Testosterone modulates cardiomyocyte Ca(2+) handling and contractile function.

The extent to which sex differences in cardiac function may be attributed to the direct myocardial influence of testosterone is unclear. In this study the effects of gonadal testosterone withdrawal (GDX) and replacement (GDX+T) in rats, on cardiomyocyte shortening and intracellular Ca(2+) handling was investigated (0.5 Hz, 25 oC). At all extracellular [Ca(2+)] tested (0.5-2.0 mM), the Ca(2+) transient amplitude was significantly reduced (by approximately 50 %) in myocytes of GDX rats two weeks post-gonadectomy. The time course of Ca(2+) transient decay was significantly prolonged in GDX myocytes (tau, 455+/-80 ms) compared with intact (279+/-23 ms) and GDX+T (277+/-19 ms). Maximum shortening of GDX myocytes was markedly reduced (by more than 60 %) and relaxation significantly delayed (by more than 35 %) compared with intact and GDX+T groups. Thus testosterone replacement completely reversed the cardiomyocyte hypocontractility induced by gonadectomy. These results provide direct evidence for a role of testosterone in regulating functional Ca(2+) handling and contractility in the heart.

Effects of gender on intracellular.

The present study investigated the effects of gender on intracellular [Ca2+] ([Ca2+]i) in freshly isolated rat cardiac myocytes. Changes in [Ca2+]i in response to varied extracellular [Ca2+], different stimulus frequencies and addition of caffeine and isoprenaline were monitored using fura-2 in both male and female cardiac myocytes. Increasing extracellular [Ca2+] and stimulus frequency resulted in significant increases in peak [Ca2+] and the amplitude of the Ca2+ transient in both male and female cardiac myocytes. However, as extracellular [Ca2+] was raised, peak [Ca2+] and the amplitude of the Ca2+ transient increased significantly more in male than female cardiac myocytes. In addition a significant difference between male and female cells at each stimulus frequency was apparent. The time course of decay of the Ca2+ transient was significantly slower in female cardiac myocytes when compared with male cardiac myocytes, along with significantly slowed times to peak shortening and 50% relaxation, and a reduced extent of shortening. There was no significant difference in the amplitude of caffeine-induced [Ca2+]i responses between male and female cells, however, [Ca2+]i increased more readily in male cells than in female cells when isoprenaline was added. The data demonstrate that, under a variety of conditions, intracellular [Ca2+] rises to higher levels in cardiac myocytes from male as compared to female rats.

Stimulus interval-dependent differences in Ca2+ transients and contractile responses of diabetic rat cardiomyocytes.

OBJECTIVE: The aim of this study was to gain further insights into the consequences of insulin-dependent diabetes mellitus on cardiomyocyte calcium handling. METHODS: The effects of steady state and transient changes in stimulus frequency on the intracellular Ca2+ transient and cell shortening were examined in left ventricular cardiomyocytes isolated from the hearts of control and streptozotocin-induced diabetic rats. RESULTS: During steady state stimulation diabetic rat cardiomyocytes displayed a slower decay of the Ca2+ transient and longer times for maximum cell shortening and re-lengthening. At 1.5 mM extracellular [Ca2+], increasing stimulus frequency over the range 0.2-1.0 Hz led to an increase in resting and peak [Ca2+]i as well as the amplitude of the transient in both the control and diabetic groups. At frequencies greater than 0.4 Hz the amplitude of the transient was significantly depressed in diabetic rat cells and this was not normalized by increasing extracellular [Ca2+] to 2.5 mM. Recovery of sarcoplasmic reticulum (SR) Ca2+ release was measured from the time course of restitution of the intracellular Ca2+ transient. In both control and diabetic rat cardiomyocytes recovery of the transient occurred in two phases. In diabetic rat myocytes, the initial rapid phase of restitution at intervals <1 s was markedly slowed. The fraction of Ca2+ recirculating between the SR and the cytosol was estimated from the decline in amplitude of transients following post-rest potentiation. There was no difference in this fraction between control and diabetic rat cells either at 1.5 or 2.5 mM extracellular [Ca2+]. CONCLUSION: The blunted frequency response of diabetic rat cardiomyocytes at frequencies greater than 0.4 Hz is consistent with reduced SR Ca2+ uptake leading to reduced SR Ca2+ content and subsequent release. At stimulus intervals greater than 1 Hz this is likely to be exacerbated by slower recovery of SR Ca2+ release. Despite the evidence for depressed SR Ca2+ uptake, the relative amount of Ca2+ recirculating within diabetic rat cardiomyocytes remains unaltered. This is most likely due to an accompanying reduction in Ca2+ efflux from the cell due either to depressed Na+/Ca2+ exchanger activity, or an elevation in intracellular Na+ levels.

