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.     

Targeted gene transfer increases contractility and decreases oxygen cost of contractility in normal rat hearts

The aim of this study was to examine how global cardiac gene transfer of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) can influence left ventricular (LV) mechanical and energetic function, especially in terms of O(2) cost of LV contractility, in normal rats. Normal rats were randomized to receive an adenovirus carrying the SERCA2a (SERCA) or beta-galactosidase (beta-Gal) gene or saline by a catheter-based technique. LV mechanical and energetic function was measured in cross-circulated heart preparations 2-3 days after the infection. The end-systolic pressure-volume relation was shifted upward, end-systolic pressure at 0.1 ml of intraballoon water volume was higher, and equivalent maximal elastance, i.e., enhanced LV contractility, was higher in the SERCA group than in the normal, beta-Gal, and saline groups. Moreover, the LV relaxation rate was faster in the SERCA group. There was no significant difference in myocardial O(2) consumption per beat-systolic pressure-volume area relation among the groups. Finally, O(2) cost of LV contractility was decreased to subnormal levels in the SERCA group but remained unchanged in the beta-Gal and saline groups. This lowered O(2) cost of LV contractility in SERCA hearts indicates energy saving in Ca(2+) handling during excitation-contraction coupling. Thus overexpression of SERCA2a transformed the normal energy utilization to a more efficient state in Ca(2+) handling and superinduced the supranormal contraction/relaxation due to enhanced Ca(2+) handling.     

Mechanism of enhanced cardiac function in mice with hypertrophy induced by overexpressed Akt

Transgenic mice with cardiac-specific overexpression of active Akt (TG) not only exhibit hypertrophy but also show enhanced left ventricular (LV) function. In 3-4-month-old TG, heart/body weight was increased by 60% and LV ejection fraction was elevated (84 +/- 2%, p < 0.01) compared with nontransgenic littermates (wild type (WT)) (73 +/- 1%). An increase in isolated ventricular myocyte contractile function (% contraction) in TG compared with WT (6.1 +/- 0.2 versus 3.5 +/- 0.2%, p < 0.01) was associated with increased Fura-2 Ca2+ transients (396 +/- 50 versus 250 +/- 24 nmol/liter, p < 0.05). The rate of relaxation (+dL/dt) was also enhanced in TG (214 +/- 15 versus 98 +/- 18 microm/s, p < 0.01). L-type Ca2+ current (ICa) density was increased in TG compared with WT (-9.0 +/- 0.3 versus 7.2 +/- 0.3 pA/pF, p < 0.01). Sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) protein levels were increased (p < 0.05) by 6.6-fold in TG, which could be recapitulated in vitro by adenovirus-mediated overexpression of Akt in cultured adult ventricular myocytes. Conversely, inhibiting SERCA with either ryanodine or thapsigargin affected myocyte contraction and relaxation and Ca2+ channel kinetics more in TG than in WT. Thus, myocytes from mice with overexpressed Akt demonstrated enhanced contractility and relaxation, Fura-2 Ca2+ transients, and Ca2+ channel currents. Furthermore, increased protein expression of SERCA2a plays an important role in mediating enhanced LV function by Akt. Up-regulation of SERCA2a expression and enhanced LV myocyte contraction and relaxation in Akt-induced hypertrophy is opposite to the down-regulation of SERCA2a and reduced contractile function observed in many other forms of LV hypertrophy.     

