The steady-state force–Ca²⁺ relationship in intact lobster (Homarus americanus) cardiac muscle

Abstract

The heart of the decapod crustacean is activated by regular impulse bursts from the cardiac ganglion. The cardiac pump function depends on ganglionic burst frequency, burst duration, and burst impulse frequency. Here, we activated isolated lobster cardiac ostial muscle (Orbicularis ostii muscle, OOM) by stimulus trains in vitro in order to characterize the response of the contractile apparatus to [Ca²⁺]_i . We employed stimulus trains that generate a steady state between the [Ca²⁺]_i and force in order to estimate the Ca²⁺ sensitivity of myofilaments. Force and [Ca²⁺]_i transients were simultaneously recorded using a silicon strain gauge and the fluorescence of iontophoretically microinjected fura-2 salt. We examined the effects of tetanus duration (TD), the interval between trains, and 6 μM cyclopiazonic acid, an inhibitor of the SR Ca²⁺ pump, on the steady-state force–[Ca²⁺]_i relationship. The instantaneous force–[Ca²⁺]_i relationships appeared sigmoidal (EC ₅₀ and Hill coefficient, 98.8±32.7 nM and 2.47±0.20, mean ± SD, respectively), as did the curves superimposed after 500 ms following the start of stimulation, indicating that the force–[Ca²⁺]_i relationship had reached a steady state at that time. Also, the maximum activated force (F _max) was estimated using the steady-state force–[Ca²⁺]_i relationship. Prolonged stimulus trains, decreasing the interval between recurrent trains from 5 to 2.5 s, and cyclopiazonic acid each increased the measured EC ₅₀ without changing F _max. The EC ₅₀ correlated strongly with averaged [Ca²⁺]_i over time. We conclude that the steady-state force–[Ca²⁺]_i relationships in the OOM indicate cooperation between force generation and Ca²⁺ binding by the myofilaments. Our data also suggest the existence of a novel Ca²⁺-dependent mechanism which reduces Ca²⁺ sensitivity and accelerates relaxation of lobster cardiac muscle myofilaments.