Altered SR protein expression associated with contractile dysfunction in diabetic rat hearts

The goal of this study was to examine whether alteration of sarcoplasmic reticulum (SR) protein levels is associated with early-onset diastolic and late-onset systolic dysfunction in streptozotocin (STZ)-induced diabetic rat hearts. Four-week diabetic rat hearts exhibited slow relaxation, whereas 6-wk diabetic rat hearts exhibited slow and depressed contraction. Total phospholamban level was increased, and phosphorylated level was decreased in 4- and 6-wk diabetic rat hearts. Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) protein level was unchanged in 4-wk but decreased in 6-wk diabetic rat hearts. Only the apparent affinity of SR Ca2+ uptake for Ca2+ was decreased in 4-wk diabetic rat hearts, but the apparent affinity and the maximum rate was decreased in 6-wk diabetic rat hearts. Insulin treatment of the diabetic rats normalized SR protein expression and function. It was concluded that an increase in nonphosphorylated phospholamban and a decrease in the apparent affinity of SR Ca2+ pump for Ca2+ are associated with early-onset diastolic dysfunction and decreases in SERCA2 protein level and apparent affinity and maximum velocity of SR Ca2+ pump are associated with late-onset systolic dysfunction in diabetic rats.     

Diabetic alterations in cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban protein expression.

Diabetic cardiomyopathy has been suggested to be caused by abnormal intracellular Ca2+ homeostasis in the myocardium, which is partly due to a defect in calcium transport by the cardiac sarcoplasmic reticulum (SR). In the present study, the underlying mechanism for this functional derangement was investigated with respect to SR Ca2+-ATPase and phospholamban (the inhibitor of SR Ca2+-ATPase). The maximal Ca2+ uptake and the affinity of Ca2+-ATPase for Ca2+ were decreased, and exogenous phosphorylation level of phospholamban was higher in streptozotocin-induced diabetic rat SR. Levels of both mRNA and protein of phospholamban were significantly increased in the diabetic hearts, whereas those of SR Ca2+-ATPase were significantly decreased. Consequently, the relative phospholamban/Ca2+-ATPase ratio was 1.88 in the diabetic hearts, and these changes were correlated with changes in the rates of SR Ca2+ uptake. However, phosphatase pretreatment of phospholamban for dephosphorylation of the sites phosphorylated in vivo did not change the levels of subsequent phospholamban phosphorylation in either control or diabetic rat hearts. The above data indicated that the increased phospholamban phosphorylation was not due to autonomic dysfunction but possibly due to increased phospholamban expression. These findings suggest that reduction of the SR Ca2+-ATPase level would contribute to decreased rates of SR Ca2+ uptake and that this function is further impaired by the enhanced inhibition by phospholamban due to its increased expression in the diabetic heart.     

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.     

Analysis of sarcoplasmic reticulum Ca2+ transport and Ca2+ ATPase enzymatic properties using mouse cardiac tissue homogenates

Recent studies have focused on developing transgenic mouse models to explore the physiological roles of sarcoplasmic reticulum (SR) calcium handling proteins. The goal of this study was to develop methodology to measure SR Ca2+ transport function and enzymatic properties of SR Ca2+ ATPase (SERCA) in individual mouse hearts. We describe here the procedures to specifically measure SR Ca2+ uptake, the formation and decomposition of SERCA phosphoenzyme intermediate (E-P) in mouse cardiac homogenates. The specificity of SERCA enzymatic activity in cardiac homogenates was established by (a) the selective inhibition of SERCA enzyme by inhibitor-thapsigargin, and (b) comparison of the kinetic parameters of SERCA activity between homogenates and isolated microsomes. Here we show that the apparent affinity of SERCA for Ca2+ and ATP, the time to reach steady-state levels of E-P, and the rate of E-P decomposition (turnover rate of SERCA enzyme) are similar in homogenates and microsomes. These studies demonstrate that SERCA Ca2+ transport and enzymatic properties can be accurately measured in mouse cardiac tissue homogenates. Additionally, we show that frozen cardiac homogenates can be used without significant loss of enzymatic activity. In conclusion, we have developed and established the methods to employ tissue homogenates to study SR Ca2+ transport function in individual mouse hearts.     