Altered dose response to beta-agonists in SERCA1a-expressing hearts ex vivo and in vivo

In this study we evaluated the contractile characteristics of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)1a-expressing hearts ex vivo and in vivo and in particular their response to beta-adrenergic stimulation. Analysis of isolated, work-performing hearts revealed that transgenic (TG) hearts develop much higher maximal rates of contraction and relaxation than wild-type (WT) hearts. Addition of isoproterenol only moderately increased the maximal rate of relaxation (+20%) but did not increase contractility or decrease relaxation time in TG hearts. Perfusion with varied buffer Ca(2+) concentrations indicated an altered dose response to Ca(2+). In vivo TG hearts displayed fairly higher maximal rates of contraction (+ 25%) but unchanged relaxation parameters and a blunted but significant response to dobutamine. Our study also shows that the phospholamban (PLB) level was decreased (-40%) and its phosphorylation status modified in TG hearts. This study clearly demonstrates that increases in SERCA protein level alter the beta-adrenergic response and affect the phosphorylation of PLB. Interestingly, the overall cardiac function in the live animal is only slightly enhanced, suggesting that (neuro)hormonal regulations may play an important role in controlling in vivo heart function.     

Cardiac dysfunction in terms of left ventricular mechanical work and energetics in hypothyroid rats

We hypothesized that cardiac dysfunction in hypothyroidism is mainly caused by the impairment of Ca(2+) handling in excitation-contraction coupling. To prove this hypothesis, we investigated left ventricular (LV) mechanical work and energetics without interference of preload and afterload in an excised, blood-perfused whole heart preparation from hypothyroid rats. We found that LV inotropism and lusitropism were significantly depressed, and these depressions were causally related to decreased myocardial oxygen consumption for Ca(2+) handling and for basal metabolism. The oxygen costs of LV contractility for Ca(2+) and for dobutamine in the hypothyroid rats did not differ from those in age-matched normal rats. The expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) significantly decreased and that of phospholamban significantly increased. The present results revealed that changes in LV energetics associated with decreased mechanical work in hypothyroid rats are mainly caused by the impairment of Ca(2+) uptake via SERCA2. We conclude that the impairment of Ca(2+) uptake plays an important role in the pathogenesis of cardiac dysfunction in hypothyroidism.     

Sarcolipin overexpression in rat slow twitch muscle inhibits sarcoplasmic reticulum Ca2+ uptake and impairs contractile function

Sarcolipin (SLN) is an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) in vitro, but its function in vivo has not been defined. NF-SLN cDNA (SLN tagged N-terminally with a FLAG epitope) was introduced into rat soleus muscle in one hindlimb by plasmid injection and electrotransfer. Western blotting showed expression and co-immunoprecipitation showed physical interaction between NF-SLN and SERCA2a. Contractile properties and SERCA2a function were assessed and compared with vector-injected contralateral soleus muscles. NF-SLN reduced both peak twitch force (P(t)) (123.9 +/- 12.5 versus 69.8 +/- 8.9 millinewtons) and tetanic force (P(o)) (562.3 +/- 51.0 versus 300.7 +/- 56.9 millinewtons) and reduced both twitch and tetanic rates of contraction (+dF/dt) and relaxation (-dF/dt) significantly. Repetitive stimulation (750-ms trains at 50 Hz once every 2 s for 3 min) showed that NF-SLN increased susceptibility to fatigue. These changes in contractile function were observed in the absence of endogenous phospholamban, and NF-SLN had no effect on either SERCA2a or SERCA1a expression levels. NF-SLN also decreased maximal Ca(2+) transport activity at pCa 5 by 31% with no significant change in apparent Ca(2+) affinity (6.36 +/- 0.07 versus 6.39 +/- 0.08 pCa units). These results show that NF-SLN expression impairs muscle contractile function by inhibiting SERCA function and diminishing sarcoplasmic reticulum Ca(2+) stores.     

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.     

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.     

