Desmin Degradation Research Articles

Enhancement technology improves palatability of normal and callipyge lambs

The objective of this research was to determine if BPI Processing Technology (BPT) improved palatability of normal (NN) and callipyge (CN) lamb meat and to determine the mechanism by which palatability was improved. Ten ewe and 10 wether lambs of each phenotype were slaughtered, and carcass traits were assessed by a trained evaluator. The LM was removed at 2 d postmortem. Alternating sides served as controls (CON) or were treated with BPT. Muscles designated BPT were injected to a target 120% by weight with a patented solution containing water, ammonium hydroxide, carbon monoxide, and salt. Muscle pH, cooking loss, Warner-Bratzler shear force (WBS), sarcomere length, cooked moisture retention, and desmin degradation were measured. A trained sensory panel and a take-home consumer panel evaluated LM chops. Callipyge had a heavier BW and HCW, less adjusted fat thickness, reduced yield grades, and greater conformation scores than NN (P < 0.05). For LM, NN had shorter sarcomeres, smaller WBS values, greater juiciness ratings, more off-flavors, reduced consumer ratings for raw characteristics (like of portion size, like of color, like of leanness, overall like of appearance) and greater consumer ratings for eating characteristics (like of juiciness, like of flavor) than CN (P < 0.05). For LM, BPT had greater cooked moisture retention, smaller WBS values, greater juiciness ratings, less off-flavors, and greater consumer ratings for raw characteristics (like of portion size, like of color, overall like of appearance) and eating characteristics (like of juiciness, like of flavor) than CON (P < 0.05). Significant phenotype × treatment interactions occurred for LM muscle pH, desmin degradation, tenderness, consumer like of texture/tenderness, and consumer overall like of eating quality (P < 0.05). For LM, BPT increased muscle pH more for NN than CN (P < 0.01) and increased desmin degradation for NN but decreased desmin degradation for CN (P < 0.01). The BPT enhancement improved LM tenderness ratings for CN more than NN (P < 0.05). For consumer like of texture/tenderness, BPT improved ratings for CN more than NN (P < 0.01). For consumer overall like of eating quality, BPT improved ratings for CN more than NN (P < 0.05). In summary, BPT had little to no effect on sarcomere length and desmin degradation, but improved palatability of NN and CN lamb by increasing cooked moisture retention, improving consumer acceptability of CN to near-normal levels.

Desmin and troponin T are degraded faster in type IIb muscle fibers than in type I fibers during postmortem aging of porcine muscle

A novel approach was applied in this study to directly evaluate the effect of muscle fiber type on postmortem protein degradation. Porcine muscle fibers were isolated from longissimus muscle at day 1, 3, and 6 postmortem. Fibers were sorted by immunochemical myosin heavy chain isoform typing. Western blot analysis of fibers pooled separately into type I or IIb showed that the relative amounts of 39- and 50-kDa desmin degradation fragments at day 6, and 28- to 31-kDa fragments of troponin T fast type isoform (fTnT) at day 1 and 6 postmortem were higher in type IIb than in type I fibers. At day 6 troponin T slow type isoform (sTnT) was less degraded than fTnT in type I fibers. These results indicated greater rate and extent of proteolysis in type IIb than in type I fibers and higher susceptibility of fTnT to proteolysis than that of sTnT isoform.

Changes in water‐holding capacity and textural properties of chicken gizzard stored at 4°C

The water-holding capacity (WHC), and toughness (shear force) of chicken gizzard were evaluated during postmortem storage for 4.5, 7, 12, 24, 48, 72 and 96 h at 4 degrees C. Degradation of the cytoskeletal proteins desmin, talin and vinculin were monitored by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and Western blotting during the same designated storage period. The WHC of the gizzards decreased significantly from 12 h to 72 h of storage, but by 96 h the WHC was restored to the level measured after storage for 12 h. The shear force value of the gizzards increased rapidly until 12 h and then decreased until 24 h, with a further slight decrease by 48 h. Degradation products of desmin, talin and vinculin appeared at 96 h, 12 h and 48 h postmortem, respectively. The intensity of immunolabeling for desmin, talin and vinculin after storage for 96 h decreased to 51%, 25% and 52% of the initial value. The appearance of desmin degradation products was accompanied by an increase in WHC. This suggests that the postmortem degradation of desmin is involved in the increase of WHC in chicken gizzard during storage at 4 degrees C, and talin and vinculin may be involved.

