Adaptations in human muscle sarcoplasmic reticulum to prolonged submaximal training.

In this study, we employed single-leg submaximal cycle training, conducted over a 10-wk period, to investigate adaptations in sarcoplasmic reticulum (SR) Ca(2+)-regulatory proteins and processes of the vastus lateralis. During the final weeks, the untrained volunteers (age 21.4 +/- 0.3 yr; means +/- SE, n = 10) were exercising 5 times/wk and for 60 min/session. Analyses were performed on tissue extracted by needle biopsy approximately 4 days after the last training session. Compared with the control leg, the trained leg displayed a 19% reduction (P < 0.05) in homogenate maximal Ca(2+)-ATPase activity (192 +/- 11 vs. 156 +/- 18 micromol. g protein(-1). min(-1)), a 4.3% increase (P < 0.05) in pCa(50), defined as the Ca(2+) concentration at half-maximal activity (6.01 +/- 0.05 vs. 6.26 +/- 0.07), and no change in the Hill coefficient (1.75 +/- 0.15 vs. 1.76 +/- 0.21). Western blot analysis using monoclonal antibodies (7E6 and A52) revealed a 13% lower (P < 0.05) sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) 1 in trained vs. control in the absence of differences in SERCA2a. Training also resulted in an 18% lower (P < 0.05) SR Ca(2+) uptake and a 26% lower (P < 0.05) Ca(2+) release. It is concluded that a downregulation in SR Ca(2+) cycling in vastus lateralis occurs with aerobic-based training, which at least in the case of Ca(2+) uptake can be explained by reduction in Ca(2+)-ATPase activity and SERCA1 protein levels.     

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   

Abnormal sarcoplasmic reticulum Ca2+-sequestering properties in skeletal muscle in chronic obstructive pulmonary disease

The objective of this study was to investigate the hypothesis that alterations in sarcoplasmic reticulum (SR) Ca(2+)-cycling properties would occur in skeletal muscle in patients with moderate to severe chronic obstructive pulmonary disease (COPD). To investigate this hypothesis, tissue samples were obtained from the vastus lateralis of 8 patients with COPD [age 65.6 +/- 3.2 yr; forced expiratory volume in 1 s (FEV(1))/forced vital capacity (FVC) = 44 +/- 2%; mean +/- SE] and 10 healthy age-matched controls (CON, age 67.5 +/- 2.5 yr; FEV(1)/FVC = 77 +/- 2%), and homogenates were analyzed for a wide range of SR properties. Compared with CON, COPD displayed (in mumol.g protein(-1).min(-1)) a 16% lower maximal Ca(2+)-ATPase activity [maximal velocity (V(max)), 158 +/- 10 vs. 133 +/- 7, P < 0.05] and a 17% lower Ca(2+) uptake (4.65 +/- 0.039 vs. 3.85 +/- 0.26, P < 0.05) that occurred in the absence of differences in Ca(2+) release. The lower V(max) in COPD was also accompanied by an 11% lower (P < 0.05) Ca(2+) sensitivity, as measured by the Hill coefficient (defined as the relationship between Ca(2+)-ATPase activity and free cytosolic Ca(2+) concentration for 10-90% V(max)). For the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms, SERCA1a was 16% higher (P < 0.05) and SERCA2a was 14% lower (P < 0.05) in COPD. It is concluded that moderate to severe COPD results in abnormalities in SR Ca(2+)-ATPase properties that cannot be explained by changes in the SERCA isoform phenotypes. The reduced catalytic properties of SERCA in COPD suggest a disturbance in Ca(2+) cycling, possibly resulting in impairment in Ca(2+)-mediated mechanical function and/or second messenger regulated processes.     

