Determination Of Sarcomere Length Research Articles

Sarcomere length measurement reliability in single myofibrils

Sarcomere length non-uniformities occur at all structural levels of skeletal muscles and have been associated with important mechanical properties. Changes in sarcomere length non-uniformities in the nano- and sub-nanometer range have been used to explain muscle properties and contractile mechanisms. Typically, these measurements rely on light microscopy with a limited spatial resolution. One critical aspect in sarcomere length determination is the relatively arbitrary choice of intensity thresholds used to delineate sarcomere structures, such as A-bands or Z-lines. In experiments, these structures are typically distorted, intensity profiles vary, and baselines drift, resulting in asymmetric intensity patterns, causing changes in the centroid location of these structures depending on threshold choice, resulting in changes of sarcomere lengths. The purpose of this study was to determine the changes in (half-) sarcomere lengths associated with small changes in the A-band threshold choice. Sarcomere and half-sarcomere length changes for minute variations in A-band threshold were 28 nm (±28 nm) and 18 nm (±22 nm), respectively, and for the entire feasible range of thresholds across A-bands were 123 nm (±88 nm) and 99 nm (±105 nm), respectively. We conclude from these results that (half-) sarcomere lengths in the nanometer range obtained with light microcopy are noise, and the functional implications associated with such data should be discarded. We suggest that a functional resolution for sarcomere length of 100 nm (0.1 µm) is reasonable and 50 nm (0.05 µm) might be possible under ideal conditions.

Live imaging of contracting muscles with wide-field second harmonic generation microscopy using a high power laser.

Wide-field second harmonic generation (SHG) microscopy was developed using a high-power (> 4 W) and high-repetition-rate (MHz range) laser oscillator to achieve fast SHG imaging over a large area (400 µm × 400 µm). The microscope was used for high spatial resolution imaging of contracting muscles in live Drosophila melanogaster larvae. Anisotropic and isotropic bands of striated muscle were distinguished, allowing accurate determination of sarcomere length and SHG intensity from individual sarcomeres. Therefore, wide-field SHG microscopy has applications in basic contractility research and studying arrhythmias, muscular dystrophies and pharmaceutical effects on the muscle contraction dynamics of sarcomeres.

The Effects of Hot vs. Cold Boning on Eating Quality of New Zealand Grass Fed Beef

Sides of the 40 selected carcasses were alternately assigned to both conventional chilling and cold boning (CB) or hot boning (HB). Fabrication of CB sides took place after overnight chilling (20 h postmortem) and subsequent carcass grading, while fabrication of HB sides occurred on the day of slaughter within 60 min of exsanguination. Five muscles were removed, including the longissimus lumborum (LL), longissimus thoracis (LT), psoas major (PM), gluteus medius (GM), and semimembranosus (SM) for consumer evaluation, compositional analysis, and sarcomere length determination. Cold boning muscles had longer (P < 0.05) sarcomere lengths compared to HB muscles, expect for the GM and SM. Chilling treatment and postmortem aging (7, 21, or 35 d postmortem) had an impact on eating quality, but these results varied by muscle. Cold shortening could be responsible for the differences in sarcomere length and ultimately differences in tenderness between HB and CB muscles. Based on consumer evaluations, HB is not recommended for LT or PM due to the reduction (P ≤ 0.02) of tenderness and overall liking scores for these subprimals; however, HB and early removal of muscles like the SM benefited (P < 0.01) from an eating quality standpoint for all palatability traits, and was neutral (P ≥ 0.21) for tenderness, juiciness, flavor, and overall liking of the LL and GM. Postmortem aging did not affect (P ≥ 0.07) eating quality of the GM and PM, but improved (P < 0.01) tenderness of LT and SM, as well as the tenderness, juiciness, flavor liking, and overall liking of the LL.