Identification of ryanodine receptor isoforms in prostate DU-145, LNCaP, and PWR-1E cells

We previously reported that left ventricular (LV) slices from isoproterenol (ISO)-induced hypertrophied rat hearts showed an increase of energy expenditure due to remodeling of Ca(2+) handling in excitation-contraction coupling, i.e., suppressed SERCA2a activity and enhanced Na(+)/Ca(2+)exchanger-1 (NCX-1) activity. Na(+)/H(+) exchanger-1 (NHE-1) inhibitor (NHEI) has been demonstrated to exert beneficial effects in the development of cardiac remodeling. We hypothesized that a novel NHE-1 selective inhibitor, BIIB723 prevents remodeling of Ca(2+) handling in LV slices of ISO-induced hypertrophied rat hearts mediated by inhibiting NCX-1 activity. The significant shortening in duration of multi-cellular Ca(2+) transient in ISO group was normalized in ISO+BIIB723 group. The significant increase in amplitude of multi-cellular Ca(2+) waves (CaW) generated at high [Ca(2+)](o) of LV slices in ISO group was also normalized in ISO+BIIB723 group. However, the enhanced NCX-1 activity was not antagonized by BIIB723. We recently reported that ISO-induced down-regulation of a Ca(2+) handling protein, SERCA2a, was normalized by BIIB723. Therefore, it seems likely that BIIB723 normalized shortened multi-cellular Ca(2+) transient duration and increased CaW amplitude in LV slices mediated via normalization of SERCA2a activity. Furthermore, the results presented here suggest the multi-cellular Ca(2+) transient duration and CaW amplitude in LV slices might be better indices reflecting SERCA2a activity than SERCA2a protein expression level.     

Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes.

Previous studies in adult myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated abnormal contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) homeostasis and decreased sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) expression and activity, but sarcoplasmic reticulum Ca(2+) leak was unchanged. In the present study, we investigated whether SERCA2 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. Compared with sham-operated hearts, 3-wk MI hearts exhibited significantly higher left ventricular end-diastolic and end-systolic volumes but lower fractional shortening and ejection fraction, as measured by M-mode echocardiography. Seventy-two hours after adenovirus-mediated gene transfer, SERCA2 overexpression in 3-wk MI myocytes did not affect Na(+)-Ca(2+) exchanger expression but restored the depressed SERCA2 levels toward those measured in sham myocytes. In addition, the reduced sarcoplasmic reticulum Ca(2+) uptake in MI myocytes was improved to normal levels by SERCA2 overexpression. At extracellular Ca(2+) concentration of 5 mM, the subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored to normal by SERCA2 overexpression. However, at 0.6 mM extracellular Ca(2+) concentration, the supernormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were exacerbated by SERCA2 overexpression. We conclude that SERCA2 overexpression was only partially effective in ameliorating contraction and [Ca(2+)](i) transient abnormalities in our rat model of ischemic cardiomyopathy. We suggest that other Ca(2+) transport pathways, e.g., Na(+)-Ca(2+) exchanger, may also play an important role in contractile and [Ca(2+)](i) homeostatic abnormalities in MI myocytes.     

Temperature dependence of cardiac sarcoplasmic reticulum Ca²⁺-ATPase from rainbow trout Oncorhynchus mykiss

In this work, the temperature dependence of the sarco-endoplasmic reticulum Ca(2+) -ATPase (SERCA2) activity from rainbow trout Oncorhynchus mykiss cardiac ventricles was measured and compared with the mammalian SERCA2 isoform. The rate of ATP-dependent Ca(2+) transport catalysed by O. mykiss vesicles was totally abolished by thapsigargin and the Ca(2+) ionophore A(23187) . At warm temperatures (25 and 30° C), the SERCA2 from O. mykiss ventricles displayed the same rate of Ca(2+) uptake. At 35° C, the activity of the O. mykiss enzyme decreased after 20 min of reaction time. The rate of Ca(2+) uptake catalysed by the mammalian SERCA2 was temperature dependent exhibiting its maximal activity at 35° C. In contrast to the rate of Ca(2+) uptake, the rate of ATP hydrolysis catalysed by O. mykiss SERCA2 was not significantly different at 25 and 35° C, but the rate of ATP hydrolysis catalysed by the rat Rattus norvegicus SERCA2 isoform at 35° C was two-fold higher than at 25° C. At low temperatures (5 to 20° C), the rate of Ca(2+) uptake from O. mykiss SR was less temperature dependent than the R. norvegicus isoform, being able to sustain a high activity even at 5° C. The mean ±s.e. Q(10) values calculated from 25 to 35° C for ATP hydrolysis were 1·112 ± 0·026 (n = 3) and 2·759 ± 0·240 (n = 5) for O. mykiss and R. norvegicus, respectively. Taken together, the results show that the O. mykiss SERCA2 was not temperature dependent over the 10 to 25° C temperature interval commonly experienced by the animal in vivo. The Q(10) value of SERCA2 was significantly lower in O. mykiss than R. norvegicus which may be key for cardiac function over the wide environmental temperatures experienced in this eurythermal fish.     

