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See detailNeuronal nitric oxide synthase signaling in the heart is regulated by the sarcolemmal calcium pump 4b.
Oceandy, Delvac; Cartwright, Elizabeth J.; Emerson, Michael et al

in Circulation (2007), 115(4), 483-92

BACKGROUND: Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the ... [more ▼]

BACKGROUND: Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the isoform 4b of plasma membrane calcium/calmodulin-dependent ATPase (PMCA4b) through direct interaction mediated by a PDZ domain (PSD 95, Drosophilia Discs large protein and Zona occludens-1) on nNOS and a cognate ligand on PMCA4b. It remains unknown, however, whether this interaction has physiological relevance in the heart in vivo. METHODS AND RESULTS: We generated 2 strains of transgenic mice overexpressing either human PMCA4b or PMCA ct120 in the heart. PMCA ct120 is a highly active mutant form of the pump that does not interact with or modulate nNOS function. Calcium was extruded normally from PMCA4b-overexpressing cardiomyocytes, but in vivo, overexpression of PMCA4b reduced the beta-adrenergic contractile response. This attenuated response was not observed in ct120 transgenic mice. Treatment with a specific nNOS inhibitor (N omega-propyl-L-arginine) reduced the beta-adrenergic response in wild-type and ct120 transgenic mice to levels comparable to those of PMCA4b transgenic animals. No differences in lusitropic response were observed in either transgenic strain compared with wild-type littermates. CONCLUSIONS: These data demonstrate the physiological relevance of the interaction between PMCA4b and nNOS and suggests its signaling role in the heart. [less ▲]

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See detailConditional neuronal nitric oxide synthase overexpression impairs myocardial contractility.
Burkard, Natalie; Rokita, Adam G.; Kaufmann, Susann G. et al

in Circulation Research (2007), 100(3), 32-44

The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS(-/-) mice or from pharmacological inhibition of nNOS are ... [more ▼]

The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS(-/-) mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca(2+)-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [(3)H]-l-arginine to [(3)H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17+/-3% decrease of +dp/dt(max) compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca(2+)ATPase and additionally with L-type Ca(2+)- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I(Ca,L) density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca(2+)-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca(2+)-channels and in turn reduces Ca(2+)-transients which accounts for the negative inotropic effect. [less ▲]

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See detailInhibition of nuclear import of calcineurin prevents myocardial hypertrophy.
Hallhuber, Matthias; Burkard, Natalie; Wu, Rongxue et al

in Circulation Research (2006), 99(6), 626-35

The time that transcription factors remain nuclear is a major determinant for transcriptional activity. It has recently been demonstrated that the phosphatase calcineurin is translocated to the nucleus ... [more ▼]

The time that transcription factors remain nuclear is a major determinant for transcriptional activity. It has recently been demonstrated that the phosphatase calcineurin is translocated to the nucleus with the transcription factor nuclear factor of activated T cells (NF-AT). This study identifies a nuclear localization sequence (NLS) and a nuclear export signal (NES) in the sequence of calcineurin. Furthermore we identified the nuclear cargo protein importinbeta(1) to be responsible for nuclear translocation of calcineurin. Inhibition of the calcineurin/importin interaction by a competitive peptide (KQECKIKYSERV), which mimicked the calcineurin NLS, prevented nuclear entry of calcineurin. A noninhibitory control peptide did not interfere with the calcineurin/importin binding. Using this approach, we were able to prevent the development of myocardial hypertrophy. In angiotensin II-stimulated cardiomyocytes, [(3)H]-leucine incorporation (159%+/-9 versus 111%+/-11; P<0.01) and cell size were suppressed significantly by the NLS peptide compared with a control peptide. The NLS peptide inhibited calcineurin/NF-AT transcriptional activity (227%+/-11 versus 133%+/-8; P<0.01), whereas calcineurin phosphatase activity was unaffected (298%+/-9 versus 270%+/-11; P=NS). We conclude that calcineurin is not only capable of dephosphorylating NF-AT, thus enabling its nuclear import, but the presence of calcineurin in the nucleus is also important for full NF-AT transcriptional activity. [less ▲]

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See detailCalcium transport in cardiovascular health and disease--the sarcolemmal calcium pump enters the stage.
Cartwright, Elizabeth J.; Schuh, Kai; Neyses, Ludwig UL

in Journal of molecular and cellular cardiology (2005), 39(3), 403-6

Calcium is known to be one of the most important ionic regulators of the heart, where it has a crucial role in contraction-relaxation. Within a single beat of the cardiomyocyte there is a 100-fold ... [more ▼]

