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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Cardiovascular Pharmacology
Levosimendan preserves the contractile responsiveness of hypoxic human myocardium via mitochondrial KATP channel and potential pERK 1/2 activation Paul F. Soeding a,b,⁎, Peter J. Crack c, Christine E. Wright a, James A. Angus a, Colin F. Royse a a b c
Cardiovascular Therapeutics Unit, Department of Pharmacology, University of Melbourne, Parkville, Australia Department of Anaesthesia and Pain Medicine, Royal Melbourne Hospital, Parkville, Australia Department of Pharmacology, University of Melbourne, Parkville, Australia
a r t i c l e
i n f o
Article history: Received 17 October 2010 Received in revised form 10 December 2010 Accepted 15 December 2010 Available online 13 January 2011 Keywords: Human right atrium levosimendan Hypoxia–reoxygenation Prosurvival mediator Apoptosis
a b s t r a c t This study investigated the role of levosimendan, a mitochondrial KATP channel opener, during hypoxia– reoxygenation injury in human isolated tissue. The activation of preconditioning pathways, and the release of mitochondrial cytochrome c were determined. Human right atrial trabeculae were mounted in an organ bath, electrically paced and contractile force measured. Tissue was subjected to hypoxia–reoxygenation, and isoproterenol concentration–response experiments were performed as an index of contractile viability. The intracellular activities of Akt, ERK 1/2, P38, caspase 3, and cytochrome c were assayed by western blot. Following hypoxia–reoxygenation, the maximal contractile response of trabeculae to isoproterenol was significantly increased with levosimendan pretreatment compared to the hypoxia–reoxygenation control (0.88 ± 0.02 versus 0.60 ± 0.01 g, P b 0.01). This enhanced response was blocked by 5-hydroxydecoanate (0.54 ± 0.09 g, P b 0.01). A significant increase in both phosphorylated and total ERK 1/2 and P38 occurred at 60 min reoxygenation, compared to control tissue. No difference was observed in phosphorylated or total Akt, though there was a trend for increased levels in hypoxic tissue. Cytochrome c was detected at 60 min post reoxygenation, in both levosimendan treated and untreated tissue. No increase in cleaved-caspase 3 activity was observed. Our findings suggest that levosimendan preserves the contractile force to isoproterenol after hypoxia–reoxygenation, a response mediated via mKATP channel activation. The significant increase in the activity of prosurvival mediators ERK 1/2 and P38 following hypoxia indicates a potential mechanism of action for levosimendan-induced cardioprotection. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In cardiac surgery, cardiopulmonary bypass subjects the myocardium to a varying degree of ischemia–reperfusion injury, and is responsible for the transient myocardial dysfunction observed on bypass separation. Restoration of normal myocardial perfusion following cardioplegic arrest, or similarly in patients with acute coronary syndrome, can paradoxically lead to myocyte death, a phenomenon termed lethal reperfusion-induced injury (Kloner, 2004). Activation of apoptotic or necrotic cell death pathways during the early phase of reperfusion is seen as an important contributor to lethal reperfusion-induced injury (Gottlieb et al., 1994; Maulik et al., 1998; Stephanou et al., 2001) Mitochondria play a central role in the triggering of apoptosis (Green and Reed, 1998). Hypoxic stress alters outer membrane permeability,
⁎ Corresponding author. Cardiovascular Therapeutics Unit, Department of Pharmacology, University of Melbourne, Victoria 3010, Australia. Tel.: +61 3 8344 5673; fax: +61 3 8344 5193. E-mail address:
[email protected] (P.F. Soeding). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.12.035
leading to mitochondrial matrix calcium overload, severe organelle dysfunction and disruption. The release of mitochondrial cytochrome c into the cytosol is postulated to activate apoptosis (Brookes et al., 2004; Halestrap et al., 2004; Hausenloy et al., 2004). Secondly the activation of important prosurvival mediators may influence mitochondrial permeability and function; and include the mitoxgen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK 1/2), P38 MAP kinase (P38), phosphoinositide 3-kinase (PI-3), serine/threonine-specific kinase (Akt), and c-Jun N-terminal kinase (JNK 1/2) (Engelbrecht et al., 2004; Ma et al., 1999; Saurin et al., 2000; Yue et al., 1998). Levosimendan, a known mitochondrial KATP channel opener (Kopustinskiene et al., 2001), has been demonstrated to have a preconditioning-like effect on myocardial function (Kersten et al., 2000) and thereby has the potential to protect the heart during ischemia–reperfusion. During cardiac surgery levosimendan administration is reported to reduce myocardial damage (Tritapepe et al., 2006) and improve cardiac function following cardiopulmonary bypass (De Hert et al., 2007). Whether levosimendan acts predominantly as an inotrope via calcium sensitization, vasodilator or cardioprotective agent at clinically used concentrations is unknown.
