Key role of endothelium in the eNOS-dependent cardioprotection with exercise training.

Modulation of endothelial nitric oxide synthase (eNOS) activation is recognized as a main trigger of the cardioprotective effects of exercise training on heart vulnerability to ischemia-reperfusion (IR). However, this enzyme is expressed both in coronary endothelial cells and cardiomyocytes and the contribution of each one to such cardioprotection has never been challenged. The aim of this study was to investigate the role of eNOS from the cardiomyocytes vs. the endothelium in the exercise cardioprotection. Male Wistar rats were assigned to a chronic aerobic training (Ex) (vs. sedentary group; Sed) and we investigated the role of eNOS in the effects of exercise on sensitivity to IR or anoxia-reoxygenation (A/R) at whole heart, isolated cardiomyocytes and left coronary artery (LCA) levels. We observed that exercise increased eNOS activation (Ser1177 phosphorylation) and protein S-nitrosylation in whole heart but not at cardiomyocyte level, suggesting the specific target of endothelial cells by exercise. Consistently, in isolated cardiomyocytes submitted to the A/R procedure, exercise reduced cell death and improved cells contractility, but independently of the eNOS pathway. Next, to evaluate the contribution of endothelial cells in exercise cardioprotection, LCA were isolated before and after an IR procedure performed on Langendorff hearts. Exercise improved basal relaxation sensitivity to acetylcholine and markedly reduced the alteration of endothelium-dependent coronary relaxation induced by IR. Furthermore, inactivation of coronary endothelial cells activity just before IR, obtained with a bolus of Triton X-100, totally suppressed cardioprotective effects of exercise on both left ventricular functional recovery after IR and infarct size, whereas no effect of Triton X-100 was observed in Sed group. In conclusion, these results show that coronary endothelial cells rather than cardiomyocytes play a key role in the eNOS-dependent cardioprotection of exercise.

Neuregulin 1 improves glucose tolerance in adult and old rats.

AIM: Studies both in vitro and ex vivo of rodent skeletal muscle have highlighted the potential involvement of neuregulin 1 (NRG1) in glucose metabolism regulation, yet nothing is known of the role of NRG1 in systemic glucose homoeostasis. For this reason, it was hypothesized that systemic delivery of NRG1 might improve glucose tolerance and that the effect might be age-dependent. METHODS: Glucose tolerance tests were performed in 6-month-old (adult) and 22-month-old (old) male Wistar rats 15min after a single injection of either NRG1 (50µg/kg) or saline (controls). Skeletal muscle and liver samples were also collected 30min after the acute NRG1 or saline treatment, while the phosphorylation status of ErbB receptors and AKT was assessed by Western blotting. RESULTS: Acute NRG1 treatment decreased the glycaemic response to an oral glucose load in both adult and old rats. NRG1 injection did not activate ErbB receptors in skeletal muscle, whereas phosphorylation of ErbB3 and AKT was markedly increased in the liver of NRG1-treated adult and old rats compared with controls. CONCLUSION: This study shows that NRG1 has a possible glucose-lowering effect in the liver and via an ErbB3/AKT signaling pathway. This NRG1 effect is also maintained in old rats, suggesting that the NRG1/ErbB signaling pathway might represent a promising therapeutic target in insulin resistance states.

Chronic clenbuterol treatment compromises force production without directly altering skeletal muscle contractile machinery.

