Temporary fatigue and altered extracellular matrix in skeletal muscle during progression of heart failure in rats.

Patients with congestive heart failure (CHF) experience increased skeletal muscle fatigue. The mechanism underlying this phenomenon is unknown, but a deranged extracellular matrix (ECM) might be a contributing factor. Hence, we examined ECM components and regulators in a rat postinfarction model of CHF. At various time points during a 3.5 mo-period after induction of CHF in rats by left coronary artery ligation, blood, interstitial fluid (IF), and muscles were sampled. Isoflurane anesthesia was employed during all surgical procedures. IF was extracted by wicks inserted intermuscularly in a hind limb. We measured cytokines in plasma and IF, whereas matrix metalloproteinase (MMP) activity and collagen content, as well as the level of glycosaminoglycans and hyaluronan were determined in hind limb muscle. In vivo fatigue protocols of the soleus muscle were performed at 42 and 112 days after induction of heart failure. We found that the MMP activity and collagen content in the skeletal muscles increased significantly at 42 days after induction of CHF, and these changes were time related to increased skeletal muscle fatigability. These parameters returned to sham levels at 112 days. VEGF in IF was significantly lower in CHF compared with sham-operated rats at 3 and 10 days, but no difference was observed at 112 days. We conclude that temporary alterations in the ECM, possibly triggered by VEGF, are related to a transient development of skeletal muscle fatigue in CHF.

Comigration of two autophagosome-associated dehydrogenases on two-dimensional polyacrylamide gels.

Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (approximately 37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (approximately 35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-alpha-hydroxysteroid dehydrogenase (3alphaHSD). Silver staining indicated that this 3alphaHSD form selectively bound to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3alphaHSD demonstrated immunostaining of four 3alphaHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3alphaHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3alphaHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.