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In this study we investigated the effect of levosimendan on contractile function in human isolated atrial tissue, following hypoxia–reoxygenation, and tested whether mKATP channels are involved. We measured the response to the β-adrenoceptor agonist isoproterenol as a ‘contractility viability index’ and the effects thereon after various pretreatments. Secondly the study evaluated whether pretreatment with levosimendan had an effect on preconditioning pathways including MAP kinases ERK 1/2, P38, MAPKa/h, PI-3 kinase/ Akt, the release of mitochondrial cytochrome c and the activation of apoptosis. 2. Material and methods 2.1. Tissue preparation, dissection and mounting Human discarded right atrial appendages were obtained from patients undergoing routine cardiac surgery at the Royal Melbourne Hospital (Ethics approval 2006/006). Patients gave informed consent but were excluded from this study if they were on oral hypoglycemic agents. At surgery, the discarded tip of the right atrial appendage was excised and immediately placed in pre-oxygenated ice-cold Krebs' solution (mM): NaCl 119; KCl 4.7; KH2PO4 1.18; MgSO4 1.17; NaHCO3 25; CaCl2 2.0; Na EDTA 0.026; and glucose 5.5 and transported to the laboratory within 5–10 min. Individual trabeculae (≤1 mm thick) were dissected free, threaded with stainless steel hooks at each end and mounted vertically in 15 ml organ baths. The upper hook was attached to a strain gauge transducer. Experiments were conducted in an isolated bio-cabinet (Gelman class 2, GHA 180). Baths contained Krebs solution (as above), saturated with carbogen at 34 °C. Each appendage provided an average of 4–6 trabeculae. Following a 60 min stabilisation period the tissue was then stimulated with square wave pulses (1 Hz, 5 ms duration, 20% above threshold voltage) via platinum wire field electrodes. The resting passive force was adjusted to 0.5 g, ensuring muscle contraction was measured at the peak of the length–tension curve, Lmax (Serone and Angus, 1999). A positive contractile response to isoproterenol 1 μM indicated tissue viability and tissues that failed to contract with a force ≥ 0.2 g (2 mN) were rejected. The tissue bath solution was replaced with fresh Krebs' solution and resting force of stimulated trabeculae continuously measured. Experiments then proceeded after a stable period, usually 30–60 min, of baseline force (Baseline). Responses were recorded on a PowerLab 8SP data acquisition system (AD Instruments, Bella Vista NSW, Australia) and Chart v5.5 software. 2.2. Hypoxia–reoxygenation protocol Five types of experiments were performed, including three with drug pretreatment: levosimendan 0.3 μM, levosimendan 0.3 μM and 5-hydroxydecoanate (5-HD) 800 μM and 5-HD 800 μM alone; and two without drug pretreatment (normoxia and hypoxia alone). 5-HD is regarded as a specific mK ATP antagonist, known to block mitochondrial channels at concentrations of 50–500 μM (Wang et al., 2001) and has been used in organ bath preparations at concentrations of 800 μM (Hanouz et al., 2002; Yvon et al., 2003). Drug pretreatment occurred 15 min before the onset of hypoxia and each drug remained within the organ bath without further washing for the remainder of the experimental protocol. In the Hypoxia alone group trabeculae were subjected to hypoxia without pretreatment, and a normoxia time control group (Normoxia) did not receive hypoxia exposure. Trabeculae were subjected to a 60 min period of hypoxia, by replacement of carbogen with a N2/CO2 (95:5%) gas mixture. In baths saturated with carbogen, solution pH was 7.47 ± 0.02, pCO2 28 ± 2 mm Hg, pO2 497 ± 27 mm Hg, compared to the hypoxic N2/ CO2 saturated solution, with pH 7.51 ± 0.01, pCO2 26 ± 1 mm Hg, pO2 65 ± 8 mm Hg. Following the hypoxic period, tissue was
