Heptanol triggers cardioprotection via mitochondrial mechanisms and mitochondrial potassium channel opening in rat hearts.

AIM: To investigate mechanisms behind heptanol (Hp)-induced infarct size reduction and in particular if protection by pre-treatment with Hp is triggered through mitochondrial mechanisms. METHODS: Langendorff perfused rat hearts, isolated mitochondria and isolated myocytes were used. Infarct size, mitochondrial respiration, time to mitochondrial permeability transition pore (MPTP) opening and AKT and glycogen synthase kinase 3 beta (GSK-3ß) phosphorylation were examined. RESULTS: Pre-treatment with Hp reduced infarct size from 29.7 ± 3.4% to 12.6 ± 2.1%. Mitochondrial potassium channel blockers 5-hydroxy decanoic acid (5HD) blocking mitoK(ATP) and paxilline (PAX) blocking mitoK(Ca) abolished cardioprotective effect of Hp (Hp + 5HD 36.7 ± 2.9% and Hp + PAX 40.2 ± 2.8%). Hp significantly reduced respiratory control ratio in both subsarcolemmal and interfibrillar mitochondria in a dose-dependent manner (0.5-5.0 mm). The ADP oxygen ratio was also significantly reduced by Hp (2 mm). Laser scanning confocal microscopy of tetramethylrhodamine-loaded isolated rat myocytes using line scan mode showed that Hp increased time to MPTP opening. Western blot analysis showed that pre-treatment with Hp increased phosphorylation of AKT and GSK-3ß before ischaemia and after 30 min of global ischaemia. CONCLUSION: Pre-treatment with Hp protects the heart against ischaemia-reperfusion injury. This protection is most likely mediated via mitochondrial mechanisms which initiate a signalling cascade that converges on inhibition of opening of MPTP.

Repeated simulated ischemia and protection against gap junctional uncoupling.

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The aim of this study was to investigate the effect of early and delayed preconditioning on gap junction communication, connexin abundance, and phosphorylation in cultured neonatal rat cardiac myocytes. Prolonged ischemia followed 5 minutes after preconditioning in the early protocol, whereas 20 hours separated preconditioning and prolonged ischemia in the delayed preconditioning protocol. Gap junctional intercellular communication (GJIC) was assessed by Lucifer yellow dye transfer. An initial reduction in communication in response to sublethal ischemia was observed. This may be one mechanism whereby neighboring cells are protected from damaging substances produced during the first phase of subsequent regional ischemia in early preconditioning protocols. With respect to delayed preconditioning, the transient decrease in GJIC disappeared prior to prolonged ischemia, indicating that other mechanisms are responsible for delayed protection. Both early and delayed preconditioning preserved intercellular coupling after prolonged ischemia and this correlated with presence of less connexin43 dephosphorylation assessed by immunoblot.

Mechanism of hypoxic preconditioning in guinea pig papillary muscles.

UNLABELLED: Brief ischemia or hypoxia has been found to protect the heart against subsequent long-lasting ischemia and to improve contractile dysfunction as well to reduce cell necrosis and the incidence of lethal arrhythmias. This phenomenon, termed preconditioning (PC) has been demonstrated in different species. However, little is known about PC in guinea pigs. Moreover, electrophysiological changes underlying protection have not been studied so far in conjuntion with force recovery in a setting of PC. The aim of the study was to study PC in a guinea pig papillary muscle, using recovery of contractility after long hypoxic challenge as the main end-point of protection, and to investigate concomitant electrophysiological alterations. In guinea pig papillary muscle preparations contracting isometrically (paced at 2 Hz), transmembrane action potentials (AP) and developed force (DF) were recorded by conventional microelectrode technique and a force transducer. In addition, effective refractory periods (ERP) were determined. Hypoxia was induced by superfusion with 100% N2 (pO2 < 5 kPa) and pacing at 3,3 Hz. In the control group, long hypoxia lasted for 45 min and was followed by 30 min reoxygenation. In the PC group, muscles were subjected to 5 min hypoxia followed by 10 min recovery prior to sustained hypoxia/reoxygenation. RESULTS: Long hypoxia induced a similar depression of DF in both, PC and control groups. However, a loss of contractile activity occured earlier in the PC group. AP duration and ERP decreased faster and were significantly shorter after PC. Upon reoxygenation, preconditioned muscles showed significantly better recovery of function (DF 86% of prehypoxic value vs. 36% in controls; p < 0,05). AP and ERP were completely restored in both, PC and control groups. Guinea pig papillary muscle can be preconditioned with a brief hypoxic challenge against contractile dysfunction upon long-lasting hypoxia/reoxygenation. Shortening of AP and loss of contractility occured more quickly during hypoxia and may participate in the protective effect of preconditioning. Possible mechanisms might involve facilitated opening of K(ATP)-dependent channels.

The role of glycolysis in myocardial calcium control.

Because glycolysis is thought to be important for maintenance of cellular ion homeostasis, the aim of the present study was to examine the role of glycolysis in the control of cytosolic calcium ([Ca2+]i) and cell shortening during conditions of increased calcium influx. Thus, [Ca2+]i and unloaded cell shortening were measured in fura-2/AM loaded rat ventricular myocytes. All cells were superfused with Tyrode's solution containing glucose and pyruvate (to preserve oxidative metabolism), and glycolysis was inhibited by iodoacetate (IAA, 100 microM). Calcium influx was increased, secondary to an increase in intracellular sodium, by addition of veratrine (1 microgram/ml), or directly by either elevating [Ca2+]o from 2 to 5 mM or by exposing the cells to isoproterenol (1 to 100 nm). Veratrine exposure caused a time-dependent increase in both diastolic and systolic [Ca2+]i that resulted in cellular calcium overload and hypercontraction. The rate of increase in [Ca2+]i was more rapid in IAA-treated than in untreated myocytes, leading to a 13+/-3 v 5+/-2% increase (P<0.05) in diastolic [Ca2+]i after 5 min of exposure. The corresponding increases in systolic [Ca2+]i were 43+/-6 and 24+/-5% (P<0.05). Elevated [Ca2+]o resulted in increased [Ca2+]i transient amplitudes and cell shortening. These responses were each attenuated by inhibiting glycolysis, so that the increase was 38+/-5 v 68+/-9% ([Ca2+]i transient amplitude, P<0.05) and 41+/-11 v 91+/-18% (cell shortening, P<0.05). Inhibition of glycolysis did not, however, affect the increase in calcium transient or cell shortening during addition of isoproterenol. We conclude that glycolysis plays an essential role in the maintenance of intracellular calcium homeostasis during severe calcium overload. Glycolysis was also essential for signalling the inotropic effect that accompanied elevation in extracellular calcium, while the changes in intracellular calcium following administration of isoproterenol were not influenced by glycolysis in the present model.