Differential expression of calcineurin and SR Ca2+ handling proteins in equine muscle fibers during early postnatal growth.

During early postnatal development, the myosin heavy chain (MyHC) expression pattern in equine gluteus medius muscle shows adaptation to movement and load,resulting in a decrease in the number of fast MyHC fibers and an increase in the number of slow MyHC fibers. In the present study we correlated the expression of MyHC isoforms to the expression of sarcoplasmic(endo)reticulum Ca2+-ATPase 1 and 2a (SERCA), phospholamban (PLB), calcineurin A (CnA), and calcineurin B (CnB). Gluteus medius muscle biopsies were taken at 0, 2, 4, and 48 weeks and analyzed using immunofluorescence. Both SERCA isoforms and PLB were expressed in almost all fiber types at birth. From 4 weeks of age onward, SERCA1 was exclusively expressed in fast MyHC fibers and SERCA2a and PLB in slow MyHC fibers. At all time points, CnA and CnB proteins were expressed at a basal level in all fibers, but with a higher expression level in MyHC type 1 fibers. From 4 weeks onward, expression of only CnA was also higher in MyHC type 2a and 2ad fibers. We propose a double function of calcineurin in calcium homeostasis and maintenance of slow MyHC fiber type identity. Although equine muscle is already functional at birth, expression patterns of the monitored proteins still show adaptation, depending on the MyHC fiber type.     

Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12

Our model of phospholamban (PLB) regulation of the cardiac Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca(2+)-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca(2+)-bound state. PLB and Ca(2+) binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca(2+). Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB. Site-specific cysteine variants of PLB were co-expressed with SERCA2a, and the effects of PKA phosphorylation and 2D12 on Ca(2+)-ATPase activity and cross-linking to SERCA2a were monitored. In Ca(2+)-ATPase assays, PKA phosphorylation and 2D12 partially and completely reversed SERCA2a inhibition by decreasing K(Ca) values for enzyme activation, respectively. In cross-linking assays, cross-linking of PKA-phosphorylated PLB to SERCA2a was inhibited at only two of eight sites when conducted in the absence of Ca(2+) favoring E2. However, at a subsaturating Ca(2+) concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. K(Ca) values for cross-linking inhibition were decreased nearly 2-fold at all sites by PLB phosphorylation, demonstrating that phosphorylated PLB binds more weakly to SERCA2a than dephosphorylated PLB. In parallel assays, 2D12 blocked PLB cross-linking to SERCA2a at all eight sites regardless of Ca(2+) concentration. Our results demonstrate that 2D12 restores maximal Ca(2+)-ATPase activity by physically disrupting the binding interaction between PLB and SERCA2a. Phosphorylation of PLB by PKA weakens the binding interaction between PLB and SERCA2a (yielding more PLB-free SERCA2a molecules at intermediate Ca(2+) concentrations), only partially restoring Ca(2+) affinity and Ca(2+)-ATPase activity.     

Variation of phospholamban in slow-twitch muscle sarcoplasmic reticulum between mammalian species and a link to the substrate specificity of endogenous Ca(2+)-calmodulin-dependent protein kinase

Systematic immunological and biochemical studies indicate that the level of expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase regulatory protein phospholamban (PLB) in mammalian slow-twitch fibers varies from zero, in the rat, to significant levels in the rabbit, and even higher in humans. The lack of PLB expression in the rat, at the mRNA level, is shown to be exclusive to slow-twitch skeletal muscle, and not to be shared by the heart, thus suggesting a tissue-specific, in addition to a species-specific regulation of PLB. A comparison of sucrose density-purified SR of rat and rabbit slow-twitch muscle, with regard to protein compositional and phosphorylation properties, demonstrates that the biodiversity is two-fold, i.e. (a) in PLB membrane density; and (b) in the ability of membrane-bound Ca(2+)-calmodulin (CaM)-dependent protein kinase II to phosphorylate both PLB and SERCA2a (slow-twitch isoform of Ca(2+)-ATPase). The basal phosphorylation state of PLB at Thr-17 in isolated SR vesicles from rabbit slow-twitch muscle, colocalization of CaM K II with PLB and SERCA2a at the same membrane domain, and the divergent subcellular distribution of PKA, taken together, seem to argue for a differential heterogeneity in the regulation of Ca(2+) transport between such muscle and heart muscle.     

