Rate-dependent changes in cell shortening, intracellular Ca²⁺ levels and membrane potential in single, isolated rainbow trout (Oncorhynchus mykiss) ventricular myocytes

The effects of increasing stimulation frequency (from 0.2 to 1.4 Hz) on the contractility, intracellular Ca²⁺ concentration ([Ca²⁺](i)) and membrane potential of single ventricular myocytes isolated from the heart of rainbow trout (Oncorhynchus mykiss) were measured. Cell shortening, expressed as a percentage of resting cell length, was our index of contractility. The fluorescent Ca²⁺ indicator Fura-2 was used to monitor changes in [Ca²⁺](i). Action potentials and L-type Ca²⁺ currents (I(Ca)) were recorded using the whole-cell patch-clamp technique. Experiments were performed at 15 °C. Increasing the stimulation frequency caused a significant increase in diastolic [Ca²⁺](i) and a significant decrease in diastolic cell length and membrane potential. During systole, there was a significant fall in the amplitude of the [Ca²⁺](i) transient, cell shortening and action potential with a decrease in the duration of the action potential at both 20% and 90% repolarisation. Caffeine was used to assess the Ca²⁺ content of the sarcoplasmic reticulum. We observed that sarcoplasmic reticulum Ca²⁺ load was greater at 1.0 Hz than at 0.6 Hz, despite a smaller electrically evoked [Ca²⁺](i) transient. The amplitude of I(Ca) was found to decrease with increased stimulation frequency. At 0.6 Hz, electrically evoked [Ca²⁺](i) transients in the presence of 10 mmol l^-1 caffeine or 10 μmol l^-1 ryanodine and 2 μmol l^-1 thapsigargin were reduced by approximately 15%. We have described the changes in contractility, [Ca²⁺](i) and action potential configuration in a fish cardiac muscle system. Under the conditions tested (0.6 Hz, 15°C), we conclude that the sarcoplasmic reticulum contributes at least 15% of the Ca²⁺ associated with the [Ca²⁺](i) transient. The rate-dependent decrease in contraction amplitude appears to be associated with the fall in the amplitude of the [Ca²⁺](i) transient. This, in turn, may be influenced by changes in the action potential configuration via mechanisms such as altered Ca²⁺ efflux and Ca²⁺ influx. In support of our conclusions, we present evidence that there is a rate-dependent decrease in Ca²⁺ influx via I(Ca) but that the Ca²⁺ load of the sarcoplasmic reticulum is not reduced at increased contraction frequencies.