Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Ca, M, Po, R, B||ELISA, ICC, IHC, IP, WB||M||Purified||Monoclonal Antibody|
|Presentation||Purified mouse monoclonal IgG1 in buffer containing 20 mM Tris-HCI, pH 7.4, 0.15 M NaCl with 0.05% sodium azide. Frozen solution.|
|Safety Information according to GHS|
|Material Size||50 µg|
|Anti-Phospholamban, clone A1||2474884|
|Anti-Phospholamban, clone A1 - 2137996||2137996|
|Anti-Phospholamban, clone A1 - 2387806||2387806|
|Anti-Phospholamban, clone A1 - 2436413||2436413|
|Anti-Phospholamban, clone A1 - 0606032063||0606032063|
|Anti-Phospholamban, clone A1 - 14658||14658|
|Anti-Phospholamban, clone A1 - 16717||16717|
|Anti-Phospholamban, clone A1 - 17778||17778|
|Anti-Phospholamban, clone A1 - 1988739||1988739|
|Anti-Phospholamban, clone A1 - 21871||21871|
|Reference overview||Application||Pub Med ID|
|Inhibition of miR-25 improves cardiac contractility in the failing heart.|
Wahlquist, C; Jeong, D; Rojas-Muñoz, A; Kho, C; Lee, A; Mitsuyama, S; van Mil, A; Park, WJ; Sluijter, JP; Doevendans, PA; Hajjar, RJ; Mercola, M
Nature 508 531-5 2014
Heart failure is characterized by a debilitating decline in cardiac function, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy. MicroRNAs (miRNAs) are dysregulated in heart failure but whether they control contractility or constitute therapeutic targets remains speculative. Using high-throughput functional screening of the human microRNAome, here we identify miRNAs that suppress intracellular calcium handling in heart muscle by interacting with messenger RNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a (also known as ATP2A2). Of 875 miRNAs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas adeno-associated virus 9 (AAV9)-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 markedly halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR oligonucleotide. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggest that it might be targeted therapeutically to restore function.
|Long term ablation of protein kinase A (PKA)-mediated cardiac troponin I phosphorylation leads to excitation-contraction uncoupling and diastolic dysfunction in a knock-in mouse model of hypertrophic cardiomyopathy.|
Dweck, D; Sanchez-Gonzalez, MA; Chang, AN; Dulce, RA; Badger, CD; Koutnik, AP; Ruiz, EL; Griffin, B; Liang, J; Kabbaj, M; Fincham, FD; Hare, JM; Overton, JM; Pinto, JR
The Journal of biological chemistry 289 23097-111 2014
The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca(2+) decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca(2+)-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca(2+) transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca(2+) content. This abnormal Ca(2+) handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na(+)-Ca(2+) exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.
|Pak1 is required to maintain ventricular Ca²⁺ homeostasis and electrophysiological stability through SERCA2a regulation in mice.|
Wang, Y; Tsui, H; Ke, Y; Shi, Y; Li, Y; Davies, L; Cartwright, EJ; Venetucci, L; Zhang, H; Terrar, DA; Huang, CL; Solaro, RJ; Wang, X; Lei, M
Circulation. Arrhythmia and electrophysiology 7 938-48 2014
Impaired sarcoplasmic reticular Ca(2+) uptake resulting from decreased sarcoplasmic reticulum Ca(2+)-ATPase type 2a (SERCA2a) expression or activity is a characteristic of heart failure with its associated ventricular arrhythmias. Recent attempts at gene therapy of these conditions explored strategies enhancing SERCA2a expression and the activity as novel approaches to heart failure management. We here explore the role of Pak1 in maintaining ventricular Ca(2+) homeostasis and electrophysiological stability under both normal physiological and acute and chronic β-adrenergic stress conditions.Mice with a cardiomyocyte-specific Pak1 deletion (Pak1(cko)), but not controls (Pak1(f/f)), showed high incidences of ventricular arrhythmias and electrophysiological instability during either acute β-adrenergic or chronic β-adrenergic stress leading to hypertrophy, induced by isoproterenol. Isolated Pak1(cko) ventricular myocytes correspondingly showed aberrant cellular Ca(2+) homeostasis. Pak1(cko) hearts showed an associated impairment of SERCA2a function and downregulation of SERCA2a mRNA and protein expression. Further explorations of the mechanisms underlying the altered transcriptional regulation demonstrated that exposure to control Ad-shC2 virus infection increased SERCA2a protein and mRNA levels after phenylephrine stress in cultured neonatal rat cardiomyocytes. This was abolished by the Pak1-knockdown in Ad-shPak1-infected neonatal rat cardiomyocytes and increased by constitutive overexpression of active Pak1 (Ad-CAPak1). We then implicated activation of serum response factor, a transcriptional factor well known for its vital role in the regulation of cardiogenesis genes in the Pak1-dependent regulation of SERCA2a.These findings indicate that Pak1 is required to maintain ventricular Ca(2+) homeostasis and electrophysiological stability and implicate Pak1 as a novel regulator of cardiac SERCA2a through a transcriptional mechanism.
