Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|M, R, H, B, Ca, Po, Rb||WB||Rb||Affinity Purified||Polyclonal Antibody|
|Application||This Anti-phospho-Phospholamban (Ser16) Antibody is validated for use in WB for the detection of phospho-Phospholamban (Ser16).|
|Safety Information according to GHS|
|Material Size||100 µg|
References | 26 Available | See All References
|Reference overview||Application||Pub Med ID|
|Atrial fibrillation complicated by heart failure induces distinct remodeling of calcium cycling proteins. |
Lugenbiel, P; Wenz, F; Govorov, K; Schweizer, PA; Katus, HA; Thomas, D
PloS one 10 e0116395 2015
Atrial fibrillation (AF) and heart failure (HF) are two of the most common cardiovascular diseases. They often coexist and account for significant morbidity and mortality. Alterations in cellular Ca2+ homeostasis play a critical role in AF initiation and maintenance. This study was designed to specifically elucidate AF-associated remodeling of atrial Ca2+ cycling in the presence of mild HF. AF was induced in domestic pigs by atrial burst pacing. The animals underwent electrophysiologic and echocardiographic examinations. Ca2+ handling proteins were analyzed in right atrial tissue obtained from pigs with AF (day 7; n = 5) and compared to sinus rhythm (SR) controls (n = 5). During AF, animals exhibited reduction of left ventricular ejection fraction (from 73% to 58%) and prolonged atrial refractory periods. AF and HF were associated with suppression of protein kinase A (PKA)RII (-62%) and Ca2+-calmodulin-dependent kinase II (CaMKII) δ by 37%, without changes in CaMKIIδ autophosphorylation. We further detected downregulation of L-type calcium channel (LTCC) subunit α2 (-75%), sarcoplasmic reticulum Ca2+-ATPase (Serca) 2a (-29%), phosphorylated phospholamban (Ser16, -92%; Thr17, -70%), and phospho-ryanodine receptor 2 (RyR2) (Ser2808, -62%). Na+-Ca2+ exchanger (NCX) levels were upregulated (+473%), whereas expression of Ser2814-phosphorylated RyR2 and LTCCα1c subunits was not significantly altered. In conclusion, AF produced distinct arrhythmogenic remodeling of Ca2+ handling in the presence of tachycardia-induced mild HF that is different from AF without structural alterations. The changes may provide a starting point for personalized approaches to AF treatment.
|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.
|Noncanonical EF-hand motif strategically delays Ca2+ buffering to enhance cardiac performance. |
Wang, W; Barnabei, MS; Asp, ML; Heinis, FI; Arden, E; Davis, J; Braunlin, E; Li, Q; Davis, JP; Potter, JD; Metzger, JM
Nature medicine 19 305-12 2013
EF-hand proteins are ubiquitous in cell signaling. Parvalbumin (Parv), the archetypal EF-hand protein, is a high-affinity Ca(2+) buffer in many biological systems. Given the centrality of Ca(2+) signaling in health and disease, EF-hand motifs designed to have new biological activities may have widespread utility. Here, an EF-hand motif substitution that had been presumed to destroy EF-hand function, that of glutamine for glutamate at position 12 of the second cation binding loop domain of Parv (ParvE101Q), markedly inverted relative cation affinities: Mg(2+) affinity increased, whereas Ca(2+) affinity decreased, forming a new ultra-delayed Ca(2+) buffer with favorable properties for promoting cardiac relaxation. In therapeutic testing, expression of ParvE101Q fully reversed the severe myocyte intrinsic contractile defect inherent to expression of native Parv and corrected abnormal myocardial relaxation in diastolic dysfunction disease models in vitro and in vivo. Strategic design of new EF-hand motif domains to modulate intracellular Ca(2+) signaling could benefit many biological systems with abnormal Ca(2+) handling, including the diseased heart.