High-oxygen modified atmosphere packaging system induces lipid and myoglobin oxidation and protein polymerization

Beef steaks from longissimus lumborum, semimembranosus, and adductor muscles (n=10; respectively) were cut at 24h postmortem, randomly assigned to either high-oxygen modified atmosphere packaging (HiOx-MAP; 80% O(2), 20% CO(2)) or vacuum (VAC), and displayed for 9days at 1 degrees C. HiOx-MAP packaged beef steaks had a rapid increase in lipid oxidation and a decrease in color stability during display. The steaks in HiOx-MAP had significantly lower tenderness and juiciness scores, and higher off-flavor scores compared to steaks in VAC. HiOx-MAP condition did not affect the postmortem degradation of troponin-T or desmin. Furthermore, autolysis of micro-calpain was not influenced by packaging. SDS-PAGE, immunoblotting, and diagonal-PAGE revealed oxidative cross-linking of myosin heavy chain in meat packaged in HiOx-MAP. These results suggest that the HiOx-MAP system may negatively affect meat quality characteristics by inducing lipid and myoglobin oxidation and cross-linking/aggregation of myosin by protein oxidation.

Ageing of large cuts of beef loin in vacuum or high oxygen modified atmosphere – Effect on shear force, calpain activity, desmin degradation and protein oxidation

The aim was to investigate the effect of ageing large beef cuts directly in high oxygen modified atmosphere (MA) and how ageing time in vacuum influence meat quality when followed by retail packaging in high oxygen MA. Large cuts (10 cm long) of beef loin (LD) were aged for up to 25 days postmortem in different ageing systems at 4 degrees C. Ageing solely in high oxygen modified atmosphere (MA, 80% O(2)+20% CO(2)) for 5 or 10 days and ageing in vacuum for 5 or 15 days followed by high oxygen MA for 5 or 10 days were compared with ageing in vacuum for 5, 15 and 25 days. Warner-Bratzler shear force, purge and cooking losses, calpain activity and desmin and carbonyl contents were measured. Shear force decreased to the same level when ageing this large beef cut solely in high oxygen MA for 5 or 10 days as when ageing in vacuum. The activity of mu-calpain disappeared, the activity of m-calpain decreased and purge loss increased between 5 and 10 days, but cooking loss and the contents of desmin and carbonyls were unaffected. The ageing time in vacuum before packaging of this large beef cut in high oxygen MA did not affect the ultimate shear force. The m-calpain activity decreased and the content of carbonyls increased compared with solely in vacuum after 15 days of total ageing, but there was no difference in the content of desmin or cooking loss between these ageing systems at the same ageing time.

Stress relaxation behaviour and structural changes of muscle tissues from Gilthead Sea Bream ( Sparus aurata L.) following high pressure treatment

Sea Bream muscle tissue was subjected to different high pressure treatments, and rheological changes were monitored during storage by means of the stress relaxation test. The best fit was obtained by application of the three term Maxwell exponential model, followed by the Nussinovich model. The application of 300 and 400 MPa pressures appeared to enable preservation of elasticity and stiffness of fish muscle during storage, compared to untreated samples. On the contrary, samples treated at 200 MPa underwent a decrease in elasticity during storage. The water holding capacity of dorsal muscle was also assessed, and it was found to decrease with increasing pressures. Immunoblot studies performed on the main structural proteins revealed that a pronounced time-dependent degradation of desmin, observed in untreated samples, could be prevented by treatment at 400 MPa. Taken together, our results strongly suggest that high pressure treatments inactivate degrading enzymes acting on proteins that are related to tissue integrity preservation, texture quality, and water holding capacity.

