Short Sarcomere Lengths Research Articles

Fast myosin binding protein C knockout in skeletal muscle alters length-dependent activation and myofilament structure.

In striated muscle, the sarcomeric protein myosin-binding protein-C (MyBP-C) is bound to the myosin thick filament and is predicted to stabilize myosin heads in a docked position against the thick filament, which limits crossbridge formation. Here, we use the homozygous Mybpc2 knockout (C2-/-) mouse line to remove the fast-isoform MyBP-C from fast skeletal muscle and then conduct mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers present deficits in force production and calcium sensitivity. Structurally, passive C2-/- fibers present altered sarcomere length-independent and -dependent regulation of myosin head conformations, with a shift of myosin heads towards actin. At shorter sarcomere lengths, the thin filament is axially extended in C2-/-, which we hypothesize is due to increased numbers of low-level crossbridges. These findings provide testable mechanisms to explain the etiology of debilitating diseases associated with MyBP-C.

Meat Characteristics, Expression of Myosin Heavy Chain and Metabolism-Related Genes in Thai Native Pigs.

This study investigated the meat quality, expression of myosin heavy chain (MyHC) and metabolism-related genes, ribonucleotides and fatty acids in Longissimus thoracis of Thai native pigs (TNPs) from different geographical regions (GR). Forty-one 9-10-month-old castrated TNPs (BW 60 kg), consisting of 18, 11 and 12 pigs from Northern (NT), Southern (ST) and Northeastern (NE) regions, respectively, were slaughtered. GR did not affect (p > 0.05) the expression of MyHC, phosphoglycerate mutase 1, cytosolic glycerol-3-phosphate dehydrogenase, triosephosphate isomerase 1 and adipocyte fatty acid binding protein genes. The trend of MyHC was MyHC IIx > MyHC IIb > MyHC IIa > MyHC I. The NT loin had higher (p < 0.05) glycogen, C18:2n6, C20:4n6 and cooking loss, lower inosine, inosine monophosphate and hypoxanthine and a shorter sarcomere length than the ST and NE loins. The ST loin had a lower (p < 0.05) a* compared to other loins. Principal component analysis established significant relationships between the TNP and specific meat quality traits. This finding suggests that GR affected the meat quality, ribonucleotides and selected fatty acids in TNPs. These results provide relevant information that can be used to optimize the use of Thai native pork.

Measuring sarcomere dynamics following immunofluorescent labelling of constitutive structural and contractile proteins.

Myofibrils are subcellular organelles made of sarcomeres. Characterizing the interaction between constitutive proteins may rationalize mechanisms of muscle contraction. Labelling of titin with immunofluorescent antibodies can reveal segmental elongation of domains while subverting shortcomings including inconsistent intensities and asymmetricity of striations (A-band, I-band) inherent to bright field or phase contrast microscopy methods. Considering antibody labelling has been associated with impairments of sarcomere dynamics, we investigated the effect of labelling α-actinin (Z-disc), myomesin (M-line), and I-band titin epitopes to determine how specific antibodies and solution condition parameters might compromise passive force and sarcomere lengths (SLs). Myofibrils were harvested and purified from psoas of New Zealand White rabbits. Labelled samples were incubated with anti-α-actinin, anti-myomesin, and AlexaFluor488 antibodies. Samples were flushed with relaxing solution, restrained in needle-nanolever systems, and observed under 100x magnification (65 nm/pixel). Myofibrils (labelled, n > 6, 150 sarcomeres; controls n = 7, 141 sarcomeres) were stretched passively from SL ranges of 2.5-2.6 μm (relaxed position; RP) to 3.1-3.25 μm (stretched position; SP). Investigating SPs, forces of 66 ± 22 and 60 ± 23 nN/μm2 were obtained in control and labelled myofibrils. No statistical significance was identified, indicating antibodies did not constrain dynamics in limited passive stretch conditions. Standard deviation of individual SLs decreased from 0.15 to 0.09 μm (39%) and 0.17 to 0.13 μm (24%) in RP1 and SP1. Decrease in SL non-uniformity was pronounced in RPs, indicating antibodies may have stiffened lattice structures to induce subtle friction. SL measurement precision was improved in fluorescent compared to phase-contrast trials by 21%. Antibodies had minimal influence at longer lengths but became increasingly constraining at shorter SLs. These results inform increasingly invasive experiments to identify how titin participates in muscle contraction.

