Doxycycline inducible expression of SERCA2a improves calcium handling and reverts cardiac dysfunction in pressure overload-induced cardiac hypertrophy

Delayed cardiac relaxation in failing hearts has been attributed to reduced activity and/or expression of sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a). Although constitutive overexpression of SERCA2a has proven effective in preventing cardiac dysfunction, it is unclear whether increasing SERCA2a expression in hearts with preexisting hypertrophy will be therapeutic. To test this hypothesis, we generated a binary transgenic (BTG) system that allows tetracycline-inducible, cardiac-specific SERCA2a expression. In this system (tet-on SERCA2a), a FLAG-tagged SERCA2a transgene is expressed in the presence of doxycycline (Dox) but not in the absence of Dox (2.3-fold more mRNA, 45% more SERCA2a protein). Calcium transients measured in isolated cardiac myocytes from nonbanded Dox-treated BTG mice showed an accelerated calcium decline and an increased systolic Ca2+ peak. Sarcoplasmic reticulum (SR) calcium loading was increased by 45% in BTG mice. In the presence of pressure overload (aortic banding), echocardiographic analysis revealed that expression of SERCA2a-FLAG caused an improvement in fractional shortening. SERCA2a-FLAG expression alleviated the resultant cardiac dysfunction. This was illustrated by an increase in the rate of decline of the calcium transient. Cell shortening and SR calcium loading were also improved in cardiac myocytes isolated from banded BTG mice after SERCA2a overexpression. In conclusion, we generated a novel transgenic mouse that conditionally overexpresses SERCA2a. This model is suitable for both long- and short-term studies of the effects of controlled SERCA2a expression on cardiac function. In addition, inducible overexpression of SERCA2a improved cardiac function and calcium handling in mice with established contractile dysfunction.     

Overexpression of SERCA2a accelerates repolarisation in rabbit ventricular myocytes

Overexpression of the sarcoplasmic reticulum Ca ATPase (SERCA2a) produces positive inotropism and it has been proposed as a promising strategy to counteract defective excitation-contraction coupling in the failing heart. However, the effects of overexpressing SERCA2a on action potential duration (APD), which can affect diastolic parameters in the heart, is unknown. We, therefore, investigated the relationship between SERCA2a overexpression and APD in adult rabbit ventricular myocytes which were cultured for 48 h. Overexpression of SERCA2a was achieved by infection with an adenovirus carrying both SERCA2a and GFP independently driven by CMV promoters, Ad.SERCA2a. Myocytes infected with Ad.GFP only and/or non-infected myocytes were used as controls. Electrophysiological measurements were taken using switch clamping with 15-25 M Omega resistance microelectrodes. In Ad.SERCA2a infected myocytes, APD was significantly reduced compared with both groups of control cells at 0.5 Hz (APD50 (ms) non-infected: 481+/-98, n=12; Ad.GFP: 464+/-85, n=11; Ad.SERCA2a: 285+/-69, n=13 (mean+/-S.E.M.) and at 1 Hz (APD50 (ms) non-infected: 375+/-64, n=22; Ad.GFP: 363+/-47, n=18; Ad.SERCA2a: 231+/-54, n=24). Using AP voltage-clamping, we recorded a 0.2 mM Cd-sensitive current which can be ascribed to Ca current flowing during the AP. The integral of this current was reduced in Ad.SERCA2a myocytes compared with control (non-infected charge (pC): 27.5+/-4.2, n=8; Ad.SERCA2a: 15.5+/-4.1, n=11; P<0.01). Using AP clamping during the loading protocol, to take into account changes in APD, SR Ca content (assessed by integrating a 20 mM caffeine-induced inward current) was significantly larger in Ad.SERCA2a compared with both controls (SR Ca content (microM/l non-mitochondrial volume): non-infected: 25.5+/-7, n=8; Ad.GFP: 25.7+/-11, n=6; Ad.SERCA2a: 80.5+/-19, n=8). In conclusion, this study shows that SR Ca content is increased despite decreased Ca entry after overexpression of SERCA2a, and this can lead to positive inotropism. This effect coupled with shorter APD may be a useful therapeutic modality in heart failure.     

Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility

The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.     

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).     

Overexpression of TRPC3 increases apoptosis but not necrosis in response to ischemia-reperfusion in adult mouse cardiomyocytes

An increase in cytosolic Ca2+ via a capacitative calcium entry (CCE)-mediated pathway, attributed to members of the transient receptor potential (TRP) superfamily, TRPC1 and TRPC3, has been reported to play an important role in regulating cardiomyocyte hypertrophy. Increased cytosolic Ca2+ also plays a critical role in mediating cell death in response to ischemia-reperfusion (I/R). Therefore, we tested the hypothesis that overexpression of TRPC3 in cardiomyocytes will increase sensitivity to I/R injury. Adult cardiomyocytes isolated from wild-type (WT) mice and from mice overexpressing TRPC3 in the heart were subjected to 90 min of ischemia and 3 h of reperfusion. After I/R, viability was 51 +/- 1% in WT mice and 42 +/- 5% in transgenic mice (P < 0.05). Apoptosis assessed by annexin V was significantly increased in the TRPC3 group compared with WT (32 +/- 1% vs. 21 +/- 3%; P < 0.05); however, there was no significant difference in necrosis between groups. Treatment of TRPC3 cells with the CCE inhibitor SKF-96365 (0.5 microM) significantly improved cellular viability (54 +/- 4%) and decreased apoptosis (15 +/- 4%); in contrast, the L-type Ca2+ channel inhibitor verapamil (10 microM) had no effect. Calpain-mediated cleavage of alpha-fodrin was increased approximately threefold in the transgenic group following I/R compared with WT (P < 0.05); this was significantly attenuated by SKF-96365. The calpain inhibitor PD-150606 (25 microM) attenuated the increase in both alpha-fodrin cleavage and apoptosis in the TPRC3 group. Increased TRPC3 expression also increased sensitivity to Ca2+ overload stress, but it did not affect the response to TNF-alpha-induced apoptosis. These results suggest that CCE mediated via TRPC may play a role in cardiomyocyte apoptosis following I/R due, at least in part, to increased calpain activation.     

Inhibitors of SERCA and mitochondrial Ca-uniporter decrease velocity of calcium waves in rat cardiomyocytes

Spontaneous calcium waves in isolated rat cardiomyocytes were investigated by confocal laser scanning microscopy using the fluorescent Ca(2+)-indicator fluo-4 AM. With increasing calcium overload propagation velocities reinforced. The calcium wavespeed was significantly diminished by drugs which interfere with the calcium uptake of both the sarcoplasmic reticulum (SR) and mitochondria, respectively. Stepwise addition of thapsigargin, a highly specific inhibitor of SERCA, decreased the wavespeed and allowed the determination of flux control coefficients which were found to be increasing from 0.15-0.75 in dependence on calcium overload. Kd was estimated to be between 0.4 and 0.6 nM TG. At 5 mM TG wavespeed was significantly reduced by almost 50%. Spontaneous calcium waves did not occur in bathing solutions with more than 20 nM thapsigargin. Calcium wave velocity was also reduced in the presence of the oxygen-bridged dinuclear ruthenium amine complex RU 360 which specifically blocks the mitochondrial Ca2+ uptake. The observed effects are likely due to a reduction of the ryanodine receptor's open probability. It is suggested that the intracellular Ca2+ signaling depends on both SR lumenal and cytosolic calcium concentration.     

