Decreased Sarcomere Length Research Articles

Myosin S2 stabilizer peptide decreases recovery rates of contraction.

A peptide, called the Stabilizer, designed to increase the energy required to unzip the myosin S2 coiled coil was tested on myofibrils, skinned trabeculae, and live cardiomyocytes. The Stabilizer bound to myosin S2 with submicromolar affinity and positive cooperativity measured by both TIRF and MST, which suggests a modification in the myosin S2 structure. Myofibril ATPase activity was activated at pCa 9 and inhibited at pCa 4.5, consistent with an inhibition of actin-activated ATPase activity. In vitro motility assays of actin sliding velocity were partially depressed in the presence of the Stabilizer. Both force development rates and regulatory light chain orientation (measured by fluorescence polarization) were also slowed by the Stabilizer following a slack-re-stretch maneuver in skinned trabeculae. To test perturbations of living cardiomyocytes, the Stabilizer peptide was chemically esterified with a cell penetrating molecule to facilitate translocation across the cell membrane and removal of the targeting molecule once inside the cell. Confocal microscopy imaging showed sarcomere labeling with fluorescently tagged targeted Stabilzer in live cardiomyocytes. The Stabilizer decreased sarcomere length shortening in living cardiomyocytes. These data are consistent with a reduced contractility by the Stabilizer, which may result from the reinforcement of the myosin S2 coiled coil structure mitigating myosin's flexibility and probability of cross-bridge formation. (Supported by NIH/NHLBI R01HL149164.)

Effect of postmortem pH on the physical, biochemical, and sensory characteristics of bovine longissimus thoracis et lumborum muscle.

From a large feeding trial study consisting of 299 bulls and steers, 15 carcasses exhibited stress-related syndromes manifested by atypical color and pH which were then selected for subsequent analysis. Samples of longissimus thoracis et lumborum muscle with postmortem pH in the range of 5.5-6.9 were subjected to a 14-day aging period at 2°C. Sensory panel tenderness, connective tissue, juiciness, and flavor intensity of high pH (6.4-6.9) meat were significantly different (p < .05) from samples of intermediate pH(6.0-6.1) as well as normal pH (5.5). Muscles at pH 6.0-6.1 were the toughest samples. This was confirmed by Warner-Bratzler shear force (WBSF), residual force, and myofibril fragmentation index. Palatability attributes of normal pH (5.5) samples were significantly different (p < .05) from dark-cutting beef in terms of tenderness and flavor and at the high pH extreme. The increase in WBSF at pH 6.0-6.1, lack of extensive degradation of muscle proteins, and the decreased sarcomere length resulted in tougher meat than low or high pH muscles. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis of meat at the high pH extreme (6.7-6.9) revealed that the breakdown of troponin-T to 30 kD was complete while at intermediate pH (6.0-6.1) was incomplete. In addition, the appearance of a 'doublet' on high-molecular-weight resolution gels may also account for the greater tenderness experienced by sensory panelists.

Insights From Computational Modeling Into the Contribution of Mechano-Calcium Feedback on the Cardiac End-Systolic Force-Length Relationship.

In experimental studies on cardiac tissue, the end-systolic force-length relation (ESFLR) has been shown to depend on the mode of contraction: isometric or isotonic. The isometric ESFLR is derived from isometric contractions spanning a range of muscle lengths while the isotonic ESFLR is derived from shortening contractions across a range of afterloads. The ESFLR of isotonic contractions consistently lies below its isometric counterpart. Despite the passing of over a hundred years since the first insight by Otto Frank, the mechanism(s) underlying this protocol-dependent difference in the ESFLR remain incompletely explained. Here, we investigate the role of mechano-calcium feedback in accounting for the difference between these two ESFLRs. Previous studies have compared the dynamics of isotonic contractions to those of a single isometric contraction at a length that produces maximum force, without considering isometric contractions at shorter muscle lengths. We used a mathematical model of cardiac excitation-contraction to simulate isometric and force-length work-loop contractions (the latter being the 1D equivalent of the whole-heart pressure-volume loop), and compared Ca2+ transients produced under equivalent force conditions. We found that the duration of the simulated Ca2+ transient increases with decreasing sarcomere length for isometric contractions, and increases with decreasing afterload for work-loop contractions. At any given force, the Ca2+ transient for an isometric contraction is wider than that during a work-loop contraction. By driving simulated work-loops with wider Ca2+ transients generated from isometric contractions, we show that the duration of muscle shortening was prolonged, thereby shifting the work-loop ESFLR toward the isometric ESFLR. These observations are explained by an increase in the rate of binding of Ca2+ to troponin-C with increasing force. However, the leftward shift of the work-loop ESFLR does not superimpose on the isometric ESFLR, leading us to conclude that while mechano-calcium feedback does indeed contribute to the difference between the two ESFLRs, it does not completely account for it.

