Extramitochondrial domain rich in carbonic anhydrase activity improves myocardial energetics

Abstract

CO₂ is produced abundantly by cardiac mitochondria. Thus an efficient means for its venting is required to support metabolism. Carbonic anhydrase (CA) enzymes, expressed at various sites in ventricular myocytes, may affect mitochondrial CO₂ clearance by catalyzing CO₂ hydration (to H⁺ and HCO₃⁻), thereby changing the gradient for CO₂ venting. Using fluorescent dyes to measure changes in pH arising from the intracellular hydration of extracellularly supplied CO₂, overall CA activity in the cytoplasm of isolated ventricular myocytes was found to be modest (2.7-fold above spontaneous kinetics). Experiments on ventricular mitochondria demonstrated negligible intramitochondrial CA activity. CA activity was also investigated in intact hearts by ¹³C magnetic resonance spectroscopy from the rate of H¹³CO₃⁻ production from ¹³CO₂ released specifically from mitochondria by pyruvate dehydrogenase-mediated metabolism of hyperpolarized [1-¹³C]pyruvate. CA activity measured upon [1-¹³C]pyruvate infusion was fourfold higher than the cytoplasm-averaged value. A fluorescent CA ligand colocalized with a mitochondrial marker, indicating that mitochondria are near a CA-rich domain. Based on immunoreactivity, this domain comprises the nominally cytoplasmic CA isoform CAII and sarcoplasmic reticulum-associated CAXIV. Inhibition of extramitochondrial CA activity acidified the matrix (as determined by fluorescence measurements in permeabilized myocytes and isolated mitochondria), impaired cardiac energetics (indexed by the phosphocreatine-to-ATP ratio measured by ³¹P magnetic resonance spectroscopy of perfused hearts), and reduced contractility (as measured from the pressure developed in perfused hearts). These data provide evidence for a functional domain of high CA activity around mitochondria to support CO₂ venting, particularly during elevated and fluctuating respiratory activity. Aberrant distribution of CA activity therefore may reduce the heart’s energetic efficiency.

Cardiac ventricular myocytes express several carbonic anhydrase (CA) isoforms, which reside in the cytosol (CAII), mitochondria (CAV), sarcolemma (CAIV, CAXIV) and sarcoplasmic reticulum (SR) membrane (CAIV, CAIX, CAXIV) (1, 2). By catalyzing CO₂ hydration and the reverse reaction between H⁺ and HCO₃⁻ ions, these enzymes help to keep CO₂, HCO₃⁻ and H⁺ at chemical equilibrium (3). CA catalysis has been proposed to facilitate transmembrane H⁺ and HCO₃⁻ fluxes responsible for regulating intracellular pH (pH_i) (4–6). The interaction between CA and a membrane-bound transporter has been dubbed the “transport metabolon,” although its physiological importance remains contentious (7). For example, transmembrane H⁺/HCO₃⁻ fluxes near resting pH_i may be too small to require CA activity.

A more substantial flux of CA substrate in the heart is mitochondrial CO₂ production by the tricarboxylic acid cycle and the enzyme complex pyruvate dehydrogenase (PDH). Under resting conditions, the heart emits ∼3 mmol CO₂ L⁻¹⋅min⁻¹ (based on a myocardial O₂ consumption of 0.1 mL⋅min⁻¹⋅g⁻¹), and this rate can rise several-fold with increased workload. Locally, at the level of individual mitochondria, CO₂ production fluctuates as a result of oscillating mitochondrial activity (8) regulated by Ca²⁺ transients (9–11) and redox state (12, 13). CO₂ vents out of the mitochondrial matrix, across the cytoplasm, and into the extracellular space, crossing at least three membranes (including two mitochondrial membranes plus the sarcolemma) and a distance of several microns which varies as a result of heterogeneous (14) and time-dependent capillary perfusion (15). This distance includes myoplasm that, because of macromolecular hindrance (16–18), particularly in the radial direction (19, 20), moderately restricts CO₂ diffusion. Mitochondrial membranes (21), like many other membranes (but with exceptions; see ref. 22), are significantly more permeable to CO₂ than to H⁺ or HCO₃⁻ ions, particularly when enriched with gas channels (23). The transmembrane [CO₂] gradient therefore is critical in determining mitochondrial CO₂ efflux. CO₂ venting could be supported by CA-catalyzed buffering of extramitochondrial CO₂ (24), particularly when local CO₂ production is elevated and fluctuating.

The activity of extracellular-facing CA isoforms has been shown to accelerate CO₂ exchange between the intra- and extracellular compartments in the heart (25). However, it has yet to be determined whether CO₂ transport from the matrix to the cytoplasm is facilitated by a particular distribution of CA activity. Previous studies have demonstrated modest CA activity in the cytoplasm (2.6-fold acceleration of CO₂ hydration) (3) and negligible CA activity in mitochondria (26). Such a distribution of CA activity could facilitate CO₂ venting from mitochondria and increase the mitochondrial membrane pH gradient (a component of the proton motive force and a driving force for H⁺-coupled solute transport across the inner membrane) (27). A possible link between CA and respiration may be important in cardiac diseases that involve a change in CA isoform expression or activity, such as phenylephrine-induced hypertrophy (28) or dilated cardiomyopathy (29).

We hypothesize that CA activity near (but not within) mitochondria facilitates CO₂ venting and improves myocardial energetics. The distribution of CA activity was investigated using two different approaches to measuring CA catalysis. In the first, pH-sensitive fluorescent dyes assayed cytoplasm-averaged CA activity evoked by rapid CO₂ entry across the sarcolemma (3). In the second, magnetic resonance spectroscopy (MRS) measured CA-catalyzed hydration of ¹³CO₂ released by PDH-mediated mitochondrial decarboxylation of the high-signal MRS tracer, hyperpolarized [1-¹³C]pyruvate (30–32). The effects of CA activity on energetics were then studied in terms of mitochondrial matrix pH (by fluorescence) and high-energy phosphate metabolites (by ³¹P MRS). We demonstrate that an extramitochondrial CA domain is an important component of mitochondrial function in the heart, with a capacity to enhance myocardiac energetics.

Results

Ventricular Mitochondria Have Negligible Carbonic Anhydrase Activity.

Mitochondrial CA activity was investigated in intact and lysed ventricular mitochondria and in ventricular myocytes subjected to saponin permeabilization, which restricts activity measurements to the mitochondrial compartment. Activity was determined from the rate of acidification after the addition of CO₂-containing solution.

Percoll-purified rat ventricular mitochondria (33) (tested for viability; Fig. S1) were lysed in mitochondrial lysate buffer (MLB), and the medium pH was measured at 4 °C using a Hamilton pH electrode. The CA-catalyzed reaction was triggered by adding CO₂-saturated water (1:2 vol/vol; Fig. 1A). A kinetic model described in SI Computational Methods was used to best-fit the experimental time courses with a CO₂ hydration constant (k_h) (34). Spontaneous k_h was determined in the presence of the potent CA inhibitor acetazolamide (ATZ) (Fig. S2A). CA activity was expressed as a ratio of catalyzed-to-spontaneous k_h. In mitochondrial lysates, CA activity was 1.31 ± 0.11 per mg of total protein. For comparison, CA activity in whole-heart lysates was 2.71 ± 0.08 per mg of total protein, that is, 5.5-fold [(2.71–1)/(1.31–1)] greater than in mitochondrial lysates.

