Sarcoplasmic Reticulum (SR) Ca2+-ATPase Research Articles

Selective SERCA2a activator as a candidate for chronic heart failure therapy

BackgroundThe sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) depression substantially contributes to diastolic dysfunction in heart failure (HF), suggesting that SERCA2a stimulation may be a mechanism-based HF therapy. Istaroxime is a drug endowed with both a SERCA2a stimulatory activity and a Na+/K+ pump inhibitory activity for acute HF treatment. Its main metabolite PST3093 shows a more favorable therapeutic profile as compared to the parent drug, but it is still unsuitable for chronic usage. Novel PST3093 derivatives have been recently developed for oral (chronic) HF treatment; compound 8 was selected among them and here characterized.MethodsEffects of compound 8 were evaluated in a context of SERCA2a depression, by using streptozotocin-treated rats, a well-known model of diastolic dysfunction. The impact of SERCA2a stimulation by compound 8 was assessed at the cellular level ad in vivo, following i.v. infusion (acute effects) or oral administration (chronic effects).ResultsAs expected from SERCA2a stimulation, compound 8 induced SR Ca2+ compartmentalization in STZ myocytes. In-vivo echocardiographic analysis during i.v. infusion and after repeated oral administration of compound 8, detected a significant improvement of diastolic function. Moreover, compound 8 did not affect electrical activity of healthy guinea-pig myocytes, in line with the absence of off-target effects. Finally, compound 8 was well tolerated in mice with no evidence of acute toxicity.ConclusionsThe pharmacological evaluation of compound 8 indicates that it may be a safe and selective drug for a mechanism-based treatment of chronic HF by restoring SERCA2a activity.Graphical

A polymorphism in histidine-rich calcium binding protein as second hit in phospholamban cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Netherlands Cardio Vascular Research Initiative (CVON): the Dutch Heart Foundation, Dutch Federation of University Medical Center, the Netherlands Organization for Health Re-search and Development and the Royal Netherlands Academy of Sciences Introduction Sudden cardiac death (SCD) is one of the severe manifestations in carriers with a phospholamban (PLN p.Arg14del) cardiomyopathy. Prediction whether a patient with this pathogenic variant will be at risk for SCD is difficult. PLN has an important role in cardiac calcium homeostasis as regulator of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). It has been shown that the p.Arg14del pathogenic variant leads to Ca2+ overload in cardiomyocytes. Recently, it was found that dilated cardiomyopathy (DCM) patients who have a polymorphism in histidine-rich calcium binding protein (HRC Ser96Ala, rs3745297), displayed an increased risk for malignant arrhythmias and SCD. HRC resides within the SR, where it acts as a regulator of Ca2+ homeostasis. This Ser96Ala gene variant is widespread, as 60% of the general population bears at least one copy of this allele. Objective To explore the effect of the HRC Ser96Ala polymorphism on ventricular arrhythmias and disease expression in PLN p.Arg14del pathogenic variant carriers. Methods 337 p.Arg14del patients were included into the study; divided into wildtype (WT) (n=134, 24 index patients), heterozygous for Ser96Ala variant (n=142, 30 index patients) and homozygous for Ser96Ala variant (n=61, 11 index patients). The study was conducted according to the Declaration of Helsinki. Blood samples were genotyped on the Infinium® Global Screening Array-24 v3.0. Clinical data were subtracted from health records. Results In total 23% of PLN variant carriers were diagnosed with DCM while 11% of the variant carriers were diagnosed with arrhythmogenic cardiomyopathy. A significant difference in age of presentation (p=0.019) of p.Arg14del patients diagnosed with a DCM phenotype was found in homozygous HRC variant carriers (median 43 years, [36-47.3], n=8) compared to WT (median 49 years, [41-56.8], n=24) and heterozygous variant carriers (median 58.5 years, [51-66.5], n=22). No significant differences between the 3 groups were detected in manifestations of premature ventricular contractions (n=188, p=0.203), non-sustained ventricular tachycardia (n=248, p=0.314) and appropriate ICD shocks (n=308, p=0.901). Conclusion Although a significant difference in disease onset was found in PLN p.Arg14del patients with DCM who were homozygous for the HRC polymorphism, no correlations with arrhythmogenic parameters were found between patients with and without the HRC polymorphism. Therefore, we conclude that presence of the HRC polymorphism is not a discriminative predictor for arrhythmogenic events in PLN p.Arg14del.

