Sarcoplasmic Reticulum Ca-ATPase Research Articles

Targeting calcium regulators as therapy for heart failure: focus on the sarcoplasmic reticulum Ca-ATPase pump.

Impaired myocardial Ca2+ cycling is a critical contributor to the development of heart failure (HF), causing changes in the contractile function and structure remodeling of the heart. Within cardiomyocytes, the regulation of sarcoplasmic reticulum (SR) Ca2+ storage and release is largely dependent on Ca2+ handling proteins, such as the SR Ca2+ ATPase (SERCA2a) pump. During the relaxation phase of the cardiac cycle (diastole), SERCA2a plays a critical role in transporting cytosolic Ca2+ back to the SR, which helps to restore both cytosolic Ca2+ levels to their resting state and SR Ca2+ content for the next contraction. However, decreased SERCA2a expression and/or pump activity are key features in HF. As a result, there is a growing interest in developing therapeutic approaches to target SERCA2a. This review provides an overview of the regulatory mechanisms of the SERCA2a pump and explores potential strategies for SERCA2a-targeted therapy, which are being investigated in both preclinical and clinical studies.

Poison peptides interactions with the sarcoplasmic reticulum Ca-ATPase (SERCA).

Maintaining cellular integrity requires maintenance of protein quality, so when damaged/misfolded proteins fail to be repaired, they are degraded via proteolysis. Proteolysis increases in heart disease, overwhelming the proteosome, resulting in an accumulation of protein fragments in the cell. We propose that undegraded transmembrane protein fragments found in heart failure may persist in the membrane and interact with SERCA, competing with and displacing the physiological regulatory micropeptide phospholamban. These interactions with “poison peptides” may contribute to calcium mishandling in heart failure. To test this hypothesis, we performed mass spectrometry of membrane fractions isolated from failing human myocardium. We detected approximately 1000 protein fragments of interest in the failing heart, then narrowed our selection to high-abundance transmembrane protein fragments ranging from 20-40 amino acids. We used fluorescence resonance energy transfer (FRET) to test whether seven candidate poison proteins could interact with SERCA. Some peptides did not interact with SERCA at all, some bound weakly, and some bound with a high affinity. The highest affinity candidates bound SERCA as avidly as some native regulatory micropeptides. The candidate peptides tested here include transmembrane fragments of phospholamban (PLB), phospholemman (PLM), calcium voltage-gated channel subunit alpha1 E (CACNA1E) and sarcolemma associated protein (SLMAP). We also tested whether the putative poison peptides altered the structure of SERCA, using time-correlated single photon counting to quantify intramolecular FRET in a “2-color SERCA” construct labeled with mCyRFP-1 and mMaroon1. Overall, the results suggest that transmembrane peptide fragments generated in heart failure can interact with SERCA and may disrupt calcium handling in the failing heart.

Dynamic regulation of SERCA by phospholamban and DWORF

The sarcoplasmic reticulum Ca-ATPase (SERCA) plays a central role in the cardiac cycle and is tightly regulated through interactions with membrane micropeptides. In the heart, phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) activates SERCA. Together, these competing regulators adjust Ca transport to meet changing cardiac demands between rest and exercise. Here, we used fluorescence resonance energy transfer (FRET) to measure how these regulatory interactions change as SERCA cycles through different intermediate conformations in its enzymatic cycle.