Novel findings on ultrastructural protection of skeletal muscle fibers during hibernation of Daurian ground squirrels: Mitochondria, nuclei, cytoskeleton, glycogen

We examined ultrastructure protective phenomena and mechanisms of slow and fast muscles in hibernating Daurian ground squirrels (Spermophilus dauricus). Some degenerative changes such as slightly decreased sarcomere length and vacuolization occurred in hibernation, but periaxonal capsular borders in intrafusal fibers remained distinct and the arrangement of extrafusal fibers and Z-lines unscathed. In soleus samples, the number of glycogenosomes more than tripled during hibernation. The expression of phosphorylated glycogen synthase remained unaltered while that of glycogen phosphorylase decreased during hibernation. The number of extensor digitorum longus glycogenosomes decreased and the expression of phosphorylated glycogen synthase decreased, while glycogen phosphorylase expression remained unaltered. The nuclei number remained unchanged. Kinesin and desmin, preventors of nuclear loss and damage, were maintained or just slightly reduced in hibernation. The single-fiber mitochondrial concentration and sub-sarcolemmal mitochondrial number increased in both muscle types. The expression of vimentin, which anchors mitochondria and maintains Z-line integrity, was increased during and after hibernation. Also, dynamin-related protein 1, mitochondrial fission factor, and adenosine triphosphate synthase were elevated in both muscle types. These findings confirm a remarkable ultrastructure preservation and show an unexpected increase in mitochondrial capacity in hibernating squirrels.

Novel Roles for Catestatin in Cardiac Metabolism and Physiology

More than 50 million people suffer from cardiometabolic syndrome in the US. Catestatin (CST), a peptide derived form chromogranin A, acts as an antihypertensive, cardioprotective, pro‐angiogenic, and insulin‐sensitizing peptide. The role of CST in cardioprotection and substrate utilization under normal and stressed conditions is not well understood. We hypothesized that CST is a key regulator of cardiac metabolism and stress adaptation. We investigated wild‐type (WT) and CST‐KO mice to evaluate cardiac function and metabolism. CST‐KO mice display hypertension, are hyperadrenergic, insulin resistant and show increased fasting plasma insulin and leptin. Fatty acid uptake and oxidation was higher in hearts of CST‐KO mice. Ultrastructural studies revealed more glycogen deposition, decreased sarcomere length, and altered mitochondria in CST‐KO mice. We have seen that in the hearts of CST‐KO mice, pAMPK as well as insulin‐stimulated phosphorylation of Akt, and GSK‐3β were decreased. High fat diet (HFD) did not uncover differences in glycogen synthesis, but less glycogen was stored in CST‐KO‐HFD hearts. Comparison of injuries after ischemia reperfusion and ischemic preconditioning showed that protection of hearts from CST‐KO mice was not possible. The same holds true after HFD with worse recovery for CST KO mice. Our studies characterize the crucial role of CST in cardiac physiology and metabolism and suggest CST as a therapeutic target for modulating cardiometabolic disease.

