Journal: PLoS ONE
Article Title: Preserved Cardiac Function despite Marked Impairment of cAMP Generation
Figure Lengend Snippet: AC6mut Design, Expression, Activity and Cellular Distribution. A . The diagram depicts the site of substitution of alanine (ala) for aspartic acid (asp) at position 426 in the C1 domain (intracellular loop) in the construction of AC6mut. The substitution inhibits Mg 2+ binding and alters the efficiency of Gsα-mediated activation of the catalytic core, which impairs the enzymatic activity of AC6, resulting in reduced cAMP production. M1 and M2, transmembrane domains of AC6; C1 and C2, cytoplasmic domains of AC6, which form the catalytic core; βAR, β-adrenergic receptor; βΥ and α, components of the guanosine 5′-triphosphate (GTP)-binding protein, Gs. B . AC6mut mRNA expression was assessed by qRT-PCR using primers common to endogenous AC6 and transgene AC6mut. Primers for detecting GAPDH mRNA were used for internal control of the qRT-PCR reaction. AC6mut mRNA was increased 62-fold vs endogenous AC6. Animal number in bars +SE; Student's t-test, unpaired, 2 tails. C . AC6mut protein was detected in immunoblotting using anti-AC5/6 antibody and confirmed using anti-AU1 tag antibody. AC6mut protein was increased 17-fold vs endogenous AC6. D . Cyclic AMP production in isolated cardiac myocytes from AC6mut and control mice, before (Basal) and after stimulation with isoproterenol (Iso; 10 µM, 10 min) or NKH477 (NKH; 10 µM, 10 min), using cAMP Enzymeimmunoassay. Cardiac myocytes from AC6mut mice (M vs C, control) showed impaired cAMP production in response to Iso and NKH477, a forskolin analog. Bars denote mean +SE; p values from 1-way ANOVA followed by Bonferroni post test (n = 6, each group). E . Double immunofluorescence staining of AC6mut protein in cardiac myocytes isolated from AC6mut vs control mice using anti-AU1 antibody (red); anti-caveolin 3 (Cav-3) antibody (green, for caveolae); anti-protein disulphide-isomerase (PDI) antibody (green, for sarcoplasmic reticulum); and anti-lamin A antibody (green, for nuclear envelope). Nucleus is blue. AC6mut transgene was detected in caveolae, SR, and nuclear envelope.
Article Snippet: Cyclic AMP was measured using the cAMP Biotrak Enzymeimmunoassay System (GE Healthcare) as previously reported .
Techniques: Expressing, Activity Assay, Binding Assay, Activation Assay, Quantitative RT-PCR, Isolation, Mouse Assay, Double Immunofluorescence Staining