Abstract 14141: Role of Pak1 in Sinus Node Function

Introduction: Sinus node (SN) dysfunction (SND) and atrial arrhythmia frequently occur simultaneously with a hazard ratio of 4.2 for new onset atrial fibrillation (AF) in SND patients. We demonstrated previously that loss of p21-activated kinase 1 (Pak1), a serine/threonine kinase increases the propensity for AF by increasing cellular production of reactive oxygen species (ROS). Since pacemaker current (HCN4) is negatively regulated by cellular ROS we hypothesized that loss of Pak1 leads to attenuated SN function. Methods: Heart rate regulation was quantified in wild type (WT) and Pak1 deficient mice (Pak1 -/- ) in vivo and the isolated Langendorff perfused heart using ECGs, bipolar and multi-electrode array derived atrial electrograms. Unpaired t-test was used to compare means between groups. Results: In vivo WT and Pak1 -/- animals exhibited no difference in basal heart rate (WT: 450 ± 26 bpm, n=16; Pak1 -/- : 440 ± 24 bpm, n=13), but intrinsic HR after autonomic blockage, was reduced in Pak1 -/- (WT: 399 ± 32 bpm, n=16; Pak1 -/- : 365 ± 34 bpm, n=13, p<0.05). The result was confirmed in the Langendorff configuration (WT: 359 ± 47 bpm, n=12; Pak1 -/- : 293 ± 47 bpm, n=21, p<0.01) suggesting attenuated SN impulse generation. To determine the contribution of the Ca clock, hearts were perfused with cyclopiazonic acid (5 μM) a blocker of the sarcoplasmic reticulum Ca ATPase. CPA attenuated the HR in WT and Pak1 -/- (WT: -19.6 ± 6.6 % n=2 ; Pak1 -/- : -15.8 ± 7.5 %, n=5) but did not eliminate the difference in HR (WT: 320 ± 37 bpm, n=2; Pak1 -/- : 255 ± 26 bpm, n=5, p<0.05). Block of HCN4 by ivabradine (3 μM), reduced the HR in WT and Pak1 -/- (WT: -45.1 ± 6.3 %, n=6; Pak1 -/- : -22.2 ± 8.6 %, n=7, p<0.001) and eliminated differences in spontaneous activity (WT: 186 ± 43 bpm, n=6; Pak1 -/- : 234 ± 41 bpm, n=7). In vivo the change in HR in response to ß-blocker propranolol (WT: -13.5 ± 2.5 %, n=6; Pak1 -/- : -18.4 ± 1.5 %; n=4, p<0.01) and muscarinic receptor blocker atropine (WT: 9.7 ± 5.0%, n=10; Pak1 -/- : 4.6 ± 4.4 %; n=9, p<0.05) revealed that the attenuated SN impulse generation in Pak1 -/- mice was compensated by an increased sympathetic and attenuated parasympathetic tone. Conclusion: Attenuated Pak1 expression results in suppressed SN impulse generation likely by ROS dependent attenuation of HCN4 expression.

Excitation Contraction Coupling in Hypertrophy and Failing Heart Cells

The contraction of the heart is dependent on a process named the excitation-contraction coupling (E-C coupling). In hypertrophy and failing heart models, the expression, phosphorylation and function of key calcium handling proteins involved in E-C coupling are altered. It’s important to figure out the relationship changes between calcium channel activity and calcium release from sarcoplasmic reticulum (SR). This review will therefore focus on novel components of E-C coupling dysfunction in hypertrophy and failing heart, such as L-type Ca2+ channel (LCC), ryanodine receptor type-2 channel (RyR2) and SR Ca ATPase (SERCA), and how these molecular modifications altered excitation-contraction coupling. A lot of literature was well read and sorted. Recent findings in E-C coupling during hypertrophy and heart failure were focused on. Most importantly, the electrophysiological and signal pathway data was carefully analyzed. This review summarizes key principles and highlights novel aspects of E-C coupling changes during hypertrophy and heart failure models. Although LCC activity changed little, the loss of notch in action potential, reduced Ca2+ transient amplitude and desynchronized Ca2+ sparks resulted in a decreased contraction strength in hypertrophy and heart failure models. What’s more, L-type Ca2+ current becomes ineffective in triggering RyR2 Ca2+ release from SR and the SR uptake is reduced in some models. It has great meanings in understanding the E-C coupling changes during different heart diseases. Theses novel changes suggest potential therapeutic approaches for certain types of hypertrophy and heart failure.

Myocardial Redox Hormesis Protects the Heart of Female Mice in Sepsis.