Calcium is known to be one of the most important ionic regulators of the heart, where it has a crucial role in contraction-relaxation. Within a single beat of the cardiomyocyte there is a 100-fold increase in the cytosolic free Ca(2+) level, this must be returned to its original concentration in order to maintain the normal physiological function of the cell. Two of the mechanisms involved in returning the Ca(2+) concentration back to resting levels are located at the sarcolemma; the sodium/calcium exchanger (NCX) and the sarcolemmal calcium pump. Compared to the NCX the sarcolemmal calcium pump extrudes significantly less calcium from the cardiomyocyte and has long been thought to be involved in the maintenance of low diastolic calcium levels. This review will outline recent evidence suggesting that the sarcolemmal calcium pump may in fact play a key role in signal transduction in the cardiovascular system. [less ▲]

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See detailTargeted proteolysis sustains calcineurin activation.
Burkard, Natalie; Becher, Jan; Heindl, Cornelia et al

in Circulation (2005), 111(8), 1045-53

BACKGROUND: Calcineurin (CnA) is important in the regulation of myocardial hypertrophy. We demonstrated that targeted proteolysis of the CnA autoinhibitory domain under pathological myocardial workload ... [more ▼]

BACKGROUND: Calcineurin (CnA) is important in the regulation of myocardial hypertrophy. We demonstrated that targeted proteolysis of the CnA autoinhibitory domain under pathological myocardial workload leads to increased CnA activity in human myocardium. Here, we investigated the proteolytic mechanism leading to activation of CnA. METHODS AND RESULTS: In patients with diseased myocardium, we found strong nuclear translocation of CnA. In contrast, in normal human myocardium, there was a cytosolic distribution of CnA. Stimulation of rat cardiomyocytes with angiotensin (Ang) II increased calpain activity significantly (433+/-11%; P<0.01; n=6) and caused proteolysis of the autoinhibitory domain of CnA. Inhibition of calpain by a membrane-permeable calpain inhibitor prevented proteolysis. We identified the cleavage site of calpain in the human CnA sequence at amino acid 424. CnA activity was increased after Ang II stimulation (310+/-29%; P<0.01; n=6) and remained high after removal of Ang II (214+/-17%; P<0.01; n=6). Addition of a calpain inhibitor to the medium decreased CnA activity (110+/-19%; P=NS; n=6) after removal of Ang II. Ang II stimulation of cardiomyocytes also translocated CnA into the nucleus as demonstrated by immunohistochemical staining and transfection assays with GFP-tagged CnA. Calpain inhibition and therefore suppression of calpain-mediated proteolysis of CnA enabled CnA exit from the nucleus. CONCLUSIONS: Ang II stimulation of cardiomyocytes increased calpain activity, leading to proteolysis of the autoinhibitory domain of CnA. This causes an increase in CnA activity and results in nuclear translocation of CnA. Loss of the autoinhibitory domain renders CnA constitutively nuclear and active, even after removal of the hypertrophic stimulus. [less ▲]

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See detailPlasma membrane Ca2+ ATPase 4 is required for sperm motility and male fertility.
Schuh, Kai; Cartwright, Elizabeth J.; Jankevics, Eriks et al

in The Journal of biological chemistry (2004), 279(27), 28220-6

Calcium and Ca(2+)-dependent signals play a crucial role in sperm motility and mammalian fertilization, but the molecules and mechanisms underlying these Ca(2+)-dependent pathways are incompletely ... [more ▼]

Calcium and Ca(2+)-dependent signals play a crucial role in sperm motility and mammalian fertilization, but the molecules and mechanisms underlying these Ca(2+)-dependent pathways are incompletely understood. Here we show that homozygous male mice with a targeted gene deletion of isoform 4 of the plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA), which is highly enriched in the sperm tail, are infertile due to severely impaired sperm motility. Furthermore, the PMCA inhibitor 5-(and-6)-carboxyeosin diacetate succinimidyl ester reduced sperm motility in wild-type animals, thus mimicking the effects of PMCA4 deficiency on sperm motility and supporting the hypothesis of a pivotal role of the PMCA4 on the regulation of sperm function and intracellular Ca(2+) levels. [less ▲]