Estrogen supplement prevents the calcium hypersensitivity of cardiac myofilaments in ovariectomized rats

Our previous biochemical and mechanical studies have demonstrated an increase in Ca2+ sensitivity of cardiac myofilaments in ovariectomized rats. To test whether the body weight gain associated with ovariectomy contributed some effects to the changes in myofibrillar functions, the relations of pCa (-log Ca2+ molar concentration) to actomyosin adenosine triphosphatase (ATPase) activity of isolated myofibrillar preparations from 10-week pair-fed ovariectomized rats were compared with those from sham-operated controls. Despite similar body weights, the maximum myofibrillar ATPase activity was significantly lower in pair-fed ovariectomized rats as compared to that of sham-operated controls. In addition, the pCa-actomyosin ATPase relationship of pair-fed ovariectomized hearts still demonstrated a significant leftward shift in pCa50 (-log half-maximally Ca2+ activation) from that of sham-operated controls. To find out which hormone was responsible for the observed increase in myofibrillar Ca2+ sensitivity, different sex hormone supplemental regimens were administered to ovariectomized rats. Subcutaneous injection of estrogen (5 microg/rat) or estrogen plus progesterone (1 mg/rat) three times a week could effectively prevent the changes in body weight, heart weight, and uterine weight of the ovariectomized animals. Moreover, supplements of either estrogen or progesterone could prevent a decrease in maximum ATPase activity. In contrast, only the estrogen replacement could abolish the Ca2+ hypersensitivity of the myofilaments in these ovariectomized rats. These results suggest differential cardio-regulatory effects of ovarian sex hormones on the Ca2+ activation of the myofilaments.     

Temperature dependence of the Ca2+-ATPase (SERCA2) in the ventricles of tuna and mackerel

Recent physiological studies on the cardiovascular performance of tunas suggest that the elevated heart rates of these fish may rely on increased use of intracellular sarcoplasmic reticulum (SR) Ca2+ stores. In this study, we compare the cellular cardiac performance in endothermic tunas (bluefin, albacore, yellowfin) and their ectothermic sister taxa (mackerel) in response to acute temperature change. The cardiac sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) plays a major role during cardiac excitation-contraction (E-C) coupling, transporting Ca2+ from the cytosol into the lumen of the SR and thus promoting the relaxation of the muscle. Measurements of oxalate-supported Ca2+ uptake in SR-enriched ventricular vesicles indicated that tunas were capable of sustaining a rate of Ca2+ uptake that was significantly higher than the mackerel. Among tunas, the cold-tolerant bluefin had the highest rates of SR Ca2+ uptake and ATPase activity. The differences among Ca2+ uptake and ATP hydrolysis rates do not seem to result from intrinsic differences between the SERCA2 present in the different tunas, as shown by their similar temperature sensitivities and similar values for activation energy. Western blots reveal that increased SERCA2 protein content is associated with the higher Ca2+ uptake and ATPase activities seen in bluefin ventricles compared with albacore, yellowfin, and mackerel. We hypothesize that a key step in the evolution of high heart rate and high metabolic rate in tunas is increased activity of the SERCA2 enzyme. We also suggest that high levels of SERCA2 in bluefin tuna hearts may be important for retaining cardiac function at cold temperatures.     

Increased inhibition of SERCA2 by phospholamban in the type I diabetic heart

The sarcoplasmic reticulum (SR) plays a critical role in mediating cardiac contractility and its function is abnormal in the diabetic heart. However, the mechanisms underlying SR dysfunction in the diabetic heart are not clear. Because protein phosphorylation regulates SR function, this study examined the phosphorylation state of phospholamban, a key SR protein that regulates SR calcium (Ca2+) uptake in the heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (STZ; 65 mg kg(-1) i.v.), and the animals were humanely killed after 6 weeks and cardiac SR function was examined. Depressed cardiac performance was associated with reduced SR Ca2+-uptake activity in diabetic animals. The reduction in SR Ca2+-uptake was consistent with a significant decrease in the level of SR Ca2+-pump ATPase (SERCA2a) protein. The level of phospholamban (PLB) protein was also decreased, however, the ratio of PLB to SERCA2a was increased in the diabetic heart. Depressed SR Ca2+-uptake was also due to a reduction in the phosphorylation of PLB by the Ca2+-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA). Although the activities of the SR-associated Ca2+-calmodulin-dependent protein kinase (CaMK), cAMP-dependent protein kinase (PKA) were increased in the diabetic heart, depressed phosphorylation of PLB could partly be attributed to an increase in the SR-associated protein phosphatase activities. These results suggest that there is increased inhibition of SERCA2a by PLB and this appears to be a major defect underlying SR dysfunction in the diabetic heart.     