|Lifelong exposure to bisphenol a alters cardiac structure/function, protein expression, and DNA methylation in adult mice. |
Patel, BB; Raad, M; Sebag, IA; Chalifour, LE
Toxicological sciences : an official journal of the Society of Toxicology 133 174-85 2013
Bisphenol A (BPA) is an estrogenizing endocrine disruptor compound of concern. Our objective was to test whether lifelong BPA would impact cardiac structure/function, calcium homeostasis protein expression, and the DNA methylation of cardiac genes. We delivered 0.5 and 5.0 µg/kg/day BPA lifelong from gestation day 11 or 200 µg/kg/day from gestation day 11 to postnatal day 21 via the drinking water to C57bl/6n mice. BPA 5.0 males and females had increased body weight, body mass index, body surface area, and adiposity. Echocardiography identified concentric remodeling in all BPA-treated males. Systolic and diastolic cardiac functions were essentially similar, but lifelong BPA enhanced male and reduced female sex-specific differences in velocity of circumferential shortening and ascending aorta velocity time integral. Diastolic blood pressure was increased in all BPA females. The calcium homeostasis proteins sarcoendoplasmic reticulum ATPase 2a (SERCA2a), sodium calcium exchanger-1, phospholamban (PLB), phospho-PLB, and calsequestrin 2 are important for contraction and relaxation. Changes in their expression suggest increased calcium mobility in males and reduced calcium mobility in females supporting the cardiac function changes. DNA methyltransferase 3a expression was increased in all BPA males and BPA 0.5 females and reduced in BPA 200 females. Global DNA methylation was increased in BPA 0.5 males and reduced in BPA 0.5 females. BPA induced sex-specific altered DNA methylation in specific CpG pairs in the calsequestrin 2 CpG island. These results suggest that continual exposure to BPA impacts cardiac structure/function, protein expression, and epigenetic DNA methylation marks in males and females.
|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.
|SERCA Cys674 sulphonylation and inhibition of L-type Ca2+ influx contribute to cardiac dysfunction in endotoxemic mice, independent of cGMP synthesis. |
Hobai, IA; Buys, ES; Morse, JC; Edgecomb, J; Weiss, EH; Armoundas, AA; Hou, X; Khandelwal, AR; Siwik, DA; Brouckaert, P; Cohen, RA; Colucci, WS
American journal of physiology. Heart and circulatory physiology 305 H1189-200 2013
The goal of this study was to identify the cellular mechanisms responsible for cardiac dysfunction in endotoxemic mice. We aimed to differentiate the roles of cGMP [produced by soluble guanylyl cyclase (sGC)] versus oxidative posttranslational modifications of Ca(2+) transporters. C57BL/6 mice [wild-type (WT) mice] were administered lipopolysaccharide (LPS; 25 μg/g ip) and euthanized 12 h later. Cardiomyocyte sarcomere shortening and Ca(2+) transients (ΔCai) were depressed in LPS-challenged mice versus baseline. The time constant of Ca(2+) decay (τCa) was prolonged, and sarcoplasmic reticulum Ca(2+) load (CaSR) was depressed in LPS-challenged mice (vs. baseline), indicating decreased activity of sarco(endo)plasmic Ca(2+)-ATPase (SERCA). L-type Ca(2+) channel current (ICa,L) was also decreased after LPS challenge, whereas Na(+)/Ca(2+) exchange activity, ryanodine receptors leak flux, or myofilament sensitivity for Ca(2+) were unchanged. All Ca(2+)-handling abnormalities induced by LPS (the decrease in sarcomere shortening, ΔCai, CaSR, ICa,L, and τCa prolongation) were more pronounced in mice deficient in the sGC main isoform (sGCα1(-/-) mice) versus WT mice. LPS did not alter the protein expression of SERCA and phospholamban in either genotype. After LPS, phospholamban phosphorylation at Ser(16) and Thr(17) was unchanged in WT mice and was increased in sGCα1(-/-) mice. LPS caused sulphonylation of SERCA Cys(674) (as measured immunohistochemically and supported by iodoacetamide labeling), which was greater in sGCα1(-/-) versus WT mice. Taken together, these results suggest that cardiac Ca(2+) dysregulation in endotoxemic mice is mediated by a decrease in L-type Ca(2+) channel function and oxidative posttranslational modifications of SERCA Cys(674), with the latter (at least) being opposed by sGC-released cGMP.