|Impaired contractile function due to decreased cardiac myosin binding protein C content in the sarcomere.|
Cheng, Y; Wan, X; McElfresh, TA; Chen, X; Gresham, KS; Rosenbaum, DS; Chandler, MP; Stelzer, JE
American journal of physiology. Heart and circulatory physiology 305 H52-65 2013
Mutations in cardiac myosin binding protein C (MyBP-C) are a common cause of familial hypertrophic cardiomyopathy (FHC). The majority of MyBP-C mutations are expected to reduce MyBP-C expression; however, the consequences of MyBP-C deficiency on the regulation of myofilament function, Ca²⁺ homeostasis, and in vivo cardiac function are unknown. To elucidate the effects of decreased MyBP-C expression on cardiac function, we employed MyBP-C heterozygous null (MyBP-C+/-) mice presenting decreases in MyBP-C expression (32%) similar to those of FHC patients carrying MyBP-C mutations. The levels of MyBP-C phosphorylation were reduced 53% in MyBP-C+/- hearts compared with wild-type hearts. Skinned myocardium isolated from MyBP-C+/- hearts displayed decreased cross-bridge stiffness at half-maximal Ca²⁺ activations, increased steady-state force generation, and accelerated rates of cross-bridge recruitment at low Ca²⁺ activations (less than 15% and less than 25% of maximum, respectively). Protein kinase A treatment abolished basal differences in rates of cross-bridge recruitment between MyBP-C+/- and wild-type myocardium. Intact ventricular myocytes from MyBP-C+/- hearts displayed abnormal sarcomere shortening but unchanged Ca²⁺ transient kinetics. Despite a lack of left ventricular hypertrophy, MyBP-C+/- hearts exhibited elevated end-diastolic pressure and decreased peak rate of LV pressure rise, which was normalized following dobutamine infusion. Furthermore, electrocardiogram recordings in conscious MyBP-C+/- mice revealed prolonged QRS and QT intervals, which are known risk factors for cardiac arrhythmia. Collectively, our data show that reduced MyBP-C expression and phosphorylation in the sarcomere result in myofilament dysfunction, contributing to contractile dysfunction that precedes compensatory adaptations in Ca²⁺ handling, and chamber remodeling. Perturbations in mechanical and electrical activity in MyBP-C+/- mice could increase their susceptibility to cardiac dysfunction and arrhythmia.
