Phosphorylation of troponin I by protein kinase A accelerates relaxation and crossbridge cycle kinetics in mouse ventricular muscle.

Phosphorylation of cardiac myofibrils by cAMP-dependent protein kinase (PKA) can increase the intrinsic rate of myofibrillar relaxation, which may contribute to the shortening of the cardiac twitch during beta-adrenoceptor stimulation. However, it is not known whether the acceleration of myofibrillar relaxation is due to phosphorylation of troponin I (TnI) or of myosin binding protein-C (MyBP-C). To distinguish between these possibilities, we used transgenic mice that overexpress the nonphosphorylatable, slow skeletal isoform of TnI in the myocardium and do not express the normal, phosphorylatable cardiac TNI: The intrinsic rate of relaxation of myofibrils from wild-type and transgenic mice was measured using flash photolysis of diazo-2 to rapidly decrease the [Ca(2+)] within skinned muscles from the mouse ventricles. Incubation with PKA nearly doubled the intrinsic rate of myofibrillar relaxation in muscles from wild-type mice (relaxation half-time fell from approximately 150 to approximately 90 ms at 22 degrees C) but had no effect on the relaxation rate of muscles from the transgenic mice. In parallel studies with intact muscles, we assessed crossbridge kinetics indirectly by determining f(min) (the frequency for minimum dynamic stiffness) during tetanic contractions. Stimulation of beta-adrenoceptors with isoproterenol increased f(min) from 1.9 to 3.1 Hz in muscles from wild-type mice but had no effect on f(min) in muscles from transgenic mice. We conclude that the acceleration of myofibrillar relaxation rate by PKA is due to phosphorylation of TnI, rather than MyBP-C, and that this may be due, at least in part, to faster crossbridge cycle kinetics.

Effects of 1- or -adrenoceptor stimulation on work-loop and isometric contractions of isolated rat cardiac trabeculae.

1. We studied the effects of alpha1- or beta-adrenoceptor stimulation on the contractility of isolated rat ventricular trabeculae at 24 degrees C using the work-loop technique, which simulates the cyclical changes in length and force that occur during the cardiac cycle. Some muscles were injected with fura-2 to monitor the intracellular Ca2+ transient. 2. Comparison of twitch records revealed that peak force was greater and was reached earlier in work-loop contractions than in corresponding isometric contractions. This was attributed to the changes in muscle length and velocity during work-loop contractions, since the Ca2+ transients were largely unaffected by the length changes. 3. Stimulation of alpha1-adrenoceptors (with 100 microM phenylephrine) increased net work, power production, the frequency for maximum work, and the frequency for maximum power production (fopt). The increase in net work was due to the positive inotropic effect of phenylephrine, which was similar at all frequencies investigated (0. 33-4.5 Hz). The increase in fopt was attributed to an abbreviation of twitch duration induced by alpha1-stimulation at higher frequencies (> 1 Hz), even though the twitch became longer at 0.33 Hz. 4. beta-Adrenoceptor stimulation (with 5 microM isoprenaline) produced marked increases in net work, power output, the frequency for net work, and fopt. These effects were attributed both to the positive inotropic effect of beta-stimulation, which was greater at higher frequencies, and to the reduction in twitch duration. beta-stimulation also abolished the frequency-dependent acceleration of twitch duration. 5. The increase in power output and fopt with alpha1- as well as beta-adrenoceptor stimulation suggested that both receptor types may contribute to the effects of catecholamines, released during stress or exercise, although the greater effects of beta-stimulation are likely to predominate.

Positive force- and [Ca2+]i-frequency relationships in rat ventricular trabeculae at physiological frequencies.