Cellular and molecular determinants of altered Ca2+ handling in the failing rabbit heart: primary defects in SR Ca2+ uptake and release mechanisms

Myocytes from the failing myocardium exhibit depressed and prolonged intracellular Ca(2+) concentration ([Ca(2+)](i)) transients that are, in part, responsible for contractile dysfunction and unstable repolarization. To better understand the molecular basis of the aberrant Ca(2+) handling in heart failure (HF), we studied the rabbit pacing tachycardia HF model. Induction of HF was associated with action potential (AP) duration prolongation that was especially pronounced at low stimulation frequencies. L-type calcium channel current (I(Ca,L)) density (-0.964 +/- 0.172 vs. -0.745 +/- 0.128 pA/pF at +10 mV) and Na(+)/Ca(2+) exchanger (NCX) currents (2.1 +/- 0.8 vs. 2.3 +/- 0.8 pA/pF at +30 mV) were not different in myocytes from control and failing hearts. The amplitude of peak [Ca(2+)](i) was depressed (at +10 mV, 0.72 +/- 0.07 and 0.56 +/- 0.04 microM in normal and failing hearts, respectively; P < 0.05), with slowed rates of decay and reduced Ca(2+) spark amplitudes (P < 0.0001) in myocytes isolated from failing vs. control hearts. Inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a revealed a greater reliance on NCX to remove cytosolic Ca(2+) in myocytes isolated from failing vs. control hearts (P < 0.05). mRNA levels of the alpha(1C)-subunit, ryanodine receptor (RyR), and NCX were unchanged from controls, while SERCA2a and phospholamban (PLB) were significantly downregulated in failing vs. control hearts (P < 0.05). alpha(1C) protein levels were unchanged, RyR, SERCA2a, and PLB were significantly downregulated (P < 0.05), while NCX protein was significantly upregulated (P < 0.05). These results support a prominent role for the sarcoplasmic reticulum (SR) in the pathogenesis of HF, in which abnormal SR Ca(2+) uptake and release synergistically contribute to the depressed [Ca(2+)](i) and the altered AP profile phenotype.     

Decreased cardiac SERCA2 expression, SR Ca uptake, and contractile function in hypothyroidism are attenuated in SERCA2 overexpressing transgenic rats

The sarco/endoplasmic reticulum (SR) Ca(2+)-ATPase SERCA2a has a key role in controlling cardiac contraction and relaxation. In hypothyroidism, decreased expression of the thyroid hormone (TH)-responsive SERCA2 gene contributes to slowed SR Ca(2+) reuptake and relaxation. We investigated whether cardiac expression of a TH-insensitive SERCA2a cDNA minigene can rescue SR Ca(2+) handling and contractile function in female SERCA2a-transgenic rats (TG) with experimental hypothyroidism. Wild-type rats (WT) and TG were rendered hypothyroid by 6-N-propyl-2-thiouracil treatment for 6 wk; control rats received no treatment. In vivo measured left ventricular (LV) hemodynamic parameters were compared with SERCA2a expression and function in LV tissue. Hypothyroidism decreased LV peak systolic pressure, dP/dt(max), and dP/dt(min) in both WT and TG. However, loss of function was less in TG. Thus slowed relaxation in hypothyroidism was found to be 1.5-fold faster in TG compared with WT (P < 0.05). In parallel, a 1.4-fold higher V(max) value of homogenate SR Ca(2+) uptake was observed in hypothyroid TG (P < 0.05 vs. hypothyroid WT), and the hypothyroidism-caused decline of LV SERCA2a mRNA expression in TG by -24% was markedly less than the decrease of -49% in WT (P < 0.05). A linear relationship was observed between the SERCA2a/PLB mRNA ratio values and the V(max) values of SR Ca(2+) uptake when the respective data of all experimental groups were plotted together (r = 0.90). The data show that expression of the TH-insensitive SERCA2a minigene compensates for loss of expressional activity of the TH-responsive native SERCA2a gene in the female hypothyroid rat heart. However, SR Ca(2+) uptake and in vivo heart function were only partially rescued.     

