Plasma Membrane Calcium Pump (PMCA4)-Neuronal Nitric-oxide Synthase Complex Regulates Cardiac Contractility through Modulation of a Compartmentalized Cyclic Nucleotide Microdomain

Mohamed, Tamer; Oceandy, Delvac; Zi, Min; Prehar, Sukhpal; Alatwi, Nasser; Wang, Yanwen; Shaheen, Mohamed A.; Abouleisa, Riham; Schelcher, Celine; Hegab, Zeinab; Baudoin, Florence; Emerson, Michael; Mamas, Mamas A.; Benedetto, Giulietta Di; Zaccolo, Manuela; Lei, Ming; Cartwright, Elizabeth J.; Neyses, Ludwig

doi:10.1074/jbc.m111.290411

Cited by 75 publications

(57 citation statements)

References 44 publications

(45 reference statements)

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“…Growing evidence indicates that PMCA isoforms regulate calcium-dependent signal transduction through protein-protein interactions, and fine-tuning of localized calcium signals [4,7,[18][19][20].…”

Section: Discussionmentioning

confidence: 99%

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263

Curry

Roberts‐Thomson

Monteith

2016

Biochemical and Biophysical Research Communications

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PMCA2 overexpression in some breast cancers suggests that this calcium pump isoform may play a role in breast pathophysiology. To investigate PMCA2 as a potential drug target for breast cancer therapy, we assessed the functional consequence of PMCA2 silencing on cell death pathways and calcium signals in the basal-like MDA-MB-231 breast cancer cell line. Silencing PMCA2 expression alone has no effect on MDA-MB-231 cell viability, however, PMCA2 silencing promotes calcium-induced cell death initiated with the calcium ionophore ionomycin.Assessment of cytoplasmic calcium responses generated with various agents including ionomycin demonstrates that in MDA-MB-231 cells, PMCA2 does not play a major role in shaping global calcium signals. We also examined the ability of PMCA2 silencing to modulate caspase-dependent cell death triggered by a Bcl-2 inhibitor that is in clinical development for the treatment of various cancers, ABT-263 (Navitoclax). Despite the lack of effect on global calcium responses, PMCA2 silencing augmented Bcl-2 inhibitor (ABT-263)-mediated MDA-MB-231 breast cancer cell death. These studies provide evidence that PMCA2 inhibitors could sensitize PMCA2-positive breast cancers to cell death initiators that work through mechanisms involving the Bcl-2 survival pathway.

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Section: Discussionmentioning

confidence: 99%

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263

Curry

Roberts‐Thomson

Monteith

2016

Biochemical and Biophysical Research Communications

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“…The exact way in which PMCA4 modulates vascular nNOS activity is unknown, but nNOS activity is calcium/calmodulin dependent and a role for PMCA4 in regulating localised Ca 2+ concentrations in a microdomain has been suggested . More recent investigation of the role of PMCA4 in cardiac signalling has supported this supposition, pointing to PMCA4 having an important role as a structural molecule in the heart Mohamed et al, 2011). Overexpression of PMCA4 in arterial smooth muscle has been shown to significantly reduce nNOS activity .…”

Section: Transgenic Expression Of Plasma Membrane Calcium Atpasementioning

confidence: 95%

Plasma membrane calcium ATPases (PMCAs) as potential targets for the treatment of essential hypertension

Little

Cartwright

Neyses

et al. 2016

Pharmacology & Therapeutics

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The incidence of hypertension, the major modifiable risk factor for cardiovascular disease, is increasing. Thus, there is a pressing need for the development of new and more effective strategies to prevent and treat hypertension. Development of these relies on a continued evolution of our understanding of the mechanisms which control blood pressure (BP). Resistance arteries are important in the regulation of total peripheral resistance and BP; changes in their structure and function are strongly associated with hypertension. Anti-hypertensives which both reduce BP and reverse changes in resistance arterial structure reduce cardiovascular risk more than therapies which reduce BP alone. Hence, identification of novel potential vascular targets which modify BP is important. Hypertension is a multifactorial disorder which may include a genetic component. Genome wide association studies have identified ATP2B1, encoding the calcium pump plasma membrane calcium ATPase 1 (PMCA1), as having a strong association with BP and hypertension. Knockdown or reduced PMCA1 expression in mice has confirmed a physiological role for PMCA1 in BP and resistance arterial regulation. Altered expression or inhibition of PMCA4 has also been shown to modulate these parameters. The mechanisms whereby PMCA1 and 4 can modulate vascular function remain to be fully elucidated but may involve regulation of intracellular calcium homeostasis and/or comprise a structural role. However, clear physiological links between PMCA and BP, coupled with experimental studies directly linking PMCA1 and 4 to changes in BP and arterial function, suggest that they may be important targets for the development of new pharmacological modulators of BP.