The isometric force-frequency relationship of isolated rat ventricular trabeculae (diameter <250 micrometer) was examined at 24, 30, and 37 degreesC at stimulation frequencies (0.1-12 Hz) encompassing the physiological range. Some muscles were microinjected with fura PE3 to monitor the diastolic and systolic intracellular concentration of Ca2+ ([Ca2+]i). At a near-physiological external Ca2+ concentration ([Ca2+]o) of 1 mM, a positive force-frequency relationship was demonstrated at all temperatures. The force-frequency relationship became negative at high frequencies (e. g., >6 Hz at 30 degreesC) at 1 mM [Ca2+]o or at low frequencies at 8 mM [Ca2+]o. The twitch and Ca2+ transient became shorter as stimulation frequency increased; these changes were related to changes in systolic, rather than diastolic, [Ca2+]i and were not blocked by inhibitors of Ca2+/calmodulin-dependent protein kinase II. The positive force-frequency relationship of rat trabeculae was caused by a frequency-dependent loading of the sarcoplasmic reticulum (SR) with Ca2+. We suggest that at high frequencies, or under conditions of Ca2+ overload, this loading saturates. Processes that tend to decrease SR Ca2+ release will then predominate, resulting in a negative force-frequency relationship.

Roles of Ca2+ and crossbridge kinetics in determining the maximum rates of Ca2+ activation and relaxation in rat and guinea pig skinned trabeculae.

We examined the influences of Ca2+ and crossbridge kinetics on the maximum rate of force development during Ca2+ activation of cardiac myofibrils and on the maximum rate of relaxation. Flash photolysis of diazo-2 or nitrophenyl-EGTA was used to produce a sudden decrease or increase, respectively, in [Ca2+] within Triton-skinned trabeculae from rat and guinea pig hearts (22 degrees C). Trabeculae from both species had similar Ca2+ sensitivities, suggesting that the rate of dissociation of Ca2+ from troponin C (k(off)) is similar in the 2 species. However, the rate of relaxation after diazo-2 photolysis was 5 times faster in the rat (16.1 +/- 0.9 s(-1), mean +/- SEM, n = 11) than in the guinea pig (2.99 +/- 0.26 s(-1), n = 7). This indicates that the maximum relaxation rate is limited by crossbridge kinetics rather than by k(off). The maximum rates of rapid activation by Ca2+ after nitrophenyl-EGTA photolysis (k(act)) and of force redevelopment after forcible crossbridge dissociation (k(act)) were similar and were approximately 5-fold faster in rat (k(act)= 14.4 +/- 0.9 s(-1), k(tr)= 13.0 +/- 0.6 s(-1)) than in guinea pig (k(act)= 2.57 +/- 0.14 s(-1), k(tr)= 2.69 +/- 0.30 s(-1)) trabeculae. This too may be mainly due to species differences in crossbridge kinetics. Both k(act) and k(tr) increased as [Ca2+] increased. This Ca2+ dependence of the rates of force development is consistent with current models for the Ca2+ activation of the crossbridge cycle, but these models do not explain the similarity in the maximal rates of activation and relaxation within a given species.

Changes in force and cytosolic Ca2+ concentration after length changes in isolated rat ventricular trabeculae.

1. Changes in cytosolic [Ca2+] ([Ca2+]i) 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 Ca2+ transient. The rapid rise of force was unaffected by inhibiting the sarcoplasmic reticulum (SR) with ryanodine and cyclopiazonic acid. 3. The force-[Ca2+]i 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 Ca2+ 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 Ca2+ transient, since there was no change in the myofibrillar force-[Ca2+]i 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 [Ca2+]i, 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 [Ca2+]i. Evidence from tetani suggests the slow force responses result from increased Ca2+ loading of the cell during the action potential.

Differential effects of the Ca2+ sensitizers caffeine and CGP 48506 on the relaxation rate of rat skinned cardiac trabeculae.

