Physical and functional association of lactate dehydrogenase (LDH) with skeletal muscle mitochondria

Abstract

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2. However, subsequent addition of NAD(+) significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD(+)-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.

Keywords: Immunohistochemistry; Lactate Shuttle; Lactic Acid; Mitochondrial Metabolism; NAD; Pyruvate; Respiration; Respiratory Chain; Respirometry; Skeletal Muscle Metabolism.

FIGURE 1.

Mitochondrial respiratory O₂ flux (JO₂) in red and white saponin-permeabilized rat gastrocnemius muscle fibers supported by components of the LDH reaction. A, addition of 5 mm lactate to either red or white fibers (5 mm ADP + 4 mm malate) had no affect on JO₂. Addition of NAD⁺ significantly increased JO₂. 10 μm cytochrome c further increased JO₂ in RG. The rate was subsequently abolished to pre-NAD⁺ levels following addition of the pyruvate transporter inhibitor, CHC. n = 6. ***, significantly different, p < 0.001. B, respiration normalized as a percentage of glutamate-stimulated JO₂ indicate no differences in proportional respiration between RG and WG. There was a non-significant trend for increased JO₂ after the addition of LDH to the fibers. n = 3. WG, significantly greater than ADP + malate, lactate, and CHC JO₂; aa, p < 0.01; aaa, p < 0.001; greater than CHC JO₂; bb, p < 0.01; bbb, p < 0.001. RG, significantly greater than ADP + malate, lactate, and CHC JO₂; c, p < 0.05; ccc, p < 0.001; greater than CHC JO₂; d, p < 0.05; ddd, p < 0.001. Data are mean ± S.D.

FIGURE 2.

Respiratory kinetics for lactate in permeabilized red (RG) and white (WG) saponin-permeabilized rat gastrocnemius muscle fibers. A, net mitochondrial respiratory O₂ flux (JO₂) in red (closed symbols) and white fibers (open symbols) supported by increasing concentrations of lactate (0.5–15 mm) titrated atop 5 mm ADP + 4 mm malate and 500 μm NAD⁺. Michaelis-Menten kinetic curves: WG, Y = 4.681 · X/(12.08 + X); RG, Y = 10.03 · X/(4.615 + X). B, apparent K_m (K_m,app) of RG and WG mitochondria for lactate, as computed from kinetic curves. C, maximal net JO₂ (V_max) of RG and WG mitochondria supported by lactate, with 5 mm ADP + 4 mm malate and 500 μm NAD⁺, as computed form kinetic curves. n = 3. **, main effect for muscle fiber type, p < 0.01; *, significantly different from WG, p < 0.05. Data are mean ± S.D.

FIGURE 3.

Lactate oxidation in isolated SS and IMF mitochondria from rat red gastrocnemius. Isolated mitochondrial respiratory O₂ flux (JO₂ as a % of glutamate-stimulated JO₂) was supported by 5 mm ADP + 4 mm malate. Addition of 5 mm lactate did not increase JO₂ in either SS or IMF mitochondria. However, 500 μm NAD⁺ increased JO₂ in SS mitochondria only. 10 μm cytochrome c further increased JO₂. The NAD⁺-dependent increase in JO₂ was abolished with 500 μm CHC. n = 3. SS, significantly greater than ADP + malate, lactate, and CHC JO₂; aaa, p < 0.001, significantly greater than all other conditions; bbb, p < 0.001, significantly greater than lactate JO₂; cc, p < 0.01. IMF, significantly greater than all other conditions; dd, p < 0.01. Data are mean ± S.D.

FIGURE 4.

High-resolution confocal laser scanning microscopic imaging of immunolabeled LDH (red in A and B), IMM proteins (green in A) and Bcl-2 (green in B). Antibodies against Bcl-2 were used as a label of the outer mitochondrial membrane. Antibodies against the respiratory chain proteins were used to label the IMM. Pearson correlation coefficients for colocalization were 0.88 for IMM with LDH, and 0.73 for Bcl-2 with LDH. The scatter plots show the intensity of the red (Ch-1) and green (Ch-2) pixels.

FIGURE 5.

Confocal laser scanning microscopic imaging of immunolabeled IMM and LDH of red gastrocnemius skeletal muscle fibers. Antibodies against proteins of the mitochondrial respiratory chain were used as a label for the IMM, along with antibodies against LDH. A, labeling of the intact fibers. B, labeled permeabilized fibers exhibiting attenuated LDH signal in the presence of the IMM signal. Note that the LDH signal was preserved in the SS mitochondria, which appear as larger elongated organelles at the cell edge. C, augmentation of the LDH signal by addition of the exogenous LDH (approximately 25 units/ml of LDH) + gentle wash. D, loss of the LDH staining in mitochondria with trypsin treatment.

FIGURE 6.

Confocal laser scanning microscopic imaging of immunolabeled Bcl-2 and LDH of red gastrocnemius skeletal muscle fibers. Antibodies against Bcl-2 were used as a label of the outer mitochondrial membrane, along with antibodies against LDH. A, labeling of the intact muscle fibers. The mitochondrial network is easily recognizable, as is the colocalization of Bcl-2 (Aa) and LDH staining (Ab). An overlay of the images are shown in Ac. B, permeabilized fibers, showing preservation of the colocalization between the Bcl-2 (Ba) and LDH (Bb). C, area of the same preparation shown in panel B, but showing the region with the lost Bcl-2 signal (Ca), whereas the LDH signal is present (Cb).

FIGURE 7.

Effect of protease treatment (10 μm trypsin for 15 min at 37 °C) on mitochondrial respiratory O₂ flux (JO₂) in red saponin-permeabilized (30 μg/ml) rat gastrocnemius muscle fibers supported by components of the LDH reaction. No significant difference was observed between trypsin-treated mitochondria versus control. As with untreated fibers, trypsin-treated fibers responded to lactate + NAD⁺. n = 3. Control, significantly greater than ADP + malate, and lactate JO₂, aa, p < 0.01; aaa, p < 0.001, significantly less than cytochrome c JO₂; b, p < 0.05, significantly greater than all other JO₂; ccc, p < 0.001. Protease, significantly greater than ADP + malate, and lactate JO₂; d, p < 0.05; dd, p < 0.01; ddd, p < 0.001, less than cytochrome c JO₂; ee, p < 0.01, significantly greater than all other JO₂; fff, p < 0.001. Data are mean ± S.D.

FIGURE 8.

Immunoblot for LDH and IMM protein complex II in permeabilized muscle fibers. Red gastrocnemius fibers permeabilized with 30 μg/ml of saponin and treated with 10 μm trypsin for 15 min revealed that LDH remains present even after trypsin treatment (lane 2). Commercially available purified LDH is shown without (lane 3) or with (lane 4) trypsin treatment.