Maintenance of resting tension in the american eel (Anguilla rostrata L.) heart is dependent upon exogenous fuel and the sarcoplasmic reticulum

The relationship between extracellular glucose and management of cell Ca(2+) in the heart of the American eel (Anguilla rostrata) was indirectly assessed by monitoring the performance of isolated ventricular strips at 20 degrees C. Twitch force increased in ventricular strips under specific conditions of 30 bpm pacing and an extracellular Ca(2+) challenge from 1.5 to 9.5 mM. The response was independent of any exogenous metabolic fuel in the medium. Resting tension was maintained when glucose was available, but in the absence of a metabolic fuel, resting tension increased in response to the increase in extracellular Ca(2+) level. When ventricular strips were treated with iodoacetate to inhibit glycolysis, a Ca(2+) challenge resulted in a decrease in twitch force in association with an approximately equivalent increase in resting tension even in the presence of exogenous glucose. However, when pyruvate (5 mM) was substituted as a metabolic fuel, twitch force increased as a function of extracellular Ca(2+), and resting tension was maintained in the presence of iodoacetate. Therefore, there is a need for an extracellular fuel but not a specific metabolic requirement for glucose to maintain the performance characteristics, which are presumably related to the management of intracellular Ca(2+) levels. Ventricular strips were treated with ryanodine to inhibit Ca(2+) release and uptake by the sarcoplasmic reticulum (SR). Ryanodine treatment impaired postrest potentiation at high extracellular Ca(2+) levels. In the presence of ryanodine, the protective effect of glucose on the increase in resting tension in the face of an extracellular Ca(2+) challenge was eliminated. Considered together, the results reveal that the heart of the American eel has a requirement for an extracellular fuel to manage intracellular Ca(2+) at high Ca(2+) loads, and that the SR plays a role in the beat-to-beat regulation of Ca(2+) at a frequency of 30 bpm, high Ca(2+) load, and 20 degrees C.     

Relaxation of diaphragm muscle.

Relaxation is the process by which, after contraction, the muscle actively returns to its initial conditions of length and load. In rhythmically active muscles such as diaphragm, relaxation is of physiological importance because diaphragm must return to a relatively constant resting position at the end of each contraction-relaxation cycle. Rapid and complete relaxation of the diaphragm is likely to play an important role in adaptation to changes in respiratory load and breathing frequency. Regulation of diaphragm relaxation at the molecular and cellular levels involves Ca(2+) removal from the myofilaments, active Ca(2+) pumping by the sarcoplasmic reticulum (SR), and decrease in the number of working cross bridges. The relative contribution of these mechanisms mainly depends on sarcomere length, muscle tension, and the intrinsic contractile function. Increased capacity of SR to take up Ca(2+) can arise from increased density of active SR pumping sites or in slow-twitch fibers from phosphorylation of phospholamban, whereas impaired coupling between ATP hydrolysis and Ca(2+) transport into the SR or intracellular acidosis reduces SR Ca(2+) pump activity. In experimental conditions of decreased contractile performance, slowed, enhanced, or unchanged relaxation rates have been reported in vitro. In vivo, a slowing in the rate of decline of the respiratory pressure is generally considered an early reliable index of respiratory muscle fatigue. Impaired relaxation rate may, in turn, favor mismatch between blood flow and metabolic demand, especially at high breathing frequencies.     

Modeling short-term interval-force relations in cardiac muscle

This study employs two modeling approaches to investigate short-term interval-force relations. The first approach is to develop a low-order, discrete-time model of excitation-contraction coupling to determine which parameter combinations produce the degree of postextrasystolic potentiation seen experimentally. Potentiation is found to increase 1) for low recirculation fraction, 2) for high releasable fraction, i.e., the maximum fraction of Ca(2+) released from the sarcoplasmic reticulum (SR) given full restitution, and 3) for strong negative feedback of the SR release on sarcolemmal Ca(2+) influx. The second modeling approach is to develop a more detailed single ventricular cell model that simulates action potentials, Ca(2+)-handling mechanisms, and isometric force generation by the myofilaments. A slow transition from the adapted state of the ryanodine receptor produces a gradual recovery of the SR release and restitution behavior. For potentiation, a small extrasystolic release leaves more Ca(2+) in the SR but also increases the SR loading by two mechanisms: 1) less Ca(2+)-induced inactivation of L-type channels and 2) reduction of action potential height by residual activation of the time-dependent delayed rectifier K(+) current, which increases Ca(2+) influx. The cooperativity of the myofilaments amplifies the relatively small changes in the Ca(2+) transient amplitude to produce larger changes in isometric force. These findings suggest that short-term interval-force relations result mainly from the interplay of the ryanodine receptor adaptation and the SR Ca(2+) loading, with additional contributions from membrane currents and myofilament activation.     