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See detailRegulation of vascular tone in animals overexpressing the sarcolemmal calcium pump.
Schuh, Kai; Quaschning, Thomas; Knauer, Sebastian et al

in The Journal of biological chemistry (2003), 278(42), 41246-52

The mechanisms governing vascular smooth muscle tone are incompletely understood. In particular, the role of the sarcolemmal calcium pump PMCA (plasma membrane calmodulin-dependent calcium ATPase), which ... [more ▼]

The mechanisms governing vascular smooth muscle tone are incompletely understood. In particular, the role of the sarcolemmal calcium pump PMCA (plasma membrane calmodulin-dependent calcium ATPase), which extrudes Ca2+ from the cytosol, and its importance compared with the sodium/calcium exchanger remain speculative. To test whether the PMCA is a regulator of vascular tone, we generated transgenic mice overexpressing the human PMCA4b under control of the arterial smooth muscle-specific SM22alpha promoter. This resulted in an elevated systolic blood pressure compared with littermate controls. In PMCA-overexpressing mice, endothelium-dependent relaxation of norepinephrine-preconstricted aortic rings to acetylcholine did not differ from wild type controls (76 +/- 8% versus 79 +/- 8% of maximum relaxation; n = 12, n.s.). De-endothelialized aortas of transgenic mice exhibited stronger maximum contraction to KCl (100 mmol/liter) compared with controls (86 +/- 6% versus 68 +/- 7% of reference KCl contraction at the beginning of the experiment; p <0.05). Preincubation of de-endothelialized vessels with the nitric oxide synthase (NOS) inhibitor l-NAME (l-N(G)-nitroarginine methyl ester) (10-5 mol/liter) resulted in a stronger contraction to KCl (p <0.05 versus without l-NAME), thus unmasking vasodilatory effects of inherent NO production. Maximum contraction to KCl after preincubation with l-NAME did not differ between PMCA mice and controls. In analogy to the results in PMCA-overexpressing mice, contractions of de-endothelialized aortas of neuronal NOS-deficient mice to KCl were significantly increased compared with controls (151 +/- 5% versus 131 +/- 6% of reference KCl contraction; p <0.05). In conclusion, our data suggest a model in which the sarcolemmal Ca2+ pump down-regulates activity of the vascular smooth muscle Ca2+/calmodulin-dependent neuronal NOS by a functionally relevant interaction. Therefore, the PMCA represents a novel regulator of vascular tone. [less ▲]

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See detailInteraction of the plasma membrane Ca2+ pump 4b/CI with the Ca2+/calmodulin-dependent membrane-associated kinase CASK.
Schuh, Kai; Uldrijan, Stjepan; Gambaryan, Stepan et al

in The Journal of biological chemistry (2003), 278(11), 9778-83

Spatial and temporal regulation of intracellular Ca(2+) is a key event in many signaling pathways. Plasma membrane Ca(2+)-ATPases (PMCAs) are major regulators of Ca(2+) homeostasis and bind to PDZ (PSD-95 ... [more ▼]

Spatial and temporal regulation of intracellular Ca(2+) is a key event in many signaling pathways. Plasma membrane Ca(2+)-ATPases (PMCAs) are major regulators of Ca(2+) homeostasis and bind to PDZ (PSD-95/Dlg/ZO-1) domains via their C termini. Various membrane-associated guanylate kinase family members have been identified as interaction partners of PMCAs. In particular, SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bind to the C-terminal tails of PMCA "b" splice variants. Additionally, it has been demonstrated that PMCA4b interacts with neuronal nitric-oxide synthase and that isoform 2b interacts with Na(+)/H(+) exchanger regulatory factor 2, both via a PDZ domain. CASK (calcium/calmodulin-dependent serine protein kinase) contains a calmodulin-dependent protein kinase-like domain followed by PDZ, SH3, and guanylate kinase-like domains. In adult brain CASK is located at neuronal synapses and interacts with various proteins, e.g. neurexin and Veli/LIN-7. In kidney it is localized to renal epithelia. Surprisingly, interaction with the Tbr-1 transcription factor, nuclear transport, binding to DNA T-elements (in a complex with Tbr-1), and transcriptional competence has been shown. Here we show that the C terminus of PMCA4b binds to CASK and that both proteins co-precipitate from brain and kidney tissue lysates. Immunofluorescence staining revealed co-expression of PMCA, CASK, and calbindin-d-28K in distal tubuli of rat kidney sections. To test if physical interaction of both proteins results in functional consequences we constructed a T-element-dependent reporter vector and investigated luciferase activity in HEK293 lysates, previously co-transfected with PMCA4b expression and control vectors. Expression of wild-type PMCA resulted in an 80% decrease in T-element-dependent transcriptional activity, whereas co-expression of a point-mutated PMCA, with nearly eliminated Ca(2+) pumping activity, had only a small influence on regulation of transcriptional activity. These results provide evidence of a new direct Ca(2+)-dependent link from the plasma membrane to the nucleus. [less ▲]