During heart failure, force production by the heart decreases. This may be overcome by Ca2+-sensitizing drugs, which increase myofibril Ca2+ sensitivity without necessarily altering intracellular Ca2+ concentration. However, Ca2+ sensitizers slow the relaxation of intact cardiac muscle. We used diazo-2, a caged chelator of Ca2+, to study the effects of the Ca2+ sensitizers caffeine and CGP 48506 on the intrinsic relaxation rate of cardiac myofibrils. Trabeculae from rat right ventricles were skinned by 1% Triton X-100 and were activated in a 10-microL bath. In steady state experiments, CGP 48506 (10 micromol/L) shifted the force-pCa curve leftward by 0.41+/-0.03 pCa units (mean+/-SEM, n=6). An identical shift was induced by caffeine (20 mmol/L). Photolysis of diazo-2 by a flash of light (160 mJ, 310 to 400 nm) caused an immediate decrease in Ca2+-activated force produced by the trabeculae. Relaxation was fitted by a double-exponential decay, and the rate constants were found to be independent of force and preflash Ca2+ concentration. The initial fast rate, corresponding to myofibrillar relaxation, was increased from 17.3+/-2.0 to 30.9+/-3.7 s(-1) (n=4) by caffeine but was unaffected by CGP 48506 (16.6+/-1.7 and 14.4+/-2.3 s(-1) in the absence and presence of drug, respectively; n=5). Thus, myofibril relaxation need not be slowed by Ca2+-sensitizing agents but can even be accelerated. Despite similarities in their effects on myofibril Ca2+ sensitivity, caffeine and CGP 48506 affect the myofibrils at least partly via different mechanisms.

Ca(2+)- and caffeine-induced Ca2+ release from the sarcoplasmic reticulum in rat skinned trabeculae: effects of pH and Pi.

OBJECTIVE: Our aims were: (1) to examine the effect of pH (7.4-6.5) on Ca2+ release from the sarcoplasmic reticulum (SR) of cardiac muscle, and (2) to see if these effects were altered by phosphate (Pi). METHODS: Rat ventricular trabeculae were permeabilised with saponin. Ca(2+)-induced Ca2+ release (CICR) from the SR was triggered by flash photolysis of nitr-5. Under similar loading conditions, SR Ca2+ loading was assessed using caffeine to release the Ca2+ in the SR. Force and fluo-3 fluorescence (a measure of the cytosolic [Ca2+]) were monitored. RESULTS: SR Ca2+ loading was optimal at pH 7.1 and was significantly reduced at pH 7.4, 6.8 and 6.5. CICR was the same at pH 7.4 as at pH 7.1, but was reduced, by more than Ca2+ loading, in acidic solutions. These differential effects on loading and CICR suggested that Ca2+ activation of the Ca2+ release channel was decreased (by > 50%) as pH was lowered from 7.4 to 6.5. A direct effect on the Ca2+ release channel was confirmed by the finding that Ca2+ release was slower in acidic solutions. Acidosis also slowed the re-uptake of Ca2+ into the SR after CICR, which may account for the reduced Ca2+ loading at low pH. As observed previously, Pi (20 mM) by itself decreased SR Ca2+ loading. However, the inhibitory effects of acidosis and Pi on SR Ca2+ loading were independent. CONCLUSIONS: A fall of pH over the range 7.4-6.5 directly inhibits the SR Ca2+ release channel. In addition, acidosis inhibits SR Ca2+ accumulation by a mechanism independent of that of Pi. Both effects of acidosis would act to decrease SR Ca2+ release and so would contribute to the negative inotropic actions of intracellular acidosis in intact cardiac muscle.

Photolysis of the novel inotropes EMD 57033 and EMD 57439: evidence that Ca2+ sensitization and phosphodiesterase inhibition depend upon the same enantiomeric site.

