Cardiovascular Pharmacology
Levosimendan preserves the contractile responsiveness of hypoxic human myocardium via mitochondrial KATP channel and potential pERK 1/2 activation

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Abstract

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 < 0.01). This enhanced response was blocked by 5-hydroxydecoanate (0.54 ± 0.09 g, P < 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.

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, 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.

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.

Section snippets

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

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 > 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 < 0.01). Trabeculae not subjected to

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 post-hypoxia, the peak contractile

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.

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