Effects of sprint training on contractility and [Ca(2+)](i) transients in adult rat myocytes.

The effects of 6-8 wk of high-intensity sprint training (HIST) on rat myocyte contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients were investigated. Compared with sedentary (Sed) myocytes, HIST induced a modest (5%) but significant (P < 0.0005) increase in cell length with no changes in cell width. In addition, the percentage of myosin heavy chain alpha-isoenzyme increased significantly (P < 0.02) from 0.566 +/- 0.077% in Sed rats to 0.871 +/- 0.006% in HIST rats. At all three (0.6, 1.8, and 5 mM) extracellular Ca(2+) concentrations ([Ca(2+)](o)) examined, maximal shortening amplitudes and maximal shortening velocities were significantly (P < 0.0001) lower and half-times of relaxation were significantly (P < 0.005) longer in HIST myocytes. HIST myocytes had significantly (P < 0.0001) higher diastolic [Ca(2+)](i) levels. Compared with Sed myocytes, systolic [Ca(2+)](i) levels in HIST myocytes were higher at 0.6 mM [Ca(2+)](o), similar at 1.8 mM [Ca(2+)](o), and lower at 5 mM [Ca(2+)](o). The amplitudes of [Ca(2+)](i) transients were significantly (P < 0.0001) lower in HIST myocytes. Half-times of [Ca(2+)](i) transient decline, an estimate of sarcoplasmic reticulum (SR) Ca(2+) uptake activity, were not different between Sed and HIST myocytes. Compared with Sed hearts, Western blots demonstrated a significant (P < 0.03) threefold decrease in Na(+)/Ca(2+) exchanger, but SR Ca(2+)-ATPase and calsequestrin protein levels were unchanged in HIST hearts. We conclude that HIST effected diminished myocyte contractile function and [Ca(2+)](i) transient amplitudes under the conditions studied. We speculate that downregulation of Na(+)/Ca(2+) exchanger may partly account for the decreased contractility in HIST myocytes.     

Coronary perfusion and muscle lengthening increase cardiac contraction: different stretch-triggered mechanisms

An increase in coronary perfusion, transversal stretch of the myocardium, increases developed force (F(dev)) (Gregg effect) through activation of stretch-activated ion channels (SACs). Lengthening of the muscle, longitudinal stretch of the myocardium, causes an immediate increase in F(dev) followed by a slow F(dev) increase (Anrep effect). In isometrically contracting perfused papillary muscles of Wistar rats, we investigated whether both effects were based on similar stretch-induced mechanisms by measuring F(dev) and intracellular Ca(2+) concentration ([Ca(2+)](i)) after a muscle length increase from 85% to 95% L(max) (length at which maximal isometric force develops) at low and high coronary perfusion before and after inhibition of SACs with gadolinium (10 micromol/l Gd(3+)). The increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect was of similar magnitude as the Anrep effect (from 3.5 +/- 0.8 to 3.9 +/- 1.2 mN/mm(2) and from 3.0 +/- 0.7% to 3.8 +/- 0.9% normalized [Ca(2+)](i), means +/- SE). SAC blockade completely blunted the increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect; however, it did not affect the Anrep effect. The slow force response, but not the calcium response, was augmented by an increase in coronary perfusion. Therefore, increased coronary perfusion, transversal stretch of the myocardium, and muscle lengthening, longitudinal stretch of the myocardium, increase myocardial contraction in the rat through different stretch-triggered mechanisms.     