Paradoxical effects of prior activity on human sarcoplasmic reticulum Ca2+-ATPase response to exercise.

To investigate the effects of intermittent heavy exercise (HE) on sarcoplasmic reticulum (SR) maximal Ca2+-ATPase activity (Vmax) and Ca2+ uptake, a continuous two-stage standardized cycling test was performed before and after HE by untrained men [peak aerobic power (Vo -->Vo2 peak) = 42.9 +/- 2.7 ml. kg-1 x min-1]. The HE consisted of 16 bouts of cycling performed for 6 min each hour at 90% Vo2 peak. Tissue was obtained from the vastus lateralis by needle biopsy before and during each cycle test. Before HE, reductions (P < 0.05 micromol. g protein-1x min-1) of 16 and 31% were observed in Vmax and Ca2+ uptake, respectively, after 40 min of the standardized test. Resting Vmax and Ca2+ uptake were depressed (P < 0.05) by 19 and 30%, respectively, when measured 36-48 h after HE. During the standardized test, after HE, Vmax increased (P < 0.05) by 20%, whereas no change was observed in Ca2+ uptake. The HE protocol resulted in small increases (P < 0.05) and decreases (P < 0.05) in sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 2a and SERCA1 expression, respectively, as determined by Western blotting techniques. These results indicate that SR Ca2+-sequestering function in response to a prolonged exercise test depends on prior activity status, such that rested muscles exhibit a decrease and prior exercised muscles, an increase in Ca2+-ATPase activity. Moreover, it appears that changes in SERCA content can occur in response to a sustained session of intermittent exercise.     

Effects of fatigue and training on sarcoplasmic reticulum Ca(2+) regulation in human skeletal muscle.

Little is known about fatigue and training effects on sarcoplasmic reticulum (SR) function in human muscle, and we therefore investigated this in eight untrained controls (UT), eight endurance-trained (ET), and eight resistance-trained athletes (RT). Muscle biopsies (vastus lateralis) taken at rest and after 50 maximal quadriceps contractions (180 degrees/s, 0.5 Hz) were analyzed for fiber composition, metabolites and maximal SR Ca(2+) release, Ca(2+) uptake, and Ca(2+)-ATPase activity. Fatigue reduced (P < 0.05) Ca(2+) release (42.1 +/- 3.8%, 43.4 +/- 3.9%, 31.3 +/- 6.1%), Ca(2+) uptake (43.0 +/- 5.2%, 34.1 +/- 4.6%, 28.4 +/- 2.8%), and Ca(2+)-ATPase activity (38.6 +/- 4.2%, 48.5 +/- 5.7%, 29.6 +/- 5.0%), in UT, RT, and ET, respectively. These decreases were correlated with fatigability and with type II fiber proportion (P < 0.05). Resting SR measures were correlated with type II proportion (r > or = 0.51, P < 0.05). ET had lower resting Ca(2+) release, Ca(2+) uptake, and Ca(2+)-ATPase (P < 0.05) than UT and RT (P < 0.05), probably because of their lower type II proportion; only minor effects were found in RT. Thus SR function is markedly depressed with fatigue in controls and in athletes, is dependent on fiber type, and appears to be minimally affected by chronic training status.     

Exercise training attenuates coronary smooth muscle phenotypic modulation and nuclear Ca2+ signaling

Physical inactivity is an independent risk factor for coronary heart disease, yet the mechanism(s) of exercise-related cardioprotection remains unknown. We tested the hypothesis that coronary smooth muscle after exercise training would have decreased mitogen-induced phenotypic modulation and enhanced regulation of nuclear Ca(2+). Yucatan swine were endurance exercise trained (EX) on a treadmill for 16-20 wk. EX reduced endothelin-1-induced DNA content by 40% compared with sedentary (SED) swine (P < 0.01). EX decreased single cell peak endothelin-1-induced cytosolic Ca(2+) responses compared with SED by 16% and peak nuclear Ca(2+) responses by 33% (P < 0.05), as determined by confocal microscopy. On the basis of these results, we hypothesized that sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and intracellular Ca(2+) stores in native smooth muscle are spatially localized to dissociate cytosolic Ca(2+) and nuclear Ca(2+). Subcellular localization of SERCA in living and fixed cells revealed a distribution of SERCA near the sarcolemma and on the nuclear envelope. These results show that EX enhances nuclear Ca(2+) regulation, possibly via SERCA, which may be one mechanism by which coronary smooth muscle cells from EX are less responsive to mitogen-induced phenotypic modulation.     