Effect of milrinone on left ventricular relaxation and Ca(2+) uptake function of cardiac sarcoplasmic reticulum

Milrinone, a phosphodiesterase 3 (PDE3) inhibitor, is known to enhance left ventricular (LV) contractility by an inhibition of the breakdown of cAMP through the mechanism inhibiting PDE3. However, it is unclear whether milrinone also exerts positive lusitropy, like dobutamine. Here, we assessed the effects of milrinone on in vivo LV relaxation, as well as the Ca(2+)-ATPase activity and the Ca(2+) uptake function of the cardiac sarcoplasmic reticulum (SR), compared with the effect of dobutamine on those functions. After dobutamine (3 microg x kg(-1) x min(-1)) was administered, the peak value of the first derivative of LV pressure (+dP/dt) increased by 46%, whereas the time constant (tau) of LV pressure decay decreased by 6.9%, respectively. After milrinone (10 microg/kg) was administered, the peak +dP/dt increased to a similar extent as dobutamine (46%), whereas tau decreased much more than dobutamine (19.9%; P < 0.05). In LV crude homogenate, the thapsigargin-sensitive, Ca(2+)-ATPase activity-cAMP relationships was significantly less increased by milrinone compared with dobutamine (P < 0.05), indicating the higher sensitivity of the SR Ca(2+)-ATPase activity on cAMP by milrinone than by dobutamine. In the SR vesicles purified from LV muscles, the addition of cAMP increased the SR Ca(2+) uptake in a dose-dependent fashion, and the PDE3 inhibitors (milrinone and cGMP) significantly augmented this response (P < 0.05). Hence, milrinone substantially improved LV relaxation in association with an acceleration of the SR Ca(2+)-ATPase activity and the SR Ca(2+) uptake. This acceleration might be due to an inhibition of the membrane-bound PDE3 in the SR, leading to a local elevation of cAMP.     

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.     

Modeling Ca2+ dynamics of mouse cardiac cells points to a critical role of SERCA's affinity for Ca2+

The SERCA2a isoform of the sarco/endoplasmic reticulum Ca(2+) pumps is specifically expressed in the heart, whereas SERCA2b is the ubiquitously expressed variant. It has been shown previously that replacement of SERCA2a by SERCA2b in mice (SERCA2(b/b) mice) results in only a moderate functional impairment, whereas SERCA activity is decreased by a 40% lower SERCA protein expression and by increased inhibition by phospholamban. To find out whether the documented kinetic differences in SERCA2b relative to SERCA2a (i.e., a twofold higher apparent Ca(2+) affinity, but twofold lower maximal turnover rate) can explain these compensatory changes, we simulated Ca(2+) dynamics in mouse ventricular myocytes. The model shows that the relative Ca(2+) transport capacity of SERCA2a and SERCA2b depends on the SERCA concentration. The simulations point to a dominant effect of SERCA2b's higher Ca(2+) affinity over its lower maximal turnover rate. The results suggest that increased systolic and decreased diastolic Ca(2+) levels in unstimulated conditions could contribute to the downregulation of SERCA in SERCA2(b/b) mice. In stress conditions, Ca(2+) handling is less efficient by SERCA2b than by SERCA2a, which might contribute to the observed hypertrophy in SERCA2(b/b) mice. Altogether, SERCA2a might be a better compromise between performance in basal conditions and performance during β-adrenergic stress.     