Muscle active force-length curve explained by an electrophysical model of interfilament spacing

The active isometric force-length relation (FLR) of striated muscle sarcomeres is central to understanding and modeling muscle function. The mechanistic basis of the descending arm of the FLR is well explained by the decreasing thin:thick filament overlap that occurs at long sarcomere lengths. The mechanistic basis of the ascending arm of the FLR (the decrease in force that occurs at short sarcomere lengths), alternatively, has never been well explained. Because muscle is a constant-volume system, interfilament lattice distances must increase as sarcomere length shortens. This increase would decrease thin and thick-filament electrostatic interactions independently of thin:thick filament overlap. To examine this effect, we present here a fundamental, physics-based model of the sarcomere that includes filament molecular properties, calcium binding, sarcomere geometry including both thin:thick filament overlap and interfilament radial distance, and electrostatics. The model gives extremely good fits to existing FLR data from a large number of different muscles across their entire range of measured activity levels, with the optimized parameter values in all cases lying within anatomically and physically reasonable ranges. A local first-order sensitivity analysis (varying individual parameters while holding the values of all others constant) shows that model output is most sensitive to a subset of model parameters, most of which are related to sarcomere geometry, with model output being most sensitive to interfilament radial distance. This conclusion is supported by re-running the fits with only this parameter subset being allowed to vary, which increases fit errors only moderately. These results show that the model well reproduces existing experimental data, and indicate that changes in interfilament spacing play as central a role as changes in filament overlap in determining the FLR, particularly on its ascending arm.

Physicochemical Properties and Consumer Acceptance of Sous Vide Cooked Lobster Tails

ABSTRACT Sous vide is becoming increasingly popular in the food industry because it results in flavorful, evenly cooked products. This study aimed to better understand the effects of sous vide on physicochemical properties and consumer acceptance of lobster. Shucked lobster tails were sous vide-cooked to internal temperatures of 55, 60, and 65°C for 208, 45, and 10 minutes, respectively, and compared to boiled controls. Samples were evaluated for shear-force, color (L*,a*,b*), salt soluble protein content, moisture content, water holding capacity, weight loss, sarcomere length, and thermal stability. Sous vide-cooked samples were more tender, with greater protein solubility and shorter sarcomere length than the controls. Despite some differences in physicochemical characteristics among the sous vide-cooked samples, hedonic testing confirmed that there were no significant differences in consumer acceptability response to the sous vide cooking parameters. These results help clarify the effects of sous vide cooking on the preparation of consistently high-quality lobster products.

Stabilizing Ryanodine Receptors Improves Left Ventricular Function in Juvenile Dogs With Duchenne Muscular Dystrophy

BackgroundDuchenne muscular dystrophy is associated with progressive deterioration in left ventricular (LV) function. The golden retriever muscular dystrophy (GRMD) dog model recapitulates the pathology and clinical manifestations of Duchenne muscular dystrophy. Importantly, they develop progressive LV dysfunction starting at early age. ObjectivesThe authors tested the cardioprotective effect of chronic administration of the ARM036, a small molecule that stabilizes the closed conformation of the cardiac sarcoplasmic reticulum ryanodine receptor/calcium release channel (RyR2) in young GRMD-dogs. MethodsTwo-month-old GRMD-dogs were treated with ARM036 or placebo for 4 months. Healthy-dogs of the same genetic background served as controls. Cardiac function was evaluated by conventional and 2-dimensional speckle-tracking echocardiography. Cardiac cellular and molecular analyses were performed at 6 months old. ResultsConventional echocardiography showed normal LV dimensions and ejection fraction in 6-month-old GRMD dogs. Interestingly, 2-dimensional speckle-tracking echocardiography revealed decreased global longitudinal strain and the presence of hypokinetic segments in placebo-treated GRMD dogs. Single-channel measurements revealed higher RyR2 open probability at low resting Ca2+ in GRMD cardiomyocytes than in controls. ARM036 prevented those in vivo and in vitro dysfunctions in GRMD dogs. Myofilament Ca2+-sensitivity was increased in permeabilized GRMD cardiomyocytes at short sarcomere length. ARM036 had no effect on this parameter. Cross-bridge cycling kinetics were altered in GRMD myocytes and recovered with ARM036 treatment, which coincided with the level of myosin binding protein-C-S glutathionylation. ConclusionsGRMD-dogs exhibit early LV dysfunction associated with altered myofilament contractile properties. These abnormalities were prevented pharmacologically by stabilizing RyR2 with ARM036.