Fig. 1.

CA activity in intact and lysed mitochondria. (A) CA activity measured by the rate of medium acidification upon the addition of 0.33 mL CO₂-saturated water at 4 °C to a 0.67-mL suspension of mitochondrial or whole-heart lysates prepared in MLB (n = 5 each). Spontaneous kinetics was determined in the presence of the broad-spectrum CA inhibitor ATZ (100 μM). (B) Matrix pH of intact mitochondria suspended in MSB, measured using BCECF at room temperature (n = 6). The addition of CO₂-saturated MSB (1:4 vol/vol) acidified the matrix by CO₂ hydration. The experiment was repeated in the presence of ATZ (100 μM). (C) (i) Ventricular myocytes (n = 8) were superfused at 37 °C with Hepes-buffered IS, permeabilized by means of 15-s treatment with 0.005% saponin (Sap) and then superfused with CO₂/HCO₃⁻-buffered IS. (ii) Experiments were repeated in 100 µM ATZ (n = 9).

Measurements on mitochondrial lysates may report CA activity with intra- or extramitochondrial catalytic sites. To measure matrix-facing CA activity only, suspensions of intact mitochondria in mitochondrial storage buffer (MSB) were loaded with the membrane-permeant acetoxymethyl (AM) ester of the ratiometric pH-sensitive fluorescent dye BCECF [2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein]. After dye loading, mitochondria were washed in dye-free solution to ensure that BCECF reported matrix pH. Upon addition of CO₂-saturated buffer, BCECF reported a fall in pH arising from CO₂ hydration in the matrix. ATZ [permeable across mitochondrial membranes (26)] did not affect pH_i time courses, indicating negligible matrix CA activity. In contrast, the rapid rate of intracellular acidification measured in BCECF-loaded human red cells (positive control for CA activity) was inhibited by ATZ (Fig. S3A).

To study mitochondrial CA activity in the native environment of the myocyte, cells were loaded with the ratiometric pH dye cSNARF-1 [5-(and-6)-Carboxy SNARF-1] for 40 min (ensuring adequate mitochondrial dye loading) and then permeabilized by brief (15-s) exposure to 0.005% saponin in Hepes-buffered internal solution (IS). Permeabilization releases cytoplasmic cSNARF-1, and the residual dye reports matrix pH. Exposure to CO₂/HCO₃⁻-buffered IS reduced matrix pH, resulting from CO₂ influx and hydration, followed by the attainment of a new steady state (Fig. 1 C, i). ATZ did not reduce the initial slope, adding to the evidence for negligible intramitochondrial CA activity (Fig. 1 C, ii).

Cytoplasm of Ventricular Myocytes Has Modest CA Activity.

CA activity in the cytoplasm of ventricular myocytes was measured from the rate of H⁺ production triggered by a rise in intracellular [CO₂]. CO₂ was introduced into isolated myocytes (under continuous superfusion at 37 °C) by switching from Hepes-buffered normal Tyrode (NT) to CO₂/HCO₃⁻-buffered NT. The progress of the intracellular CA-catalyzed reaction was measured using cSNARF-1 fluorescence (Fig. 2A). Dye loading was restricted to 10 min to minimize partitioning into organelles. The degree of subcellular sequestration of cSNARF-1 was determined at the end of each experiment by permeabilizing myocytes with saponin. Upon permeabilization, cSNARF-1 fluorescence (interpolated to the isosbestic wavelength of 605 nm) decreased by 83 ± 2%, indicating that the fluorescence signal in intact myocytes was predominantly cytosolic. The rate of pH_i change upon CO₂ entry (or exit) is a quantitative measure of CA activity (Fig. S3D). Because pH_i was acquired over the entire cell, this experimental protocol measured cytosol-averaged CA activity. Using a computational model (34), described in SI Computational Methods, k_h was determined to be 0.58 ± 0.08/s and 0.58 ± 0.12/s upon the addition and subsequent removal of CO₂, respectively (n = 17). The measured pH_i changes were not rate-limited by the much faster extracellular solution exchange rate (Fig. S3B), and the rate constants obtained were not at the upper limit of the method’s resolving power (Fig. S3C). Transient exposure to CO₂/HCO₃⁻ was repeated in the presence of ATZ (100 µM) to measure spontaneous kinetics (0.20 ± 0.02/s and 0.23 ± 0.02/s on CO₂ addition and removal, respectively), which were in agreement with uncatalyzed kinetics (35, 36). Thus, CA activity across the cytosol accelerated CO₂ hydration by a factor of 2.7 ± 0.3.

Fig. 2.

Intracellular CA activity is concentrated near mitochondria. (A) Measuring CA activity in isolated, intact myocytes (n = 17) under superfusion at 37 °C. Switching from Hepes-buffered NT to CO₂/HCO₃⁻-buffered NT (and back) evokes a pH_i change proportional to the intracellular CA activity. The experiment was repeated in the presence of the CA inhibitor ATZ (100 µM). Thick cyan lines show best-fit simulation (CA activity of 2.7). (B) Measuring cardiac CA activity in rats infused in vivo with metabolic tracer. (i) Time course of ¹³C-labeled pyruvate, CO₂, and HCO₃⁻ measured by MRS of rats infused with 80 µmol of hyperpolarized [1-¹³C]pyruvate (n = 8). (ii) The experiment was repeated on rats pretreated with 25 µmol ATZ 15 min before the infusion of [1-¹³C]pyruvate (n = 6). Continuous traces show the best-fit model simulation (CA activity 11.4). (C) Measuring cardiac CA activity in Langendorff-perfused rat hearts. Hearts were perfused with 0.6 mM [1-¹³C]pyruvate, and ¹³C-labeled metabolites were measured by MRS (n = 5); the experiment was repeated with 100 μM ATZ (n = 5). For clarity, only ¹³CO₂ and H¹³CO₃⁻ time courses are shown. The [1-¹³C]pyruvate signal attained a peak at ∼30 s. The H¹³CO₃⁻ signal was quenched (Q₁–Q₄) every 20 s to trigger ¹³CO₂ hydration. The initial rate of H¹³CO₃⁻ reappearance was obtained from exponential best-fit. (D) Apparent CA activity measured under different experimental protocols. Black bars: From the initial appearance of H¹³CO₃⁻-signal. Membrane-impermeable CA inhibitor C23 was infused at 2 µmol, and the Cl⁻/HCO₃⁻ exchange inhibitor DIDS (n = 3) was infused at 25 µmol to rats in vivo; control experiments were repeated on Langendorff-perfused hearts ex vivo (n = 8). Gray bars: After sequential H¹³CO₃⁻-quenching reactions, once every 20 s. White bar: Upon addition of CO₂ from the superfusate.

