C1 - Nucleotide signalling in the heart

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Nucleotide Signalling Experiments (1)

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Zacollo & Pozzan aims

Zacollo & Pozzan methods

Zacollo & Pozzan results

Lygren aims

Lygren methods

Lygren results

Mongillo aims

Mongillo methods

Mongillo results

Beca aims

Beca methods

Beca results

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Determine the effects of AKAP18d siRNA knockdown on SR Ca uptake/release.

AKAP18d facilitates formation of signalling complex, disruption prevents PLN from being phosphorylated & dissociating from SERCA.

SR depletion with SERCA inhibitor BHQ, Ca added to EC compartment. siRNA AKAP18d knockdown -> attenuates reuptake.

Investigated the roles of PDE3A&B in regulating basal contractility and SR Ca content.

Neonatal rat cardiac myocytes. [cAMP] assessed by FRET from cyan->yellow GFP. effects of PDE inhibitor IBMX also assessed.

PDE4 inhibitor disrupted cAMP signalling at rest & after B-AR activation significantly more than did PDE3 inhibition.

Studied roles of PDE3&4 in cAMP signalling at rest and after B-AR activation.

[cAMP] measured w/ FRET (as above), PDE3 & PDE4 inhibitors applied at rest and after iso.

PDE3A predominant enzyme, associates with the AKAP18d complex described by Lygren et al

Isolated lagendorff hearts, contractility & relaxation enhanced in PDE3A KO mice but not PDE4A KO mice. Co-immunoprecipitation showed that PDE3A colocalises with SERCA2, PLN, AKAP18d, & PP2A

Studied the spatio-temporal changes in cAMP following B-AR activation

cAMP localised to microdomains @ T-tubules/SR membrane, ~1um. IBMX disrupts localisation of signalling.


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