Mitochondrial A‐kinase anchoring proteins in cardiac ventricular myocytes

Figures

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  FIGURE 1. mRNA expression of mitochondria‐associated AKAPs in ARVM. Bar graphs show the mean ± S.E.M. expression value of AKAPs from ARVM normalized to the expression of the endogenous housekeeping gene, GAPDH; n = 7 independent RNA extractions analyzed in triplicate for each extraction

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  FIGURE 2. Expression levels of mitochondria‐associated AKAPs in ARVM. (a) Ponceau S (Pon‐S) staining as a loading control. Lanes represent total cell lysate (T), the cytosolic fraction (C), and the mitochondria enriched fraction (M) from ARVMs. (b) Tubulin was used as a marker for the cytosolic fraction and TOMM20 as the specific mitochondrial marker. (c) PVDF membrane was probed for indicated AKAPs. Molecular weight markers are placed adjacent to the blots. n ≥ 3 independent extractions

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  FIGURE 3. Co‐localization of D‐AKAP1 and mitochondria in ARVM. Representative confocal images of ARVM showing mitochondrial marker (a) TOMM20 and (b) D‐AKAP1. (c) Merged image of TOMM20 (red), D‐AKAP1 (green), and DAPI (blue) shows that D‐AKAP1 overlaps the mitochondrial marker. PCC = 0.77 ± 0.03; N = 4 independent isolations with n = 6 from each isolation. Magnified images are shown below each corresponding image with the appropriate scale bar

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  FIGURE 4. Co‐localization of D‐AKAP2 and mitochondria in ARVM. Confocal images of ARVM showing mitochondrial marker (a) TOMM20 and (b) D‐AKAP2, presenting a punctate pattern and overlap with mitochondria. (c) Merged images representing TOMM20 (red), D‐AKAP2 (green), and DAPI (blue). PCC = 0.72 ± 0.03; N = 4 independent isolations with n = 6 from each isolation. Magnified images are shown below each corresponding image with the appropriate scale bar

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  FIGURE 5. Co‐ labeling of ACBD3 with mitochondria in ARVM. Labeling of the mitochondrial marker, (a) TOMM20 and (b) ACBD3 show a similar pattern of immunoreactivity. (c) Merged images of TOMM20 (red), ACBD3 (green), and DAPI (blue) show that ACBD3 is distributed in a diffuse manner generating a PCC of 0.56 ± 0.02; N = 4 independent isolations with n = 6 from each isolation. Magnified images are shown below each corresponding image with the appropriate scale bar

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  FIGURE 6. Co‐ labeling of OPA1 with mitochondria in ARVM. Labeling of the mitochondrial marker, (a) TOMM20 and (b) OPA1. (c) Merged images of TOMM20 (red), OPA1 (green), and DAPI (blue). PCC = 0.58 ± 0.03; N = 4 independent isolations with n = 6 from each isolation. Magnified images are shown below each corresponding image with the appropriate scale bar

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  FIGURE 7. Expression levels of PKA subunits in mitochondria. Exemplar immunoblot for the expression of PKA regulatory, RIα/β, RIIα, RIIβ, and catalytic (Cat‐α/β/γ) subunits are shown in total cell lysate (T), the cytosolic fraction (C), and mitochondria enriched fraction (M); n ≥ 3 independent protein extractions

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  FIGURE 8. PKA RI subunit and mitochondria co‐labeling. Representative confocal images of ARVM showing mitochondrial marker (a) TOMM20 and (b) PKA RI regulatory subunit. (c) Composite image showing strong overlap of TOMM20 (red) and PKA RI subunit (green) in the subsarcolemmal mitochondria. PKA RI antibody is also seen to label transverse striations. PCC = 0.69 ± 0.02, MOC = 0.80 ± 0.03; N = 4 independent isolations with n = 6 from each isolation. Magnified images are shown below each corresponding image with the appropriate scale bar

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  FIGURE 9. Co‐localization of PKA RIIα subunit with mitochondria. Representative confocal images of ARVM showing mitochondrial marker (a) TOMM20 and (b) PKA RII‐α subunit. (c) Merged images of TOMM20 (red), PKA RIIα subunit (green), and DAPI (blue). PCC = 0.63 ± 0.02, MOC = 0.80 ± 0.02; N = 4 independent isolations with n = 6 from each isolation. Magnified images are shown below each corresponding image with the appropriate scale bar

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  FIGURE 10. Co‐localization of PKA RIIβ subunit with mitochondria. Representative confocal images of ARVM showing mitochondrial marker (a) TOMM20 and (b) PKA RII‐β subunit. (c) Merged image shows moderate colocalization between TOMM20 (red), PKA RII‐β (green), and DAPI (blue). PCC = 0.61 ± 0.02, MOC = 0.73 ± 0.02; N = 4 independent isolations/experiments with n = 6 from each isolation. Magnified images are shown below each corresponding image with the appropriate scale bar