Impacts of hanging method and high pre-rigor temperature and duration on quality attributes of ovine muscles

This study evaluated effects of high pre-rigor temperature and duration and suspension of lamb sides on quality traits and protein denaturation in two muscles [semimembranosus (SM) and longissimus thoracis et lumborum (LTL)]. Twenty-four lamb carcasses, within each of 3 slaughter days, were used to assign eight carcasses to one of four pre-rigor temperature treatments: chilled at 2°C directly after slaughter, or held at 37°C in water for 1.5, 3.0 or 4.5 h before transfer to a 2°C chiller. At ~15 min post slaughter, one side of each carcass was suspended from the Achilles tendon, whereas the other side was suspended by the aitch bone and the leg tied down to the ribs. The sides subjected to aitch bone hanging had an increased sarcomere length in the SM, but decreased sarcomere length in the LTL. For the LTL, the time of exposure to high pre-rigor temperature had a significant effect on measures of protein denaturation and related meat quality traits such as purge and colour, although tenderness (shear force) after 2 days of aging was not affected. For the SM, the high temperature treatment also resulted in increase in measures of protein denaturation and thus negatively influenced meat quality traits such as purge, colour and shear force after aging. However, these effects on purge and shear force in the SM were significantly mitigated by the aitchbone hanging treatment. The results of the present experiment indicate that pre-rigor aitchbone hanging of muscles can counteract the negative effects of high pre-rigor temperature on both water loss and meat tenderness.

Nogo-A Modulates Calcium Handling and Contractility in Cardiomyocytes

Key features in the progression from left ventricular hypertrophy to heart failure include the dysregulation of cardiomyocyte Ca2+ handling and the impairment of contractile function. Knockdown of Nogo-A has previously been shown to inhibit hypoxia/reoxygenation-induced apoptosis in cardiomyocytes in part by preventing impairment of intracellular Ca2+ regulation. In this study, we determined whether altering Nogo-A expression can affect cardiomyocyte Ca2+ handling and contractility in non-stressed cardiomyocytes. Compared to control, knockdown of Nogo-A in neonatal rat ventricular myocytes (NRVMs) significantly increased the amplitude of [Ca2+]i transients, increased SR load, and decreased the time constant of [Ca2+]i decay (tau) evoked by electrical stimulation. Diastolic [Ca2+]i, time to peak, and [Ca2+]i decay of caffeine-induced Ca2+ release from the SR were not affected by Nogo-A knockdown. Conversely, over-expression of Nogo-A in adult rabbit cardiomyocytes significantly decreased the amplitude of [Ca2+]i transients, decreased SR load, and increased the time constant of [Ca2+]i decay. Diastolic [Ca2+]i, time to peak, and [Ca2+]i decay of caffeine-induced Ca2+ release from the SR were not affected by Nogo-A overexpression. In addition to the impairment of Ca2+ handling, Nogo-A over-expression significantly decreased sarcomere length (SL) shortening, increased the time to 90% relaxation (TR90), and decreased relaxation velocity. Contraction velocity was not affected by Nogo-A over-expression. Overall, alteration of Nogo-A levels affects cardiomyocyte Ca2+ handling and contractility, primarily by modulating Ca2+ reuptake into the SR. Together, these data indicate that Nogo-A may serve as a potential therapeutic target in the treatment or prevention of heart failure.

Ultrafine particles increase the production of reactive oxygen species (ROS) and decrease contractile function in rat adult ventricular myocytes (ALVM)

Increases in the concentration of airborne fine particles (&lt; 0.1 µm, UFP) are positively correlated with increased cardiovascular mortality. UFP produced by the combustion of fossil fuels and wastes contain stable free radicals capable of redox cycling and producing oxidative stress. After inhalation, UFP transit from the lung to the heart raising the possibility that UFP produces oxidative stress in the heart by a direct action. This study determined whether radical‐containing UFP produce oxidative stress and contractile deficits in ALVM. Rat ALVM were treated with SiCuO UFP containing dichlorobenzene (DCB230, 8.8 and 17.6 µg/cm2) for 24 hr. DCB230 increased ROS production (measured by dihydroethidium staining) in ALVM by 50% relative to control. The higher dose killed all the cells. An IonOPtix system was used to assess contractile function in treated and control ALVM. Compared to control, DCB230‐treated ALVM showed decreased sarcomere length shortening percent and decreased the maximal velocities of shortening and re‐lengthening during stimulation (1Hz). In conclusion, treatment with UFP directly increased ROS production and decreased contractile function in ALVM. Support RR018766