Clenbuterol is a ß2 -adrenergic receptor agonist known to induce skeletal muscle hypertrophy and a slow-to-fast phenotypic shift. The aim of the present study was to test the effects of chronic clenbuterol treatment on contractile efficiency and explore the underlying mechanisms, i.e. the muscle contractile machinery and calcium-handling ability. Forty-three 6-week-old male Wistar rats were randomly allocated to one of six groups that were treated with either subcutaneous equimolar doses of clenbuterol (4 mg kg(-1) day(-1) ) or saline solution for 9, 14 or 21 days. In addition to the muscle hypertrophy, although an 89% increase in absolute maximal tetanic force (Po ) was noted, specific maximal tetanic force (sPo) was unchanged or even depressed in the slow twitch muscle of the clenbuterol-treated rats (P < 0.05). The fit of muscle contraction and relaxation force kinetics indicated that clenbuterol treatment significantly reduced the rate constant of force development and the slow and fast rate constants of relaxation in extensor digitorum longus muscle (P < 0.05), and only the fast rate constant of relaxation in soleus muscle (P < 0.05). Myofibrillar ATPase activity increased in both relaxed and activated conditions in soleus (P < 0.001), suggesting that the depressed specific tension was not due to the myosin head alteration itself. Moreover, action potential-elicited Ca(2+) transients in flexor digitorum brevis fibres (fast twitch fibres) from clenbuterol-treated animals demonstrated decreased amplitude after 14 days (-19%, P < 0.01) and 21 days (-25%, P < 0.01). In conclusion, we showed that chronic clenbuterol treatment reduces contractile efficiency, with altered contraction and relaxation kinetics, but without directly altering the contractile machinery. Lower Ca(2+) release during contraction could partially explain these deleterious effects.

Palmitoyl-carnitine increases RyR2 oxidation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes: Role of adenine nucleotide translocase.

Long chain fatty acids bind to carnitine and form long chain acyl carnitine (LCAC), to enter into the mitochondria. They are oxidized in the mitochondrial matrix. LCAC accumulates rapidly under metabolic disorders, such as acute cardiac ischemia, chronic heart failure or diabetic cardiomyopathy. LCAC accumulation is associated with severe cardiac arrhythmia including ventricular tachycardia or fibrillation. We thus hypothesized that palmitoyl-carnitine (PC), alters mitochondrial function leading to Ca(2+) dependent-arrhythmia. In isolated cardiac mitochondria from C57Bl/6 mice, application of 10µM PC decreased adenine nucleotide translocase (ANT) activity without affecting mitochondrial permeability transition pore (mPTP) opening. Mitochondrial reactive oxygen species (ROS) production, measured with MitoSOX Red dye in isolated ventricular cardiomyocytes, increased significantly under PC application. Inhibition of ANT by bongkrekic acid (20 µM) prevented PC-induced mitochondrial ROS production. In addition, PC increased type 2 ryanodine receptor (RyR2) oxidation, S-nitrosylation and dissociation of FKBP12.6 from RyR2, and therefore increased sarcoplasmic reticulum (SR) Ca(2+) leak. ANT inhibition or anti-oxidant strategy (N-acetylcysteine) prevented SR Ca(2+) leak, FKBP12.6 depletion and RyR2 oxidation/S-nitrosylation induced by PC. Finally, both bongkrekic acid and NAC significantly reduced spontaneous Ca(2+) wave occurrences under PC. Altogether, these results suggest that an elevation of PC disturbs ANT activity and alters Ca(2+) handling in a ROS-dependent pathway, demonstrating a new pathway whereby altered FA metabolism may contribute to the development of ventricular arrhythmia in pathophysiological conditions.

[Consequences of mechanical ventilation on diaphragmatic function]. / Conséquences de la ventilation mécanique sur le diaphragme.

INTRODUCTION: Mechanical ventilation is associated with ventilator-induced diaphragmatic dysfunction (VIDD) in animal models and also in humans. BACKGROUND: The main pathophysiological pathways implicated in VIDD seems to be related to muscle inactivity but may also be the consequence of high tidal volumes. Systemic insults from side effects of medication, infection, malnutrition and hypoperfusion also play a part. The diaphragm is caught in the cross-fire of ventilation-induced and systemic-induced dysfunctions. Intracellular consequences of VIDD include oxidative stress, proteolysis, impaired protein synthesis, autophagy activation and excitation-contraction decoupling. VIDD can be diagnosed at the bedside using non-invasive magnetic stimulation of the phrenic nerves which is the gold standard. Other techniques involve patient's participation such as respiratory function tests or ultrasound examination. CONCLUSION AND PERSPECTIVES: At this date, only spontaneous ventilatory cycles and perhaps phrenic nerve stimulation appear to diminish the severity of VIDD in humans but several pathways are currently being examined using animal models. Specific pharmacological options are currently under investigation in animal models.