Electric currents applied during refractory period enhance contractility and systolic calcium in the ferret heart

We investigated the mechanism of positive inotropism of electric currents applied during the absolute refractory period. Ten Langendorff-perfused ferret hearts were instrumented to measure isovolumic left ventricular pressure (LVP) and the aequorin luminescence. Biphasic square-wave electric currents (+/-20 mA, total duration 30 ms) were delivered between pairs of electrodes. Six hearts were perfused at different extracellular Ca(2+) concentrations ([Ca(2+)](o); 1, 2, 4, and 8 mM). These signals increased LVP from 50.0 +/- 9.4 to 70.1 +/- 14.7, from 67.5 +/- 11.0 to 79.0 +/- 15.6, from 79.3 +/- 21.0 to 87.1 +/- 22.8, and from 84.6 +/- 24.0 to 91.8 +/- 28.5 mmHg at the respective [Ca(2+)](o) (P < 0.05). Peak free intracellular [Ca(2+)] ([Ca(2+)](i)) increased from 0.52 +/- 0.13 to 1.37 +/- 0.23, from 0.76 +/- 0.23 to 1.73 +/- 0.14, from 1.10 +/- 0.24 to 2.05 +/- 0.33, and from 1.41 +/- 0.36 to 2.24 +/- 0.36 microM/ml, respectively (P < 0.001). With the use of 1 mg/l propranolol with 1 mM [Ca(2+)](o), LVP and [Ca(2+)](i) were increased significantly from 48.7 +/- 8.18 to 56.3 +/- 6.11 mmHg and from 0.61 +/- 0.11 to 1.17 +/- 0.20 microM, respectively (P < 0.05). In conclusion, positive inotropism of such electrical currents was due to increased peak [Ca(2+)](i) and Ca(2+) responsiveness of the myofilaments did not change significantly.     

Overexpression of Na+/Ca2+ exchanger gene attenuates postinfarction myocardial dysfunction

We monitored myocardial function in postinfarcted wild-type (WT) and transgenic (TG) mouse hearts with overexpression of the cardiac Na(+)/Ca(2+) exchanger. Five weeks after infarction, cardiac function was better maintained in TG than WT mice [left ventricular (LV) systolic pressure: WT, 41 +/- 2; TG, 58 +/- 3 mmHg; P < 0.05; maximum rising rate of LV pressure (+dP/dt(max)): WT, 3,750 +/- 346; TG, 5,075 +/- 334 mmHg/s; P < 0.05]. The isometric contractile response to beta-adrenergic stimulation was greater in papillary muscles from TG than WT mice (WT, 13.2 +/- 0.9; TG, 16.3 +/- 1.0 mN/mm(2) at 10(-4) M isoproterenol). The sarcoplasmic reticulum (SR) Ca(2+) content investigated by rapid cooling contractures in papillary muscles was greater in TG than WT mouse hearts. We conclude that myocardial function is better preserved in TG mice 5 wk after infarction, which results from enhanced SR Ca(2+) content via overexpression of the Na(+)/Ca(2+) exchanger.     

Gender differences in sarcoplasmic reticulum calcium loading after isoproterenol

Males exhibit enhanced myocardial ischemia-reperfusion injury versus females under hypercontractile conditions associated with increased sarcoplasmic reticulum (SR) Ca2+. We therefore examined whether there were gender differences in SR Ca2+. We used NMR Ca2+ indicator 1,2-bis(2-amino-5,6-difluorophenoxy)-ethane-N,N,N',N'-tetraacetic acid to measure SR Ca2+ in perfused rabbit hearts. Isoproterenol increased SR Ca2+ in males from a baseline of 1.13 +/- 0.07 to 1.52 +/- 0.24 mM (P < 0.05). Female hearts had basal SR Ca2+ that was not significantly different from males (1.04 +/- 0.03 mM), and addition of isoproterenol to females resulted in a time-averaged SR Ca2+ (0.97 +/- 0.07 mM) that was significantly less than in males. To confirm this difference, we measured caffeine-induced release of SR Ca2+ with fura-2 in isolated ventricular myocytes. Ca2+ release after caffeine in untreated male myocytes was 377 +/- 41 nM and increased to 650 +/- 55 nM in isoproterenol-treated myocytes (P < 0.05). Ca2+ release after caffeine addition in untreated females was 376 +/- 27 nM and increased to 503 +/- 49 nM with isoproterenol, significantly less than in male myocytes treated with isoproterenol (P < 0.05). Treatment of female myocytes with NG-nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase (NOS), resulted in higher SR Ca2+ release than that measured in females treated only with isoproterenol and was not significantly different from that measured in males with isoproterenol. Female myocytes also have significantly higher levels of neuronal NOS. This gender difference in SR Ca2+ handling may contribute to reduced ischemia-reperfusion injury observed in females.     