BIOCHEMICAL AND PHYSICOCHEMICAL CHANGES IN GOAT MEAT DURING POSTMORTEM AGING

ABSTRACT This study was conducted to evaluate the effect of postmortem aging on pH, temperature, drip loss, sarcomere length, myofibrillar fragmentation index (MFI), myofibrillar protein solubility, released calcium and myofibrillar adenosinetriphosphatase (ATPase) activity in longissimus dorsi (LD), biceps femoris (BF), semimembranosus (SM) and semitendinosus (ST) muscles of goat maintained at 5C up to 20 days postmortem. The results showed that the rates of pH decline, temperature and sarcomere length decreased during aging. There was a significant increase in drip loss of LD, BF, SM and ST at 3, 6 and 9 days, respectively. The increased MFI followed a pattern of LD &gt; ST &gt; BF &gt; SM at 20 days postmortem. The myofibrillar ATPase activity rapidly decreased between 0.1 and 0.2 M ionic strength in all the muscles. The biochemical index of myofibrillar aging (BIMA) value of LD, BF and SM muscles was constant after 9 days postmortem. The results obtained here describe a close relationship between rate of pH decline, decreased sarcomere length and increased MFI and BIMA value during postmortem aging. Therefore, the biochemical indices described in this article could be used as indicators of the degree of aging of the goat myofibrillar structure.

Induced Deletion of the N-Cadherin Gene in the Heart Leads to Dissolution of the Intercalated Disc Structure

The structural integrity of the heart is maintained by the end-to-end connection between the myocytes called the intercalated disc. The intercalated disc contains different junctional complexes that enable the myocardium to function as a syncytium. One of the junctional complexes, the zonula adherens or adherens junction, consists of the cell adhesion molecule, N-cadherin, which mediates strong homophilic cell-cell adhesion via linkage to the actin cytoskeleton. To determine the function of N-cadherin in the working myocardium, we generated a conditional knockout containing loxP sites flanking exon 1 of the N-cadherin (Cdh2) gene. Using a cardiac-specific tamoxifen-inducible Cre transgene, N-cadherin was deleted in the adult myocardium. Loss of N-cadherin resulted in disassembly of the intercalated disc structure, including adherens junctions and desmosomes. The mutant mice exhibited modest dilated cardiomyopathy and impaired cardiac function, with most animals dying within two months after tamoxifen administration. Decreased sarcomere length and increased Z-line thickness were observed in the mutant hearts consistent with loss of muscle tension because N-cadherin was no longer available to anchor myofibrils at the plasma membrane. Ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous ventricular tachycardia, confirming that the deaths were arrhythmic in nature. A significant decrease in the gap junction protein, connexin 43, was observed in the N-cadherin-depleted hearts. This animal model provides the first demonstration of the hierarchical relationship of the structural components of the intercalated disc in the working myocardium, thus establishing N-cadherin's paramount importance in maintaining the structural integrity of the heart.

The Cardiac Troponin C Isoform and the Length Dependence of Ca2+Sensitivity of Tension in Myocardium

The Ca2+sensitivity of tension in cardiac muscle is length dependent, such that the sensitivity is diminished with decreasing sarcomere length below 2.4μm. This length dependence of Ca2+sensitivity of tension also forms the basis for the Frank–Starling mechanism in the heart. The fast-twitch skeletal muscle has a much lower length dependence of Ca2+sensitivity. In a recent study of skinned cardiotrabeculae, we indicated that the exchange of endogenous cardiac troponin C (TnC) for skeletal troponin C also resulted in a major reduction in the length dependence to the level of skeletal muscle. These findings suggested that cardiac troponin C has a key role in the length-sensing mechanism. The present investigation supports this conclusion and delineates the specific domain in cardiac TnC responsible for the length effect. Chimeras splicing either 41, 61, or 96 N-terminal cardiac amino acids with the remaining skeletal residues have indicated that while Ca2+binding in all three constructs is similar to that in wild type cardiac TnC, the functional responsiveness of the 96-cardiac residue construct is improved over the other two. This 96-cardiac residue construct yielded a tension response indistinguishable from that of wild-type cardiac TnC. A tryptophan variant of the chimera indicated fluorescence characteristics indistinguishable from cardiac troponin C. The findings provide further support for the idea that cardiac troponin Cin situis modified in response to sarcomere length change and thereby participates in the Frank–Starling mechanism. Moreover, the study indicates that the tropinin C length-sensing attribute originates within the N-terminal domain constituted by these 96 residues.