Structural basis for sarcolipin's regulation of muscle thermogenesis by the sarcoplasmic reticulum Ca2+-ATPase.

The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) plays a central role in muscle contractility and nonshivering thermogenesis. SERCA is regulated by sarcolipin (SLN), a single-pass membrane protein that uncouples Ca2+ transport from ATP hydrolysis, promoting futile enzymatic cycles and heat generation. The molecular determinants for regulating heat release by the SERCA/SLN complex are unclear. Using thermocalorimetry, chemical cross-linking, and solid-state NMR spectroscopy in oriented phospholipid bicelles, we show that SERCA’s functional uncoupling and heat release rate are dictated by specific SERCA/SLN intramembrane interactions, with the carboxyl-terminal residues anchoring SLN to the SR membrane in an inhibitory topology. Systematic deletion of the carboxyl terminus does not prevent the SERCA/SLN complex formation but reduces uncoupling in a graded manner. These studies emphasize the critical role of lipids in defining the active topology of SLN and modulating the heat release rate by the SERCA/SLN complex, with implications in fat metabolism and basal metabolic rate.

SERCA Inhibition as a Potential Therapeutic Target for Breast Cancer

Chemotherapy is both a palliative treatment for many cancers and adjuvant treatment before, during, or after local treatment (surgery and/or radiation therapy) in single or combination regimens. Despite current treatments, approximately 1 in 39 women (3%) still die from breast cancer. A major complication with systemic therapy is drug resistance which limits the efficacy of clinical chemotherapy. Hence, current research aims to uncover novel targets for breast cancer therapy. Various studies have shown altered calcium signaling in breast cancer cells, therefore, calcium modulators may be potential therapeutic targets. Studies have also shown that Thapsigargin (TG) and cyclopiazonic acid (CPZ), two potent and specific inhibitors of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA), can impair cancer cell growth. However, TG and CPZ also impair non-malignant cells, which results in significant toxicity, thus preventing use in chemotherapy. Using spectrometry UV-visible measurements to assess calcium loading and ATPase activity in SR microsomes, our lab has found that various drugs which display cell protective effects in ischemia also inhibit SERCA. They include novel benzothiazepines (BZTs; K201, CGP37157, PH00095) and have shown to inhibit SERCA in a calcium dependent mechanism. They decreased SR uptake when cytosolic Ca2+ levels ([Ca2+]c) are low, similar to competitive inhibition of Ca2+ uptake, which produces mild store depletion. Contrarily, TG and CPZ block Ca2+ uptake at all [Ca2+]c and largely deplete SR stores. Using various SERCA inhibitors, we sought out to determine their effects on breast cancer cell lines of different subtypes. MTS assay studies show that TG and CPZ significantly reduce cell viability in MCF7 and MCF10A cell lines while having no significant effects on triple negative breast cancer (TNBC) cell line MDAMB231. Preliminary studies also show no significant effect by TG on BT20 cells, another TNBC cell line. BZT K201 also significantly reduced cell viability in MCF7 cells but not MDAMB231. Immunoblot shows MCF7 cells have significantly higher SERCA2 expression than MDAMB231. We observe no significant differences in plasma membrane calcium channel ATPase (PMCA) protein expression. Preliminary RT-PCR studies estimate MCF7 cells have a higher fold change in SERCA genes. Thus, targeting SERCA may serve as a potential therapeutic target for breast cancer. Here, we find that SERCA inhibitors TG, CPZ, and BZT K201 reduce cell viability in MCF7 cells. Still, these studies indicate that SERCA inhibition may only be efficacious in certain breast cancer cell types which do not express a TNBC phenotype. TNBC cell lines (MDAMB231, BT20) are not significantly affected by SERCA inhibition, which may be due to higher dependence for signaling on plasmalemma calcium channels and transporters. Additional studies are required to confirm the expression profiles of calcium transporters among cell lines, including those in the plasmalemma (PMCA, calcium channels), which may help to understand if alternative sources of Ca2+ for intracellular signaling make TNBC cell lines more resistant to SERCA inhibitors.

