategy by testing the ability of the fast skeletal troponin activator, CK-2066260, to restore diaphragm fiber strength. We observed that, upon exposure to CK-2066260, fast-twitch diaphragm fibers from critically ill patients regained strength at calcium concentrations that reflect activation during daily live activities to levels found in untreated fibers from control patients (Figure 1C). Because approximately 50% of fibers and total fiber area in the human diaphragm consists of fast-twitch fibers (Figure E3), fast skeletal troponin activators might significantly improve in vivo diaphragm strength. The potential of fast troponin activators is further strengthened by the notion that these drugs do not affect cardiac function (4), which would be an undesirable side effect in critically ill patients. The analog of CK-2066260, tirasemtiv (formerly CK-2017357), is currently under study in patients with amyotrophic lateral sclerosis (clinical trial no. NCT01709149). What causes weakness of diaphragm muscle fibers in critically ill patients? It seems plausible that the observed diaphragm weakness was acquired during ICU stay, as we used strict exclusion criteria to rule out that our study patients had pre-existing diaphragm weakness. Also, during their stay in the ICU, patients received nutrition according to an optimized nutrition algorithm (7). A commonly suggested concept is that mechanical ventilation per se rapidly induces weakness and atrophy of muscle fibers due to contractile inactivity of the diaphragm (8–12). The critically ill patients we studied received MV for 28–603 hours before biopsy, a time frame that was associated with significant reductions in the CSA of diaphragm fibers in braindead organ donors (8, 13). Thus, the diaphragm muscle fiber atrophy and weakness that we observed may, at least partly, be explained by mechanical ventilation per se. Other ICU-related phenomena that could contribute to diaphragm muscle weakness include underlying disease, such as sepsis (14, 15). Clearly, to elucidate the main factors that contribute to the observed diaphragm muscle fiber weakness requires studies with larger cohorts of various patient groups.

Diabetes, perioperative ischaemia and volatile anaesthetics: consequences of derangements in myocardial substrate metabolism

Volatile anaesthetics exert protective effects on the heart against perioperative ischaemic injury. However, there is growing evidence that these cardioprotective properties are reduced in case of type 2 diabetes mellitus. A strong predictor of postoperative cardiac function is myocardial substrate metabolism. In the type 2 diabetic heart, substrate metabolism is shifted from glucose utilisation to fatty acid oxidation, resulting in metabolic inflexibility and cardiac dysfunction. The ischaemic heart also loses its metabolic flexibility and can switch to glucose or fatty acid oxidation as its preferential state, which may deteriorate cardiac function even further in case of type 2 diabetes mellitus.Recent experimental studies suggest that the cardioprotective properties of volatile anaesthetics partly rely on changing myocardial substrate metabolism. Interventions that target at restoration of metabolic derangements, like lifestyle and pharmacological interventions, may therefore be an interesting candidate to reduce perioperative complications. This review will focus on the current knowledge regarding myocardial substrate metabolism during volatile anaesthesia in the obese and type 2 diabetic heart during perioperative ischaemia.

General anesthesia with sevoflurane decreases myocardial blood volume and hyperemic blood flow in healthy humans

BACKGROUND:Preservation of myocardial perfusion during general anesthesia is likely important in patients at risk for perioperative cardiac complications. Data related to the influence of general anesthesia on the normal myocardial circulation are limited. In this study, we investigated myocardial microcirculatory responses to pharmacological vasodilation and sympathetic stimulation during general anesthesia with sevoflurane in healthy humans immediately before surgical stimulation. METHODS:Six female and 7 male subjects (mean age 43 years, range 28–61) were studied at baseline while awake and during the administration of 1 minimum alveolar concentration sevoflurane. Using myocardial contrast echocardiography, myocardial blood flow (MBF) and microcirculatory variables were assessed at rest, during adenosine-induced hyperemia, and after cold pressor test–induced sympathetic stimulation. MBF was calculated from the relative myocardial blood volume multiplied by its exchange frequency (&bgr;) divided by myocardial tissue density (&rgr;T), which was set at 1.05 g·mL−1. RESULTS:During sevoflurane anesthesia, MBF at rest was similar to baseline values (1.05 ± 0.28 vs 1.05 ± 0.32 mL·min−1·g−1; P = 0.98; 95% confidence interval [CI], −0.18 to 0.18). Myocardial blood volume decreased (P = 0.0044; 95% CI, 0.01–0.04) while its exchange frequency (&bgr;) increased under sevoflurane anesthesia when compared with baseline. In contrast, hyperemic MBF was reduced during anesthesia compared with baseline (2.25 ± 0.5 vs 3.53 ± 0.7 mL·min−1·g−1; P = 0.0003; 95% CI, 0.72–1.84). Sympathetic stimulation during sevoflurane anesthesia resulted in a similar MBF compared to baseline (1.53 ± 0.53 and 1.55 ± 0.49 mL·min−1·g−1; P = 0.74; 95% CI, −0.47 to 0.35). CONCLUSIONS:In otherwise healthy subjects who are not subjected to surgical stimulation, MBF at rest and after sympathetic stimulation is preserved during sevoflurane anesthesia despite a decrease in myocardial blood volume. However, sevoflurane anesthesia reduces hyperemic MBF, and thus MBF reserve, in these subjects.

