Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects

1998 ◽  
Vol 274 (4) ◽  
pp. H1335-H1347 ◽  
Author(s):  
Li Li ◽  
Guoxiang Chu ◽  
Evangelia G. Kranias ◽  
Donald M. Bers
Keyword(s):  
Cardiac Myocyte ◽  
Ventricular Myocytes ◽  
Flux Analysis ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Ca Transport ◽  
Pump Activity ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Exchange Activity

Increases in heart rate are accompanied by acceleration of relaxation. This effect is apparent at the single myocyte level and depends on sarcoplasmic reticulum (SR) Ca transport and Ca/calmodulin dependent protein kinase [CaMKII; see R. A. Bassani, A. Mattiazzi, and D. M. Bers. Am. J. Physiol. 268 ( Heart Circ. Physiol. 37): H703–H712, 1995]. Because phosphorylation of phospholamban (PLB) by CaMKII can stimulate SR Ca transport, it is a plausible candidate mechanism. We examined this issue using ventricular myocytes isolated from wild-type (WT) mice and those in which the PLB gene was ablated by gene targeting (PLB-KO). During steady-state (SS) stimulation, twitch relaxation and intracellular Ca concentration ([Ca]i) decline were significantly faster than after a rest in both WT and PLB-KO myocytes. Furthermore, the CaMKII inhibitor KN-93 (1 μM) abolished the stimulation-dependent acceleration of twitch [Ca]i decline in PLB-KO. This indicates that neither PLB nor its phosphorylation are required for the CaMKII-dependent acceleration of the SS twitch [Ca]i decline and relaxation. Other quantitative aspects of Ca transport in WT and PLB-KO myocytes were also examined. As expected, the time constant (τ) of [Ca]i decline during the SS twitch is much faster in PLB-KO than in WT myocytes (112 ± 6 vs. 188 ± 14 ms, P < 0.0001). There was also an increase in SS SR Ca load, based on the change of [Ca]i during rapid caffeine-induced contractures (CafC) with Na/Ca exchange blocked (565 ± 74 nM for WT, 1118 ± 133 nM for PLB-KO, P < 0.01). Accounting for cytosolic Ca buffering, this implies a 37% increase in SR Ca content. The τ for [Ca]idecline of the CafC with Na present indicated slower extrusion by Na/Ca exchange in the PLB-KO mouse (2.2 ± 0.2 s in WT vs. 3.2 ± 0.2 s in PLB-KO, P < 0.01), although exchanger protein expression was unchanged. Integrated Ca flux analysis in WT and PLB-KO myocytes, respectively, shows that 90 and 96% of Ca during twitch relaxation is removed by the SR Ca-ATPase, 9 and 3.4% by Na/Ca exchange, and 0.5 and 0.1% by slow mechanisms (mitochondria Ca uniporter and sarcolemmal Ca-ATPase). We conclude that the PLB-KO myocytes retain a CaMKII-dependent acceleration of SS twitch [Ca]i decline. The PLB-KO (vs. WT) myocytes also have higher SR Ca pump activity, higher SR Ca load, and reduced Na/Ca exchange activity.

Abstract 190: Protein Phosphatase 2Ce Is a Novel Phospholamban Phosphatase that Exacerbates Cell Death and Suppresses Cardiac Contractility

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Toru Akaike ◽  
Gang Lu ◽  
Yibin Wang ◽  
Hongmei Ruan
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Protein Phosphatase ◽  
Ventricular Myocytes ◽  
Cardiac Contractility ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
Phospholamban Phosphorylation