Mice challenged with lipopolysaccharide develop cardiomyopathy in a sex and redox-dependent fashion. Here we extended these studies to the cecal ligation and puncture (CLP) model.We compared male and female FVB mice (wild type, WT) and transgenic littermates overexpressing myocardial catalase (CAT). CLP induced 100% mortality within 4 days, with similar mortality rates in male and female WT and CAT mice. 24 h after CLP, isolated (Langendorff) perfused hearts showed depressed contractility in WT male mice, but not in male CAT or female WT and CAT mice. In WT male mice, CLP induced a depression of cardiomyocyte sarcomere shortening (ΔSS) and calcium transients (ΔCai), and the inhibition of the sarcoplasmic reticulum Ca ATPase (SERCA). These deficits were associated with overexpression of NADPH-dependent oxidase (NOX)-1, NOX-2, and cyclooxygenase 2 (COX-2), and were partially prevented in male CAT mice. Female WT mice showed unchanged ΔSS, ΔCai, and SERCA function after CLP. At baseline, female WT mice showed partially depressed ΔSS, ΔCai, and SERCA function, as compared with male WT mice, which were associated with NOX-1 overexpression and were prevented in CAT female mice.In conclusion, in male WT mice, septic shock induces myocardial NOX-1, NOX-2, and COX-2, and redox-dependent dysregulation of myocardial Ca transporters. Female WT mice are resistant to CLP-induced cardiomyopathy, despite increased NOX-1 and COX-2 expression, suggesting increased antioxidant capacity. Female resistance occurred in association with NOX-1 overexpression and signs of increased oxidative signaling at baseline, indicating the presence of a protective myocardial redox hormesis mechanism.

Assessment of Na+/K+ ATPase Activity in Small Rodent and Human Skeletal Muscle Samples.

In skeletal muscle, the Na/K ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism; however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for the determination of NKA activity in small rodent muscle samples (5-10 mg) and in human skeletal muscle. Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize "background noise." We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as the addition of 25 μM of blebbistatin, a specific myosin ATPase inhibitor, considerably minimized "background noise" (threefold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. The specificity of the assay was demonstrated after the addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, the addition of blebbistatin did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively. The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.

Live-Cell FRET Biosensors for High-Throughput Screening Targeting the SERCA2a/Plb Complex

We have engineered cell-based high-throughput screening (HTS) methods using time-resolved fluorescence energy transfer (TR-FRET) sensitive to binding and structural dynamics of the SERCA2a/PLB complex. The membrane protein complex between the sarcoplasmic reticulum Ca-ATPase 2a (SERCA2a) and its regulator phospholamban (PLB) is a validated therapeutic target for reversing contractile dysfunction caused by aberrant calcium handling in the heart. Unfortunately, efforts to discover compounds with specificity for this complex have yet to yield an efficacious drug. GFP-SERCA2a (donor) was co-expressed in the endoplasmic reticulum of HEK293 cells with and without RFP-PLB (acceptor), and FRET was measured in a unique fluorescence lifetime microplate reader (FLT-PR), which increases the throughput of high-precision FLT measurements by several orders of magnitude. A triplicate screen against a small-molecule library containing 1500 compounds (LOPAC = Library of Pharmaceutically Active Compounds) identified ten hit compounds that reproducibly changed FRET by >3 SD and alter SERCA's ATPase activity. One compound increases SERCA2a Ca affinity and activity at physiological calcium levels in cardiac SR, but not in skeletal SR, suggesting that the compound is acting specifically on the SERCA2a/PLB complex (Stroik et al., Nature Scientific Reports 8:12560). These properties characterize a drug that could reverse calcium mishandling, as required for treatment of heart disease. We will present recent developments in the development and applications of live-cell FRET biosensors. This work was supported by NIH grants (GM27906, HL129814, AR07612, and DA037622).

Epigallocatechin-3 gallate prevents pressure overload-induced heart failure by up-regulating SERCA2a via histone acetylation modification in mice.

