It has been known since the work of Parmley & Chuck
(1973) that a reduction in length of isolated cardiac muscle
produces a rapid decrease of twitch force, followed by a
further, slow fall of force over 10 min or so. If length is
increased, there are corresponding rapid and slow increases
in twitch force (reviewed by Allen & Kentish, 1985;
Lakatta, 1992). The rapid changes in force are thought to
form the basis of the length—tension (Frank—Starling)
mechanism in the whole heart. The slow force responses to a
length change are also likely to be important for the
regulation of cardiac function, since they have been seen in
blood-perfused isolated hearts (e.g. Tucci, Bregagnollo,
Spadaro, Cicogna & Ribeiro, 1984; Burkhoff, de Tombe,
Hunter & Kass, 1991) and in vivo in anaesthetized dogs
(Lew, 1993). Indeed, in vivo they probably underlie the
‘Anrep effect’, by which an increase in ventricular volume
due to a rise in aortic pressure is followed by a secondary,
slow increase in myocardial performance, such that the
ventricular volume falls towards its original size. However,
the mechanism of the slow force responses has remained
obscure. Allen & Kurihara (1982) showed that the slow
increase in force after stretch of isolated papillary muscles
was due, at least in part, to a slow rise in the magnitude of
the intracellular Ca¥ transient. This slow enhancement of
the Ca¥ transient could have been due to a potentiation of
excitation—contraction coupling, or it could have risen
indirectly by a rise in diastolic Ca¥ concentration, which
would then lead to greater Ca¥ sequestration by the
sarcoplasmic reticulum (SR). Subsequently, Nichols (1985)
reported that diastolic length was the important factor
controlling the slow force response to a length change in
papillary muscles. Thus it seemed likely that stretch did
increase the diastolic Ca¥ concentration, perhaps by
activation of the stretch-activated channels reported in
cardiac cells by a number of workers (e.g. Craelius, 1993).
However, it has proved difficult to establish whether
diastolic Ca¥ concentration does indeed increase after
muscle stretch. Allen, Nichols & Smith (1988) used aequorin
to confirm that it was the diastolic length of papillary
muscles that controlled the size of the systolic Ca¥
Journal of Physiology (1998), 506.2, pp.431—444
431
Changes in force and cytosolic Ca¥ concentration after length
changes in isolated rat ventricular trabeculae
Jonathan C. Kentish and Antoni Wrzosek
Department of Pharmacology, United Medical and Dental Schools, St Thomas’s Hospital,
London SE1 7EH, UK
(Received 7 August 1997; accepted 12 September 1997)
1. Changes in cytosolic [Ca¥] ([Ca¥]é) were measured in isolated rat trabeculae that had been
micro-injected with fura_2 salt, in order to investigate the mechanism by which twitch force
changes following an alteration of muscle length.
2. A step increase in length of the muscle produced a rapid potentiation of twitch force but not
of the Ca¥ transient. The rapid rise of force was unaffected by inhibiting the sarcoplasmic
reticulum (SR) with ryanodine and cyclopiazonic acid.
3. The force—[Ca¥]é relationship of the myofibrils in situ, determined from twitches and tetanic
contractions in SR-inhibited muscles, showed that the rapid rise of force was due primarily
to an increase in myofibrillar Ca¥ sensitivity, with a contribution from an increase in the
maximum force production of the myofibrils.
4. After stretch of the muscle there was a further, slow increase of twitch force which was due
entirely to a slow increase of the Ca¥ transient, since there was no change in the myofibrillar
force—[Ca¥]é relationship. SR inhibition slowed down, but did not alter the magnitude of, the
slow force response.
5. During the slow rise of force there was no slow increase of diastolic [Ca¥]é, whether or not
the SR was inhibited. The same was true in unstimulated muscles.
6. We conclude that the rapid increase in twitch force after muscle stretch is due to the length-
dependent properties of the myofibrils. The slow force increase is not explained by length
dependence of the myofibrils or the SR, or by a rise in diastolic [Ca¥]é. Evidence from tetani
suggests the slow force responses result from increased Ca¥ loading of the cell during the
action potential.
7257
Keywords: Calcium, Cardiac myocyte, Force

transient, but the insensitivity of aequorin to resting levels
of Ca¥ made it difficult to measure the diastolic Ca¥
concentration. More recent studies have used fura_2, which
has the appropriate Kd to measure diastolic levels of Ca¥
(•200 nÒ; Grynkiewicz, Poenie & Tsien, 1985). In a
preliminary study, Steele & Smith (1993) reported that the
diastolic Ca¥ concentration in guinea-pig trabeculae did
increase during the slow force responses. In contrast, Hongo,
White, Le Guennec & Orchard (1996) found diastolic Ca¥
concentration was unchanged during the slow force response
in rat myocytes. In addition to this discrepancy, in both
studies the preparations were loaded with the acetoxymethyl
(AM) form of fura_2, which has the disadvantage that
fura_2 AM enters, and gives a fluorescence signal from,
intracellular organelles such as mitochondria. In addition,
there can be a Ca¥-independent fluorescence signal from
partly hydrolysed fura_2 AM. Both factors may obscure true
changes in the Ca¥ concentration of the cytosol (Backx &
ter Keurs, 1993).
Another source of uncertainty is that it is not known whether
the magnitude of the slow force responses can be attributed
entirely to changes in the systolic Ca¥ transient, or whether
there is also a contribution from a change in the Ca¥
sensitivity of the myofibrils.