Close proximity between residue 30 of phospholamban and cysteine 318 of the cardiac Ca2+ pump revealed by intermolecular thiol cross-linking

Phospholamban (PLB) is a 52-amino acid inhibitor of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SERCA2a), which acts by decreasing the apparent affinity of the enzyme for Ca(2+). To localize binding sites of SERCA2a for PLB, we performed Cys-scanning mutagenesis of PLB, co-expressed the PLB mutants with SERCA2a in insect cell microsomes, and tested for cross-linking of the mutated PLB molecules to SERCA2a using 1,6-bismaleimidohexane, a 10-A-long, homobifunctional thiol cross-linking agent. Of several mutants tested, only PLB with a Cys replacement at position 30 (N30C-PLB) cross-linked to SERCA2a. Cross-linking occurred specifically and with high efficiency. The process was abolished by micromolar Ca(2+) or by an anti-PLB monoclonal antibody and was inhibited 50% by phosphorylation of PLB by cAMP-dependent protein kinase. The SERCA2a inhibitors thapsigargin and cyclopiazonic acid also completely prevented cross-linking. The two essential requirements for cross-linking of N30C-PLB to SERCA2a were a Ca(2+)-free enzyme and, unexpectedly, a micromolar concentration of ATP or ADP, demonstrating that N30C-PLB cross-links preferentially to the nucleotide-bound, E2 state of SERCA2a. Sequencing of a purified proteolytic fragment in combination with SERCA2a mutagenesis identified Cys(318) of SERCA2a as the sole amino acid cross-linked to N30C-PLB. The proximity of residue 30 of PLB to Cys(318) of SERCA2a suggests that PLB may interfere with Ca(2+) activation of SERCA2a by a protein interaction occurring near transmembrane helix M4.     

Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33

To study PLB (phospholamban) inhibition of the cardiac Ca(2+) pump [SERCA2a (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2a)], a fusion protein (SER-20G-PLB) was engineered by tethering SERCA2a with PLB through a 20-glycine residue chain, allowing the PLB tether to either bind to or dissociate from the inhibition site on SERCA2a. When expressed in insect cells, SER-20G-PLB produced active Ca(2+) uptake, which was stimulated by the anti-PLB antibody, both similar to that which occurred with the control sample co-expressing WT (wild-type)-SERCA2a and WT-PLB. The K(Ca) values of Ca(2+)-dependent ATPase were similar for SER-20G-PLB (0.29±0.02 μM) and for the control sample (0.30±0.02 μM), both greater than 0.17±0.01 μM for WT-SERCA2a expressed alone. Thus SER-20G-PLB retains a fully active Ca(2+) pump, but its apparent Ca(2+) affinity was decreased intrinsically by tethered PLB at a 1:1 molar stoichiometry. Like WT-PLB, SER-20G-PLB ran as both monomers and homo-pentamers on SDS/PAGE. As Ca(2+) concentrations increase from 0 to the micromolar range, the proportion of non-inhibiting pentamers increased from 32% to 52%, suggesting that Ca(2+) activation of the pump completely dissociates the PLB tether from the inhibition site on SERCA2a, with concurrent association of PLB pentamers. Collectively, the regulation of SERCA2a is achieved through the Ca(2+)-dependent equilibria involving PLB association and dissociation from SERCA2a, and assembling and disassembling of SER-20G-PLB pentamers.     

Role of leucine 31 of phospholamban in structural and functional interactions with the Ca2+ pump of cardiac sarcoplasmic reticulum

The ability of two loss-of-function mutants, L31A and L31C, of phospholamban (PLB) to bind to and inhibit the Ca(2+) pump of cardiac sarcoplasmic reticulum (SERCA2a) was investigated using a molecular cross-linking approach. Leu(31) of PLB, located at the cytoplasmic membrane boundary, is a critical amino acid shown previously to be essential for Ca(2+)-ATPase inhibition. We observed that L31A or L31C mutations of PLB prevented the inhibition of Ca(2+)-ATPase activity and disabled the cross-linking of N27C and N30C of PLB to Lys(328) and Cys(318) of SERCA2a. Although L31C-PLB failed to cross-link to any Cys or Lys residue of wild-type SERCA2a, L31C did cross-link with high efficiency to T317C of SERCA2a with use of the homobifunctional sulfhydryl cross-linking reagent, 1,6-bismaleimidohexane. This places Leu(31) of PLB within 10 angstroms of Thr(317) of SERCA2a in the M4 helix. Thus, contrary to previous suggestions, PLB with loss-of-function mutations at Leu(31) retains the ability to bind to SERCA2a, despite losing inhibitory activity. Cross-linking of L31C-PLB to T317C-SERCA2a occurred only in the absence of Ca(2+) and in the presence of nucleotide and was prevented by thapsigargin and by anti-PLB antibody, demonstrating for a fourth cross-linking pair that PLB interacts near M4 only when the Ca(2+) pump is in the Ca(2+)-free, nucleotide-bound E2 conformation, but not in the E2 state inhibited by thapsigargin. L31I-PLB retained full functional and cross-linking activity, suggesting that a bulky hydrophobic residue at position 31 of PLB is essential for productive interaction with SERCA2a. A model for the three-dimensional structure of the interaction site is proposed.     