Effects of anoxia on force, intracellular calcium and lactate production of urinary bladder smooth muscle from control and diabetic rats.

PURPOSE: To examine the effects of inhibiting oxidative metabolism on lactate production (J(Lac)), force and [Ca2+]i in longitudinal smooth muscle from urinary bladders of control and diabetic rats. MATERIALS AND METHODS: Strips of longitudinal smooth muscle were isolated from urinary bladders of diabetic rats and their age-matched controls. Force and [Ca2+]i were measured simultaneously in muscle strips loaded with the calcium indicator, fura-2. Separate muscle strips were used to determine J(Lac) by standard enzymatic assay. The muscles were stimulated to contract with 65 mM K+ or 1 microM carbachol (CCh) in the presence of 2.5 mM Ca2+ and either 5, 10 or 25 mM glucose. Oxidative metabolism was inhibited either by replacing O2 in solution with N2, or by addition of 2 mM NaCN. RESULTS: J(Lac) was significantly less in diabetic muscles than control muscles under both normoxic and anoxic conditions. During stimulation under anoxic conditions, the diabetic muscles were less able to maintain force than the controls. Despite a marked decline in force in both diabetic and control muscles under anoxic conditions, [Ca2+]i remained elevated to levels that were in fact higher than those observed during stimulation under normoxic conditions. Increasing the glucose concentration had no significant effect during normoxia, however, under anoxic conditions, the higher concentration improved force maintenance in both control and diabetic muscles. There were no apparent effects of the glucose concentration on [Ca2+]i in either diabetic or control muscles. CONCLUSION: The results reveal that urinary bladder smooth muscle from diabetic rats has a reduced ability to maintain contraction under anoxic conditions. This most likely reflects a greater energy limitation as evidenced by the reduced J(Lac) in diabetic muscles. In both diabetic and control muscles there was a marked dissociation between force and [Ca2+]i when oxidative metabolism was inhibited. This may indicate preferential use of glycolytically produced ATP for maintenance of [Ca2+]i homeostasis under these conditions.

Effects of streptozotocin-induced diabetes mellitus on intracellular calcium and contraction of longitudinal smooth muscle from rat urinary bladder.

PURPOSE: To examine the effect of diabetes on [Ca2+]i and contractility in longitudinal smooth muscle of the urinary bladder. MATERIALS AND METHODS: Longitudinal smooth muscle strips were isolated from the urinary bladders of rats with STZ-induced diabetes as well as age matched controls. Force and [Ca2+]i were measured simultaneously in muscle strips loaded with the calcium indicator, fura-2. Contractions were initiated by electrical field stimulation (EFS) at various frequencies, as well as by high K+, carbachol (CCh) and cyclopiazonic acid (CPA) in the presence of varying concentrations of extracellular Ca2+. RESULTS: In unstimulated muscles, there was no significant difference in resting [Ca2+]i between the control and diabetic groups. However, the muscle strips from the diabetic animals produced higher force levels in response to EFS, high K+, CCh and CPA than those from control animals. The higher force development in the diabetic muscles was not associated with greater increases in [Ca2+]i, which in fact tended to be lower during stimulation in the diabetic tissues. When stimulated by CCh in the presence of nifedipine, both control and diabetic muscles exhibited a nifedipine-resistant component of contraction, however, this was significantly larger in the diabetic muscles. CONCLUSION: The results suggest that there are no major impairments in either intracellular calcium regulation or contractile function in bladder smooth muscle after 8-weeks of STZ-induced diabetes. However, a non-specific enhancement of force production was seen, which was not associated with increases in [Ca2+]i. These changes imply that the apparent sensitivity to [Ca2+]i is enhanced in bladder smooth muscle from diabetic rats.