Changes in Muscle Stress and Sarcomere Adaptation in Mice Following Ischemic Stroke.

While abnormal muscle tone has been observed in people with stroke, how these changes in muscle tension affect sarcomere morphology remains unclear. The purpose of this study was to examine time-course changes in passive muscle fiber tension and sarcomeric adaptation to these changes post-ischemic stroke in a mouse model by using a novel in-vivo force microscope. Twenty-one mice were evenly divided into three groups based on the time point of testing: 3 days (D3), 10 days (D10), and 20 days (D20) following right middle cerebral artery ligation. At each testing time, the muscle length, width, and estimated volume of the isolated soleus muscle were recorded, subsequently followed by in-vivo muscle tension and sarcomere length measurement. The mass of the soleus muscle was measured at the end of testing to calculate muscle density. Two-way ANOVA with repeated measures was used to examine the differences in each of the dependent variable among the three time-point groups and between the two legs. The passive muscle stress of the impaired limbs in the D3 group (27.65 ± 8.37 kPa) was significantly lower than the less involved limbs (42.03 ± 18.61 kPa; p = 0.05) and the impaired limbs of the D10 (48.92 ± 14.73; p = 0.03) and D20 (53.28 ± 20.54 kPa; p = 0.01) groups. The soleus muscle density of the impaired limbs in the D3 group (0.69 ± 0.12 g/cm3) was significantly lower than the less involved limbs (0.80 ± 0.09 g/cm3; p = 0.04) and the impaired limbs of the D10 (0.87 ± 0.12 g/cm3; p = 0.02) and D20 (1.00 ± 0.14 g/cm3; p < 0.01) groups. The D3 group had a shorter sarcomere length (2.55 ± 0.26 μm) than the D10 (2.83 ± 0.20 μm; p = 0.03) and D20 group (2.81 ± 0.15 μm; p = 0.04). These results suggest that, while ischemic stroke may cause considerable changes in muscle tension and stress, sarcomere additions under increased mechanical loadings may be absent or disrupted post-stroke, which may contribute to muscle spasticity and/or joint contracture commonly observed in patients following stroke.

The effects of inorganic phosphate on contractile function of slow skeletal muscle fibres are length-dependent

Muscle operates across a wide range of sarcomere lengths. Inorganic phosphate (Pi) diminishes force output of striated muscle, with greater influence at short relative to long sarcomere lengths in fast skeletal and cardiac muscle fibres. The purpose of this study was to fill a gap in the literature regarding the length-dependent effects of Pi on contractile function of slow skeletal muscle fibres. Permeabilized slow skeletal muscle fibres from rabbit soleus were assessed at average sarcomere lengths of 2.0, 2.4, or 2.8 μm, with and without 20 mM Pi added to activating solutions (22±1 °C). The magnitude of Pi-induced reductions in peak force (43 ± 7% at 2.0 μm, 38 ± 7% at 2.4 μm, and 31 ± 8% at 2.8 μm) and peak stiffness (41 ± 9% at 2.0 μm, 36 ± 8% at 2.4 μm, and 26 ± 9% at 2.8 μm) were length dependent. Peak stiffness was less affected by Pi than peak force. Pi diminished the Ca2+-sensitivity of the force-pCa and stiffness-pCa relationships to a greater extent at 2.8 μm than 2.0 μm. Comparable results were obtained from a cooperative model of Ca2+ and myosin binding to regulated actin. In conclusion, Pi is more detrimental to the peak force output of slow skeletal muscle fibres held at short relative to long sarcomere lengths, whereas Pi has a greater effect on the Ca2+-sensitivity of force production at long relative to short sarcomere lengths. Stiffness data suggest that Pi-induced reductions in force are primarily due to fewer bound cross-bridges, with a lesser contribution attributable to lower average force per cross-bridge.

Age-related changes in human single muscle fibre passive elastic properties are sarcomere length dependent.