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.     

Defective Ca(2+) handling proteins regulation during heart failure

Abnormal release of Ca(2+) from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction in heart failure (HF). We previously demonstrated that RyR2 macromolecular complexes from HF rat were significantly more depleted of FK506 binding protein (FKBP12.6). Here we assessed expression of key Ca(2+) handling proteins and measured SR Ca(2+) content in control and HF rat myocytes. Direct measurements of SR Ca(2+) content in permeabilized cardiac myocytes demonstrated that SR luminal [Ca(2+)] is markedly lowered in HF (HF: DeltaF/F(0) = 26.4+/-1.8, n=12; control: DeltaF/F(0) = 49.2+/-2.9, n=10; P<0.01). Furthermore, we demonstrated that the expression of RyR2 associated proteins (including calmodulin, sorcin, calsequestrin, protein phosphatase 1, protein phosphatase 2A), Ca(2+) ATPase (SERCA2a), PLB phosphorylation at Ser16 (PLB-S16), PLB phosphorylation at Thr17 (PLB-T17), L-type Ca(2+) channel (Cav1.2) and Na(+)- Ca(2+) exchanger (NCX) were significantly reduced in rat HF. Our results suggest that systolic SR reduced Ca(2+) release and diastolic SR Ca(2+) leak (due to defective protein-protein interaction between RyR2 and its associated proteins) along with reduced SR Ca(2+) uptake (due to down-regulation of SERCA2a, PLB-S16 and PLB-T17), abnormal Ca(2+) extrusion (due to down-regulation of NCX) and defective Ca(2+) -induced Ca(2+) release (due to down-regulation of Cav1.2) could contribute to HF.     

Muscle sarcoplasmic reticulum calcium regulation in humans during consecutive days of exercise and recovery.

The study investigated the hypothesis that three consecutive days of prolonged cycle exercise would result in a sustained reduction in the Ca(2+)-cycling properties of the vastus lateralis in the absence of changes in the sarcoplasmic (endoplasmic) reticulum Ca(2+)-ATPase (SERCA) protein. Tissue samples were obtained at preexercise (Pre) and postexercise (Post) on day 1 (E1) and day 3 (E3) and during recovery day 1 (R1), day 2 (R2), and day 3 (R3) in 12 active but untrained volunteers (age 19.2 +/- 0.27 yr; mean +/- SE) and analyzed for changes (nmol.mg protein(-1).min(-1)) in maximal Ca(2+)-ATPase activity (V(max)), Ca(2+) uptake and Ca(2+) release (phase 1 and phase 2), and SERCA isoform expression (SERCA1a and SERCA2a). At E1, reductions (P < 0.05) from Pre to Post in V(max) (150 +/- 7 vs. 121 +/- 7), Ca(2+) uptake (7.79 +/- 0.28 vs. 5.71 +/- 0.33), and both phases of Ca(2+) release (phase 1, 20.3 +/- 1.3 vs. 15.2 +/- 1.1; phase 2, 7.70 +/- 0.60 vs. 4.99 +/- 0.48) were found. In contrast to V(max), which recovered at Pre E3 and then remained stable at Post E3 and throughout recovery, Ca(2+) uptake remained depressed (P < 0.05) at E3 Pre and Post and at R1 as did phase 2 of Ca(2+) release. Exercise resulted in an increase (P < 0.05) in SERCA1a (14% at R2) but not SERCA2a. It is concluded that rapidly adapting mechanisms protect V(max) following the onset of regular exercise but not Ca(2+) uptake and Ca(2+) release.     