Effects of pyruvate on intracellular Ca2+ regulation in cardiac myocytes from normal and diabetic rats.

1. Pyruvate has been shown to enhance the contractile performance of cardiac muscle when provided as an alternative substrate to glucose. The aims of the present study were to determine whether the inotropic effects of pyruvate are due to increased mobilization of intracellular Ca2+ ([Ca2+]i) and to compare the effects of pyruvate on [Ca2+]i levels in myocytes from normal and diabetic animals. 2. Fura-2 was used to monitor [Ca2+]i in ventricular myocytes isolated from control and streptozotocin-treated male Wistar rats. The experiments were performed at 25 degrees C, with an extracellular [Ca2+] of 1.5 mmol/L and either 10 mmol/L glucose or 10 mmol/L pyruvate as the substrate. 3. In myocytes from both control and diabetic rats, increasing the stimulus frequency from 0.33 to 2.0 Hz resulted in significant increases in resting and peak [Ca2+]i as well as in the amplitude of the [Ca2+]i transient, irrespective of substrate. Compared with glucose, pyruvate significantly increased resting and peak [Ca2+]i and the amplitude of the [Ca2+]i transient at each stimulus frequency in myocytes from both control and diabetic animals. However, the extent of potentiation of the [Ca2+]i transient amplitude produced by pyruvate was significantly less in myocytes from the diabetic rats. 4. The rate of restitution of the [Ca2+]i transient was used as an index of the rate of Ca2+ cycling by the sarcoplasmic reticulum (SR). Pyruvate enhanced the rate of restitution in control but not diabetic rat cells. 5. The time course of decay of the [Ca2+]i transient was analysed as a measure of the rate of removal of Ca2+ from the cytosol. Pyruvate tended to increase the rate of decay in cells from control but not diabetic animals. The rate of decay was slower in cells from diabetic animals compared with controls. 6. The data reveal that pyruvate increases SR Ca2+ cycling, leading to greater Ca2+ release and an increase in the amplitude of the [Ca2+]i transient. Therefore, it seems highly likely that increased [Ca2+]i mobilization is responsible for the previously reported positive inotropic actions of pyruvate. These effects of pyruvate are attenuated in diabetic rat cells, which may reflect an impaired capacity of mitochondria in diabetic hearts to oxidize pyruvate, thus limiting potential energetic benefits.

Force, membrane potential and cytoplasmic Ca2+ responses to cyclic nucleotides in rat anococcygeus muscle.

Simultaneous recordings of membrane potential and force, and cytoplasmic calcium ([Ca2+]i) and force were made in rat anococcygeus to determine whether membrane hyperpolarisation plays a role in cyclic nucleotide-induced relaxation. In the presence of phenylephrine (0.2 microM), which evoked sustained contraction, an elevation in [Ca2+]i, and depolarisation, nitroprusside (5 microM) caused 96+/-3% relaxation, 77+/-3% decrease in suprabasal [Ca2+]i, and 16+/-2 mV hyperpolarisation. Forskolin (1 microM) caused 98+/-1% relaxation, 92+/-2% decrease in suprabasal [Ca2+]i, and 18+/-1 mV hyperpolarisation. These responses persisted in the presence of a variety of K+ channel blockers or in ouabain. The decrease in [Ca2+]i preceded the commencement of relaxation whereas the onset of hyperpolarisation lagged behind. Thus, cyclic nucleotide-mediated relaxation in rat anococcygeus is not dependent on hyperpolarisation mediated by the opening of K+ channels. Rather, it is suggested that the decrease in [Ca2+]i gives rise to hyperpolarisation, which reflects a decline in the Ca2+ dependent conductance(s) activated by phenylephrine.

Force and intracellular Ca2+ during cyclic nucleotide-mediated relaxation of rat anococcygeus muscle and the effects of cyclopiazonic acid.