In situ SR function in postinfarction myocytes.

Previous studies have shown lower systolic intracellular Ca(2+) concentrations ([Ca(2+)](i)) and reduced sarcoplasmic reticulum (SR)-releasable Ca(2+) contents in myocytes isolated from rat hearts 3 wk after moderate myocardial infarction (MI). Ca(2+) entry via L-type Ca(2+) channels was normal, but that via reverse Na(+)/Ca(2+) exchange was depressed in 3-wk MI myocytes. To elucidate mechanisms of reduced SR Ca(2+) contents in MI myocytes, we measured SR Ca(2+) uptake and SR Ca(2+) leak in situ, i.e., in intact cardiac myocytes. For sham and MI myocytes, we first demonstrated that caffeine application to release SR Ca(2+) and inhibit SR Ca(2+) uptake resulted in a 10-fold prolongation of half-time (t(1/2)) of [Ca(2+)](i) transient decline compared with that measured during a normal twitch. These observations indicate that early decline of the [Ca(2+)](i) transient during a twitch in rat myocytes was primarily mediated by SR Ca(2+)-ATPase and that the t(1/2) of [Ca(2+)](i) decline is a measure of SR Ca(2+) uptake in situ. At 5.0 mM extracellular Ca(2+), systolic [Ca(2+)](i) was significantly (P 相似文献   

Sprint training normalizes Ca(2+) transients and SR function in postinfarction rat myocytes.

Previous studies have shown that myocytes isolated from sedentary (Sed) rat hearts 3 wk after myocardial infarction (MI) undergo hypertrophy, exhibit altered intracellular Ca(2+) concentration ([Ca(2+)](i)) dynamics and abnormal contraction, and impaired sarcoplasmic reticulum (SR) function manifested as prolonged half-time of [Ca(2+)](i) decline. Because exercise training elicits positive adaptations in cardiac contractile function and myocardial Ca(2+) regulation, the present study examined whether 6-8 wk of high-intensity sprint training (HIST) would restore [Ca(2+)](i) dynamics and SR function in MI myocytes toward normal. In MI rats, HIST ameliorated myocyte hypertrophy as indicated by significant (P 相似文献   

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).     

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.     

Early effects of metabolic inhibition on intracellular Ca2+ in toad pacemaker cells: involvement of Ca2+ stores

The early effects of metabolic inhibition on intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) current, and sarcoplasmic reticulum (SR) Ca(2+) content were studied in single pacemaker cells from the sinus venosus of the cane toad. The amplitude of the spontaneous elevations of systolic [Ca(2+)](i) (Ca(2+) transients) was reduced after 5-min exposure to 2 mM NaCN from 338 +/- 30 to 189 +/- 37 nM (P < 0.005, n = 9), and the spontaneous firing rate was reduced from 27 +/- 2 to 12 +/- 4 beats/min (P < 0.002, n = 9). It has been proposed that CN(-) acts by inhibition of cytochrome P-450, resulting in a reduction of cAMP and Ca(2+) current. To test this proposal, we used clotrimazole, a cytochrome P-450 inhibitor, which also decreased the Ca(2+) transients and firing rate. CN(-) caused an insignificant fall of Ca(2+) current (23 +/- 11%) but a substantial reduction of SR Ca(2+) content (by 65 +/- 5%), whereas clotrimazole produced a larger reduction of Ca(2+) current and did not affect the SR Ca(2+) content. Thus the main effect of CN(-) does not seem to be through inhibition of cytochrome P-450. In conclusion, CN(-) appears to reduce Ca(2+) release from the SR mainly by reducing SR Ca(2+) content. A likely cause of the decreased SR content is reduced Ca(2+) uptake by the SR pump.     