Maximal inhibition of SERCA2 Ca(2+) affinity by phospholamban in transgenic hearts overexpressing a non-phosphorylatable form of phospholamban

Phospholamban is a phosphoprotein in the cardiac sarcoplasmic reticulum (SR) which regulates the apparent Ca(2+) affinity of the SR Ca(2+)-ATPase (SERCA2). To determine the levels of phospholamban which are associated with maximal inhibition of SERCA2, several lines of transgenic mice were generated which expressed increasing levels of a non-phosphorylatable form of phospholamban (S16A,T17A) specifically in the heart. This mutant form of phospholamban was chosen to prevent phosphorylation as a compensatory mechanism in vivo. Quantitative immunoblotting revealed increased phospholamban protein levels of 1.8-, 2.6-, 3.7-, and 4.7-fold in transgenic hearts compared with wild types. There were no changes in the expression levels of SERCA2, calsequestrin, calreticulin, and ryanodine receptor. Assessment of SR Ca(2+) uptake in hearts of transgenic mice indicated increases in the inhibition of the affinity of SERCA2 for Ca(2+) with increased phospholamban expression. Maximal inhibition was obtained at phospholamban expression levels of 2.6-fold or higher. Transgenic hearts with functional saturation in phospholamban:SERCA2 (>/=2.6:1) exhibited increases in beta-myosin heavy chain expression, associated with cardiac hypertrophy. These findings demonstrate that overexpression of a non-phosphorylatable form of phospholamban in transgenic mouse hearts resulted in saturation of the functional phospholamban:SERCA2 ratio at 2.6:1 and suggest that approximately 40% of the SR Ca(2+) pumps are functionally regulated by phospholamban in vivo.     

Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2+)-ATPase.

Chronic excitation, at 2 Hz for 6-7 weeks, of the predominantly fast-twitch canine latissimus dorsi muscle promoted the expression of phospholamban, a protein found in sarcoplasmic reticulum (SR) from slow-twitch and cardiac muscle but not in fast-twitch muscle. At the same time that phospholamban was expressed, there was a switch from the fast-twitch (SERCA1) to the slow-twitch (SERCA2a) Ca(2+)-ATPase isoform. Antibodies against Ca(2+)-ATPase (SERCA2a) and phospholamban were used to assess the relative amounts of the slow-twitch/cardiac isoform of the Ca(2+)-ATPase and phospholamban, which were found to be virtually the same in SR vesicles from the slow-twitch muscle, vastus intermedius; cardiac muscle; and the chronically stimulated fast-twitch muscle, latissimus dorsi. The phospholamban monoclonal antibody 2D12 was added to SR vesicles to evaluate the regulatory effect of phospholamban on calcium uptake. The antibody produced a strong stimulation of calcium uptake into cardiac SR vesicles, by increasing the apparent affinity of the Ca2+ pump for calcium by 2.8-fold. In the SR from the conditioned latissimus dorsi, however, the phospholamban antibody produced only a marginal effect on Ca2+ pump calcium affinity. These different effects of phospholamban on calcium uptake suggest that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch muscles and that phospholamban may have some other function in slow-twitch and chronically stimulated fast-twitch muscle.     

Acute, nongenomic effect of thyroid hormones in preventing calcium overload in newborn rat cardiocytes

In this study, we examined the acute effects of thyroid hormones (TH) T(3) and T(4), leading to improvement of myocardial function through activation of Ca(2+) extrusion mechanisms and, consequently, prevention of intracellular calcium overload. Extracellular calcium elevation from 1.8 to 3.8 mM caused immediate increase in intracellular calcium level ([Ca(2+)](i)) in newborn cardiomyocyte cultures. Administration of 10 or 100 nM T(3) or T(4) rapidly (within 10 sec) decreased [Ca(2+)](i) to its control level. Similar results were obtained when [Ca(2+)](i) was elevated by decreasing extracellular Na(+) concentration, causing backward influx of Ca(2+) through Na(+)/Ca(2+) exchanger, or by administration of caffeine, releasing Ca(2+) from the sarcoplasmic reticulum (SR). Under these conditions, T(3) or T(4) decreased [Ca(2+)](i). T(3) and T(4) also exhibited protective effects during ischemia. T(3) or T(4) presence during hypoxia for 120 min in culture medium restricted the increase of [Ca(2+)](i) and prevented the pathological effects of its overload. An inhibitor of SR Ca(2+)-ATPase (SERCA2a), thapsigargin, increases [Ca(2+)](i) and in its presence neither T(3) nor T(4) had any effect on the [Ca(2+)](i) level. The reduction of [Ca(2+)](i) level by T(3) and T(4) was also blocked in the presence of H-89 (a PKA inhibitor), and by calmodulin inhibitors. The effect of TH on the reduction of [Ca(2+)](i) was prevented by propranolol, indicating that the hormones exert their effect through interaction with adrenergic receptors. These results support our hypothesis that TH prevent calcium overload in newborn rat cardiomyocytes, most likely by a direct, acute, and nongenomic effect on Ca(2+) transport into the SR.     