1. Simultaneous recordings of tension and [Ca2+]i were made in rat anococcygeus muscle strips to investigate possible mechanisms involved during cyclic nucleotide-mediated relaxation. Relaxation of pre-contracted muscles was induced by sodium nitroprusside (SNP) or forskolin and the effects of cyclopiazonic acid (CPA) on these responses were examined. 2. In muscles pre-contracted with 0.2 microM phenylephrine addition of SNP (10 microM) caused a rapid and near complete relaxation of force. This was accompanied by a decrease in [Ca2+]i, however, this was not of a comparable magnitude to the decrease in force. The level of [Ca2+]i in muscles relaxed with SNP was shown to be associated with substantially higher force levels in the absence of SNP. Forskolin (10 microM) caused a slower, essentially complete relaxation which was associated with a proportional decrease in [Ca2+]i. 3. In muscles pretreated with SNP or forskolin subsequent responses to phenylephrine were attenuated with both force and [Ca2+]i rising slowly to attain eventually levels similar to those observed when the relaxant was applied to pre-contracted muscles. 4. Exposure of the muscles to the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor, CPA (10 microM), resulted in a sustained increase in [Ca2+]i which, in most cases, was not associated with any force development. The relaxation and decrease in [Ca2+]i in response to both SNP and forskolin were attenuated and substantially slowed in the presence of CPA. Overall the extent of this attenuation was greater for SNP. For both SNP and forskolin, CPA attenuated the decrease in [Ca2+]i to a greater extent than the decrease in force. In some cases, SNP-mediated relaxation in the presence of CPA was observed with almost no detectable change in [Ca2+]i. 5. The results suggest that, in the rat anococcygeus muscle under normal circumstances, a lowering of [Ca2+]i can fully account for the relaxation induced by forskolin but not for that induced by SNP, where mechanisms independent of changes in [Ca2+]i appear to contribute. Whilst Ca2+ sequestration into the sarcoplasmic reticulum plays a role in the relaxation mediated by both SNP and forskolin other Ca2+ lowering mechanisms may also be involved, especially in the response to forskolin.

Force and intracellular Ca2+ during NANC-mediated relaxation of rat anococcygeus muscle and the effects of cyclopiazonic acid.

1. Simultaneous recordings of tension and [Ca2+]i during NANC-mediated relaxation were made in the rat anococcygeus muscle under various conditions. 2. In muscles precontracted with guanethidine, nitrergic stimulations at 2 Hz produced a rapid decrease in both the tension and [Ca2+]i. 3. The nitric oxide synthase inhibitor, NG-nitro-L-Arginine (NOLA, 100 mumol/L) completely abolished the decreases in the [Ca2+]i and force response of the NANC-mediated relaxation. 4. Noradrenergic-mediated contractions elicited by electrical field stimulation were potentiated by the addition of NOLA. In the absence of NOLA, the motor responses were larger in magnitude at 10 Hz stimulation than at 2 Hz. After NOLA, both the force response and the associated rise in [Ca2+]i were substantially increased in comparison to the control stimulations. Proportionately the potentiation of the 2 Hz response was of a far greater magnitude than that of the 10 Hz response. 5. The guanylate cyclase inhibitor methylene blue (10 mumol/L), partially inhibited the force and [Ca2+]i response of the NANC relaxation. 6. Following exposure of the muscles to the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor, cyclopiazonic acid, (10 mumol/L) the responses to NANC stimulation were inhibited. The attenuated relaxation response displayed a bi-phasic timecourse and the Ca2+ change in comparison to that of the control was markedly smaller. In some cases, a relaxation was observed with no detectable change in the [Ca2+]i. 7. The results suggest that part of the relaxation response observed with NANC-mediated relaxation in the rat anococcygeus is dependent on Ca2+ sequestration into the sarcoplasmic reticulum. However, other Ca2+ lowering mechanisms and possible Ca2+ independent mechanisms may also contribute to the NANC relaxation response.

Metabolism of toad ventricle during alterations to the inotropic state.