Calcium sparks in human ventricular cardiomyocytes from patients with terminal heart failure

Cardiomyocytes from terminally failing hearts display significant abnormalities in e-c-coupling, contractility and intracellular Ca(2+) handling. This study is the first to demonstrate the influence of end-stage heart failure on specific properties of Ca(2+) sparks in human ventricular cardiomyocytes. We investigated the frequency and characteristics of spontaneously arising Ca(2+) sparks in single isolated human myocytes from terminally failing (HF) and non-failing (NF) control myocardium by using the Ca(2+) indicator Fluo-3. The Ca(2+) sparks were recorded by line-scan images along the longitudinal axis of the myocytes at a frequency of 250Hz. After loading the sarcoplasmic reticulum (SR) with Ca(2+) by repetitive field stimulation (10 pulses at 1Hz) the frequency of the Ca(2+) sparks immediately after stimulation (t = 0s) was reduced significantly in HF compared to NF (4.15 +/- 0.42 for NF vs. 2.81 +/- 0.20 for HF sparks s(-1), P = 0.05). This difference was present constantly in line-scan recordings up to 15s duration (t = 15s: 2.75 +/- 0.65 for NF vs. 1.36 +/- 0.34 for HF sparks s(-1), P = 0.05). The relative amplitude (F/F(0)) of Ca(2+) sparks was also significantly lower in HF cardiomyocytes (1.33 +/- 0.015 NF vs. 1.19 +/- 0.003 HF, t = 0s) and during subsequent recordings of 15s. Significant differences between HF and NF were also present in calculations of specific spark properties. The time to peak was estimated at 25.75 +/-0.88ms in HF and 18.68 +/- 0.45ms in NF cardiomyocytes (P = 0.05). Half-time of decay was 66.48 +/- 1.89ms (HF) vs. 44.15 +/- 1.65ms (NF, P < 0.05), and the full width at half-maximum (FWHM) was 3.99 +/- 0.06 microm (HF) vs. 3.5 +/- 0.07 microm (NF, P < 0.05). These data support the hypothesis that even in the absence of cardiac disease, Ca(2+) sparks from human cardiomyocytes differ from previous results of animal studies with respect to the time-to-peak, half-time of decay and FWHM. The role of elevated external Ca(2+) in HF was studied by recording Ca(2+) sparks in HF cardiomyocytes with 10mmol external Ca(2+) concentration. Under these conditions, the average spark amplitude was increased from 1.19 +/- 0.003 (F/F(0), 2mmol Ca(2+)) to 1.26 +/- 0.01 (F/F(0), 10mmol Ca(2+)). We conclude that human heart failure causes distinct changes in Ca(2+) spark frequency and characteristics comparable to results established in animal models of heart failure. A reduced Ca(2+) load of the SR alone is unlikely to account for the observed differences between HF and NF and additional alterations in intracellular Ca(2+) release mechanisms must be postulated.     

Contractile and Ca2+-handling properties of the right ventricular papillary muscle in the late-gestation sheep fetus.

