Longer Sarcomere Lengths Research Articles

Collagen architecture and biomechanics of gracilis and adductor longus muscles from children with cerebral palsy.

Cerebral palsy (CP) describes some upper motoneuron disorders due to non-progressive disturbances occurring in the developing brain that cause progressive changes to muscle. While longer sarcomeres increase muscle stiffness in patients with CP compared to typically developing (TD) patients, changes in extracellular matrix (ECM) architecture can increase stiffness. Our goal was to investigate how changes in muscle and ECM architecture impact muscle stiffness, gait and joint function in CP. Gracilis and adductor longus biopsies were collected from children with CP undergoing tendon lengthening surgery for hamstring and hip adduction contractures, respectively. Gracilis biopsies were collected from TD patients undergoing anterior cruciate ligament reconstruction surgery with hamstring autograft. Muscle mechanical testing, two-photon imaging and hydroxyproline assay were performed on biopsies. Corresponding data were compared to radiographic hip displacement in CP adductors (CPA), gait kinematics in CP hamstrings (CPH), and joint range of motion in CPA and CPH. We found at matched sarcomere lengths muscle stiffness and collagen architecture were similar between TD and CP hamstrings. However, CPH stiffness (R2=0.1973), collagen content (R2=0.5099) and cross-linking (R2=0.3233) were correlated to decreased knee range of motion. Additionally, we observed collagen fibres within the muscle ECM increase alignment during muscular stretching. These data demonstrate that while ECM architecture is similar between TD and CP hamstrings, collagen fibres biomechanics are sensitive to muscle strain and may be altered at longer in vivo sarcomere lengths in CP muscle. Future studies could evaluate the impact of ECM architecture on TD and CP muscle stiffness across in vivo operating ranges. KEY POINTS: At matched sarcomere lengths, gracilis muscle mechanics and collagen architecture are similar in TD patients and patients with CP. In both TD and CP muscles, collagen fibres dynamically increase their alignment during muscle stretching. Aspects of muscle mechanics and collagen architecture are predictive of in vivo knee joint motion and radiographic hip displacement in patients with CP. Longer sarcomere lengths in CP muscle in vivo may alter collagen architecture and biomechanics to drive deficits in joint mobility and gait function.

Subzero saline chilling with or without prechilling in icy water improved chilling efficiency and meat tenderness of broiler carcasses

Freshly slaughtered carcasses need to be chilled to improve product quality, meat safety, and processing efficiency. This research investigated the effect of subzero saline chilling (SSC) on broiler carcasses with or without prechilling in icy water. Water immersion chilling at 0.5°C (WIC) or SSC at 4% NaCl/-2.41°C (SSC) was a major chilling step. For the combination of pre- and postchilling, the warm water immersion chilling (WWIC) at 10°C was used as prechilling and the WIC as postchilling (WWIC-WIC), and WIC was used as prechilling and the SSC as postchilling (WIC-SSC). The internal temperature of breast fillets was monitored during chilling. Carcasses in a prechiller were transported to a postchiller when their internal temperature reached 15°C. Chilling was completed when the carcass temperature reached 4.4°C or below, and breast fillets were harvested at 3-h postmortem to measure the pH and sarcomere length. Color (L*, a*, and b*) values were evaluated on both breast skin and skinless breast surfaces. Meat tenderness was evaluated using the breast fillets after overnight storage and cooking to an internal temperature of 76°C. The carcasses in the SSC and WIC-SSC showed shorter chilling times (85-91 min) than those (100-144 min) of WIC and WWIC-WIC. A higher chilling yield was observed for the carcasses in WIC-SSC, and a lower cooking yield was seen for the carcasses in WWIC-WIC than other chilling methods (P < 0.05). The breast fillets of broilers in the SSC and WIC-SSC showed lower shear forces and longer sarcomere length than the WIC and WWIC-WIC. No difference was found for L* and a* values, while lower b* value was observed in the SSC than the other chilling methods (P < 0.05). Based on these results, chilling of broiler carcasses in the SSC (4% NaCl/-2.41°C) with or without prechilling in WIC at 0.5°C significantly improved chilling efficiency and meat tenderness, with minor color changes on carcasses.

