Functionally Isolated Sarcoplasmic Reticulum Model: Intrinsic Regulation of SR Ca2+ Release and Tetracaine Effect

Abstract

The fraction of the sarcoplasmic reticulum (SR) Ca2+ content released at a twitch (fractional release, FR) in cardiomyocytes is regulated by the L-type Ca2+ current (ICa,L) and the SR Ca2+ content ([Ca2+]SR). In the experimental model of the functionally isolated SR (FISRM), cardiomyocytes are perfused with Na+, Ca2+-free solution, which makes the cells unexcitable and thermodynamically inhibits the sarcolemmal Na+/Ca2+ exchanger. Intracellular free Ca2+ concentration ([Ca2+]i) was measured with fluo-3 and Ca2+ transients due to SR release were evoked by 100 ms-long caffeine pulses. FISRM was used to study the intrinsic relationship between FR and [Ca2+]SR in the absence of ICa,L, the physiological trigger for SR Ca2+ release. After SR depletion by prolonged caffeine application, 1-20 electrical stimuli were applied to attain different [Ca2+]SR (from 55.3±10.0 to 123.0±10.3 μM). The variation in total [Ca2+] (Δ[Ca2+]T) during transients was estimated from [Ca2+]i and Ca2+ buffering parameters. FR (Δ[Ca2+]T/[Ca2+]SR) showed a non-linear relationship with [Ca2+]SR (from 5.4±5.4 to 82.8±7.2%, p< 0.05), as previously observed when SR Ca2+ release was triggered by ICa,L. This behavior was reproduced with a mathematical model of FISRM, after inclusion of intra-SR and cytosolic Ca2+ buffers, which resulted in more realistic simulations. In addition, FISRM was used to study the inhibitory effect of tetracaine on FR. The tetracaine concentration that caused 50% inhibition of FR (IC50) was 58.9±5.7 μM. The FISRM may be a useful experimental model to investigate in intact cells the selective effects on SR Ca2+ release of drugs that also affect sarcolemmal currents, such as tetracaine. Support: CNPq.