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Title: Protein Tyrosine Nitration in Chronic Intramuscular Parasitism: Immunohistochemical evaluation of Relationships Between Nitration, Fiber Types, and Ubiquitin

item Elsasser, Theodore
item Romo, German
item Eastridge, Janet
item Kahl, Stanislaw - Stass
item Li, Congjun - Cj
item SARTIN, J
item Fayer, Ronald
item Solomon, Morse

Submitted to: Journal of Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/20/2011
Publication Date: 5/18/2011
Citation: Elsasser, T.H., Romo, G.A., Eastridge, J.S., Kahl, S., Martinez, A., Li, C., Sartin, J.L., Cuttitta, F., Fayer, R., Solomon, M.B. 2011. Protein Tyrosine Nitration in Chronic Intramuscular Parasitism: Immunohistochemical evaluation of Relationships Between Nitration, Fiber Types, and Ubiquitin. Journal of Physiology. 1:10-18.

Interpretive Summary: Proinflammatory infections caused by different microorganisms result in weight loss and muscle weakness. We used a model of parasite infection to determine if some muscles are more sensitive to the wasting effets of disease than others. Calves were infected with a coccidian-like organism called Sarcocystis. Muscle tissues were collected from control and infected calves and analyzed by various microscopic and biochemical techniques. The data indicated that not all muscles responded equally to the infection and muscles associated with stability of posture were more affected than muscle associated with locomotion. More specifically, muscles with a higher percentage of slow contraction fibers were more affected than muscles with more fast contraction fibers. We tracked the association with particular fiber types to the density of mitochondria that each fiber type had. Fibers with more mitochondria were more affected by the response to the infection. Mitochondria generate toxic free radicals if the normal process of generating ATP is disrupted. Infection causes such a disruption of mitochondrial function and therefore generated free radicals and generated more of the radicals in the slow fibers because they have more mitochondria. The free radicals generate nitrated proteins which loose their biological function. Mitochondrial protein nitration markers may serve as a useful tools to diagnose muscle disease associated with proinflammatory response to muscle infections.

Technical Abstract: Previous studies from our laboratory demonstrated that the catabolic processes associated with the proinflammatory impact of protozoan parasitic infection in Holstein calves were significantly more evident in red postural muscle such as psoas major (PM) than locomotor muscles typified by white rectus femoris (RF). The present study was conducted to determine whether metabolic factors inherent to particular to muscle fiber types might impact the predominant catabolism of PM as evaluated during the chronic intramuscular stage of Sarcocystis cruzi infection. Specifically we addressed whether fiber catabolism can be associated with the pathological consequences of increased protein tyrosine nitration. Holstein steer calves (av.wt.= 124 kg) were assigned to control (C), or infected (I, 250,000 Sarco oocysts) groups, n=5/group. Calves were euthanized between d-56 or d-63 of infection. RF and PM were dissected and specimens prepared for metabolic fiber type (MFT, slow oxidative, SO; fast oxidative glycolytic, FOC; fast glycolytic, FG) and size determination as well as immunohistochemical localization of fast myosin heavy chain 2a and 2b, nitrotyrosine (NT),ubiquitin (UBI), and mitochondrial membrane-associated Complex V (Cv). MFT analysis indicated that PM contained twice the SO count of RF (32 v 16%, P<0.002). Morphometrically, both SO and FOG were significantly smaller in area in I while mean FG areas were not different between C and I calves. Overall muscle NT and UBI content (Western blot of myofibrillar protein fraction) increased with infection. NT and UBI were immunohistochemically localized into three distinct patterns: a) sparse peripheral fiber staining, b) dispersed dense intrafiber staining, and c) sarcocyst-specific staining. In most fibers and cystic areas, NT staining was largely colocalized with UBI and Cv pixels. The greatest intrafiber pixel density of NT was associated with fibers not staining positive for fast-type 2a/ 2b (ie., SO, mitochondrially dense (MD) MHC Type 1 fibers); The data are consistent with the concept that mitochondrially dense fibers such as Type 1/SO are more sensitive to the proinflammatory response to parasitic infection and generate the nitro-oxidative stress marker nitrotyrosine.