Mechanisms underlying increases in SR Ca2+-ATPase activity after exercise in rat skeletal muscle

Prolonged exercise followed by a brief period of reduced activity has been shown to result in an overshoot in maximal sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity [maximal velocity (V(max))] in rat locomoter muscles (Ferrington DA, Reijneveld JC, B?r PR, and Bigelow DJ. Biochim Biophys Acta 1279: 203-213, 1996). To investigate the functional significance and underlying mechanisms for the increase in V(max), we analyzed Ca(2+)-ATPase activity and Ca(2+) uptake in SR vesicles from the fast rat gastrocnemius muscles after prolonged running (RUN) and after prolonged running plus 45 min of low-intensity activity (RUN+) or no activity (REC45) and compared them with controls (Con). Although no differences were observed between RUN and Con, both V(max) and Ca(2+) uptake were higher (P < 0.05) by 43 and 63%, respectively, in RUN+ and by 35 and 34%, respectively, in REC45. The increase in V(max) was accompanied by increases (P < 0.05) in the phosphorylated enzyme intermediate measured by [gamma-(32)P]ATP. No differences between groups for each condition were found for the fluorescent probes FITC and (N-cyclohexyl-N(1)-dimethylamino-alpha-naphthyl)carbodiimide, competitive inhibitors of the nucleotide-binding and Ca(2+)-binding sites on the enzyme, respectively. Similarly, no differences for the Ca(2+)-ATPase were observed between groups in nitrotyrosine and phosphoserine residues, a measure of nitrosylation and phosphorylation states, respectively. Western blots indicated no changes in relative isoform content of sarcoendoplasmic reticulum (SERCA)1 and SERCA2a. It is concluded that the increase in V(max) of the Ca(2+)-ATPase observed in recovery is not the result of changes in enzyme nitroslyation or phosphorylation, changes in ATP and Ca(2+)-binding affinity, or changes in protein content of the Ca(2+)-ATPase.     

Measurement and modeling of Ca2+ waves in isolated rabbit ventricular cardiomyocytes

The time course and magnitude of the Ca(2+) fluxes underlying spontaneous Ca(2+) waves in single permeabilized ventricular cardiomyocytes were derived from confocal Fluo-5F fluorescence signals. Peak flux rates via the sarcoplasmic reticulum (SR) release channel (RyR2) and the SR Ca(2+) ATPase (SERCA) were not constant across a range of cellular [Ca(2+)] values. The Ca(2+) affinity (K(mf)) and maximum turnover rate (V(max)) of SERCA and the peak permeability of the RyR2-mediated Ca(2+) release pathway increased at higher cellular [Ca(2+)] loads. This information was used to create a computational model of the Ca(2+) wave, which predicted the time course and frequency dependence of Ca(2+) waves over a range of cellular Ca(2+) loads. Incubation of cardiomyocytes with the Ca(2+) calmodulin (CaM) kinase inhibitor autocamtide-2-related inhibitory peptide (300 nM, 30 mins) significantly reduced the frequency of the Ca(2+) waves at high Ca(2+) loads. Analysis of the Ca(2+) fluxes suggests that inhibition of CaM kinase prevented the increases in SERCA V(max) and peak RyR2 release flux observed at high cellular [Ca(2+)]. These data support the view that modification of activity of SERCA and RyR2 via a CaM kinase sensitive process occurs at higher cellular Ca(2+) loads to increase the maximum frequency of spontaneous Ca(2+) waves.     

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.     

Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr(671)-Leu(690); 36 microM) enhanced the rate of Ca(2+) release from normal SR (SR(N)) and from SR of MH-susceptible muscle (SR(MH)) by 10 +/- 3.2 nmole/mg/min and 76 +/- 9.7 nmole/mg/min, respectively. Ca (2+) release from SR(N) or SR(MH) was not increased by control peptide NB (Gly(689)-Lys(708)). AS (scrambled A sequence; 36 microM) did not alter Ca (2+) release from SR(N), but increased release from SR(MH) by 29 +/- 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR(MH)) were up to about fourfold more strongly activated by peptide A (> or =1 nM) than normal RyR channels (RyR(N)) at -40 mV. Neither NB or AS activated RyR(N). RyR(MH) showed an approximately 1.8-fold increase in mean current with 30 microM AS. Inhibition at +40 mV was stronger in RyR(MH) and seen with peptide A (> or = 0.6 microM) and AS (> or = 0.6 microM), but not NB. These results show that the Arg(615)Cys substitution in RyR(MH) has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca(2+) release from SR in MH-susceptible muscle.     

Postcontractile force depression in humans is associated with an impairment in SR Ca(2+) pump function

To investigate the hypothesis that intrinsic changes in sarcoplasmic reticulum (SR) Ca(2+)-sequestration function can be implicated in postcontractile depression (PCD) of force in humans, muscle tissue was obtained from the vastus lateralis and determinations of maximal Ca(2+) uptake and maximal Ca(2+)-ATPase activity were made on homogenates obtained before and after the induction of PCD. Eight untrained females, age 20.6+/-0.75 yr (mean +/- SE), performed a protocol consisting of 30 min of isometric exercise at 60% maximal voluntary contraction and at 50% duty cycle (5-s contraction and 5-s relaxation) to induce PCD. Muscle mechanical performance determined by evoked activation was measured before (0 min), during (15 and 30 min), and after (60 min) exercise. The fatiguing protocol resulted in a progressive reduction (P<0.05) in evoked force, which by 30 min amounted to 52% for low frequency (10 Hz) and 20% for high frequency (100 Hz). No force restoration occurred at either 10 or 100 Hz during a 60-min recovery period. Maximal SR Ca(2+)-ATPase activity (nmol x mg protein(-1) x min(-1)) and maximal SR Ca(2+) uptake (nmol. mg protein(-1) x min(-1)) were depressed (P<0.05) by 15 min of exercise [192+/-45 vs. 114+/-8.7 and 310+/-59 vs. 205+/-47, respectively; mean +/- SE] and remained depressed at 30 min of exercise. No recovery in either measure was observed during the 60-min recovery period. The coupling ratio between Ca(2+)-ATPase and Ca(2+) uptake was preserved throughout exercise and during recovery. These results illustrate that during PCD, Ca(2+) uptake is depressed and that the reduction in Ca(2+) uptake is due to intrinsic alterations in the Ca(2+) pump. The role of altered Ca(2+) sequestration in Ca(2) release, cytosolic-free calcium, and PCD remains to be determined.     

Ca²⁺-induced delayed afterdepolarizations are triggered by dyadic subspace Ca2²⁺ affirming that increasing SERCA reduces aftercontractions

Ca(2+)-induced delayed afterdepolarizations (DADs) are depolarizations that occur after full repolarization. They have been observed across multiple species and cell types. Experimental results have indicated that the main cause of DADs is Ca(2+) overload. The main hypothesis as to their initiation has been Ca(2+) overflow from the overloaded sarcoplasmic reticulum (SR). Our results using 37 previously published mathematical models provide evidence that Ca(2+)-induced DADs are initiated by the same mechanism as Ca(2+)-induced Ca(2+) release, i.e., the modulation of the opening of ryanodine receptors (RyR) by Ca(2+) in the dyadic subspace; an SR overflow mechanism was not necessary for the induction of DADs in any of the models. The SR Ca(2+) level is better viewed as a modulator of the appearance of DADs and the magnitude of Ca(2+) release. The threshold for the total Ca(2+) level within the cell (not only the SR) at which Ca(2+) oscillations arise in the models is close to their baseline level (～1- to 3-fold). It is most sensitive to changes in the maximum sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump rate (directly proportional), the opening probability of RyRs, and the Ca(2+) diffusion rate from the dyadic subspace into the cytosol (both indirectly proportional), indicating that the appearance of DADs is multifactorial. This shift in emphasis away from SR overload as the trigger for DADs toward a multifactorial analysis could explain why SERCA overexpression has been shown to suppress DADs (while increasing contractility) and why DADs appear during heart failure (at low SR Ca(2+) levels).     