Effect of a high dose of simvastatin on muscle mitochondrial metabolism and calcium signaling in healthy volunteers.

Statin use may be limited by muscle side effects. Although incompletely understood to date, their pathophysiology may involve oxidative stress and impairments of mitochondrial function and of muscle Ca(2+) homeostasis. In order to simultaneously assess these mechanisms, 24 male healthy volunteers were randomized to receive either simvastatin for 80 mg daily or placebo for 8 weeks. Blood and urine samples and a stress test were performed at baseline and at follow-up, and mitochondrial respiration and Ca(2+) spark properties were evaluated on a muscle biopsy 4 days before the second stress test. Simvastatin-treated subjects were separated according to their median creatine kinase (CK) increase. Simvastatin treatment induced a significant elevation of aspartate amino transferase (3.38±5.68 vs -1.15±4.32 UI/L, P<0.001) and CK (-24.3±99.1±189.3 vs 48.3 UI/L, P=0.01) and a trend to an elevation of isoprostanes (193±408 vs 12±53 pmol/mmol creatinine, P=0.09) with no global change in mitochondrial respiration, lactate/pyruvate ratio or Ca(2+) sparks. However, among statin-treated subjects, those with the highest CK increase displayed a significantly lower Vmax rotenone succinate and an increase in Ca(2+) spark amplitude vs both subjects with the lowest CK increase and placebo-treated subjects. Moreover, Ca(2+) spark amplitude was positively correlated with treatment-induced CK increase in the whole group (r=0.71, P=0.0045). In conclusion, this study further supports that statin induced muscular toxicity may be related to alterations in mitochondrial respiration and muscle calcium homeostasis independently of underlying disease or concomitant medication.

Muscle mitochondrial metabolism and calcium signaling impairment in patients treated with statins.

The most common and problematic side effect of statins is myopathy. To date, the patho-physiological mechanisms of statin myotoxicity are still not clearly understood. In previous studies, we showed that acute application in vitro of simvastatin caused impairment of mitochondrial function and dysfunction of calcium homeostasis in human and rat healthy muscle samples. We thus evaluated in the present study, mitochondrial function and calcium signaling in muscles of patients treated with statins, who present or not muscle symptoms, by oxygraphy and recording of calcium sparks, respectively. Patients treated with statins showed impairment of mitochondrial respiration that involved mainly the complex I of the respiratory chain and altered frequency and amplitude of calcium sparks. The muscle problems observed in statin-treated patients appear thus to be related to impairment of mitochondrial function and muscle calcium homeostasis, confirming the results we previously reported in vitro.

Targeting neonatal ischemic brain injury with a pentapeptide-based irreversible caspase inhibitor.

Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia-ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns.

Effects of high-altitude exercise training on contractile function of rat skinned cardiomyocyte.