Abstract P1056: Stretch Reduces Inter-filament Spacing And Triggers Alterations In Thick-filament Structure In Healthy Left-atrial Fibers

Introduction: Left-atrial dilatation is thought to play a key pathological role in facilitating remodeling in both genetic and acquired cardiac myopathies, as well as enabling the onset and maintenance of atrial fibrillation. However, despite its likely role in disease, the structural responses of the atria to stretch remain understudied. The myofilament responses to stretch have been extensively studied in ventricular tissue, where strain-dependent recruitment of reserve, OFF-state, myosin heads from the thick-filament contribute to length dependent activation (LDA, i.e., greater forcer at longer sarcomere lengths). In these experiments, the effects of stretch in both atrial sarcomere and thick-filament structure were evaluated. Methods: Myofilament structure was evaluated via x-ray diffraction at two sarcomere lengths (SL, SHORT: 2.1 μm, and LONG: 2.3 μm) in relaxed left-atrial and left-ventricular myocardial skinned fibers from Yucatan mini-pigs. In these experiments, both equatorial and meridional reflections reflecting sarcomeric structure and ordering were evaluated. In addition, the biomechanical effects of stretch were also evaluated in both skinned porcine fibers and engineered heart tissues (EHT) from atrial and ventricular hIPSc-derived myocytes via a proprietary system (MyoPod; Propria Biosciences). Results: In both atrial skinned fibers and EHTs, stretch triggered moderate increases in force, demonstrating LDA. LA fibers, stretch decreased lattice spacing but failed to alter the I 11 /I 10 equatorial intensity ratio, suggesting negligible length-dependent recruitment of OFF-state myosin heads from the thick-filament. LA fibers, however, tended to have higher I 11 /I 10 values at short SL (vs. LV fibers) suggesting an intrinsically higher population of ON-state myosin heads. In addition, stretch triggered alterations in thick filament ordering in both LA and LV fibers, as reflected by changes in both the 3 rd and 6 th order meridional reflections (M3 and M6), suggesting effects in both myosin heads and thick-filament backbone. Conclusion: Atrial stretch results in marked sarcomere structural alterations, reducing both inter-filament spacing and thick-filament ordering that likely contribute to length dependent activation. The role of these alteration in atrial pathophysiology remains to be studied

Role of creatine supplementation on the myofiber characteristics and muscle protein synthesis of grass carp (Ctenopharyngodon idellus).

To assess the role of dietary creatine on myofiber characteristics and protein synthesis in muscle, we fed grass carp (Ctenopharyngodon idellus, initial body weight: 88.47 ± 1.44 g) creatine-supplemented diets (1.84, 5.91, 8.48, and 15.44 g/kg diet) for 8 weeks. Creatine supplementation did not affect growth performance, but significantly increased creatine contents in muscle and liver. At 8.48 g/kg, creatine decreased the activities of alanine transaminase and aspartate aminotransferase in serum, and improved hardness and chewiness of muscle due to shorter myofiber mean diameter, higher myofiber density and the frequencies of the diameters of class I and III and collagen content, longer sarcomere length, and upregulated mRNA levels of slow myosin heavy chains. Creatine supplementation upregulated the mRNA expressions of myogenic regulatory factors. The 8.48 g/kg creatine-supplemented diet significantly increased the contents of protein, total amino acids (AAs), essential AAs, and free flavor AAs in muscle, the protein levels of insulin-like growth factor I, myogenic differentiation antigen, and peroxisome proliferator-activated receptor-γ coactlvator-1α in muscle, and stimulated the phosphorylation of target of rapamycin (TOR) pathway in muscle. In summary, 8.48 mg/kg creatine improved fish health and skeletal muscle growth, and increased hardness and protein synthesis in muscle of grass carp by affecting myofiber characteristics and the TOR signaling pathway. A second-order regression model revealed that the optimal dietary creatine supplementation of grass carp ranges between 8.48 and 12.04 g/kg.

Myosin motors that cannot bind actin leave their folded OFF state on activation of skeletal muscle.