Effects of zilpaterol hydrochloride with or without an estrogen-trenbolone acetate terminal implant on carcass traits, retail cutout, tenderness, and muscle fiber diameter in finishing steers1

Our objective was to determine the effects of feeding zilpaterol hydrochloride (ZH), a beta-agonist, for the final 30 d of the feeding period, with or without a terminal estrogen + trenbolone acetate (TBA) implant (Revalor-S; 24 mg of estradiol-17beta and 120 mg of TBA; REV) on meat tenderness and carcass cutout yields. Crossbred steers (n = 2,279) were divided into 6 BW blocks and 24 pens. Within each block, pens were assigned randomly to 1 of 4 treatments: 1) no terminal implant (control); 2) a terminal REV given 91 d before slaughter; 3) no terminal implant plus ZH; and 4) a terminal REV implant plus ZH (REV+ZH). All cattle received Component TE-IS (16 mg of estradiol and 80 mg of TBA) on d - 61 of the feeding period [corrected]. Zilpaterol hydrochloride was added to the diets at a concentration of 8.38 mg/kg (DM basis) during the final 30 d of the feeding period, followed by a 3-d period before slaughter in which ZH was withdrawn from the diet. Carcasses (n = 30/treatment) were selected from the 2,279 cattle and fabricated into subprimal cuts as per Institutional Meat Purchase Specifications. Strip loins were collected, cut into 2.54-cm steaks, and aged 7, 14, and 21 d, after which Warner-Bratzler shear force (WBSF), collagen content, desmin degradation, and muscle fiber diameter measurements were determined. Feeding ZH increased (P < 0.05) yield of the #112A ribeye roll, #116B chuck mock tender, #167A peeled knuckle, #169 top inside round, #171B outside round, #171C eye of round, #180 strip loin, #184 top sirloin butt, and #189A full tenderloin for ZH treatment. Longissimus muscle WBSF at 7, 14, and 21 d postmortem was increased (P < 0.001) with ZH supplementation. Desmin degradation at 7, 14, and 21 d postmortem was not affected with REV or ZH supplementation compared with controls. Zilpaterol hydrochloride had an additive effect with REV on increasing LM fiber diameter (P < 0.001). When fed to cattle that received a terminal implant of REV, ZH potentially increased LM WBSF as a result of induced muscle hypertrophy. During the 21-d aging period, WBSF decreased with aging, suggesting that carcasses from cattle supplemented with ZH might require longer aging time to ensure that acceptable levels of tenderness are reached.

Injection of marinade with actinidin increases tenderness of porcine M. biceps femoris and affects myofibrils and connective tissue

AbstractBACKGROUND: Marination of beef muscles with brine solutions containing proteolytic enzymes from fruit extracts has been shown to tenderize meat. However, the effect of marination with actinidin on tenderness of pork muscles has not been investigated. Tenderness and eating quality of porcine M. biceps femoris was investigated by Warner–Bratzler (WB) shear test and sensory evaluation after injection of brine containing up to 11 g L−1 actinidin‐containing kiwi fruit powder and 2, 5 or 9 days of storage.RESULTS: Actinidin decreased WB shear force, increased tenderness and did not affect flavour and juiciness. Injection of 2.8 g L−1 actinidin powder and storage for 2 days resulted in WB shear force values similar to control samples stored for 5 or 9 days. In samples injected with 10 g L−1 actinidin powder, degradation of desmin and percentage of heat‐soluble collagen (P &lt; 0.05) increased compared to control samples. Myofibrillar particle size tended to decrease (P &lt; 0.1) with increasing actinidin concentration. No major changes were observed by proteome analysis. Atomic force microscopy showed actinidin‐induced damage of endomysium surrounding isolated single muscle fibres.CONCLUSION: Our results indicate that actinidin tenderizes pork M. biceps femoris by affecting both the myofibrils and connective tissue. Copyright © 2009 Society of Chemical Industry

Effect of blade tenderization, aging time, and aging temperature on tenderness of beef longissimus lumborum and gluteus medius1,2