The heat produced by toad ventricle during manipulations of the inotropic state was measured using thermopiles, and some comparisons made to rat ventricle. The tension-independent heat, peak stress, and the tension-dependent heat increased when [Ca2+]o increased from 0.25 to 2 mM in Ringer. In 2 mM [Ca2+]o, tension-independent heat, peak stress, and tension-dependent heat were 3.1 +/- 0.4 mJ/g, 38.4 +/- 5.5 mN/mm2, and 0.49 +/- 0.06 units; about 25% of the tension-independent heat may relate to the Na(+)-K+ pump. At similar [Ca2+]o, rat ventricle produced a smaller tension-independent heat (1.6 +/- 0.2 mJ/g), and active heat per unit stress (0.22 +/- 0.01 units) than toad. Tension-independent heat, stress, and tension-dependent heat were increased by orciprenaline, and decreased by BDM. Ouabain increased the stress and tension-dependent heat but not the tension-independent heat. Five millimolar [Ca2+]o in HEPES buffer decreased the stress but increased the tension-dependent heat compared to 2 mM [Ca2+]o in Ringer. Ryanodine and CPA caused major reductions in force and tension-independent heat in rat, but had little effect on toad ventricle. In conclusion, our results suggest that in toad ventricle (a) the sarcoplasmic reticulum plays only a minor role in activation and relaxation, (b) the Na(+)-K+ pump contributes substantially to activation metabolism, (c) active metabolism is stimulated by increases in [Ca2+]o and (d) there is a larger tension-independent heat, a larger active metabolism per unit stress, and a lower basal metabolism than in rat papillary muscle. The energy cost of removing intracellular Ca2+ through the sarcolemma appears to be greater than uptake into sarcoplasmic reticulum.

Effects of cyclopiazonic acid on [Ca2+]i and contraction in rat urinary bladder smooth muscle.

Cyclopiazonic acid (CPA) has been reported to inhibit the Ca(2+)-ATPase of the sarcoplasmic reticulum (SR) in skeletal and smooth muscle. In the present study the effect of CPA on [Ca2+]i and force in rat urinary bladder smooth muscle was examined. The fluorescent Ca2+ indicator Fura-2 was used to monitor intracellular Ca2+, simultaneously with isometric force production. Addition of CPA to unstimulated muscles bathed in 2.5 mM Ca2+ containing Krebs solution resulted in a significant and sustained increase in [Ca2+]i from 99 +/- 7 to 273 +/- 51 nM. This increase in [Ca2+]i was dependent upon the presence of extracellular Ca2+ since when CPA was added to muscles in Ca(2+)-free media it produced only a small, transient increase in [Ca2+]i that was not sustained. Peak force levels produced by transmural stimulation, carbachol and high KCl solution were not altered by the presence of CPA, however, the increase in [Ca2+]i associated with these contractions was larger when CPA was present. In response to transmural stimulation, the times taken for both force and [Ca2+]i to rise to 50% of their peak values were attenuated in the presence of CPA. Conversely, there was no effect of CPA on the times taken for force or [Ca2+]i to fall to 50% of their stimulated values upon the cessation of stimulation. Under control conditions both carbachol and high KCl could initiate transient increases in [Ca2+]i and force in the absence of extracellular Ca2+. In the presence of CPA, the response to carbachol was virtually completely inhibited, however, the response to high KCl was only partially inhibited. The ability of CPA to inhibit the carbachol response in Ca(2+)-free media suggests that this response is due to release of Ca2+ from the SR. The incomplete inhibition of the response to KCl indicates other Ca2+ storage sites may also be mobilised by sarcolemmal depolarisation. Although the mechanism whereby CPA induces a large, sustained rise in [Ca2+]i remains unknown, the data lend support to the suggestion that depletion of intracellular Ca2+ storage sites may activate a Ca2+ entry pathway across the sarcolemma.

Cholinergic neuromuscular transmission in the longitudinal muscle of the guinea-pig ileum.