Regulation of sarcoplasmic reticulum (SR) calcium-ATPase (SERCA) activity is critical for calcium homeostasis in cardiomyocytes, and has a major impact on contractility and cellular viability of cardiomyocytes. The key regulators for SERCA activity include protein kinases, cAMP dependent protein kinase A and calcium/calmodulin dependent protein kinase II, and protein phosphatase 1. In this report, we have discovered that protein phosphatase 2Ce (PP2Ce) is a novel serine/threonine protein phosphatase specifically targeted to SR membrane in cardiomyocytes. PP2Ce was detected to interact with phosphlamban in heart. Recombinant PP2Ce protein showed a potent and specific activity towards the calcium/calmodulin dependent protein kinase II dependent phospholamban phosphorylation at threonin 17 site with no significant activity to cAMP dependent protein kinase A dependent phospholamban phosphorylation at serine 16 site. Expression of PP2Ce blunted β-adrenergic stimulated increase of phospholamban phosphorylation without affecting phosphorylation of ryanodine recepter or troponin I. PP2Ce expression reduced β-adrenergic stimulated intracellular calcium transient in isolated adult rabbit ventricular myocytes, and promoted hydrogen peroxide induced cell death in cultured neonatal rat ventricular myocytes. Transgenic mice with cardiac specific expression of PP2Ce showed no significant basal phenotype. However, in isolated perfusion heart preparation, β-adrenergic stimulated contractility was significant reduced in PP2Ce transgenic hearts comparing to wild type controls. Furthermore, we observed significantly larger infarct sizes and more impaired functional recovery following global ischemia/reperfusion injury in the transgenic hearts comparing to wild type controls. Therefore, PP2Ce is a novel component of SR calcium regulatory network that has a potentially important role in cell death regulation and cardiac contractility.

scholarly journals Overexpression of adenylyl cyclase type 5 (AC5) confers a proarrhythmic substrate to the heart

2015 ◽  
Vol 308 (3) ◽  
pp. H240-H249 ◽  
Author(s):  
Zhenghang Zhao ◽  
Gopal J. Babu ◽  
Hairuo Wen ◽  
Nadezhda Fefelova ◽  
Richard Gordan ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Ventricular Myocytes ◽  
Ryanodine Receptors ◽  
Wild Type ◽  
Therapeutic Approaches ◽  
Dependent Protein Kinase ◽  
Key Enzyme ◽  
Intracellular Calcium Ion ◽  
Dependent Protein ◽  
Species Production

Inhibition of β-adrenergic receptor (β-AR) signaling is one of the most common therapeutic approaches for patients with arrhythmias. Adenylyl cyclase (AC) is the key enzyme responsible for transducing β-AR stimulation to increases in cAMP. The two major AC isoforms in the heart are types 5 and 6. Therefore, it is surprising that prior studies on overexpression of AC5 and AC6 in transgenic (Tg) mice have not examined mediation of arrhythmogenesis. Our goal was to examine the proarrhythmic substrate in AC5Tg hearts. Intracellular calcium ion (Ca2+i) was imaged in fluo-4 AM-loaded ventricular myocytes. The sarcoplasmic reticulum (SR) Ca2+ content, fractional Ca2+ release, and twitch Ca2+ transient were significantly higher in the AC5Tg vs. wild-type (WT) myocytes, indicating Ca2+ overload in AC5Tg myocytes. Action potential (AP) duration was significantly longer in AC5Tg than in WT myocytes. Additionally, AC5Tg myocytes developed spontaneous Ca2+ waves in a larger fraction compared with WT myocytes, particularly when cells were exposed to isoproterenol. The Ca2+ waves further induced afterdepolarizations and triggered APs. AC5Tg hearts had increased level of SERCA2a, oxidized Ca2+/calmodulin-dependent protein kinase II (CaMKII), and phosphorylation of ryanodine receptors (RyR) at the CaMKII site, especially after isoproterenol treatment. This was consistent with higher reactive oxygen species production in AC5Tg myocytes after isoproterenol treatment. Isoproterenol induced more severe arrhythmias in AC5Tg than in WT mice. We conclude that AC5 overexpression promotes arrhythmogenesis, by inducing SR Ca2+ overload and hyperactivation of RyR (phosphorylation by CaMKII), which in turn induces spontaneous Ca2+ waves and afterdepolarizations.

scholarly journals Chronic Elevation of Calmodulin in the Ventricles of Transgenic Mice Increases the Autonomous Activity of Calmodulin-Dependent Protein Kinase II, Which Regulates Atrial Natriuretic Factor Gene Expression

