Extracellular Cathepsin-L Alters Ca2+ Transient Amplitude and Sarcoplasmic Reticulum Ca2+ Content in Adult Rat Cardiomyocytes

Abstract

Serum levels of Cathepsin-L (CatL), a ubiquitous cysteine protease, are increased in patients with ischaemic heart disease. It is unknown whether CatL could (i) be released by the heart following ischaemia and (ii) contribute to altered sarcoplasmic reticulum (SR)-mediated function in cardiomyocytes during cardiac disease. Ex vivo perfused rat hearts underwent 30min global ischaemia with subsequent reperfusion. CatL activity was detected (Z-LR-AMC fluorometric assay) in coronary effluent throughout the 90 min post-reperfusion period. The effect of extracellular CatL on SR-mediated Ca2+ release across a range of reported concentrations found in patients with coronary heart disease was also determined. Ventricular rat cardiomyocytes were isolated, loaded with Fura-2AM and incubated (30min) with recombinant CatL/vehicle at 0.68nM(n=23), 2.70nM(n=22) and 5.40nM (n=18). Cardiomyocytes were field-stimulated (0.5Hz) and perfused with a modified Krebs-Henseleit solution containing CatL/vehicle. CatL activity at physiological pH within the perfusate was confirmed. using epifluorescence microscopy, Ca2+ transient parameters during field-stimulation and during rapid application of 10mM caffeine were determined. Ca2+ transient amplitude was decreased in a concentration-dependent manner by the above concentrations of CatL (Control:100±4.6 (n=67) vs.79.8±7.1 (0.68nM), 68.8±4.8 (2.7nM), 42.9±5.2% (5.4nM); P 0.05). This study demonstrates that CatL is released from ischaemic hearts upon reperfusion and depresses SR-mediated Ca2+ release in field-stimulated Ca2+ transients in a concentration-dependent manner. Extracellular CatL may therefore have the potential to contribute to cardiac dysfunction in patients with heart failure.