In the present work, we investigated the influence of muscle
length on the cytosolic Ca¥ concentration ([Ca¥]é) during
systole and diastole in rat trabeculae. We iontophoresed
fura_2 salt into the myocardial cells of the trabeculae, using
the technique of Backx & ter Keurs (1993). This procedure
not only allows an unequivocal measure of cytosolic [Ca¥]é
alone, but also ensures that the fura_2 signal comes entirely
from the myocardial cells and not from the smooth muscle
and endothelial cells in the preparation. In addition, we
determined the force—[Ca¥]é relationship for the myofibrils
in situ in the trabeculae, and so could determine, for the
first time, whether changes in this relationship contributed
to the slow force responses. Finally, we examined the relative
contributions of the length-dependent changes in maximum
force production and Ca¥ sensitivity of the myofibrils to the
rapid effects on twitch force of a change in muscle length.
A preliminary account of this work has been presented
(Kentish &Wrzosek, 1995).
METHODS
Preparation of trabeculae
Male Wistar rats (•250 g) were stunned by a blow to the head and
were killed by cervical dislocation under Home Office guidelines
(Schedule 1). Their hearts were removed and washed free of blood
with Tyrode solution containing (mÒ): NaCl, 93; NaHCO×, 20;
NaµHPOÚ, 1; MgSOÚ, 1; KCl, 5; CaClµ, 1; glucose, 10; sodium
acetate, 20; insulin, 5 U l¢; oxygenated with 95% Oµ—5% COµ;
pH 7·4 at room temperature (23°C). Trabeculae (1·5—3 mm long,
90—150 ìm wide and 50—100 ìm thick), with a small piece of
mitral valve attached, were dissected from the right ventricle in
Tyrode solution containing 25 mÒ 2,3-butanedione monoxime
(BDM) to minimize muscle damage (Mulieri, Hasenfuss, Ittleman,
Blanchard & Alpert, 1989). A trabecula was then mounted in a
perfusion bath (5 mm ² 4 mm ² 5 cm) located on a stage of a
Nikon Diaphot inverted microscope and was superfused with
Tyrode solution. The valve end of the muscle was impaled on a fine
hook connected to a force transducer (SensoNor, Horten, Norway)
and the wall end was ensnared in a wire loop attached to a
Narishige micromanipulator. A digitimer D4030 (Digitimer Ltd,
Welwyn Garden City, Herts, UK) triggered an isolated stimulator
(DS2, Digitimer), which delivered pulses (5 ms; 10—20% above
threshold) at 0·33 Hz to the muscles via platinum electrodes
running along each side of the muscle bath. During an equilibratory
period of >1 h the muscles were stretched progressively to the
length where developed (‘twitch’) force was •95% of its maximum.
This initial length is termed L0. Muscles were shortened, usually to
90% of L0 (L90), by manual adjustment of the micromanipulator
(taking 3—5 s to complete).
All experiments were done at room temperature (23—25°C). For
unstimulated muscles and for twitches we used an extracellular
[Ca¥] ([Ca¥]ï) of 1 mÒ, which is close to the value of 1·3 mÒ for
the ionized [Ca¥]ï in rat blood (Forester & Mainwood, 1974;
Chambers, Braimbridge & Hearse, 1991). For tetanic stimulation
[Ca¥]ï was raised to 8 mÒ to ensure maximum Ca¥ activation of
the myofibrils.
Cell loading with fura_2
Before injection of fura_2, the 340 and 380 nm autofluorescence
signals at muscle lengths L0 and L90 were measured. Fura_2 penta-
potassium salt was then iontophoresed into myocardial cells in the
trabecula using the method of Backx & ter Keurs (1993) with small
modifications. Briefly, the tip of a microelectrode (final resistance,
50—300 MÙ) was filled with 2 mÒ fura_2 pentapotassium salt in
HµO and was backfilled with 140 mÒ KCl. The perfusion solution
was changed to Tyrode solution containing 10 mÒ BDM and
stimulation was stopped. Then the microelectrode was advanced
into a cell, which usually had a membrane potential of −55 to
−80 mV, measured using an AxoClamp-2A amplifier (Axon
Instruments, Foster City, CA, USA). When the voltage was stable,
a negative current of 4—8 nA was passed for 8—15 min (depending
on the size of the muscle) to inject fura_2 into the cell. The muscle
was then superfused with normal Tyrode solution and fura_2
fluorescence was measured. Usually two or three injections were
given at different sites along the muscle in order to ensure that
fura_2 fluorescence was 3—5 times above autofluorescence and to
increase the uniformity of dye distribution. After iontophoresis, the
muscle was stimulated at 0·33 Hz for about 1 h, during which
fura_2 spreads along the muscle via the gap junctions (Backx & ter
Keurs, 1993). We measured dye distribution by recording the 340
and 380 nm fluorescence signals through a small (300—450 ìm
square) window that was moved along the muscle. Figure 1 shows
an example of a muscle in which we gave three injections. The
uniformity of dye distribution over the typical recording distance
for the length-change experiments (•1 mm) was usually > 80%
after 1 h (Fig. 1), when the experiments were started. The dye
uniformity was increased by the continued diffusion of dye during
the experiment, although dye concentration fell due to loss of fura_2
from the cells (Fig. 1). Any slight non-uniformity of dye
distribution should not affect the results because we measured the
340 nmÏ380 nm fluorescence ratio, which is independent of dye
concentration.
Over the period required for successful multiple injections and post-
injection equilibration (2—3 h), twitch force declined by about 20%.
Although some of this decrease may have been due to buffering of
cytosolic [Ca¥]é by the fura_2 (see below), most of it was probably
J. C. Kentish and A. Wrzosek
J. Physiol. 506.2
432