Real-Time Determination of Sarcomere Length of a Single Cardiomyocyte during Contraction

Mean sarcomere length of a cardiomyocyte is an important control parameter for physiology studies on a single cell level. For instance, accurate determination of the mean sarcomere lenght in real-time is essential for performing single cardiomyocyte contraction experiments.The aim of this work is to develop an accurate and efficient computational method for determining the mean sarcomere length from transmission images of a single contracting cardiomyocyte. This algorithm has to be robust to noise in input and exact for perfectly periodic input, that is, the best algorithm must have no systematic errors.To accomplish the goal of this work we are (i) using unbiased measure of similarities to eliminate systematic errors from conventional autocorrelation function (ACF) based methods when applied to region of interest of an image, (ii) using a semi-analytical semi-numerical approach for evaluating the similarity measure to take into account spatial dependence of neighboring image pixels, (iii) and using a detrend algorithm to extract the sarcomere striation pattern content from the microscopy images.The developed mean sarcomere length estimation procedure has superior computational efficiency and estimation accuracy compared to the conventional ACF and spectral analysis based methods using Fast Fourier Transform. As shown by analyzing synthetic images with the known periodicity, the estimates obtained by the developed method are more accurate at the sub-pixel level than ones obtained using ACF analysis. When applied in practice on rat cardiomyocytes, our method was found to be robust to the choice of the region of interest that may (i) include projections of carbon fibers and nucleus, (ii) have uneven background, and (iii) be slightly disoriented with respect to average direction of sarcomere striation pattern. The developed method is implemented in open-source software, see http://iocbio.googlecode.com.

Real-time determination of sarcomere length of a single cardiomyocyte during contraction

Sarcomere length of a cardiomyocyte is an important control parameter for physiology studies on a single cell level; for instance, its accurate determination in real time is essential for performing single cardiomyocyte contraction experiments. The aim of this work is to develop an efficient and accurate method for estimating a mean sarcomere length of a contracting cardiomyocyte using microscopy images as an input. The novelty in developed method lies in 1) using unbiased measure of similarities to eliminate systematic errors from conventional autocorrelation function (ACF)-based methods when applied to region of interest of an image, 2) using a semianalytical, seminumerical approach for evaluating the similarity measure to take into account spatial dependence of neighboring image pixels, and 3) using a detrend algorithm to extract the sarcomere striation pattern content from the microscopy images. The developed sarcomere length estimation procedure has superior computational efficiency and estimation accuracy compared with the conventional ACF and spectral analysis-based methods using fast Fourier transform. As shown by analyzing synthetic images with the known periodicity, the estimates obtained by the developed method are more accurate at the subpixel level than ones obtained using ACF analysis. When applied in practice on rat cardiomyocytes, our method was found to be robust to the choice of the region of interest that may 1) include projections of carbon fibers and nucleus, 2) have uneven background, and 3) be slightly disoriented with respect to average direction of sarcomere striation pattern. The developed method is implemented in open-source software.

Postmortem titin proteolysis is influenced by sarcomere length in bovine muscle1

The calpain protease system, in particular, μ-calpain is involved in the disassembly of specific myofibrillar proteins, resulting in tenderization of meat postmortem. Given the size, complexity, and integral nature of titin to the structure of the sarcomere, it is plausible that the length of a sarcomere may alter the susceptibility of various domains of titin to cleavage by the calpains. Therefore, we hypothesized titin degradation differs in a sarcomere-length-dependent manner in beef. After slaughter, beef carcasses were split and sides were either suspended by the Achilles tendon (normal suspension, NS) or by the aitchbone (hip suspension, HS). Immediately after suspension, samples were dissected from the LM, psoas major (PM), and semitendinosus (STN) muscles to serve as 0-d controls. After 24 h, 4 steaks were removed from each muscle and randomly assigned to 1-, 4-, 7-, or 10-d aging treatments. After the assigned aging period, myofibrils were purified for determination of sarcomere length. Warner-Bratzler shear force analysis was also performed to evaluate differences in tenderness. Muscle proteins were solubilized and subjected to SDS-VAGE (vertical agarose gel electrophoresis) to evaluate titin degradation. Sarcomere lengths differed (P < 0.0001) between contralateral muscles of NS and HS carcasses. Quantification of SDS-VAGE gels revealed less (P < 0.05) intact titin in the PM muscle of NS carcasses at each aging period compared with the PM of HS carcasses. No significant differences (P > 0.05) were detected in the disappearance of intact titin among suspension methods in the LM or STN. These data demonstrate that suspension method alters proteolysis of titin and suggest an increase in sarcomere length may contribute to the susceptibility of titin to postmortem proteolysis in beef.