reoxygenated with carbogen for 15 min, and then stimulated with isoprenaline (Fig. 1). A concentration–contractile response curve was constructed by equilibrating trabeculae with cumulative concentrations of isoprenaline 1–32,000 nM. At each concentration, the maximal contractile response was measured when contractile force plateaued at 5–8 min. Increasing concentration was not associated with episodes of arrhythmia. 2.3. Intracellular mediator protocol In another series of experiments trabeculae (n = 4) were removed at different time intervals following the hypoxia–reoxygenation phase (time 0 at the end of hypoxia, then 15, 60 and 120 min during reoxygenation), weighed and immediately frozen in liquid nitrogen at −80 °C, and assayed later for intracellular markers (phosphorylated Akt, ERK and P38, cleaved-caspase 3, and cytochrome c). These trabeculae were not subjected to isoproterenol stimulation. Analysis of protein activation, from 0–120 min following reoxygenation, was aimed to provide a temporal picture of the response to hypoxia– reoxygenation. At assay, samples were ground and homogenised in lysis buffer 15 μl mg− 1 (Bio-Rad cytoplasmic protein extraction buffer 4307616, Bio-Rad Laboratories, Hercules CA, USA). Following 5 min incubation on ice, homogenates underwent centrifugation for 10 min (1000 ×g at 4 °C) and the supernatant decanted. The protein content of each homogenate was determined by spectrophotometry. In view of the small volumes used, protein determination was made by measuring the absorbance of a 1.5 μl sample aliquot at 280 nm wavelength (NanoDrop 280, Nanodrop Technologies, Wilmington, DE, USA). In addition a small number of samples was taken for protein estimation by the Bradford method. Absorbance of 300 μl samples were made at 590 nM (FLUOstar OPTIMA, BMG Labtechnologies Pty. Ltd., Offenburg, Germany). Sample measurements are referred to a standard absorbance curve based on known concentrations of bovine serum albumin (Bradford, 1976). Western blotting was done based on a previously established protocol (Taylor et al., 2005) with slight modifications. Protein extracts (approximately 3–10 μg) were separated on 12% SDS-PAGE gels and subsequently transferred to polyvinylidene difluoride membranes (Millipore Co., MA, USA). Membranes were blocked with 4% w/v skim milk in tris buffered saline containing 0.05% v/v Tween 20 (TBST; pH 7.6) for 1 h at room temperature and subsequently incubated with 1:1000 dilution of rabbit polyclonal antibodies specific for either Phospho-ERK1/2, Phospho-Akt, Phospho-P38–MAPK, ERK1/2, or P38–MAPK, cleaved caspase 3 and cytochrome c (Cell Signaling) in TBST containing 1% w/v skim milk (skim milk–TBST buffer) overnight at 4 °C. Following that, membranes were incubated with 1:1000 dilution of anti-rabbit polyclonal antibodies conjugated to horseradish peroxidase in skim milk–TBST buffer for 1 h at room temperature. β-tubulin was used as loading control and detected by incubating membranes with 1:1000 dilution of mouse monoclonal anti-β-tubulin antibodies in skim milk–TBST buffer, followed by 1:1000 dilution of anti-mouse polyclonal antibodies conjugated to horseradish peroxidase (HRP) in skim milk–TBST buffer, both antibody incubations 1 h each at room temperature. Membranes were washed in TBST 3 times after antibody incubation. Proteins were revealed using SuperSignal Chemiluminescence Substrate system (Pierce Biotechnology Inc., IL, USA), visualised by exposure to X-ray film and protein bands quantitated using NIH ImageJ densitometry analysis. 2.4. Statistical methods Force was expressed in grams, as mean ± S.E.M. Concentration– response (CR) curves were constructed from the maximum total force generated in response to drug addition. CR curves were fitted using non-linear regression analysis (Prism 5 Graphpad software 2008, Inc).