Co-ordinated expression of contractile and non-contractile features of control equine muscle fibre types characterised by immunostaining of myosin heavy chains

Combined methodologies of immunohistochemistry, histochemistry and photometric image analysis were applied: (1) to characterise control equine skeletal muscle fibres according to their myosin heavy chain (MyHC) composition and (2) to determine on a fibre-to-fibre basis the correlation between contractile [i.e. MyHC(s), myofibrillar ATPase (mATPase) and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms], metabolic [i.e. succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities, glycogen and phospholamban (PLB) contents], and morphological [i.e. cross-sectional area (CSA), capillary and nuclear densities] features of individual myofibres. An accurate delineation of MyHC-based fibre types was obtained with the immunohistochemical method developed. This protocol showed a high sensitivity and objectivity to delineate hybrid fibres with overwhelming dominance of one MyHC isoform and, furthermore, it allowed a semiquantitative delineation of fast hybrid fibres according to the predominant MyHC isoform expressed. The phenotypic differences in contractile, metabolic and morphological properties seen between fibre types were related to MyHC content. Slow fibres had the lowest mATPase activity (related to shortening velocity), the highest SDH activity (oxidative capacity), the lowest GPD activity (glycolytic metabolism) and glycogen content, the smallest CSA, the greatest capillary and nuclear densities, and expressed slow SERCA isoform and PLB, but not the fast SERCA isoform. The reverse pattern was true for pure IID/X fibres, and type IIA fibres had intermediate properties. Hybrid IIAD/X fibres had mean values intermediate to those of their respective pure phenotypes. Discrimination of fibres according to their MyHC content was possible on the basis of their contractile and non-contractile profiles. These intrafibre interdependencies suggest that, even when controlled by different mechanisms, myofibres of control horses exhibit a high degree of co-ordination in their physiological, biochemical and anatomical features.     

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

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

FKBP12.6 mice display temporal gender differences in cardiac Ca(2+)-signalling phenotype upon chronic pressure overload

Preventing Ca(2+)-leak during diastole may provide a means to improve overall cardiac function. The immunosuppressant FK506-binding protein 12.6 (FKBP12.6) regulates ryanodine receptor-2 (RyR2) gating and binds to and inhibits calcineurin (Cn). It is also involved in the pathophysiology of heart failure (HF). Here, we investigated the effects of FKBP12.6 over-expression and gender on Ca(2+)-handling proteins (RyR2, SERCA2a/PLB, and NCX), and on pro-(CaMKII, Cn/NFAT) and anti-hypertrophic (GSK3β) signalling pathways in a thoracic aortic constriction (TAC) mouse model. Wild type mice (WT) and mice over-expressing FKBP12.6 of both genders underwent TAC or sham-operation (Sham). FKBP12.6 over-expression ameliorated post-TAC survival rates in both genders. Over time, FKBP12.6 over-expression reduced the molecular signature of left ventricular hypertrophy (LVH) and the transition to HF (BNP and β-MHC mRNAs) and attenuated Cn/NFAT activation in TAC-males only. The gender difference in pro- and anti-hypertrophic LVH signals was time-dependent: TAC-females exhibited earlier pathological LVH associated with concomitant SERCA2a down-regulation, CaMKII activation, and GSK3β inactivation. Both genotypes showed systolic dysfunction, possibly related to down-regulated RyR2, but only FK-TAC-males exhibited preserved diastolic LV function. Although FKBP12.6 over-expression did not impact the vicious cycle of TAC-induced HF, this study reveals some subtle sequential and temporal gender differences in Ca(2+)-signalling pathways of pathological LVH.     

