The contractile behaviour of EGTA- and detergent-treated heart muscle

Abstract

Tension responses of rat ventricular trabeculae subjected to successive 'treatment' with EGTA and Triton X-100 are described in order to investigate the effects of chemical 'skinning' techniques. In some preparations the alkaloid saponin was also used before Triton. Ultrastructural evidence is cited that the 'EGTA-treatment' fails to render cells 'hyperpermeable', i.e. freely permeable to small ions, whereas both saponin and Triton do so. In this paper we show that contractile responses like those described previously for the 'EGTA-treated' tissue can be obtained. However, more detailed examination shows that such behaviour is quantitatively distinct from that of conventionally skinned fibres in a way that is incompatible with the notion of 'hyperpermeability'. The Ca-sensitivity after treatment with either EGTA, saponin or Triton is identical in our hands. However, this is not explained by free access of Ca (and EGTA) to the intracellular space in the EGTA-treated preparation: contractures develop with very different time courses, being fastest after Triton and only marginally slower when first exposed to saponin but a factor of five times slower after 'EGTA-treatment' alone. This applies to contractures evoked direct from Ca2+ concentration congruent to 10(-9) M to the test Ca2+ concentration at constant total buffer concentration. 'EGTA-treated' fibres develop tension when ATP or creatine phosphate (CrP) are removed from the bath. However, responses to ADP and to CrP changes persist with millimolar levels of ATP present, quite unlike the Triton-skinned muscle. Exposure to each of a variety of solutions for 24 h produce preparations showing similar behaviour: whatever the explanation for the EGTA-'skinning' phenomenon it is not dependent upon low bathing Ca2+ concentration. On the basis of the functional characteristics described here, and the structural results cited, we conclude that the cell membrane continues to function as a selective permeability barrier after 'EGTA-treatment': this treatment does not produce a model of a selectively 'skinned' cardiac cell.