Espessura da lâmina de cisalhamento na avaliação instrumental da textura da carne

A padronização da textura é um dos principais problemas relacionados à qualidade da carne bovina. Por isso, a avaliação objetiva da maciez, por métodos mecânicos, vem sendo estudada há muitos anos. Para que a avaliação instrumental da textura seja uma ferramenta efetiva nos estudos envolvendo a maciez da carne, é necessário minimizar as causas de variação envolvidas na análise. Atualmente, a força de cisalhamento Warner-Bratzler é o método mais amplamente empregado para essa finalidade. O protocolo padrão dessa técnica analítica preconiza a utilização de uma lâmina de cisalhamento de 1,016mm de espessura. Neste trabalho, foram comparados dados de textura de carne bovina obtidos com a lâmina de cisalhamento padrão Warner-Bratzler, de 1,016mm de espessura, e uma lâmina mais grossa, de 3,05mm de espessura. Após o abate, amostras de músculo Longissimus dorsi, semelhantes em relação aos teores de umidade, lipídeos e proteínas, foram submetidas a duas diferentes temperaturas de resfriamento, atingindo diferentes velocidades de queda de temperatura e, consequentemente, diferentes graus de contração do tecido muscular, o que foi confirmado pela determinação do comprimento dos sarcômeros. Os resultados demonstraram uma maior sensibilidade da lâmina padrão Warner-Bratzler na detecção de variações da textura da carne, em função da velocidade de resfriamento.

Sarcomere length influences postmortem proteolysis of excised bovine semitendinosus muscle

The interaction between sarcomere length and postmortem proteolysis as related to meat tenderness is not clear. The extent of thick and thin filament overlap alters actomyosin binding and may alter substrate availability during aging-induced tenderization. The objective of this study was to determine the influence of sarcomere length on proteolytic degradation in beef. Strips from bovine semitendinosus were either stretched 40% and restrained or allowed to shorten unrestrained in an ice bath. After rigor completion, 0.6-cm cross sections were fabricated and were randomly assigned to 2, 4, 7, or 10 d of aging treatments. Myofibrils were isolated for sarcomere length determination. Samples were collected and frozen for shear force analysis, and muscle proteins were extracted for SDS-PAGE and Western blotting analyses to determine troponin T (TnT) proteolysis. Sarcomere length was greater (P < 0.01) in stretched muscle samples compared with shortened samples (2.57 vs. 1.43 microm, respectively). Correspondingly, shear force values were greater (P < 0.05) in shortened samples than stretched samples. Western blots revealed the presence of 3 major intact TnT bands that diminished with time postmortem and 4 bands (TnT degradation products) that accumulated during postmortem storage. Quantification of intact TnT showed increased (P < 0.05) proteolysis at 4 and 7 d postmortem in samples with long sarcomeres. By 10 d, only traces of the greatest molecular weight intact TnT band were evident in both shortened and stretched samples, suggesting this TnT band may be more susceptible to proteolysis than other intact TnT bands. Degradation products of TnT appeared earlier postmortem in samples with long sarcomeres. The 30-kDa TnT fragment appeared after 7 d of postmortem storage in samples with long sarcomeres but not until 10 d in muscle containing short sarcomeres. Collectively, these data show that postmortem TnT proteolysis is sarcomere length-dependent and suggest that thick and thin filament overlap may influence the postmortem aging process in beef.

Pelvic suspension and fast post-mortem chilling: Effects on technological and sensory quality of pork – A combined NMR and sensory study