The myosin motors in resting skeletal muscle are folded back against their tails in the thick filament in a conformation that makes them unavailable for binding to actin. When muscles are activated, calcium binding to troponin leads to a rapid change in the structure of the actin-containing thin filaments that uncovers the myosin binding sites on actin. Almost as quickly, myosin motors leave the folded state and move away from the surface of the thick filament. To test whether motor unfolding is triggered by the availability of nearby actin binding sites, we measured changes in the x-ray reflections that report motor conformation when muscles are activated at longer sarcomere length, so that part of the thick filaments no longer overlaps with thin filaments. We found that the intensity of the M3 reflection from the axial repeat of the motors along the thick filaments declines almost linearly with increasing sarcomere length up to 2.8 µm, as expected if motors in the nonoverlap zone had left the folded state and become relatively disordered. In a recent article in JGP, Squire and Knupp challenged this interpretation of the data. We show here that their analysis is based on an incorrect assumption about how the interference subpeaks of the M3 reflection were reported in our previous paper. We extend previous models of mass distribution along the filaments to show that the sarcomere length dependence of the M3 reflection is consistent with <10% of no-overlap motors remaining in the folded conformation during active contraction, confirming our previous conclusion that unfolding of myosin motors on muscle activation is not due to the availability of local actin binding sites.

Myopalladin knockout mice develop cardiac dilation and show a maladaptive response to mechanical pressure overload.

Myopalladin (MYPN) is a striated muscle-specific immunoglobulin domain-containing protein located in the sarcomeric Z-line and I-band. MYPN gene mutations are causative for dilated (DCM), hypertrophic, and restrictive cardiomyopathy. In a yeast two-hybrid screening, MYPN was found to bind to titin in the Z-line, which was confirmed by microscale thermophoresis. Cardiac analyses of MYPN knockout (MKO) mice showed the development of mild cardiac dilation and systolic dysfunction, associated with decreased myofibrillar isometric tension generation and increased resting tension at longer sarcomere lengths. MKO mice exhibited a normal hypertrophic response to transaortic constriction (TAC), but rapidly developed severe cardiac dilation and systolic dysfunction, associated with fibrosis, increased fetal gene expression, higher intercalated disc fold amplitude, decreased calsequestrin-2 protein levels, and increased desmoplakin and SORBS2 protein levels. Cardiomyocyte analyses showed delayed Ca2+ release and reuptake in unstressed MKO mice as well as reduced Ca2+ spark amplitude post-TAC, suggesting that altered Ca2+ handling may contribute to the development of DCM in MKO mice.

The Super-Relaxed State and Length Dependent Activation in Porcine Myocardium.

[Figure: see text].

The muscle M3 x-ray diffraction peak and sarcomere length: No evidence for disordered myosin heads out of actin overlap.

X-ray diffraction studies of muscle have provided a wealth of information on muscle structure and physiology, and the meridian of the diffraction pattern is particularly informative. Reconditi et al. (2014. J. Physiol.https://doi.org/10.1113/jphysiol.2013.267849) performed superb experiments on changes to the M3 meridional peak as a function of sarcomere length (SL). They found that the M3 intensity dropped almost linearly as sarcomere length increased at least to about SL = 3.0 µm, and that it followed the same track as tension, pointing toward zero at the end of overlap at ∼3.6 µm. They concluded that, just as tension could only be generated by overlapped myosin heads, so ordered myosin heads contributing to the M3 intensity could only occur in the overlap region of the A-band, and that nonoverlapped heads must be highly disordered. Here we show that this conclusion is not consistent with x-ray diffraction theory; it would not explain their observations. We discuss one possible reason for the change in M3 intensity with increasing sarcomere length in terms of increasing axial misalignment of the myosin filaments that at longer sarcomere lengths is limited by the elastic stretching of the M-band and titin.

ERR\u03b3 enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes

One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.