Influence of CaCl2 and NaCl injections on the texture and flavour of beef

The present study compares the effects of pre− and post‐rigor injections of 0.1m CaCl2 or 0.15m NaCl on sensory characteristics (appearance, texture and flavour) and on physical measurements (sarcomere length, drip loss and mechanical resistance of raw myofibres) of beef meat at 2, 6 and 14 days of ageing. An injection of CaCl2 1 h post‐mortem (pH ≈6.8) greatly decreased sarcomere length and myofibrillar resistance, increased drip loss, contracted appearance and brightness, and induced abnormal flavour, mainly bitterness, whatever the ageing time. CaCl2‐treatment 1 h post‐mortem produced little effect on tenderness during ageing; it was slightly higher at day 2 and lower at day 14 than controls. CaCl2‐injection at 24 h post‐mortem did not change the sarcomere length but produced a smaller increase in drip loss and a decrease in the resistance of raw myofibres. Improved meat tenderness compared to controls occurred mainly at days 2 and 6 but, during ageing, abnormal flavour appeared by day 6. NaCl‐injection had no effect on either sarcomere length or resistance of raw myofibres, but produced higher drip loss and saltier taste at day 14 only. Thus pre‐rigor injection of CaCl2 is not recommended because of adverse effects on flavour and appearance of meat. Post‐rigor injection of CaCl2 has a beneficial effect on tenderness in the first part of the ageing period, but after complete ageing, the only overall benefit is a decrease in the variability between animals.

An assessment of the role of pH differences in determining the relative tenderness of meat from bulls and steers

Samples of M. longissimus dorsi from 16–20 month Friesian bulls or steers (80/group) were assessed for a range of meat quality characteristics after being held at ambient temperature for 24h (to avoid cold-shortening) and then at 0–2°C for 6 days. Mean ultimate pH was significantly higher for samples from the bulls (6·35 versus 5·89), and as a consequence, reflectance values were lower, sarcomere lengths were shorter, and expressed juice and cooking losses were lower. Mean Warner-Bratzler shear values did not differ between the groups, apparently because the bull values were mainly above the peak of the pH/shear force curve, while the steer values were mainly below it. There was no evidence that the relationship between shear force and pH differed for samples from the bulls and steers. Results suggested that the increased shear force with increased pH up to 6·2 was at least partly due to a decreased sarcomere length. It is suggested that some of the differences in tenderness between beef from bulls and steers, that have been reported elsewhere, may have been largely due to differences in ultimate pH values.

Effect of Post-Mortem Electrical Stimulation on Color, Texture, pH, and Cooking Losses of Hot and Cold Deboned Chicken Broiler Breast Meat

Chicken broilers were electrically stunned prior to bleeding and slaughter in the processing plant at the Spelderholt Center. A nonstimulated (NS) control was used. Post-mortem intermittent electrical stimulation (ES) for 60 s was applied using 100 V immediately after bleeding. Birds were deboned from one breast half at various times (0 to 240 min) after evisceration. The remaining half was left attached to the carcass.Breast muscle pH was significantly lowered by ES compared with pH of control birds. There was an electrical stimulation × time interaction when measuring Warner Bratzler maximum shear, slope, and work values. Electrical stimulation at 0 or 15 min actually toughened the breast muscle, but ES significantly tenderized breast muscle deboned at 120 and 240 min, compared with the control. Breast muscles from ES carcasses were significantly darker in color, with lower lightness (L*) values when hot deboned at 60 min or earlier. Post-mortem ES significantly increased redness (a*) values at all deboning times except 30 min. Cooking losses and expressible moisture were significantly increased by ES. Hot deboning significantly decreased sarcomere length, but ES had no significant effect on sarcomere length.