The effects of pre-rigor excision, which results in a fast post-mortem chilling; pelvic suspension, which results in a muscular stretching; control treatment of m. longissimus dorsi on water distribution measured by 1H NMR relaxometry and on sensory properties were investigated in two sub-studies including a total of 16 pigs. Determination of sarcomere length revealed significant effects of the treatments on the degree of contraction, as pre-rigor excision and pelvic suspension resulted in shorter and longer sarcomere length, respectively. In addition, an effect of treatment on pH measured at 24 h post-mortem was found, as pre-rigor excision was associated with a higher ultimate pH. NMR measurements revealed that water distribution in the meat was affected to a minor degree by the various treatments. However, an interaction between treatment and ageing period was observed, as the data demonstrated an effect of pre-rigor excision on water distribution in the cooked samples when meat was only aged for 2 days, but this effect was eliminated when meat was aged for 5 days. The effect of pre-rigor excision on water distribution in cooked meat after 2 days of ageing is suggested to reflect structural constraints in the meat that are eliminated during ageing. Sensory analyses demonstrated strong interactions between treatment and position on the muscle and between treatment and ageing period. However, in general pre-rigor-excised meat was sensed as significantly juicier compared with the other two treatments. Accordingly, the study demonstrates that under the present conditions the ultimate pH is more important for juiciness than the sarcomere length.

Intake, growth and carcass characteristics of lambs consuming low digestible hay and cereal grain

The influence of barley and oat grain supplements on hay dry matter intake (DMI), carcass components gain and meat quality in lambs fed a low quality basal diet was examined. Thirty five crossbred wether lambs (9 months of age) were divided into four groups. After adaptation to a basal diet of 85% oat hay and 15% lucerne hay for one week, an initial group of 11 was slaughtered. The weights of carcass components and digesta-free empty body weight (EBW) of this group was used to estimate the weight of carcass components of the other three experimental groups at the start of the experiment. The remaining three groups were randomly assigned to pens and fed ad libitum the basal diet alone (basal), basal with 300 g air dry barley grain (barley), basal with 300 g air dry oat grain (oat). Supplements were fed twice weekly (i.e., 900 g on Tuesday and 1200 g on Friday). After 13 weeks of feeding, animals were slaughtered and, at 24 h post-mortem meat quality and subcutaneous fat colour were measured. Samples of longissimus muscle were collected for determination of sarcomere length and meat tenderness. Hay DMI was reduced ( P<0.01) by both barley and oat supplements. Lambs fed barley or oat had a higher and moderate digestibility of DM, and a higher intake of CP ( P<0.05) and ME ( P<0.01) than basal lambs. Final live weight of barley and oat lambs was higher ( P<0.05) than basal, but this was not reflected in EBW or hot carcass weight. Lambs fed barley or oat had increases in protein ( P<0.01) and water ( P<0.001) in the carcass, but fat gain was not changed ( P>0.05). There were no differences in eye muscle area or fat depth (total muscle and adipose tissue depth at 12th rib, 110 mm from midline; GR) among groups. The increased levels of protein and water components in the carcass of barley and oat fed lambs, associated with improved muscle production, were small and did not alter ( P>0.05) any of the carcass/meat quality attributes compared to lambs fed a low quality forage diet. Feeding barley or oat grain at 0.9–1% of live weight daily to lambs consuming poor quality hay may not substantially improve carcass quality, but may be useful in maintaining body condition of lambs through the dry season for slaughter out of season.

Prediction of Poultry Meat Tenderness Using Razor Blade Shear, Allo‐Kramer Shear, and Sarcomere Length

ABSTRACT: This study was conducted to evaluate a new shearing method for the determination of poultry meat tenderness. Breast fillets were deboned at various postmortem times (0.25 to 24 h) to yield a vast array of tenderness levels. A trained descriptive panel was used to evaluate samples for attributes including initial hardness and chewdown hardness; instrumental measurements included Allo‐Kramer (AK) and razor blade (RB) shear and laser sarcomere length determination. The RB shear method exhibited a higher correlation to sensory attributes than the AK method, suggesting that the new razor blade shear method is more advantageous in predicting poultry meat tenderness than the standard AK shear method. This new method not only has a higher sensory predictive value, but also requires shorter sample preparation time than the AK shear test because it is conducted on intact fillets.

