Use of the Ca-Shortening Curve to Estimate the Myofilament Responsiveness to Ca2+ in Tetanized Rat Ventricular Myocytes

Abstract

Abstract:We previously estimated the myofilament responsiveness to Ca 2+ in isolated intact ventricular myocytes, using the steady-state relationship between cytosolic Ca 2+ concentration ([Ca 2+ ] i ) and cell-shortening during tetanus (Ca-L trajectory). This method was useful and easy; however, it could not be used for a high dose of Ca sensitizer because the instantaneous plots after the application of Ca sensitizer did not make a fixed point of shortening (we used 5% shortening). Therefore we must produce another method to investigate Ca 2+ responsiveness. For an estimation of a wider range of the Ca-L trajectory, we fitted the Ca-L trajectory data with the Hill equation to construct the Ca-shortening curve. To fit this curve, we measured the maximal shortening, which was on average 31.6%. The value of [Ca 2+ ] i to produce the half-maximal shortening (Ca 50 ) was dose-dependently decreased by EMD57033 (sensitization). Either isoproterenol or 3-isobutyl-1-methylxanthine increased Ca 50 (desensitization) with a concomitant increase in intracellular c-AMP. EMD57439, a selective PDE-III inhibitor, did not significantly increase the c-AMP concentration and produced little change in Ca 50 . These results are in agreement with previous reports with skinned or intact multicellular preparations. The Ca-shortening curve constructed in intact cardiac myocytes can be used to estimate the myofibrillar responsiveness to Ca 2+ in a wide range of [Ca 2+ ] i .Key words: calcium (cellular), contractile function, inotropic agents, myocytes, second messengers.The change in the myofibrillar responsiveness to Ca 2+ is one of the key mechanisms in the regulation of cardiac contraction [1]. Myofibrillar responsiveness to Ca 2+ has been investigated most extensively by the use of skinned cardiac muscles [2], in which the sarcolemmal membrane is removed either chemically or mechanically. In skinned preparations, however, intracellular signal transduction pathways that regulate myofibrillar responsiveness to Ca 2+ might be altered. Therefore it is important to estimate the myofibrillar responsiveness to Ca 2+ by using intact preparations.One approach in intact multicellular preparations is the use of tetanic contraction, which produces a quasi-steady state relation between force and [Ca 2+ ] i [3,4]. However, this approach may bring on methodological difficulties, such as an inhomogeneous activation of force and limited drug permeation through the tissue. In regard to isolated myocytes, Spurgeon et al. [5] proposed the use of the phase-plane diagram of cell length versus intracellular Ca 2+ concentration ([Ca 2+ ] i ) during the relaxation of twitch contraction. Equilibrium between cell length and [Ca 2+ ] i , however, may not always be achieved during twitch relaxation, particularly with interventions that increase the decay rate of the [Ca 2+ ] i transient (e.g., β-adrenoceptor stimulation). Previously, we reported that an instantaneous relation between [Ca 2+ ] i and cell length (Ca-L trajectory) during tetanus could estimate t...

Real-time measurement of the length of a single sarcomere in rat ventricular myocytes: a novel analysis with quantum dots

“…The heart was then excised and perfused with 2 mM Ca 2ϩ -HEPES-Tyrode solution. Ventricular myocytes were isolated by digesting the extracellular matrix with collagenase, as reported previously (38,39,46). Myocytes were suspended in Ca 2ϩ -free-HEPES-Tyrode solution for up to 12 h at ϳ3°C.…”

“…Physiological experiments were performed based on our laboratory's earlier studies (38,39,42) under epi-illumination fluorescence microscopy. First, for the observation of SPOC, skinned myocytes were immersed in relaxing solution (see above) containing 2 nM anti-␣-actinin antibody-QDs for 1 h at ϳ3°C.…”

“…Refs. 38,39,42), filled with relaxing solution, and mounted on the inverted microscope stage (IX71, Olympus, Tokyo, Japan). The edges of the myocyte were held with glass needles (diameter ϳ3 m) attached to manipulators (Narishige, Tokyo, Japan) to control SL and to maintain the myocyte length during SPOC (as in our laboratory's previous studies; e.g., Refs.…”

“…Refs. 38,39,42). Voltage was set so that ϳ20% of myocytes were stimulated and showed cyclic contractions on the Petri dish.…”

Comparing hyoscine and drotaverine effects on colon in CT colonography

“…The increased cAMP level affects cellular processes inhibiting the contractile process in SMs [25,26]. The specific way of action of drotaverine includes reduction of the Ca 2+ cytosolic level and/or desensitization of contractile apparatus [27,28]. Both processes are fundamental for the development of SM contraction/relaxation [29] in all types of SM tissues, involving both spontaneous phasic contractions and maintenance of SM tone.…”