Purveyors are concerned about the potential food safety risk of nonintact meat products and are seeking strategies to ensure adequate meat tenderness without blade tenderization. This study was conducted to determine the effects of blade tenderization and time and temperature of aging on beef longissimus lumborum (LL) and gluteus medius (GM) tenderness. Beef strip loins (n = 300) and top sirloin butts (n = 300) were assigned to storage at -0.5 or 3.3 degrees C for 12, 26, or 40 d. Cuts were blade tenderized (BT) or not blade tenderized (NBT) before steak cutting. One 2.54-cm steak from each subprimal was used for slice shear force determination and Western blotting of desmin. Desmin degradation was less (P < 0.05) in LL stored at -0.5 degrees C than LL stored at 3.3 degrees C (57 and 65%, respectively). Aging from 12 to 26 d increased (P < 0.05) proteolysis (50 to 65%) in LL. Regardless of aging time, BT reduced (P < 0.05) LL slice shear force values. Aging time did not affect (P > 0.05) slice shear force values of BT LL steaks (10.4, 9.9, and 9.4 kg for 12, 26, and 40 d aging, respectively), but reduced (P < 0.05) NBT steak slice shear force values (15.1, 13.8, and 12.3 kg for 12, 26, and 40 d aging, respectively). Greater temperature did not affect (P > 0.05) slice shear force values of BT LL steaks (10.2 and 9.6 kg for steaks aged at -0.5 and 3.3 degrees C, respectively), but improved (P < 0.05) slice shear force of NBT LL steaks (15.1 and 12.4, respectively). Aging at 3.3 degrees C increased (P < 0.05) proteolysis in GM steaks (43 and 54% for -0.5 and 3.3 degrees C, respectively). Longer aging times increased (P < 0.05) proteolysis (40, 46, and 60% for 12, 26, and 40 d aging, respectively) in GM steaks. Blade-tenderized GM steaks had dramatically less (P < 0.05) slice shear force values than NBT steaks (13.7 and 19.9 kg, respectively). Raising aging temperature from -0.5 to 3.3 degrees C reduced (17.6 vs. 16.0 kg; P < 0.05) and increasing aging time from 12 d to 40 d improved (17.9 vs. 15.2 kg; P < 0.05) slice shear force values of GM steaks. Blade tenderization and increased aging time and temperature all improved tenderness of beef LL and GM steaks, though blade tenderization provided greater improvements than increased aging time and temperature. Longer aging could potentially be used to replace blade tenderization for LL steaks, but not in GM steaks.

Comparison of palatability characteristics of beef gluteus medius and triceps brachii muscles1,2

The objective of this experiment was to evaluate triceps brachii steaks as a substitute for gluteus medius steaks in foodservice and retail applications, including the effect of aging time and USDA quality grade on the palatability of both muscles. Top sirloin butts (n = 600) and shoulder clod arm roasts (n = 600) representing US Choice and US Select quality grades were selected at 48 h postmortem and aged for 7, 14, 21, 28, 35, or 42 d. Steaks were evaluated using a trained sensory panel, slice shear force, sarcomere length, and Western blotting of desmin measurements. Sarcomere length was measured only on steaks at 14 and 42 d. Triceps brachii and gluteus medius steaks were similar in tenderness rating at 7 and 14 d, but triceps brachii steaks aged longer were more tender (P < 0.05) than were gluteus medius steaks. Triceps brachii steaks reached ultimate tenderness values by 21 d. Gluteus medius steak tenderness ratings improved through 35 d, and at 42 d were similar to those given to triceps brachii steaks at 21 d. Sarcomere lengths were longer (P < 0.05) in triceps brachii than in gluteus medius (2.09 and 1.58 mum, respectively). Significant increases in desmin degradation were detected through 42 d in both muscles (30.9, 46.3, 50.6, 51.0, 57.6, and 64.1% at d 7, 14, 21, 28, 35, and 42 for gluteus medius and 28.9, 40.8, 49.3, 59.2, 61.8, and 71.9% at d 7, 14, 21, 28, 35, and 42 for triceps brachii). At 14 d, gluteus medius had more (P < 0.05) desmin degraded than triceps brachii, but by 28 d, desmin degradation was greater (P < 0.05) in triceps brachii. Quality grade had minimal effects on palatability traits. Desmin degradation contributed to gluteus medius tenderness variation (r = 0.36) across all aging times, but not at individual aging times. Sarcomere length contributed to variation in slice shear force values of gluteus medius at 14 and 42 d (r = -0.59 and -0.48, respectively). Sarcomere length contributed to triceps brachii tenderness variation at 14 d, but not 42 d (r = 0.44 and -0.12, respectively). Desmin degradation was strongly correlated (r = 0.55) to triceps brachii tenderness ratings pooled across aging times but not at individual aging times. These data indicate that triceps brachii steaks could provide the same or improved palatability as gluteus medius steaks at the same or slightly shorter aging times.