Targeted inhibition of sarcoplasmic reticulum CaMKII activity results in alterations of Ca2+ homeostasis and cardiac contractility

Transgenic (TG) mice expressing a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide targeted to the cardiac myocyte longitudinal sarcoplasmic reticulum (LSR) display reduced phospholamban phosphorylation at Thr17 and develop dilated myopathy when stressed by gestation and parturition (Ji Y, Li B, Reed TD, Lorenz JN, Kaetzel MA, and Dedman JR. J Biol Chem 278: 25063-25071, 2003). In the present study, these animals (TG) are evaluated for the effect of inhibition of sarcoplasmic reticulum (SR) CaMKII activity on the contractile characteristics and Ca2+ cycling of myocytes. Analysis of isolated work-performing hearts demonstrated moderate decreases in the maximal rates of contraction and relaxation (+/-dP/dt) in TG mice. The response of the TG hearts to increases in load is reduced. The TG hearts respond to isoproterenol (Iso) in a dose-dependent manner; the contractile properties were reduced in parallel to wild-type hearts. Assessment of isolated cardiomyocytes from TG mice revealed 40-47% decrease in the maximal rates of myocyte shortening and relengthening under both basal and Iso-stimulated conditions. Although twitch Ca2+ transient amplitudes were not significantly altered, the rate of twitch intracellular Ca2+ concentration decline was reduced by approximately 47% in TG myocytes, indicating decreased SR Ca2+ uptake function. Caffeine-induced Ca2+ transients indicated unaltered SR Ca2+ content and Na+/Ca2+ exchange function. Phosphorylation assays revealed an approximately 30% decrease in the phosphorylation of ryanodine receptor Ser2809. Iso stimulation increased the phosphorylation of both phospholamban Ser16 and the ryanodine receptor Ser2809 but not phospholamban Thr17 in TG mice. This study demonstrates that inhibition of SR CaMKII activity at the LSR results in alterations in cardiac contractility and Ca2+ handling in TG hearts.     

The effects of calcium, temperature and phospholamban phosphorylation on the dynamics of the calcium-stimulated ATPase of canine cardiac sarcoplasmic reticulum

Highly purified sarcoplasmic reticulum (SR) has been prepared from dog hearts and has been incubated with the triplet probe erythrosinyl isothiocyanate to specifically label the Ca2+-stimulated ATPase (Ca2+-ATPase) of the SR. The rotational mobility of the Ca2+-ATPase has been studied in this erythrosin-labelled SR using time-resolved phosphorescence polarization. Qualitatively, the mobility of the cardiac Ca2+-ATPase resembles that of skeletal muscle SR Ca2+-ATPase. Addition of Ca2+ to SR affects the mobility of the Ca2+-ATPase in a way consistent with a segment of the ATPase altering its orientation relative to the plane of the membrane. Phosphorylation of phospholamban in cardiac SR by the purified catalytic subunit of cAMP-dependent protein kinase, which is known to increase the activity of the Ca2+-ATPase by deinhibition, also alters measured anisotropy. The changes observed are not compatible with dissociation of the Ca2+-ATPase from phospholamban after the latter is phosphorylated. The data are more consistent with phospholamban associating with the Ca2+-ATPase following phosphorylation, or more complex models in which only the hydrophilic domain of phospholamban binds with and dissociates from the Ca2+-ATPase.     

Phosphorylation of phospholamban in intact myocardium. Role of Ca2+-calmodulin-dependent mechanisms