Studies have revealed an age-related decrease in the ability to produce force as well as an increase in the contractile stiffness and passive stress of single muscle fibres. However, further insight into age-related changes to the passive properties of human skeletal muscles is needed. The aim of this study was to characterize single muscle fibre passive properties from young and old males across a physiologic range of sarcomere lengths (SLs). Ten young ([YM]: mean age: 25.4years) and ten old ([OM]: mean age: 68.9years) males participated. Vastus lateralis muscles were biopsied and 182 fibres were tested and analyzed (90 YM and 92 OM) using a cumulative stretch-relaxation protocol. Passive mechanical characteristics of each fibre were obtained by fitting the integral of the logistic function to experimental stress-SL data. Muscle fibres from older individuals had significantly greater passive elastic moduli at short SL (1.9-2.65μm) compared with young (p<0.05). This resulted in a significantly larger passive stress for SLs between 2.1μm and 3.55μm for older individuals when compared with young (p<0.05). These results provide novel insight into the length dependency of changes in single muscle fibre passive mechanical properties with age.

Effects of a titin mutation on force enhancement and force depression in mouse soleus muscles.

The active isometric force produced by muscles varies with muscle length in accordance with the force-length relationship. Compared with isometric contractions at the same final length, force increases after active lengthening (force enhancement) and decreases after active shortening (force depression). In addition to cross-bridges, titin has been suggested to contribute to force enhancement and depression. Although titin is too compliant in passive muscles to contribute to active tension at short sarcomere lengths on the ascending limb and plateau of the force-length relationship, recent evidence suggests that activation increases titin stiffness. To test the hypothesis that titin plays a role in force enhancement and depression, we investigated isovelocity stretching and shortening in active and passive wild-type and mdm (muscular dystrophy with myositis) soleus muscles. Skeletal muscles from mdm mice have a small deletion in the N2A region of titin and show no increase in titin stiffness during active stretch. We found that: (1) force enhancement and depression were reduced in mdm soleus compared with wild-type muscles relative to passive force after stretch or shortening to the same final length; (2) force enhancement and force depression increased with amplitude of stretch across all activation levels in wild-type muscles; and (3) maximum shortening velocity of wild-type and mdm muscles estimated from isovelocity experiments was similar, although active stress was reduced in mdm compared with wild-type muscles. The results of this study suggest a role for titin in force enhancement and depression, which contribute importantly to muscle force during natural movements.

A Proposed Mechanism for Texture Property of Woody Breast in Broilers

ObjectivesWoody breast is a myopathy observed in chicken breast meat (Pectoralis major) characterized by its tough and rubbery texture. However, the exact causation of woody breast texture is still unknown. We hypothesize that sarcoplasmic reticulum (SR) dysfunctionality early postmortem results in rapid leakage of intracellular calcium may partially contribute to the abnormal meat texture observed in woody breast meat. The objective of this preliminary study was to investigate this hypothesis.Materials and MethodsFourteen Ross line broiler breast fillets (7 severe woody breast and 7 normal) were collected at 3 h postmortem from a commercial processing plant located in the southeast United States. The 7 woody breast samples also exhibited moderate to severe white striping. The 7 normal samples did not exhibit any signs of white striping or woody breast. Each sample was trimmed, weighed, vacuum packaged and frozen at –20°C at approximately 8 h postmortem. One 1.9 cm strip across the cranial end of each fillet was fabricated and pulverized in liquid nitrogen to measure sarcomere length (Laser Scan Confocal Microscope with a 100x/NA 1.4 objective), calpain activity (immunoblotting for µ-calpain autolysis), proteolysis (immunoblotting for troponin-T degradation) and collagen content (hydroxyproline content). Purge was also collected from each sample to evaluate protein (bicinchoninic acid assay) and free calcium concentration (atomic absorption).ResultsWoody breast fillets were heavier than normal chicken breast fillets (522.9 vs. 446.9g; P &lt; 0.05). Woody breast samples tended to have shorter sarcomeres (1.70 vs. 2.02 µm; P = 0.0543) and less intact troponin-T compared to normal breast samples (relative intact troponin-T band density: 49.98 vs. 56.97%; P = 0.0515) at 8 h postmortem. It was interesting to note that no µ-calpain band was detected through immunoblotting for both the woody breast and normal samples at 8 h postmortem. Other studies have found similar results as poultry µ-calpain autolyzed at a much rapid rate than µ-calpain in mammalian species. In addition, the purge from woody breast samples also had higher levels of free calcium compared to normal samples (6.2 vs. 4.2 nmol calcium/mg protein; P &lt; 0.05). Lastly, there was more collagen present in the woody breast samples compared to normal chicken breast samples (3.89 vs. 2.08 mg collagen/g muscle tissue; P &lt; 0.05).ConclusionThe results indicated that the cause of texture abnormality of woody breast may be the combined effects of more calcium being released from the SR early postmortem resulting in shorter sarcomere length and more collagen being deposited in the chicken breast meat. Additional research with the focus on SR integrity and functionality as well as collagen crosslinks are needed to further elucidate the basic mechanism of woody breast texture formation.