Myofilament Degradation and Dysfunction of Human Cardiomyocytes in Fabry Disease

Early detection of myocardial dysfunction in Fabry disease (FD) cardiomyopathy suggests the contribution of myofilament structural alterations. Six males with untreated FD cardiomyopathy submitted to cardiac studies, including tissue Doppler imaging and left ventricular endomyocardial biopsy. Active and resting tensions before and after treatment with protein kinase A (PKA) were determined in isolated Triton-permeabilized cardiomyocytes. Cardiomyocyte cross-sectional area, glycosphingolipid vacuole area, myofibrillolysis, and extent of fibrosis were also determined. Biopsies of mitral stenosis in patients with normal left ventricles served as controls. Active tension was four times lower in FD cardiomyocytes and correlated with extent of myofibrillolysis. Resting tension was six times higher in FD cardiomyocytes than in controls. PKA treatment decreased resting tension but did not affect active force. Protein analysis revealed troponin I and desmin degradation products. FD cardiomyocytes were significantly larger and filled with glycosphingolipids. Fibrosis was mildly increased compared with controls. Tissue Doppler imaging lengthening and shortening velocities were reduced in FD cardiomyocytes compared with controls, correlating with resting and active tensions, respectively, but not with cardiomyocyte area, percentage of glycosphingolipids, or extent of fibrosis. In conclusion, myofilament degradation and dysfunction contribute to FD cardiomyopathy. Partial reversal of high resting tension after pharmacological PKA treatment of cardiomyocytes suggests potential benefits from enzyme replacement therapy and/or energy-releasing agents.

Effects of different packaging atmospheres and injection-enhancement on beef tenderness, sensory attributes, desmin degradation, and display color1,2

The objectives were to determine the effects of packaging atmosphere and injection-enhancement on tenderness, sensory traits, desmin degradation, and display color of different beef muscles. Longissimus lumborum (LL; n = 12 pairs), semitendinosus (ST; n = 12 pairs), and triceps brachii (TB; n = 24 pairs; 12 from the same carcasses as the LL and ST and 12 additional pairs) were obtained from the same USDA Select, A-maturity carcasses. On d 7 postmortem, each muscle from one side of the carcass was injection-enhanced, and each muscle from the other side was nonenhanced. Steaks 2.54-cm thick were cut from the muscles and packaged in vacuum packaging (VP), ultra-low oxygen with CO (ULO(2)CO; 0.4% CO/35% CO(2)/69.6% N(2)) modified atmosphere packaging (MAP), or high-oxygen MAP (HiO(2); 80% O(2)/20% CO(2)) and assigned to 14-d tenderness or display followed by 18- or 28-d tenderness measurement. Steaks packaged in HiO(2) MAP were in dark storage (2 degrees C) for 4 d and all other steaks for 14 d. Steaks for Warner-Bratzler shear force, sensory panel (n = 8 trained panelists), and desmin degradation were cooked to 70 degrees C. Steaks were displayed under fluorescent lighting (2,153 lx, 3,000 K) for 7 d. Trained color panelists (n = 10) assigned display color scores. Enhanced steaks had lower (P < 0.05) Warner-Bratzler shear force values than nonenhanced steaks. Sensory panelists found that nonenhanced steaks packaged in ULO(2)CO MAP or VP were more tender (P < 0.05), had more (P < 0.05) beef flavor, and had less (P < 0.05) off-flavors than steaks packaged in HiO(2) MAP. The LL and TB were more tender (P < 0.05) according to myofibrillar tenderness than the ST. Nonenhanced steaks were less (P < 0.05) juicy than enhanced steaks. The most common off-flavors associated with steaks packaged in HiO(2) MAP were oxidative or rancid. Enhanced steaks had more (P < 0.05) off-flavors than nonenhanced steaks, with typical descriptors of salty, metallic, or chemical, in addition to an undesirable mushy texture. Desmin degradation increased (P < 0.05) from 7 to 14 d postmortem and differed among muscles but was not affected (P > 0.05) by packaging or enhancement. Enhanced steaks were darker (P < 0.05) initially than nonenhanced steaks. Steaks packaged in HiO(2) MAP discolored faster (P < 0.05) and to a greater extent (P < 0.05) than steaks packaged in VP or ULO(2)CO MAP. Nonenhanced muscles packaged in VP and ULO(2)CO MAP had more stable display color and very desirable tenderness and flavor compared with those packaged in HiO(2) MAP.