Ventricular Mitochondria Are Close to a Domain Rich in CA Activity.

In an alternative approach to measuring CA activity, CO₂ was introduced into the cytoplasm by mitochondrial decarboxylation of pyruvate. This process and the subsequent CO₂ hydration to HCO₃⁻ was measured using ¹³C MRS following the administration of [1-¹³C]pyruvate to intact hearts. To improve the signal-to-noise ratio of measurements, ¹³C-labeled pyruvate was subjected to a hyperpolarization procedure (30). Labeling the first carbon of pyruvate ensured that the source of ¹³CO₂ was exclusively the mitochondrial activity of PDH (32, 37). Thus, CA activity could be measured locally in the ¹³CO₂-containing cell domain by the rate of H¹³CO₃⁻ production.

The hyperpolarized metabolic tracer [1-¹³C]pyruvate was infused i.v. into anesthetized living rats, and ¹³C spectra were acquired from a surface coil placed over the rat’s chest to detect PDH activity predominantly from the heart (32). Fig. 2 B, i shows the average time courses of ¹³C-labeled compounds measured by MRS. Experiments also were performed on animals pretreated with 25 μmol ATZ (Fig. 2 B, ii) or 2 μmol of the membrane-impermeant CA inhibitor C23 [4-(2,4,6-trimethylpyridinium-N-methylcarboxamido)-benzenesulfonamide perchlorate] (Fig. S2B) (38). The initial rate of H¹³CO₃⁻ production measures CO₂ hydration near its site of production. ATZ reduced the initial slope and the peak of the H¹³CO₃⁻ signal (normalized to peak [1-¹³C]pyruvate) by 85% and 24%, respectively, and increased the initial slope and the peak of the ¹³CO₂ signal by 70% and 88%, respectively. In contrast, C23 had no effect on the ¹³C time courses (Fig. S3E). Because the mitochondrial matrix has no detectable CA activity (Fig. 1), the effect of ATZ must arise from the activity of extramitochondrial CA enzymes.

The CO₂ hydration constant k_h, ¹³C signal decay, and PDH activity were determined by best-fitting to a computational simulation (Fig. S3F), as described in SI Computational Methods (31). ATZ and C23 had no effect on PDH activity itself (0.0049 ± 0.0004, 0.0043 ± 0.0006, and 0.0051 ± 0.0005/s in ATZ, C23, and control, respectively) or on the signal-decay constant of either [1-¹³C]pyruvate (0.14 ± 0.01, 0.13 ± 0.02, and 0.16 ± 0.01/s, respectively) or ¹³CO₂/H¹³CO₃⁻ (0.091 ± 0.08, 0.084 ± 0.04, and 0.073 ± 0.01/s, respectively). The best-fit k_h was 2.2 ± 0.2/s under control conditions and 0.19 ± 0.01/s in the presence of ATZ. Thus, CA activity accelerated CO₂ hydration in the ¹³CO₂-rich domain by a factor of 11.4 ± 0.9. This measurement of CA activity within a spatially confined region of the cell was considerably higher than the cytosol-averaged estimate (Fig. 2A) matched for pH_i, and temperature. C23 did not affect k_h (2.1 ± 0.2/s), confirming that the method was assaying intracellular CA activity only (Fig. S3E). These data suggest that a cytoplasmic domain near mitochondria is enriched in CA activity.

In the in vivo experiments described above, CAII activity in red cells could be affecting the CA activity measurements. To test this possibility, experiments were performed on rats infused with 25 μmol DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) to block HCO₃⁻ exchange between red cells and plasma and, in separate experiments, on Langendorff-mode Krebs–Henseleit buffer (KHB)–perfused hearts ex vivo. The best-fit k_h (in vivo with DIDS: 2.0 ± 0.3/s; ex vivo: 2.0 ± 0.2/s) was not different from measurements on control hearts in vivo, arguing against a contribution from red cell CA. Results from Langendorff-perfused hearts also confirm that the ¹³C time courses measured in rats in vivo were cardiac in origin.

The ¹³C-based method described so far inferred CA activity from the initial rate of appearance of hyperpolarized H¹³CO₃⁻ and ¹³CO₂ signals. CA activity was probed further using an alternative ¹³C-based protocol involving H¹³CO₃⁻-signal quenching to drive CO₂/HCO₃⁻ out of equilibrium. A radio-frequency electromagnetic pulse was applied every 20 s after the administration of hyperpolarized [1-¹³C]pyruvate to Langendorff-perfused hearts. Each quenching pulse permanently destroyed all MR signal originating from the hyperpolarized H¹³CO₃⁻ pool without directly affecting the ¹³CO₂ signal. The rate of H¹³CO₃⁻ production after each quench therefore measures the current ¹³CO₂ hydration rate (Fig. 2 C, i). This was quantified from the initial slope of the H¹³CO₃⁻ signal increase divided by the ¹³CO₂ signal amplitude and normalized to measurements from ATZ-treated hearts (100 μM; n = 5) (Fig. 2 C, ii). After the first quenching reaction (Q₁), CA activity (10.9 ± 1.2; control k_h = 2.12 ± 0.22/s; ATZ k_h = 0.19 ± 0.01/s) was similar to that determined from the initial appearance of the H¹³CO₃⁻ signal (Fig. 2B). However, subsequent quenching reactions yielded a lower CA activity (Fig. 2D), suggesting that the distribution of ¹³CO₂ signal had changed with time to a spatial domain encompassing a lower CA density. Because hyperpolarized ¹³CO₂ is washed out of the perfused heart, the amplitude of its global signal declines, as observed experimentally (Fig. 2C). During this washout period, which lasts for tens of seconds, much of the remaining hyperpolarized ¹³CO₂ permeates into neighboring and down-stream myocytes and there, it undergoes hydration to H¹³CO₃⁻, catalyzed by bulk cytoplasmic CA. Meanwhile, de novo ¹³CO₂ signal production by mitochondria decreases because of spin-lattice relaxation of hyperpolarized [1-¹³C]pyruvate [decaying to 1/e of its original value in 50–60 s (31)]. Therefore it is notable that spatial dispersal of the myocardial ¹³CO₂ signal was associated with a shift in the measured CA activity from the high initial value associated locally with mitochondria to a more modest value typical of that measured previously in superfused myocytes, when CO₂ was delivered exogenously to bulk cytoplasm (Fig. 2A). Thus, depending on the time period after acute introduction of hyperpolarized [1-¹³C]pyruvate to the perfused heart, the H¹³CO₃⁻-quenching protocol provides estimates of CA activity that are consistent initially with a high local, extramitochondrial value and subsequently with a lower more general cytoplasmic value.

Fluorescent CA Ligand Colocalizes with Mitochondria.

