Cardiac Myofibril ATPase Assay

Cardiac myofibrils were prepared, endogenous RLCs exchanged with either wildtype RLC or RLCs crosslinked to bifunctional rhodamine, and the ATPase activity under relaxing conditions determined as previously described^{22 (link)}. Briefly, cardiac myofibrils (CMFs) were prepared by homogenising freshly frozen ventricular tissue samples in myofibril buffer (composition in mmol L⁻¹: 20 imidazole, 75 KCl, 2 MgCl₂, 2 EDTA, 1 DTT, 1% (v/v) Triton X-100, pH 7.4, protease inhibitor cocktail (ROCHE), PhosStop cocktail (ROCHE)) followed by centrifugation at 5000 g for 5 min at 4 °C. CMFs were washed and homogenised three more times in the same buffer without Triton X-100. Endogenous RLCs were extracted from CMFs and reconstituted with recombinant proteins using the same protocol as described above for trabeculae. CMFs were washed three times in ATPase assay buffer (composition in mmol L⁻¹: 20 MOPS, 35 NaCl, 5 MgCl₂, 1 EGTA, 1 DTT, pH 7.0). Reactions were started by the addition of 2.5 mmol L⁻¹ ATP and samples were quenched with 0.5 volumes ice cold 25% (w/v) TCA solution. Samples were kept on ice at all times, diluted with double-deionized water and inorganic phosphate content measured using the malachite green assay according to manufacturer’s instructions (SIGMA, GILLINGHAM SP8 4XT, United Kingdom; MAK030).