Formation of the calpain-1/calpastatin complex promotes activation of calpain-1 under oxidizing conditions.

Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are inhibited by calpastatin, an endogenous inhibitor. Other factors, such as variations in pH, ionic strength, and oxidation influence calpain activity. This study aimed to determine the extent to which oxidation influences calpastatin inhibition of calpain-1. A series of order of addition assays were used to determine calpain-1 calcium activation and autolysis after exposure to an oxidizing agent (n-ethylmaleimide [NEM] or hydrogen peroxide [H2O2]. In the first series, purified calpastatin was added to the assay before or after oxidizing exposure at 165 mM NaCl, pH 6.5. In the second series, incubation buffer ionic strength (165 mM or 295 mM NaCl) was evaluated. The inhibitory activities of purified porcine calpastatin, purified human calpastatin domain I, or a subdomain B inhibitor peptide were evaluated in the third series. In the fourth series, a maleimide-polyethylene glycol molecule (MAL-PEG; MW = 5,000 Dalton) was used to evaluate the accessibility of free sulfhydryl groups and tagging of calpain-1 under each condition through a molecular weight shift assay. Results from this study indicate that autolysis of calpain-1, when used as an indicator of activation, occurred when the calpain-1/calpastatin complex was exposed to an oxidant or cysteine modifier such as NEM. However, when calpain-1 was exposed to the cysteine modifier before calpastatin, autolysis of calpain-1 did not occur or was significantly decreased (P < 0.05). Irreversible modification of cysteine residues by NEM prevented activation of calpain-1 in the absence of calpastatin, but if the cysteine modification is potentially reversible (H2O2), calpain-1 activity can be recovered. Results from this study indicate that when calpastatin is bound to calpain-1, calpain-1 activation can occur even after being exposed to a cysteine modifier (NEM) or hydrogen peroxide (H2O2). Calpain-1 is not tagged with maleimide-polyethylene glycol (MAL-PEG) in the presence of calpastatin, indicating that calpastatin blocks or covers free cysteines on calpain-1 from modification. Moreover, exposure to calpain-1/calpastatin complex with a cysteine modifier allows activation of calpain-1, indicating that the inhibitory action of calpastatin is compromised. These results indicate a regulatory role for calpastatin that is not inhibitory but protective for calpain-1.

Protein degradation in skeletal muscle is a key component of protein turnover and maintenance of muscle function. Protein degradation in postmortem muscle is commonly observed and is associated with the accumulation of degradation products and improved meat tenderness. Because there is significant evidence that calpain-1 is involved with proteolysis of muscle proteins in both situations, defining the factors that regulate calpain activity will position scientists to improve calpain-1 activity in both contexts. Calpain-1 is a neutral calcium-dependent proteinase that is inhibited by calpastatin, oxidation, and slightly acidic pH environments. Because oxidation of the calpain/calpastatin complex with hydrogen peroxide appeared to activate calpain-1, we hypothesize that calpastatin binding to calpain may protect the active site cysteine. In the current study, we tested this hypothesis and investigated how n-ethyl maleimide (NEM), an alkylating agent, affects the regulation of calpain in the presence and absence of calpastatin molecules. The results suggest that calpastatin can protect calpain-1 from reacting with maleimide-polyethylene glycol but that exposure of calpain-1/calpastatin complex to NEM or hydrogen peroxide resulted in autolysis and activation of calpain. Under some circumstances, calpastatin appears to protect calpain-1 from inhibition by modification of active site cysteine. These novel observations show a different role for calpastatin and give reason to interpret calpastatin abundance and activity data in a different light.

Maternal metabolizable protein restriction during gestation affects the vascular function of maternal and fetal placental arteries in sheep.

We hypothesized isocaloric diets low in protein would decrease the sensitivity of caruncular (CAR) and cotyledonary (COT) arteries compared to placental arteries from ewes receiving adequate metabolizable protein (MP) requirements. Pregnant ewes were fed one of three isocaloric dietary treatments that provided 60% (MP60), 80% (MP80), or 100% (MP100) of the MP requirements. Diets were fed from day 100-130 of gestation. In vitro dose response curves to bradykinin (BK), sodium nitroprusside (SNP), potassium chloride (KCl), and phenylephrine (PE) in CAR and COT arteries were performed. As MP decreased, the sensitivity to a low dose of KCl increased (P = 0.05) in the COT arteries. There was an overall treatment effect in the CAR and COT arteries for the BK dose response curve, where CAR arteries of MP80 ewes were more sensitive (P = 0.05) to BK compared with MP60 and MP100 ewes, and COT arteries of MP60 and MP80 ewes were more sensitive (P = 0.01) to BK compared with MP100 ewes. There were no treatment effects (P ≥ 0.09) on the SNP or PE dose response curves in CAR or COT arteries. The mechanism of the BK induced vasodilation needs to be elucidated. Moreover, MP restriction appears to alter placental vascular function, which could help explain the differences in nutrient flux previously reported.

Effects of ractopamine hydrochloride supplementation on feeding behavior, growth performance, and carcass characteristics of finishing steers.

Ractopamine hydrochloride (RAC) is a ß-adrenergic agonist that functions as a repartitioning agent to improve muscling in feedlot cattle. Many studies have investigated the effects of RAC on growth performance and carcass characteristics; however, there is minimal information about the influence of RAC on feeding behavior. Sixty-nine steers (body weight [BW] = 364 ± 3.9 kg) predominately of Angus and Simmental breeding were subjected to a 126-d (n = 46) or 154-d (n = 23) feeding period and randomly assigned to one of two treatment groups: supplementation to provide 0 (CON; n = 34) or 267 ± 4.9 mg/d of RAC (n = 35). Ractopamine was provided as Optaflexx 45 at 0.024% of the diet (dry matter [DM] basis; Elanco Animal Health, Greenfield, IN). Dietary treatments were fed the final 42 d in the feed yard (treatment period). Feeding behavior and growth performance were measured using radio frequency identification tags and the Insentec feeding system. Following the final day of treatment, steers were slaughtered and carcass measurements were recorded. Data were analyzed using MIXED models in SAS. There were no differences in BW, average daily gain (ADG), DM intake (DMI), gain:feed ratio (G:F), or feeding behavior during the pretreatment period (P > 0.44). Ractopamine supplementation increased G:F during the treatment period (P = 0.02) and during the total period (P = 0.03) and tended to increase ADG during the treatment and total period (P ≤ 0.08). DMI was not affected during the treatment or total period (P > 0.67). Eating time per visit, per meal, and per day were decreased (P < 0.02) in steers supplemented with RAC during the treatment period. DMI per minute was increased (P = 0.02) in steers supplemented with RAC. Hot carcass weight, dressing percentage, and 12th rib fat were not influenced by RAC supplementation. Ractopamine supplementation decreased marbling (P = 0.008) and kidney, pelvic, and heart percentage (P = 0.04) and increased longissimus muscle area (P = 0.01). These data demonstrate that RAC supplementation for 42 d improves feed efficiency, increases the rate of DMI without altering DMI, and increases muscling in finishing cattle.