|Myocardial ATGL overexpression decreases the reliance on fatty acid oxidation and protects against pressure overload-induced cardiac dysfunction. |
Kienesberger, PC; Pulinilkunnil, T; Sung, MM; Nagendran, J; Haemmerle, G; Kershaw, EE; Young, ME; Light, PE; Oudit, GY; Zechner, R; Dyck, JR
Molecular and cellular biology 32 740-50 2012
Alterations in myocardial triacylglycerol content have been associated with poor left ventricular function, suggesting that enzymes involved in myocardial triacylglycerol metabolism play an important role in regulating contractile function. Myocardial triacylglycerol catabolism is mediated by adipose triglyceride lipase (ATGL), which is rate limiting for triacylglycerol hydrolysis. To address the influence of triacylglycerol hydrolysis on myocardial energy metabolism and function, we utilized mice with cardiomyocyte-specific ATGL overexpression (MHC-ATGL). Biochemical examination of MHC-ATGL hearts revealed chronically reduced myocardial triacylglycerol content but unchanged levels of long-chain acyl coenzyme A esters, ceramides, and diacylglycerols. Surprisingly, fatty acid oxidation rates were decreased in ex vivo perfused working hearts from MHC-ATGL mice, which was compensated by increased rates of glucose oxidation. Interestingly, reduced myocardial triacylglycerol content was associated with moderately enhanced in vivo systolic function in MHC-ATGL mice and increased isoproterenol-induced cell shortening of isolated primary cardiomyocytes. Most importantly, MHC-ATGL mice were protected from pressure overload-induced systolic dysfunction and detrimental structural remodeling following transverse aortic constriction. Overall, this study shows that ATGL overexpression is sufficient to alter myocardial energy metabolism and improve cardiac function.
|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.
|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.
|Cardiac response to doxorubicin and dexrazoxane in intact and ovariectomized young female rats at rest and after swim training. |
Calvé, A; Haddad, R; Barama, SN; Meilleur, M; Sebag, IA; Chalifour, LE
American journal of physiology. Heart and circulatory physiology 302 H2048-57 2012
The impact of cancer therapies on adult cardiac function is becoming a concern as more children survive their initial cancer. Cardiovascular disease is now a significant problem to adult survivors of childhood cancer. Specifically, doxorubicin (DOX) may be particularly harmful in young girls. The objective of this study was to characterize DOX damage and determine the ability of dexrazoxane (DEX) to reduce DOX-mediated cardiac damage in sedentary and swim-trained female rats. Female Sprague-Dawley rats were left intact or ovariectomized (OVX) at weaning then injected with DEX (60 mg/kg) before DOX (3 mg/kg), DOX alone, or PBS. Rats were separated into sedentary and swim cohorts. Body weight was reduced in DOX:DEX- but not PBS- or DOX-treated rats. Echocardiographic parameters were similar in sedentary rats. Swim training revealed greater concentric remodeling in DOX-treated rats and reduced fractional shortening in DOX:DEX-treated rats. Calsequestrin 2 was reduced with DOX and increased with DOX:DEX postswim. Sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a was reduced and calsequestrin 2 reduced further by swim training only in intact rats. OVX rats were heavier and developed eccentric remodeling post-swim with DOX and eccentric hypertrophy with DOX:DEX. Changes in SERCA2a and calsequestrin 2 expression were not observed. Ovariectomized DOX- and DOX:DEX-treated rats stopped growing during swim training. DEX coinjection did not relieve DOX-mediated cardiotoxicity in intact or hormone-deficient rats. DOX-mediated reductions in growth, cardiac function, and expression of calcium homeostasis proteins were exacerbated by swim. DEX coadministration did not substantially relieve DOX-mediated cardiotoxicity in young female rats. Ovarian hormones reduce DOX-induced cardiotoxicity.