Dilated cardiomyopathy mutation (R174W) in troponin T attenuates the length-mediated increase in cross-bridge recruitment and myofilament Ca2+ sensitivity.

Alterations in length-dependent activation (LDA) may constitute a mechanism by which cardiomyopathy mutations lead to deleterious phenotypes and compromised heart function, because LDA underlies the molecular basis by which the heart tunes myocardial force production on a beat-to-beat basis (Frank-Starling mechanism). In this study, we investigated the effect of DCM-linked mutation (R173W) in human cardiac troponin T (TnT) on myofilament LDA. R173W mutation is associated with left ventricular dilatation and systolic dysfunction and is found in multiple families. R173W mutation is in the central region (residues 80-180) of TnT, which is known to be important for myofilament cooperativity and cross-bridge (XB) recruitment. Steady-state and dynamic contractile parameters were measured in detergent-skinned guinea pig left ventricular muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnTWT) or mutant TnT (TnTR174W; guinea pig analog of human R173W mutation) at two different sarcomere lengths (SL): short (1.9 µm) and long (2.3 µm). TnTR174W decreased pCa50 (-log [Ca2+]free required for half-maximal activation) to a greater extent at long than at short SL; for example, pCa50 decreased by 0.12 pCa units at long SL and by 0.06 pCa units at short SL. Differential changes in pCa50 at short and long SL attenuated the SL-dependent increase in myofilament Ca2+ sensitivity (ΔpCa50) in TnTR174W fibers; ΔpCa50 was 0.10 units in TnTWT fibers but only 0.04 units in TnTR174W fibers. Furthermore, TnTR174W blunted the SL-dependent increase in the magnitude of XB recruitment. Our observations suggest that the R173W mutation in human cardiac TnT may impair Frank-Starling mechanism.NEW & NOTEWORTHY This work characterizes the effect of dilated cardiomyopathy mutation in cardiac troponin T (TnTR174W) on myofilament length-dependent activation. TnTR174W attenuates the length-dependent increase in cross-bridge recruitment and myofilament Ca2+ sensitivity.

A Proposed Mechanism for Texture Property of Woody Breast in Broilers

ObjectivesWoody breast is a myopathy observed in chicken breast meat (Pectoralis major) characterized by its tough and rubbery texture. However, the exact causation of woody breast texture is still unknown. We hypothesize that sarcoplasmic reticulum (SR) dysfunctionality early postmortem results in rapid leakage of intracellular calcium may partially contribute to the abnormal meat texture observed in woody breast meat. The objective of this preliminary study was to investigate this hypothesis.Materials and MethodsFourteen Ross line broiler breast fillets (7 severe woody breast and 7 normal) were collected at 3 h postmortem from a commercial processing plant located in the southeast United States. The 7 woody breast samples also exhibited moderate to severe white striping. The 7 normal samples did not exhibit any signs of white striping or woody breast. Each sample was trimmed, weighed, vacuum packaged and frozen at –20°C at approximately 8 h postmortem. One 1.9 cm strip across the cranial end of each fillet was fabricated and pulverized in liquid nitrogen to measure sarcomere length (Laser Scan Confocal Microscope with a 100x/NA 1.4 objective), calpain activity (immunoblotting for µ-calpain autolysis), proteolysis (immunoblotting for troponin-T degradation) and collagen content (hydroxyproline content). Purge was also collected from each sample to evaluate protein (bicinchoninic acid assay) and free calcium concentration (atomic absorption).ResultsWoody breast fillets were heavier than normal chicken breast fillets (522.9 vs. 446.9g; P < 0.05). Woody breast samples tended to have shorter sarcomeres (1.70 vs. 2.02 µm; P = 0.0543) and less intact troponin-T compared to normal breast samples (relative intact troponin-T band density: 49.98 vs. 56.97%; P = 0.0515) at 8 h postmortem. It was interesting to note that no µ-calpain band was detected through immunoblotting for both the woody breast and normal samples at 8 h postmortem. Other studies have found similar results as poultry µ-calpain autolyzed at a much rapid rate than µ-calpain in mammalian species. In addition, the purge from woody breast samples also had higher levels of free calcium compared to normal samples (6.2 vs. 4.2 nmol calcium/mg protein; P < 0.05). Lastly, there was more collagen present in the woody breast samples compared to normal chicken breast samples (3.89 vs. 2.08 mg collagen/g muscle tissue; P < 0.05).ConclusionThe results indicated that the cause of texture abnormality of woody breast may be the combined effects of more calcium being released from the SR early postmortem resulting in shorter sarcomere length and more collagen being deposited in the chicken breast meat. Additional research with the focus on SR integrity and functionality as well as collagen crosslinks are needed to further elucidate the basic mechanism of woody breast texture formation.