Heart failure is a common, costly, and potentially fatal condition. The cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) plays a critical role in the regulation of cardiac function. Previously, low SERCA2a expression was revealed in mice with heart failure. Epigallocatechin-3-gallate (EGCG) can function as an epigenetic regulator and has been reported to enhance cardiac function. However, the underlying epigenetic regulatory mechanism is still unclear. In this study, we investigated whether EGCG can up-regulate SERCA2a via histone acetylation and play role in preventing heart failure. For this, we generated a mouse model of heart failure by performing a minimally invasive transverse aortic constriction (TAC) operation and used this to test the effects of EGCG. The TAC+EGCG group showed nearly normal cardiac function compared to that in the SHAM group. The expression of SERCA2a was decreased at both the mRNA and protein levels in the TAC group but was enhanced in the TAC+EGCG group. Levels of AcH3 and AcH3K9 were determined to decrease near the promoter region of Atp2a2 (the gene encoding SERCA-2a) in the TAC group, but were elevated in the TAC+EGCG group. Meanwhile, HDAC1 activity and binding near the Atp2a2 promoter were increased in the TAC group but decreased with EGCG addition. Further, binding levels of GATA4 and Mef2c near the Atp2a2 promoter region were reduced in TAC hearts, which might have been caused by histone hypoacetylation; this was reversed by EGCG. Together, upregulation of SERCA2a via the modification of histone acetylation plays a role in EGCG-mediated prevention of pressure overload-induced heart failure, and this might represent a novel pharmacological target for the treatment of heart failure.

Targeting protein-protein interactions for therapeutic discovery via FRET-based high-throughput screening in living cells

We have developed a structure-based high-throughput screening (HTS) method, using time-resolved fluorescence resonance energy transfer (TR-FRET) that is sensitive to protein-protein interactions in living cells. The membrane protein complex between the cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) and phospholamban (PLB), its Ca-dependent regulator, is a validated therapeutic target for reversing cardiac contractile dysfunction caused by aberrant calcium handling. However, efforts to develop compounds with SERCA2a-PLB specificity have yet to yield an effective drug. We co-expressed GFP-SERCA2a (donor) in the endoplasmic reticulum membrane of HEK293 cells with RFP-PLB (acceptor), and measured FRET using a fluorescence lifetime microplate reader. We screened a small-molecule library and identified 21 compounds (Hits) that changed FRET by >3SD. 10 of these Hits reproducibly alter SERCA2a-PLB structure and function. One compound increases SERCA2a calcium affinity in cardiac membranes but not in skeletal, suggesting that the compound is acting specifically on the SERCA2a-PLB complex, as needed for a drug to mitigate deficient calcium transport in heart failure. The excellent assay quality and correlation between structural and functional assays validate this method for large-scale HTS campaigns. This approach offers a powerful pathway to drug discovery for a wide range of protein-protein interaction targets that were previously considered “undruggable”.

Selectivity of the phospholamban ion channel investigated by single channel measurements

Phospholamban (PLN) is a small integral membrane protein, which is involved in the contractility of cardiac muscle. PLN has a dual function: the monomer inhibits Ca2+ transport by the sarcoplasmic reticulum Ca-ATPase (SERCA). In its pentameric form it generates an ion channel, which presumably contributes to a modulation of SERCA activity by balancing the charge generated by Ca2+ uptake into the sarcoplasmic reticulum. The PLN channel is characterized by a low unitary conductance, a sub-conductance and long open/closed dwell times. In this study we investigated the ion selectivity of the PLN channel. PLN is selective for monovalent cations and not permeable to divalent cations (Ca2+ and Ba2+) and anions (chloride and phosphate) over the voltage range investigated. The selectivity for monovalent cations is not determined by ionic radius but it probably involves a well-regulated mechanism of interaction between permeant ions and the binding site(s) in the PLN-generated channel. The finding that the cation selectivity follows the second Eisenman series (Rb > Cs > K > Na > Li) indicates that selectivity is mostly determined by the difference in energy required to remove water from a fully hydrated cation.