1. We studied the effects of flash photolysis on the novel enantiomeric cardiac inotropes EMD 57033 (a calcium sensitizer) and EMD 57439 (a phosphodiesterase III inhibitor) in rat isolated ventricular trabeculae. 2. In skinned trabeculae, EMD 57439 had no effect on force production, consistent with lack of an active cyclic AMP system in this preparation. In contrast, EMD 57033 potentiated force at partial and maximal activation. A single flash of near u.v. light given at partial activation (30-70%) reduced force potentiation by 52.4 +/- 5.2%. No effect was produced by flashes in the presence of EMD 57439 or in the absence of either drug. 3. The time course of relaxation induced by EMD 57033 photolysis was indistinguishable from that obtained on deactivating the muscle by rapidly lowering Ca2+ using photolysis of the caged chelator of calcium, diazo-2. 4. In intact, twitching trabeculae, EMD 57033 increased diastolic and peak force and slowed relaxation. These effects were simultaneously reduced by a light flash. In these muscles EMD 57439 reduced force, without affecting the twitch time course. These effects were also reduced by a light flash. 5. The u.v. absorbance spectra of EMD 57033 and EMD 57439 were identical. After photolysis optical density decreased substantially and the peak shifted from 320 nm to 280 nm. 6. The proton n.m.r. spectra of these compounds were identical. The main change post-photolysis was a decrease in the proton signal associated with the enantiomeric carbon atom. 7. This novel manipulation of the molecular structure of EMD 57033 and EMD 57439 within an experiment thus provides direct evidence linking calcium sensitization to a particular molecular structure. The three main effects of the sensitizer on the twitch were simultaneously abolished and may be mechanistically linked. Flash photolysis may be a useful tool for further investigations of the actions of these compounds. In particular, flash photolysis of the sensitizer represents a novel method of rapidly deactivating cardiac muscle.

Unloaded shortening velocities of rabbit masseter muscle fibres expressing skeletal or alpha-cardiac myosin heavy chains.

1. Some rabbit masseter fibres express the alpha-cardiac myosin heavy chain (MHC). To compare the biochemical and physiological properties of these fibres with other skeletal fibre types, we examined the histochemical and immunohistochemical staining characteristics, maximum velocity of shortening (V(zero)) and MHC isoform content of fibres from rabbit masseter and soleus muscles. 2. The fibre-type composition of muscle sections was determined with MHC antibodies and myofibrillar ATPase histochemistry. Fibres we designated 'type alpha-cardiac' were different from type I and type II fibres in that they stained positively with the alpha-cardiac MHC antibody and they maintained. ATPase reactivity after acid and alkali pre-incubations. Samples of superficial masseter contained a few type I fibres, with the majority of fibres classified as either type IIA or type alpha-cardiac. Soleus samples contained type I, IIA and IIC fibres. 3. The V(zero) of chemically skinned fibres was determined by the slack-test method. Each fibre was subsequently characterized as type I, IIA, IIC or alpha-cardiac from MHC identification using gel electrophoresis (SDS-PAGE). In masseter fibres the V(zero) values were (in muscle lengths s-1): type I, 0.54 +/- 0.05 (mean +/- S.D., n = 3); type IIA, 1.23 +/- 0.34 (n = 27); type alpha-cardiac, 0.78 +/- 0.08 (n = 9). In soleus fibres V(zero) values were: type I, 0.55 +/- 0.06 (n = 14); type IIA, 0.89 +/- 0.04 (n = 8); type IIC, 0.73 (n = 2). 4. We conclude that the rabbit masseter muscle contains an 'alpha-cardiac' fibre type that is distinct from other skeletal fibres. This fibre type expresses only the alpha-cardiac MHC, has unusual myofibrillar ATPase reactivity and has a V(zero) intermediate between type I and type II fibres.

Developmental differences and regional similarities in the responses of rat cardiac skinned muscles to acidosis, inorganic phosphate and caffeine.