Functionally Driven Modulation of Sarcomeric Structure and Membrane Systems in the Fast Muscles of a Copepod (Gaussia princeps).

Muscles of the mesopelagic copepod Gaussia princeps (Arthropoda, Crustacea, Calanoida) are responsible for repetitive movements of feeding and swimming appendages that are too fast to be followed by eye. This article provides a comparative functional and ultrastructural description of five muscles that have different contraction speeds and are located within different anatomical sites. All are very fast, as indicated by a thick:thin filament ratio of 3:1 and sarcomere lengths that vary between 1 and 3 μm. Measured lengths of thin and thick filaments indicate classification of the muscles into three distinct groups (short, medium, and long) and predict a difference in speed of up to threefold between fibers with the shortest and longest sarcomeres. Indeed, the kicking movement of the posterior legs (with the shortest sarcomere length) is approximately threefold faster than the simultaneous back-folding of the antennae (with the longest length). Thus, a specific relationship between speed of movement and sarcomere length is established, and we can use the latter to predict the former. Regulatory systems of contraction (sarcoplasmic reticulum [SR] and transverse [T] tubules) match the different contractile properties, varying in frequency of distribution and overall content in parallel to sarcomere variations. All muscles from appendages and body musculature show a unique disposition of contractile material, SR, and T tubules found only in copepod muscles; muscle filaments are grouped in large supermyofibrils that are riddled with frequent cylindrical shafts containing SR and T tubules. This arrangement insures a high spatial frequency of regulatory components. Anat Rec, 301:2164-2176, 2018. © 2018 Wiley Periodicals, Inc.

Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity.

Recent clinical studies have revealed a new hypertrophic cardiomyopathy-associated mutation (F87L) in the central region of human cardiac troponin T (TnT). However, despite its implication in several incidences of sudden cardiac death in young and old adults, whether F87L is associated with cardiac contractile dysfunction is unknown. Because the central region of TnT is important for modulating the muscle length-mediated recruitment of new force-bearing cross-bridges (XBs), we hypothesize that the F87L mutation causes molecular changes that are linked to the length-dependent activation of cardiac myofilaments. Length-dependent activation is important because it contributes significantly to the Frank-Starling mechanism, which enables the heart to vary stroke volume as a function of changes in venous return. We measured steady-state and dynamic contractile parameters in detergent-skinned guinea pig cardiac muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnTWT) or the guinea pig analogue (TnTF88L) of the human mutation at two different sarcomere lengths (SLs): short (1.9 µm) and long (2.3 µm). TnTF88L increases pCa50 (-log [Ca2+]free required for half-maximal activation) to a greater extent at short SL than at long SL; for example, pCa50 increases by 0.25 pCa units at short SL and 0.17 pCa units at long SL. The greater increase in pCa50 at short SL leads to the abolishment of the SL-dependent increase in myofilament Ca2+ sensitivity (ΔpCa50) in TnTF88L fibers, ΔpCa50 being 0.10 units in TnTWT fibers but only 0.02 units in TnTF88L fibers. Furthermore, at short SL, TnTF88L attenuates the negative impact of strained XBs on force-bearing XBs and augments the magnitude of muscle length-mediated recruitment of new force-bearing XBs. Our findings suggest that the TnTF88L-mediated effects on cardiac thin filaments may lead to a negative impact on the Frank-Starling mechanism.