Discovery of SERCA2a/PLB Activators and Inhibitors by Structure-Based High-throughput Screening using Live Cell FRET Biosensors

We have developed a cell-based high-throughput screening (HTS) method using time-resolved fluorescence energy transfer (TR-FRET) sensitive to binding and structural dynamics of the SERCA2a/PLB complex. Overwhelming evidence establishes the membrane protein complex between the sarcoplasmic reticulum Ca-ATPase 2a (SERCA2a) and its regulator phospholamban (PLB) as a therapeutic target for reversing cardiac contractile dysfunction caused by aberrant calcium handling. Unfortunately, efforts to discover compounds with specificity for this complex have yet to yield an efficacious drug. GFP-SERCA2a (donor) was co-expressed in the endoplasmic reticulum of HEK293 cells with and without RFP-PLB (acceptor), and FRET was measured in a fluorescence lifetime microplate reader. We carried out a triplicate screen against a small chemical library (LOPAC) and identified 21 compounds that reproducibly changed FRET by >3 SD. Dose-response of FRET and ATPase activity readouts reveal that ten hits reproducibly alter SERCA2a/PLB structure and function. One compound increases SERCA2a Ca affinity and activity (EC50 = 23 µM) at physiological calcium levels in cardiac SR, but not in skeletal SR, suggesting that the compound is acting specifically on the SERCA2a/PLB complex. These properties characterize a drug that could reverse calcium mishandling. The excellent Z’ factor and correlation between structural and functional assays validate this method to discover SERCA2a-PLB complex effectors for large-scale HTS campaigns. This work was supported by NIH grants (GM27906, HL129814, AR07612, and DA037622).

Abstract 354: Combination of Triiodothyronine and Dexamethasone with Flexible Matrigel Substrate Improves Structural and Electrophysiological Maturation of Human iPSC-derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have promise for disease modeling and cell therapy, but electrophysiological immaturity (ie. large pacemaker currents, small I K1 ) and structural immaturity (ie. lack of t-tubules) remain major limitations. Here, we test whether the combination of triiodothyronine (T 3 ) and dexamethasone (Dex) with a soft Matrigel substrate improves hiPSC-CMs maturity. T 3 & Dex or vehicle were added to cardiac induction media for 14 days. Day 30 hiPSC-CMs were plated either on hard or soft Matrigel-coated substrate for 5 days and studied using confocal imaging and voltage clamp. T 3 & Dex treatment significantly increased the width and volume of hiPSC-CMs cultured on soft but not on hard substrate ( Fig.1 ). T 3 & Dex treated hiPSC-CMs exhibited organized subcellular Ca release ( Fig.2 ) and evidence of t-tubule development, possibly as a result of increased Bin-1 and Caveolin-3 membrane trafficking. The combination of T 3 & Dex and soft substrate increased Ca spark frequency and the amount of Ca released per spark ( Fig.3 ). T 3 & Dex treatment significantly accelerated Ca transient decay under field stimulation and increased sarcoplasmic reticulum Ca-ATPase activity. Compared to vehicle, T 3 & Dex treatment significantly improved electrophysiological maturity: Pacemaker current (I f ) density was 3-fold reduced (-2.7±0.35 vs. -6.2±1.3 pA/pF) and inward rectifier current (I K1 ) density was 4-fold increased (-19.3±2.2 vs. -4.4±0.5 pA/pF). Combining T 3 & Dex and soft Matrigel substrate enhances structural and functional maturation of hiPSC-CMs, which will increase the utility of hiPSC-CM for disease modeling and cell therapy.

Liguzinediol Enhances the Inotropic Effect of Rat Hearts via Inhibition of Protein Phosphatase (PP1 and PP2A) Activities.