2000 ◽  
Vol 14 (8) ◽  
pp. 1125-1136 ◽  
Author(s):  
Josep M. Colomer ◽  
Anthony R. Means
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Transgenic Mice ◽  
Atrial Natriuretic Factor ◽  
Ventricular Myocytes ◽  
Dependent Protein Kinase ◽  
Natriuretic Factor ◽  
Dependent Protein ◽  
Autonomous Activity

Abstract Although isoforms of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been implicated in the regulation of gene expression in cultured cells, this issue has yet to be addressed in vivo. We report that the overexpression of calmodulin in ventricular myocytes of transgenic mice results in an increase in the Ca2+/calmodulin-independent activity of endogenous CaMKII. The calmodulin transgene is regulated by a 500-bp fragment of the atrial natriuretic factor (ANF) gene promoter which, based on cell transfection studies, is itself known to be regulated by CaMKII. The increased autonomous activity of CaMKII maintains the activity of the transgene and establishes a positive feedforward loop, which also extends the temporal expression of the endogenous ANF promoter in ventricular myocytes. Both the increased activity of CaMKII and transcriptional activation of ANF are highly selective responses to the chronic overexpression of calmodulin. These results indicate that CaMKII can regulate gene expression in vivo and suggest that this enzyme may represent the Ca2+-dependent target responsible for reactivation of the ANF gene during ventricular hypertrophy.

A myb-related protein required for culmination in Dictyostelium

Development ◽  
1999 ◽  
Vol 126 (12) ◽  
pp. 2813-2822 ◽  
Author(s):  
K. Guo ◽  
C. Anjard ◽  
A. Harwood ◽  
H.J. Kim ◽  
P.C. Newell ◽  
...  
Keyword(s):  
Developmental Defect ◽  
Intercellular Signaling ◽  
Wild Type ◽  
Fruiting Body Formation ◽  
Dependent Protein Kinase ◽  
Myb Gene ◽  
Repeat Domain ◽  
Intercellular Signalling ◽  
Dependent Protein ◽  
Signaling Process

The avian retroviral v-myb gene and its cellular homologues throughout the animal and plant kingdoms contain a conserved DNA binding domain. We have isolated an insertional mutant of Dictyostelium unable to switch from slug migration to fruiting body formation i.e. unable to culminate. The gene that is disrupted, mybC, codes for a protein with a myb-like domain that is recognized by an antibody against the v-myb repeat domain. During development of myb+ cells, mybC is expressed only in prestalk cells. When developed together with wild-type cells mybC- cells are able to form both spores and stalk cells very efficiently. Their developmental defect is also bypassed by overexpressing cAMP-dependent protein kinase. However even when their defect is bypassed, mybC null slugs and culminates produce little if any of the intercellular signalling peptides SDF-1 and SDF-2 that are believed to be released by prestalk cells at culmination. We propose that the mybC gene product is required for an intercellular signaling process controlling maturation of stalk cells and spores and that SDF-1 and/or SDF-2 may be implicated in this process.

Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies

2008 ◽  
Vol 294 (5) ◽  
pp. H2352-H2362 ◽  
Author(s):  
Andreas A. Werdich ◽  
Eduardo A. Lima ◽  
Igor Dzhura ◽  
Madhu V. Singh ◽  
Jingdong Li ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Myocytes ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Frequency Dependent ◽  
Physiological Range ◽  
Dependent Protein ◽  
Isolated Cardiac Myocytes

In cardiac myocytes, the activity of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is hypothesized to regulate Ca2+ release from and Ca2+ uptake into the sarcoplasmic reticulum via the phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of cytosolic Ca2+ concentration ([Ca2+]i) transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37°C). When compared with that of mice lacking PLN only, the combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca2+ release rate by more than 50% at 10 Hz. Although PLN ablation increased the rate of Ca2+ uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca2+]i transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca2+]i transient, indicating that the PLN-mediated feedback on [Ca2+]i removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes, the combined chronic CaMKII inhibition and PLN ablation slowed Ca2+ release at physiological frequencies: the frequency-dependent decay of the amplitude and shortening of the [Ca2+]i transient occurs independent of chronic CaMKII inhibition and PLN ablation, and the PLN-mediated regulation of Ca2+ uptake is diminished at higher stimulation frequencies within the physiological range.