The Ca2+ sensitivity of cardiac myofibrillar force production can be decreased by acidosis or inorganic phosphate (P(i)) and increased by caffeine. To investigate whether the source of tissue influences the potency of these agents, we compared the actions of acidosis (change of pH from 7.0 to 6.2), P(i) and caffeine (both 20 mM) on force production of skinned cardiac muscles from adult ventricle, adult atrium and neonate ventricle of the rat. Maximum Ca(2+)-activated force was reduced by all three interventions and the responses of the different muscle types to a given intervention were similar. Acidosis reduced myofibrillar Ca2+ sensitivity by 1.09 and 1.04 pCa units in adult ventricle and atrium, respectively, and P(i) reduced it by 0.19 and 0.22 pCa units. However, each effect was only one-third as great in the neonate ventricle, which showed falls of 0.33 pCa units for acidosis and 0.06 for P(i). In contrast, caffeine raised the Ca2+ sensitivity by the same amount (approximately 0.4 pCa units) in all three muscle types. The differential effect between adult and neonate seen with both acidosis and P(i) suggests some similarity in the mechanisms by which these factors decrease Ca2+ sensitivity. In contrast, the equal effects of caffeine on neonate and adult suggests that caffeine acts by a completely different mechanism. The lower pH- and P(i)-sensitivity of the neonatal ventricle can help to explain why neonatal and adult myocardium exhibit differential force responses to ischaemia (or hypoxia alone).

Effect of pH on myofilament Ca(2+)-sensitivity in alpha-toxin permeabilized guinea pig detrusor muscle.

PURPOSE: To ascertain if the inotropic effects of altered intracellular pH on detrusor smooth muscle could be explained by a change in Ca(2+)-sensitivity of the contractile proteins. MATERIALS AND METHODS: Guinea pig detrusor smooth muscle was permeabilized with alpha-toxin and exposed to solutions mimicking the composition of the intracellular compartment and of varying [Ca2+]. Isometric tension was measured during exposure to solutions of varying [Ca2+] at pH 6.3, 7.1 and 7.5. RESULTS: At pH 7.1 the pCa (-log10[Ca2+]) required for half-maximal activation (pCa50) was 6.00 +/- 0.07 at 22C. A Hill coefficient of unity suggested lack of cooperativity in myofilament Ca2+ activation. A decrease of pH from 7.1 to 6.3 had no significant effect on the pCa50 value or the maximum Ca2+ activated force. An increase to pH 7.5 decreased the pCa50 value by 0.65 +/- 0.20 units but left maximum force unaffected. CONCLUSIONS: The reduced Ca2+ sensitivity of detrusor myofilaments at alkaline pH could partly explain the negative inotropic effect of intracellular alkalosis in intact muscle. The positive inotropic effect of intracellular acidosis cannot, however, be explained by alteration to myofilament Ca2+ sensitivity.

Effects of inorganic phosphate and ADP on calcium handling by the sarcoplasmic reticulum in rat skinned cardiac muscles.

OBJECTIVE: The aim was to investigate whether, and how, increases in inorganic phosphate (Pi) and ADP, similar to those occurring intracellularly during early myocardial ischaemia, affect the calcium handling of the sarcoplasmic reticulum. METHODS: Rat ventricular trabeculae were permeabilised with saponin. The physiological process of calcium induced calcium release (CICR) from the muscle sarcoplasmic reticulum was triggered via flash photolysis of the "caged Ca2+", nitr-5. Alternatively, calcium release was induced by rapid application of caffeine to give an estimate of sarcoplasmic reticular calcium loading. The initial rate of sarcoplasmic reticular calcium pumping was also assessed by photolysis of caged ATP at saturating [Ca2+]. Myoplasmic [Ca2+] (using fluo-3) and isometric force were measured. RESULTS: Pi (2-20 mM) significantly depressed the magnitude of CICR and the associated force transient. Sarcoplasmic reticular calcium loading was inhibited even more than CICR by Pi, suggesting that reduced calcium loading could account for all of the inhibitory effect of Pi on CICR and that Pi may slightly activate the calcium release mechanism. The reduced sarcoplasmic reticular calcium loading seemed to be due to a fall in the free energy of ATP hydrolysis (delta GATP) available for the calcium pump, since equal decreases in delta GATP produced by adding both Pi and ADP in various ratios caused similar falls in the calcium loading of the sarcoplasmic reticulum. The caged ATP experiments indicated that Pi (20 mM) did not affect the rate constant of sarcoplasmic reticular calcium uptake. ADP (10 mM) alone, or with 1 mM Pi, inhibited calcium loading. In spite of this, ADP (10 mM) did not alter CICR and, when 1 mM Pi was added, ADP increased CICR above control. CONCLUSIONS: An increase in intracellular Pi reduces sarcoplasmic reticular calcium loading and thus depresses the CICR. This could be an important contributing factor in the hypoxic or ischaemic contractile failure of the myocardium. However the detrimental effect of Pi may be offset to some extent by a stimulatory action of ADP on the calcium release mechanism of CICR.