Capillary configuration and fiber shortening in muscles of the rat hindlimb: Correlation between corrosion casts and stereological measurements

It has been a matter of discussion whether the dramatic increase in capillary tortuosity visualized in shortened muscles by vascular cast represents in vivo situations. In this study, we combined vascular cast and stereological methods in the same samples, in order to obtain (1) measurements of sarcomere length in the same muscles from which corrosion casts were prepared, and (2) scanning electron micrographs of the three-dimensional arrangement of capillaries in the same muscles where capillary anisotropy was estimated by morphometry. Various rat skeletal muscles (soleus, gastrocnemius, and gracilis) were examined at lengths ranging from full shortening to full extension. We found a very good correlation between capillary geometry in material prepared for vascular casts and in muscles perfusion-fixed in situ. All muscles, cast and noncast, showed the same progressive curvilinear decrease in capillary anisotropy with decreasing sarcomere length. Capillary tortuosity visualized by corrosion casts in shortened muscles is a consequence of fiber shortening, within physiological sarcomere lengths; it does not represent an artifact related to the casting procedure.

Diaphragm in emphysematous hamsters: sarcomere adaptability.

We investigated whether the shift in the diaphragmatic length-tension curve of emphysematous animals was due to changes in either sarcomere number or sarcomere length. In vitro length-tension characteristics of the diaphragm were evaluated in control and emphysematous hamsters. Emphysema was induced by a single endotracheal instillation of elastase. Functional residual capacity, measured by means of a plethysmograph, was about twice that measured in emphysematous animals compared with control animals. Small diaphragmatic bundles were isolated and evaluated for their length-tension characteristics. The length-tension curve of diaphragms from emphysematous animals was displaced toward the left. Maximal tetanic tensions were similar in both groups, whereas optimal bundle length was significantly decreased in the emphysematous animals. Sarcomere number and sarcomere length at optimal length were then calculated for the diaphragmatic bundles. The bundles from emphysematous animals were found to have a significantly reduced number of sarcomeres, as well as a significantly decreased sarcomere length at optimal compared with control values. The total number of sarcomeres were also found to be significantly negatively correlated with the animal's functional residual capacity. From these results, we conclude that the decrease in diaphragmatic length, seen with hyperinflation, is due mainly to a loss of sarcomeres and possibly to a small decrease in sarcomere length measured at optimal bundle length.

Effect of myosin on conformational changes of F-actin in thin filament in vivo induced by calcium ions.

The measurements of ultraviolet linear dichroism has been carried out on glycerinated muscle fibers of rabbit psoas and crab leg having various sarcomere lengths and the effect of interaction of the thin filament with myosin on the dichroism has been investigated under various conditions. The dichroism spectrum of the muscle fiber has a large positive peak at 285 nm (smaller absorption of light polarized perpendicular to the fiber axis) and a small negative peak at 297.5 nm. The negative peak at 297.5 nm, which comes from the dichroism of F-actin in the thin filament, became smaller in the presence of Ca2+ than in its absence. The positive peak at 285 nm, where the thick filament has main contribution, showed no appreciable change by Ca2+. The dichroism at 292.5 nm, where the thin filament has no contribution, showed no change either. Thus, the decrease of negative dichroism at 297.5 nm by Ca2+ indicates a conformational change in F-actin in the thin filament. With decreasing sarcomere length or increasing overlapping between thin and thick filaments, the dichroism decrease at 297.5 nm by Ca2+ increased, and attained a maximum at maximum overlapping. That is the conformational change of F-actin by Ca2+ was amplified by interaction with myosin. With further decrease of the sarcomere length, the dichroism decrease by Ca2+ decreased. In the absence of Ca2+, the addition of ATP or pyrophosphate induced a large change of transmittance and transmittance anisotropy, and also a large increase of birefringence at long wavelengths.

RIGOR‐STRETCHED TURKEY MUSCLES: EFFECT OF HEAT ON FIBER DIMENSIONS AND SHEAR VALUES

Fiber dimensions and shear force values were measured in folded, prerigor and rigor stretched semi‐tendinosus and sartorius muscles from unaged adult turkey carcasses. The unheated rigor muscles can be extended in situ but the extent is significantly less than prerigor stretching. Extensibility was detected by increases in sarcomere length. Prerigor and rigor stretching caused significantly smaller fiber diameters. Heat (70°C and 82°C) decreased sarcomere length of stretched muscles, but produced no significant change in folded controls. The stretched muscles had either significantly higher shear force values (semitendinosus) or were not different (sartorius) from the folded controls. All treatments gave reduced fiber diameters on heating. The relevance of this observation to meat tenderness is discussed. Light microscope evidence showed little damage in the rigor‐stretched or heated fibers.