Proteolysis and tenderisation in reindeer ( Rangifer tarandus tarandus L.) bull longissimus thoracis muscle of varying ultimate pH

Proteolysis and tenderisation in reindeer ( Rangifer tarandus tarandus L.) M. Longissimus Thoracis were studied. Mm. Longissimus ( the part cut out between vertebrae thoracales 6–7 and vertebrae lumbales 5–6) from 12 reindeer bulls (age 1 1 2 years) were, after ultimate pH and temperature measurements, excised and then sampled at various times post mortem for determination of sarcomere length, Warner-Bratzler shear force, calpain and calpastatin activities, cathepsins B + L activities, active site titration of cystatin-like inhibitors, myofibrillar protein degradation, collagen content and heat solubility. Upon measurement of ultimate pH, the carcasses were divided into two pH groups; normal pH (5.65 ≤ pH ≤ 5.79) and high pH (pH ≥ 5.80). Temperature and pH fall were relatively rapid in all reindeer carcasses. Sarcomere lengths tended to be shorter in the carcasses of the high pH group. In the three carcasses with the highest ultimate pH values (6.11, 6.34 and 6.38), sarcomere lengths were around or below 40% of resting length (1.37μm, 1.25μm and 1.25μm, respectively) which is likely associated with the occurrence of heat shortening. Total collagen content was higher and heat solubility lower in the high pH group, which could have masked differences in tenderness. However, all reindeer longissimus muscle samples were found to be extremely tender regardless of ultimate pH. By 3 days post mortem Warner-Bratzler shear force values were varying between 2.1–4.9 kg cm 2. There was a significantly higher activity of μ-calpain in the high pH group at 1 day post mortem. No differences in shear force, myofibrillar protein degradation as observed by SDS-PAGE, m-calpain and calpastatin activities, calhepsins B+ L activities or the levels of cystatin-like inhibitors were found between the two pH groups.

Beef tenderness and sarcomere length

A wide range of muscle glycolytic rates was produced in 60 beef carcasses by applying different forms and periods of electrical stimulation immediately after decapitation; a further seven carcasses were not stimulated. The sides were subjected to normal chilling, and at 48 h one short loin per carcass was taken for tenderness evaluation and determination of sarcomere length (SL). In the 19 relatively slow-glycolysing loins—those of 3-h pH (pH 3) above 6·3—tenderness extended over almost five panel units on a 1–8 scale, and included both the tenderest two and the toughest 10 muscles of the entire study. In the 48 loins of pH 3 below 6·3, on the other hand, tenderness ranged over only 2·5 units, and on average was a full panel unit higher than that of the high-pH 3 muscles. The most striking difference between the two groups, however, was in the relationship between toughness and shortening; the correlation of panel tenderness on SL was remarkably high in the slow glycolysers ( r = 0·84), but negligible in those of faster pH decline ( r = 0·16). Thus although shortening occurred to about the same extent and over the same range in both groups, it influenced tenderness and tenderness variability only when glycolysis was quite slow. A loin pH 3 of below 6·3, however, is unusual in non-stimulated carcasses, so slow glycolysis and the very wide tenderness diversity accompanying it must be expected in commercial operations that do not use electrical stimulation.

Computerized diffractometer for measurements of sarcomere lengths in meat: An evaluation of data reduction methods

A computerized laser diffractometer was evaluated on the basis of two different theoretical models for light diffraction in muscle tissue. It was shown that the method of computing sarcomere lengths from the positions of the left and right first order diffraction lines is model dependent. Sarcomere lengths obtained by the diffractometer were compared to microscopically determined sarcomere lengths on freeze-sectioned M. sternomandibularis from a material consisting of muscle strips held at different degrees of stretch. Treating left and right diffraction lines as independent observations led to the determination of sarcomere lengths with negligible deterministic error. The possibility of utilizing the fine structure and intensity of the diffraction patterns to improve the sarcomere length determination and provide more relevant information is discussed.

A scanning light diffractometer for muscle studies.

A light diffractometer with a cosecant drive is described. It can provide the intensity spectrum of the fine structures within the first-order laser light diffraction from striated muscle. In addition, the sarcomere length associated with each fine structure can be obtained. The accuracy of the sarcomere length determination is discussed.

Sarcomere length determination using laser diffraction. Effect of beam and fiber diameter

An experimental and theoretical analysis is presented involving the effect of variation in fiber and beam diameter upon the determination of average sarcomere length in isolated single muscle fibers using laser light diffraction. The muscle diffraction phenomenon is simplified by first considering diffraction order position and intensity to be the result of grating and Bragg diffraction. It is the product of the intensity profiles, which results from these types of diffraction, that produces the diffracted order. These simplifying assumptions are then extended to the case of the real muscle. Based on these considerations and the theory that we recently presented, conditions are set forth under which grating information (i.e., sarcomere length) can be maximally expressed to yield accurate average sarcomere length values.