Comparison of postmortem changes in goose cardiac and breast muscles at 5 °C

AbstractBACKGROUD: The tenderness of goose heart is an important consideration in its utilization as a popular meat product. It is generally thought that postmortem degradation of myofibrillar proteins may improved meat tenderness. Little information, however, is available regarding the postmortem changes in goose cardiac muscle. Therefore, the postmortem proteolysis between goose cardiac and breast muscles at 5 °C is compared.RESULTS: The pH is higher (P &lt; 0.05) in cardiac samples than in breast samples. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blots results indicate that postmortem degradation of titin and desmin and the appearance of the 28 and 30 kDa components are faster in breast muscle than in cardiac muscle.CONCLUSION: Our results may suggest that goose postmortem proteolysis occurs more rapidly in breast muscle than in cardiac muscle at 5 °C. Copyright © 2008 Society of Chemical Industry

Packaging atmospheres and injection enhancement affect beef tenderness and sensory traits

Case-ready meat provides many benefits, including quality and safety. Meat packaged in high-oxygen (HiO2) modified atmosphere packaging (MAP) has a desirable bright red display color but may have increased off- flavors and decreased tenderness. According to several international research reports, steaks aged and packaged in HiO2 MAP had more off-flavor, including warmed-over flavor, and were less tender and juicy than steaks aged in vacuum packaging (VP). Research at Kansas State University found that injection-enhanced beef quadriceps muscles packaged in HiO2 MAP were less tender and had more offflavors than those in ultra-low oxygen MAP. Detrimental effects of O2 on tenderness might be caused by protein oxidation. Oxidation of beef muscle proteins early postmortem inactivates the primary enzyme (μ-calpain) necessary to break down proteins postmortem, which results in decreased myofibrillar proteolysis and limited tenderization. Injection-enhancement improves tenderness and juiciness while decreasing variation and often is used in conjunction with MAP. Several studies have reported that enhanced steaks were more tender and juicy than non-enhanced steaks. Several researchers found an increase in beef flavor associated with enhanced steaks, but others have reported a decreased or no change in beef flavor. Offflavors associated with enhanced beef include salty and oxidative. Objectives of our study were to determine the effects of packaging atmosphere and injection-enhancement on beef strip loin, eye of round, and chuck clod tenderness, sensory traits, and desmin degradation.

The effect of recombinant caspase 3 on myofibrillar proteins in porcine skeletal muscle

The objective of this study was to investigate the potential role of the caspase protease family in meat tenderisation by examining if caspase 3 was capable of causing myofibril protein degradation. Full-length human recombinant caspase 3 (rC3) was expressed in Escherichiacoli and purified. The rC3 was active in the presence of myofibrils isolated from porcine longissimus dorsi muscle (LD) and retained activity in a buffer system closely mimicking post mortem conditions. The effect of increasing concentrations of rC3, incubation temperature, as well as incubation time on the degradation of isolated myofibril proteins were all investigated in this study. Myofibril protein degradation was determined by SDS-PAGE and Western blotting. There was a visible increase in myofibril degradation with a decrease in proteins identified as desmin and troponin I and the detection of protein degradation products at approximately 32, 28 and 18 kDa with increasing concentrations of rC3. These degradation products were analysed using MALDI-TOF mass spectrometry and identified to occur from the proteolysis of actin, troponin T and myosin light chain, respectively. The production of these degradation products was not inhibited by 5 mM EDTA or semi-purified calpastatin but was inhibited by the caspase-specific inhibitor Ac-DEVD-CHO. The temperature at which isolated myofibrils were incubated with rC3 was also found to affect degradation, with increasing incubation temperatures causing increased desmin degradation and cleavage of pro-caspase 3 into its active isoform. Incubation of isolated myofibrils at 4°C for 5 days with rC3 resulted in the visible degradation of a number of myofibril proteins including desmin and troponin I. This study has shown that rC3 is capable of causing myofibril degradation, hydrolysing myofibril proteins under conditions that are similar to those found in muscle in the post mortem conditioning period.