Mutations Linked to Dilated Cardiomyopathy Perturb the Topology and Regulatory Response of Phospholamban

Phospholamban (PLN), a small 52-residue membrane protein expressed in cardiac tissue, functions as a regulatory subunit to the 110 kDa sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) calcium pump. The SERCA-PLN complex is responsible for restoring high SR Ca2+ concentrations prior to each cardiomyocyte contraction, in which PLN maintains SERCA's activity within a tight physiological window. PLN binds SERCA via a transmembrane protein-protein interaction that suppresses the affinity of SERCA's Ca2+ sites, but this effect is relieved upon phosphorylation of PLN's cytoplasmic extension by protein kinase A (PKA) upon β-adrenergic stimulation. PLN mutations that alter its activity or interfere with phosphorylation are linked to hereditary forms of dilated cardiomyopathy (DCM), although the reasons for this remain unclear due to a lack of structural data for the SERCA-PLN complex. Using structural restraints from high-resolution magic-angle spinning (MAS) and oriented sample (OS) solid-state NMR (ssNMR) spectroscopy, we have constructed dynamic models of the SERCA-PLN complex that capture a topological allosteric mechanism for PLN-driven communication of cytoplasmic regulatory stimuli to SERCA's Ca2+ binding sites. Further OS-ssNMR measurements and activity assays of DCM mutants of PLN displayed an altered topology and a dampened response to phosphorylation, which was consistent with our dynamic models and suggest disease onset may occur from an impaired β-adrenergic pathway. Overall, our recent observations by OS-ssNMR and modelling demonstrate that structural topology (i.e., helical tilt, rotation and secondary structure) is a critical determinant of SERCA regulation by PLN as well as by other homologous regulatory partners expressed in non-cardiac tissues.

Dynamics-Driven Allostery Underlies Ca2+-Mediated Release of SERCA Inhibition by Phospholamban

Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and phospholamban (PLB) are essential for intracellular Ca2+ transport in myocytes. Ca2+-dependent activation of SERCA-PLB provides a control function that regulates cytosolic and SR Ca2+ levels. Although experimental and computational studies alone have led to a greater insight into SERCA-PLB regulation, the structural mechanisms for Ca2+ binding reversing inhibition of the complex remain poorly understood. Therefore, we have performed atomistic simulations totaling 32.7 μs and cell-based intramolecular fluorescence resonance energy transfer (FRET) experiments to determine structural changes of PLB-bound SERCA in response to binding of a single Ca2+ ion. Complementary MD simulations and FRET experiments showed that open-to-closed transitions in the structure of the headpiece underlie PLB inhibition of SERCA, and binding of a single Ca2+ ion is sufficient to shift the protein population toward a structurally closed structure of the complex. Closure is accompanied by functional interactions between the N-domain β5–β6 loop and the A-domain and the displacement of the catalytic phosphorylation domain toward a competent structure. We propose that reversal of SERCA-PLB inhibition is achieved by stringing together its controlling modules (A-domain and loop Nβ5–β6) with catalytic elements (P-domain) to regulate function during intracellular Ca2+ signaling. We conclude that binding of a single Ca2+ is a critical mediator of allosteric signaling that dictates structural changes and motions that relieve SERCA inhibition by PLB. Understanding allosteric regulation is of paramount importance to guide therapeutic modulation of SERCA and other evolutionarily related ion-motive ATPases.

Resting metabolic rate and skeletal muscle SERCA and Na+ /K+ ATPase activities are not affected by fish oil supplementation in healthy older adults.

Omega‐3 polyunsaturated fatty acids (PUFAs) have unique properties purported to influence several aspects of metabolism, including energy expenditure and protein function. Supplementing with n‐3 PUFAs may increase whole‐body resting metabolic rate (RMR), by enhancing Na+/K+ ATPase (NKA) activity and reducing the efficiency of sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) activity by inducing a Ca2+ leak‐pump cycle. The purpose of this study was to examine the effects of fish oil (FO) on RMR, substrate oxidation, and skeletal muscle SERCA and NKA pump function in healthy older individuals. Subjects (n = 16 females; n = 8 males; 65 ± 1 years) were randomly assigned into groups supplemented with either olive oil (OO) (5 g/day) or FO (5 g/day) containing 2 g/day eicosapentaenoic acid and 1 g/day docosahexaenoic acid for 12 weeks. Participants visited the laboratory for RMR and substrate oxidation measurements after an overnight fast at weeks 0 and 12. Skeletal muscle biopsies were taken during weeks 0 and 12 for analysis of NKA and SERCA function and protein content. There was a main effect of time with decrease in RMR (5%) and fat oxidation (18%) in both the supplementation groups. The kinetic parameters of SERCA and NKA maximal activity, as well as the expression of SR and NKA proteins, were not affected after OO and FO supplementation. In conclusion, these results suggest that FO supplementation is not effective in altering RMR, substrate oxidation, and skeletal muscle SERCA and NKA protein levels and activities, in healthy older men and women.