Cytosol-facing CA catalytic sites were visualized in permeabilized myocytes bathed in mitochondrial respiration buffer (MRB) using the fluorescein-conjugated CA inhibitor (F-CAI) (38). After 90-s exposure to the ligand (1 mM), unbound F-CAI was washed out by superfusion. Compared with the water-soluble derivative fluorescein sulfonate (FS), F-CAI bound more stably to membranes (Fig. S4A), so that after 60-s washout, fluorescence was derived mainly from CA-bound F-CAI. A similar procedure was followed for the membrane-tagging derivative DHPE-fluorescein [DHPE-F; N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium] used as a positive control for mitochondrial membrane staining. [NB: Substantially longer (>10 min) dye loading leads to dye accumulation in the intermembrane space.]

Mitochondria were visualized using the fluorescent marker tetramethylrhodamine ethyl (TMRE) loaded before permeabilization. At 488-nm excitation, fluorescein fluorescence bleed-through to the TMRE detection range was only 11% (Fig. S4B) and was corrected for in subsequent analyses. Colocalization between TMRE and either F-CAI or DHPE-F was determined by simultaneous imaging after 60-s washout of unbound fluorescein derivative (Fig. 3A). The lack of FRET from F-CAI to TMRE indicated that F-CAI did not enter mitochondria (Fig. S4C), as expected from its membrane impermeability (38).

Fig. 3.

Mitochondria colocalize with CA enzymes. (A) TMRE-loaded myocytes were permeabilized and exposed to one of three fluorescein derivatives (1 mM) for 90 s: (i) membrane-tagging DHPE-F; (ii) the fluorescent CA inhibitor F-CAI; and (iii) FS. Fluorescein (excited at 488 nm) measured <555 nm; TMRE (excited at 555 nm) measured >600 nm. Fluorescence images (100× magnification) were captured after 60 s of washout to remove unbound fluorophores. (B) Pearson’s correlation coefficients between TMRE and fluorescein derivatives. Correlation with F-CAI was reduced in the presence of the nonfluorescent CA inhibitor ATZ (1 mM). Asterisk denotes significant difference (P < 0.05) vs. DHPE-F. (C) Western blots were performed on total myocyte lysate, and samples were taken after three consecutive washing steps in the mitochondrial purification procedure. Immunoreactivity was tested for CA isoforms XIV, VA, and CAII, the mitochondrial marker mitofilin, and the cytoplasm marker LDH-B. Samples 2 and 3 are enriched in mitochondria. CAXIV and CAII remain associated with mitochondria. (D) Immunofluorescence of mitochondrial sample for (i) CAXIV, (ii) secondary antibody only (negative control), and (iii) mitochondrial marker JC-1; (iv) overlay of CAXIV and JC-1 staining.

Correlation of the fluorescence intensity in the TMRE and fluorescein signals was quantified pixel by pixel and plotted as a binned scatter plot (Fig. S4D). Soluble FS and membrane-tagging DHPE-F were used as negative and positive controls, respectively, for mitochondrial colocalization. The high Pearson’s correlation coefficient obtained with F-CAI and its reduction in the presence of the nonfluorescent competitor ATZ (Fig. 3B) suggested that mitochondria are in close contact with a CA-rich cytosolic domain.

Extramitochondrial CAXIV and CAII Are Detected in Isolated Mitochondria.

The identity of CA isoforms associated with the CA-rich extramitochondrial domain was tested by immunoreactivity. Mitochondrial samples were prepared using the Percoll method (39), and several washing steps were applied in an attempt to purify the sample. Mitochondrial samples were then compared with lysates prepared from isolated myocytes. Lactate dehydrogenase B (LDH-B) immunoreactivity, strongly present in total myocyte lysates, diminished to undetectable levels in mitochondrial samples after the first washing step (sample #1). The washing procedure therefore had removed unbound soluble cytosolic proteins (Fig. 3 C, i). Sample #1 was positive for CAXIV (a membrane-tethered isoform) and CAII (a nominally soluble isoform) (2). The mitochondrial marker mitofilin and matrix CA isoform VA were strongly detected after the second wash (sample #2) and remained after a subsequent wash (sample #3), indicating that a highly enriched mitochondrial sample had been achieved after two rounds of washing. In the process of mitochondrial enrichment (sample #2 vs. #1), CAXIV immunoreactivity increased markedly and then remained stable following subsequent washing steps (sample #3 vs. #2; Fig. 3 C, ii). The presence of small but consistently measureable CAII immunoreactivity (Fig. 3 C, i; repeated five times) even after five washing steps (Fig. S5A) suggests that some of this cytosolic protein associates with mitochondria. Mitochondrial samples showed immunoreactivity to sarcoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2A) but not ryanodine receptor type 2 (RyR2) (Fig. S5B), suggesting that the presence of CAXIV protein in isolated mitochondria may arise from their interaction with network (rather than junctional) SR (40). The colocalization of CAXIV immunoreactivity with mitochondria was tested with immunofluorescence. CAXIV staining was concentrated within particles of regular size (diameter 0.5–1.0 μm; Fig. 3 D, i) expected of mitochondria (Fig. 3 D, iii). Furthermore, colocalization was observed in samples stained dually for CAXIV and the mitochondrial indicator JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; Fig. 3 D, iv).

Extramitochondrial CA Activity Raises Mitochondrial Matrix pH but Not Cytoplasmic pH.

The presence of an extramitochondrial CA-rich domain may affect mitochondrial matrix pH by facilitating CO₂ venting (7, 24). This possibility was studied by measuring pH in myocytes that had been saponin-permeabilized and superfused (37 °C) with mitochondrial respiration buffer (MRB) at pH 7.15 (Fig. 4A). Fluorescence from cSNARF-1–loaded permeabilized myocytes reports ensemble matrix pH. In the presence of 5 mM ATP, 80 µM ADP, and mitochondrial substrates to stimulate respiration (41), the matrix was found to acidify in response to CA inhibition with 100 µM ATZ. This effect was not associated with any significant change in mitochondrial membrane potential (Δψ) measured using JC-1 (Fig. S6). In myocytes preincubated with ATZ for 30 min before permeabilization, steady-state matrix pH was 0.11 units lower than in untreated cells.

Fig. 4.