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See detailAT2 receptor activation regulates myocardial eNOS expression via the calcineurin-NF-AT pathway.
Ritter, Oliver; Schuh, Kai; Brede, Marc et al

in FASEB journal : official publication of the Federation of American Societies for Experimental Biology (2003), 17(2), 283-5

The role of AT2-receptors has recently been subject of considerable debate. We investigated the influence of AT2-stimulation/inhibition on myocardial endothelial NO-synthase (eNOS, NOS-III) promoter ... [more ▼]

The role of AT2-receptors has recently been subject of considerable debate. We investigated the influence of AT2-stimulation/inhibition on myocardial endothelial NO-synthase (eNOS, NOS-III) promoter activity and eNOS protein expression. Stimulation of rat cardiomyocytes with angiotensin II (AngII) increased eNOS protein expression 3.3-fold. This was blocked by Cyclosporin A (CsA). Inhibition of the AT1-receptor did not reduce AngII-mediated eNOS protein expression, whereas AT2 stimulation increased it 2.4-fold and AT2 inhibition suppressed it. The modulatory effects of the AT2-receptor on eNOS expression was confirmed in mice with a genetic deletion of the AT2-receptor (AT2-KO). In gel shift assays two putative NF-AT sites in a 1.6 kb eNOS promoter fragment showed NF-AT binding and a supershift by NF-AT2(-c1)-specific antibodies. Stimulation of transfected cells with AngII or specific AT2-receptor agonists resulted in a significant increase in eNOS promoter activity, which was blocked by CsA, MCIP1, and mutation of an upstream NF-AT site. CONCLUSION: 1) AngII-stimulation of the myocardium, both in vivo and in vitro, is accompanied by increased expression of eNOS. 2) This effect is mediated by the calcineurin pathway and is induced by the AT2-receptor. 3) These results define a calcineurin/NF-AT/eNOS pathway as downstream effector of AT2-receptor activation in the myocardium. [less ▲]

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See detailCalcineurin in human heart hypertrophy.
Ritter, Oliver; Hack, Susanne; Schuh, Kai et al

in Circulation (2002), 105(19), 2265-9

BACKGROUND: In animal models, increased signaling through the calcineurin pathway has been shown to be sufficient for the development of cardiac hypertrophy. Calcineurin activity has been reported to be ... [more ▼]

BACKGROUND: In animal models, increased signaling through the calcineurin pathway has been shown to be sufficient for the development of cardiac hypertrophy. Calcineurin activity has been reported to be elevated in the myocardium of patients with congestive heart failure. In contrast, few data are available about calcineurin activity in patients with pressure overload or cardiomyopathic hypertrophy who are not in cardiac failure. METHODS AND RESULTS: We investigated calcineurin activity and protein expression in 2 different forms of cardiac hypertrophy: hypertrophic obstructive cardiomyopathy (HOCM) and aortic stenosis (AS). We found that the C-terminus of calcineurin A protein containing the autoinhibitory domain was less abundant in myocardial hypertrophy than in normal heart, which suggests the possibility of proteolysis. No new splice variants could be detected by reverse-transcription polymerase chain reaction. This resulted in a significant elevation of calcineurin enzymatic activity in HOCM and AS compared with 6 normal hearts. Increased calcineurin phosphatase activity caused increased migration of NF-AT2 (nuclear factor of activated T cells 2) in SDS-PAGE compatible with pronounced NF-AT dephosphorylation in hypertrophied myocardial tissue. CONCLUSIONS: Hypertrophy in HOCM and AS without heart failure is characterized by a significant increase in calcineurin activity. This might occur by (partial) proteolysis of the calcineurin A C-terminus containing the autoinhibitory domain. Increased calcineurin activity has functional relevance, as shown by altered NF-AT phosphorylation state. Although hypertrophy in AS and HOCM may be initiated by different upstream triggers (internal versus external fiber overload), in both cases, there is activation of calcineurin, which suggests an involvement of this pathway in the pathogenesis of human cardiac hypertrophy. [less ▲]

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