Exogenous nitric oxide triggers classic ischemic preconditioning by preventing intracellular Ca2+ overload in cardiomyocytes

The involvement of nitric oxide (NO) in the late phase of ischemic preconditioning is well established. However, the role of NO as a trigger or mediator of "classic preconditioning" remains to be determined. The present study was designed to investigate the effects of NO on calcium homeostasis in cultured newborn rat cardiomyocytes in normoxia and hypoxia. We found that treatment with the NO donor, sodium nitroprusside (SNP) induced a sustained elevation of intracellular calcium level ([Ca(2+)](i)) followed by a decrease to control levels. Elevation of extracellular calcium, which generally occurs during ischemia, caused an immediate increase in [Ca(2+)](i) and arrhythmia in cultures of newborn cardiomyocytes. Treatment with SNP decreased [Ca(2+)](i) to control levels and re-established synchronized beating of cardiomyocytes. A decrease in extracellular [Na(+)], which inhibits the Na(+)/Ca(2+) exchanger, did not prevent [Ca(2+)](i) reduction by SNP. In contrast, application of thapsigargin, an inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), increased [Ca(2+)](i), and in its presence, SNP did not reduce [Ca(2+)](i), indicating that Ca(2+) reduction is achieved via activation of SERCA2a. The results obtained suggest that activation of SERCA2a by SNP increases Ca(2+) uptake into the sarcoplasmic reticulum (SR) and prevents cytosolic Ca(2+) overload, which might explain the protective effect of SNP from hypoxic damage.     

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.     

Role of endoplasmic reticulum calcium content in prostate cancer cell growth regulation by IGF and TNFalpha

Variations in calcium concentration within the endoplasmic reticulum ([Ca(2+)](ER)) may play a role in cell growth. This study evaluates the regulation of calcium pools by growth modulators of prostate cancer (PC) cells, the insulin growth factor (IGF), and the tumor necrosis growth factor-alpha (TNFalpha) as well as evaluating the possible role of [Ca(2+)](ER) variations as signals for growth modulation. We show that IGF (5 ng/ml), which increases cell growth, induces an increase in [Ca(2+)](ER) whereas TNFalpha (1 ng/ml) which reduces cell proliferation and induces apoptosis, reduces [Ca(2+)](ER). IGF-induced [Ca(2+)](ER) increase is correlated to an overexpression of the sarcoendoplasmic calcium-ATPase 2B (SERCA2b), whereas TNFalpha-induced [Ca(2+)](ER) decrease is associated to a reduction in SERCA2b expression. Pretreatment with epidermal growth factors (EGF) or IGF does not prevent TNFalpha from affecting the induction of apoptosis, [Ca(2+)](ER) reduction and SERCA2b downregulation. Reduction in [Ca(2+)](ER) induced by thapsigargin (TG) (from 1 pM to 1 microM, 48 h) reduces LNCaP growth in a dose dependent manner and induces apoptosis when cells are treated with 1 microM TG. We also show that a transient TG application (1 pM, 1 nM, 1 microM 15 min) is insufficient to induce a long lasting decrease in [Ca(2+)](ER), since [Ca(2+)](ER) remains identical to the control for 48 h following TG application. These treatments (1 pM and 1 nM, 15 min) do not modify cell growth. However, TG (1 microM, 15 min) induces apoptosis. We thus identify [Ca(2+)](ER) and SERCA2b as a central targets for causing LNCaP PC cell life or death induced by growth modulators. Furthermore our results indicate that calcium pool contents can regulate cell growth.     

Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs.

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.     

Apelin decreases the SR Ca2+ content but enhances the amplitude of [Ca2+]i transient and contractions during twitches in isolated rat cardiac myocytes

Apelin has been reported to have a positive inotropic action in the isolated rat heart. However, the effect of apelin on sarcoplasmic reticulum (SR) Ca2+ content and its influence on intracellular Ca2+ transient during excitation-contraction coupling remains poorly understood. In the present study, we determined the effect of apelin on Ca2+ transient and contractions in isolated rat cardiomyocytes. When compared with control, treatment with apelin caused a 55.7 +/- 13.9% increase in sarcomere fraction shortening and a 43.6 +/- 4.56% increase in amplitude of electrical-stimulated intracellular Ca2+ concentration (E[Ca2+]i) transients (n = 14, P < 0.05). But SR Ca2+ content measured by caffeine-induced [Ca2+]i (C[Ca2+]i) transient was decreased 8.41 +/- 0.92% in response to apelin (n = 14, P < 0.05). Na+/Ca2+ exchanger (NCX) function was increased since half-decay time of C[Ca2+]i was decreased 16.22 +/- 1.36% in response to apelin. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity was also increased by apelin. These responses can be partially or completely blocked by chelerythrine chloride, a PKC inhibitor. In addition, to confirm our data, we used indo-1 as another Ca2+ indicator and rapid cooling as another way to measure SR Ca2+ content, and we observed similar results. So we conclude that apelin has a positive inotropic effect on isolated myocytes, and increased amplitude of E[Ca2+]i is at least partially involved in the mechanism. NCX function and SERCA activity are increased by apelin, and the SR Ca2+ content is decreased by apelin during twitches. PKC played an important role in these signaling mechanisms.     

Possible role of acylphosphatase,Bcl-2 and Fas/Fas-L system in the early changes of cardiac remodeling induced by volume overload

To identify early adaptive processes of cardiac remodeling (CR) in response to volume overload, we investigated the molecular events that may link intracellular Ca(2+) homeostasis alterations and cardiomyocyte apoptosis. In swine heart subjected to aorto-cava shunt for 6, 12, 24, 48 and 96 h sarcoplasmic reticulum (SR) Ca(2+) pump activity was reduced until 48 h (-30%), but a recovery of control values was found at 96 h. The decrease in SR Ca(2+)-ATPase (SERCA2a) expression at 48 h, was more marked (-60%) and not relieved by a subsequent recovery, while phospholamban (PLB) concentration and phosphorylation were unchanged at all the considered times. Conversely, acylphosphatase activity and expression significantly increased from 48 to 96 h (+40%). Bcl-2 expression increased significantly from 6 to 24 h, but at 48 h, returned to control values. At 48 h, microscopic observations showed that overloaded myocardium underwent substantial damage and apoptotic cell death in concomitance with an enhanced Fas/Fas-L expression. At 96 h, apoptosis appeared attenuated, while Fas/Fas-L expression was still higher than control values and cardiomyocyte hypertrophy became to develop. These data suggest that in our experimental model, acylphosphatase could be involved in the recovery of SERCA2a activity, while cardiomyocyte apoptosis might be triggered by a decline in Bcl-2 expression and a concomitant activation of Fas.     

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.     

Effects of Na+/Ca2+ exchange induced by SR Ca2+ release on action potentials and afterdepolarizations in guinea pig ventricular myocytes