The role of troponin C in modulating the Ca2+ sensitivity of mammalian skinned cardiac and skeletal muscle fibres.

1. We investigated the effects of acidosis, inorganic phosphate (Pi) and caffeine on the Ca2+ affinity of isolated fast-twitch skeletal and cardiac troponin C (TnC), labelled with fluorescent probes to report Ca2+ binding to the regulatory sites. We also measured the effects of these interventions on the maximum force development and the Ca2+ sensitivity of skinned fibres from fast-twitch skeletal muscle and cardiac muscle, as has been done previously. The two types of experiment were carried out under similar solution conditions, so that we could assess the contribution of any direct actions on TnC to the modulation of Ca2+ sensitivity in the skinned muscle fibres. 2. In skinned fibres, acidosis (decreasing pH from 7.0 to 6.2) and Pi (20 mM) suppressed maximum force to the same extent within a given muscle type, but had greater effects on cardiac fibres compared with skeletal fibres. Caffeine (20 mM) depressed maximum force equally in cardiac and skeletal muscle. Thus, the fall of force induced by acidosis or Pi may involve a different mechanism from that induced by caffeine. 3. Skinned skeletal fibres were more Ca2+ sensitive than cardiac fibres by 0.29 pCa units (pCa = -log10[Ca2+]). Isolated skeletal TnC also had a greater Ca2+ affinity than cardiac TnC, by 0.20 pCa units. These results suggest that the Ca2+ sensitivity of skinned fibres is at least partly determined by the type of TnC present. 4. Acidosis reduced the Ca2+ sensitivity of force in skinned fibres profoundly and had a 2-fold greater effect in cardiac muscle than skeletal muscle (falls in pCa for 50% activation, pCa50, were 1.09 and 0.55, respectively). Acidosis also reduced the Ca2+ affinity of TnC, again having double the effect on the pCa50 for cardiac TnC (0.58) as on that for skeletal TnC (0.28). The greater effect of acidosis on cardiac skinned fibres, compared with skeletal, may be partly explained, therefore, by the type of TnC present, and one-half of the effect on fibres may be attributed to the direct effect of H+ on TnC. 5. Pi reduced the Ca2+ sensitivity of force in skeletal and cardiac skinned fibres by 0.30 and 0.19 pCa units, respectively. However, the Ca2+ affinity of isolated cardiac and skeletal TnC was unaffected by Pi, indicating that the decrease in muscle Ca2+ sensitivity is not mediated by a direct action of Pi on TnC. 6. Caffeine increased the Ca2+ sensitivity of cardiac skinned fibres by 0.31 pCa units, which was 3 times greater than for the skeletal fibres (0.09 pCa units).(ABSTRACT TRUNCATED AT 400 WORDS)

Differential effects of length on maximum force production and myofibrillar ATPase activity in rat skinned cardiac muscle.