OBJECTIVE: Previous studies have questioned whether there is an improved cardiac function after high-altitude training. Accordingly, the present study was designed specifically to test whether this apparent blunted response of the whole heart to training can be accounted for by altered mechanical properties at the cellular level. METHODS: Adult rats were trained for 5 weeks under normoxic (N, NT for sedentary and trained animals, respectively) or hypobaric hypoxic (H, HT) conditions. Cardiac morphology and function were evaluated by echocardiography. Calcium Ca2+ sensitivity of the contractile machinery was estimated in skinned cardiomyocytes isolated from the left ventricular (LV) sub-epicardium (Epi) and sub-endocardium (Endo) at short and long sarcomere lengths (SL). RESULTS: Cardiac remodelling was harmonious (increase in wall thickness with chamber dilatation) in NT rats and disharmonious (hypertrophy without chamber dilatation) in HT rats. Contrary to NT rats, HT rats did not exhibit enhancement in global cardiac performance evaluated by echocardiography. Stretch- dependent Ca2+ sensitization of the myofilaments (cellular index of the Frank-Starling mechanism) increased from Epi to Endo in N rats. Training in normoxic conditions further increased this stretch-dependent Ca2+ sensitization. Chronic hypoxia did not significantly affect myofibrilar Ca2+ sensitivity. In contrast, high-altitude training decreased Ca2+ sensitivity of the myofilaments at both SL, mostly in Endo cells, resulting in a loss of the transmural gradient of the stretch-dependent Ca2+ sensitization. Expression of myosin heavy chain isoforms was affected both by training and chronic hypoxia but did not correlate with mechanical data. CONCLUSIONS: Training at sea level increased the transmural gradient of stretch-dependent Ca2+ sensitization of the myofilaments, accounting for an improved Frank-Starling mechanism. High-altitude training depressed myofilament response to Ca2+, especially in the Endo layer. This led to a reduction in this transmural gradient that may contribute to the lack of improvement in LV function via the Frank-Starling mechanism.

Defects in ryanodine receptor calcium release in skeletal muscle from post-myocardial infarct rats.

Defective calcium (Ca2+) signaling and impaired contractile function have been observed in skeletal muscle secondary to impaired myocardial function. However, the molecular basis for these muscle defects have not been identified. In this study, we evaluated the alterations of the ryanodine-sensitive Ca2+ release channels (RyR1) by analyzing global and local Ca2+ signaling in a rat postmyocardial infarction (PMI) model of myocardial overload. Ca2+ transients, measured with multiphoton imaging in individual fibers within a whole extensor digitorum longus (EDL) muscle, exhibited significantly reduced amplitude and a prolonged time course in PMI. Spatio-temporal properties of spontaneous Ca2+ sparks in fibers isolated from PMI EDL muscles were also significantly altered. In addition, RyR1 from PMI skeletal muscles were PKA-hyperphosphorylated and depleted of the FK506 binding protein (FKBP12). These data show that PMI skeletal muscles exhibit altered local Ca2+ signaling, associated with hyperphosphorylation of RyR1. The observed changes in Ca2+ signaling may contribute to defective excitation-contraction coupling in muscle that can contribute to the reduced exercise capacity in PMI, out of proportion to the degree of cardiac dysfunction.

Is titin the length sensor in cardiac muscle? Physiological and physiopathological perspectives.

One of the most salient physiological characteristics of cardiac muscle is that a dilated heart pumps more vigorously, a phenomenon known as the Frank-Starling relationship (see Allen and Kentish, 1985). At least two cellular mechanisms participate in this phenomenon: the reduction of the interfilament lattice spacing which favors the formation of cross-bridges (Wang and Fuchs, 1995) and the increased affinity of troponin C (TnC) for calcium (Ca2+) (Babu et al., 1988). In the latter case, it has been established that TnC itself is not the length sensor (Moss et al., 1991). The intracellular structure(s) able to sense changes in cell length has always been challenged and is still not known. We previously observed on intact isolated cardiac cells that active tension is more closely related to passive tension than to sarcomere length per se (Cazorla et al., 1997). This might have some physiological implications in the working heart since we found that sub-epicardial cells are more supple than sub-endocardial cells. In the present work on skinned cells, we studied the relationship between different levels of passive tension (modulated by a mild trypsin digestion) and the shift in pCa50 of tension-pCa relations induced by a stretch of cells from 1.9 to 2.3 microns sarcomere length. A significant correlation was obtained between passive tension and the stretch-induced shift in pCa50, or stretch-sensitivity of the active force. These observations led us to assume that titin might play a role in sensing cell length to modulate the contractile activity. Besides, it is known that myocardial infarcted cells are less sensitive to stretch. We propose that, in such a rat model, alterations of titin might participate in heart failure.