1. Brief transmural stimuli, 0.5-1 ms, initiated contractions of the longitudinal muscle taken from the guinea-pig ileum that were recorded isometrically. In separate preparations similar stimuli were found to initiate excitatory junction potentials which were recorded using intracellular recording electrodes. All of these responses were abolished by either tetrodotoxin, omega-conotoxin or hyoscine. 2. The contractions produced by increasing [K+]o were blocked by nifedipine, 1 x 10(-7) M; nicardipine, 1 x 10(-7) M; verapamil, 1 x 10(-5) M or diltiazem, 1 x 10(-5) M. In these solutions brief stimuli continued to initiate contractions: this indicates that neuronally released acetylcholine continues to trigger a contraction when muscle voltage-dependent calcium channels appear to have been blocked. 3. When membrane potential recordings were made from the smooth muscle layer, brief transmural stimuli initiated excitatory junction potentials that triggered muscle action potentials. Although muscle action potentials were abolished by low concentrations of a range of organic calcium antagonists, excitatory junction potentials persisted and continued to initiate contractions of reduced amplitude. 4. When the internal concentration of calcium ions, [Ca2+]i, was measured using fura-2, brief transmural stimuli caused an increase in [Ca2+]i. Part of this response, which occurred at a time corresponding to the unblocked excitatory junction potential, persisted in the presence of the organic calcium antagonist nifedipine. 5. Two explanations appear possible. Neuronally released acetylcholine may simultaneously activate non-selective cation channels and cause the release of Ca2+ from an internal store. Alternatively, neuronally released acetylcholine may cause an increase in [Ca2+]i which is separate from that which accompanies the activation of voltage-dependent calcium channels. At this stage there is little other anatomical or electrophysiological evidence to support this view.

Intracellular Ca2+, force and activation heat in rabbit papillary muscle: effects of 2,3-butanedione monoxime.

We have investigated the effects of 2,3-butanedione monoxime (BDM) and mannitol on Ca2+ metabolism in rabbit cardiac muscle. Simultaneous measurements of force and intracellular Ca2+ were made in right ventricular papillary muscles loaded with the fluorescent Ca2+ indicator fura-2. At a BDM concentration of 2 mM, peak isometric force was only 52% of control values and this was reduced to 18% at a concentration of 5 mM. The peak of the Ca2+ transient decreased by 8% at 2 mM BDM and by 18% at the higher concentration. In the presence of 362 mM mannitol peak isometric force decreased by 78% and there was a tendency for the peak of the Ca2+ transient to increase. A combination of 362 mM mannitol with 5 mM BDM completely inhibited force production despite peak Ca2+ levels that were no different from control values. In myothermic experiments under similar conditions the latency release protocol of Gibbs et al. (1988) and the BDM protocol of Alpert et al. (1989) were used to derive independent estimates of tension-independent (activation) heat in the same muscle. For both protocols the heat-stress relationship was well fitted by first-order linear regression. The activation heat estimate was significantly higher when measured with the latency release technique (2.31 mJ/g) compared with the BDM protocol (1.24 mJ/g). Our results confirm that in rabbit cardiac muscle low concentrations of BDM (2 mM) cause a marked inhibition of force development with little apparent effect on peak Ca2+ levels. Therefore, the lower activation heat estimates under these conditions may not be due to a reduced intracellular Ca2+ concentration. It is possible that the higher activation heat values obtained with protocols not involving chemical interventions may include the energy usage contributed by cellular processes that presumably do not occur in the presence of BDM and mannitol.

Contractile and metabolic properties of longitudinal smooth muscle from rat urinary bladder and the effects of aging.

Longitudinal smooth muscle strips taken from the urinary bladders of Sprague-Dawley rats, aged approximately 6 months and 24 months, were examined under a variety of conditions. Force development in response to electrical field stimulation and to cumulative addition of Ca2+ in the continual presence of 64 mM. KCl was the same in both adult and aged preparations. In response to cumulative additions of carbachol, however, it was observed that there was a shift to the right of the dose-response curve and a decrease in maximal force in the aged muscle strips. The maximal velocity of shortening was significantly lower in muscles from aged animals than in those from young adult animals. The metabolic tension cost during isometric contraction was, however, the same in both groups suggesting that the decline in Vus is largely due to factors not influencing energetic cost. The aged muscles also exhibited a lower basal metabolic rate and a reduced contribution of aerobic glycolysis to total metabolic energy production during both quiescence and stimulation under normoxic conditions than did muscles from young adult animals. They were, however, able to increase their rate of lactate production to the same levels as young adult muscles when stimulated under anoxic conditions.

Contractile activation properties of ventricular myocardium from hypothyroid, euthyroid and juvenile rats.