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3.2. Effect of reoxygenation Upon reoxygenation, contractile force increased significantly in all groups. Trabeculae pretreated with levosimendan had a significantly greater contractile response on reoxygenation (0.25 ± 0.06 g) increasing to 59 ± 12% baseline, compared to the Hypoxia group (0.10 ± 0.02 g) and Levo + 5-HD + Hypoxia group (0.11 ± 0.03 g; P b 0.05 one-way ANOVA). Levosimendan pretreated tissue also tended to have an increased contractile force compared to the 5-HD + Hypoxia group (0.13 ± 0.02 g), but the difference did not reach significance.
3.3. Response to isoproterenol
Fig. 1. A. Hypoxia protocol: following harvest, human right atrial trabeculae were electrically paced and equilibrated with isoprenaline 1 μM (ISO) for viability. Following rewash and baseline stabilisation (baseline), trabeculae were pretreated with levosimendan (0.3 μM), 5-hydroxydecanoic acid (800 μM) or both for 15 min, then subjected to hypoxia 60 min, followed by reoxygenation for 15 min before equilibration with isoproterenol 1–32,000 nM. B. Measurement of protein expression in trabeculae subjected to hypoxia–reoxygenation. Levosimendan 0.3 μM pretreatment 15 min before hypoxia. 0, 30, 60, 120 min protein assay measurements.
The maximal contractile response to isoproterenol was significantly decreased in trabeculae subjected to hypoxia (Fig. 2, Table 1), compared to the response observed in normoxic trabeculae (Normoxia 0.88 ± 0.02 g versus hypoxia alone 0.60 ± 0.01 g, P b 0.05). There was no significant difference in the sensitivity to isoproterenol in both these groups; pEC50 7.49 ± 0.22 and 7.39 ± 0.34, respectively. Post-hypoxic trabeculae pretreated with levosimendan responded to isoproterenol with a maximum contraction force and contractile range similar to normoxic tissue without levosimendan (Levo + Hypoxia 0.78 ± 0.04 g, P b 0.05); this was also significantly greater than the maximum contractile force seen with the hypoxia alone group (P b 0.05). This increased contractile response with levosimendan pretreatment was not observed when 5-HD was equilibrated with levosimendan (Levo + 5-HD 0.54 ± 0.09 g compared to Levo + Hypoxia, P b 0.01).
The EC50 was estimated as the drug concentration producing 50% maximal response. Differences in maximal contractile force and response range were tested by one-way analysis of variance with Dunnetts post-hoc analysis. Assay experiments were performed using 6 trabeculae for each group per time interval. During hypoxia– reoxygenation, differences between the pretreatment and time control groups were tested by repeated-measures analysis of variance using the Greenhouse–Geisser correction (Ludbrook, 1994). Analysis was performed on the raw data using SPSS version 15 (SPSS Inc., Illinois, USA). P b 0.05 was considered statistically significant. 2.5. Drugs Levosimendan was supplied by Abbott Australia Pty. Ltd. in its proprietary form Simdax 2.5 g/ml. 5-HD (0.1 M) was prepared in Krebs solution. (5-Hydroxydecanoic acid, sodium salt, was supplied by Sigma-Aldrich, St. Louis, MO, USA). 3. Results 3.1. Contractile response following hypoxia The baseline contraction force was similar in all groups ranging from 0.35 ± 0.07 to 0.46 ± 0.08 g (P N 0.05). Trabeculae pretreated with levosimendan 0.3 μM or with 5-HD 800 μM alone had no direct effect on baseline contractile force. A 60 min period of hypoxia significantly reduced the contractile force in all tissues (Fig. 2) to 15 ± 3% (Hypoxia alone), 21± 4% (Levo+ Hypoxia), 23 ± 5% (Levo + 5-HD+ Hypoxia) and 29 ± 5% (5-HD+ Hypoxia) of baseline respective contractile force (P b 0.01). Trabeculae not subjected to hypoxia (Normoxia) had a gradual decline in contractile force to 61± 10% of baseline, before stimulation with isoproterenol.
Fig. 2. Effect of levosimendan 0.3 μM on human right atrial trabeculae contractile force with isoproterenol stimulation following hypoxia–reoxygenation. **Effect of hypoxia on baseline contraction force, decline in normoxic tissue contractile force from baseline at 90 min (P b 0.01 Students t test). *Contractile force of levosimendan-pretreated tissue compared to other atria on reoxygenation (P b 0.05, one way ANOVA). † Maximal contractile force (Emax) Normoxia time control and Levo + Hypoxia group compared to Hypoxia alone and Levo + 5-HD (5-hydroxydecanoate 800 μM) + Hypoxia groups, oneway ANOVA. Error bars are ± 1 S.E.M. on Baseline, P, H and av S.E.M. from rm ANOVA on each curve. Baseline following harvesting and stabilisation, P pretreatment, H following hypoxia, and R 15 min after reoxygenation.