Effect of CA activity on mitochondrial pH, cytoplasmic pH, energetics, and contractility. (A) Experiments on saponin-permeabilized ventricular myocytes superfused in MRB. (i) Effect of ATZ (100 µM) on pH measured with membrane-tagging DHPE-F (reporting pH in intermembrane space; n = 34; blue trace), glycoprotein-binding WGA-fluorescein (reporting pH near sarcolemma and T system; n = 28; green trace), and cSNARF-1 (reporting pH in matrix; n = 31; black trace). To stimulate respiration, experiments with DHPE-F and WGA-F were performed using higher (300 µM) ADP. Significant acidification (P < 0.01) was recorded with cSNARF-1 and DHPE-F. (ii) Steady-state matrix pH in myocytes pretreated with 100 µM ATZ for 30 min before permeabilization (n = 25) vs. control (n = 41) (P = 0.007). (B) Relationship between exogenously supplied extramitochondrial CAII and matrix pH in suspensions of cSNARF-1–loaded mitochondria in state III respiration. Best-fit to Hill curve. Experiments with 10 nM CAII were repeated with 100 μM ATZ. Error bars show coefficient of variation. (n = 6–10 repeats/dose.) (C) Effect of ATZ (100 μM) on cytoplasmic pH measured in cSNARF-1–loaded myocytes superfused with CO₂/HCO₃⁻-buffered NT containing 2 mM pyruvate (n = 25). (D) Cytoplasmic pH measured in perfused hearts by ³¹P MRS under baseline conditions at the end of 15-min Ca²⁺ stress (double perfusate [Ca²⁺]) and upon recovery (n = 8). Experiments were repeated with 100 μM ATZ (n = 8). Two-way ANOVA: P = 0.008 for Ca²⁺ stress; P = 0.68 for ATZ. (E) Effect of ATZ (100 μM) on Mag-Indo-1 fluorescence ratio measured in myocytes (n = 20) superfused with CO₂/HCO₃⁻-buffered NT containing 2 mM pyruvate. The effect of FCCP (1 μM) confirmed the inverse [Mg²⁺]_i–[ATP]_i relationship. (F) PCr/ATP ratio measured in perfused hearts (n = 4 control, n = 6 with ATZ) using ³¹P MRS. Two-way ANOVA for ATZ: P = 0.009. (G) Developed pressure in the left ventricle of perfused hearts sequentially exposed to 35 µM C23 (not significant) and 100 µM ATZ plus 35 µM C23 (P = 0.01). (H) Cartoon showing the CA-rich domain near mitochondria and its role in facilitating CO₂ venting from the matrix. Extramitochondrial CA enzymes include cytoplasmic and SR-associated isoforms.

In separate experiments, pH was measured ratiometrically using fluorescein derivatives, which target different compartments of the cell. DHPE-F applied to permeabilized cells for 10 min concentrates in the intermembrane space (IMS) (42), as validated by the pH response to carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and electron transport chain inhibitors (Fig. S7A). Following a similar loading procedure, wheat germ agglutinin-conjugated fluorescein (WGA-F) binds to the partially disrupted sarcolemmal and T-system and the nucleus, further away from mitochondria (Fig. S7 B, ii). Bulk solution pH was reduced to 7.0 to improve the sensitivity of fluorescein signal to changes in pH. It was necessary to stimulate respiration with 300 µM ADP to resolve the potentially small changes in pH measured with WGA-F and DHPE-F. Although the pH reported by WGA-F was close to bulk pH, DHPE-F measured a more acidic level, consistent with its targeting to the IMS. Upon CA inhibition with ATZ, DHPE-F, but not WGA-F, reported a decrease in pH (by 0.04 pH units; Fig. 4 A, i), suggesting that the site of CA activity is between the IMS and the bulk cytoplasm. Inhibition of CA activity therefore leads to the accumulation of acid in the matrix and, to a lesser extent, in the IMS.

The effect of ATZ on matrix pH was studied further by flow cytometry of isolated, cSNARF-1–loaded mitochondria at room temperature (Fig. S8A). Mitochondria were incubated under state III respiration (i.e., ADP stimulated, with excess substrate) to enhance CO₂ production. Extramitochondrial CA activity was varied experimentally by adding up to 250 nM bovine red cell CAII. CA activity in these suspensions was determined in separate experiments from the rate of medium acidification upon the addition of CO₂-saturated water. CO₂ hydration was accelerated 84% per nanomolar CAII at 4 °C (Fig. S8B). Based on the enzyme's temperature sensitivity (35), CAII activity increases CO₂ hydration 313%/nM at 25 °C. CAII at 3.2 nM produced a milieu that accelerated CO₂ hydration 11-fold, i.e., the level measured in the intact extramitochondrial domain of myocytes (Fig. 2B). Raising extramitochondrial CA activity was found to increase the population-average matrix pH by up to 0.12 units in an ATZ-sensitive manner (Fig. 4B). The half-maximal effect was attained with 0.4 nM CAII (equivalent to 2.2-fold CA catalysis). Thus, the extramitochondrial CA activity normally present in intact myocytes would be sufficient to raise matrix pH.

To test for effects of CA activity on resting cytoplasmic pH, intact ventricular myocytes were loaded for 5 min with cSNARF-1 and superfused with CO₂/HCO₃⁻-buffered NT solution containing 2 mM pyruvate. Permeabilization performed at the end of each experiment confirmed a low (<15%) degree of dye sequestration into mitochondria. Resting cytoplasmic pH was not affected by the addition of 100 µM ATZ, even after 20 min exposure (Fig. 4C). Cytoplasmic pH also was measured in Langendorff-perfused hearts by ³¹P MRS [using the frequency difference between phosphocreatine (PCr) and inorganic phosphate (P_i) peaks]. After 30 min perfusion to attain steady state, cytoplasmic pH was not different in control and hearts treated with 100 µM ATZ (Fig. 4D). To test for effects of ATZ during increased workload, positive inotropy was imposed by doubling perfusate [Ca²⁺] from 1.35 mM to 2.7 mM for 15 min (Ca²⁺ stress). This maneuver reduced cytoplasmic pH, but ATZ had no additional effect.

CA Activity Improves Myocardial Energetics.

The effect of CA activity on increasing the pH gradient across the inner mitochondrial membrane (ΔpH) may regulate mitochondrial function by changing the proton motive force (PMF) and the driving force for transport processes such as pyruvate and P_i uptake and Ca²⁺ removal (27). The relationship between CA activity and energetics was investigated in isolated myocytes (superfused with CO₂/HCO₃⁻-buffered NT containing 2 mM pyruvate) by measuring intracellular [Mg²⁺] levels, which are inversely related to intracellular [ATP] (43). Exposure to 100 µM ATZ resulted in a decrease followed by a slow recovery and overshoot in free [Mg²⁺] measured using Mag-Indo-1. However, these changes were less than 4%, suggesting that [ATP] is highly buffered. The impermeant CA inhibitor C23 did not evoke this biphasic pattern of [Mg²⁺], indicating that the metabolic response was caused by intracellular CA isoforms.

An important buffer that keeps [ATP] nearly constant is the creatine kinase equilibrium involving phosphocreatine (PCr). To assess the state of the heart’s energetic reserve, the ratio of PCr to ATP was determined from ³¹P MR spectra of Langendorff-perfused hearts. The addition of 100 µM ATZ decreased the PCr/ATP ratio significantly (Fig. 4F). Given the constancy of ATP and the decrease in PCr/ATP in response to ATZ, the creatine kinase equilibrium predicts a substantial increase in free cytoplasmic [ADP] (44). The overall decrease in free energy available in high-energy phosphates can contribute to impaired contractility (45). Developed pressure (DP) was unaffected in the presence of 35 µM C23 but decreased within 40 min of perfusion in the additional presence of 100 µM ATZ (Fig. 4G), indicating that extracellular-facing CA sites do not underlie the observed responses to ATZ. The ATZ effect was not associated with a change in the normalized rate of rise or fall of pressure (dP/dt): [dP/dt]_max/DP: 21.1 ± 1.3 s⁻¹ vs. 21.7 ± 1.4 s⁻¹; [dP/dt]_min/DP: 17.4 ± 0.9 s⁻¹ vs. 17.2 ± 1.0 s⁻¹ for C23 and C23+ATZ, respectively.