Modulation of the cardiac Na+-Ca2+ exchanger by cytoplasmic protons: Molecular mechanisms and physiological implications

A precise temporal and spatial control of intracellular Ca2+ concentration is essential for a coordinated contraction of the heart. Following contraction, cardiac cells need to rapidly remove intracellular Ca2+ to allow for relaxation. This task is performed by two transporters: the plasma membrane Na+-Ca2+ exchanger (NCX) and the sarcoplasmic reticulum (SR) Ca2+‐ATPase (SERCA). NCX extrudes Ca2+ from the cell, balancing the Ca2+entering the cytoplasm during systole through L-type Ca2+ channels. In parallel, following SR Ca2+ release, SERCA activity replenishes the SR, reuptaking Ca2+ from the cytoplasm.The activity of the mammalian exchanger is fine-tuned by numerous ionic allosteric regulatory mechanisms. Micromolar concentrations of cytoplasmic Ca2+ potentiate NCX activity, while an increase in intracellular Na+ levels inhibits NCX via a mechanism known as Na+-dependent inactivation. Protons are also powerful inhibitors of NCX activity. By regulating NCX activity, Ca2+, Na+ and H+ couple cell metabolism to Ca2+ homeostasis and therefore cardiac contractility. This review summarizes the recent progress towards the understanding of the molecular mechanisms underlying the ionic regulation of the cardiac NCX with special emphasis on pH modulation and its physiological impact on the heart.

The Cardiotoxicity Induced by Arsenic Trioxide is Alleviated by Salvianolic Acid A via Maintaining Calcium Homeostasis and Inhibiting Endoplasmic Reticulum Stress.

Arsenic trioxide (ATO) has been verified as a breakthrough with respect to the management of acute promyelocytic leukemia (APL) in recent decades but associated with some serious adverse phenomena, particularly cardiac functional abnormalities. Salvianolic acid A (Sal A) is a major effective component in treating ATO-induced cardiotoxicity. Therefore, the objective of our study was to assess whether Sal A had protective effects by the regulation of calcium homeostasis and endoplasmic reticulum (ER) stress. For the in vivo study, BALB/c mice were treated with ATO and/or Sal A via daily tail vein injections for two weeks. For the in vitro study, we detected the effects of ATO and/or Sal A in real time using adult rat ventricular myocytes (ARVMs) and an IonOptix MyoCam system. Our results showed that Sal A pretreatment alleviated cardiac dysfunction and Ca2+ overload induced by ATO in vivo and vitro. Moreover, Sal A increased sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) activity and expression, alleviated [Ca2+]ER depletion, and decreased ER stress-related protein expression. Sal A protects the heart from ATO-induced injury and its administration correlates with the modulation of SERCA, the recovery of Ca2+ homeostasis, and the down-regulation of ER stress-mediated apoptosis.

Restoration of SERCA ATPase prevents oxidative stress-related muscle atrophy and weakness

Molecular targets to reduce muscle weakness and atrophy due to oxidative stress have been elusive. Here we show that activation of Sarcoplasmic Reticulum (SR) Ca2+ ATPase (SERCA) with CDN1163, a novel small molecule allosteric SERCA activator, ameliorates the muscle impairment in the CuZnSOD deficient (Sod1-/-) mouse model of oxidative stress. Sod1-/- mice are characterized by reduced SERCA activity, muscle weakness and atrophy, increased oxidative stress and mitochondrial dysfunction. Seven weeks of CDN1163 treatment completely restored SERCA activity and reversed the 23% reduction in gastrocnemius mass and 22% reduction in specific force in untreated Sod1-/- versus wild type mice. These changes were accompanied by restoration of autophagy protein markers to the levels found in wild-type mice. CDN1163 also reversed the increase in mitochondrial ROS generation and oxidative damage in muscle tissue from Sod1-/- mice. Taken together our findings suggest that the pharmacological restoration of SERCA is a promising therapeutic approach to counter oxidative stress-associated muscle impairment.

