We have measured the distribution of intracellular calcium concentration in isolated single muscle fibres from Xenopus laevis using the fluorescent calcium indicator fura-2 with digital imaging fluorescence microscopy. Under control conditions, resting and tetanic calcium were uniform throughout a fibre. When fatigue was produced using a prolonged, high-frequency tetanus, the distribution of calcium within muscle fibres became non-uniform, with greater levels near the outer parts of a fibre than near the centre. This non-uniform distribution of calcium was rapidly abolished by lowering the stimulation frequency. When fatigue was produced using a series of repeated intermittent tetani, tetanic calcium showed an initial small increase, followed by a decrease as stimulation was continued. The distribution of calcium remained uniform under these conditions. Calcium distribution was also uniform during recovery from intermittent tetanic stimulation. Although fibres varied considerably in their fatigue resistance, the time for tension to fall to 50% was correlated with the reduction in tetanic calcium seen at this time. These results indicate that there are at least two patterns of reduced calcium release that can contribute to the development of fatigue. The appearance of a calcium gradient is consistent with impaired t-tubular conduction, while a uniform reduction of calcium is likely to be due to the action of metabolic factors on systems controlling calcium homeostasis within the cell.

1. In this study we have investigated the effects of a novel inotropic agent, pimobendan (UDCG 115-BS), on skinned and intact ventricular muscle from ferrets. 2. Pimobendan (20 or 100 mumol/l) increased tension at a given free [Ca2+] when applied to skinned ventricular muscle, i.e. it increased the Ca2+ sensitivity of the myofibrils. 3. Tension and intracellular free Ca2+ [( Ca2+]i) were measured simultaneously in intact papillary muscles using the aequorin technique. When 25 mumol/l pimobendan was added to the superfusing solution, a slowly developing positive ionotropic effect was produced, which was accompanied by an increase in the size of the systolic rise in [Ca2+]i (Ca2+ transients) with a similar time course. 4. In order to determine whether pimobendan increased the Ca2+ sensitivity of myofibrils in an intact papillary muscle, we compared the increase in Ca2+ transients and tension observed in response to changes in extracellular [Ca2+] with those observed in response to pimobendan. The result of this comparison was that in intact muscle pimobendan caused no apparent increase in myofibrillar Ca2+ sensitivity. 5. Pimobendan caused an abbreviation of the time course of the Ca2+ transients, but the twitch was slightly prolonged. 6. When isoprenaline was added to the superfusing solution, a positive inotropic effect was produced, which was accompanied by a marked increase in the size of the Ca2+ transients. Isoprenaline caused an abbreviation of the time course of both the Ca2+ transients and the twitch. When the Ca2+ sensitivity of the intact myofibrils was determined as described above, isoprenaline caused a desensitization. Pimobendan produced a sensitization when compared with isoprenaline. 7. These results are consistent with the hypothesis that pimobendan produces an inotropic effect in isolated cardiac muscle which is mediated both by an increase in Ca2+ sensitivity and by an increase in adenosine 3':5'-cyclic monophosphate due to its phosphodiesterase-inhibiting activity. Such a combination of activities may be particularly advantageous for an inotropic agent.