gamma-aminobutyric acid type B receptors are expressed and functional in mammalian cardiomyocytes.

gamma-Hydroxybutyrate (GHB), an anesthetic adjuvant analog of gamma-aminobutyrate (GABA), depresses cell excitability in hippocampal neurons by inducing hyperpolarization through the activation of a prominent inwardly rectifying K(+) (Kir3) conductance. These GABA type B (GABA(B))-like effects are clearly shown at high concentrations of GHB corresponding to blood levels usually reached during anesthesia and are mimicked by the GABA(B) agonist baclofen. Recent studies of native GABA(B) receptors (GABA(B)Rs) have favored the concept that GHB is also a selective agonist. Furthermore, cloning has demonstrated that GABA(B)Rs assemble heteromeric complexes from the GABA(B)R1 and GABA(B)R2 subtypes and that these assemblies are activated by GHB. The surprisingly high tissue content, together with anti-ischemic and protective effects of GHB in the heart, raises the question of a possible influence of GABA(B) agonists on excitable cardiac cells. In the present study, we provide electrophysiological evidence that GHB activates an inwardly rectifying K(+) current in rat ventricular myocytes. This effect is mimicked by baclofen, reversibly inhibited by GABA(B) antagonists, and prevented by pertussis toxin pretreatment. Both GABA(B)R1 and GABA(B)R2 are detected in cardiomyocytes by Western blotting and are shown to coimmunoprecipitate. Laser scanning confocal microscopy discloses an even distribution of the two receptors in the sarcolemma and along the transverse tubular system. Hence, we conclude that GABA(B)Rs are distributed not only in neuronal tissues but also in the heart, where they can be activated and induce electrophysiological alterations through G-protein-coupled inward rectifier potassium channels.

Two mechanisms for termination of individual Ca2+ sparks in skeletal muscle.

Ca(2+) sparks are brief, localized elevations of myoplasmic [Ca(2+)] caused by release of increments of Ca(2+) via sarcoplasmic reticulum Ca(2+) release channels in muscle. The properties of individual sparks provide information regarding the opening of sarcoplasmic reticulum Ca(2+) channels within functioning cells. Here we use high-speed confocal microscopy to show that individual Ca(2+) sparks activated by membrane depolarization in single frog skeletal muscle fibers can be terminated prematurely by repolarization. Thus, either voltage sensor deactivation on repolarization or release channel inactivation during continued depolarization can terminate the Ca(2+) release channel activity underlying voltage-activated Ca(2+) sparks in skeletal muscle.

A repetitive mode of activation of discrete Ca2+ release events (Ca2+ sparks) in frog skeletal muscle fibres.

1. Ca2+ release events (Ca2+ 'sparks'), which are believed to arise from the opening of a sarcoplasmic reticulum (SR) Ca2+ release channel or a small cluster of such channels that act as a release unit, have been measured in single, frog (Rana pipiens) skeletal muscle fibres. 2. Under conditions of extremely low rates of occurrence of Ca2+ sparks we observed, within individual identified triads, repetitive Ca2+ release events which occurred at a frequency more than 100-fold greater than the prevailing average event rate. Repetitive sparks were recorded during voltage-clamp test depolarizations after a brief (0.3-2 s) repriming interval in fibres held at 0 mV and in chronically depolarized, 'notched' fibres. 3. These repetitive events are likely to arise from the re-opening of the same SR Ca2+ release channel or release unit operating in a repetitive gating mode ('rep-mode'), rather than from the random activation of multiple, independent channels or release units within a triad. A train of rep-mode events thus represents a series of Ca2+ sparks arising from a single location within the fibre. Rep-mode events are activated among different triads in a random manner after brief repriming. The frequency of repetitive events among all identified events during voltage-clamp depolarization to 0 mV after brief repriming was 3.9 +/- 1.3 %. The occurrence of repetitive events was not related to exposure of the fibre to laser illumination. 4. The events observed within a rep-mode train exhibited a relatively uniform amplitude. Analysis of intervals between identified events in triads exhibiting rep-mode trains indicated similar variations of fluorescence as in neighbouring, quiescent triads, suggesting there was not a significant number of small, unidentified events at the triads exhibiting rep-mode activity. 5. The distribution of rep-mode interspark intervals exhibited a paucity of events at short intervals, consistent with the need for recovery from inactivation before activation of the next event in a repetitive train. The mean interspark interval of repetitive sparks during voltage-clamp depolarizations was 88 +/- 5 ms, and was independent of membrane potential. 6. The individual Ca2+ sparks within a rep-mode train were similar in average amplitude and spatiotemporal extent to singly occurring sparks, suggesting a common mechanism for termination of the channel opening(s) underlying both types of events. The average properties of the sparks did not vary during a train. The relative amplitude of a spark within a rep-mode was not correlated with its rise time. 7. Repetitive Ca2+ release events represent a mode of gating of SR Ca2+ release channels which may be significant during long depolarizations and which may be influenced by the biochemical state of the SR ryanodine receptor Ca2+ release channels.