Contrast-enhanced ultrasound for myocardial perfusion imaging.

Ultrasound contrast agents are gas-filled microbubbles that enhance visualization of cardiac structures, function and blood flow during contrast-enhanced ultrasound (CEUS). An interesting cardiovascular application of CEUS is myocardial contrast echocardiography, which allows real-time myocardial perfusion imaging. The intraoperative use of this technically challenging imaging method is limited at present, although several studies have examined its clinical utility during cardiac surgery in the past. In the present review we provide general information on the basic principles of CEUS and discuss the methodology and technical aspects of myocardial perfusion imaging.

Level of agreement between heart rate variability and pulse rate variability in healthy individuals.

Background and objective According to international standards, autonomic function is assessed by heart rate variability (HRV) calculated from R–R intervals obtained with an electrocardiogram (ECG). However, intra-operative movement artefacts and electrical interference may complicate R-wave detection. Pulse rate variability (PRV) derived from continuous blood pressure measurements may provide a feasible alternative for HRV. We aimed to investigate the level of agreement between PRV and traditional HRV using a novel beat-to-beat non-invasive blood pressure monitoring device. Methods In this prospective observational study, R–R intervals and non-invasive blood pressure waveforms were recorded simultaneously from 20 healthy male individuals at rest. HRV and PRV were analysed offline by spectral analysis, which divides the signal into its composing frequencies. Spearmans correlation coefficient, intra-class correlation coefficients and Bland–Altman analysis were used to study the level of agreement between HRV and PRV. Results The correlation coefficient between HRV and PRV was 0.99 (P < 0.001). Level of agreement was excellent with a mean difference of 1% in the very low frequency and low-frequency band and 14% in the high-frequency band. Reliability of both HRV and PRV was moderate to high. Conclusion Our data show that PRV derived from non-invasive blood pressure waveforms corresponds well with traditional HRV derived from ECG. These results indicate that under standard conditions, blood pressure waveforms may replace HRV in healthy individuals and that the use of PRV in the peri-operative setting should be further evaluated.

High fat diet-induced glucose intolerance impairs myocardial function, but not myocardial perfusion during hyperaemia: a pilot study

BackgroundGlucose intolerance is a major health problem and is associated with increased risk of progression to type 2 diabetes mellitus and cardiovascular disease. However, whether glucose intolerance is related to impaired myocardial perfusion is not known. The purpose of the present study was to study the effect of diet-induced glucose intolerance on myocardial function and perfusion during baseline and pharmacological induced hyperaemia.MethodsMale Wistar rats were randomly exposed to a high fat diet (HFD) or control diet (CD) (n = 8 per group). After 4 weeks, rats underwent an oral glucose tolerance test. Subsequently, rats underwent (contrast) echocardiography to determine myocardial function and perfusion during baseline and dipyridamole-induced hyperaemia (20 mg/kg for 10 min).ResultsFour weeks of HFD feeding resulted in glucose intolerance compared to CD-feeding. Contractile function as represented by fractional shortening was not altered in HFD-fed rats compared to CD-fed rats under baseline conditions. However, dipyridamole increased fractional shortening in CD-fed rats, but not in HFD-fed rats. Basal myocardial perfusion, as measured by estimate of perfusion, was similar in CD- and HFD-fed rats, whereas dipyridamole increased estimate of perfusion in CD-fed rats, but not in HFD-fed rats. However, flow reserve was not different between CD- and HFD-fed rats.ConclusionsDiet-induced glucose intolerance is associated with impaired myocardial function during conditions of hyperaemia, but myocardial perfusion is maintained. These findings may result in new insights into the effect of glucose intolerance on myocardial function and perfusion during hyperaemia.