Thyroid hormones differentially regulate the distribution of rabbit skeletal muscle Ca(2+)-ATPase (SERCA) isoforms in light and heavy sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) is composed of two fractions, the heavy fraction that contains proteins involved in Ca2+ release, and the light fraction enriched in Ca(2+)-ATPase (SERCA), an enzyme responsible for Ca2+ transport from the cytosol to the lumen of SR. It is known that in red muscle thyroid hormones regulate the expression of SERCA 1 and SERCA 2 isoforms. Here we show the effects of thyroid hormone on SERCA expression and distribution in light and heavy SR fractions from rabbit white and red muscles. In hyperthyroid red muscle there is an increase of SERCA 1 and a decrease of SERCA 2 expression. This is far more pronounced in the heavy than in the light SR fraction. As a result, the rates of Ca(2+)- ATPase activity and Ca(2+)-uptake by the heavy vesicles are increased. In hypothyroidism we observed a decrease in SERCA 1 and no changes in the amount of SERCA 2 expressed. This promoted a decrease of both Ca(2+)-uptake and Ca(2+)-ATPase activity. While the major differences in hyperthyroidism were found in the heavy SR fraction, the effects of hypothyroidism were restricted to light SR fraction. In white muscle we did not observe any significant changes in either hypo- or hyperthyroidism in both SR fractions. Thus, the regulation of SERCA isoforms by thyroid hormones is not only muscle specific but also varies depending on the subcellular compartment analyzed. These changes might correspond to the molecular basis of the altered contraction and relaxation rates detected in thyroid dysfunction.     

Effects of buthionine sulfoximine treatment on diaphragm contractility and SR Ca2+ pump function in rats.

The purpose of this study was to examine the effects of glutathione (GSH) depletion and cellular oxidation on rat diaphragm contractility and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in vitro under basal conditions and following fatiguing stimulation. Buthionine sulfoximine (BSO) treatment (n = 10) for 10 days (20 mM in drinking water) reduced (P < 0.05) diaphragm GSH content (nmol/mg protein) and the ratio of GSH to glutathione disulfide (GSH/GSSG) by 91% and 71%, respectively, compared with controls (CTL) (n = 10). Western blotting showed that Hsp70 expression in diaphragm was not increased (P > 0.05) with BSO treatment. As hypothesized, basal peak twitch force (g/mm(2)) was increased (P < 0.05), and fatigability in response to repetitive stimulation (350-ms trains at 100 Hz once every 1 s for 5 min) was also increased (P < 0.05) in BSO compared with CTL. Both Ca(2+) uptake and maximal SERCA activity (mumol.g protein(-1).min(-1)) measured in diaphragm homogenates that were prepared at rest were increased (P < 0.05) with BSO treatment, an effect that could be partly explained by a twofold increase (P < 0.05) in SERCA2a expression with BSO. In response to the 5-min stimulation protocol, both Ca(2+) uptake and maximal SERCA activity were increased (P < 0.05) in CTL but not (P > 0.05) in BSO diaphragm. We conclude that 1) cellular redox state is more optimal for contractile function and fatigability is increased in rat diaphragm following BSO treatment, 2) SERCA2a expression is modulated by redox signaling, and 3) regulation of SERCA function in working diaphragm is altered following BSO treatment.     

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