Phospholamban, a putative regulator of cardiac sarcoplasmic reticulum Ca2+ transport, has been shown to be phosphorylated in vitro by cAMP-dependent protein kinase and an intrinsic Ca2+-calmodulin-dependent protein kinase activity. This study was conducted to determine if Ca2+-calmodulin-dependent phosphorylation of phospholamban occurs in response to physiologic increases in intracellular Ca2+ in intact myocardium. Isolated guinea pig and rat ventricles were perfused with 32Pi after which membrane vesicles were isolated from individual hearts by differential centrifugation. Administration of isoproterenol (10 nM) to perfused hearts stimulated 32P incorporation into phospholamban, Ca2+-ATPase activity, and Ca2+ uptake of sarcoplasmic reticulum isolated from these hearts. These biochemical changes were associated with increases in contractility and shortening of the t 1/2 of relaxation. Elevated extracellular Ca2+ produced comparable increases in contractility but failed to stimulate phospholamban phosphorylation or Ca2+ transport and did not alter the t 1/2 of relaxation. Inhibition of trans-sarcolemmal Ca2+ influx by perfusing the ventricles with reduced extracellular Ca2+ (50 microM) attenuated the increases in 32P incorporation produced by 10 nM isoproterenol. Trifluoperazine (10 microM) also attenuated isoproterenol-induced increases in 32P incorporation into phospholamban. In both cases, Ca2+ transport was reduced to a degree comparable to the reduction in phospholamban phosphorylation. These results suggest that direct physiologic increases in intracellular Ca2+ concentration do not stimulate phospholamban phosphorylation in intact functioning myocardium. Ca2+-calmodulin-dependent phosphorylation of phospholamban may occur in response to agents which stimulate cAMP-dependent mechanisms in intact myocardium.     

Intermolecular conformational coupling and free energy exchange enhance the catalytic efficiency of cardiac muscle SERCA2a following the relief of phospholamban inhibition

Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by beta1-agonists involves cAMP- and PKA-dependent phosphorylation of phospholamban (PLB), which relieves the inhibitory effects of PLB on SERCA2a. To investigate the mechanism of SERCA2a activation, we compared the kinetic properties of SERCA2a expressed with (+) and without (-) PLB in High Five insect cell microsomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle SR. Both native SERCA1 and expressed SERCA2a without PLB exhibited high-affinity (10-50 microM) activation of pre-steady-state catalytic site dephosphorylation by ATP, steady-state accumulation of the ADP-sensitive phosphoenzyme (E1P), and a rapid phase of EGTA-induced phosphoenzyme (E2P) hydrolysis. In contrast, SERCA2a in native cardiac SR vesicles and expressed SERCA2a with PLB lacked the high-affinity activation by ATP and the rapid phase of E2P hydrolysis, and exhibited low steady-state levels of E1P. The results indicate that the kinetic differences in Ca2+ transport between skeletal and cardiac SR are due to the presence of phospholamban in cardiac SR, and not due to isoform-dependent differences between SERCA1 and SERCA2a. Therefore, the results are discussed in terms of a model in which PLB interferes with SERCA2a oligomeric interactions, which are important for the mechanism of Ca2+ transport in skeletal muscle SERCA1 [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 43, 4400-4416]. We propose that intermolecular coupling of SERCA2a molecules during catalytic cycling is obligatory for the changes in Ca2+ transport activity that accompany the relief of PLB inhibition of the cardiac SR Ca2+-ATPase.     

Ca2+-overload inhibits the cardiac SR Ca2+-calmodulin protein kinase activity

There is increasing evidence to suggest that Ca2+-calmodulin dependent protein kinase (CaMK) regulates the sarcoplasmic reticulum (SR) function and thus plays an important role in modulating the cardiac performance. Because intracellular Ca2+-overload is an important factor underlying cardiac dysfunction in a heart disease, its effect on SR CaMK was examined in the isolated rat heart preparations. Ca2+-depletion for 5 min followed by Ca2+-repletion for 30 min, which is known to produce intracellular Ca2+-overload, was observed to attenuate cardiac function as well as SR Ca2+-uptake and Ca2+-release activities. Attenuated SR function in the heart was associated with reduced CaMK phosphorylation of the SR Ca2+-cycling proteins such as Ca2+-release channel, Ca2+-pump ATPase, and phospholamban, decreased CaMK activity, and depressed levels of SR Ca2+-cycling proteins. These results indicate that alterations in cardiac performance and SR function following the occurrence of intracellular Ca2+-overload may partly be due to changes in the SR CaMK activity.     