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3.4. Activation of pERK 1/2 The mean weight for an individual trabecula was 6.51 ± 0.28 mg, and protein content of resuspended supernatant (approx 60–80 μl) was 0.80 ± 0.04 μg μl− 1. Trabeculae subjected to hypoxia and pretreated with levosimendan showed a significant increase in ERK 1/2 at 60 min reoxygenation. Levosimendan increased both phosphorylated ERK 1/2 (23.3 ± 10.1 × 105 versus 0.42 ± 0.36 × 105 units, P b 0.05) and total ERK 1/2 (295.0 ± 97.1 × 105 versus 45.1 ± 25.2 × 105 units, P b 0.05) compared to normoxic tissue. This increase did not reach significance when compared to hypoxia alone (Fig. 3). At other time intervals phosphorylated ERK 1/2 levels were not significantly different to normoxia or hypoxia groups: 0.5 ± 0.3 (baseline), 0.5 ± 0.2 (0 min), 4.6 ± 3.2 (15 min) and 5.0 ± 1.3 × 104 units (120 min). Similarly there was no increase in total ERK 1/2 levels in levosimendan pretreated tissue: 1.2 ± 0.4 (baseline), 11.5 ± 2.0 (0 min), no data (15 min), and 8.2 ± 3.0 × 104 units (120 min). 3.5. Activation of P38 Similarly at 60 min reoxygenation (Fig. 4) there was a significant increase in phosphorylated P38 compared to normoxic tissue (6.5 ± 0.3 versus 1.3 ± 0.1 × 103 units, P b 0.05) but not to untreated hypoxic tissue (2.0 ± 0.9 × 103 units, P N 0.05). There was no significant difference in total P38 activity in both untreated and levosimendanpretreated tissue. No activity was detected at 0, 15 or 120 min. 3.6. Activation of pAkt Phosphorylated Akt was not detected at the 0, 15, 60 and 120 min intervals following hypoxia and reoxygenation. Despite a trend for increased levels of total Akt at 60 min in tissue exposed to hypoxia (Fig. 5) these values did not attain significance (35.5 ± 5.2, 24.7 ± 15.3 and 7.3 ± 2.3 × 104 units for levosimendan pretreated, untreated hypoxic and normoxic groups respectively). This suggests Akt activity was increased but without detection of phosphorylated Akt, possibly indicating rapid activation and decay of the phosphorylated form. 3.7. Proapoptotic indicators cytochrome c and cleaved caspase 3 Cytochrome c was detected at 60 min post reoxygenation in both levosimendan treated and untreated tissues (Fig. 6). In untreated tissue, the increase in cytochrome c was significantly greater than in normoxic tissue, but not for levosimendan pretreated tissues. Cytochrome c was not detected at 0 or 15 min reoxygenation, and at 120 min levels had decreased to baseline. No increase in cleavedcaspase 3 activity was detected at any of the time intervals. The decrease in cytochrome c levels could indicate either protein breakdown or reflect binding with cytosolic proteins, decreasing the sensitivity to antibody binding.
Table 1 The effect of levosimendan and 5-HD pretreatment on the contractile response to isoproterenol in human atrial trabeculae. pEC50 Normoxia (n = 15) Levo + hypoxia (n = 16) 5-HD + hypoxia (n = 10) Levo + 5-HD + hypoxia (n = 10) Hypoxia (n = 15) a b
7.49 ± 0.22 7.67 ± 0.29 7.54 ± 0.22 7.58 ± 0.53 7.39 ± 0.34
Emax (g)
Range (g) a
0.88 ± 0.05 0.78 ± 0.05b 0.64 ± 0.04 0.54 ± 0.05 0.60 ± 0.05
0.66 ± 0.07 0.64 ± 0.06 0.55 ± 0.07 0.45 ± 0.08 0.49 ± 0.08
P b 0.05 Normoxia vs Hypoxia, Levo + 5-HD + Hypoxia. Levo + Hypoxia vs Hypoxia, Levo + 5-HD + Hypoxia; one-way ANOVA.