|The effects of neuregulin on cardiac Myosin light chain kinase gene-ablated hearts.|
Chang, AN; Huang, J; Battiprolu, PK; Hill, JA; Kamm, KE; Stull, JT
PloS one 8 e66720 2013
Activation of ErbB2/4 receptor tyrosine kinases in cardiomyocytes by neuregulin treatment is associated with improvement in cardiac function, supporting its use in human patients with heart failure despite the lack of a specific mechanism. Neuregulin infusion in rodents increases cardiac myosin light chain kinase (cMLCK) expression and cardiac myosin regulatory light chain (RLC) phosphorylation which may improve actin-myosin interactions for contraction. We generated a cMLCK knockout mouse to test the hypothesis that cMLCK is necessary for neuregulin-induced improvement in cardiac function by increasing RLC phosphorylation.The cMLCK knockout mice have attenuated RLC phosphorylation and decreased cardiac performance measured as fractional shortening. Neuregulin infusion for seven days in wildtype mice increased cardiac cMLCK protein expression and RLC phosphorylation while increasing Akt phosphorylation and decreasing phospholamban phosphorylation. There was no change in fractional shortening. In contrast, neuregulin infusion in cMLCK knockout animals increased cardiac performance in the absence of cMLCK without increasing RLC phosphorylation. In addition, CaMKII signaling appeared to be enhanced in neuregulin-treated knockout mice.Thus, Neuregulin may improve cardiac performance in the failing heart without increasing cMLCK and RLC phosphorylation by activating other signaling pathways.
|Anthrax lethal toxin induces acute diastolic dysfunction in rats through disruption of the phospholamban signaling network.|
Golden, HB; Watson, LE; Nizamutdinov, D; Feng, H; Gerilechaogetu, F; Lal, H; Verma, SK; Mukhopadhyay, S; Foster, DM; Dillmann, WH; Dostal, DE
International journal of cardiology 168 3884-95 2013
Anthrax lethal toxin (LT), secreted by Bacillus anthracis, causes severe cardiac dysfunction by unknown mechanisms. LT specifically cleaves the docking domains of MAPKK (MEKs); thus, we hypothesized that LT directly impairs cardiac function through dysregulation of MAPK signaling mechanisms.In a time-course study of LT toxicity, echocardiography revealed acute diastolic heart failure accompanied by pulmonary regurgitation and left atrial dilation in adult Sprague-Dawley rats at time points corresponding to dysregulated JNK, phospholamban (PLB) and protein phosphatase 2A (PP2A) myocardial signaling. Using isolated rat ventricular myocytes, we identified the MEK7-JNK1-PP2A-PLB signaling axis to be important for regulation of intracellular calcium (Ca(2+)(i)) handling, PP2A activation and targeting of PP2A-B56α to Ca(2+)(i) handling proteins, such as PLB. Through a combination of gain-of-function and loss-of-function studies, we demonstrated that over-expression of MEK7 protects against LT-induced PP2A activation and Ca(2+)(i) dysregulation through activation of JNK1. Moreover, targeted phosphorylation of PLB-Thr(17) by Akt improved sarcoplasmic reticulum Ca(2+)(i) release and reuptake during LT toxicity. Co-immunoprecipitation experiments further revealed the pivotal role of MEK7-JNK-Akt complex formation for phosphorylation of PLB-Thr(17) during acute LT toxicity.Our findings support a cardiogenic mechanism of LT-induced diastolic dysfunction, by which LT disrupts JNK1 signaling and results in Ca(2+)(i) dysregulation through diminished phosphorylation of PLB by Akt and increased dephosphorylation of PLB by PP2A. Integration of the MEK7-JNK1 signaling module with Akt represents an important stress-activated signalosome that may confer protection to sustain cardiac contractility and maintain normal levels of Ca(2+)(i) through PLB-T(17) phosphorylation.