1. The fall of maximum Ca(2+)-activated force of cardiac myofibrils at short muscle lengths could be due to a reduction of cross-bridge cycling or to development of an opposing (restoring) force. To try to distinguish between these possibilities, we measured simultaneously myofibrillar force development and MgATPase activity (a measure of cross-bridge cycling) in rat skinned trabeculae at different muscle lengths. ATPase activity was measured photometrically from the utilization of NADH in a coupled enzyme assay. Muscle length was varied to give estimated 0.2 micron changes in sarcomere length (SL) over the range 1.4-2.4 microns. 2. Both Ca(2+)-activated force development and ATPase activity were optimal at a muscle length (Lo) where the resting SL was 2.2 microns. At Lo the maximum ATPase activity at 21 degrees C was 0.56 +/- 0.05 mM s-1 (mean +/- S.E.M., n = 6), which was equivalent to an ATP turnover per myosin S1 head of 3.3 s-1. 3. The relationship between ATPase activity and SL was curved, with rather little change in ATPase activity over the SL range 2.0-2.4 microns, but significant falls at 1.8 microns and below. At 65% of Lo (corresponding to a mean active SL of approximately 1.4 microns), the ATPase activity was only 50% of its value at 2.2 microns SL. 4. Force development decreased linearly as SL was reduced below 2.2 microns. Force fell by more than ATPase activity, particularly at SL 1.6 and 1.8 microns. 5. The fall of ATPase activity indicates that some of the decline of force production at short SL results from a fall in the net rate of cross-bridge cycling. This is probably the result of double overlap of thin filaments. However, the differential effect on force and ATPase reveals that, in the intermediate range of SL, decreased cross-bridge cycling can account for only part of the fall of force; the remainder is probably due to an increase in a restoring force, which may arise from deformation of the connective tissue in the muscle preparations used.

Isoprenaline reverses the slow force responses to a length change in isolated rabbit papillary muscle.

An alteration in the length of isolated cardiac muscle produces an immediate change in twitch force, then a slow further change in the same direction. We have found that the slow changes in force in rabbit papillary muscles are blocked or reversed by the beta-agonist, isoprenaline (1 microM). The abolition of the slow responses by isoprenaline was not due to saturation of the myofibrils with Ca2+, as the blockade continued if the extracellular [Ca2+] was reduced in the presence of isoprenaline so that twitch force was < 50% maximal. Ryanodine (1 microM) did not block the slow responses, suggesting that the sarcoplasmic reticulum does not mediate the responses. These results suggest that changes of intracellular [cAMP] may mediate, or at least modulate, the slow force responses to a length change in cardiac muscle.

Combined inhibitory actions of acidosis and phosphate on maximum force production in rat skinned cardiac muscle.

Possible interactions between the effects of pH and phosphate (Pi) on the maximum force development of cardiac myofibrils were investigated in rat skinned trabeculae in solutions of different pH (7.4-6.2) and [Pi] (where [] denote concentration). At pH 7.0 there was an inverse linear relationship between force and log [Pi] over the [Pi] range 0.2-20 mM; its slope (-0.46/decade) was twice that found previously for skeletal muscle [21]. Acidosis depressed force substantially, but the relative change of force was unaffected by Pi addition (0, 5, 20 mM); there was no evidence for the synergism between acidosis and Pi that would be expected if some of the inhibition by acidosis was due to protonation of Pi to the putative inhibitory form, H2PO4-. It was taken into account that even without Pi addition, there was enough Pi inside the muscle from various sources to produce significant changes in [H2PO4-] as the pH was varied. The results suggest that H+ and Pi inhibit maximum force development of cardiac myofibrils independently, by different mechanisms. From this it is argued that H+ and Pi may be released at different steps in the crossbridges cycle. In the myocardium Pi and H+ probably exert tonic inhibitory influences on cardiac myofibrils under all conditions.

Effects of changes of pH on the contractile function of cardiac muscle.