It has been demonstrated that liguzinediol (2,5-dihydroxymethyl-3,6-dimethylpyrazine, LZDO), a derivative of ligustrazine from Ligusticum wallichii Franch, exerts positive inotropy in isolated rat heart mediated by the sarcoplasmic reticulum Ca-ATPase (SERCA2a). Here, we further explore the underlying mechanism of the positive inotropic effect of LZDO in rat hearts. In vivo and ex vivo rat heart experiments, biochemistry, and Western blot techniques were used to analyze the rat heart contractility, and SERCA2a activity, phospholamban (PLB) phosphorylation, and protein phosphatase (PP1 and PP2A) activities in rat left ventricular myocytes, respectively. LZDO (20 mg/kg) significantly increased the inotropy of rat heart in vivo. In isolated rat heart experiments, LZDO (100 μM) restored the decreased inotropy induced by caffeine (0.5 mM); however, calyculin A (4 nM), an inhibitor of PP1 and PP2A, eliminated the inotropic effect of LZDO (100 μM). Moreover, LZDO (1, 10, and 100 μM) significantly enhanced SERCA2a activity and increased the levels of phosphorylated PLB on both serine-16 (Ser-16) and threonine-17 (Thr-17). In addition, LZDO (100 μM) significantly inhibited the activities of PP1 and PP2A. The positive inotropic effects of LZDO on in vivo and ex vivo rat hearts seem to be mediated through inhibition of PP1/PP2A, which may suppress dephosphorylated PLB and enhance SERCA2a activity. LZDO may prove effective in treating heart failure in clinical settings based on its unique biological mechanism.

Heart Failure Re-Distributes Phospholamban between Nuclear Membranes and Sarcoplasmic Reticulum in Cardiomyocytes

We recently documented that phospholamban (PLB), a sarcoplasmic reticulum (SR) resident protein regulator of cardiac SR Ca-ATPase (SERCA2a), is localized to the nuclear envelope (NE) of cardiomyocytes (CMs). It is well-known that heart failure (HF) remodeling involves altered protein expression of PLB and SERCA2a; however, with the demonstrated compartmentalization of PLB accumulation, we asked whether HF alters trafficking between ER/SR subcompartments affecting steady-state intracellular distribution of PLB and SERCA. In this study, we examined heart tissue from non-failing and pacing-induced HF dogs with the monoclonal PLB (2D12 or 1F1) and SERCA (2A7-A1) antibodies. Confocal immunofluorescence microscopy was used to compare subcellular concentration of PLB and SERCA. At least 3 regions from NE and SR in 10 CMs were analyzed for each dog, n = 4 dogs). Both PLB and SERCA antibodies stained SR and NE in these CMs. Fluorescence intensity ratios between NE and SR for PLB was 2.05±0.82 (mean ± SD) in control dog CMs, consistent with results just reported. In HF CMs we found a highly decreased ratio of PLB between NE and SR to 1.26 ± 0.34 (n = 8 dogs). In contrast to re-distribution of PLB, intensity ratios for SERCA in NE and SR remained unchanged (1.01±0.14 in Control, and 0.98±0.26 in HF). Furthermore, while there was only minor levels of phosphoPLB in Control CMs, we found that phosphoPLB (S16) were very significantly higher in NE of failed CMs. The decreased PLB relative to SERCA in the NE of CMs suggests that retention and accumulation of PLB in the NE is decreased in HF, leading to a smaller, possibly hyper-phosphorylated steady-state of PLB concentration in the NE.

Distinct Myocardial Mechanisms Underlie Cardiac Dysfunction in Endotoxemic Male and Female Mice.

