beta-Adrenergic receptor stimulated Ncx1 upregulation is mediated via a CaMKII/AP-1 signaling pathway in adult cardiomyocytes - PubMed (original) (raw)

beta-Adrenergic receptor stimulated Ncx1 upregulation is mediated via a CaMKII/AP-1 signaling pathway in adult cardiomyocytes

Santhosh K Mani et al. J Mol Cell Cardiol. 2010 Feb.

Abstract

The Na(+)-Ca(2+) exchanger gene (Ncx1) is upregulated in hypertrophy and is often found elevated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. beta-Adrenergic receptor (beta-AR) signaling plays an important role in the regulation of calcium homeostasis in the cardiomyocyte, but chronic activation in periods of cardiac stress contributes to heart failure by mechanisms which include Ncx1 upregulation. Here, using a Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIdelta(c)) null mouse, we demonstrate that beta-AR-stimulated Ncx1 upregulation is dependent on CaMKII. beta-AR-stimulated Ncx1 expression is mediated by activator protein 1 (AP-1) factors and is independent of cAMP-response element-binding protein (CREB) activation. The MAP kinases (ERK1/2, JNK and p38) are not required for AP-1 factor activation. Chromatin immunoprecipitation demonstrates that beta-AR stimulation activates the ordered recruitment of JunB homodimers, which then are replaced by c-Jun homodimers binding to the proximal AP-1 elements of the endogenous Ncx1 promoter. In conclusion, this work has provided insight into the intracellular signaling pathways and transcription factors regulating Ncx1 gene expression in a chronically beta-AR-stimulated heart.

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Figures

Figure 1. Stimulation of either β1-AR or β2-AR agonist is sufficient to induce the upregulation of Ncx1

A) Freshly isolated adult feline cardiomyocytes were infected with adenovirus expressing 1831Ncx1 promotor-luciferace reporter construct (MOI 1.5). Cells were treated with or without β1 and β2-AR agonist 0.1 μM isoproterenol (Iso), β1-AR agonist 1 μM dobutamine (dob) or β2-AR agonist 1μM salbutamol (Sal) for 24 hours. Cardiomyocytes were also pretreated with propranonal (Prop) for 30 minutes and then subjected to β1-AR and β2AR agonist treatment for 24 hours. Cells were lysed in reporter buffer and luciferase activity was determined. The values are expressed as relative luciferase activity normalized to GFP levels (RLU/GFP). * p < 0.001 when compared to control, (experiments performed in duplicate from 6 independent cell isolations) # p<0.001 when compared to β-AR stimulations, (experiments performed in duplicate from 6 independent cell isolations). B) Isoproterenol (30 mg /kg/day) or vehicle control was continuously infused into 1831Ncx1 promoter-luciferase transgenic mice. After 72 hours, the mice were euthanized and luciferase activity was determined as described in the methods. Relative luciferace units were normalized to protein levels (RLU/Protein). Values are the mean ± S.E.M (n=4 mice for each condition). *p<0.05 when compared to control. C) Isoproterenol (3.0 mg/kg/day) or vehicle control was continuously infused into adult male rats by osmotic pump. After 72 hours of β-adrenergic infusion, the rats were euthanized and total RNA isolated from the hearts. Relative endogenous Ncx1 transcript levels were quantified by real time RT-PCR. Relative Ncx1 mRNA levels were extrapolated from a standard curve and were normalized to 18S rRNA. Values are the mean ± S.E.M (n=4 rats for each condition). *p<0.01 when compared to control.

Figure 2. PKA activation is not required for Ncx1 upregulation

Adult cardiomyocytes were infected with 1831Ncx1 promoter-luciferase adenovirus (MOI 1.5). A) Cells were subsequently treated with 1 μM dobutamine or 10 μM forskolin for 48 hours. B) Infected cardiomyocytes were treated with or without 1μM dobutamine for 24 hours and/or PKA inhibitor H-89 (20 μM). Cells were lysed in reporter buffer and luciferase activity was determined. The values are expressed as relative luciferase activity normalized to GFP levels (RLU/GFP). Values are the mean ± S.E.M (experiments performed in triplicate from 3 independent cell isolations). * p < 0.05 when compared to control and # p < 0.05 when compared to dobutamine treated.

Figure 3. CaMKII mediates β-AR-stimulated Ncx1 expression

A) Isolated adult cardiomyocytes were infected with the 1831Ncx1 adenovirus (MOI 1.5). Infected cardiomyocytes were treated with or without 1μM dobutamine for 24 hours and/or CaMKII inhibitor KN93 (10 μM) or analog to KN93, KN92 (10 μM) as indicated. Cells were lysed in reporter buffer and luciferase activity was determined. The values are expressed as relative luciferase activity normalized to GFP levels (RLU/GFP). * p < 0.05 when compared to control and # p < 0.05 when compared to dobutamine treated (experiments performed in duplicate from 6 independent cell isolations). B) Cell lysates from each condition were run on separate SDS-PAGE gels followed by Western blotting with antibodies against either phospho-T287 CaMKII or CaMKII. The graph represents normalized P-CaMKII levels determined by Western analysis from four separate experiments. Values are the mean ± S.E.M, * p < 0.05 when compared to control and # p < 0.05 when compared to dobutamine treated.

