Fresh control hearts were perfused with cardioplegic solution (Plegisol®, Medline, Arnhem, Holland, modified with increased KCl to 23 mM) in anesthetized animals. The hearts were exercised and cooled (4°C) in cold cardioplegia for 30 min before further preparation. Trabecular muscles, free from side branches, were identified in the right ventricular wall, and carefully dissected. Sutures (6/0 silk) were tied at each end, and the preparations were transferred to 50 ml open organ glass baths filled with Krebs’ solution at 37°C gassed with 95/5% O2/CO2 giving a pH of 7.4. The muscles were mounted vertically, with one end attached to a fixed pin in the bath, and the other connected to a Grass FT03 force transducer. Stimulation was applied using a Grass S48 stimulator at 0.5 Hz, 0.5 ms pulse duration and supramaximal voltage. The muscles were allowed to accommodate for about 20 min. When stable contractions could be elicited, the preparations were stretched to a length giving maximal force (about 1.3 × slack length). Initial maximal force responses were recorded on all preparations. The trabecular preparations varied in absolute active force generation between 3 and 10 mN depending on size. Preparations giving less than 3 mN or having increased basal tone during accommodation were excluded. Ischemia/hypoxia was induced by transfer to Krebs’ solution made without glucose, and with the bath gassed with 95/5% N2/CO2. Using a polarographic O2 electrode (MLT1120, ADInstruments), the O2 pressure in the bath was monitored. With N2/CO2 gassing, PO2 reached below 7 mmHg in about 4 min. The ischemia/hypoxia was maintained for 30 min during which stimulation was continued. Thereafter, normal Krebs’ solution and O2 were reintroduced, and the recovery phase of the muscles was followed for 30 min. Samples for ATP/PCr measurements were taken after accommodation and at the end of the ischemia/hypoxia period.
Different solutions were examined during the hypoxia period: glucose-free Krebs’, modified Plegisol®, glucose-free Krebs’ with 23 mM KCl or 16 mM MgCl2. To examine the effect of MYK-461, the compound was introduced 20 min prior to, and maintained during the hypoxia/ischemia period in the Krebs’ solution. DMSO (<0.1%) was used as a solvent control for the MYK-461 experiments.
Different solutions were examined during the hypoxia period: glucose-free Krebs’, modified Plegisol®, glucose-free Krebs’ with 23 mM KCl or 16 mM MgCl2. To examine the effect of MYK-461, the compound was introduced 20 min prior to, and maintained during the hypoxia/ischemia period in the Krebs’ solution. DMSO (<0.1%) was used as a solvent control for the MYK-461 experiments.
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