Fig 1: Mitochondrial function of soluble adenylyl cyclase (sAC).(A) Quantitative ELISA measurements of cAMP inside isolated mitochondria (pmol/mg). cAMP is measured following treatment with indicated concentrations of bicarbonate (HCO3-), which activates sAC, and forskolin, which activates transmembrane adenylyl cyclases (tmACs) (n = 6, 6, 5 for 0, 10, and 15 mM [HCO3-], respectively, mitochondria are isolated independently from four hearts). (B) The dependence of ATP production on [HCO3-] and [IBMX]. Sigmoidal fits to the data are shown. (C) The sensitivity of ATP production to phosphodiesterase inhibition (with 50 µM IBMX) at different concentrations of [HCO3-]. (D) Airyscan super-resolution images of isolated cardiac mitochondria immuno-labeled for CAXIV (red, left), Tom20 (blue, center), and merged images (right). (E) Fluorescence profile of CAXIV (red) and Tom20 (blue) for individual mitochondrial images as in D. Mean peak intervals (in µm) are indicated (n = 32 mitochondria). (F) Airyscan super-resolution images of isolated cardiac mitochondria immuno-labeled for CAXIV (red, left), sAC (yellow, center), and merged images (right). (G) Fluorescence profile of CAXIV (red) and sAC (yellow) for individual mitochondrial images as in F (n = 32). For (B, C), n = 12–16 independent experiments per group, mitochondria are isolated independently from four hearts. Data in (A–C) are mean ± standard error of the mean (SEM). One-way two-tailed analysis of variance (ANOVA) with Bonferroni correction in (A, C). *p < 0.05, **p < 0.01, ***p < 0.001. (H) Structure of a typical mitochondria from a rat cardiomyocyte showing the intimate arrangement and packing of the cristae and matrix and the IMS. The image is a slice from an electron microscopic tomogram with membranes traced for clarity and quantitation. As shown in the accompanying video of a similar tomogram (Figure 2—video 1), crista compartments are formed by closely spaced parallel membranes that extend out of the plane of this image. The functional ‘intermembrane space’ (IMS) consists of the narrow peripheral space between the inner boundary membrane (IBM) and the outer mitochondrial membrane (OMM) plus the spaces inside the cristae that connect to the peripheral space through narrow (20–40 nm) crista junctions (represented by small white circles) (Frey and Mannella, 2000). Stereological analysis (Smith and Page, 1976) indicates the cristae contain 85% of the total IMS in this mitochondrion, a value that increases to 91% for a mitochondrion with similar crista packing and an area ×2.5 larger. Scale bar is 250 nm. Figure 2—source data 1.Numeric source data for Figure 2.