Figure 4. CaMKII is required for β-AR-stimulated Ncx1 expression

β-AR agonist isoproterenol (30 mg/kg/day) or vehicle was injected by IP to wild-type and CaMKIIδ-/-mice for 72 hour. NCX levels (Upper panel) and CaMKIIδ expression levels (lower panel) were measured by Western blot. The graph represents the NCX1 protein level normalized to total protein. Values are the mean ± S.E.M, n=4 mice for each group * p < 0.008 verses wild-type).

Figure 5. CREB does not mediate β-AR-stimulated Ncx1 expression

A) Depicts the AP-1 and CRE binding elements in the Ncx1 promoter. B) Cell lysate samples for each condition were run on separate SDS-PAGE gels followed by Western blotting with antibodies against either phospho-S113 CREB or CREB, (experiments performed from 4 independent cell isolations). C) Diagram of Ncx1 promoter depicts the presence of CRE elements and the proximal, middle (mid) and distal primers used for ChIP. D) ChIP shows that CREB does not associate with Ncx1 promoter. Freshly isolated adult cardiomyocytes were treated with dobutamine (1 μM) and/or KN93 (10 μM) for 2 hr. Following crosslinking and sonication, cell extracts were immunoprecipitated with CREB antibody. A negative control using rabbit IgG as the precipitating antibody was run to demonstrate the specificity of the ChIP assay. Immune complexes were eluted, decrosslinked and then analyzed by PCR with primer sets specific for the proximal, mid and distal regions of the Ncx1 promoter. Input chromatin was subjected to PCR as a positive control (experiments performed from 3 independent cell isolations).

Figure 6. Identification of AP1 sites mediating β-AR-stimulated Ncx1 upregulation

Schematic of Ncx1 promoter illustrating the AP1 consensus sites, the deletions and site-specific point mutations used to evaluate the role of AP-1 elements in β-AR-stimulated upregulation of Ncx1(Upper panel). Adenoviral constructs for these mutations were made and used to infect freshly isolated adult cardiomyocytes at a MOI of 1.5. Cells were stimulated with or without 1 μM dobutamine for 24 hours (Lower panel). Cells were lysed in reporter buffer and luciferase activity was determined. The values are expressed as relative luciferase activity normalized to GFP levels (RLU/GFP) * p < 0.001 when compared to control, # p<0.001 when compared to β-AR stimulations (experiments performed in triplicate from 4 independent cell isolations).

Figure 7. MAP Kinases are not required for β-AR-stimulated upregulation of Ncx1

Isolated adult cardiomyocytes were infected with the 1831Ncx1 adenovirus (MOI 1.5). Cells were then incubated for 24 hours with 1 μM dobutamine in the presence or absence of: A) the MEK inhibitor 5 μM U0126 or co-infected with MKP3 adenovirus (MOI 4), B) the p38 inhibitor 10 μM SB202190 (SB) or co-infected with DNp38 adenovirus (MOI 4), C) the JNK inhibitor 10 μM SP600125 (SP). Cells from each treatment were lysed in reporter buffer and luciferase activity was determined. The values are expressed as relative luciferase activity normalized to GFP levels (RLU/GFP) * p < 0.05 when compared to control, (experiments performed in duplicate from 4 independent cell isolations in each case).

Figure 8. β-AR-stimulation initiates binding of AP1 transcription factors c-Jun and JunB to the endogenous Ncx1 proximal promoter

A) Diagram of Ncx1 promoter depicts the presence of AP1 elements and the proximal, middle (mid) and distal primers used for ChIP. B) Freshly isolated adult cardiomyocytes were treated with dobutamine (1 μM) for times indicated. Following crosslinking and sonication, cell extracts were immunoprecipitated with JunB, and c-Jun antibodies. A negative control using rabbit IgG as the precipitating antibody was run to demonstrate the specificity of the ChIP assay. Immune complexes were eluted, decrosslinked and then analyzed by PCR with primer sets specific for the proximal, mid and distal regions of the Ncx1 promoter. Input chromatin was subjected to PCR as a positive control (experiments performed from 7 different cell isolations).

Figure 9. Inhibition of CaMKII blunts c-Jun binding of the Ncx1 promoter

ChIP assays were performed from primary adult feline cardiomyocytes in the presence or absence of CaMK inhibitor KN93 (10 μM) and/or dobutamine (1 μM) for 2 hours using c-Jun antibody. PCR was performed with Ncx1 proximal promoter primers. Input chromatin was subjected to PCR to control for variations in immunoprecipitation starting material. Rabbit IgG was used as a negative control for nonspecific binding, (experiments performed from 4 different cell isolations).

References

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beta-Adrenergic receptor stimulated Ncx1 upregulation is mediated via a CaMKII/AP-1 signaling pathway in adult cardiomyocytes - PubMed (original) (raw)