It has been known for over 100 years that acidosis decreases the contractility of cardiac muscle. However, the mechanisms underlying this decrease are complicated because acidosis affects every step in the excitation-contraction coupling pathway, including both the delivery of Ca2+ to the myofilaments and the response of the myofilaments to Ca2+. Acidosis has diverse effects on Ca2+ delivery. Actions that may diminish Ca2+ delivery include 1) inhibition of the Ca2+ current, 2) reduction of Ca2+ release from the sarcoplasmic reticulum, and 3) shortening of the action potential, when such shortening occurs. Conversely, Ca2+ delivery may be increased by the prolongation of the action potential that is sometimes observed and by the rise of diastolic Ca2+ that occurs during acidosis. This rise, which will increase the uptake and subsequent release of Ca2+ by the sarcoplasmic reticulum, may be due to 1) stimulation of Na+ entry via Na(+)-Ca2+ exchange; 2) direct inhibition of Na(+)-Ca2+ exchange; 3) mitochondrial release of Ca2+; and 4) displacement of Ca2+ from cytoplasmic buffer sites by H+. Acidosis inhibits myofibrillar responsiveness to Ca2+ by decreasing the sensitivity of the contractile proteins to Ca2+, probably by decreasing the binding of Ca2+ to troponin C, and by decreasing maximum force, possibly by a direct action on the cross bridges. Thus the final amount of force developed by heart muscle during acidosis is the complex sum of these changes.

Calcium release from cardiac sarcoplasmic reticulum induced by photorelease of calcium or Ins(1,4,5)P3.

The ability of Ca2+ or inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to release Ca2+ from cardiac sarcoplasmic reticulum (SR) was investigated using saponin-skinned ventricular trabeculae from rats. To overcome diffusion delays, rapid increases in the concentrations of Ca2+ and Ins(1,4,5)P3 were produced by laser photolysis of "caged Ca2+" (Nitr-5) and "caged Ins(1,4,5)P3". Photolysis of Nitr-5 to produce a small jump in [Ca2+] from pCa 6.8 to 6.4 induced a large and rapid force response (t1/2 = 0.89 s at 12 degrees C); the source of the Ca2+ that activated the myofibrils was judged to be the SR, since it was blocked by 0.1 mM ryanodine or 5 mM caffeine. A smaller, slower, and less consistent release of SR Ca2+ was produced by photorelease of Ins(1,4,5)P3. The results demonstrate that these caged compounds can be used to study excitation-contraction coupling in skinned multicellular preparations of cardiac muscle. The data are consistent with a major role for Ca2(+)-induced Ca2+ release in cardiac activation, whereas the role for Ins(1,4,5)P3 may be to modulate, rather than directly stimulate, SR Ca2+ release.

Ca2+, pH and the regulation of cardiac myofilament force and ATPase activity.

When the pH surrounding myofilaments of striated muscle is reduced there is an inhibition of both the actin-myosin reaction as well as the Ca2+-sensitivity of the myofilaments. Although the mechanism for the effect of acidic pH on Ca2+-sensitivity has been controversial, we have evidence for the hypothesis that acidic pH reduces the affinity of troponin C (TNC) for Ca2+. This effect of acidic pH depends not only on a direct effect of protons on Ca2+-binding to TNC, but also upon neighboring thin filament proteins, especially TNI, the inhibitory component of the TN complex. Using fluorescent probes that report Ca2+-binding to the regulatory sites of skeletal and cardiac TNC, we have shown, for example, that acidic pH directly decreases the Ca2+-affinity of TNC, but only by a relatively small amount. However, with TNC in whole TN or in the TNI-TNC complex, there is about a 2-fold enhancement of the effects of acidic pH on Ca2+-binding to TNC. Acidic pH decreases the affinity of skeletal TNI for skeletal TNC, and also influences the micro-environment of a probe positioned at Cys-133 of TNI, a region of interaction with TNC. Other evidence that the effects of acidic pH on Ca2+-TNC activation of myofilaments are influenced by TNI comes from studies with developing hearts. In contrast, to the case with the adult preparations, Ca2+-activation of detergent extracted fibers prepared from dog or rat hearts in the peri-natal period are weakly affected by a drop in pH from 7.0 to 6.5. This difference in the effect of acidic pH appears to be due to a difference in the isoform population of TNI, and not to differences in isotype population or amount of TNC.