Effects of ryanodine in skinned cardiac cells

Ryanodine (1 X 10(-5) M) did not affect the Ca2+ sensitivity of the myofilaments of skinned (sarcolemma removed by microdissection) cardiac cells from the rat ventricle. Ryanodine (1 X 10(-5) M) inhibited three types of Ca2+ release from the sarcoplasmic reticulum (SR), which have different mechanisms: 1) Ca2+-induced release of Ca2+ triggered by a rapid and transient increase of [free Ca2+] at the outer surface of the SR; 2) caffeine-induced release of Ca2+; 3) spontaneous cyclic release of Ca2+ occurring in the continuous presence of a [free Ca2+] sufficient to overload the SR. These results suggest that the three types of Ca2+ release are through the same channel across the SR membrane, although the gating mechanisms are different for the three types. Ryanodine also diminished the rate of Ca2+ accumulation into the SR. Even in the presence of 1 X 10(-5) M ryanodine the SR accumulated Ca2+ that could be released when the SR was sufficiently overloaded with Ca2+. Thus, ryanodine pretreatment did not permit the direct activation of the myofilaments by externally applied Ca2+. The approximately 1000-fold difference in the effective concentrations of ryanodine in intact vs. skinned cardiac cells suggests that low concentrations of ryanodine act in the intact cardiac tissues through processes or on structures that are destroyed by the skinning procedure. No significant differences were observed in the effects of ryanodine in skinned cardiac cells from different adult mammalian species.     

Role of extracellular Ca2+ in diaphragmatic contraction: effects of ouabain, monensin, and ryanodine.

The effects of zero extracellular Ca2+ on the contractility of rat diaphragmatic strips in vitro were studied in conjunction with various pharmacological agents known to influence the intracellular Ca2+ concentration: the Na+ ionophore, monensin, and the Na(+)-K+ pump inhibitor, ouabain, which enhance [Ca2+]i, caffeine, which induces Ca2+ release from the sarcoplasmic reticulum (SR), and ryanodine, which prevents Ca2+ retention by the SR. The effect of increasing [Ca2+]i on diaphragmatic contraction was assessed by comparing contractions induced by 120 mM K+ in the small muscle strips before and after the addition of ouabain or monensin. Monensin (20 microM) and ouabain (1-100 microM) augmented contractions up to threefold. Treatment of diaphragm strips with 3 nM ryanodine increased baseline tension 360% above the original resting tension but only if the diaphragm was electrically stimulated concurrently; 100 microM ryanodine induced contracture in quiescent tissue. High K+ contractures were of greater magnitude in the presence of ryanodine compared with control, and relaxation time was prolonged by greater than 200%. Ca(2+)-free conditions ameliorated these actions of ryanodine. Ryanodine reduced contractions induced by 10 mM caffeine and nearly abolished them in Ca(2+)-free solution. The data demonstrate that extracellular Ca2+ is important in certain types of contractile responses of the diaphragm and suggest that the processes necessary to utilize extracellular Ca2+ are present in the diaphragm.     

Short-term in vivo inhibition of nitric oxide synthase with L-NAME influences the contractile function of single left ventricular myocytes in rats

The main purpose of this study was to investigate the effects of short-term L-NAME treatment on the contractile function of left ventricle (LV) myocytes and the expression of proteins related to Ca(2+) homeostasis. Data from Wistar rats treated with L-NAME (L group, n = 20; 0.7?g/L in drinking water; 7?days) were compared with results from untreated controls (C group, n = 20). Cardiomyocytes from the L group showed increased (p?< 0.05) fractional shortening (23%) and maximum rate of shortening (20%) compared with the C group. LV from the L group also showed increased (p?< 0.05) expression of the ryanodine receptor 2 and Na(+)/Ca(2+) exchanger proteins (76% and 83%, respectively; p?< 0.05). However, the L and C groups showed similar in vivo hemodynamic parameters of cardiac function. In conclusion, short-term NOS inhibition determines an increased expression of Ca(2+) regulatory proteins, which contributes to improving cardiomyocyte contractile function, preserving left ventricular function.     

Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.     

Cardiac hypertrophy and impaired relaxation in transgenic mice overexpressing triadin 1

Triadin 1 is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum (SR), which forms a quaternary complex with the ryanodine receptor (Ca(2+) release channel), junctin, and calsequestrin. To better understand the role of triadin 1 in excitation-contraction coupling in the heart, we generated transgenic mice with targeted overexpression of triadin 1 to mouse atrium and ventricle, employing the alpha-myosin heavy chain promoter to drive protein expression. The protein was overexpressed 5-fold in mouse ventricles, and overexpression was accompanied by cardiac hypertrophy. The levels of two other junctional SR proteins, the ryanodine receptor and junctin, were reduced by 55% and 73%, respectively, in association with triadin 1 overexpression, whereas the levels of calsequestrin, the Ca(2+)-binding protein of junctional SR, and of phospholamban and SERCA2a, Ca(2+)-handling proteins of the free SR, were unchanged. Cardiac myocytes from triadin 1-overexpressing mice exhibited depressed contractility; Ca(2+) transients decayed at a slower rate, and cell shortening and relengthening were diminished. The extent of depression of cell shortening of triadin 1-overexpressing cardiomyocytes was rate-dependent, being more depressed under low stimulation frequencies (0.5 Hz), but reaching comparable levels at higher frequencies of stimulation (5 Hz). Spontaneously beating, isolated work-performing heart preparations overexpressing triadin 1 also relaxed at a slower rate than control hearts, and failed to adapt to increased afterload appropriately. The fast time inactivation constant, tau(1), of the l-type Ca(2+) channel was prolonged in transgenic cardiomyocytes. Our results provide evidence for the coordinated regulation of junctional SR protein expression in heart independent of free SR protein expression, and furthermore suggest an important role for triadin 1 in regulating the contractile properties of the heart during excitation-contraction coupling.     

Effects of temperature and calcium availability on cardiac contractility in Synbranchus marmoratus, a neotropical teleost

An isometric muscle preparation was used to investigate the importance of the ventricular sarcoplasmic reticulum (SR) and extracellular Ca2+ (1.25 up to 11.25 mM) to force generation at 25 degrees C (acclimation temperature), 15 and 35 degrees C. The post-rest tension and force-frequency relationship were conducted with and without 10 microM ryanodine in the bathing medium. Increments in extracellular Ca2+ resulted in increases in twitch force development only at 35 degrees C. A significant post-rest potentiation was recorded for the control preparations at 25 degrees C (100% to 119.8+/-4.1%). However, this post-rest potentiation was inhibited by ryanodine only at 25 degrees C (100% to 97.6+/-1.5%). At 35 degrees C, force remained unchanged in the control preparations, but a significant post-rest decay was recorded in the presence of ryanodine (100% to 76.6+/-4.6%) while at 15 degrees C, ryanodine was not able to preventing the post-rest potentiation observed in the control preparations. The increases in the imposed contraction frequency caused a decline of the force at 25 and 35 degrees C and ryanodine decreased significantly peak tension at both temperatures. The findings suggest a high or medium calcium turnover, possibly related to the presence of a functional SR, whose functionality is diminished when temperature is decreased.     