Time course of individual Ca2+ sparks in frog skeletal muscle recorded at high time resolution.

Discrete Ca2+ release events (Ca2+ "sparks") were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 microseconds per line). Fibers loaded with the Ca2+ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at -90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca2+ sparks at approximately 30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5-2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 +/- 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca2+ ions are being released by the sarcoplasmic reticulum Ca2+ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca2+ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.

Modulation of the frequency of spontaneous sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks) by myoplasmic [Mg2+] in frog skeletal muscle.

The modulation by internal free [Mg2+] of spontaneous calcium release events (Ca2+ "sparks") from the sarcoplasmic reticulum (SR) was studied in depolarized notched frog skeletal muscle fibers using a laser scanning confocal microscope in line-scan mode (x vs. t). Over the range of [Mg2+] from 0.13 to 1.86 mM, decreasing the [Mg2+] induced an increase in the frequency of calcium release events in proportion to [Mg2+]-1.6. The change of event frequency was not due to changes in [Mg-ATP] or [ATP]. Analysis of individual SR calcium release event properties showed that the variation in event frequency induced by the change of [Mg2+] was not accompanied by any changes in the spatiotemporal spread (i.e., spatial half width or temporal half duration) of Ca2+ sparks. The increase in event frequency also had no effect on the distribution of event amplitudes. Finally, the rise time of calcium sparks was independent of the [Mg2+], indicating that the open time of the SR channel or channels underlying spontaneous calcium release events was not altered by [Mg2+] over the range tested. These results suggest that in resting skeletal fibers, [Mg2+] modulates the SR calcium release channel opening frequency by modifying the average closed time of the channel without altering the open time. A kinetic reaction scheme consistent with our results and those of bilayer and SR vesicle experiments indicates that physiological levels of resting Mg2+ may inhibit channel opening by occupying the site for calcium activation of the SR calcium release channel.

Voltage dependence of the pattern and frequency of discrete Ca2+ release events after brief repriming in frog skeletal muscle.

Applying a brief repolarizing pre-pulse to a depolarized frog skeletal muscle fiber restores a small fraction of the transverse tubule membrane voltage sensors from the inactivated state. During a subsequent depolarizing test pulse we detected brief, highly localized elevations of myoplasmic Ca2+ concentration (Ca2+ "sparks") initiated by restored voltage sensors in individual triads at all test pulse voltages. The latency histogram of these events gives the gating pattern of the sarcoplasmic reticulum (SR) calcium release channels controlled by the restored voltage sensors. Both event frequency and clustering of events near the start of the test pulse increase with test pulse depolarization. The macroscopic SR calcium release waveform, obtained from the spark latency histogram and the estimated open time of the channel or channels underlying a spark, exhibits an early peak and rapid marked decline during large depolarizations. For smaller depolarizations, the release waveform exhibits a smaller peak and a slower decline. However, the mean use time and mean amplitude of the individual sparks are quite similar at all test depolarizations and at all times during a given depolarization, indicating that the channel open times and conductances underlying sparks are essentially independent of voltage. Thus, the voltage dependence of SR Ca2+ release is due to changes in the frequency and pattern of occurrence of individual, voltage-independent, discrete release events.