|Cardiac remodeling and myocardial dysfunction in obese spontaneously hypertensive rats.|
Linz, D; Hohl, M; Mahfoud, F; Reil, JC; Linz, W; Hübschle, T; Juretschke, HP; Neumann-Häflin, C; Rütten, H; Böhm, M
Journal of translational medicine 10 187 2012
The additive effects of obesity and metabolic syndrome on left ventricular (LV) maladaptive remodeling and function in hypertension are not characterized.We compared an obese spontaneously hypertensive rat model (SHR-ob) with lean spontaneously hypertensive rats (SHR-lean) and normotensive controls (Ctr). LV-function was investigated by cardiac magnetic resonance imaging and invasive LV-pressure measurements. LV-interstitial fibrosis was quantified and protein levels of phospholamban (PLB), Serca2a and glucose transporters (GLUT1 and GLUT4) were determined by immunohistochemistry.Systolic blood pressure was similar in SHR-lean and SHR-ob (252 ± 7 vs. 242 ± 7 mmHg, p = 0.398) but was higher when compared to Ctr (155 ± 2 mmHg, p less than 0.01 for both). Compared to SHR-lean and Ctr, SHR-ob showed impaired glucose tolerance and increased body-weight. In SHR-ob, LV-ejection fraction was impaired vs. Ctr (46.2 ± 1.1 vs. 59.6 ± 1.9%, p = 0.007). LV-enddiastolic pressure was more increased in SHR-ob than in SHR-lean (21.5 ± 4.1 vs. 5.9 ± 0.81 mmHg, p = 0.0002) when compared to Ctr (4.3 ± 1.1 mmHg, p less than 0.0001 for both), respectively. Increased LV-fibrosis together with increased myocyte diameters and ANF gene expression in SHR-ob were associated with increased GLUT1-protein levels in SHR-ob suggestive for an upregulation of the GLUT1/ANF-axis. Serca2a-protein levels were decreased in SHR-lean but not altered in SHR-ob compared to Ctr. PLB-phosphorylation was not altered.In addition to hypertension alone, metabolic syndrome and obesity adds to the myocardial phenotype by aggravating diastolic dysfunction and a progression towards systolic dysfunction. SHR-ob may be a useful model to develop new interventional and pharmacological treatment strategies for hypertensive heart disease and metabolic disorders.
|Fructose diet treatment in mice induces fundamental disturbance of cardiomyocyte Ca2+ handling and myofilament responsiveness.|
Mellor, KM; Wendt, IR; Ritchie, RH; Delbridge, LM
American journal of physiology. Heart and circulatory physiology 302 H964-72 2012
High fructose intake has been linked to insulin resistance and cardiac pathology. Dietary fructose-induced myocardial signaling and morphological alterations have been described, but whether cardiomyocyte function is influenced by chronic high fructose intake is yet to be elucidated. The goal of this study was to evaluate the cardiomyocyte excitation-contraction coupling effects of high dietary fructose and determine the capacity for murine cardiomyocyte fructose transport. Male C57Bl/6J mice were fed a high fructose diet for 12 wk. Fructose- and control-fed mouse cardiomyocytes were isolated and loaded with the fura 2 Ca(2+) fluorescent dye for analysis of twitch and Ca(2+) transient characteristics (4 Hz stimulation, 37°C, 2 mM Ca(2+)). Myocardial Ca(2+)-handling protein expression was determined by Western blot. Gene expression of the fructose-specific transporter, GLUT5, in adult mouse cardiomyocytes was detected by real-time and conventional RT-PCR techniques. Diastolic Ca(2+) and Ca(2+) transient amplitude were decreased in isolated cardiomyocytes from fructose-fed mice relative to control (16 and 42%, respectively), coincident with an increase in the time constant of Ca(2+) transient decay (24%). Dietary fructose increased the myofilament response to Ca(2+) (as evidenced by a left shift in the shortening-Ca(2+) phase loop). Protein expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), phosphorylated (P) phospholamban (Ser(16)), and P-phospholamban (Thr(17)) was reduced, and protein phosphatase 2A expression increased, in fructose-fed mouse hearts. Hypertension and cardiac hypertrophy were not evident. These findings demonstrate that fructose diet-associated myocardial insulin resistance induces profound disturbance of cardiomyocyte Ca(2+) handling and responsiveness in the absence of altered systemic loading conditions.