The force-generating capacity of cardiomyocytes rapidly changes during gestation and early postnatal life coinciding with a transition in cardiomyocyte nucleation in both mice and rats. Changes in nucleation, in turn, appear to coincide with important changes in the excitation-contraction coupling architecture. However, it is not clear whether similar changes are observed in other mammals in which this transition occurs prenatally, such as sheep. Using small (70-300 microM diameter) chemically skinned cardiomyocyte bundles from the right ventricular papillary muscle of sheep fetuses at 126-132 and 137-140 days (d) gestational age (GA), we aimed to examine whether changes in cardiomyocyte nucleation during late gestation coincided with developmental changes in excitation-contraction coupling parameters (e.g., Ca(2+) uptake, Ca(2+) release, and force development). All experiments were conducted at room temperature (23 +/- 1 degrees C). We found that the proportion of mononucleate cardiomyocytes decreased significantly with GA (126-132 d, 45.7 +/- 4.7%, n = 7; 137-140 d, 32.8 +/- 1.6%, n = 6; P < 0.05). When we then examined force development between the two groups, there was no significant difference in either the maximal Ca(2+)-activated force (6.73 +/- 1.54 mN/mm(2), n = 14 vs. 6.55 +/- 1.25 mN/mm(2), n = 7, respectively) or the Ca(2+) sensitivity of the contractile apparatus (pCa at 50% maximum Ca(2+)-activated force: 126-132 d, 6.17 +/- 0.06, n = 14; 137-140 d, 6.24 +/- 0.08, n = 7). However, sarcoplasmic reticulum (SR) Ca(2+) uptake rates (but not Ca(2+) release) increased with GA (P < 0.05). These data reveal that during late gestation in sheep when there is a major transition in cardiomyocyte nucleation, SR Ca(2+) uptake rates increase, which would influence total SR Ca(2+) content and force production.     

Mibefradil improves beta-adrenergic responsiveness and intracellular Ca(2+) handling in hypertrophied rat myocardium

The present study investigated the effects of mibefradil, a novel T-type channel blocker, on ventricular function and intracellular Ca(2+) handling in normal and hypertrophied rat myocardium. Ca(2+) transient was measured with the bioluminescent protein, aequorin. Mibefradil (2 microM) produced nonsignificant changes in isometric contraction and peak systolic intracellular Ca(2+) concentration ([Ca(2+)](i)) in normal rat myocardium. Hypertrophied papillary muscles isolated from aortic-banded rats 10 weeks after operation demonstrated a prolonged duration of isometric contraction, as well as decreased amplitudes of developed tension and peak Ca(2+) transient compared with the sham-operated group. Additionally, diastolic [Ca(2+)](i) increased in hypertrophied rat myocardium. The positive inotropic effect of isoproterenol stimulation was blunted in hypertrophied muscles despite a large increase in Ca(2+) transient amplitude. Afterglimmers and corresponding aftercontractions were provoked with isoproterenol (10(-5) and 10(-4) M) stimulation in 4 out of 16 hypertrophied muscles, but were eliminated in the presence of mibefradil (2 microM). In addition, hypertrophied muscles in the presence of mibefradil had a significant improvement of contractile response to isoproterenol stimulation and a reduced diastolic [Ca(2+)](I), although a mild decrease of peak Ca(2+)-transient was also shown. However, verapamil (2 microM) did not restore the inotropic and Ca(2+) modulating effects of isoproterenol in hypertrophied myocardium. Mibefradil partly restores the positive inotropic response to beta-adrenergic stimulation in hypertrophied myocardium from aortic-banded rats, an effect that might be useful in hypertrophied myocardium with impaired [Ca(2+)](i) homeostasis.     

Sex-based differences in myocardial contractile reserve

Recent studies have identified sex differences in heart function that may affect the risk of developing heart failure. We hypothesized that there are fundamental differences in calcium (Ca) regulation in cardiac myocytes of males and premenopausal females. Isometric force transients (n = 45) were measured at various stimulation frequencies to define the force frequency responses (FFR) (0.5, 1.0, 1.5, and 2.0 Hz) during either changes in bath Ca ([Ca]o) (1.0, 1.75, 3.5, and 7.0 mM) or length-tension (20, 40, 60, 80, and 100% L(max)) in right ventricle trabeculae from normal male (MT) and premenopausal female (FT) cats. Force-Ca measurements were also obtained in chemically skinned trabeculae. Under basal conditions (0.5 Hz, 1.75 mM Ca, 80% L(max)) both MT and FT achieved similar developed forces (DF) (MT 11 +/- 1, FT = 10 +/- 1 mN/mm2). At low rates and lengths, there is no sex difference. At higher preloads and rates, there is a separation in DF in MT and FT. At basal [Ca]o both MT and FT exhibited positive FFR (2.0 Hz, 1.75 mM Ca: MT 38 +/- 3, FT 21 +/- 4 mN/mm2); however, at higher [Ca]o, MT achieved greater DF (2.0 Hz, 7.0 mM Ca: MT 40 +/- 3 and FT = 24 +/- 4 mN/mm2). We detected no sex difference in myofilament Ca sensitivity at a sarcomere length of 2.1 mum. However, rapid cooling contractures indicated greater sarcoplasmic reticulum (SR) Ca load in MT at higher frequencies. Despite virtually identical contractile performance under basal conditions, significant sex differences emerge under conditions of increased physiological stress. Given the lack of sex differences in myofilament Ca sensitivity, these studies suggest fundamental sex differences in cellular Ca regulation to achieve contractile reserve, with myocardium from males exhibiting higher SR Ca load.     