Role of the C-terminus mobile domain of cardiac troponin I in the regulation of thin filament activation in skinned papillary muscle strips

The C-terminus mobile domain of cTnI (cTnI-MD) is a highly conserved region which stabilizes the actin-cTnI interaction during the diastole. Upon Ca2+-binding to cTnC, cTnI-MD participates in a regulatory switching that involves cTnI to switch from interacting with actin toward interacting with the Ca2+-regulatory domain of cTnC. Despite many studies targeting the cTnI-MD, the role of this region in the length-dependent activation of cardiac contractility is yet to be determined. The present study investigated the functional consequences of losing the entire cTnI-MD in cTnI(1–167) truncation mutant, as it was exchanged for endogenous cTnI in skinned rat papillary muscle fibers. The influence of cTnI-MD truncation on the extent of the N-domain of cTnC hydrophobic cleft opening and the steady-state force as a function of sarcomere length (SL), cross-bridge state, and [Ca2+] was assessed using the simultaneous in situ time-resolved FRET and force measurements at short (1.8 μm) and long (2.2 μm) SLs. Our results show the significant role of cTnI-MD in the length dependent thin filament activation and the coupling between thin and thick filament regulations affected by SL. Our results also suggest that cTnI-MD transmits the effects of SL change to the core of troponin complex.

Altered myofilament structure and function in dogs with Duchenne muscular dystrophy cardiomyopathy

AimDuchenne Muscular Dystrophy (DMD) is associated with progressive depressed left ventricular (LV) function. However, DMD effects on myofilament structure and function are poorly understood. Golden Retriever Muscular Dystrophy (GRMD) is a dog model of DMD recapitulating the human form of DMD. ObjectiveThe objective of this study is to evaluate myofilament structure and function alterations in GRMD model with spontaneous cardiac failure. Methods and resultsWe have employed synchrotron X-rays diffraction to evaluate myofilament lattice spacing at various sarcomere lengths (SL) on permeabilized LV myocardium. We found a negative correlation between SL and lattice spacing in both sub-epicardium (EPI) and sub-endocardium (ENDO) LV layers in control dog hearts. In the ENDO of GRMD hearts this correlation is steeper due to higher lattice spacing at short SL (1.9μm). Furthermore, cross-bridge cycling indexed by the kinetics of tension redevelopment (ktr) was faster in ENDO GRMD myofilaments at short SL. We measured post-translational modifications of key regulatory contractile proteins. S-glutathionylation of cardiac Myosin Binding Protein-C (cMyBP-C) was unchanged and PKA dependent phosphorylation of the cMyBP-C was significantly reduced in GRMD ENDO tissue and more modestly in EPI tissue. ConclusionsWe found a gradient of contractility in control dogs' myocardium that spreads across the LV wall, negatively correlated with myofilament lattice spacing. Chronic stress induced by dystrophin deficiency leads to heart failure that is tightly associated with regional structural changes indexed by increased myofilament lattice spacing, reduced phosphorylation of regulatory proteins and altered myofilament contractile properties in GRMD dogs.

Spatial and age-related changes in the microstructure of dystrophic and healthy diaphragms.

Duchenne muscular dystrophy (DMD) is a progressive degenerative disease that results in fibrosis and atrophy of muscles. The main cause of death associated with DMD is failure of the diaphragm. The diaphragm is a dome-shaped muscle with a fiber microstructure that differs across regions of the muscle. However, no studies to our knowledge have examined spatial variations of muscle fibers in dystrophic diaphragm or how aging affects those variations in DMD. In this study, diaphragms were obtained from mdx and healthy mice at ages three, seven, and ten months in the dorsal, midcostal, and ventral regions. Through immunostaining and confocal imaging, we quantified sarcomere length, interstitial space between fibers, fiber branching, fiber cross sectional area (CSA), and fiber regeneration measured by centrally located nuclei. Because DMD is associated with chronic inflammation, we also investigated the number of macrophages in diaphragm muscle cross-sections. We saw regional differences in the number of regenerating fibers and macrophages during the progression of DMD in the mdx diaphragm. Additionally, the number of regenerating fibers increased with age, while CSA and the number of branching fibers decreased. Dystrophic diaphragms had shorter sarcomere lengths than age-matched controls. Our results suggest that the dystrophic diaphragm in the mdx mouse is structurally heterogeneous and remodels non-uniformly over time. Understanding regional changes in dystrophic diaphragms over time will facilitate the development of targeted therapies to prevent or minimize respiratory failure in DMD patients.