|Aromatase deficiency confers paradoxical postischemic cardioprotection. |
Bell, JR; Mellor, KM; Wollermann, AC; Ip, WT; Reichelt, ME; Meachem, SJ; Simpson, ER; Delbridge, LM
Endocrinology 152 4937-47 2011
The conventional view is that estrogen confers female cardioprotection. Estrogen synthesis depends on androgen availability, with aromatase regulating conversion of testosterone to estradiol. Extragonadal aromatase expression mediates estrogen production in some tissues, but a role for local steroid conversion has not yet been demonstrated in the heart. This study's goal was to investigate how aromatase deficiency influences myocardial function and ischemic resilience. RT-PCR analysis of C57Bl/6 mouse hearts confirmed cardiac-specific aromatase expression in adult females. Functional performance of isolated hearts from female aromatase knockout (ArKO) and aromatase wild-type mice were compared. Left ventricular developed pressures were similar in aerobic perfusion, but the maximal rate of rise of ventricular pressure was modestly reduced in ArKO hearts (3725 ± 144 vs. 4272 ± 154 mm Hg/sec, P less than 0.05). After 25 min of ischemia, the recovery of left ventricular developed pressure was substantially improved in ArKO (percentage of basal at 60 min of reperfusion, 62 ± 8 vs. 30 ± 6%; P less than 0.05). Hypercontracture was attenuated (end diastolic pressure, 25 ± 5 vs. 51 ± 1 mm Hg; P less than 0.05), and lactate dehydrogenase content of coronary effluent was reduced throughout reperfusion in ArKO hearts. This was associated with a hyperphosphorylation of phospholamban and a reduction in phosphorylated Akt. Immediately after reperfusion, ArKO hearts exhibited increased incidence of ventricular premature beats (194 ± 70 vs. 46 ± 6, P less than 0.05). These observations indicate more robust functional recovery, reduced cellular injury, and modified cardiomyocyte Ca(2+) handling in aromatase-deficient hearts. Our findings indicate that androgen-to-estrogen conversion may be of pathophysiologic importance to the heart and challenge the notion that estrogen deficiency is deleterious. These studies suggest the possibility that aromatase suppression may offer inotropic benefit in the acute ischemia/reperfusion setting with appropriate arrhythmia management.
|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.
|β-adrenergic receptor blockade reduces endoplasmic reticulum stress and normalizes calcium handling in a coronary embolization model of heart failure in canines. |
George, I; Sabbah, HN; Xu, K; Wang, N; Wang, J
Cardiovascular research 91 447-55 2011
Alterations in calcium homeostasis in the endoplasmic/sarcoplasmic reticulum (ER) cause ER stress that ultimately may affect ventricular function. However, the role of ER stress in β-blocker therapy for congestive heart failure (CHF) has not been studied. This study examined ER stress in CHF and evaluated its role in β-blocker therapy in a canine model of ischaemic CHF.CHF was created by daily coronary embolization in chronically instrumented dogs. After oral administration of β-blocker metoprolol or vehicle for 12 weeks, Ca(2+) transport proteins including sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), ryanodine receptor (RyR2), Na(+)-Ca(2+) exchanger (NCX1), Ca(2+) storage protein calreticulin (CRT), and phospholamban were evaluated by Western blot analysis. Cellular levels of ER stress marker, phosphorylated eukaryotic initiation factor 2α (eIF2α-P), were also examined. Compared with the vehicle-treated group, metoprolol caused significantly improved cardiac function, restored the proteins of SERCA2a, NCX1, and CRT, increased phosphorylated phospholamban, reversed protein kinase A hyperphosphorylation of RyR2, and resulted in normalized ER stress marker eIF2α-P and reduced DNA damage.Our results suggest that ER stress could be induced by abnormal Ca(2+) homeostasis in CHF. The restoration of calcium-handling protein function and resultant decrease in ER stress might, in part, explain the beneficial effects of β-blockade observed in CHF. Whether this mechanism occurs in other animal CHF models or human CHF warrants further study.
