The [Ca²⁺] within the SR of cardiac muscle ([Ca²⁺]_SR) may modulate the activity of SR Ca²⁺ ATPase (SERCA2a) and the Ca²⁺ release channels (ryanodine receptor). However, direct measurement of [Ca²⁺]_SR in adult myocytes has not been possible due to the lack of a specific indicator. A mutated aequorin chimera has been used to measure free [Ca²⁺]_SR in cultured myotubes. Targeting of the photoprotein was achieved by generation of a construct that consisted of cDNA encoding the SR resident protein calsequestrin fused to the aequorin cDNA (p srAEQ) (Brini et al. 1997). To allow expression of this protein in adult cardiac myocytes, an adenovirus was generated from the aequorin-calsequestin plasmid (Ad srAEQ). Male Wistar rats were killed by cervical dislocation and the hearts removed and perfused with collagenase/protease solution to isolate ventricular cardiomyocytes. Isolated cardiomyocytes were infected with the Ad srAEQ. Calcium dynamics were studied in populations of cells 2 days post-infection. Cardiomyocytes were permeabilised by brief exposure to 0.01 mg ml^-1 β-escin and subsequently bathed in a mock intracellular solution (mM): 130 K⁺, 30 Na⁺, 1 Mg²⁺, 25 Hepes, 100 Cl^– , 0.05 EGTA, 5 ATP, 10 CrP and 0.9 Mg; pH 7.0. Aequorin was reconstituted by 15 min incubation with 5 mM coelenterazine n. Cardiomyocytes (5 X 10⁵ in 200 µl) were placed in a luminometer and the aequorin light emission was monitored while the bathing [Ca²⁺] was increased from < 1 nM to 380 nM in the presence of 10 mM EGTA. Total luminescence was assayed by subsequently adding 1 % Triton X-100/100 mM CaCl₂. The fractional luminescence was converted to [Ca²⁺] using a previously published calibration curve (Brini et al. 1997). As shown in Fig. 1, these calculations suggest that [Ca²⁺]_SR increases from approximately 5 to 50 mM. The increase in signal was completely inhibited by previous incubation with thapsigargin (125 mg ml^-1). These measurements indicate a [Ca²⁺]_SR of 55 ± 6 mM (mean ± S.E.M., n = 14; this corresponds to a fractional luminescence of 1.55 ± 0.2 X 10^-3) in the presence of [Ca²⁺]_cy 380 nM.

This work was financially supported by BHF. The SR aequorin construct was a gift from Dr R. Rizzuto, University of Ferrara, Italy.

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