Contractile activation properties of intact and chemically skinned ventricular myocardium preparations were studied in juvenile (3-4 weeks old), adult euthyroid and adult hypothyroid rats. The rats were made hyperthyroid by treatment with iodine-131 and propylthiouracil. The ventricular muscle of euthyroid rats contains a mixture of isozymes of myosin while the myocardium of juvenile and hypothyroid rats are relatively pure in regard to V1 and V3 types of myosin respectively. No significant differences were found in either the maximum Ca2+ activated or rigor force developed by "chemically skinned" preparations in either the juvenile or hypothyroid groups compared with euthyroid adults, suggesting that there is no difference between myocardia with different isozymes of myosin in the intrinsic capacity to generate force. In the hypothyroid (V3) preparations there was a significant shift in the force/pCa relation to the left compared with the euthyroid adult (mixture of V1 and V3 isozymes). The force/pCa relation for the juvenile lay in between that for the hypothyroid and euthyroid adults. The greater apparent Ca2+ sensitivity to activation in the hypothyroid group may relate to a slower cross-bridge cycling rate or altered Ca2+ kinetics in ventricular myocardium with exclusively V3 isozyme. In intact papillary muscles differences were found in the dependence of force on extracellular [Ca2+] such that a higher extracellular [Ca2+] was required for muscles from hypothyroid animals to attain maximum twitch force than those from juveniles. The force/frequency relations also differed, with the hypothyroid group being better able to sustain force as stimulation frequency increased than the juvenile group.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical, energetic, and biochemical changes in long-term volume overload of rabbit heart.

The mechanical and energetic consequences of long-term volume-overload (VOL) hypertrophy have been investigated in rabbits and compared with the consequence in sham-operated controls (SOC). Hypertrophy was induced by creating an aortocaval shunt, and the mechanical, biochemical, and energetic properties of the compensated heart were examined approximately 12 wk later. At 27 degrees C and a stimulus frequency of 1 Hz there were no significant changes in peak stress development, 10-90% rise times, shortening velocity, work, and mechanical power output. There was, however, a prolongation of contractile duration. The inverse relationship between peak stress and cross-sectional area was unchanged in the VOL and SOC groups. Polarographic and myothermic experiments were made on papillary muscles. Hypertrophy produced a small increment in basal metabolism. In isometric studies there were no significant changes in either the activation heat magnitude or the slope of the heat-stress relationship. In isotonic contractions there was no change in work output or total enthalpy (heat + work), and as a result mechanical efficiency was unchanged. A force-length-area (FLA) analysis of the isotonic data showed no significant change in intercept or FLA contractile efficiency. Biochemical studies showed no significant difference in the myosin isoenzyme profile at the time of death. The Ca(2+)-stimulated adenosinetriphosphatase activity of the sarcoplasmic reticulum was unchanged as were the enzymatic activities of mitochondrial citrate synthase and alpha-ketoglutarate dehydrogenase. Interestingly essentially the same data were obtained from the hearts of four animals in failure and from the hearts of seven compensated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered pattern of post-rest contractions in hypertrophied rabbit ventricle.

The pattern of contractions elicited after rest periods of 0.25-10 min duration was investigated in right ventricular papillary muscles from control and hypertrophied rabbit hearts. Hypertrophy was induced by pressure overload following coarctation of the pulmonary artery. In control hearts, the first post-rest contraction was always of a smaller amplitude than the preceding steady-state (0.5 Hz stimulation) contractions, and the amplitude of this first post-rest contraction decreased as the rest interval increased. In contrast, the amplitude of the first post-rest contraction of muscles from hypertrophied hearts exceeded the steady-state amplitude for rest durations of up to at least 2 min. In the hypertrophied muscles, force in the first post-rest contraction (expressed as a percentage of the pre-rest steady-state) was potentiated compared to the control muscles at all rest intervals studied. There was no significant difference in the second post-rest contraction between control and hypertrophied muscles at any rest interval. Following the second post-rest contraction, force increased monotonically toward the steady-state levels in all the muscles. The recovery of force was, however, somewhat faster in the hypertrophied muscles. Upon resumption of 1-Hz stimulation following rest intervals of 2 min or greater, pulsus alternans were invariably observed in the hypertrophied muscles but never in the control muscles. These differences in the non-steady-state contractile behavior of ventricular muscle from normal and hypertrophied hearts are suggestive of some alteration in the normal pattern of Ca2+ translocation in pressure overload hypertrophy of rabbit ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical, energetic, and biochemical changes in long-term pressure overload of rabbit heart.

