Ferret Ventricular Myocytes Research Articles

Relaxation in ferret ventricular myocytes: unusual interplay among calcium transport systems.

Transport systems responsible for removing Ca2+ from the myoplasm during relaxation in isolated ferret ventricular myocytes were studied using caffeine-induced contractures. Internal calcium concentration ([Ca2+]i) was measured with the fluorescent calcium indicator indo-1, and the results were compared with our recent detailed characterizations in rabbit and rat myocytes. Relaxation and [Ca2+]i decline during a twitch in ferret myocytes were fast and similar to that in rat myocytes (i.e. half-time, t 1/2 approximately 100-160 ms). During a caffeine-induced contracture (SR Ca2+ accumulation prevented), relaxation was still relatively fast (t 1/2 = 0.57 s) and similar to relaxation in rabbit supported mainly by a strong Na(+)-Ca2+ exchange. When both the SR Ca2+ uptake and Na(+)-Ca2+ exchange are blocked (by caffeine and 0 Na+, 0 Ca2+ solution) relaxation in the ferret myocyte is remarkably fast (approximately 5-fold) compared with rabbit and rat myocytes. The decline of the Cai2+ transient was also fast under these conditions. These values were similar to those in rat under conditions where relaxation is due primarily to Na(+)-Ca2+ exchange. Additional inhibition of either the sarcolemmal Ca(2+)-ATPase or mitochondrial Ca2+ uptake caused only modest slowing of the relaxation of caffeine-induced contracture in 0 Na+, 0 Ca2+ (t 1/2 increased to approximately 3 s). In rabbit myocytes the relaxation t 1/2 is slowed to 20-30 s by these procedures. Even when the systems responsible for slow relaxation in rabbit ventricular myocytes are inhibited (i.e. sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake) along with the SR Ca(2+)-ATPase and Na(+)-Ca2+ exchange, relaxation and [Ca2+]i decline in ferret myocytes remain rapid compared with rabbit myocytes. Ca2+ taken up by mitochondria in rabbit myocytes during a caffeine contracture in 0 Na+, 0 Ca2+ solution gradually returns to the SR after caffeine removal, but this component appears to be much smaller in ferret myocytes under the same conditions. We tested for possible residual Ca2+ transport by each of the four systems which suffice to explain Ca2+ removal from the cytoplasm in rabbit (SR Ca(2+)-ATPase, Na(+)-Ca2+ exchange, sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ uptake). We conclude that there is an additional calcium transport system at work in ferret myocytes. For this additional system, our results are most compatible with a trans-sarcolemmal Ca2+ transport, but neither a cation exchanger nor a Ca(2+)-ATPase with characteristics like that in other cardiac cells. This additional system appears able to transport Ca2+ nearly as fast as the Na(+)-Ca2+ exchange in rat ventricular myocytes.

Paradoxical Twitch Potentiation After Rest in Cardiac Muscle: Increased Fractional Release of SR Calcium

Rest interval dependent changes in contractile force (rest decay and rest potentiation) were studied in rabbit, rat and ferret ventricular muscle and myocytes. The SR Ca content was assessed by rapid cooling contractures or caffeine induced contractures. Intracellular Ca transients, action potentials and Ca current were also recorded. Rest decay of twitches ventricle are roughly paralleled by a decline in SR Ca content. Rat ventricle exhibits primarily rest potentiation, which is not necessarily paralleled by an increased SR Ca content. Ferret ventricle exhibits both rest potentiation and rest decay. However, the SR Ca content in ferret appears to decline monotonically throughout the rest. It is demonstrated that the rest potentiation is not due to an increase in Ca current or in action potential duration. We conclude that there is increase in the fraction of SR Ca content which is released the time that rest potentiation develops. The differences in post-rest contractile function among different cardiac preparations can be described by a simple unifying mechanistic model. In this model there is an exponential time dependent recovery of the ability of the SR to release Ca (e.g. recovery from inactivation) which can be considered to increase the fraction os SR Ca release in response to activation. This fractional SR Ca release is multiplied by the SR Ca content (which may decline exponentially) to provide a measure of the Ca available for activation of contractile force.