Abstract 16191: Phospholamban Regulates Perinuclear/nuclear Calcium Dynamics in Cardiomyocytes

Introduction: Phospholamban (PLB) regulates cardiac sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA2a), thus modulating SR Ca 2+ dynamics. Recent studies demonstrated that SERCA is involved in Ca 2+ uptake into the lumen of nuclear envelope (NE) of cardiomyocytes (CMs). However, the regulatory role of PLB on Ca 2+ uptake into NE remains unknown. Hypothesis: We hypothesize that PLB is also responsible for modulating nuclear Ca 2+ dynamics. Methods: Confocal immunofluorescence microscopy was used to determine subcellular expression of PLB. By using fluo-4 based confocal line-scan Ca 2+ imaging, we measured spontaneous Ca 2+ waves (SCWs) across both cytoplasmic and nuclear regions in isolated permeabilized mouse CMs. Results: Several anti-PLB antibodies strongly stained PLB at both SR and the perinuclear membranes in CMs. A PLB peptide (residues 1-31) eliminated all these anti-PLB antibody stains. To identify the functional role of PLB expressed in the perinuclear membranes, we took advantage of our recently established method that a Fab fragment of anti-PLB monoclonal antibody (Fab) reversed PLB inhibition specifically and increased SR Ca 2+ uptake and release. SCWs through the nuclear regions had typically relative low fluorescent amplitude (F/F 0 ) and slow decay time (t 1/2 ) compared to that in the cytoplasmic region. At the free intracellular Ca 2+ concentration ([Ca 2+ ] i ) of 400 nM, Fab (100 μg/mL) significantly enhanced F/F 0 and decreased t 1/2 of SCWs in both cytoplasmic and nuclear regions. After addition of Fab, F/F 0 of SCWs through the nuclear regions increased from 0.91±0.16 to 1.27±0.19 (n=9, p<.05) while t 1/2 decreased from 137.6±18.5 ms to 105.0±11.3 ms, (p<.05). Similar effects were also observed after phosphorylation of PLB by addition of 20 μM cAMP (F/F 0 =1.43±0.11 vs. 1.04±0.14 in control, p<.05; t 1/2 =107.82±10.9 ms vs. 139.21±20.1 ms in control, n=6, p<.05). At high [Ca 2+ ] i of 1000 nM where PLB does not inhibit SERCA2a, addition of cAMP or Fab had no significant effect on SCWs in both cytoplasmic and nuclear regions. Conclusions: We demonstrated that PLB is expressed in and around NE. Acute removal of PLB inhibition increased perinuclear/nuclear Ca 2+ uptake and release. PLB is critically involved in nuclear Ca 2+ signaling modulation.

Acute reversal of phospholamban inhibition facilitates the rhythmic whole-cell propagating calcium waves in isolated ventricular myocytes

Phospholamban (PLB) inhibits the activity of cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a). Phosphorylation of PLB during sympathetic activation reverses SERCA2a inhibition, increasing SR Ca2+ uptake. However, sympathetic activation also modulates multiple other intracellular targets in ventricular myocytes (VMs), making it impossible to determine the specific effects of the reversal of PLB inhibition on the spontaneous SR Ca2+ release. Therefore, it remains unclear how PLB regulates rhythmic activity in VMs.Here, we used the Fab fragment of 2D12, a monoclonal anti-PLB antibody, to test how acute reversal of PLB inhibition affects the spontaneous SR Ca2+ release in normal VMs. Ca2+ sparks and spontaneous Ca2+ waves (SCWs) were recorded in the line-scan mode of confocal microscopy using the Ca2+ fluorescent dye Fluo-4 in isolated permeabilized mouse VMs. Fab, which reverses PLB inhibition, significantly increased the frequency, amplitude, and spatial/temporal spread of Ca2+ sparks in VMs exposed to 50nM free [Ca2+]. At physiological diastolic free [Ca2+] (100–200nM), Fab facilitated the formation of whole-cell propagating SCWs. At higher free [Ca2+], Fab increased the frequency and velocity, but decreased the decay time of the SCWs. cAMP had little additional effect on the frequency or morphology of Ca2+ sparks or SCWs after Fab addition. These findings were complemented by computer simulations. In conclusion, acute reversal of PLB inhibition alone significantly increased the spontaneous SR Ca2+ release, leading to the facilitation and organization of whole-cell propagating SCWs in normal VMs. PLB thus plays a key role in subcellular Ca2+ dynamics and rhythmic activity of VMs.