Differences in mechanisms of SR dysfunction in ischemic vs. idiopathic dilated cardiomyopathy

We examined 1) contractile properties and the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient in cardiac myocytes and 2) sarcoplasmic reticulum (SR) Ca(2+) uptake and release function in myocardium from patients with end-stage heart failure caused by ischemic (ICM) vs. idiopathic dilated cardiomyopathy (DCM). The amplitude of cell motion was decreased 43 +/- 6% in ICM and 68 +/- 7% in DCM compared with that in normal organ donors (DN). Time to peak of shortening was increased 43 +/- 15% in DCM, but not in ICM. Prolongation of the relaxation time was more predominant in ICM. In DCM the systolic [Ca(2+)](i) was decreased 27 +/- 9% and diastolic [Ca(2+)](i) was increased 36 +/- 11%. In ICM the diastolic [Ca(2+)](i) was increased 59 +/- 12% but the systolic [Ca(2+)](i) was unchanged. A significant decrease of the ATP-dependent SR Ca(2+) uptake rate associated with the reduction of the SR Ca(2+)-ATPase protein level was found in ICM. In contrast, the significant decrease in SR Ca(2+) release rate was distinct in DCM. The large amount of Ca(2+) retained in the SR associated with a significant decrease in the maximum reaction velocity and increase in the Michaelis-Menten constant in the caffeine concentration-response curve suggests a fundamental abnormality in the SR Ca(2+) release channel gating property in DCM. We conclude that potentially important differences exist in the intracellular Ca(2+) homeostasis and excitation-contraction coupling in ICM vs. DCM. The SR Ca(2+) release dysfunction may play an important pathogenetic role in the abnormal Ca(2+) homeostasis in DCM, and the SR Ca(2+) uptake dysfunction may be responsible for the contractile dysfunction in ICM.     

肾上腺髓质素对大鼠损伤性心肌肌浆网功能的改善

通过观察下述五个指标,评价肾上腺髓质素(adrenomedullin,Adm)对大鼠损伤性心肌肌浆网功能的改善程度左心室压力最大变化速率(±dp/dtmax)、肌浆网钙摄取和释放及钙泵活性.皮下注射异丙肾上腺素(isoproterenol,ISO,69μmol/kg体重)制备大鼠心肌损伤坏死模型.摘取心脏后用Adm灌流,观察左心室压力最大变化速率(±dp/dtmax);制备并提纯心肌肌浆网(sarcoplasmicreticulum,SR)膜,测定SRCa2+摄取和释放速率、SR钙泵活性和钙通道蛋白～3H-ryanodine受体的最大结合量.结果发现,5×10-5mol/LAdm灌流能使ISO损伤的大鼠心脏左室±dp/dtmax分别增加16.9%(2?135±281vs1?980±302)和29.2%(1?375±267vs1?064±355,均P<0.05);SRCa2+摄取和释放率分别增加23.0%(15.0±1.4vs12.2±1.2)和43.5%(6.6±1.0vs4.6±0.6,均P<0.01);SRCa2+-ATPase活性和～3H-ryanodine受体最大结合量(Bmax)分别增加24.2%(P<0.01)和42.2%(P<0.05).提示Adm对ISO诱导的大鼠心肌损伤具有保护作用,其机制可能与Adm增加SRCa2+-ATPase活性、增加～3H-ryanodine所致SRCa2+摄取和释放升高有关.外源性给予Adm对损伤心肌可能具有临床治疗作用.     