|AFos inhibits phenylephrine-mediated contractile dysfunction by altering phospholamban phosphorylation. |
Jeong MY, Walker JS, Brown RD, Moore RL, Vinson CS, Colucci WS, Long CS
Am J Physiol Heart Circ Physiol 298 H1719-26. Epub 2010 Apr 2. 2010
Using neonatal rat ventricular myocytes, we previously reported that the expression of a dominant negative form of the c-Fos proto-oncogene (AFos) inhibited activator protein 1 activity and blocked the induction of the pathological gene profile stimulated by phenylephrine (PE) while leaving growth unaffected. We now extend these observations to the adult rat ventricular myocyte (ARVM) to understand the relationship between gene expression, growth, and function. Ventricular myocytes were isolated from adult rats and infected with adenovirus expressing beta-galactosidase (control) or AFos. The cells were subsequently treated with PE, and protein synthesis, gene program, calcium transients, and contractility were evaluated. As seen with the neonatal rat ventricular myocytes, in control cells PE stimulated an increase in protein synthesis, induced the pathological gene profile, and exhibited both depressed contractility and calcium transients. Although ARVMs expressing AFos still had PE-induced growth, pathological gene expression as well as contractility and calcium handling abnormalities were inhibited. To determine a possible mechanism of the preserved myocyte function in AFos-expressing cells, we examined phospholamban (PLB) and sarco(endo)plasmic reticulum calcium-ATPase proteins. Although there was no change in total PLB or sarco(endo)plasmic reticulum calcium-ATPase expression in response to PE treatment, PE decreased the phosphorylation of PLB at serine-16, an observation that was prevented in AFos-expressing cells. In conclusion, although PE-induced growth was unaffected in AFos-expressing ARVMs, the expression of the pathological gene profile was inhibited and both contractile function and calcium cycling were preserved. The inhibition of functional deterioration was, in part, due to the preservation of PLB phosphorylation.Full Text Article
|Novel bradykinin signaling in adult rat cardiac myocytes through activation of p21-activated kinase. |
Yunbo Ke,Katherine A Sheehan,E Eroume A Egom,Ming Lei,R John Solaro
American journal of physiology. Heart and circulatory physiology 298 2010
Although bradykinin (BK) is known to exert effects on the myocardium, its intracellular signaling pathways remain poorly understood. Experiments in other cell types indicated that p21-activated kinase-1 (Pak1), a Ser/Thr kinase downstream of small monomeric G proteins, is activated by BK. We previously reported that the expression of active Pak1 in adult cardiac myocytes induced activation of protein phosphatase 2A and dephosphorylation of myofilament proteins (Ke et al. Circ Res 94: 194-200, 2004). In experiments reported here, we tested the hypothesis that BK signals altered protein phosphorylation in adult rat cardiac myocytes through the activation and translocation of Pak1. Treatment of myocytes with BK resulted in the activation of Pak1 as demonstrated by increased autophosphorylation at Thr423 and a diminished striated localization, which is present in the basal state. BK induced dephosphorylation of both cardiac troponin I and phospholamban. Treatment of isolated myocytes with BK also blunted the effect of isoproterenol to enhance peak Ca(2+) and relaxation of Ca(2+) transients. Protein phosphatase 2A was demonstrated to associate with both Pak 1 and phospholamban. Our studies indicate a novel signaling mechanism for BK in adult rat cardiac myocytes and support our hypothesis that Pak 1 is a significant regulator of phosphatase activity in the heart.Full Text Article
|Inhibition of protein kinase C-beta by ruboxistaurin preserves cardiac function and reduces extracellular matrix production in diabetic cardiomyopathy. |
Connelly, KA; Kelly, DJ; Zhang, Y; Prior, DL; Advani, A; Cox, AJ; Thai, K; Krum, H; Gilbert, RE
Circulation. Heart failure 2 129-37 2009
Heart failure is a common cause of morbidity and mortality in diabetic patients that frequently manifests in the absence of impaired left ventricular systolic function. In contrast to the strong evidence base for the treatment of systolic heart failure, the treatment of heart failure with preserved left ventricular function is uncertain, and therapeutic targets beyond blockade of the renin-angiotensin-aldosterone and beta-adrenergic systems are being sought. One such target is the beta-isoform of protein kinase C (PKC), implicated in both the complications of diabetes and in cardiac dysfunction in the nondiabetic setting.Using a hemodynamically validated rodent model of diabetic diastolic heart failure, the (mRen-2)27 transgenic rat, we sought to determine whether selective inhibition of PKC-beta would preserve cardiac function and reduce structural injury. Diabetic rats were randomized to receive either vehicle or the PKC-beta inhibitor, ruboxistaurin (20 mg/kg per d) and followed for 6 weeks. Compared with untreated animals, ruboxistaurin-treated diabetic rats demonstrated preserved systolic and diastolic function, as measured by the slope of preload recruitable stroke work relationship (Pless than 0.05) and the slope of the end-diastolic pressure volume relationship (Pless than 0.01). Collagen I deposition and cardiomyocyte hypertrophy were both reduced in diabetic animals treated with ruboxistaurin (Pless than 0.01), as was phosphorylated-Smad2, an index of transforming growth factor-beta activity (Pless than 0.01 for all, versus untreated diabetic rats).PKC-ss inhibition attenuated diastolic dysfunction, myocyte hypertrophy, and collagen deposition and preserved cardiac contractility. PKC-beta inhibition may represent a novel therapeutic strategy for the prevention of diabetes-associated cardiac dysfunction.