Influence of Woody Breast Myopathy on Sarcomere Length and Tensile Strength in Commercial Broiler Pectoralis major Muscle

Woody breast syndrome is characterized by degenerative changes at the muscle fiber level and accumulation of connective tissue between the fibers. This study explored effects of the syndrome on muscle properties by focusing on a comparison of the sarcomere lengths between normal and woody breast muscles, including cranial and middle parts, surface and deeper layers, electrically stimulated and nonstimulated muscles, and their combinations. Tensile strengths longitudinally and transversally to the muscle fiber direction in the cranial and middle parts of the muscles were determined. The overall sarcomere lengths of woody breasts were longer than normal muscles (1.93 μ m vs. 1.88 μm; P &amp;lt; 0.05). The surface layer had overall longer sarcomere lengths than the deeper layer (cranial surface vs. cranial deeper layer: 1.97 vs. 1.89 μ m; middle surface vs. middle deeper layer: 1.93 vs.1.84 μ m; P &amp;lt; 0.05). Compared with normal breast muscles, woody breast muscles had longer sarcomeres in the surface layer; however, sarcomere length did not differ in the deeper layer. Electrically stimulated chicken breasts generally had longer sarcomere lengths (2.00 vs. 1.82 μm; P &amp;lt; 0.001). There was no significant difference in tensile strength between normal and focal woody breast (mild local lesion, usually in the cranial end) samples when fiber direction or sample location was studied (P &amp;gt; 0.05), but there was a clear difference between normal and focal versus diffuse (severe woody breast lesion throughout the muscle) samples (P &amp;lt; 0.001). Tensile strength was much greater in diffuse woody breast muscles when extended longitudinally or transversely to the fiber direction. In conclusion, although this study did not show sarcomere lengths in living muscle, it suggests an imbalance in sarcomere lengths in different parts of the breast muscle, which may induce a reduction in the functionality and strength of the muscle.

A Comparative Study of Sheep Breeds: Fattening Performance, Carcass Characteristics, Meat Chemical Composition and Quality Attributes.

Fattening performance, Carcass characteristics, chemical composition, and meat quality were evaluated in three sheep breeds: Awassi, Harri, and Najdi. Forty-five lambs of similar weight and age were raised for 90 days under similar conditions. The Harri and Najdi breeds had higher dressing-out percentages than Awassi sheep. The Awassi and Harri breeds had thicker backfat than the Najdi breed. No significant difference was found in moisture, protein, and intramuscular fat among the breeds. However, the Harri breed had a higher ash content than the Awassi and Najdi breeds. The Najdi breed had higher ultimate pH and lower cooking loss than the Awassi and Harri breeds. Awassi and Harri sheep had a higher myofibril fragmentation index, longer sarcomere length, and lower hardness and chewiness than Najdi sheep. Subjectively, no significant differences were detected between the breeds, except for flavor intensity while the Awassi sheep were rated in between and not significantly different. In conclusion, breed affected carcass characteristics, meat composition, and the quality of sheep. The dressing yield was higher in Harri and Najdi than Awassi sheep. Awassi sheep showed superior meat quality characteristics followed by Harri sheep. However, Najdi sheep had the best cooking loss percentage and flavor intensity score.

Structural Changes in the Myosin Motors Induced by Stretch and Phosphorylation of the Myosin Regulatory Light Chain in Rat Cardiac Trabeculae

The contractility of cardiac muscle is potentiated by phosphorylation of the myosin regulatory light chain (RLC) and by length-dependent activation, but the molecular pathways mediating these effects remain unclear. Here we studied the role of structural changes in the thick filament in the activation of cardiac muscle by stretch and RLC phosphorylation. We used fluorescence polarization from bifunctional sulphorhodamine probes on the N- and C-lobes of the myosin RLC to monitor changes in the orientation of the myosin motors induced by increasing the sarcomere length in the range 2.0-2.3 µm in relaxed and partially calcium-activated demembranated cardiac trabeculae, at different levels of RLC phosphorylation. We show that under relaxing or diastolic conditions at near physiological temperature and lattice spacing the RLC lobe orientations are consistent with about one third of the myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. This folded conformation is disrupted by cooling relaxed trabecula, but not by RLC phosphorylation or stretch at diastolic [Ca2+]. These results indicate that in near-physiological conditions the myosin filament is not switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in diastole, as suggested previously. However, stretching cardiac trabecula at near-systolic [Ca2+] triggers a stress-dependent activation of the thick filament and a delayed increase in active force. Physiological levels of RLC phosphorylation potentiate both the stress-dependent release of myosin motors from the folded state and the stretch-activated force. We conclude that the increase in heart contractility induced by RLC phosphorylation and stretch can be explained by an inter-filament signaling pathway involving both thin filament sensitization and thick filament mechano-sensing (Supported by Wellcome Trust, BHF, UK).