Hypertrophic cardiomyopathy mutation in cardiac troponin T (R95H) attenuates length-dependent activation in guinea pig cardiac muscle fibers.

The central region of cardiac troponin T (TnT) is important for modulating the dynamics of muscle length-mediated cross-bridge recruitment. Therefore, hypertrophic cardiomyopathy mutations in the central region may affect cross-bridge recruitment dynamics to alter myofilament Ca2+ sensitivity and length-dependent activation of cardiac myofilaments. Given the importance of the central region of TnT for cardiac contractile dynamics, we studied if hypertrophic cardiomyopathy-linked mutation (TnTR94H)-induced effects on contractile function would be differently modulated by sarcomere length (SL). Recombinant wild-type TnT (TnTWT) and the guinea pig analog of the human R94H mutation (TnTR95H) were reconstituted into detergent-skinned cardiac muscle fibers from guinea pigs. Steady-state and dynamic contractile measurements were made at short and long SLs (1.9 and 2.3 µm, respectively). Our results demonstrated that TnTR95H increased pCa50 (-log of free Ca2+ concentration) to a greater extent at short SL; TnTR95H increased pCa50 by 0.11 pCa units at short SL and 0.07 pCa units at long SL. The increase in pCa50 associated with an increase in SL from 1.9 to 2.3 µm (ΔpCa50) was attenuated nearly twofold in TnTR95H fibers; ΔpCa50 was 0.09 pCa units for TnTWT fibers but only 0.05 pCa units for TnTR95H fibers. The SL dependency of rate constants of cross-bridge distortion dynamics and tension redevelopment was also blunted by TnTR95H Collectively, our observations on the SL dependency of pCa50 and rate constants of cross-bridge distortion dynamics and tension redevelopment suggest that mechanisms underlying the length-dependent activation cardiac myofilaments are attenuated by TnTR95HNEW & NOTEWORTHY Mutant cardiac troponin T (TnTR95H) differently affects myofilament Ca2+ sensitivity at short and long sarcomere length, indicating that mechanisms underlying length-dependent activation are altered by TnTR95H TnTR95H enhances myofilament Ca2+ sensitivity to a greater extent at short sarcomere length, thus attenuating the length-dependent increase in myofilament Ca2+ sensitivity.

Omecamtiv Mecarbil Abolishes Length-Mediated Increase in Guinea Pig Cardiac Myofiber Ca2+ Sensitivity

Omecamtiv mecarbil (OM) is a pharmacological agent that augments cardiac contractile function by enhancing myofilament Ca2+ sensitivity. Given that interventions that increase myofilament Ca2+ sensitivity have the potential to alter length-dependent activation (LDA) of cardiac myofilaments, we tested the influence of OM on this fundamental property of the heart. This is significant not only because LDA is prominent in cardiac muscle but also because it contributes to the Frank-Starling law, a mechanism by which the heart increases stroke volume in response to an increase in venous return. We measured steady-state and dynamic contractile indices in detergent-skinned guinea pig (Cavia porcellus) cardiac muscle fibers in the absence and presence of 0.3 and 3.0 μM OM at two different sarcomere lengths (SLs), short SL (1.9 μm) and long SL (2.3 μm). Myofilament Ca2+ sensitivity, as measured by pCa50 (−log of [Ca2+]free concentration required for half-maximal activation), increased significantly at both short and long SLs in OM-treated fibers when compared to untreated fibers; however, the magnitude of increase in pCa50 was twofold greater at short SL than at long SL. A consequence of this greater increase in pCa50 at short SL was that pCa50 did not increase any further at long SL, suggesting that OM abolished the SL dependency of pCa50. Furthermore, the SL dependency of rate constants of cross-bridge distortion dynamics (c) and force redevelopment (ktr) was abolished in 0.3-μM-OM-treated fibers. The negative impact of OM on the SL dependency of pCa50, c, and ktr was also observed in 3.0-μM-OM-treated fibers, indicating that cooperative mechanisms linked to LDA were altered by the OM-mediated effects on cardiac myofilaments.