|Cardiac-directed parvalbumin transgene expression in mice shows marked heart rate dependence of delayed Ca2+ buffering action. |
Day, SM; Coutu, P; Wang, W; Herron, T; Turner, I; Shillingford, M; Lacross, NC; Converso, KL; Piao, L; Li, J; Lopatin, AN; Metzger, JM
Physiological genomics 33 312-22 2008
Relaxation abnormalities are prevalent in heart failure and contribute to clinical outcomes. Disruption of Ca2+ homeostasis in heart failure delays relaxation by prolonging the intracellular Ca2+ transient. We sought to speed cardiac relaxation in vivo by cardiac-directed transgene expression of parvalbumin (Parv), a cytosolic Ca2+ buffer normally expressed in fast-twitch skeletal muscle. A key feature of Parv's function resides in its Ca2+/Mg2+ binding affinities that account for delayed Ca2+ buffering in response to the intracellular Ca2+ transient. Cardiac Parv expression decreased sarcoplasmic reticulum Ca2+ content without otherwise altering intracellular Ca2+ homeostasis. At high physiological mouse heart rates in vivo, Parv modestly accelerated relaxation without affecting cardiac morphology or systolic function. Ex vivo pacing of the isolated heart revealed a marked heart rate dependence of Parv's delayed Ca2+ buffering effects on myocardial performance. As the pacing frequency was lowered (7 to 2.5 Hz), the relaxation rates increased in Parv hearts. However, as pacing rates approached the dynamic range in humans, Parv hearts demonstrated decreased contractility, consistent with Parv buffering systolic Ca2+. Mathematical modeling and in vitro studies provide the underlying mechanism responsible for the frequency-dependent fractional Ca2+ buffering action of Parv. Future studies directed toward refining the dose and frequency-response relationships of Parv in the heart or engineering novel Parv-based Ca2+ buffers with modified Mg2+ and Ca2+ affinities to limit systolic Ca2+ buffering may hold promise for the development of new therapies to remediate relaxation abnormalities in heart failure.
|Protein kinase A type I and type II define distinct intracellular signaling compartments. |
Di Benedetto, G; Zoccarato, A; Lissandron, V; Terrin, A; Li, X; Houslay, MD; Baillie, GS; Zaccolo, M
Circulation research 103 836-44 2008
Protein kinase A (PKA) is a key regulatory enzyme that, on activation by cAMP, modulates a wide variety of cellular functions. PKA isoforms type I and type II possess different structural features and biochemical characteristics, resulting in nonredundant function. However, how different PKA isoforms expressed in the same cell manage to perform distinct functions on activation by the same soluble intracellular messenger, cAMP, remains to be established. Here, we provide a mechanism for the different function of PKA isoforms subsets in cardiac myocytes and demonstrate that PKA-RI and PKA-RII, by binding to AKAPs (A kinase anchoring proteins), are tethered to different subcellular locales, thus defining distinct intracellular signaling compartments. Within such compartments, PKA-RI and PKA-RII respond to distinct, spatially restricted cAMP signals generated in response to specific G protein-coupled receptor agonists and regulated by unique subsets of the cAMP degrading phosphodiesterases. The selective activation of individual PKA isoforms thus leads to phosphorylation of unique subsets of downstream targets.