|Changes in myofilament proteins, but not Ca²⁺ regulation, are associated with a high-fat diet-induced improvement in contractile function in heart failure.|
Cheng, Y; Li, W; McElfresh, TA; Chen, X; Berthiaume, JM; Castel, L; Yu, X; Van Wagoner, DR; Chandler, MP
American journal of physiology. Heart and circulatory physiology 301 H1438-46 2011
Pathological conditions such as diabetes, insulin resistance, and obesity are characterized by elevated plasma and myocardial lipid levels and have been reported to exacerbate the progression of heart failure (HF). Alterations in cardiomyocyte Ca(2+) regulatory properties and myofilament proteins have also been implicated in contractile dysfunction in HF. However, our prior studies reported that high saturated fat (SAT) feeding improves in vivo myocardial contractile function, thereby exerting a cardioprotective effect in HF. Therefore, we hypothesized that SAT feeding improves contractile function by altering Ca(2+) regulatory properties and myofilament protein expression in HF. Male Wistar rats underwent coronary artery ligation (HF) or sham surgery (SH) and were fed normal chow (SHNC and HFNC groups) or a SAT diet (SHSAT and HFSAT groups) for 8 wk. Contractile properties were measured in vivo [echocardiography and left ventricular (LV) cannulation] and in isolated LV cardiomyocytes. In vivo measures of contractility (peak LV +dP/dt and -dP/dt) were depressed in the HFNC versus SHNC group but improved in the HFSAT group. Isolated cardiomyocytes from both HF groups were hypertrophied and had decreased percent cell shortening and a prolonged time to half-decay of the Ca(2+) transient versus the SH group; however, SAT feeding reduced in vivo myocyte hypertrophy in the HFSAT group only. The peak velocity of cell shortening was reduced in the HFNC group but not the HFSAT group and was positively correlated with in vivo contractile function (peak LV +dP/dt). The HFNC group demonstrated a myosin heavy chain (MHC) isoform switch from fast MHC-α to slow MHC-β, which was prevented in the HFSAT group. Alterations in Ca(2+) transients, L-type Ca(2+) currents, and protein expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase and phosphorylated phospholamban could not account for the changes in the in vivo contractile properties. In conclusion, the cardioprotective effects associated with SAT feeding in HF may occur at the level of the isolated cardiomyocyte, specifically involving changes in myofilament function but not sarcoplasmic reticulum Ca(2+) regulatory properties.
|Endotoxin-induced uveitis is primarily dependent on radiation-resistant cells and on MyD88 but not TRIF.|
Kezic J, Taylor S, Gupta S, Planck SR, Rosenzweig HL, Rosenbaum JT
Journal of leukocyte biology 90 305-11. Epub 2011 May 24. 2011
TLR4 activation by LPS (endotoxin) is mediated by the MyD88 and TRIF intracellular signaling pathways. We determined the relative activation of these pathways in murine ocular tissue after LPS exposure. Additionally, we explored whether BM-derived or non-BM-derived cells were the major contributors to EIU. Mice deficient in TRIF or MyD88 and their congenic (WT) controls received 250 ng ultrapure LPS ivt at 0 h. Ocular inflammation was assessed by histological analysis at 4, 6, and 24 h, and additionally, in MyD88(-/-) mice, intravital microscopy was performed at 4 h and 6 h to assess adherent, rolling, and infiltrating cells in the iris vasculature and tissue. Cytokines associated with the MyD88 and TRIF intracellular signaling pathways were analyzed in ocular tissue at 4 h. BM chimeric mice (WT→WT, TLR4(-/-)→WT, WT→TLR4(-/-)) received 250 ng LPS by ivt injection, and ocular tissues were examined by histology at 6 h. Lack of MyD88 resulted in a markedly diminished cellular response and reduced production of MyD88-related cytokines 4 h post-LPS treatment. In contrast, lack of TRIF led to reduced production of TRIF-related cytokines and no change in the cellular response to LPS. Therefore, the MyD88 pathway appears to be the dominant TLR4 pathway in EIU. Only WT → TLR4(-/-) chimeric mice were resistant to EIU, and this suggests, surprisingly, that non-BM-derived (radiation-resistant) cells in the eye play a greater role than BM-derived cells.