Insight into the Effects of Sous Vide on Cathepsin B and L Activities, Protein Degradation and the Ultrastructure of Beef.

This study aimed to evaluate the effects of sous vide cooking (SV) on beef tenderness and its underlying potential mechanism. Beef semimembranosus (SM) were subjected to SV treatments at 45 °C, 55 °C and 65 °C for 4 h. Compared with control samples (CK, cooked at 75 °C until a core temperature of 72 °C was attained), SV treatment significantly promoted the release of cathepsin B and cathepsin L from lysosomes and decreased the shear force of beef SM (p < 0.05). In comparison with CK, samples treated with SV had more hydrolysis of myosin heavy chain and obtained higher myofibrillar fragmentation index, collagen solubility as well as longer sarcomere length (p < 0.05). The current study showed that the proteolysis of myofibrillar protein and collagen induced by cathepsin B and cathepsin L, and the limited longitudinal shrinkage together contributed to the improvement of beef tenderness upon SV.

Myosin heavy chain isoforms expression, calpain system and quality characteristics of different muscles in goats

Myosin heavy chain (MHC) isoforms in goat muscles and their possible relationships with meat quality have not been fully elucidated. This study characterized the MHC isoforms in different caprine muscles using sodium dodecyl sulphate glycerol gel electrophoresis (SDS-GGE). The relationships between MHC isoforms, calpain systems and meat quality characteristics of different muscles in goats were examined. Four muscles, namely infraspinatus (IF), longissimus dorsi (LD), psoas major (PM) and supraspinatus (SS) were obtained from ten Boer crossbred bucks (7–10 months old; 26.5 ± 3.5 kg, BW). The percentages of MHC I, MHC IIa and MHC IIx in SS, IF, PM and LD were 47.2, 38.3, 32.1, 11.9; 28.0, 42.1, 33.0, 36.4; and 24.8, 19.6, 34.9 and 51.7, respectively. IF and SS had higher levels of calpastatin, total collagen and insoluble collagen contents than did PM and LD. PM had longer sarcomere length than did other muscles. LD had higher collagen solubility, troponin-T degradation products and glycogen content than did other muscles. These results infer that variable fiber-type composition could account partially for the differences in the physicochemical properties of goat muscles.

PH Variability and its Relationship with Sarcomere Length and Free Calcium in Beef from Commercial Cattle in Puerto Rico