Impaired sarcoplasmic reticulum function leads to contractile dysfunction and cardiac hypertrophy

Sarcoplasmic reticulum (SR)-mediated Ca(2+) sequestration and release are important determinants of cardiac contractility. In end-stage heart failure SR dysfunction has been proposed to contribute to the impaired cardiac performance. In this study we tested the hypothesis that a targeted interference with SR function can be a primary cause of contractile impairment that in turn might alter cardiac gene expression and induce cardiac hypertrophy. To study this we developed a novel animal model in which ryanodine, a substance that alters SR Ca(2+) release, was added to the drinking water of mice. After 1 wk of treatment, in vivo hemodynamic measurements showed a 28% reduction in the maximum speed of contraction (+dP/dt(max)) and a 24% reduction in the maximum speed of relaxation (-dP/dt(max)). The slowing of cardiac relaxation was confirmed in isolated papillary muscles. The late phase of relaxation expressed as the time from 50% to 90% relaxation was prolonged by 22%. After 4 wk of ryanodine administration the animals had developed a significant cardiac hypertrophy that was most prominent in both atria (right atrium +115%, left atrium +100%, right ventricle +23%, and left ventricle +13%). This was accompanied by molecular changes including a threefold increase in atrial natriuretic factor mRNA and a sixfold increase in beta-myosin heavy chain mRNA. Sarcoplasmic endoplasmic reticulum Ca(2+) mRNA was reduced by 18%. These data suggest that selective impairment of SR function in vivo can induce changes in cardiac gene expression and promote cardiac growth.     

Cardiac function of two ecologically distinct Neotropical freshwater fish: Curimbata, Prochilodus lineatus (Teleostei, Prochilodontidae), and trahira, Hoplias malabaricus (Teleostei, Erythrinidae)

An isometric muscle preparation was used to investigate the importance of the ventricular sarcoplasmic reticulum (SR) and extracellular Ca(2+) (2.5 up to 10.5 mM) to force generation at 25 degrees C (acclimation temperature) in two ecologically distinct Neotropical teleost fish: Curimbata (active species), and trahira (sedentary species). The post-rest force was studied with and without 10 muM ryanodine in the medium. The positive inotropism observed for both species in response to increases on extracellular Ca(2+) reflected a greater Ca(2+) influx through sarcolemma, as well as an increase in Ca(2+) liberation from the SR by the Ca(2+)-induced Ca(2+) release mechanism. The significant post-rest potentiation recorded for the curimbata and trahira control preparations (3.22+/-0.24 to 6.55+/-0.77 mN mm(-2) and 0.74+/-0.07 to 2.26+/-0.26 mN mm(-2), respectively), was completely inhibited by the addition of ryanodine to the bathing medium, suggesting a potential functionality of SR for both species. Considering the differences in these species habitats, modes of life and levels of activity and the fact of a probable SR Ca(2+) cycling in a physiological temperature, we suggest that the functionality of the SR in these species is probably related to their phylogeny.     

Sarcoplasmic reticulum Ca2+ transport and gene expression in congestive heart failure are modified by imidapril treatment

This study was designed to test the hypothesis that blockade of the renin-angiotensin system improves cardiac function in congestive heart failure by preventing changes in gene expression of sarcoplasmic reticulum (SR) proteins. We employed rats with myocardial infarction (MI) to examine effects of an angiotensin-converting enzyme inhibitor, imidapril, on SR Ca(2+) transport, protein content, and gene expression. Imidapril (1 mg.kg(-1).day(-1)) was given for 4 wk starting 3 wk after coronary artery occlusion. Infarcted rats exhibited a fourfold increase in left ventricular end-diastolic pressure, whereas rates of pressure development and decay were decreased by 60 and 55%, respectively. SR Ca(2+) uptake and Ca(2+) pump ATPase, as well as Ca(2+) release and ryanodine receptor binding activities, were depressed in the failing hearts; protein content and mRNA levels for Ca(2+) pump ATPase, phospholamban, and ryanodine receptor were also decreased by approximately 55-65%. Imidapril treatment of infarcted animals improved cardiac performance and attenuated alterations in SR Ca(2+) pump and Ca(2+) release activities. Changes in protein content and mRNA levels for SR Ca(2+) pump ATPase, phospholamban, and ryanodine receptor were also prevented by imidapril treatment. Beneficial effects of imidapril on cardiac function and SR Ca(2+) transport were not only seen at different intervals of MI but were also simulated by another angiotensin-converting enzyme inhibitor, enalapril, and an ANG II receptor antagonist, losartan. These results suggest that blockade of the renin-angiotensin system may increase the abundance of mRNA for SR proteins and, thus, may prevent the depression in SR Ca(2+) transport and improve cardiac function in congestive heart failure due to MI.     

