Cathepsin-L (CatL) is a lysosomal cysteine protease which can be released from a variety of cell types into the extracellular space. CatL is increased in the serum of patients with heart disease1 and has been linked to post-infarct extracellular matrix (ECM) remodelling2. However, whether extracellular CatL can directly alter cardiac function remains unknown. Sarcoplasmic reticulum (SR)-mediated Ca2+ release in cardiomyocytes is a major determinant of cardiac function and is modified during cardiac disease. The aim of this study was to determine whether extracellular CatL can directly alter SR-mediated Ca2+ release in cardiomyocytes. Adult male Wistar rats (~250g) were killed in accordance with Schedule 1 of the Animal (Scientific Procedures) Act 1986 and ventricular cardiomyocytes isolated. Recombinant mammalian CatL activity at physiological pH was confirmed using a Z-LR-AMC fluorometric assay. Spontaneous Ca2+ wave characteristics in Fluo-3-loaded cardiomyocytes incubated for 30min with 5.4nM CatL/vehicle were measured by confocal microscopy. In separate experiments, Ca2+ transient parameters in field stimulated (0.5Hz) Fura-2AM loaded cardiomyocytes incubated for 30min and subsequently perfused with a modified Krebs-Henseleit solution containing 5.4nM CatL/vehicle were measured using epifluoresence microscopy. SR Ca2+ content in these cells was determined by the amplitude of caffeine-induced Ca2+ release after 6min of field stimulation. Data are presented as mean±SEM, CatL vs. control; statistical comparisons were made by Student’s paired t-test. Confocal microscopy revealed that Ca2+ wave frequency was increased by CatL (0.16±0.02 vs. 0.12±0.02, CatL:n=73, control:n=70)) and time constant (tau) of Ca2+ wave decay significantly shortened (69±3 vs. 79±4ms; P<0.05). Field stimulated Ca2+ transient amplitude was decreased by CatL (0.22±0.03 vs. 0.51±0.06µM, CatL:n=18, control:n=16;P<0.05) via both a reduced transient peak [Ca2+]i (0.31±0.029 vs. 0.58±0.068µM, P<0.05) and minimum [Ca2+]i (73.4±4.89 vs. 93.5±6.40nM; P<0.05). The maximum rate of rise (dCa2+/dtmax), was slower (3.8±0.4 vs. 9.6±1.2µM.s-1; P<0.05) as was the maximum rate of fall, (dCa2+/dtmin), (0.1±0.02 vs. 0.6±0.1µM.s-1; P<0.05). The tau of Ca2+ transient decay was prolonged (2.4±0.2 vs. 0.9±0.1s; P<0.05). The amplitude of the caffeine-induced Ca2+ transient was reduced by CatL (0.5±0.1 vs. 0.8±0.1µM; P<0.05) and the tau prolonged (1.1±0.07 vs. 0.9±0.04s; P<0.05) suggesting reduced SR Ca2+ content and slowed cellular extrusion of Ca2+ via sarcolemmal fluxes. This study demonstrates for the first time that extracellular CatL reduces Ca2+ transient amplitude and increases Ca2+ wave frequency. Therefore CatL may not only play a role in ECM remodelling but also has the potential to directly alter cardiac function during heart disease.