ObjectivesResearch conducted at the University of Puerto Rico noted that beef with elevated pH values (&gt; 5.86) resulted in more tender meat (P ≤ 0.05). It has been established that proteolytic degradation mechanisms can be influenced by pH and calcium concentration in muscle. Beef with pH values ≥ 5.86 is classified as Dark Firm and Dry (DFD) but there are negative implications associated with greater pH values. However, observations indicating increased tenderness with increased pH raise the question: can variations in pH be associated with differences in sarcomere length (SL) and free calcium concentration (FCC)? Therefore, the objectives of this project were to: (1) document pH distribution; (2) determine the incidence of DFD; and (3) evaluate the relationship between pH, SL, and FCC in commercial cattle harvested in Puerto Rico.Materials and MethodsLongissimus lumborum samples (n = 51) were obtained and background information was noted including number of permanent incisors (PI), type (Dairy or Beef), and gender. The pH values were used to categorize beef into the following groups: Low (≤ 5.40), Normal (5.41 to 5.59), High (5.60 to 5.85) and DFD (≥ 5.86). Meat was flash frozen, powdered, and placed on a microscope slide and a Helium-Neon laser was used to determine SL. A subset of samples was sent off and prepared at the University of Nebraska-Lincoln for FCC quantification (Ward Laboratories; Kearney, NE) with an inductively coupled plasma emission spectrometer (iCAP 6500 Radial; Thermo Electron, Cambridge, UK). All statistical analyses were conducted in SAS (9.4). The Proc FREQ was used to determine pH category distributions and incidence of DFD. The Proc GLIMMIX and Tukey adjustment (α = 0.05) were used to determine the effects of number of PI, type, and gender on pH category, SL and FCC. The Proc CORR was used to evaluate the relationship between pH category, SL and FCC.ResultsThe pH category distribution for the current samples was as follows: 3.92% Low, 41.18% Normal, 35.29% High and 19.61% DFD. The SL ranged from 1.69 to 1.46 mm with an average of 1.53 mm. The FCC ranged from 132.19 to 31.39 mM with an average of 64.23 mM. Longer sarcomeres were detected in cattle with eight and zero PI (1.57 and 1.56 mm, respectively); cattle with two and four PI had intermediate SL (1.53 and 1.52 mm, respectively), and cattle with six PI had the shortest sarcomeres (1.51 mm; P = 0.03). Dairy cattle had longer sarcomeres relative to beef cattle (1.56 vs. 1.52 mm; P = 0.02). Dairy cattle tended to have increased FCC relative to beef cattle (70.72 vs. 58.38 mM; P = 0.08). Also, FCC tended to be greater within the Normal and Low pH categories relative to the High and DFD categories (72.36 vs. 57.31 mM; P = 0.06). The SL and FCC had no relationship (P &gt; 0.05) within the Low, Normal and High pH categories. However, DFD beef had longer SL (0.78; P = 0.01), while having decreased FCC (–0.66; P = 0.04).ConclusionOver half (54.90%) of the beef samples analyzed fell into the High and DFD pH categories, with nearly 20% being classified as DFD. Although, a clear relationship was not established between SL and FCC within the Low, Normal or High pH categories, the results indicate that the increased pH in samples surpassing the DFD threshold correspond to longer sarcomeres and decreased free calcium.

Vascular Rinsing and Chilling Carcasses: Effects on Quality Attributes and Metabolic Changes in Beef

ObjectivesRinse &amp; Chill® (RC) is a process applied early postmortem that provides the ability to manipulate muscle metabolism and can have a positive impact on meat quality traits. This study aimed to evaluate the effect of RC on pH decline, shear force, sarcomere length and cooking losses on different cull dairy cow carcass grades. Investigate the ability of different substrates to modulate contractile response as an indirect measure of metabolic activity on beef early postmortem.Materials and MethodsFor each carcass grade (lean, LE; light, LI), ten carcasses were conventionally chilled (CC) and twelve carcasses were chilled using RC technology (MPSC Inc.). The RC process involved infusion of a chilled isotonic solution (98.5% water; balance: glucose, phosphates, and maltose) through the vascular system, beginning in the arterial and exiting the venous side of the vasculature. Shear force and cooking losses were measured on Longissimus dorsi steaks aged (7 d). Sarcomere length (SL) was determined by a laser diffraction method. Animal served as the experimental unit and data were analyzed with a PROC MIXED procedure. For contraction measurements, a muscle-fiber bundle from the Sternomandibularis muscles (n = 14) was collected from cull dairy cows in a commercial packing plant, 15 min after bleeding. The muscle bundle was attached to a force transducer (FT-302, iWorx, Dover, NH). Stimulation electrodes were used to elicit a supramaximal electrical stimulus at a frequency of 50 V, 0.1 Hz (HCS-100 stimulator, iWorx). Muscle weight was standardized, and length was adjusted to obtain maximum twitch-tension output. After 3 min of rest in a test solution, 200 stimuli were given, and the contractile response was recorded. Four solutions were tested (A = RC, B = Fructose, C = Sodium phosphate, D = Dipotassium phosphate; substrates added at 1% except fructose 1.5%). Descriptive means for initial peak twitch force, final peak twitch force, percentage decline and percentage half-time decline were calculated to determine the response associated with each solution.ResultsRC reduced (P &lt; 0.05) shear force by 51.9% (6.79 kgf CC) and 55.8% (8.50 kgf CC) for LI and LE cows, respectively. LI cows were more tender than LE for CC (6.79 vs. 8.50 kgf; P &lt; 0.05). RC compared to CC had longer SLs (LE: 1.80 vs. 1.44µ; P &lt; 0.05) and LI (1.80 vs. 1.40µ; P &lt; 0.05). Purge and cooking losses were not affected by chilling method. The contractile responses of the muscle after the exposure to the solutions were slightly different. The average percentage decline of peak twitch force was higher for solution B, followed by solutions A, D, C (54.8%, 53.5%, 48.0%, 43.4%, respectively). Furthermore, the same pattern was observed for the average percentage decline at half time of the test (82.5%, 80.4%, 78.1% and 74.7%, respectively).ConclusionPacking plants that harvest cull dairy cows have the potential to dramatically improve tenderness and thereby merchandize a greater amount of whole muscle cuts as a result of the application of the Rinse &amp; Chill®. This improvement may be associated with accelerating postmortem glycolysis, thereby limiting cold shortening, although enhanced proteolysis may also be involved. Continuous electrical stimulation of isolated muscle-fiber bundles while being soaked in selected test solutions led to decreased and somewhat varied contractile force responses suggesting the potential to modify muscle metabolism.