The mechanical and energetic consequences of long-term pressure-overload (POL) hypertrophy have been investigated in rabbits and compared with sham-operated controls (SOC). Hypertrophy was induced by banding the pulmonary artery of young rabbits and examining the mechanical, biochemical, and energetic properties of the compensated heart 10-16 wk later. Experiments were undertaken on papillary muscles from the hypertrophic hearts. At 27 degrees C and a stimulus frequency of 1 Hz there was a modest depression of peak stress development but no significant changes in isometric rise times and one-half widths or in isotonic maximum velocity of shortening and power output. The inverse relationship between peak stress and cross-sectional area (CSA) was practically identical in the POL and SOC groups. Both polarographic and myothermic investigations were made on papillary muscles. Hypertrophy nearly halved basal metabolism, and in isometric contractions there was increased isometric economy due to a combination of a lower stress cost and a reduced activation heat. Hypertrophy did significantly depress the extent of shortening leading to a reduced work output per beat. In isotonic contractions the reduced work output was offset by a reduced energy output such that there was no significant change in suprabasal mechanical efficiency. Biochemical studies showed that the transition of myosin isoenzymes to the V3 form was essentially complete in the POL group, but that the SOC group was also predominantly V3 when the animals were killed. There was a significant 30% decline in the Ca2(+)-stimulated adenosinetriphosphatase activity of the sarcoplasmic reticulum. It is concluded that in long-term compensated hypertrophy of rabbit hearts there are only a few mechanical and energetic differences between control and hypertrophic muscles. The changes that can be detected appear to predominantly reflect disturbances in cellular Ca2+ regulation.

The effect of increasing extracellular potassium concentration on the resting heart rate of the isolated rat papillary muscle.

The effect of elevating extracellular K+ concentration on the basal metabolism of the isolated rat left ventricular papillary muscle has been investigated. The preparation was mounted on a thermopile and connected to a force transducer, to allow simultaneous measurement of muscle heat production and force. The resting heat rate (RHR) of the quiescent preparation was measured as an index of basal metabolism. Throughout all of the experiments, the muscles were maintained under a resting force of 10 mN and all measurements of RHR were made at times when there was no active force present above this passive level. Elevating the extracellular K+ concentration from 5.9 to 20, 40, then 80 mM produced graded increases in the RHR. The increase in RHR produced by 40 mM K+ was observed to be time-dependent, its effect being significantly greater at 5-7 h than at 2-4 h after cardiectomy. Averaged over all times, the percentage increases in RHR produced by 20, 40, and 80 mM K+ in the presence of 2 mM Ca2+ were 6.4 +/- 2.0%, 28.7 +/- 2.3%, and 51.3 +/- 8.9% (mean +/- SEM) respectively. The high K(+)-induced increase in basal metabolism was also shown to be Ca2(+)-dependent, the increase in RHR produced by 40 mM K+ being greater the higher the extracellular Ca2+ concentration (0.5-8.0 mM). The addition of verapamil was found to partially inhibit the K(+)-induced increase in resting metabolism. These results show that elevation of the extracellular K+ concentration produces a graded increase in the RHR that is Ca2(+)-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Smooth muscle energetics: testing theories of crossbridge regulation.

Our results indicate that the kinetic "latch" model of Hai & Murphy is not very sensitive to the proportion of ATP assigned to crossbridges relative to that ascribed to MLC phosphorylation/dephosphorylation. Thus the basis for the relatively low efficiency of smooth muscle, attributed to high MLC phosphorylation/dephosphorylation in this model, remains open to question. Moreover, this model, or any model with mixed populations of crossbridges with differing cycle rates and/or high MLC phosphorylation/dephosphorylation rates is unlikely to account for the observed linearity of JATP and stress reported for many smooth muscles. Our studies comparing the heat production of intact and skinned smooth muscle indicate that the ATPase associated with myosin phosphorylation/dephosphorylation is unlikely to be a major factor in the tension cost of intact smooth muscle. Thus it would appear that energetics places considerable constraints on current theories of crossbridge regulation. Our modelling (Paul, 1989) suggests that it may be time to reevaluate Bozler's original hypothesis that a high attachment:detachment rate ratio for smooth muscle actin-myosin interaction may be sufficient to explain the energetics of smooth muscle.