Effects of extracellular ATP on ICa, [Ca2+]i, and contraction in isolated ferret ventricular myocytes

The effects of extracellular ATP on the voltage-activated "L-type" Ca current (ICa), action potential, resting and transient intracellular Ca2+ levels, and cell contraction were examined in enzymatically isolated myocytes from the right ventricles of ferrets. With the use of the whole cell patch-clamp technique, extracellular ATP (10(-7) to 10(-3) M) inhibited ICa in a time- and concentration-dependent manner. ATP decreased the peak amplitude of ICa without altering the residual current at the end of 500-ms clamp steps. The concentration-response relationship for ATP inhibition of ICa was well described by a conventional Michaelis-Menten relationship with a half-maximal inhibitory concentration of 1 microM and a maximal effect of 50%. Consistent with its inhibitory effect on ICa, ATP hyperpolarized the plateau phase and shortened the action potential duration. In fura-2-loaded myocytes, extracellular ATP did not change the resting myoplasmic Ca2+ levels; however, when current was elicited under voltage-clamp conditions, ATP both decreased the myoplasmic intracellular Ca2+ transient and inhibited the degree of cell shortening. Our results suggest that ATP could be a genuine and potent extracellular modulator of cardiac function in ferret ventricular myocardium.

Possible involvement of a chloride conductance in the transient outward current of whole-cell voltage-clamped ferret ventricular myocytes

The transient outward current was studied, using the whole-cell patch-clamp technique, in isolated ventricular cells from the ferret heart. In the presence of 4-aminopyridine and cadmium chloride which respectively blocked the Ca-insensitive and the Ca-dependent outward currents, a residual transient outward current was observed in about 30% of the cells tested. This current was suppressed in external hypochloride solution, completely inhibited by SITS (3 mM) and reversed at the equilibrium potential for chloride ions. This suggests the presence of a chloride permeability which could contribute to the repolarization phase of the cardiac action potential.

Voltage sensor of excitation-contraction coupling in skeletal muscle.

Voltage sensor of excitation-contraction coupling in skeletal muscle.

The length, width and volume of isolated rat and ferret ventricular myocytes during twitch contractions and changes in osmotic strength

The length and width of rat and ferret ventricular myocytes have been measured using a linear photodiode array; the volume of the myocytes has been calculated based on the assumption that the cells were elliptical cylinders. During a twitch contraction, there was a decrease in cell length, but no significant change in the calculated cell volume, because the cells increased in width. Inotropic interventions not only resulted in a greater shortening of the cell during each contraction, but also a greater increase in cell width. Changes in cell length, width and volume on changing the osmotic strength of the bathing solution have also been investigated. The increase in volume in hypotonic solution, and the decrease in hypertonic solution, were the result of changes in the cell width; there were no significant changes in the cell length. It is concluded from the latter experiment that the lateral compliance of a cell is greater than its longitudinal compliance, and, therefore, during a twitch contraction, when the cell shortens, the displacement of the cell contents from the two ends of the cell and the expansion of the cell laterally will not act as a large force to oppose shortening.

An efficient isolation procedure of Ca‐tolerant ventricular myocytes from ferret heart for applications in electrophysiological studies

A simple procedure which provides a large yield of isolated ferret ventricular myocytes is described. The enzymatic dissociation was performed by perfusion of the whole heart with the "Langendorff method" at 37 degrees C, without an incubation period. Special attention was given to the period of perfusion with Ca-free or low-calcium containing solutions and to the proportion of both collagenase and elastase used. The viability and calcium tolerance of the isolated cells were tested by ultrastructural and electrophysiological studies. Photo-microscopy showed that 60 to 80% of the isolated cells had an elongated shape (18 microns in diameter, 150 microns in length) and did not beat spontaneously in normal Tyrode solution. The morphological and ultrastructural integrity of these cells was shown in SEM by their smooth surface with regularly spaced T-tubule openings and in TEM by the regular distribution of the transverse tubular system, mitochondrium and sarcomeres. Using the whole-cell patch-clamp technique, they had a resting membrane potential of -72 mV, two types ("Purkinje like" and "ventricular like") of action potentials could be elicited and they were correctly affected by well-known modulators of calcium channels. This technique was successfully applied to the rat heart and could be used for heart dissociation of small mammals. It can simultaneously provide isolated cells of different regions of the heart and can be easily and routinely used by any investigator.

Rapid regulation of the 'second inward current' by intracellular calcium in isolated rat and ferret ventricular myocytes.