Serca Located in the Junctional SR Shapes Calcium Release in Cardiac Myocytes

Calcium-induced calcium release is in principle an all-or-nothing mechanism and self-propagation of Ca2+ waves is a pathological manifestation of this positive feedback loop. However, since local release of Ca2+ only elicits elementary Ca2+ release events (Ca2+ sparks) that do not develop into Ca2+ waves, Ca2+ release can be graded. This local control is possible because of the spatial arrangement of L-type Ca2+ channels and clusters of ryanodine receptors at the sites of Ca2+ release (dyads) (Stern 1992). We propose that the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) also contributes to local control by limiting diffusion of Ca2+. Six days following conditional SERCA knockout, Ca2+ sparks exhibited broadened geometry and slowed kinetics (increase in spark width, time to peak, and duration by 33%, 33%, and 51%, respectively). At this time point, SERCA protein levels are reduced by 53% and SR Ca2+ content is decreased by 25% (Stokke et al. 2010). To determine the precise localization of SERCA we employed cryo immuno-gold electron microscopy on sections from the mouse papillary muscle. Normal cardiac myocytes showed preferential SERCA expression near the Z-lines (nearly twofold higher labeling density at the Z-line than the A-band) with SERCA molecules clearly located in the junctional SR in close proximity to the dyads. The conditional SERCA knockout resulted in preferential loss of labeling at these locations. From resin-embedded cross-sections of papillary muscles, we observed more abundant SR in the I-band compared to the A-band (by factor of 1.7 and 1.6 for control and KO respectively). These results suggest that SERCA contributes to local control of Ca2+ release by limiting diffusion of Ca2+ from the dyad. Thus, the processes of Ca2+ release and re-uptake are closely linked by a population of SERCA molecules in the junctional SR.

EPR Accessibility Measurements of the SERCA-PLB Complex using Site-Directed Spin Labeling

We investigate the structure formed byof phospholamban (PLB) bound to the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) using site-directed spin labeling. SERCA is a 994 residue transmembrane protein that helps control Ca2+ concentration in the SR relative to the cytoplasm in order to facilitate muscle relaxation. PLB is a 52 residue regulatory muscle protein found in cardiac muscle which, upon binding, has been shown to inhibit SERCA activity. However, phosphorylation of PLB at Ser-16 partially relieves this inhibition while still maintaining a combined structureconserving the complex. The structural mechanism behind this relief of inhibition is largely unknown. Using EPR accessibility measurements, we have shown that phosphorylation of PLB at Ser-16 alters the binding interface between its transmembrane helix and the PLB binding groove on SERCA, likely breaking the interactions that lead to inhibition.

Long-term obesity promotes alterations in diastolic function induced by reduction of phospholamban phosphorylation at serine-16 without affecting calcium handling.

Few studies have evaluated the relationship between the duration of obesity, cardiac function, and the proteins involved in myocardial calcium (Ca(2+)) handling. We hypothesized that long-term obesity promotes cardiac dysfunction due to a reduction of expression and/or phosphorylation of myocardial Ca(2+)-handling proteins. Thirty-day-old male Wistar rats were distributed into two groups (n = 10 each): control (C; standard diet) and obese (Ob; high-fat diet) for 30 wk. Morphological and histological analyses were assessed. Left ventricular cardiac function was assessed in vivo by echocardiographic evaluation and in vitro by papillary muscle. Cardiac protein expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a), calsequestrin, L-type Ca(2+) channel, and phospholamban (PLB), as well as PLB serine-16 phosphorylation (pPLB Ser(16)) and PLB threonine-17 phosphorylation (pPLB Thr(17)) were determined by Western blot. The adiposity index was higher (82%) in Ob rats than in C rats. Obesity promoted cardiac hypertrophy without alterations in interstitial collagen levels. Ob rats had increased endocardial and midwall fractional shortening, posterior wall shortening velocity, and A-wave compared with C rats. Cardiac index, early-to-late diastolic mitral inflow ratio, and isovolumetric relaxation time were lower in Ob than in C. The Ob muscles developed similar baseline data and myocardial responsiveness to increased extracellular Ca(2+). Obesity caused a reduction in cardiac pPLB Ser(16) and the pPLB Ser(16)/PLB ratio in Ob rats. Long-term obesity promotes alterations in diastolic function, most likely due to the reduction of pPLB Ser(16), but does not impair the myocardial Ca(2+) entry and recapture to SR.