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.     

The onset of left ventricular diastolic dysfunction in SHR rats is not related to hypertrophy or hypertension

Left ventricular (LV) diastolic dysfunction, particularly relaxation abnormalities, are known to be associated with the development of LV hypertrophy (LVH). Preliminary human and animal studies suggested that early LV diastolic dysfunction may be revealed independently of LVH. However, whether LV diastolic dysfunction is compromised before the onset of hypertension and LVH remains unknown. We therefore evaluated LV diastolic function in spontaneously hypertensive rats (SHR) at different ages and tested whether LV diastolic dysfunction is associated with abnormal intracellular calcium homeostasis. LV systolic and diastolic functions were evaluated by invasive and echocardiographic methods in 3-week-old (without hypertension) and 5-week-old (with hypertension) SHR and Wistar-Kyoto control rats. Basal intracytoplasmic calcium and sarcoplasmic reticulum (SR) Ca(2+) contents were measured in cardiomyocytes using fura-2 AM. Sarco(endo)plasmic Ca(2+)-ATPase isoform 2a (SERCA 2a) and phospholamban (PLB) expressions were quantified by Western blot and quantitative RT-PCR techniques. LV relaxation dysfunction was observed in 3-week-old SHR rats before onset of hypertension and LVH. An increase in basal intracytoplasmic Ca(2+) and a decrease in SR Ca(2+) release were demonstrated in SHR. Decreased expression of SERCA 2a and Ser16 PLB (p16-PLB) protein levels was also observed in SHR rats, whereas mRNA expression was not decreased. For the first time, we have shown that LV myocardial dysfunction precedes hypertension in 3-week-old SHR rats. This LV myocardial dysfunction was associated with high diastolic [Ca(2+)](i) possibly due to decreased SERCA 2a and p16-PLB protein levels. Diastolic dysfunction may be a potential predictive marker of arterial hypertension in genetic hypertension syndromes.     

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.     

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.     

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.     

Inhibition of human SERCA3 by PL/IM430. Molecular analysis of the interaction

The monoclonal antibody PL/IM430 has previously been reported to uncouple Ca(2+) transport from ATP hydrolysis in platelet membranes (Hack, N., Wilkinson, J. M., and Crawford, N. (1988) Biochem. J. 250, 355-361). More recently, we have demonstrated that this antibody is specific for human SERCA3 (Poch, E., Leach, S., Snape, S., Cacic, T., MacLennan, D. H., and Lytton, J. (1998) Am. J. Physiol. 275, C1449-C1458). In this paper, we have extended the analysis of the PL/IM430-SERCA3 interaction. Using HEK293 cells to express human SERCA3a, we were able to measure both ATP-mediated, oxalate-dependent (45)Ca(2+) uptake and Ca(2+)-dependent ATP hydrolysis activities due exclusively to SERCA3. Treatment with PL/IM430 inhibited both activities almost identically, with a maximal inhibition of 81 and 73% and a half-maximal concentration of 8.3 and 5.9 microg/ml, for Ca(2+) uptake and ATP hydrolysis, respectively. We conclude that PL/IM430 does inhibit SERCA3 activity but does not uncouple Ca(2+) transport from ATP hydrolysis. Using a combination of partial proteolysis, GST fusion protein expression, and mutation of residues that differ between rat and human SERCA3, we have identified human SERCA3 amino acids Pro(8) and Glu(192) as essential to forming the PL/IM430 epitope. PL/IM430 thus recognizes a linearly noncontiguous set of amino acids within the actuator domain of human SERCA3. We propose that PL/IM430 inhibits SERCA3 activity by sterically preventing movement of the actuator domain into a catalytically critical position in the E2 conformation of the enzyme.