Calpain inhibition attenuates right ventricular contractile dysfunction after acute pressure overload

Right ventricular contractile failure from acute RV pressure overload is an important cause of morbidity and mortality, but the mechanism of RV failure in this setting is incompletely defined. We hypothesized that RV dysfunction from acute RV pressure overload is, in part, due to activation of calpain, and that calpain inhibition would therefore attenuate RV dysfunction. Anesthetized, open chest pigs were treated with the calpain inhibitor MDL-28170 or with inactive vehicle, and then subjected to acute RV pressure overload for 90 min. RV contractile function was assessed by the regional Frank–Starling relation. RV myocardial tissue was analyzed for evidence of calpain activation and calpain-mediated proteolysis. RV pressure overload caused severe contractile dysfunction, along with significant alterations in the endogenous calpain inhibitor calpastatin typical of calpain activation. MDL-28170 attenuated RV free wall dysfunction by more than 50%. However, there were no differences in degradation of spectrin, desmin, troponin-I or SERCA2 between SHAM operated pigs and pigs subjected to acute RV pressure overload, or between vehicle and MDL-28170 treated pigs. Acute RV pressure overload causes calpain activation, and RV contractile dysfunction from acute RV pressure overload is attenuated by the calpain inhibitor MDL-28170; however, the effect is not explained by inhibition of calpain-mediated degradation of spectrin, desmin, troponin-I or SERCA2. Because this is the first report of any agent that can directly attenuate RV contractile dysfunction in acute RV pressure overload, further investigation of the mechanism of action of MDL-28170 in this setting is warranted.

H2O2activation of HSP25/27 protects desmin from calpain proteolysis in rat ventricular myocytes

Ischemia-reperfusion-induced Ca(2+) overload results in activation of calpain-1 in the heart. Calpain-dependent proteolysis contributes to myocardial dysfunction and cell death. Previously, preischemic treatment with low doses of H(2)O(2) was shown to improve postischemic function and reduce myocardial infarct size. Our aim was to determine the mechanism by which H(2)O(2) protects the heart. We hypothesized that H(2)O(2) causes the activation of p38 MAPK which initiates translocation of heat shock protein 25/27 (HSP25/27) to the myofilament Z disk. We further hypothesized that HSP25/27 shields structural proteins, particularly desmin, from calpain-induced proteolysis. To address this hypothesis, we first determined that an ischemia-reperfusion-induced decrease in desmin content could be blocked by H(2)O(2) pretreatment of hearts from rats. We next determined that ventricular myocytes that underwent Ca(2+) overload also demonstrated a calpain-dependent disruption of desmin that could be reduced by H(2)O(2)/p38 MAPK activation. Furthermore, myocytes acutely treated with H(2)O(2) exhibited a decrease in cleavage of desmin upon exposure to exogenous calpain-1 compared with myocytes not pretreated with H(2)O(2). The H(2)O(2)-induced attenuation of desmin degradation by calpain-1 was blocked by inhibition of p38 MAPK. In a final series of experiments, we demonstrated that cardiac myofilaments exposed to recombinant phosphorylated HSP27, but not nonphosphorylated HSP27, had a significant reduction in the calpain-induced degradation of desmin compared with non-HSP27-treated myofilaments. These findings are consistent with the hypothesis that H(2)O(2)-induced activation of p38 MAPK and subsequent HSP25/27 translocation attenuates desmin degradation brought about by calpain-1 activation in ischemia-reperfused hearts.

