Protein tyrosine nitration in chronic intramuscular parasitism: immunohistochemical evaluation of relationships between nitration, and fiber type-specific responses to infection

Abstract

The present study was conducted to determine whether preferential muscle catabolism [psoas major (PM) > rectus femoris (RF)] observed during the chronic intramuscular stage of Sarcocystis cruzi infection could be associated with the pathological consequences of increased protein tyrosine nitration in fibers characteristically more metabolically active due to higher mitochondrial density. Holstein calves were assigned to control (C), or S. cruzi-infected (I) groups, n=5/group. Calves were euthanized on day 63 of infection. Samples of RF and PM were prepared for metabolic fiber typing (MFT: slow oxidative, SO – Type I; fast oxidative glycolytic, FOG - Type IIa; fast glycolytic, FG – Type IIb), fiber area, and immunohistochemical localization of fast myosin heavy chain 2a and 2b, nitrotyrosine (NT), and mitochondrial Complex V ATP-synthase. MFT analysis documented that PM contained twice the number of SO fibers compared to RF (32 v 16%, P<0.002). SO and FOG fibers (Both higher in mitochondrial density than FG fibers) in both PM and RF were significantly smaller in area in I calves with mean FG areas not different between C and I. Muscle NT content (Western blot of myofibrillar protein fraction) increased with infection; NT was immunohistochemically localized into three distinct patterns in fibers: i) sparse fiber staining, ii) dense punctuate intrafiber staining, and iii) pericystic staining. By image analysis, the greatest punctuate intrafiber pixel density of NT was associated with SO fibers from I calves with the NT colocalizing with mitochondrial Complex V – F1F0 ATP synthase. More fibers were positive for the colocalization in PM than RF (P<0.04). The data are consistent with the concept that fibers rich in mitochondria possessing more inherent oxidative energy capacity generate more nitrated proteins than glycolytic fibers and as such are more affected by the proinflammatory response to infections like Sarcocystosis.