In male mice, sepsis-induced cardiomyopathy develops as a result of dysregulation of myocardial calcium (Ca) handling, leading to depressed cellular Ca transients (ΔCai). ΔCai depression is partially due to inhibition of sarcoplasmic reticulum Ca ATP-ase (SERCA) via oxidative modifications, which are partially opposed by cGMP generated by the enzyme soluble guanylyl cyclase (sGC). Whether similar mechanisms underlie sepsis-induced cardiomyopathy in female mice is unknown.Male and female C57Bl/6J mice (WT), and mice deficient in the sGC α1 subunit activity (sGCα1), were challenged with lipopolysaccharide (LPS, ip). LPS induced mouse death and cardiomyopathy (manifested as the depression of left ventricular ejection fraction by echocardiography) to a similar degree in WT male, WT female, and sGCα1 male mice, but significantly less in sGCα1 female mice. We measured sarcomere shortening and ΔCai in isolated, externally paced cardiomyocytes, at 37°C. LPS depressed sarcomere shortening in both WT male and female mice. Consistent with previous findings, in male mice, LPS induced a decrease in ΔCai (to 30 ± 2% of baseline) and SERCA inhibition (manifested as the prolongation of the time constant of Ca decay, τCa, to 150 ± 5% of baseline). In contrast, in female mice, the depression of sarcomere shortening induced by LPS occurred in the absence of any change in ΔCai, or SERCA activity. This suggested that, in female mice, the causative mechanism lies downstream of the Ca transients, such as a decrease in myofilament sensitivity for Ca. The depression of sarcomere shortening shortening after LPS was less severe in female sGCα1 mice than in WT female mice, indicating that cGMP partially mediates cardiomyocyte dysfunction.These results suggest, therefore, that LPS-induced cardiomyopathy develops through distinct sex-specific myocardial mechanisms. While in males LPS induces sGC-independent decrease in ΔCai, in female mice LPS acts downstream of ΔCai, possibly via sGC-dependent myofilament dysfunction.

Dyssynchronous calcium removal in heart failure-induced atrial remodeling.

We tested the hypothesis that in atrial myocytes from a rabbit left ventricular heart failure (HF) model, spatial inhomogeneity and temporal dyssynchrony of Ca removal during excitation-contraction coupling together with increased Na/Ca exchange (NCX) activity generate a substrate for proarrhythmic Ca release. Ca removal occurs via Ca reuptake into the sarcoplasmic reticulum and extrusion via NCX exclusively in the cell periphery since rabbit atrial myocytes lack transverse tubules. Ca removal kinetics were assessed by the time constant τ of decay of local peripheral subsarcolemmal (SS) and central (CT) action potential (AP)-induced Ca transients (CaTs) recorded in confocal line scan mode (using Fluo-4). Spatial and temporal dyssynchrony of Ca removal was quantified by CV TAU, defined as the standard deviation of local τ along the transverse cell axis divided by mean τ. In normal cells CT CaT decline was slower compared with the SS domain, while in HF cells decline was accelerated, became equal in SS and CT regions, and a significant increase of CV TAU indicated an increased Ca removal dyssynchrony. In HF atrial cells NCX upregulation was accompanied by an overall higher incidence of spontaneous Ca waves and a higher propensity of arrhythmogenic Ca waves, defined as waves that triggered APs due to NCX-mediated membrane depolarization. NCX inhibition normalized CV TAU in HF atrial cells and decreased the propensity of Ca waves. In summary, HF atrial myocytes show accelerated but dyssynchronous diastolic Ca removal and altered sarcoplasmic reticulum Ca-ATPase (SERCA) and NCX activity that result in increased susceptibility to arrhythmia.

Sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) reduces the migratory capacity of CCL21-treated monocyte-derived dendritic cells.

The migration of dendritic cells (DCs) to secondary lymphoid organs depends on chemoattraction through the interaction of the chemokine receptors with chemokines. However, the mechanism of how lymphoid chemokines attract DCs to lymphoid organs remains unclear. Here, we demonstrate the mechanism of DC migration in response to the lymphoid chemokine CCL21. CCL21-mediated DC migration is controlled by the regulation of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) expression rather than through the activation of mitogen-activated protein kinases CCL21-exposed mature DCs (mDCs) exhibited decreased SERCA2 expression but not decreased phospholamban (PLB) or Hax-1 expression, which are known to be SERCA2-interacting proteins. In addition, CCL21 did not affect the mRNA levels of SERCA2 or its interacting protein Hax-1. Interestingly, SERCA2 expression was inversely related to DC migration in response to chemokine stimulation. The migratory capacity of CCL21-treated mDCs was decreased by the phospholipase C inhibitor U73122 and by the protein kinase C inhibitor BAPTA-AM. The migratory capacities of mDCs were increased in response to SERCA2 siRNA expression but were decreased by SERCA2 overexpression. In addition, DCs treated with a SERCA2-specific inhibitor (cyclopiazonic acid) had significantly increased migratory capacities as mDCs regardless of SERCA2 expression. Moreover, SERCA2 expression was dependent on DC maturation induced by cytokines or Toll-like receptor agonists. Therefore, the migratory capacities differed in differentially matured DCs. Taken together, these results suggest that SERCA2 contributes to the migration of CCL21-activated DCs as an important feature of the adaptive immune response and provide novel insights regarding the role of SERCA2 in DC functions.