Individual sarcomere length determination from isolated cardiac cells using high-resolution optical microscopy and digital image processing

Discrete sarcomere lengths have been determined from dynamically contracting isolated cardiac cells with a high-speed, high-resolution direct optical imaging system. Calcium-tolerant cardiac cells from the rat are isolated by perfusion with collagenase and hyaluronidase. Individual sarcomere lengths can be determined by directly imaging the cell's striation pattern onto a solid-state charge-coupled device (CCD) detector interfaced with a digital computer. The precision of detection in a real light microscopic optical system is discussed in relation to the type of image detector, optical contract enhancement techniques, and digital image processing. The optical performance of the direct striation pattern image apparatus has been determined empirically with test grids under standard bright-field and Nomarski-differential interference contrast (DIC) conditions for application to real muscle imaging. Discrete striation positions of isolated cells have been detected and followed with high precision during phasic contraction-relaxation cycles down to average sarcomere lengths as short as 1.43 +/- 0.053 microns. The maximum rates of contraction and relaxation are rapid and synchronous in time course along the length of the cell. These results indicate that direct optical imaging can provide an accurate means to monitor discrete striations and sarcomere lengths along the length of Ca2+-tolerant heart cells.

STRUCTURAL CHANGES IN ELECTRICALLY STIMULATED BEEF MUSCLE

ABSTRACTThe right sides of five steers were electrically stimulated with 50 impulses of 0.5‐1.0 set duration, 100 volts (machine setting), 5 amps, 50‐60 cycles per set, producing an approximate 440 volt potential difference between electrodes; the left sides were used as untreated controls. Longissimus muscle samples were removed 20‐24 hr postmortem from both sides of each carcass for palatability, cookery and sarcomere length determination as well as for light and electron microscopy. Steaks from electrically stimulated sides were less juicy, more tender and more flavorful, had less organoleptically detectable connective tissue and lower shear force values, and sustained greater cooking losses than did samples from control sides. Mean sarcomere length did not differ significantly between control and electrically stimulated samples. Light micrographs of electrically stimulated samples revealed contracture bands throughout the myofibers along with stretched areas on either side of the contracture bands. Electron micrographs also showed contracture bands along with physical disruption of the myofibrils on either side of the bands. These data suggest that electrical stimulation (performed in the manner described here) may improve tenderness by physical disruption and the formation of contracture bands and not by prevention of cold shortening.

RESPONSE TO ELECTRICAL STIMULATION AND POST‐MORTEM CHANGES IN TURKEY PECTORALIS MAJOR MUSCLE

SUMMARY– Muscle samples were obtained from 20 turkey hens 5 min postexsanguination for the determination of sarcomere length, response to electrical stimulation, and time‐course of rigor mortis. Additional samples were obtained from the opposite (undisturbed) pectoralis major muscle at 1, 2, 6 and 24 hr post‐mortem for the determination of sarcomere length. Birds were divided into three groups based on the time to complete rigor mortis: (1) slow, &gt; 125 min; (2) fast, &lt;50 min; and (3) intermediate, 50–125 min. Birds that displayed a long time course of rigor mortis had a low threshold of response (9v) and a long duration of response (106 seel to electrical stimulation. Conversely, muscles which become inextensible within 50 min displayed a high threshold (123v) and low duration (8 sec). Muscles with intermediate rates of rigor had intermediate threshold (58v) and duration (50 sec). Increase in sarcomere length occurred within 1 hr post‐mortem in the fast‐rigor group, 2 hr in the intermediate‐rigor group and 6 hr in slow‐rigor birds. A similar experiment conducted on 24 toms indicated that postexsanguination struggling resulted in a decreased time course of rigor, elevated threshold and shortened duration of response to electrical stimulation. Strugglers displayed a more rapid (P&lt;.01) post‐mortem increase in sarcomere length than non‐strugglers. The results suggested that in some presently‐used commercial turkey processes, some of the carcasses are frozen with muscles in a prerigor, short‐sarcomere state.