Effects of high myoplasmic L-lactate concentration on E-C coupling in mammalian skeletal muscle.

The effects of high myoplasmic L-lactate concentrations (20-40 mM) at constant pH (7.1) were investigated on contractile protein function, voltage-dependent Ca(2+) release, and passive Ca(2+) leak from the sarcoplasmic reticulum (SR) in mechanically skinned fast-twitch (extensor digitorum longus; EDL) and slow-twitch (soleus) fibers of the rat. L-Lactate (20 mM) significantly reduced maximum Ca(2+)-activated force by 4 +/- 0.5% (n = 5, P < 0.05) and 5 +/- 0.4% (n = 6, P < 0.05) for EDL and soleus, respectively. The Ca(2+) sensitivity was also significantly decreased by 0.06 +/- 0. 002 (n = 5, P < 0.05) and 0.13 +/- 0.01 (n = 6, P < 0.001) pCa units, respectively. Exposure to L-lactate (20 mM) for 30 s reduced depolarization-induced force responses by ChCl substitution by 7 +/- 3% (n = 17, P < 0.05). This inhibition was not obviously affected by the presence of the lactate transport blocker quercetin (10 microM), or the chloride channel blocker anthracene-9-carboxylic acid (100 microM). L-Lactate (20 mM) increased passive Ca(2+) leak from the SR in EDL fibers (the integral of the response to caffeine was reduced by 16 +/- 5%, n = 9, P < 0.05) with no apparent effect in soleus fibers (100 +/- 2%, n = 3). These results indicate that the L-lactate ion per se has negligible effects on either voltage-dependent Ca(2+) release or SR Ca(2+) handling and exerts only a modest inhibitory effect on muscle contractility at the level of the contractile proteins.     

Effects of Na(+)/Ca(2+) exchanger downregulation on contractility and [Ca(2+)](i) transients in adult rat myocytes

Postmyocardial infarction (MI) rat myocytes demonstrated depressed Na(+)/Ca(2+) exchange (NCX1) activity, altered contractility, and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients. We investigated whether NCX1 downregulation in normal myocytes resulted in contractility changes observed in MI myocytes. Myocytes infected with adenovirus expressing antisense (AS) oligonucleotides to NCX1 had 30% less NCX1 at 3 days and 66% less NCX1 at 6 days. The half-time of relaxation from caffeine-induced contracture was twice as long in ASNCX1 myocytes. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase abundance, SR Ca(2+) uptake, resting membrane potential, action potential amplitude and duration, L-type Ca(2+) current density and cell size were not affected by ASNCX1 treatment. At extracellular Ca(2+) concentration ([Ca(2+)](o)) of 5 mM, ASNCX1 myocytes had significantly lower contraction and [Ca(2+)](i) transient amplitudes and SR Ca(2+) contents than control myocytes. At 0.6 mM [Ca(2+)](o), contraction and [Ca(2+)](i) transient amplitudes and SR Ca(2+) contents were significantly higher in ASNCX1 myocytes. At 1.8 mM [Ca(2+)](o), contraction and [Ca(2+)](i) transient amplitudes were not different between control and ASNCX1 myocytes. This pattern of contractile and [Ca(2+)](i) transient abnormalities in ASNCX1 myocytes mimics that observed in rat MI myocytes. We conclude that downregulation of NCX1 in adult rat myocytes resulted in decreases in both Ca(2+) influx and efflux during a twitch. We suggest that depressed NCX1 activity may partly account for the contractile abnormalities after MI.     