Induced NCX1 overexpression attenuates pressure overload-induced pathological cardiac remodelling.

Although increased Na(+)/Ca(2+) exchanger 1 (NCX1) expression is observed during heart failure (HF), the pathological role of NCX1 during the progression of HF remains unclear. We examined alterations of NCX1 expression and activity in hearts after transverse aortic constriction (TAC) surgery and explored whether NCX1 influences pressure overload-induced pathological cardiac remodelling. We generated novel transgenic mice in which NCX1 expression is controlled by a cardiac-specific, doxycycline (DOX)-dependent promoter. In the absence of DOX, TAC surgery caused substantial chamber dilation with a gradual decrease in contractility by 16 weeks. Cardiomyocytes showed a decline in contractility with abnormal Ca(2+) handling during excitation-contraction (E-C) coupling. Reduced NCX1 activity was observed 8 weeks after TAC and was still apparent at 17 weeks. Induced NCX1 overexpression by DOX treatment starting 8 weeks after TAC returned NCX1 activity to pre-TAC levels and prevented chamber dilation with cardiac dysfunction. DOX treatment not only upregulated NCX1 expression in TAC-operated hearts but also returned L-type Ca(2+) channel and sarcoplasmic reticulum (SR) Ca(2+) ATPase expression levels to those in sham-operated hearts. In DOX-treated myocytes, contractility, T-tubule integrity, synchrony of Ca(2+) release from the SR, and Ca(2+) handling during E-C coupling was preserved 16 weeks after TAC surgery. In addition, DOX treatment attenuated the down-regulation of survival signalling and up-regulation of apoptosis signalling 16 weeks after TAC surgery. Induced overexpression of NCX1 attenuated pressure overload-induced pathological cardiac remodelling. Thus, maintaining NCX1 activity may be a potential therapeutic strategy for preventing the progression of HF.

Exogenous hydrogen sulphide ameliorates diabetic cardiomyopathy in rats by reversing disordered calcium-handling system in sarcoplasmic reticulum.

Hydrogen sulphide (H2 S) has been found to be involved in cardiovascular diseases, but the exact mechanism has not been clarified. The purpose of this study was to investigate whether sodium hydrogen sulphide (NaHS), the donor of H2 S, can improve diabetic cardiomyopathy by reversing disordered calcium-handling system in sarcoplasmic reticulum (SR). Sprague Dawley rats were injected with streptozotocin (STZ, 60 mg/kg, i.p.) to build diabetic model. Treatment groups included: aminoguanidine group (AG, 100 mg/kg, p.o.) and NaHS group (5 mg/kg per day, s.c.). Cardiac dysfunction and myocardial hypertrophy were found in diabetic model (DM) group, along with increased ROS levels and upregulated mRNA and protein expressions of NADPH p22(phox) , endothelin A receptor (ETA ) and protein kinase Cε (PKCε). Expressions of calcium-handling proteins in SR including FK506-binding proteins (FKBP12.6), sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) and calsequestrin 2 (CASQ2) were downregulated in DM group, accompanied by elevated concentration of diastolic free calcium in high glucose-incubated cardiomyocytes, indicating of calcium leak. After treated by NaHS, these abnormalities were attenuated significantly. Exogenous H2 S played a protective role in diabetic cardiomyopathy by inhibiting abnormal calcium-handling system in SR and ET-NADPH oxidase-PKCε pathway.