Discussion

Intracellular Carbonic Anhydrase Activity Is Highly Compartmentalized.

The 13 catalytically active CA isoforms are commonly categorized as cytosolic (CAI, II, III, VII and XIII), mitochondrial (VA and VB), secreted (VI), and membrane-associated (IV, IX, XII, XIV, XV). The complex architecture of cardiac myocytes features H⁺, HCO₃⁻, and CO₂ fluxes at discrete loci, and therefore it is important to understand the distribution of CA activity at fine spatial resolution. This study demonstrates that ventricular mitochondria (a major source of CO₂) have negligible CA activity in their matrix but are surrounded by a domain of high CA activity.

Our finding of negligible intramitochondrial CA activity is in agreement with previous measurements performed on dilute suspensions of broken and intact cardiac mitochondria (26). The protocols used herein (Fig. 1 B and C) restricted pH recordings to within the mitochondria, eliminating the possibility that excessive dilution of mitochondrial matter is the cause of apparently low CA activity. High CA activity in the extramitochondrial domain was inferred from the observation that the cytoplasm-averaged CO₂ hydration rate (measured using a pH-sensitive dye dissolved in the cytoplasm; Fig. 2A) was fourfold lower than the rate of conversion of mitochondrially released ¹³CO₂ to H¹³CO₃⁻ (Fig. 2B). ¹³CO₂ production was confined to mitochondria by infusing hearts with [1-¹³C]pyruvate, a substrate metabolized by pyruvate dehydrogenase to form ¹³CO₂ exclusively in the matrix (32, 37). Cytoplasm-averaged CA activity agrees with previous measurements of intracellular CA catalysis (3), but CA activity near mitochondria has not been quantified previously.

Colocalization of a CA-binding fluorescent drug with the mitochondrial marker TMRE suggests that CA catalytic sites are present near mitochondria (Fig. 3A). The use of a membrane-impermeant fluorescent CA ligand on surface-permeabilized cells precluded binding to CA sites inside organelles such as in the SR lumen. The extramitochondrial CA-rich domain appears to consist of CAXIV. Levels of this isoform increased during mitochondrial purification (Fig. 3C), and immunofluorescence demonstrated its colocalization with mitochondria (Fig. 3D). Because CAXIV is present on the network (longitudinal) SR membrane (2), the extramitochondrial CA domain may be constructed on an SR framework. Immunoreactivity for SERCA2a (but not RyR2) in isolated mitochondria (Fig. S5) further supports the mounting evidence for a close structural and functional relationship between mitochondria and specific compartments of the SR (46, 47). Little is known about the specific role of SR-associated CAXIV in tissues. In contrast, CAXIV activity at the surface membrane has been shown to buffer the brain’s extracellular space (48), facilitate lactic acid transport in skeletal muscle (49), and enhance Cl⁻/HCO₃⁻ exchange in cardiac myocytes, particularly from hypertrophied hearts (50). Purified samples of mitochondria were consistently positive for CAII immunoreactivity, despite the complete washout of the cytosolic protein LDH-B (Fig. 3C). Previous studies have demonstrated CAII binding to the cell membrane (5–7, 51), and a similar interaction may take place with the mitochondrial membranes. An association between CAII/CAXIV and mitochondrial membranes may explain why modest CA activity was detected in mitochondrial lysates (Fig. 1A) but not in the matrix of intact mitochondria (Fig. 1B). It should be noted that CA activity associated with isolated mitochondria is likely to be lower than in the intact environment of cells because of membrane disruption caused by the isolation procedure.

Extramitochondrial CA Activity Improves the Efficiency of Mitochondrial Respiration.

Inhibition of CA activity with ATZ evoked a slow but sustained acidification of the mitochondrial matrix by ∼0.1 pH units at steady state (Fig. 4 A and B). Matrix acidification was associated with a concurrent (albeit smaller) acidification of the intermembrane space (Fig. 4A), suggesting that the ATZ effect cannot be explained by the inhibition of the electron transport chain (which would have produced opposite pH effects on either side of the inner membrane). The pH in the bulk cytoplasm (Fig. 4C) or near the sarcolemma (Fig. 4A) did not acidify in response to ATZ, indicating that the drug targets CA enzymes close to the mitochondrial CO₂ source.

The alkalinizing effect of extramitochondrial CA activity on cell-averaged matrix pH can be explained in terms of facilitated CO₂ venting from mitochondria. The activity of individual mitochondria oscillates and can vary considerably in magnitude (8–13); therefore CO₂ production will not be in a steady state locally (although the ensemble flux may appear so). Extramitochondrial CA activity can convert [CO₂] changes into relatively much smaller [HCO₃⁻] changes (in the presence of pH buffers). This chemical conversion helps maintain a lower [CO₂] at the outer surface of respiring mitochondria, improving mitochondrial [CO₂] venting and shortening the time interval during which matrix [H⁺] overshoots, thereby reducing the time-averaged ensemble matrix [H⁺]. Anchoring CA enzymes in the aqueous compartment near mitochondria, along the path of CO₂ diffusion, increases the available catalytic potential for CO₂ buffering. Extramitochondrial CA activity also may dampen the amplitude of cytoplasmic [CO₂] fluctuations arising from temporal changes in capillary perfusion (14, 15). In summary, by chemically buffering cytoplasmic [CO₂], extramitochondrial CA activity allows a more stable matrix pH and hence ATP production. Extramitochondrial CA activity can be particularly important for matching energy supply with demand. During systole–diastole cycles, changes in cytoplasmic [Ca²⁺] have been proposed to regulate mitochondrial respiration in order to keep the ATP supply constant (52, 53). This matching would be disrupted if matrix pH were allowed to fall after each systolic rise in energy demand. It is important to note that CA activity inside mitochondria would not achieve the same beneficial effects as extramitochondrial CA, perhaps explaining why mitochondria of metabolically active tissue (such as heart) have no detectable matrix CA activity (26, 54).

The CO₂ diffusion coefficient in cardiac cytoplasm is likely to be reduced by the highly organized network of proteins (16–18), particularly in the radial direction because of anisotropy (19, 20), giving an estimated radial tortuosity factor of three- to 11-fold. CA catalysis has been demonstrated (24) to facilitate CO₂ diffusion in aqueous compartments by allowing a parallel flux of HCO₃⁻. However, over the short mitochondria–sarcolemma distances, the modest CA activity measured in cardiac cytoplasm is unlikely to produce a major facilitation of cytoplasmic CO₂ diffusion.