μ-Calpain is essential for postmortem proteolysis of muscle proteins1,2

The objective of this investigation was to test the hypothesis that -calpain is largely responsible for postmortem proteolysis of muscle proteins. To accomplish this objective, we compared proteolysis of known muscle proteins in muscles of wild type and micro-calpain knockout mice during postmortem storage. Knockout mice (n = 6) were killed along with control mice (n = 6). Hind limbs were removed and stored at 4 degrees C. Muscles were dissected at 0, 1, and 3d postmortem and subsequently analyzed for degradation of nebulin, dystrophin, metavinculin, vinculin, desmin, and troponin T. In a separate experiment, hind limb muscles from knockout (n = 4) and control mice (n = 4) were analyzed at 0, 1, and 3 d postmortem using casein zymography to confirm that mu-calpain activity was knocked out in muscle and to determine whether or not m-calpain is activated in murine postmortem muscle. Cumulatively, the results of the first experiment indicated that postmortem proteolysis was largely inhibited in micro-calpain knockout mice. The results of the second experiment established the absence of micro-calpain in the muscle tissue of knockout mice and confirmed the results of an earlier study that m-calpain is active in postmortem murine muscle. The results of the current study show that even in a species in which m-calpain is activated to some extent postmortem, micro-calpain is largely responsible for postmortem proteolysis. This observation excludes a major role for any of the other members of the calpain family or any other proteolytic system in postmortem proteolysis of muscle proteins. Therefore, understanding the regulation of micro-calpain in postmortem muscle should be the focus of further research on postmortem proteolysis and tenderization of meat.

Contribution of postmortem changes of integrin, desmin and μ-calpain to variation in water holding capacity of pork

The purpose of this study was to examine the relationship between integrin, desmin, μ-calpain and water holding capacity in fresh pork. High levels of intact integrin at one day postmortem were negatively correlated with day 1 ( P < 0.05) and days 1–5 (cumulative) drip loss ( P < 0.05). High levels of intact integrin at five days postmortem were negatively correlated with days 1–7 (cumulative) purge loss ( P < 0.05). Intensity of intact desmin at one day postmortem was positively correlated with days 1–7 purge loss ( P < 0.01). There were positive correlations between intensity of intact desmin at day 7 and day 1 ( P < 0.01), days 1–5 drip loss ( P < 0.01) and days 1–7 purge loss ( P < 0.05). Autolysis of μ-calpain was associated with the degradation of desmin and drip or purge loss postmortem. Our results indicate that low levels of degradation of integrin and high levels of desmin degradation were associated with low drip loss values in fresh pork.

Effect of oxidation, pH, and ionic strength on calpastatin inhibition of μ- and m-calpain

The objective of this study was to evaluate the effect of oxidation on mu- and m-calpain activity at varying pH and ionic strength conditions in the presence of calpastatin. In 2 separate experiments, purified porcine skeletal muscle mu- or m-calpain (0.45 units of caseinolytic activity) was incubated in the presence of calpastatin (0, 0.15, or 0.30 units) at pH 7.5, 6.5, or 6.0 with either 165 or 295 mM NaCl. The reactions were initiated with the addition of CaCl2 (100 microM for mu-calpain; 1 mM for m-calpain). In Experiment 1, mu- or m-calpain was incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-AMC (170 microM). Either 0 or 16 mu microM H2O2 was added to each assay. Activity was measured at 60 min. In Experiment 2, calpain was incubated with highly purified porcine myofibrils (4 mg/mL) under conditions described. Either 0 or 100 microM H2O2 was added immediately prior to the addition of calpain. Degradation of desmin was determined on samples collected at 2, 15, 60, and 120 min. Results from Experiment 1 indicated that oxidation decreased (P < 0.01) activity of mu-calpain. Mu-calpain had the greatest (P < 0.01) activity at pH 6.5, and m-calpain had the greatest (P < 0.01) activity at pH 7.5 at 60 min. m-Calpain activity was not detected at pH 6.0. Mu- and m-calpain activity were lower (P < 0.01) at 295 mM NaCl than at 165 mM NaCl at all pH conditions. Oxidation lowered (P < 0.01) calpastatin inhibition of mu-and m-calpain at all pH and ionic strength combinations. In Experiment 2, oxidation decreased proteolytic activity of mu-calpain against desmin at pH 6.0 (P < 0.05 at 15, 60, and 120 min) and decreased m-calpain at all pH conditions. However, desmin degradation by mu-calpain was not as efficiently inhibited by calpastatin at pH 7.5 and as at pH 6.5 (P = 0.03 at 60 min) when oxidizing conditions were created. This is consistent with the results from Experiment 1, which indicated that oxidation decreased the ability of calpastatin to inhibit mu-calpain. These studies provide evidence that oxidation influences calpain activity and inhibition of calpains by calpastatin differently under varying environmental conditions. The results suggest that, at the higher pH conditions used, calpastatin may limit the possibility of oxidation-induced inactivation of mu-calpain.