Involvement of L-type calcium channel and SERCA2a in myocardial dysfunction induced by obesity

Obesity has been shown to impair myocardial performance. Nevertheless, the mechanisms underlying the participation of calcium (Ca(2+) ) handling on cardiac dysfunction in obesity models remain unknown. L-type Ca(2+) channels and sarcoplasmic reticulum (SR) Ca(2+) -ATPase (SERCA2a), may contribute to the cardiac dysfunction induced by obesity. The purpose of this study was to investigate whether myocardial dysfunction in obese rats is related to decreased activity and/or expression of L-type Ca(2+) channels and SERCA2a. Male 30-day-old Wistar rats were fed standard (C) and alternately four palatable high-fat diets (Ob) for 15 weeks. Obesity was determined by adiposity index and comorbidities were evaluated. Myocardial function was evaluated in isolated left ventricle papillary muscles under basal conditions and after inotropic and lusitropic maneuvers. L-type Ca(2+) channels and SERCA2a activity were determined using specific blockers, while changes in the amount of channels were evaluated by Western blot analysis. Phospholamban (PLB) protein expression and the SERCA2a/PLB ratio were also determined. Compared with C rats, the Ob rats had increased body fat, adiposity index and several comorbidities. The Ob muscles developed similar baseline data, but myocardial responsiveness to post-rest contraction stimulus and increased extracellular Ca(2+) was compromised. The diltiazem promoted higher inhibition on developed tension in obese rats. In addition, there were no changes in the L-type Ca(2+) channel protein content and SERCA2a behavior (activity and expression). In conclusion, the myocardial dysfunction caused by obesity is related to L-type Ca(2+) channel activity impairment without significant changes in SERCA2a expression and function as well as L-type Ca(2+) protein levels.     

Differential regulation of SR calcium transporters by thyroid hormone in rat atria and ventricles

Thyroid hormone exerts positive inotropic effects on the heart mediated in part by its regulation of calcium transporter proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2), phospholamban (PLB), and Na(+)/Ca(2+) exchanger (NCX). To further understand the potential cardiac chamber-specific effects of thyroid hormone action, we compared the triiodo-L-thyronine (T(3)) responsiveness of calcium transporter proteins in atrial versus ventricular tissues. Rats were rendered hypothyroid by ingestion of propylthiouracil, and a subgroup of animals was treated with T(3) for 7 days (7 microg/day by constant infusion). Atrial and left ventricular (LV) tissue homogenates were analyzed for expression of SERCA2, PLB, and NCX proteins by Western blot analysis. SERCA2 protein significantly decreased by 50% in hypothyroid LV and was normalized by T(3) treatment. In contrast, SERCA2 protein in atria was unaltered in the hypothyroid state. PLB protein expression significantly increased by 1.6- and 5-fold in the hypothyroid LV and atria, respectively, and returned to euthyroid levels with T(3) treatment. Expression of NCX protein showed a greater response to T(3) treatment in atria tissue than in ventricular tissue. Sarcoplasmic reticulum calcium cycling is determined in part by the ratio of SERCA2 to PLB. This ratio was sixfold higher in the atria compared with LV, suggesting that PLB may play a minor role in the regulation of SERCA2 function in normal atria. We conclude that calcium transporter proteins are responsive to thyroid hormone in a chamber-specific manner, with atria showing a greater change in protein content in response to T(3). The differential effect on atria may account for the occurrence of atrial rather than ventricular arrhythmias in response to even mild degrees of thyrotoxicosis.     

Sarco/endoplasmic reticulum Ca2+ (SERCA)-pumps: link to heart beats and calcium waves.

Mobilization of endoplasmic reticulum Ca2+ is pivotal to the ability of a cell to send or respond to stimuli. Ca(2+)-Mg(2+)-ATPases, termed SERCA pumps, sequester Ca2+ into the sarco/endoplasmic reticulum. There are several SERCA protein isoforms encoded by three genes. This paper summarizes the structure, function, tissue and subcellular distribution, and regulation of various SERCA isoforms. Then it attempts to link divergence in the signal transduction processes of cells to the types and levels of SERCA proteins they express and to how the cells regulate their SERCA pump activity. The paper examines possible linkages between SERCA pumps and receptor-activated Ca2+ entry, SERCA isoform localization and Ca(2+)-waves, and the role of SERCA pumps in nuclear Ca2+ in cell proliferation and apoptosis. Then it uses available information on cardiac function and chronic stimulation of the fast-twitch muscle to answer a series of basic questions on the regulation of SERCA activity and expression and their linkage to signal transduction. Finally, it discusses the possibility that neurons exhibit complex Ca(2+)-waves whose interactions have the potential to explain the operational basis of neural networks. A series of unanswered questions emerge based on this synthesis, including the unsettling issue of whether all the isoforms are needed to achieve the divergence in signal transduction or if there is a degree of redundancy in the system.     