Mice null for calsequestrin 1 exhibit deficits in functional performance and sarcoplasmic reticulum calcium handling

In skeletal muscle, the release of calcium (Ca(2+)) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca(2+) release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca(2+) buffering as well as its potential for modulating RyR1, the L-type Ca(2+) channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca(2+)]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca(2+) content and SR Ca(2+) release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca(2+) release with single action potentials and a collapse of the Ca(2+) release with repetitive trains. Under voltage clamp, SR Ca(2+) release flux and total SR Ca(2+) release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca(2+) release flux appears to be solely due to elimination of the slowly decaying component of SR Ca(2+) release, whereas the rapidly decaying component of SR Ca(2+) release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca(2+)] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca(2+)](free) in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca(2+) buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca(2+) release.     

Gender differences in the modulation of cardiac gene expression by dietary conjugated linoleic acid isomers

In an earlier study, we showed that dietary conjugated linoleic acid (CLA) isomers can exert differential effects on heart function in male and female rats, but the underlying mechanisms for these actions are not known. Cardiomyocyte Ca2+ cycling is a key event in normal cardiac contractile function and defects in Ca2+ cycling are associated with cardiac dysfunction and heart disease. We therefore hypothesized that abnormalities in the sarcolemmal (SL) and sarcoplasmic reticulum (SR)-mediated regulation of intracellular Ca2+ contribute to altered cardiac contractile function of male and female rats owing to dietary CLA isomers. Healthy male and female Sprague-Dawley rats were fed different CLA isomers, (cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12)) individually and in combination (50:50 mix as triglyceride or fatty acids) from 4 to 20 weeks of age. We determined the mRNA levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) 2a, ryanodine receptor, phospholamban, calsequestrin, Na+-Ca2+-exchanger (NCX), and L-type Ca2+ channel in the left ventricle (LV) by RT-PCR. The SR function was assessed by measurement of Ca2+-uptake and -release. Significant gender differences were seen in the LV NCX, L-type Ca2+ channel, and ryanodine receptor mRNA expression levels in control male and female rats. Dietary CLA isomers in the various forms induced changes in the mRNA levels of SERCA 2a, NCX, and L-type Ca2+ channel in the LV of both male and female hearts. Whereas protein contents of the Ca2+ cycling proteins were altered, changes in SR Ca2+-uptake and -release were also detected in both male and female rats in response to dietary CLA. The results of this study demonstrate that long-term dietary supplementation can modulate cardiac gene expression and SR function in a gender-related manner and may, in part, contribute to altered cardiac contractility.     

Effects of temperature and calcium availability on ventricular myocardium from rainbow trout

We studied the mechanical and electrophysiological properties of ventricular myocardium from rainbow trout (Oncorhynchus mykiss) in vitro at 4, 10, and 18 degrees C from fish acclimated at 10 degrees C. Temperature alone did not significantly alter the contractile force of the myocardium, but the time to peak tension and time to 80% relaxation were prolonged at 4 degrees C and shortened at 18 degrees C. The duration of the action potential was also prolonged at 4 degrees C and progressively shortened at higher temperatures. An alteration of the stimulation frequency did not affect contraction amplitude at any temperature. Calcium influx via L-type calcium channels was increased by raising extracellular calcium concentration (?Ca(2+)(o)) or including Bay K 8644 (Bay K) and isoproterenol in the bathing medium. These treatments significantly enhanced the contractile force at all temperatures. Calcium channel blockers had a reverse-negative inotropic effect. Unexpectedly, the duration of the action potential at 10 degrees C was shortened as ?Ca(2+)(o) increased. However, Bay K prolonged the plateau phase at 4 degrees C. Caffeine, which promotes the release of sarcoplasmic reticulum (SR) calcium, increased contractile force eightfold at all three temperatures, but the SR blocker ryanodine was only inhibitory at 4 degrees C. Our results suggest that contractile force in ventricular myocardium from Oncorhynchus mykiss is primarily regulated by sarcolemmal calcium influx and that ventricular contractility is maintained during exposure to a wide range of temperatures.     

Sarcoplasmic reticulum Ca(2+) release and muscle fatigue.