Fig. 3. Western blot analysis of phosphorylated (Ph ERK) and total ERK 1/2 protein expression in human isolated right atrial trabeculae subjected to hypoxia–reoxygenation treated with or without levosimendan. A. Densitometric analysis of band mean intensity showing an increased protein expression of phosphorylated ERK 1/2 in trabeculae subjected to hypoxia and pretreated with levosimendan (P b 0.05). B. Densitometric analysis of band mean intensity showing an increased protein expression of total ERK 1/2 in trabeculae subjected to hypoxia and pretreated with levosimendan (P b 0.05). C. Representative Western blot shown for phosphorylated and total ERK, and β-actin control (n = 4 per group).
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Fig. 5. Western blot analysis of total Akt protein expression in human isolated right atrial trabeculae subjected to hypoxia–reoxygenation treated with or without levosimendan. A. Densitometric analysis of band mean intensity showing an increased protein expression of total Akt in trabeculae subjected to hypoxia and pretreated with levosimendan (P b 0.05). B. Representative Western blot shown for phosphorylated (Ph Akt) and total ERK, and β-actin control (n = 4 per group).
Fig. 4. Western blot analysis of phosphorylated (Ph P38) and total P38 protein expression in human isolated right atrial trabeculae subjected to hypoxia–reoxygenation treated with or without levosimendan. A. Densitometric analysis of band mean intensity showing an increased protein expression of phosphorylated P38 in trabeculae subjected to hypoxia and pretreated with levosimendan (P b 0.05). B. Densitometric analysis of band mean intensity showing an increased protein expression of total P38 in trabeculae subjected to hypoxia and pretreated with levosimendan (P b 0.05). C. Representative Western blot shown for phosphorylated and total P38 and β-actin control (n = 4 per group).
4. Discussion This study of simulated ischemia–reperfusion demonstrates that the pretreatment of human atrial trabeculae with levosimendan attenuates the contractile force deficit induced by hypoxia, results in
stronger contraction on reoxygenation, and causes a greater contractile response to isoproterenol. The range of contractile responsiveness to isoproterenol, or contractile viability, is increased compared to untreated tissue. These two key measures of muscle function posthypoxia, the peak contractile force with isoproterenol stimulation and the contractile response range of the CR curve, both reflect the inotropic viability of trabeculae post-injury. Hypoxic injury alters cellular function, depresses contractility, and can decrease the inotropic responsiveness to adrenoceptor agonists (Peters et al., 1997). Our use of isoproterenol-stimulated contraction reflects an integrated myocyte response, involving receptor activation and coupling, intracellular signalling, myofilament responsiveness, calcium release and reuptake, as well as cellular metabolic integrity. All trabeculae exhibited a response range with similar sensitivity, indicating that the myocyte β-adrenoceptor activation with isoproterenol remained intact. Tissue pretreated with 5-HD had a contractile response similar to the hypoxia alone group, and with pretreatment of levosimendan, there was no restoration of peak contractile force. Even with the variation in baseline contraction on reoxygenation, the concentration–response viability and the peak contraction force were of the same order, with both values similar in normoxic and levosimendan pretreated trabeculae, and in hypoxia alone and 5-HD treated trabeculae. This suggests that 5-HD prevented the positive action of
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Fig. 6. Western blot analysis of cytochrome c and cleaved caspase 3 protein expression in human isolated right atrial trabeculae subjected to hypoxia–reoxygenation, treated with or without levosimendan. A. Densitometric analysis of band mean intensity showing an increased protein expression of cytochrome c in trabeculae subjected to hypoxia and pretreated with levosimendan (P b 0.05). B. Representative Western blot shown for cytochrome c and β-actin control (n = 4 per group). C. Representative Western blot shown for cleaved caspase 3 and β-actin control (n = 4 per group).