Abstract 905: Length Dependent Activation in Porcine Cardiac Myofilaments is Modulated by Mavacamten

Length dependent activation (LDA) is a property of muscle where increased sarcomere length (SL) leads to increased force of contraction. Despite its key role in both normal and pathological states, the molecular mechanisms underlying LDA are not understood. Previous studies suggest that increased titin-based passive tension at longer SL triggers structural changes in the troponin complex leading to increased Ca 2+ sensitivity. Stretch also appears to release myosin heads from the OFF or super-relaxed state (SRX) increasing the number of available heads for cross-bridge formation, and, hence, maximum force. The small molecule inhibitor of myosin, mavacamten (mava) has been shown to stabilize the SRX state. Here, we used both stretch and mava on permeabilized porcine myocardium for force/pCa measurements coupled with x-ray diffraction to interrogate the mechanisms underlying LDA. Increasing SL from 2.0 to 2.3 um increases Ca 2+ sensitivity and elevates both diastolic and maximal tension (i.e. LDA). Mava abolished LDA while blunting the passive stiffness/tension relationship, suggesting increased compliance. The equatorial x-ray reflection intensity ratio, I 1,1 /I 1,0 , increases with stretch, indicating increased myosin head displacement towards actin. Stretch also decreases the intensities of both the m3 meridional and the MyBPC reflections, suggesting stretch-induced disordering of the thick-filament. Surprisingly, these findings in pig skinned muscle are opposite to those previously reported in intact rat muscle despite robust LDA in both preparations. Consistent with previous studies, stretch increases thick filament strain (indicated by changes in the m6 meridional spacing) and changes in the structure of the troponin complex (indicated by increased intensity of the third order troponin reflection), suggesting that the mechanism for increased Ca 2+ sensitivity are similar in both muscle systems. Interestingly, all diffraction pattern changes that occurred with stretch moved in the opposite direction in the presence of mava. Together, these data indicate that mava is not only capable of stabilizing the SRX state of myosin, but can modulate inter-filament interactions responsible for increasing Ca 2+ sensitivity and force.

Vascular Rinsing and Chilling Carcasses: Effects on Quality Attributes and Metabolic Changes in Beef