Distinct Functional Effects of Phospholamban Phosphorylation States

We have studied the differential effects of phospholamban (PLB) phosphorylation states on the activity of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). It is known that PLB inhibits SERCA and that PLB phosphorylation at S16 and/or T17 relieves this inhibition in cardiac SR. However, the functional effects of the two PLB phosphorylation sites have not been compared quantitatively. Therefore, we co-reconstituted SERCA1a with synthetic PLB, pS16-PLB, pT17-PLB, or pS16/pT17-PLB in phospholipid vesicles and measured Ca2+-dependent SERCA activity. Inhibition was maximally relieved by phosphorylation at S16, followed by doubly phosphorylated PLB (pS16/pT17-PLB) and pT17-PLB. Thus, T17 phosphorylation shows the least capacity for SERCA inhibition relief, and actually decreases the effect of S16 phosphorylation on the same PLB. Quantitative Western blots were used to demonstrate that all four PLB phosphorylation states are present in cardiac SR. Cardiac SR was treated with PLB antibodies and protein kinase A (PKA) to quantify the effects of PLB phosphorylation states on SERCA inhibition in a biological milieu. These results show that each PLB phosphorylation state uniquely alters Ca2+ homeostasis. These effects are relevant to cardiovascular health, disease and treatment.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Identifying cellular mechanisms of zinc-induced relaxation in isolated cardiomyocytes

We tested several molecular and cellular mechanisms of cardiomyocyte contraction-relaxation function that could account for the reduced systolic and enhanced diastolic function observed with exposure to extracellular Zn(2+). Contraction-relaxation function was monitored in isolated rat and mouse cardiomyocytes maintained at 37°C, stimulated at 2 or 6 Hz, and exposed to 32 μM Zn(2+) or vehicle. Intracellular Zn(2+) detected using FluoZin-3 rose to a concentration of ∼13 nM in 3-5 min. Peak sarcomere shortening was significantly reduced and diastolic sarcomere length was elongated after Zn(2+) exposure. Peak intracellular Ca(2+) detected by Fura-2FF was reduced after Zn(2+) exposure. However, the rate of cytosolic Ca(2+) decline reflecting sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) activity and the rate of Na(+)/Ca(2+) exchanger activity evaluated by rapid Na(+)-induced Ca(2+) efflux were unchanged by Zn(2+) exposure. SR Ca(2+) load evaluated by rapid caffeine exposure was reduced by ∼50%, and L-type calcium channel inward current measured by whole cell patch clamp was reduced by ∼70% in cardiomyocytes exposed to Zn(2+). Furthermore, ryanodine receptor (RyR) S2808 and phospholamban (PLB) S16/T17 were markedly dephosphorylated after perfusing hearts with 50 μM Zn(2+). Maximum tension development and thin-filament Ca(2+) sensitivity in chemically skinned cardiac muscle strips were not affected by Zn(2+) exposure. These findings suggest that Zn(2+) suppresses cardiomyocyte systolic function and enhances relaxation function by lowering systolic and diastolic intracellular Ca(2+) concentrations due to a combination of competitive inhibition of Ca(2+) influx through the L-type calcium channel, reduction of SR Ca(2+) load resulting from phospholamban dephosphorylation, and lowered SR Ca(2+) leak via RyR dephosphorylation. The use of the low-Ca(2+)-affinity Fura-2FF likely prevented the detection of changes in diastolic Ca(2+) and SERCA2a function. Other strategies to detect diastolic Ca(2+) in the presence of Zn(2+) are essential for future work.

Molecular Details of SERCA Regulation by Phospholamban Revealed by Paramagnetic Relaxation Enhancements and Solid-State NMR

Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) ia a 110 kDa membrane protein that regulates cardiomyocyte relaxation by transporting Ca2+ into the SR. SERCA is regulated by PLN, a 52-resdiue membrane protein consisting of a helical, inhibitory transmembrane domain and a regulatory cytoplasmic domain. The cytoplasmic domain is in equilibrium between a folded T state and an unfoled R state. Phosphorylation of PLN at S16 relieves the inhibition of SERCA and, based on studies in animal models, mimicking the effects of PLN phosphorylation is a promising path for the development of therapeutics against heart failure.Here we co-reconstituted SERCA and PLN in lipid bilayers under fully functional conditions and utilized solid state NMR to probe the molecular mechanisms of SERCA regulation. Asymmetric spin labeling and isotopic labeling of SERCA, PLN and lipids in combination with magic angle spinning experiments provided paramagnetic relaxation enhancements (PREs) that mapped the structure of the membrane protein complex. PREs were combined with oriented restraints from separated local field experiments, chemical shift perturbations and torsion angles from backbone chemical shifts to determine a structural model of the PLN/SERCA complex. The transmembrane domain of PLN remains anchored to the ATPase, while the cytoplasmic domain is membrane-associated in the T state and interacts transiently with the phosphorylation and nucleotide domains of SERCA in the R state. Phosphorylation increases the R state population and the affinity of the R state for SERCA. These results explain the non-inhibitory character of PLN mutants with increased R state population and provide crucial information for future design of therapeutics to increase SERCA function. In addition, the methodological advances described here further open the landscape to conducting structural studies of membrane protein complexes of molecular weights beyond 100 kDa.