Accelerated CO₂ hydration by extramitochondrial CA may be expected to acidify the cytoplasm, but this acidification was not observed (Fig. 4 C and D). A likely explanation may be that any changes in cytoplasmic pH are too small to resolve. Mitochondria occupy a third of the cell volume, and their intrinsic buffering capacity is threefold lower than cytoplasmic buffering (55). If CA activity raises matrix pH by 0.1 units, changes in cytoplasmic pH would be below resolving power (∼0.01). Additionally, the pH of the cytoplasm is regulated directly by pH-sensing acid/base transporters, such as Na⁺/H⁺ exchange (56).

The inner membrane pH gradient contributes to the ATP-generating PMF and the driving force for Ca²⁺ extrusion (by Ca²⁺/H⁺ exchange), phosphate uptake (on P_i-H⁺ cotransport), and pyruvate uptake (on H⁺-monocarboxylate transport) (27). CA inhibition was found to decrease ΔpH without affecting mitochondrial membrane potential (Δψ) (Fig. S6), reiterating the notion that these components of PMF can be regulated independently (27). A decrease in PMF, P_i, and pyruvate uptake is predicted to decrease ATP production, whereas matrix Ca²⁺ accumulation would stimulate respiration (27, 52, 53). Measurements of [Mg²⁺]_i, an inverse indicator of [ATP]_i, suggested that CA inhibition did not change ATP levels by more than 4% (Fig. 4E). However, the small but reproducible biphasic [Mg²⁺] time course suggests that the cell had undergone changes in its energy metabolism (possibly a transient stimulation by matrix [Ca²⁺] accumulation followed by inhibition as substrate uptake and PMF decrease). This pattern was not observed with the impermeant inhibitor C23 (Fig. S9).

The absence of a substantial effect of CA inhibition on ATP levels would be in agreement with energetic buffering by PCr. This energetic reserve (expressed in terms of PCr/ATP ratio) was reduced by ATZ (Fig. 4F), suggesting a role for CA activity in improving cardiac energetics. Interestingly, reduced energetics have been reported in skeletal muscle treated with CA inhibitors (57) or from CAIII-knockout animals (58). At constant pH_i, a down-stream effect of a decreased PCr/ATP ratio is reduced cardiac contractility (45). This was observed experimentally as the reduction of developed pressure with ATZ. Only intracellular CA isoforms participate in this effect, because the impermeant CA inhibitor C23 did not mimic the ATZ response (Fig. 4G). Studies on skeletal muscle have demonstrated that CA inhibitors also can affect contraction through changes to Ca²⁺ signaling. SR-associated CA activity has been proposed to accelerate the kinetics of Ca²⁺ transients by buffering [H⁺] changes arising from SR-membrane Ca²⁺ fluxes, thereby reducing single-twitch tension (59). This mechanism is unlikely to underlie the observed effect of ATZ on contractility. First, in perfused hearts, CA activity allowed stronger contractions. Second, the normalized rate of pressure rise was not affected by CA inhibition, suggesting that the underlying Ca²⁺ transient had not changed its time course. Indeed, studies on cardiac tissue inferred that SR-associated CA activity does not affect Ca²⁺ flux as markedly as has been demonstrated in skeletal muscle (60).

A reduced myocardial PCr/ATP ratio is characteristic of heart failure and correlates with the New York Heart Association classes of heart disease (61) and with indices of systolic (61) and diastolic (62) function and predicted prognosis (63). Off-target effects of clinically used membrane-permeant CA-inhibiting drugs (64), down-regulated CA activity, or aberrant subcellular CA distribution may lead to inefficient CO₂ venting and a mismatch between energy supply and demand and may contribute toward the heart failure phenotype. Interestingly, tachycardia-induced heart failure has been associated with reduced total CA activity (29), although whether extramitochondrial CA activity also is affected remains to be tested. Further work is warranted to explore whether the activity or size of the extramitochondrial CA domain changes in the failing heart.

Conclusions

We have shown that a cytoplasmic domain of elevated CA activity surrounds mitochondria and facilitates the venting of CO₂ generated by aerobic respiration (Fig. 4H). This CA-rich domain alkalinizes mitochondrial matrix pH and improves the myocardial energetic reserve. Our results demonstrate the importance of CA activity and distribution in supporting the heart’s high metabolic rate. The dependence of CO₂ efflux on the subcellular distribution of CA activity exposes the system’s potential to impair respiratory efficiency and contribute to the development of heart disease.

Materials and Methods

All investigations conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (65) and the UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act, 1986.

Solutions.

All chemicals, unless stated otherwise, were purchased from Sigma-Aldrich (UK). The following buffers were used (all values are in mM). KHB: NaCl (118), KCl (4.7), 1.2 MgSO₄ (1.2), CaCl₂ (1.25), KH₂PO₄ (1.2), NaHCO₃ (25), bubbled with 5% CO₂/95% O₂, pH 7.4 at 37 °C. Hyperpolarized ¹³C dissolution buffer (13CB): Na₂EDTA (0.3), Tris base (20), NaOH (60), heated to 180 °C and pressurized to 10 bar. Hepes-buffered NT: KCl (4.5), glucose (11), NaCl (130), Hepes (20), CaCl₂ (1), MgCl₂ (1), pH 7.4 at 37 °C (with 4 M NaOH). CO₂/HCO₃⁻-buffered NT: Hepes replaced with NaHCO₃ (22) and solution bubbled with 5% CO₂ (balanced with air) at 37 °C (pH 7.4). Hepes-buffered IS: KCl (135), NaCl (10), Hepes (20), K-glutamate (2), K-malate (2), KH₂PO₄ (5), 2,3-butanedione monoxime (15), EGTA (5), CaCl₂ (2; free [Ca²⁺] = 90 nM), MgCl₂ (0.5), pH 7.2 at 37 °C. CO₂/HCO₃⁻-buffered IS: Hepes replaced with NaHCO₃ (14) and solution bubbled with 5% CO₂ gas (balanced with air) at 37 °C. Lysate CA assay buffer: K-gluconate (135), MgCl (1), Hepes (20), MES (20), pH 8 at 4 °C with KOH. Mitochondrial respiration buffer (MRB): KCl (35), K-Gluconate (100), K-Phosphate (1), K-ADP (0.08 or 0.3), Mg-ATP (5), K-glutamate (2), K-malate (2), Na-succinate (2), pyruvic acid (1), NaHCO₃ (10), EGTA (5), MgCl₂ (1.4), CaCl₂ (1.4; free [Ca²⁺] = 50 nM), bubbled with 5% CO₂/95% O₂. Mitochondrial isolation buffer (MIB): sucrose (300), EGTA (2), Tris⋅HCl (10), pH 7.1. Mitochondrial storage buffer (MSB): K-aspartate (100), KCl (30), KH₂PO₄ (5), K-glutamate (2), K-malate (2), NaCl (10), Hepes (10), EGTA (5), ATP (4), CaCl₂ (2; free [Ca²⁺] = 90 nM), MgCl₂ (1), pH 7.2 at 25 °C. Mitochondrial lysis buffer (MLB): K-gluconate (100), KCl (30), MgCl₂ (1), Hepes (20), MES (20), pH 8. State III respiration buffer: K-gluconate (100), KCl (30), NaCl (10), K-glutamate (2), K-malate (2), KH₂PO₄ (5), ADP (0.25), EGTA (5), MgCl₂ (1), CaCl₂ (2; free [Ca²⁺] = 90 nM), pH 7.2. radioimmunoprecipitation assay (RIPA) lysis buffer: Na-deoxycholate (1g/L), Triton X-100 (1% vol/vol), NaCl (150), Tris-base (20), pH 7.3), protease inhibitor mixture. Phosphate-buffered saline (PBS) was obtained from Sigma-Aldrich. Free [Ca²⁺] was calculated using CaBuf software (G. Droogmans, Leuven, Belgium; available at ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip).