levosimendan in restoring the baseline force of contraction, and enhanced the action of isoproterenol. This implies that the effect of levosimendan on contractile viability post-hypoxia is likely to be mediated by mKATP channel activation. Activation of the mKATP channel is known to have a potent protective effect against ischemic injury in the heart (Eliseev et al., 2004; Kicinska and Szewczyk, 2003) and is implicated in protection against apoptosis and cell death (Akao et al., 2002; Eliseev et al., 2004; Murata et al., 2001; Tai et al., 2003; Teshima et al., 2003a; Teshima et al., 2003b). Levosimendan has been demonstrated to activate potassium flux into the myocardial mitochondrial matrix at therapeutic plasma levels (Kopustinskiene et al., 2004). Influx of potassium into the organelle is postulated to depolarise the membrane, prevent intermembrane expansion and matrix shrinkage (Dos Santos et al., 2002). This action stabilises the organelle, prevents opening of the mitochondrial permeability transition pore (MPTP), with subsequent release of cytochrome c and activation of caspase 3 (Halestrap et al., 2004).
Compared to untreated normoxic tissue, this study has demonstrated a significant increase in the activity of the prosurvival kinase MAP ERK 1/2 in human myocardium pretreated with levosimendan and subjected to hypoxia. Levosimendan pretreatment increased both phosphorylated ERK 1/2 and total ERK 1/2 activity, compared to untreated tissue subjected to hypoxia. However, since this increase did not reach significance, the comparison between treated and untreated tissue post-hypoxia is not as defined. It is not possible to state that phosphorylation of ERK 1/2 was significantly potentiated during hypoxia, since levosimendan resulted in simultaneous increases in both phosphorylated and total ERK 1/2 levels. The upregulation of total ERK 1/2 though, was associated with a stable βactin immunoblot and increased phosphorylated ERK 1/2. It could be argued that treatment with levosimendan during hypoxia resulted in a significant upregulation of total ERK 1/2 which subsequently resulted in greater stimulation of phosphorylated ERK 1/2 formation, even though the ratio of phosphorylated to total ERK 1/2 was not drastically altered. This increase in ERK 1/2 activity was detected 60 min following reperfusion indicating activation occurred within the first hour of reoxygenation. The signalling proteins ERK 1/2 are known preconditioning mediators, and activation during reoxygenation identifies a potential mechanism of action in levosimendan-induced cardioprotection (Mackay and Mochly-Rosen, 1999; Saurin et al., 2000; Yue et al., 1998). Activation of ERK 1/2 is reported to occur in the first minutes of reperfusion and is believed to be an important mediator in cellular protection (Bolli, 2000; Fryer et al., 2001; Hausenloy et al., 2005a; Yue et al., 1998). This pathway has been termed the reperfusion injury salvage kinase (RISK) pathway indicating its importance in limiting injury on reperfusion (Hausenloy and Yellon, 2004). Activation of the RISK pathway is postulated to stabilise mitochondria, in particular induce closure of the MPTP, mediated by ERK-induced phosphorylation, eNOS activation, PKCε and effects on glycogen synthase kinase 3β (Hausenloy et al., 2005b). This finding of increased ERK 1/2 activity is in agreement with a recent study, reporting levosimendan to induce pERK activation in guinea pig isolated hearts, subjected to ischemia–reperfusion (du Toit et al., 2008). Pretreating hearts with levosimendan (0.1 μM) was found to increase the activity of pERK 1/2 on reperfusion, and significantly reduce infarct size. Activation of this prosurvival kinase was postulated to pharmacologically precondition the myocardium and reduce reperfusion injury. Other pathways have been also been implicated in studies of ischemia–reperfusion. In rabbits subjected to coronary artery ligation, the reduction in infarct size with levosimendan was mediated by mKATP activation and nitric oxide (Das and Sarkar, 2007); this was associated with preserved myocardial ATP and high energy phosphate stores. Similarly using an in vivo rat model, levosimendan given 5 min before reperfusion, was shown to reduce infarct size, and involve the PI3 pathway and activation of the mKATP channel (Honisch et al.). These studies indicate the potential of levosimendan in the setting of reperfusion during acute coronary ischemia. The data also indicate that levosimendan pretreatment has a tendency to potentiate P38 phosphorylation, a kinase also implicated in the preconditioning response. Activation of the P38–MAPK