New perspectives on the role of SERCA2's Ca2+ affinity in cardiac function

Cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (SR) Ca2+ transport ATPase (SERCA2a). The SR Ca2+ -uptake activity not only determines the speed of Ca(2+) removal during relaxation, but also the SR Ca2+ content and therefore the amount of Ca2+ released for cardiomyocyte contraction. The Ca2+ affinity is the major determinant of the pump's activity in the physiological Ca2+ concentration range. In the heart, the affinity of the pump for Ca2+ needs to be controlled between narrow borders, since an imbalanced affinity may evoke hypertrophic cardiomyopathy. Several small proteins (phospholamban, sarcolipin) adjust the Ca2+ affinity of the pump to the physiological needs of the cardiomyocyte. It is generally accepted that a chronically reduced Ca2+ affinity of the pump contributes to depressed SR Ca2+ handling in heart failure. Moreover, a persistently lower Ca2+ affinity is sufficient to impair cardiomyocyte SR Ca2+ handling and contractility inducing dilated cardiomyopathy in mice and humans. Conversely, the expression of SERCA2a, a pump with a lower Ca2+ affinity than the housekeeping isoform SERCA2b, is crucial to maintain normal cardiac function and growth. Novel findings demonstrated that a chronically increased Ca2+ affinity also may trigger cardiac hypertrophy in mice and humans. In addition, recent studies suggest that some models of heart failure are marked by a higher affinity of the pump for Ca2+, and hence by improved cardiomyocyte relaxation and contraction. Depressed cardiomyocyte SR Ca2+ uptake activity may therefore not be a universal hallmark of heart failure.     

Transient ventricular fibrillation and myosin heavy chain isoform profile

The present paper deals with spontaneous ventricular defibrillation in mammals and the possibility to facilitate its occurrence. Clinical and experimental evidence suggest that in the majority of cases, ventricular fibrillation (VF) is permanent, requiring defibrillation by electric shock. However, a growing number of reports show that VF can terminate spontaneously in various mammals, including human beings.The mechanisms involved in spontaneous ventricular defibrillation are controversial. Available reports imply that intracellular Ca2+ overload is the key event triggering VF and preventing its reversal. Since the sarcoplasmatic reticulum is the main intracellular Ca2+ regulating organelle and the activity of the cardiac SR Ca2+ ATPase (SERCA 2a) is its prime element of Ca2+ sequestration, spontaneous ventricular defibrillation likely requires high level of SERCA 2a activity. We suggest that mammalian hearts with high SERCA 2a activity defibrillate spontaneously and those with low activity only after its enhancement. Since high SERCA 2a activity is co-expressed with the myosin heavy chain (MHC) isoform V1, we assumed that those hearts preferentially expressing V1 MHC are able to defibrillate spontaneously. Hearts with small amounts of V1 MHC and correspondingly lower level of SERCA 2a activity can only defibrillate following administration of compounds that augment SERCA 2a activity and prevent intracellular Ca2+ overload.     

On the mechanism of the reduction by thyroid hormone of beta-adrenergic relaxation rate stimulation in rat heart.

The effects of beta-adrenergic stimulation on the relaxation rate and the Ca2+-transport rate in sarcoplasmic reticulum of hypothyroid, euthyroid and hyperthyroid rat hearts were studied. Administration of isoproterenol (0.1 microM) to perfused, electrically stimulated hearts (5 Hz) caused a decrease in the half-time of relaxation (RT 1/2) the extent of which depended on the thyroid status, i.e. hypothyroid (-24%), euthyroid (-19%) or hyperthyroid (-8%). A similar decreasing effect was found for the stimulation of Ca2+ transport in isolated SR by cyclic AMP and protein kinase, i.e. hypothyroid (75%), euthyroid (37%) and hyperthyroid (20%). These alterations were not due to differences in endogenous protein kinase activity or cyclic AMP production. Estimations of Ca2+-ATPase and phospholamban (PL) content of the sarcoplasmic reticulum were obtained by measurement of the phosphorylated forms of Ca2+-ATPase (E-P) and phospholamban (PL-P) followed by electrophoresis and autoradiography. A 3-fold decrease of PL-P, accompanied by a 2-fold increase of E-P per mg of protein was observed in sarcoplasmic reticulum preparations in the direction hypothyroid----hyperthyroid. Consequently the E-P/PL-P ratio increased from 0.32 (hypothyroid), through 0.81 (euthyroid) to 1.69 (hyperthyroid). In spite of certain limitations inherent to quantification of Ca2+-ATPase and phospholamban by their phosphorylated products, these data provide strong evidence that during thyroid-hormone mediated cardiac hypertrophy, with concomitant proliferation of the sarcoplasmic reticulum, the relative amount of phospholamban decreases with respect to Ca2+-ATPase. This could provide an explanation for the observed gradual diminishment of the beta-adrenergic effect on the relaxation rate when cardiac tissue is exposed to increasing amounts of thyroid hormone.     

Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2(+)-ATPase with phospholamban in phospholipid vesicles

The Ca2(+)-ATPase in cardiac sarcoplasmic reticulum (SR) is under regulation by phospholamban, an oligomeric proteolipid. To determine the molecular mechanism by which phospholamban regulates the Ca2(+)-ATPase, a reconstitution system was developed, using a freeze-thaw sonication procedure. The best rates of Ca2+ uptake (700 nmol/min/mg reconstituted vesicles compared with 800 nmol/min/mg SR vesicles) were observed when cholate and phosphatidylcholine were used at a ratio of cholate/phosphatidylcholine/Ca2(+)-ATPase of 2:80:1. The EC50 values for Ca2+ were 0.05 microM for both Ca2+ uptake and Ca2(+)-ATPase activity in the reconstituted vesicles compared with 0.63 microM Ca2+ in native SR vesicles. Inclusion of phospholamban in the reconstituted vesicles was associated with a significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0. However, phosphorylation of phospholamban by the catalytic subunit of the cAMP-dependent protein kinase reversed the inhibitory effect on the Ca2+ pump. Similar findings were observed when a peptide, corresponding to amino acids 1-25 of phospholamban, was used. These findings indicate that phospholamban is an inhibitor of the Ca2(+)-ATPase in cardiac SR and phosphorylation of phospholamban relieves this inhibition. The mechanism by which phospholamban inhibits the Ca2+ pump is unknown, but our findings with the synthetic peptide suggest that a direct interaction between the Ca2(+)-ATPase and the hydrophilic portion of phospholamban may be one of the mechanisms for regulation.     

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.     

SERCA2a: a prime target for modulation of cardiac contractility during heart failure

Heart failure is one of the leading causes of sudden death in developed countries. While current therapies are mostly aimed at mitigating associated symptoms, novel therapies targeting the subcellular mechanisms underlying heart failure are emerging. Failing hearts are characterized by reduced contractile properties caused by impaired Ca²⁺ cycling between the sarcoplasm and sarcoplasmic reticulum (SR). Sarcoplasmic/endoplasmic reticulum Ca²⁺ATPase 2a (SERCA2a) mediates Ca²⁺ reuptake into the SR in cardiomyocytes. Of note, the expression level and/or activity of SERCA2a, translating to the quantity of SR Ca²⁺ uptake, are significantly reduced in failing hearts. Normalization of the SERCA2a expression level by gene delivery has been shown to restore hampered cardiac functions and ameliorate associated symptoms in pre-clinical as well as clinical studies. SERCA2a activity can be regulated at multiple levels of a signaling cascade comprised of phospholamban, protein phosphatase 1, inhibitor-1, and PKCα. SERCA2 activity is also regulated by post-translational modifications including SUMOylation and acetylation. In this review, we will highlight the molecular mechanisms underlying the regulation of SERCA2a activity and the potential therapeutic modalities for the treatment of heart failure. [BMB Reports 2013; 46(5): 237-243]     

Upregulation of beta 1-adrenergic receptors in ovariectomized rat hearts

Changes in cardiac myofilament Ca(2+) activation have been demonstrated in ovariectomized rats. The underlying mechanisms responsible for these changes, however, are unknown. Accordingly, we measured both density and binding affinity of cardiac beta(1)-adrenergic receptors in sarcolemmal preparations from 10-week ovariectomized rats, pair-fed ovariectomized rats, and sham-operated control rats. Receptor protein content was also measured by immunoblotting. Deprivation of ovarian sex hormones for 10 weeks induced a significant upregulation of beta(1)-adrenergic receptors without affecting binding affinity. The same magnitude of receptor upregulation was also detected in pair-fed ovariectomized hearts. To determine which hormone is responsible for the observed increase in beta(1)-adrenergic receptor density, various sex hormone supplemental regimens were administered to ovariectomized rats. Subcutaneous injection of estrogen (5 microg/rat), progesterone (1 mg/rat), or estrogen plus progesterone three times a week all effectively prevented the upregulation of the beta(1)-adrenoceptors. Western blot analyses using polyclonal antibody of beta(1)-adrenergic receptors revealed the same pattern of changes in the protein content of the receptors in these various groups of experimental hearts as those obtained from the receptor binding assay. These results suggest a possible direct suppressive effect of ovarian sex hormones on the expression of cardiac beta(1)-adrenergic receptors.