The Role of Heart Failure-Causing Phospholamban R9C Mutation in SERCA Dysfunction and Cardiac Ca2+ Mishandling

The naturally occurring, heterozygous Arg9-to-Cys (R9C) missense mutation of phospholamban (PLB) triggers dilated cardiomyopathy, heart failure and premature death in humans. However, the fundamental molecular mechanism underlying the cardiotoxic role of PLBR9C in regulation of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and cardiomyocyte Ca2+ handling is not clear. The dynamic equilibrium between PLB monomer and pentamer plays a critical role in SERCA regulation. We compared the effect of R9C and pentamer-destabilizing transmembrane triple cysteine mutation (SSS) on PLB pentameric assembly. We co-expressed Cer- or YFP-tagged PLBWT, PLBR9C, PLBSSS, or PLBR9C+SSS in AAV-293 cells and measured intrapentameric Fluorescence Resonance Energy Transfer (FRET). PLBR9C and PLBR9C+SSS exhibited a significant increase in oligomerization as compared to PLBWT and PLBSSS, which was further enhanced by application of 100μM H2O2. In addition, we investigated the stability of PLB pentamer in WT, R9C, SSS, or R9C+SSS background by molecular dynamic simulation studies. Importantly, co-expression of CFP-PLBR9C and YFP-PLBR9C in adult rabbit ventricular myocytes exhibited a large and rapid increase in oligomerization over time after exposure to 100μM H2O2 as compared to PLBWT. To investigate the effect of PLBR9C on SR Ca2+ kinetics, we recorded Ca2+ transients in electrically paced adult cardiomyocytes overexpressing CFP-PLBR9C or CFP-PLBWT. PLBR9C overexpressing cardiomyocytes exhibited faster SR Ca2+ uptake as compared to PLBWT, and a blunted Ca2+ uptake/pacing frequency response. We propose that R9C mutation in PLB increases the stability of the PLB pentameric assembly by crosslinking of adjacent cysteine thiols via disulfide bonds, and these effects are enhanced during oxidative stress. The resultant decrease in availability of PLB monomeric species leads to decrease in inhibition of SERCA, inability to respond to stress, and eventual heart failure.

Phospholamban ablation in hearts expressing the high affinity SERCA2b isoform normalizes global Ca2+ homeostasis but not Ca2+-dependent hypertrophic signaling

Cardiomyocytes from failing hearts exhibit reduced levels of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) and/or increased activity of the endogenous SERCA inhibitor phospholamban. The resulting reduction in the Ca(2+) affinity of SERCA impairs SR Ca(2+) cycling in this condition. We have previously investigated the physiological impact of increasing the Ca(2+) affinity of SERCA by substituting SERCA2a with the higher affinity SERCA2b pump. When phospholamban was also ablated, these double knockouts (DKO) exhibited a dramatic reduction in total SERCA levels, severe hypertrophy, and diastolic dysfunction. We presently examined the role of cardiomyocyte Ca(2+) homeostasis in both functional and structural remodeling in these hearts. Despite the low SERCA levels in DKO, we observed near-normal Ca(2+) homeostasis with rapid Ca(2+) reuptake even at high Ca(2+) loads and stimulation frequencies. Well-preserved global Ca(2+) homeostasis in DKO was paradoxically associated with marked activation of the Ca(2+)-dependent nuclear factor of activated T-cell-calcineurin pathway known to trigger hypertrophy. No activation of the MAP kinase signaling pathway was detected. These findings suggest that local changes in Ca(2+) homeostasis may play an important signaling role in DKO, perhaps due to reduced microdomain Ca(2+) buffering by SERCA2b. Furthermore, alterations in global Ca(2+) homeostasis can also not explain impaired in vivo diastolic function in DKO. Taken together, our results suggest that normalizing global cardiomyocyte Ca(2+) homeostasis does not necessarily protect against hypertrophy and heart failure development and that excessively increasing SERCA Ca(2+) affinity may be detrimental.