Efforts to examine the relevant mechanisms involved in skeletal muscle fatigue are focusing on Ca(2+) handling within the active muscle cell. It has been demonstrated time and again that reductions in sarcoplasmic reticulum (SR) Ca(2+) release resulting from increased or intense muscle contraction will compromise tension development. This review seeks to accomplish two related goals: 1) to provide an up-to-date molecular understanding of the Ca(2+)-release process, with considerable attention devoted to the SR Ca(2+) channel, including its associated proteins and their regulation by endogenous compounds; and 2) to examine several putative mechanisms by which cellular alterations resulting from intense and/or prolonged contractile activity will modify SR Ca(2+) release. The mechanisms that are likely candidates to explain the reductions in SR Ca(2+) channel function following contractile activity include elevated Ca(2+) concentrations, alterations in metabolic homeostasis within the "microcompartmentalized" triadic space, and modification by reactive oxygen species.     

Pharmacological blockade of sarcoplasmic reticulum induces a negative lusitropic effect

The relaxation and the inter-beat mechanical tension are termed lusitropic functions. It is generally assumed that they are primarily determined by Ca(2+) homeostasis of cardiac cell and by interactions of Ca(2+) with the contractile machinery. In the present study we studied the effects of various pharmacological interventions on the excitation-contraction coupling in right ventricular papillary muscles of adult rabbits at various stimulation rates. The maximal force of isometric contraction (MG, a.u.), the time to peak of isometric contraction (TTP, ms), the maximal speed of relaxation (dF/dt(relax)), the diastolic tension (DT, a.u.) and the total tension (MG+DT, a.u.) were measured. To affect excitation-contraction coupling, caffeine (5 mmol x l(-1)), ryanodine (1 micromol x l(-1)) and dantrolene sodium (50 micromol x l(-1)) were used. Whereas caffeine and ryanodine elicited a pronounced negative lusitropic effect, the effect of dantrolene was less dramatic with preserved frequency dependence. The results indicate that the key element for affecting the lusitropic functions is the ryanodine receptor of the sarcoplasmic reticulum (SR). The lusitropic effects of dantrolene, that affects cardiac excitation-contraction coupling but only minimally the ryanodine receptors of SR, were considerably less pronounced. The findings agree with the assumption that the lusitropic disturbances are closely related to the defects of SR ryanodine receptors of cardiac myocytes.     

CaMKII-dependent reactivation of SR Ca(2+) uptake and contractile recovery during intracellular acidosis

In hearts, intracellular acidosis disturbs contractile performance by decreasing myofibrillar Ca(2+) response, but contraction recovers at prolonged acidosis. We examined the mechanism and physiological implication of the contractile recovery during acidosis in rat ventricular myocytes. During the initial 4 min of acidosis, the twitch cell shortening decreased from 2.3 +/- 0.3% of diastolic length to 0.2 +/- 0.1% (means +/- SE, P < 0.05, n = 14), but in nine of these cells, contractile function spontaneously recovered to 1.5 +/- 0.3% at 10 min (P < 0.05 vs. that at 4 min). During the depression phase, both the diastolic intracellular Ca(2+) concentration ([Ca(2+)](i)) and Ca(2+) transient (CaT) amplitude increased, and the twitch [Ca(2+)](i) decline prolonged significantly (P < 0.05). In the cells that recovered, a further increase in CaT amplitude and a reacceleration of twitch [Ca(2+)](i) decline were observed. The increase in diastolic [Ca(2+)](i) was less extensive than the increase in the cells that did not recover (n = 5). Blockade of sarcoplasmic reticulum (SR) function by ryanodine (10 microM) and thapsigargin (1 microM) or a selective inhibitor of Ca(2+)-calmodulin kinase II, 2-[N- (2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methyl benzylamine (1 microM) completely abolished the reacceleration of twitch [Ca(2+)](i) decline and almost eliminated the contractile recovery. We concluded that during prolonged acidosis, Ca(2+)-calmodulin kinase II-dependent reactivation of SR Ca(2+) uptake could increase SR Ca(2+) content and CaT amplitude. This recovery can compensate for the decreased myofibrillar Ca(2+) response, but may also cause Ca(2+) overload after returning to physiological pH(i).