Superfusion.

Superfusion was performed in a 1-mL Perspex chamber with a coverslip base, mounted on an inverted Zeiss Observer.Z1 microscope. Superfusate was delivered at 2 mL/min at 37 °C. A solution switcher allowed the superfusate to be changed rapidly between two lines.

Isolation of Myocytes.

Myocytes were isolated following the methods in ref. 3. Myocytes from 15-wk-old Wistar hearts were isolated enzymatically with Blendzyme mixture (Roche) and stored in DMEM for use within 10 h.

Isolation of Mitochondria.

Mitochondria were isolated using the methods described in ref. 39, with modifications. Hearts of 15-wk-old male Wistar rats were Langendorff-perfused with Hepes-buffered NT for 5 min to remove blood and then homogenized manually (Dounce Potter homogenizer) in ice-cold MIB containing 0.1 mg/mL protease XXIV. Tissue was centrifuged at 7,500 × g for 7 min at 4 °C. A sample of the pellet (labeled sample #1) was collected, washed in PBS, and lysed in RIPA for Western blotting. The remainder of the pellet was washed twice and resuspended in MIB containing protease inhibitors (Roche). The suspension was homogenized for 3 min and then centrifuged at 700 × g for 10 min at 4 °C. The supernatant was centrifuged at 7,000 × g for 10 min, and the pellet obtained was resuspended in MIB + 25% (wt/vol) Percoll. The suspension was centrifuged at 17,000 × g for 10 min for 4 °C, and the pellet obtained was resuspended in MIB. This suspension was centrifuged at 7,000 × g for 10 min at 4 °C, and the pellet was washed in MSB. A sample of this suspension was collected and labeled sample #2. The remainder was centrifuged at 7,000 × g for 10 min at 4 °C, and the pellet was washed in MSB. A sample of this pellet was collected and labeled sample #3. The procedure was repeated for samples #4 and #5, if required. Immunofluorescence and functional activity measurements were performed on sample #3. All samples (lysed in RIPA buffer containing protease inhibitors) were used for Western blot analysis.

Western Blotting and Immunofluorescence.

PAGE and Western blotting were performed according protocols recommended for our choice of primary antibodies against LDH-B (rabbit mAb; Novus), CAII (rabbit mAb; Novus), CAXIV [rabbit polyclonal antibody (pAb); Novus], CAVA (R18, goat pAb; Santa Cruz), Mitofilin (Y17, goat pAb; Santa Cruz), RyR2 (rabbit pAb; Millipore), Serca2a (mouse mAb; Novus Biologicals). Goat anti-rabbit and anti-mouse and rabbit anti-goat secondary antibodies (Dako) and ECL (Pierce) were used to visualize the signal. Sample #3 from mitochondrial isolation was fixed in 4% (wt/wt) paraformaldehyde in PBS for 30 min (4 °C), followed by antigen blocking in 3% BSA for 30 min and with primary antibody against CAXIV (rabbit pAb, Novus) for 90 min at 37 °C. Signals were visualized with donkey anti-rabbit Alexa Fluor 555 secondary antibody.

Fluorescence Measurements.

Fluorescence from suspensions of cells or mitochondria was measured using an Hitachi F4500 cuvette spectrofluorometer at 25 °C. Fluorescence imaging of isolated cells or mitochondria was performed on a Zeiss LSM700 confocal microscope, integrated with a superfusion system, at 37 °C. A 40× oil-immersion objective was used for fluorescence pH measurements. For immunofluorescence and colocalization experiments, a 100× objective was used instead. Flow cytometry was performed on a Beckman Coulter SC Quanta (25 °C). See SI Materials and Methods for dye-loading procedures and wavelength settings.

Hyperpolarized ¹³C MRS.

Hyperpolarized [1-¹³C]pyruvate was prepared and administered, and ¹³C spectra were acquired as described previously (30–32) and in SI Materials and Methods. For in vivo imaging, a ¹³C pulse-acquire MRS sequence was used in an intact, ∼300-g male Wistar rat with a surface coil on its chest [the heart is the dominant source of PDH activity in the chest (32)]. Hyperpolarized [1-¹³C]pyruvate (80 μmol) was infused i.v. over 10 s, and 30 spectra were acquired over 1 min. Metabolism of this substrate to ¹³CO₂ occurs exclusively in the matrix by pyruvate dehydrogenase (37). Experiments were repeated on rats infused with 2 μmol of the membrane-impermeant CA inhibitor C23 (38), 25 μmol of the membrane-permeant CA inhibitor ATZ, or 25 μmol DIDS to block HCO₃⁻ exchange between red cells and plasma, each dissolved in 1 mL saline and injected 15 min before [1-¹³C]pyruvate. Assuming C23 retention in the extracellular space (20% of 300 g) and ATZ distribution in the total body water (60% of 300 g), concentrations of drugs would exceed their inhibitory constants over the MRS time frame (C23 K_i = 14 µM, ATZ K_i = 53 nM; Fig. S2). ¹³C MR spectra were analyzed as described in SI Materials and Methods. Ex vivo measurements were performed on excised rat hearts that were Langendorff perfused with KHB containing [1-¹³C]pyruvate, as described previously (31, 66) and in SI Materials and Methods.

H¹³CO₃⁻ Quenching.

Hearts were perfused in Langendorff mode with KHB containing [1-¹³C]pyruvate, and metabolically generated ¹³CO₂ and H¹³CO₃⁻ were detected using ¹³C MRS with 1-s temporal resolution for 2 min. At 20-s intervals during ¹³C spectral acquisition, radio-frequency saturation was applied to the H¹³CO₃⁻ resonance to quench the H¹³CO₃⁻ signal using a cascade of eight SNEEZE pulses (SI Materials and Methods).

Cardiac Energetics and Contractile Function.

Hearts were Langendorff perfused with KHB, and ³¹P spectra were acquired to measure the PCr, P_i, and γ-ATP resonances as described in SI Materials and Methods. Cytoplasmic pH was calculated from the P_i chemical shift (67). Contractile function was measured by the insertion into the left ventricle of a water-filled balloon attached to a pressure transducer. Contractility was monitored in each heart perfused with the following for 40 min each: (i) perfusion buffer, (ii) buffer with 35 µM C23, and (iii) buffer with 35 µM C23 and 100 µM ATZ. Contractile function was reported as the average DP over the final 10 min of each perfusion series.