Age-dependent suppression of SERCA2a mRNA in pediatric atrial myocardium

Differential expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban (PLB) has been shown in heart failure and atrial arrhythmias. We investigated the influence of volume overload and age on their expression in pediatric atrial myocardium. Right atrial specimens from 18 children with volume overloaded right atrium (VO) and 12 patients without overload were studied. Each group was further divided into patients less than and older than 12 months of age. Only in the younger patients SERCA2a was significantly reduced in the VO group. In younger patients PLB mRNA level tended to be lower in VO. The PLB:SERCA protein ratio was significantly reduced in the VO group. Age itself did not influence the SERCA2a and PLB expression, if the hemodynamic overload was not taken into account. This study is the first to show a combined influence of volume overload and age on atrial SERCA2a expression.     

Expression patterns of sarco/endoplasmic reticulum Ca(2+)-ATPase and inositol 1,4,5-trisphosphate receptor isoforms in vascular endothelial cells.

Expression patterns of sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) and inositol 1,4,5-trisphosphate receptor (IP3R) isoforms were studied in endothelial cells at the mRNA level by ratio RT-PCR technique and subsequent restriction-enzyme analysis. Three types of cells have been used in the present study: rat adrenal medulla microvascular endothelial cells (RAMEC), rat aortic endothelial cells (RAEC), and human umbilical vein endothelial cells (HUVEC). Our data show the presence of multiple SERCA and IP3R isoforms in each type of endothelial cells. Freshly isolated HUVEC were an exception in this respect since they contained only SERCA3 without SERCA2b messengers. The expression patterns changed upon cell proliferation: SERCA3 and IP3R-1 messengers decreased, while IP3R-3 increased with culturing. Upon cell differentiation, induced by culturing the cells on Matrigel, the expression pattern of the IP3R changed even further in all endothelial cell types: IP3R-1 was reduced in all three cell kinds, while IP3R-3 raised significantly in RAEC and RAMEC. In HUVEC the expression of SERCA returned, upon differentiation, to the levels observed in the freshly isolated cells. Thus, the plasticity of expression of various SERCA and IP3R isoforms shows that possibly different Ca2+ pools may play distinct roles in cell proliferation and differentiation.     

Sarco(endo)plasmic reticulum calcium pump isoforms in paralyzed rat slow muscle

To assess the influence of paralysis on the expression of phenotypic protein isoforms related to muscle relaxation, the effects of spinal cord transection (ST) on sarco(endo)plasmic reticulum calcium ATPase (SERCA) pump isoform protein levels in the slow rat soleus were measured. Western blotting using SERCA isoform specific antibodies demonstrated a rapid up-regulation (7 days post ST) of the fast fiber type-specific isoform (SERCA1). In contrast, the slow fiber type-specific isoform, SERCA2, was decreased with a slower time-course. The up-regulation of SERCA1 protein preceded the up-regulation of fast myosin heavy chain (MyHC) (i.e., MyHC-II). Immunohistochemical analyses of single muscle fibers showed that 15 days after ST there was a pronounced increase in the proportion of slow MyHC fibers with SERCA1 confirming that SERCA1 was up-regulated in the slow fibers of the soleus prior to MyHC-II. These data suggest that the expression of the SERCA isoforms (particularly SERCA1) may serve as more sensitive markers of phenotypic adaptation in response to altered levels of contractile activity than the MyHC isoforms. In addition, since the expression of SERCA isoforms was dissociated from MyHC isoforms, regulation of gene expression for these two different protein systems must involve different signaling events and/or synthetic processes.     

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.     

The Effect of Passive Movement on Denervated Soleus Highlights a Differential Nerve Control on SERCA and MyHC Isoforms

The sarco-endoplasmic reticulum Ca²⁺ ATP-ase (SERCA) and myosin heavy chain (MyHC) levels were measured in hindlimb-denervated and selectively denervated rat soleus muscles. Selective denervation allowed passive movement of the soleus, whereas hindlimb denervation rendered it to passivity. To minimize chronic effects, we followed the changes only for 2 weeks. Selective denervation resulted in less muscle atrophy, a faster slow-to-fast transition of MyHC isoforms, and less coordinated expressions of the slow vs fast isoforms of MyHC and SERCA. Generally, expression of the slow-twitch type SERCA2a was found to be less dependent, whereas the slow-twitch type MyHC1 was the most dependent on innervation. Our study shows that passive movement is able to ameliorate denervation-induced atrophy of the soleus and that it also accentuates the dyscoordination in the expression of the corresponding slow and fast isoforms of MyHC and SERCA. (J Histochem Cytochem 56:1013–1022, 2008)     

Phospholamban binds with differential affinity to calcium pump conformers

To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.