Cultured equine satellite cells as a model system to assess leucine stimulated protein synthesis in horse muscle.

Leucine has been shown to stimulate the mammalian/mechanistic target of rapamycin (mTOR) signaling pathway which plays numerous key regulatory roles in cell growth, survival, and metabolism including protein synthesis in a number of species. However, previous work with equine satellite cells has suggested distinct species differences in regards to physiological effects and the magnitude of responses to growth factors and regulators. Because there is limited research available regarding the role of leucine in regulating equine skeletal muscle protein synthesis, the objective of this study was to evaluate the effect of leucine on the mTOR signaling pathway in cultured equine satellite. Protein synthesis was evaluated by measuring the incorporation of [3H] Phenylalanine (3HPhe) in equine satellite cell myotube cultures treated with a leucine titration ranging from 0 to 408 µM. Our results show a 1.8-fold increase (P < 0.02) in protein synthesis at levels slightly greater than those found in the general circulation, 204 and 408 µM when compared to a no leucine control (0 µM). Puromycin incorporation, a nonradioactive surface sensing of translation (SUnSET) methodology, was also measured in cells treated with leucine (LEU; 408 µM), a no-leucine control (CON), and a puromycin-negative vehicle (PURO-). These results demonstrated a 180% increase (P = 0.0056) in puromycin incorporation in LEU compared to CON cultures. To evaluate the mTOR signaling pathway, equine satellite cell myotube cultures were treated with leucine (LEU; 408 µM) or a no-leucine control (CON) in the presence or absence of rapamycin (LR and CR, respectively), an inhibitor of mTOR. The mTOR inhibitor, rapamycin, suppressed phosphorylation of mTOR (P < 0.01) and rS6 (P < 0.01) with an increase in phosphorylation of rS6 in leucine-treated cultures observed when compared to control cultures (P < 0.05). Similarly, there was a 27% increase (P < 0.005) in the hyperphosphorylated γ-form of 4E-BP1 compared to total 4E-BP1 in LEU compared to CON cultures with leucine-induced phosphorylation of 4E-BP1 completely blocked by rapamycin with a smaller decrease observed in CR compared to CON cultures. The major finding of this study was that leucine activated the mTOR translation initiation pathway and increased transcription of global proteins in cultured equine satellite cells. Use of the cell culture system with primary equine muscle cell lines provides the opportunity to distinguish the impact of leucine on muscle and protein synthesis, independent of systemic interactions.

Evaluation of blood cardiac troponin I concentrations obtained with a cage-side analyzer to differentiate cats with cardiac and noncardiac causes of dyspnea.

OBJECTIVE: To determine whether measurement of blood cardiac troponin I (cTnI) concentrations with a cage-side analyzer could be used to differentiate cardiac from noncardiac causes of dyspnea in cats. DESIGN: Prospective, multicenter study. ANIMALS: 44 client-owned cats with dyspnea and 37 healthy staff-owned cats. PROCEDURES: Affected cats were examined because of dyspnea; treatment was administered in accordance with the attending clinician's discretion. Cats were judged to have a cardiac or noncardiac cause of dyspnea on the basis of results of physical examination, thoracic radiography, and echocardiography. Blood cTnI concentrations were determined with a cage-side analyzer on samples collected within 12 hours after admission of affected cats. Concentrations for healthy cats were obtained for comparison. RESULTS: 5 enrolled cats were excluded from the study because of concurrent cardiac and respiratory disease. Of the remaining 39 cats with dyspnea, 25 had a cardiac cause and 14 had a noncardiac cause. The 25 cats with a cardiac cause of dyspnea had a significantly higher blood cTnI concentration than did the 37 healthy cats or the 14 cats with a noncardiac cause of dyspnea. CONCLUSIONS AND CLINICAL RELEVANCE: Measurement of cTnI concentrations with a cage-side assay in emergency settings may be useful for differentiating cardiac from noncardiac causes of dyspnea in cats.