In cardiac cells, evoked Ca2+ releases or spontaneous Ca2+ waves activate the inward Na+/Ca2+ exchange current (INaCa), which may modulate membrane excitability and arrhythmogenesis. In this study, we examined changes in membrane potential due to INaCa elicited by sarcoplasmic reticulum (SR) Ca2+ release in guinea pig ventricular myocytes using whole cell current clamp, fluorescence, and confocal microscopy. Inhibition of INaCa by Na+-free, Li+-containing Tyrode solution reversibly abbreviated the action potential duration at 90% repolarization (APD90) by 50% and caused SR Ca2+ overload. APD90 was similarly abbreviated in myocytes exposed to the Na+/Ca2+ exchange inhibitor KB-R7943 (5 microM) or after inhibition of SR Ca2+ release with ryanodine (20 microM). In the absence of extracellular Na+, spontaneous SR Ca2+ releases caused minimal changes in resting membrane potential. After the myocytes were returned to Na+-containing solution, the potentiated intracellular Ca2+ concentration ([Ca2+]i) transients dramatically prolonged APD90 and [Ca2+]i oscillations caused delayed and early afterdepolarizations (DADs and EADs). Laser-flash photolysis of caged Ca2+ mimicked the effects of spontaneous [Ca2+]i oscillations, confirming that APD prolongation, DADs, and EADs could be ascribed to intracellular Ca2+ release. These results suggest that Na+/Ca2+ exchange is a major physiological determinant of APD and that INaCa activation by spontaneous SR Ca2+ release/oscillations, depending on the timing, can account for both DADs and EADs during SR Ca2+ overload.     

Effect of indomethacin on force responses and sarcoplasmic reticulum function in skinned skeletal muscle fibers and cytosolic [Ca2+] in myotubes

In this study, the effects of phospholipase A2 (PLA2) inhibitors on excitation-contraction coupling (ECC) and sarcoplasmic reticulum (SR) function were examined in skinned extensor digitorum longus (EDL) muscle fibers of the rat. The nonspecific PLA2 inhibitor indomethacin (200 microM) significantly increased the peak (approximately 2-fold, P = 0.02) and the width (approximately 6-fold, P = 0.008) of depolarization-induced force responses (DIFRs) elicited in the fibers (n = 4). Exposure of the skinned EDL fibers to indomethacin (200 microM) (n = 7) and another PLA2 inhibitor quinacrine (200 microM) (n = 5) resulted in the return of large DIFRs after use-dependent rundown. However, aristolochic acid (100 microM), an inhibitor of secretory PLA2, failed to return DIFRs after rundown. Indomethacin did not protect against the loss of DIFRs induced by exposure to elevated myofibrillar [Ca2+]. Indomethacin (200 microM) produced a small but significant increase in the Ca2+ sensitivity of the contractile apparatus of skinned EDL fibers and the maximum force production. Indomethacin (200 microM) also had significant effects on SR function, increasing SR Ca2+ loading in the skinned fibers (117.2 +/- 3.0% of controls, P = 0.0008, n = 8) and inducing intracellular Ca2+ release in isolated intact flexor digitorum brevis (FDB) fibers (n = 7) and C2C12 myotubes (n = 6). These data suggest that intracellular PLA2 may be an important modulator of ECC in skeletal muscle.     

Effects of N-n-butyl haloperidol iodide on the rat myocardial sarcoplasmic reticulum Ca(2+)-ATPase during ischemia/reperfusion

We have previously shown that N-n-butyl haloperidol iodide (F(2)), a newly synthesized compound, reduces ischemia/reperfusion (I/R) injury by preventing intracellular Ca(2+) overload through inhibiting L-type calcium channels and outward current of Na(+)/Ca(2+) exchanger. This study was to investigate the effects of F(2) on activity and protein expression of the rat myocardial sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) during I/R to discover other molecular mechanisms by which F(2) maintains intracellular Ca(2+) homeostasis. In an in vivo rat model of myocardial I/R achieved by occluding coronary artery for 30-60min followed by 0-120min reperfusion, treatment with F(2) (0.25, 0.5, 1, 2 and 4mg/kg, respectively) dose-dependently inhibited the I/R-induced decrease in SERCA activity. However, neither different durations of I/R nor different doses of F(2) altered the expression levels of myocardial SERCA2a protein. These results indicate that F(2) exerts cardioprotective effects against I/R injury by inhibiting I/R-mediated decrease in SERCA activity by a mechanism independent of SERCA2a protein levels modulation.     

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.