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Physical and functional association of lactate dehydrogenase with mitochondria in intact and permeabilized skeletal muscle fibers
Author(s) -
Kane Daniel A.,
White Adrienne E.,
Elustondo Pia,
Hughes Meghan C.,
Brebner Karen,
Pavlov Evgeny
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.lb765
Subject(s) - colocalization , mitochondrion , lactate dehydrogenase , nad+ kinase , skeletal muscle , biochemistry , biology , myofibril , chemistry , microbiology and biotechnology , anatomy , enzyme
To clarify whether mitochondria oxidize lactate, mitochondrial respiratory oxygen flux ( J O 2 ) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat red (RG) and white (WG) gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter J O 2 in either RG or WG. However, subsequent addition of NAD + significantly increased J O 2 , and was abolished by the inhibitor of mitochondrial pyruvate transport, α‐hydroxycinnamate. In parallel experiments with subsarcolemmal (SS) and intramyofibrillar (IMF) mitochondria isolated from RG, only SS exhibited significant NAD + ‐ dependent lactate oxidation. To determine whether lactate dehydrogenase (LDH) is physically associated with mitochondria in RG, confocal microscopy was used to detect immunohistochemically stained LDH and mitochondrial proteins. LDH clearly colocalized with mitochondria in intact fibers. In permeabilized fibers, most of the LDH signal was lost in IMF but not SS regions. The LDH signal was absent in SS regions following trypsin treatment; however, colocalization was augmented in both SS and IMF regions with exogenous LDH + washing, suggesting LDH preferentially associates with muscle mitochondria. Collectively, these results suggest that LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.