Increased Ca(2+) sparks and sarcoplasmic reticulum Ca(2+) stores potentially determine the spontaneous activity of pulmonary vein cardiomyocytes

Pulmonary veins (PVs) contain cardiomyocytes with spontaneous activity that may be responsible for PV arrhythmia. Abnormal Ca(2+) regulation is known to contribute to PV arrhythmogenesis. The purpose of this study was to investigate whether PV cardiomyocytes with spontaneous activity have different intracellular Ca(2+) ([Ca(2+)](i)) transients, Ca(2+) sparks and responses to isoproterenol and ryanodine receptor modulators (magnesium and FK506) than do PV cardiomyocytes without spontaneous activity and left atrial (LA) cardiomyocytes. Through fluorescence and confocal microscopy, we evaluated the [Ca(2+)](i) transients and Ca(2+) sparks in isolated rabbit PV and LA cardiomyocytes. PV cardiomyocytes with spontaneous activity had larger [Ca(2+)](i) transients and sarcoplasmic reticulum (SR) Ca(2+) stores than PV cardiomyocytes without spontaneous activity or LA cardiomyocytes. PV cardiomyocytes with spontaneous activity also had a higher incidence and frequency of Ca(2+) sparks, and had Ca(2+) sparks with larger amplitudes than other cardiomyocytes. Magnesium (5.4 mM) reduced the [Ca(2+)](i) transient amplitude and beating rate in PV cardiomyocytes with spontaneous activity. However, in contrast with other cardiomyocytes, low doses (1.8 mM) of magnesium did not reduce the [Ca(2+)](i) transients amplitude in PV cardiomyocytes with spontaneous activity. FK506 (1 muM) diminished the SR Ca(2+) stores in PV cardiomyocytes with spontaneous activity to a lesser extent than that in other cardiomyocytes. Isoproterenol (10 nM) increased the [Ca(2+)](i) transient amplitude to a lesser extent in LA cardiomyocytes than in PV cardiomyocytes with or without spontaneous activity. In conclusion, our results suggest that enhanced [Ca(2+)](i) transients, increased Ca(2+) sparks and SR Ca(2+) stores may contribute to the spontaneous activity of PV cardiomyocytes.     

Enhanced calcium mobilization in rat ventricular myocytes during the onset of pressure overload-induced hypertrophy

Early cardiovascular changes evoked by pressure overload (PO) may reveal adaptive strategies that allow immediate survival to the increased hemodynamic load. In this study, systolic and diastolic Ca(2+) cycling was analyzed in left ventricular rat myocytes before (day 2, PO-2d group) and after (day 7, PO-7d group) development of hypertrophy subsequent to aortic constriction, as well as in myocytes from time-matched sham-operated rats (sham group). Ca(2+) transient amplitude was significantly augmented in the PO-2d group. In the PO-7d group, intracellular Ca(2+) concentration ([Ca(2+)](i)) was reduced during diastole, and mechanical twitch relaxation (but not [Ca(2+)](i) decline) was slowed. In PO groups, fractional sarcoplasmic reticulum (SR) Ca(2+) release at a twitch, SR Ca(2+) content, SR Ca(2+) loss during diastole, and SR-dependent integrated Ca(2+) flux during twitch relaxation were significantly greater than in sham-operated groups, whereas the relaxation-associated Ca(2+) flux carried by the Na(+)/Ca(2+) exchanger was not significantly changed. In the PO-7d group, mRNA levels of cardiac isoforms of SR Ca(2+)-ATPase (SERCA2a), phospholamban, calsequestrin, ryanodine receptor, and NCX were not significantly altered, but the SERCA2a-to-phospholamban ratio was increased 2.5-fold. Moreover, greater sensitivity to the inotropic effects of the beta-adrenoceptor agonist isoproterenol was observed in the PO-7d group. The results indicate enhanced Ca(2+) cycling between SR and cytosol early after PO imposition, even before hypertrophy development. Increase in SR Ca(2+) uptake may contribute to enhancement of excitation-contraction coupling (augmented SR Ca(2+) content and release) and protection against arrhythmogenesis due to buildup of [Ca(2+)](i) during diastole.