Repriming and activation alter the frequency of stereotyped discrete Ca2+ release events in frog skeletal muscle.

1. Brief localized elevations in myoplasmic [Ca2+] (Ca2+ sparks) in individual sarcomeres of voltage-clamped frog skeletal muscle fibres were examined by laser scanning confocal microscopy. 2. Fibres held at 0 mV were briefly repolarized to -90 mV (repriming pulse) to restore only a small fraction of sarcoplasmic reticulum (SR) calcium release. Subsequent depolarization to 0 mV (test pulse) caused the appearance of small numbers of Ca2+ sparks at different sarcomeres from pulse to pulse. Increasing the repriming time resulted in an increase in the frequency of occurrence of the Ca2+ sparks. 3. The amplitude and spatio-temporal extent of the Ca2+ sparks were independent of the repriming time and test pulse voltage. Ca2+ sparks recorded during small depolarizations of fibres held at -90 mV had a similar amplitude and spatio-temporal extent as those recorded after brief repriming of the same fibre held at 0 mV. 4. We conclude that stereotyped Ca2+ sparks underlie calcium release at all voltages and all extents of repriming. The amplitude of Ca2+ release is thus graded by the frequency but not by the amplitude or spatio-temporal extent of the individual SR Ca2+ release events.

Orthophosphate salts induce calcium current recovery from blockade by gadolinium in isolated guinea-pig ventricular myocytes.

Using the whole-cell configuration of the patch-clamp technique, we studied the incomplete recovery of the high voltage-activated calcium current (ICa,L) from a complete blockade by 10 microM gadolinium. The study was performed on isolated guinea-pig ventricular myocytes. ICa,L recovery depended on the duration of application of Gd3+: for a mean application time of 1.63 +/- 0.45 min, a recovery to 67 +/- 22% of the initial current amplitude (n = 12 cells) was observed; when the application lasted 5.1 +/- 1.2 min ICa,L recovered to 27 +/- 13% (n = 14 cells). The partial recovery of ICa,L was accompanied by a slowing of the inactivation phase of the current. IF, during the incomplete recovery, cells were exposed to a solution containing 330 microM of an orthophosphate salt, such as NaH2PO4 or KH2PO4, the current amplitude increased to 80 +/- 13% of the initial current (n = 10 cells) in a reversible manner. However, the slowing of the inactivation phase was maintained. Our results show that the partial recovery of ICa,L from blockade by gadolinium is due to an interaction of the blocker with an extracellular part of the channel, possibly one involved in voltage-dependent inactivation.

Effect of ryanodine on cardiac calcium current and calcium channel gating current.

The effects of 100 microM ryanodine on the L-type calcium channel were studied using the pacth-clamp technique in isolated guinea pig ventricular myocytes. The inactivation kinetics of the calcium current were slowed down in the presence of ryanodine in agreement with the blockade of the release of calcium from the sarcoplasmic reticulum by the drug. The I-V and steady-state inactivation curves of the calcium current were shifted to negative values by ryanodine. A similar shift was observed in the activation and inactivation curves of the intramembrane charge movement associated with the calcium channel. Due to this shift, ryanodine slightly reduced the maximal amount of displaced charge although it did not modify the transition from the inactivated to the activated state (i.e., charge movement repriming). This result is in notable contrast with that obtained in skeletal muscle, where it has been found that ryanodine interferes with charge movement repriming. These results provide additional evidence of the postulated differences between the architecture of the excitation-contraction coupling system in cardiac and skeletal muscle.