|Single histidine-substituted cardiac troponin I confers protection from age-related systolic and diastolic dysfunction. |
Palpant, NJ; Day, SM; Herron, TJ; Converso, KL; Metzger, JM
Cardiovascular research 80 209-18 2008
Contractile dysfunction associated with myocardial ischaemia is a significant cause of morbidity and mortality in the elderly. Strategies to protect the aged heart from ischaemia-mediated pump failure are needed. We hypothesized that troponin I-mediated augmentation of myofilament calcium sensitivity would protect cardiac function in aged mice.To address this, we investigated transgenic (Tg) mice expressing a histidine-substituted form of adult cardiac troponin I (cTnI A164H), which increases myofilament calcium sensitivity in a pH-dependent manner. Serial echocardiography revealed that Tg hearts showed significantly improved systolic function at 4 months, which was sustained for 2 years based on ejection fraction and velocity of circumferential fibre shortening. Age-related diastolic dysfunction was also attenuated in Tg mice as assessed by Doppler measurements of the mitral valve inflow and lateral annulus Doppler tissue imaging. During acute hypoxia, cardiac contractility significantly improved in aged Tg mice made evident by increased stroke volume, end systolic pressure, and +dP/dt compared with non-transgenic mice.This study shows that increasing myofilament function by means of a pH-responsive histidine button engineered into cTnI results in enhanced baseline heart function in Tg mice over their lifetime, and during acute hypoxia improves survival in aged mice by maintaining cardiac contractility.Full Text Article
|Functional, structural and molecular aspects of diastolic heart failure in the diabetic (mRen-2)27 rat. |
Connelly, KA; Kelly, DJ; Zhang, Y; Prior, DL; Martin, J; Cox, AJ; Thai, K; Feneley, MP; Tsoporis, J; White, KE; Krum, H; Gilbert, RE
Cardiovascular research 76 280-91 2007
Diabetic cardiomyopathy is an increasingly recognized cause of cardiac failure despite preserved left ventricular systolic function. Given the over-expression of angiotensin II in human diabetic cardiomyopathy, we hypothesized that combining hyperglycaemia with an enhanced tissue renin-angiotensin system would lead to the development of diastolic dysfunction with adverse remodeling in a rodent model.Homozygous (mRen-2)27 rats and non-transgenic Sprague Dawley (SD) rats were randomized to receive streptozotocin (diabetic) or vehicle (non-diabetic) and followed for 6 weeks. Prior to tissue collection, animals underwent pressure-volume loop acquisition.Diabetic Ren-2 rats developed impairment of both active and passive phases of diastole, accompanied by reductions in SERCA-2a ATPase and phospholamban along with activation of the fetal gene program. Structural features of diabetic cardiomyopathy in the Ren-2 rat included interstitial fibrosis, cardiac myocyte hypertrophy and apoptosis in conjunction with increased activity of transforming growth factor-beta (pless than 0.01 compared with non-diabetic Ren-2 rats for all parameters). No significant functional or structural derangements were observed in non-transgenic, SD diabetic rats.These findings indicate that the combination of enhanced tissue renin-angiotensin system and hyperglycaemia lead to the development of diabetic cardiomyopathy. Fibrosis, and myocyte hypertrophy, a prominent feature of this model, may be a consequence of activation of the pro-sclerotic cytokine, transforming growth factor-beta, by the diabetic state.
|Differential activation of stress-response signaling in load-induced cardiac hypertrophy and failure. |
Rothermel, BA; Berenji, K; Tannous, P; Kutschke, W; Dey, A; Nolan, B; Yoo, KD; Demetroulis, E; Gimbel, M; Cabuay, B; Karimi, M; Hill, JA
Physiological genomics 23 18-27 2005
Hypertrophic growth of the myocardium occurs in most forms of heart failure and may contribute to the pathogenesis of the failure state. Little is known about the regulatory mechanisms governing the often-coexisting phenotypes of hypertrophy, systolic failure, and diastolic stiffness that characterize clinical disease. We hypothesized that intracellular signaling pathways are differentially activated by graded degrees of hemodynamic stress. To test this, we developed models of graded pressure stress in mice and used them to directly compare compensated hypertrophy and pressure-overload heart failure. Surgical interventions were designed to be similar, on either side of a threshold separating compensated from decompensated responses. Our findings revealed two dramatically different hypertrophic phenotypes with only modest differences in the activation of relevant intracellular signaling pathways. Furthermore, we uncovered a functional requirement of calcineurin signaling in each model such that calcineurin suppression blunted hypertrophic growth. Remarkably, in each case, suppression of calcineurin signaling was not associated with clinical deterioration or increased mortality. Profiles of stress-response signaling and Ca2+ handling differ between the steady-state, maintenance phases of load-induced cardiac hypertrophy and failure. This information may be useful in identifying novel targets of therapy in chronic disease.