1. Single cells were isolated from the ventricles of ferret and rat hearts. Cells were voltage clamped using a single conventional microelectrode. Membrane voltage, membrane currents and cell length were monitored. 2. The current elicited by decreasing the membrane potential from a holding potential of -40 or -45 mV to potentials more positive than -20 mV was abolished by D600, by Cd2+ and by removal of Ca2+ from the cell superfusate. This current activated within 20 ms and inactivated over several hundred milliseconds; it had a bell-shaped current-voltage relation, and was maximal at about +10 mV. It is concluded that this is the fast Ca2+ current ICa. 3. Increasing bathing [Ca2+] (Ca2+o) led to the appearance of transient inward currents (Iti). If ICa was triggered during Iti, it was reduced in magnitude, and inactivated more slowly. 4. The sarcoplasmic reticulum inhibitor ryanodine (1 mumol/l) abolished Iti, and reduced twitch contraction, but had no direct effect on the magnitude of ICa, although its rate of inactivation was slowed. 5. Iti produced by depolarization of the holding potential, or by lowering bathing [K+] or [Na+], led to similar changes to those described in paragraph 3. 6. Gradually increasing diastolic cytoplasmic [Ca2+] (Ca2+i) by rapid stimulation in the presence of ryanodine, by lowering bathing [K+], or lowering bathing [Na+], led to a parallel decrease of ICa. 7. The effects of lowering bathing [Na+] could be abolished by using an electrode-filling solution containing EGTA. 8. In some ferret cells a slow component of the second inward current was observed. The size of this current was directly related to the size of the twitch: changes in the size of the twitch produced by changing the pattern of stimulation or application of ryanodine were paralleled by changes in the size of this current, but had no effect on the size of ICa. 9. It is concluded that the magnitude of ICa can be decreased by an increase of either resting Ca2+i, or the spontaneous increase of Ca2+ which underlies Iti, but it is not affected by the size of the stimulated calcium transient (although the time course of inactivation is dependent on the calcium transient). The size of the slow component of the second inward current, however, is directly related to the size of the twitch and may, therefore, be activated by Ca2+.

The negative inotropic effect of acetylcholine on ferret ventricular myocardium.

1. The effects of acetylcholine (ACh) on developed tension and intracellular Ca2+ concentration (as measured with aequorin) were studied in ferret papillary muscles, and on twitch shortening, the action potential and membrane currents in ferret ventricular myocytes. 2. Addition of ACh to ferret papillary muscles resulted in decreases in developed tension and the intracellular Ca2+ transient, both of which then partially recovered in the continued presence of ACh ('fade' of the response). On wash-off of ACh both developed tension and the intracellular Ca2+ transient increased above control ('rebound') before returning to control values. 3. Addition of ACh to ferret ventricular myocytes resulted in a membrane hyperpolarization of 2 +/- 0.5 mV (mean +/- S.E.M.; n = 9), a decrease in action potential duration to 23 +/- 6% of control and a decrease in twitch shortening to 31 +/- 5% of control. In the continued presence of ACh these responses to ACh faded. Thirty seconds after the maximal effect of ACh, action potential duration had partially recovered to 34 +/- 6% of control and twitch shortening to 46 +/- 7% of control. 4. The effects of ACh on twitch shortening could be mimicked under voltage clamp by varying voltage clamp pulse duration to simulate the ACh-induced changes in action potential duration. 5. When ACh was applied during a train of voltage clamp pulses of constant duration, 81% of the cells showed less than a 20% decrease in Ca2+ current and twitch shortening. However in 19% of the cells twitch shortening and the apparent Ca2+ current decreased by more than 30%. 6. In the 81% of cells, the normal decrease in twitch shortening was wholly the result of the shortening of the action potential. This in turn was the result of an increase in an outward background current which increased the rate of repolarization during the action potential. The ACh-induced background current reversed at -89 +/- 2 mV and showed inward-going rectification; these properties suggest that it was carried by K+. 7. In the 19% of cells, the normal decrease in twitch shortening was only partly the result of the shortening of the action potential (due to both the increase in outward background current as well as the apparent decrease in Ca2+ current). In these cells the decrease in twitch shortening may also have been partly the direct result of the apparent decrease of Ca2+ current.

Use-dependent changes and ultra-slow inactivation of the Ca 2+ current in ferret ventricular myocytes : M.R. Boyett, M.S. Kirby. Department of Physiology, University of Leeds, Leeds LS2 9JT, U.K.

Use-dependent changes and ultra-slow inactivation of the Ca 2+ current in ferret ventricular myocytes : M.R. Boyett, M.S. Kirby. Department of Physiology, University of Leeds, Leeds LS2 9JT, U.K.