pathway leads to phosphorylation of Hsp27, a well established P38–MAPK substrate, and mediator of cytoprotection (Gaitanaki et al., 2003; Krajewski et al., 1999). Activation of the pAkt pathway was also not demonstrated despite evidence indicating PI-3–Akt to be a mediator of cell survival, and protective during ischemia–reperfusion injury (Bell and Yellon, 2003; Toth et al., 2003). Akt is reported to inhibit mitochondrial cytochrome c release and maintain mitochondrial membrane potential, independent of the pro-apoptotic BCL-2 family member BAD (Kennedy et al., 1999) and is also associated with inactivation of caspase 9 (Datta et al., 1999). A limitation in the study of human myocardium is the low sample volume and protein content in each sample extract. Multiple gels
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were plated for five antibody probes, with each well receiving only 4–6 μg of protein sample. The efficacy of protein transfer with low mass samples onto the membrane ultimately determines the success of immunodetection, and may explain failure to detect pAkt. For this reason the measurement of total p38, Akt and ERK 1/2, as well as β-actin were used as internal controls. The data suggest that total Akt however, may be upregulated by hypoxia and further study could explore this. Hypoxia resulted in increased cytochrome c levels, evident at 60 min reoxygenation. Since cytochrome c was not elevated in normoxic tissue, the detection of an increase in hypoxic tissue reflects cell damage in both treated and untreated groups. Since these were not significantly different, a limitation of injury with levosimendan pretreatment, on the basis of cytochrome c levels, could not be demonstrated. The release of cytochrome c is associated with the initiation of cell death primarily through the activation of caspase 3. In this study there was no detection of cleaved caspase 3, and the reason for this could be explained by a longer time frame of 6–8 h being required for caspase activation. This study indicates that levosimendan has the potential to upregulate the RISK pathway, attenuate ischemic injury, and limit myocardial infarction in patients undergoing cardiac surgery. In patients with acute coronary syndromes, levosimendan may also limit myocardial damage during the reperfusion phase of injury (Staat et al., 2005). During cardiac surgery, therapeutic inhibition of apoptosis may protect against ischemia–reperfusion injury, improve myocyte recovery and maintain contractile function. 5. Conclusions Our findings have shown that in human tissue exposed to hypoxia, levosimendan preserves both the contractile responsiveness and peak contraction force to isoproterenol. This action on contractile function indicates that myocytes pretreated with levosimendan withstand hypoxia more favourably than untreated tissue, an effect mediated by mKATP channel activation and potential upregulation of the prosurvival pathway ERK 1/2. Acknowledgements We thank Mark Ross-Smith and Dr. Moses Zhang for their assistance. Levosimendan was supplied by Abbott Australia. References Akao, M., Teshima, Y., Marban, E., 2002. Antiapoptotic effect of nicorandil mediated by mitochondrial ATP-sensitive potassium channels in cultured cardiac myocytes. J. Am. Coll. Cardiol. 40, 803–810. Bell, R.M., Yellon, D.M., 2003. Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3K. Akt and eNOS. J. Mol. Cell. Cardiol. 35, 185–193. Bolli, R., 2000. The late phase of preconditioning. Circ. Res. 87, 972–983. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Brookes, P.S., Yoon, Y., Robotham, J.L., Anders, M.W., Sheu, S.S., 2004. Calcium, ATP, and ROS: a mitochondrial love–hate triangle. Am. J. Physiol. Cell Physiol. 287, C817–C833. Das, B., Sarkar, C., 2007. Pharmacological preconditioning by levosimendan is mediated by inducible nitric oxide synthase and mitochondrial KATP channel activation in the in vivo anesthetized rabbit heart model. Vascul. Pharmacol. 47, 248–256. Datta, S.R., Brunet, A., Greenberg, M.E., 1999. Cellular survival: a play in three Akts. Genes Dev. 13, 2905–2927. De Hert, S.G., Lorsomradee, S., Cromheecke, S., Van der Linden, P.J., 2007. The effects of levosimendan in cardiac surgery patients with poor left ventricular function. Anesth. Analg. 104, 766–773. Dos Santos, P., Kowaltowski, A.J., Laclau, M.N., Seetharaman, S., Paucek, P., Boudina, S., Thambo, J.B., Tariosse, L., Garlid, K.D., 2002. Mechanisms by which opening the mitochondrial ATP-sensitive K(+) channel protects the ischemic heart. Am. J. Physiol. Heart Circ. Physiol. 283, H284–H295.
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