Mutation of the Gs protein alpha subunit NH2 terminus relieves an attenuator function, resulting in constitutive adenylyl cyclase stimulation

1990 ◽  
Vol 10 (6) ◽  
pp. 2931-2940
Author(s):  
S Osawa ◽  
L E Heasley ◽  
N Dhanasekaran ◽  
S K Gupta ◽  
C W Woon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
G Proteins ◽  
Fold Increase ◽  
Alpha Subunit ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Gs Protein ◽  
Camp Levels

G-proteins couple hormonal activation of receptors to the regulation of specific enzymes and ion channels. Gs and Gi are G-proteins which regulate the stimulation and inhibition, respectively, of adenylyl cyclase. We have constructed two chimeric cDNAs in which different lengths of the alpha subunit of Gs (alpha s) have been replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). One chimera, referred to as alpha i(54)/s' replaces the NH2-terminal 61 amino acids of alpha s with the first 54 residues of alpha i. Within this sequence there are 7 residues unique to alpha s, and 16 of the remaining 54 amino acids are nonhomologous between alpha i and alpha s. The second chimera, referred to as alpha i/s(Bam), replaces the first 234 amino acids of alpha s with the corresponding 212 residues of alpha i. Transient expression of alpha i(54)/s in COS-1 cells resulted in an 18- to 20-fold increase in cyclic AMP (cAMP) levels, whereas expression of either alpha i/s(Bam) or the wild-type alpha s polypeptide resulted in only a 5- to 6-fold increase in cellular cAMP levels. COS-1 cells transfected with alpha i showed a small decrease in cAMP levels. Stable expression of the chimeric alpha i(54)/s polypeptide in Chinese hamster ovary (CHO) cells constitutively increased both cAMP synthesis and cAMP-dependent protein kinase activity. CHO clones expressing transfected alpha i/s(Bam) or the wild-type alpha s and alpha i cDNAs exhibited cAMP levels and cAMP-dependent protein kinase activities similar to those in control CHO cells. Therefore, the alpha i(54)/s chimera behaves as a constitutively active alpha s polypeptide, whereas the alpha i/s(Bam) polypeptide is regulated similarly to wild-type alpha s. Expression in cyc-S49 cells, which lack expression of wild-type alpha s, confirmed that the alpha i(54)/s polypeptide is a highly active alpha s molecule whose robust activity is independent of any change in intrinsic GTPase activity. The difference in phenotypes observed upon expression of alpha i(54)/s or alpha i/s(Bam) indicates that the NH2-terminal moieties of alpha s and alpha i function as attenuators of the effector enzyme activator domain which is within the COOH-terminal half of the alpha subunit. Mutation at the NH2 terminus of alpha s relieves the attenuator control of the Gs protein and results in a dominant active G-protein mutant.

Revertants of an S49 cell mutant that expresses altered cyclic AMP-dependent protein kinase

1982 ◽  
Vol 2 (10) ◽  
pp. 1229-1237
Author(s):  
T van Daalen Wetters ◽  
P Coffino
Keyword(s):  
Protein Kinase ◽  
Regulatory Subunit ◽  
Mutational Event ◽  
Wild Type ◽  
Cell Mutant ◽  
Dependent Protein Kinase ◽  
Cell Extracts ◽  
Counter Selection ◽  
Dependent Protein ◽  
Gene Lesion

Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive (Bt2cAMPS) revertants were isolated from a resistant S49 cell mutant carrying a structural gene lesion in the regulatory subunit of cAMP-dependent protein kinase (cA-PK). This was accomplished with a counter-selection in which, first, Bt2cAMP was used to reversibly arrest revertants, and then a sequence of treatments with bromodeoxyuridine, 33258 Hoechst dye, and white light was used to kill cycling mutant cells. Reversion rates in nonmutagenized cultures could not be accurately measured, but spontaneous revertants do occur and with frequencies of less than 10(-7) to 10(-5). The mutagens ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitro-soguanidine (MNNG), and ICR191 increased the reversion frequency. In all cases, reversion to Bt2cAMP sensitivity was associated with restoration of wild-type levels and apparent activation constant for cAMP of cA-PK. MNNG induced revertants whose cell extracts contained cA-PK activity distinguishable from that of wild type by thermal liability. EMS did not. The counter-selection effectively isolates rare phenotypes and is therefore a useful tool in further somatic genetic experiments. The association of reversion with alterations in cA-PK function supports all previous data from this and other laboratories implicating cA-PK as the intracellular mediator of cAMP effects. Reversion is probably the result of a mutational event. Induction of reversion by ICR191 suggests the existence of a novel mechanism for generating revertants in somatic cells.

Abstract 1078: β-Adrenergic Stimulation of Ca/Calmodulin-Dependent Protein Kinase II (CaMKII) Overexpressing Myocytes Increases Sarcoplasmic Reticulum (SR) Calcium Leak Causing KN-93 Sensitive Arrhythmias

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Daniel P Wadsack ◽  
Michael Kohlhaas ◽  
Adam G Rokita ◽  
Stefan Neef ◽  
Lars S Maier
Keyword(s):  
Control Level ◽  
Adrenergic Stimulation ◽  
Dependent Protein Kinase ◽  
Inotropic Effects ◽  
Positive Inotropic Effects ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Ca Homeostasis ◽  
Sr Calcium Leak ◽  
Inotropic Stimulation

CaMKII is associated with hypertrophy, heart failure and alters intracellular Ca homeostasis. An increased SR Ca leak due to phosphorylation of SR Ca release channels by CaMKII leads to decreased SR Ca content and impaired contractility. This loss of Ca from the SR may also contribute to arrhythmias. We investigated whether β-adrenergic stimulation with isoproterenol (ISO) normalizes SR Ca content and whether inhibiting CaMKII reduces arrhythmias. CaMKII-overexpressing rabbit and mouse myocytes were investigated. Cell shortening, Ca fluorescence (fluo-3) and the incidence of arrhythmias were assessed. An arrhythmia-score differentiated between: early-spike-arrhythmias (ESA), late-spike-arrhythmias (LSA) and permanent arrhythmias (PA). ISO (37°C) had significantly different effects on myocytes with acute (24 h, rabbit, n=34) or chronic (22 w, mouse, n=34) CaMKII overexpression vs corresponding control myocytes (LacZ, n=21 or WT n=34). CaMKII overexpression lead to an ISO concentration-dependent (10 −10 -10 −5 mol/L) inotropic but compared to WT (or LacZ, respectively) impaired shortening and Ca transients (two-way ANOVA, P <0.05). A similar difference between CaMKII-overexpressing (n=17) and WT (n=19) myocytes was also seen during a shortening-frequency protocol (stepwise increase from 0.1– 4 Hz, two-way ANOVA, P <0.05). Arrhythmias spontaneously occurred in CaMKII-overexpressing mouse myocytes. With β-inotropic stimulation (10 −6 mol/L ISO) arrhythmias were increased 6.4-fold. Appearance of ESA and PA could be significantly reduced by KN-93 (1 μmol/L). At a basal stimulation rate of 1 Hz and 10 −7 mol/L ISO, PA could be dramatically reduced by half from control-level 21.43% (KN-92, inactive derivative, n=42) down to 10.87% (KN-93, n=46) arrhythmic events. ESA could be reduced almost 4-fold from 16.67% (KN-92) to 4.35% in the presence of KN-93. We conclude from these data that increasing ISO concentrations exerts positive inotropic effects but cannot normalize altered Ca handling in CaMKII-overexpressing myocytes. This may be due to an increased SR Ca leak under these conditions thus contributing to the arrhythmias observed. CaMKII inhibition clearly can reduce arrhythmias in the presence of β-adrenergic stimulation with ISO.

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