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“…Western blot analyses using anti-GFP (Abcam) and anti-GAPDH (Abcam) (loading control) were carried out as described before [17].…”

Section: Isolation Of Adult and Neonatal Cardiomyocytes And Western Bmentioning

confidence: 99%

“…Identified inhibitors which had no effect on the coupled enzyme assay components were then confirmed with a fresh solid bought from Sigma-Aldrich and dose response curves for these compounds were carried out. To test the specificity of the most potent identified inhibitor for PMCA4 over other ATPases expressed in the heart, we used membrane microsomes derived from HEK293 cells overexpressing PMCA4, SERCA2a or PMCA1 [7,17] and purified Na + /K + ATPase (Sigma-Aldrich). The inhibitory effect was tested over a range of concentrations in each microsome using the modified coupled enzyme assay [18].…”

Section: Identification Of a Novel Pmca4 Inhibitormentioning

confidence: 99%

Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes

Mohamed

Abouleisa

Baudoin

et al. 2013

Journal of Molecular and Cellular Cardiology

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Isoform 4 of the plasma membrane calcium/calmodulin dependent ATPase (PMCA4) has recently emerged as an important regulator of several key pathophysiological processes in the heart, such as contractility and hypertrophy. However, direct monitoring of PMCA4 activity and assessment of calcium dynamics in its vicinity in cardiomyocytes are difficult due to the lack of molecular tools. In this study, we developed novel calcium fluorescent indicators by fusing the GCaMP2 calcium sensor to the N-terminus of PMCA4 to generate the PMCA4-GCaMP2 fusion molecule. We also identified a novel specific inhibitor of PMCA4, which might be useful for studying the role of this molecule in cardiomyocytes and other cell types. Using an adenoviral system we successfully expressed PMCA4-GCaMP2 in both neonatal and adult rat cardiomyocytes. This fusion molecule was correctly targeted to the plasma membrane and co-localised with caveolin-3. It could monitor signal oscillations in electrically stimulated cardiomyocytes. The PMCA4-GCaMP2 generated a higher signal amplitude and faster signal decay rate compared to a mutant inactive PMCA4 mut GCaMP2 fusion protein, in electrically stimulated neonatal and adult rat cardiomyocytes. A small molecule library screen enabled us to identify a novel selective inhibitor for PMCA4, which we found to reduce signal amplitude of PMCA4-GCaMP2 and prolong the time of signal decay (Tau) to a level comparable with the signal generated by PMCA4 mut GCaMP2. In addition, PMCA4-GCaMP2 but not the mutant form produced an enhanced signal in response to β-adrenergic stimulation. Together, the PMCA4-GCaMP2 and PMCA4 mut GCaMP2 demonstrate calcium dynamics in the vicinity of the pump under active or inactive conditions, respectively. In summary, the PMCA4-GCaMP2 together with the novel specific inhibitor provides new means with which to monitor calcium dynamics in the vicinity of a calcium transporter in cardiomyocytes and may become a useful tool to further study the biological functions of PMCA4. In addition, similar approaches could be useful for studying the activity of other calcium transporters during excitation-contraction coupling in the heart.

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Plasma Membrane Calcium Pump (PMCA4)-Neuronal Nitric-oxide Synthase Complex Regulates Cardiac Contractility through Modulation of a Compartmentalized Cyclic Nucleotide Microdomain

Abstract: Background:Previously we have shown that PMCA4 interacts with nNOS. Results: In PMCA4Ϫ/Ϫ mice, plasma membrane-associated nNOS protein was delocalized to the cytosol with no change in total

Cited by 75 publications

References 44 publications

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263

Plasma membrane calcium ATPases (PMCAs) as potential targets for the treatment of essential hypertension

Development and characterization of a novel fluorescent indicator protein PMCA4-GCaMP2 in cardiomyocytes

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