|Transgenic overexpression of the Ca2+-binding protein S100A1 in the heart leads to increased in vivo myocardial contractile performance. |
Most, P; Remppis, A; Pleger, ST; Löffler, E; Ehlermann, P; Bernotat, J; Kleuss, C; Heierhorst, J; Ruiz, P; Witt, H; Karczewski, P; Mao, L; Rockman, HA; Duncan, SJ; Katus, HA; Koch, WJ
The Journal of biological chemistry 278 33809-17 2003
S100A1, a Ca2+-sensing protein of the EF-hand family, is most highly expressed in myocardial tissue, and cardiac S100A1 overexpression in vitro has been shown to enhance myocyte contractile properties. To study the physiological consequences of S100A1 in vivo, transgenic mice were developed with cardiac-restricted overexpression of S100A1. Characterization of two independent transgenic mouse lines with approximately 4-fold overexpression of S100A1 in the myocardium revealed a marked augmentation of in vivo basal cardiac function that remained elevated after beta-adrenergic receptor stimulation. Contractile function and Ca2+ handling properties were increased in ventricular cardiomyocytes isolated from S100A1 transgenic mice. Enhanced cellular Ca2+ cycling by S100A1 was associated both with increased sarcoplasmic reticulum Ca2+ content and enhanced sarcoplasmic reticulum Ca2+-induced Ca2+ release, and S100A1 was shown to associate with the cardiac ryanodine receptor. No alterations in beta-adrenergic signal transduction or major cardiac Ca2+-cycling proteins occurred, and there were no signs of hypertrophy with chronic cardiac S100A1 overexpression. Our findings suggest that S100A1 plays an important in vivo role in the regulation of cardiac function perhaps through interacting with the ryanodine receptor. Because S100A1 protein expression is down-regulated in heart failure, increasing S100A1 expression in the heart may represent a novel means to augment contractility.
|Characterization and quantitation of phospholamban and its phosphorylation state using antibodies. |
Mayer, E J, et al.
Biochem. Biophys. Res. Commun., 267: 40-8 (2000) 2000
Quantitative immunoassays to discriminate and quantitate phospholamban and its phosphorylation states in heart homogenates were developed using known amounts of protein determined by amino acid analysis. Synthetic 1-52 phospholamban, the hydrophilic 1-25 peptide, and 1-25 phosphopeptides containing P-Ser(16), P-Thr(17), and dually phosphorylated (P-Ser(16), P-Thr(17)) were used to calibrate immunoblot systems. In addition, synthetic 1-52 peptide was phosphorylated using cAMP-dependent protein kinase (P-Ser(16)) or Ca(2+)-calmodulin protein kinase (P-Thr(17)) and then separated from unphosphorylated 1-52 by HPLC prior to quantitation. Further, canine cardiac sarcoplasmic reticulum was phosphorylated in vitro using [gamma-(32)P]-ATP with cAMP-dependent protein kinase and/or Ca(2+)-calmodulin-dependent protein kinase as well as sequential phosphorylation in both orders to assess the veracity of antibody recognition of phosphorylated forms. Western blots proved useful in characterizing the reactivity of the different antibodies to phospholamban and phosphorylated phospholamban, but were inefficient for accurate quantitation and problems with antibody recognition of dually phosphorylated phospholamban were found. mAb 1D11 recognized all forms of phospholamban, polyclonal antibodies 285 and PS-16 were highly selective for P-Ser(16) phospholamban but had diminished reactivity to diphosphorylated (P-Ser(16), P-Thr(17)) phospholamban, and polyclonal antibody PT-17, although selective for P-Thr(17) phospholamban, generated very weak signals on Western blots and reacted poorly with diphosphorylated phospholamban. Results in quantitative immunodot blot experiments were even more compelling. None of the phosphorylation specific antibodies reacted with the diphospho 1-25 phospholamban peptide. Transgenic mouse hearts expressing varying levels of PLB and ferret heart biopsy samples taken before and after isoproterenol perfusion were analyzed. In all samples containing phospholamban, a basal level of Ser(16) phosphorylation (about 4% of the total PLB population) and a lesser amount of Thr(17) phosphorylation was observed. Upon isoproterenol perfusion, Ser(16) phosphorylation increased only to 17% of the total phospholamban population with a similar change in Thr(17) phosphorylation. This suggests that phospholamban phosphorylation may serve as an electrostatic switch that dissociates inactive calcium pump complexes into catalytically active units. Thus, direct correlations between phospholamban phosphorylation state and contractile parameters may not be valid.