ObjectivesRinse & Chill® (RC) is a process applied early postmortem that provides the ability to manipulate muscle metabolism and can have a positive impact on meat quality traits. This study aimed to evaluate the effect of RC on pH decline, shear force, sarcomere length and cooking losses on different cull dairy cow carcass grades. Investigate the ability of different substrates to modulate contractile response as an indirect measure of metabolic activity on beef early postmortem.Materials and MethodsFor each carcass grade (lean, LE; light, LI), ten carcasses were conventionally chilled (CC) and twelve carcasses were chilled using RC technology (MPSC Inc.). The RC process involved infusion of a chilled isotonic solution (98.5% water; balance: glucose, phosphates, and maltose) through the vascular system, beginning in the arterial and exiting the venous side of the vasculature. Shear force and cooking losses were measured on Longissimus dorsi steaks aged (7 d). Sarcomere length (SL) was determined by a laser diffraction method. Animal served as the experimental unit and data were analyzed with a PROC MIXED procedure. For contraction measurements, a muscle-fiber bundle from the Sternomandibularis muscles (n = 14) was collected from cull dairy cows in a commercial packing plant, 15 min after bleeding. The muscle bundle was attached to a force transducer (FT-302, iWorx, Dover, NH). Stimulation electrodes were used to elicit a supramaximal electrical stimulus at a frequency of 50 V, 0.1 Hz (HCS-100 stimulator, iWorx). Muscle weight was standardized, and length was adjusted to obtain maximum twitch-tension output. After 3 min of rest in a test solution, 200 stimuli were given, and the contractile response was recorded. Four solutions were tested (A = RC, B = Fructose, C = Sodium phosphate, D = Dipotassium phosphate; substrates added at 1% except fructose 1.5%). Descriptive means for initial peak twitch force, final peak twitch force, percentage decline and percentage half-time decline were calculated to determine the response associated with each solution.ResultsRC reduced (P < 0.05) shear force by 51.9% (6.79 kgf CC) and 55.8% (8.50 kgf CC) for LI and LE cows, respectively. LI cows were more tender than LE for CC (6.79 vs. 8.50 kgf; P < 0.05). RC compared to CC had longer SLs (LE: 1.80 vs. 1.44µ; P < 0.05) and LI (1.80 vs. 1.40µ; P < 0.05). Purge and cooking losses were not affected by chilling method. The contractile responses of the muscle after the exposure to the solutions were slightly different. The average percentage decline of peak twitch force was higher for solution B, followed by solutions A, D, C (54.8%, 53.5%, 48.0%, 43.4%, respectively). Furthermore, the same pattern was observed for the average percentage decline at half time of the test (82.5%, 80.4%, 78.1% and 74.7%, respectively).ConclusionPacking plants that harvest cull dairy cows have the potential to dramatically improve tenderness and thereby merchandize a greater amount of whole muscle cuts as a result of the application of the Rinse & Chill®. This improvement may be associated with accelerating postmortem glycolysis, thereby limiting cold shortening, although enhanced proteolysis may also be involved. Continuous electrical stimulation of isolated muscle-fiber bundles while being soaked in selected test solutions led to decreased and somewhat varied contractile force responses suggesting the potential to modify muscle metabolism.

Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats.

Force production by actin-myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower β-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca2+-activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases β-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of β-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and β-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that β-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.

Analysis of Titin in Red and White Muscles: Crucial Role on Muscle Contractions Using a Fish Model.

Several studies have compared molecular components between red and white skeletal muscles in mammals. However, mammalian skeletal muscles are composed of mixed types of muscle fibers. In the current study, we analyzed and compared the distributions of titin, lipid, phosphate ions, and fatty acid levels in red and white muscles using a fish model (Tilapia), which is rich in red and white muscles, and these are well separated. Oil-red O staining showed that red muscle had more-abundant lipids than did white muscle. A time-of-flight secondary-ion mass spectrometric (TOF-SIMS) analysis revealed that red muscle possessed high levels of palmitic acid and oleic acid, but white muscle contained more phosphate ions. Moreover, elastica-van Gieson (EVG) and Mito-Tracker green FM staining showed that collagen and elastic fibers were highly, respectively, distributed in connective tissues and mitochondria in red muscle. An electron micrographic analysis indicated that red muscle had a relatively higher number of mitochondria and longer sarcomere lengths and Z-line widths, while myofibril diameters were thicker in white muscle. Myofibrillar proteins separated by SDS-PAGE showed that the major giant protein, titin, was highly expressed in white muscle than in red muscle. Furthermore, ratios of titin to myosin heavy chain (MHC) (titin/MHC) were about 1.3 times higher in white muscle than red muscle. We postulated that white muscle is fit for short and strong contractile performance due to high levels of titin and condensed sarcomeres, whereas red muscle is fit for low intensity and long-lasting activity due to high levels of lipids and mitochondria and long sarcomeres.