Unloaded Shortening Research Articles

EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.

Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. Screening of 157 kinase inhibitors in wild-type (n=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (n=24 HCM and n=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (n=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.

The Force-Velocity Profile for Jumping: What It Is and What It Is Not.

Force-velocity profiling has been proposed in the literature as a method to identify the overall mechanical characteristics of lower extremities. A force-velocity profile is obtained by plotting for jumps at different loads the effective work as a function of the average push-off velocity, fitting a straight line to the results, and extrapolating this line to find the theoretical maximum isometric force and unloaded shortening velocity. Here we investigated whether the force-velocity profile and its characteristics can be related to the intrinsic force-velocity relationship. We used simulation models of various complexity, ranging from a simple mass actuated by a linearly damped force to a planar musculoskeletal model comprising four segments and six muscle-tendon complexes. The intrinsic force-velocity relationship of each model was obtained by maximizing the effective work during isokinetic extension at different velocities. Several observations were made. First, at the same average velocity, less effective work can be done during jumping than during isokinetic lower extremity extension at this velocity. Second, the intrinsic relationship is curved; fitting a straight line and extrapolating it seem arbitrary. Third, the maximal isometric force and the maximal velocity corresponding to the profile are not independent. Fourth, they both vary with inertial properties of the system. For these reasons, we concluded that the force-velocity profile is specific for the task and is just what it is: the relationship between effective work and an arbitrary estimate of average velocity; it does not represent the intrinsic force-velocity relationship of the lower extremities.

One size does not fit all: diversity of length-force properties of obliquely striated muscles.

Obliquely striated muscles occur in 17+ phyla, likely evolving repeatedly, yet the implications of oblique striation for muscle function are unknown. Contrary to the belief that oblique striation allows high force output over extraordinary length ranges (i.e. superelongation), recent work suggests diversity in operating length ranges and length-force relationships. We hypothesize oblique striation evolved to increase length-force relationship flexibility. We predict that superelongation is not a general characteristic of obliquely striated muscles and instead that length-force relationships vary with operating length range. To test these predictions, we measured length-force relationships of five obliquely striated muscles from inshore longfin squid, Doryteuthis pealeii: tentacle, funnel retractor and head retractor longitudinal fibers, and arm and fin transverse fibers. Consistent with superelongation, the tentacle length-force relationship had a long descending limb, whereas all others exhibited limited descending limbs. The ascending limb at 0.6P0 was significantly broader (P<0.001) for the tentacle length-force relationship (0.43±0.04L0; where L0 is the preparation length that produced peak isometric stress, P0) than for the arm (0.29±0.03L0), head retractor (0.24±0.06L0), fin (0.20±0.04L0) and funnel retractor (0.27±0.03L0). The fin's narrow ascending limb differed significantly from those of the arm (P=0.004) and funnel retractor (P=0.012). We further characterized the tentacle preparation's maximum isometric stress (315±78 kPa), maximum unloaded shortening velocity (2.97±0.55L0s-1) and ultrastructural traits (compared with the arm), which may explain its broader length-force relationship. Comparison of obliquely striated muscles across taxa revealed length-force relationship diversity, with only two species exhibiting superelongation.

Posttetanic potentiation improves neuromuscular efficiency of mouse muscle in vitro.

Neuromuscular efficiency is defined as the ratio of work output to stimulation rate. The purpose of these experiments was to test the hypothesis that neuromuscular efficiency would be increased in proportion to posttetanic potentiation, that is, the stimulation-induced increase in work output displayed by rodent fast-twitch muscle. To this end, extensor digitorum longus muscles from wild-type and skeletal myosin light chain kinase knockout (skMLCK-/- ) mice were surgically isolated and suspended in vitro (25°C). Concentric force development during shortening at 70% of maximal unloaded shortening velocity was tested at stimulus frequencies between 10 and 80 Hz both before and after a potentiating tetanus. A strong genotype-dependent difference in the potentiation of concentric work output was observed; concentric work output of wild-type muscles was increased by 51%-88% while that of skMLCK-/- muscles was increased by only 20%-34% across the frequencies tested. As a result, comparison of work - frequency plots revealed that the frequency required for peak and 50% peak unpotentiated work of wild-type muscles was decreased from ~80 to 52 Hz and from ~48 to 21 Hz, respectively. By contrast, the frequency required for peak and 50% peak unpotentiated work of skMLCK-/- muscles was decreased from ~80 to 68 Hz and from ~51 to 41 Hz, respectively. Thus, wild-type muscles with the ability to phosphorylate myosin displayed larger increases in neuromuscular efficiency than skMLCK-/- muscles (25-30 vs 10-15 Hz, respectively). This suggests that the presence of myosin phosphorylation may ameliorate or mitigate fatigue mechanisms associated with high-frequency stimulation rates.

Comparing contractile properties within an integrated group of muscles: The abdominal wall

The abdominal wall (external oblique (EO), internal oblique (IO), rectus abdominis (RA), and transverse abdominis (TrA)) is a functionally and anatomically integrated group of muscles. While the passive mechanical properties of the individual abdominal muscles have been studied previously, their contractile properties have yet to be described. Muscle samples were taken from the EO, IO, RA, and TrA of 6 Sprague-Dawley rats. Single muscle fibres were isolated from each sample, chemically permeabilized and tested in a Ca2+ (pCa 4.2) bath to determine their contractile properties: specific force, active modulus, unloaded shortening velocity, and rate of force redevelopment. Myosin heavy chain (MHC) isoforms were identified by gel electrophoresis to determine the fibre type of each tested fibre, as well as larger bundles of fibres. No type I fibres and only two type IIa fibres were tested, therefore type IIx and IIb fibres were combined for statistical analysis. There were no significant differences between muscles for specific force, active modulus, and unloaded shortening velocity (p > 0.05). Rate of force redevelopment was statistically significant (p = 0.029), with TrA being 62 % greater than EO, suggesting faster cross-bridge transitioning between low and high force-generating states in the TrA. The functional significance of this difference is unclear and will need to be studied further.

Activation of the myosin motors in fast-twitch muscle of the mouse is controlled by mechano-sensing in the myosin filaments.

Myosin motors in resting muscle are inactivated by folding against the backbone of the myosin filament in an ordered helical array and must be released from that conformation to engage in force generation. Time‐resolved X‐ray diffraction from single fibres of amphibian muscle showed that myosin filament activation could be inhibited by imposing unloaded shortening at the start of stimulation, suggesting that filaments were activated by mechanical stress. Here we improved the signal‐to‐noise ratio of that approach using whole extensor digitorum longus muscles of the mouse contracting tetanically at 28°C. Changes in X‐ray signals associated with myosin filament activation, including the decrease in the first‐order myosin layer line associated with the helical motor array, increase in the spacing of a myosin‐based reflection associated with packing of myosin tails in the filament backbone, and increase in the ratio of the 1,1 and 1,0 equatorial reflections associated with movement of motors away from the backbone, were delayed by imposing 10‐ms unloaded shortening at the start of stimulation. These results show that myosin filaments are predominantly activated by filament stress, as in amphibian muscle. However, a small component of filament activation at zero load was detected, implying an independent mechanism of partial filament activation. X‐ray interference measurements indicated a switch‐like change in myosin motor conformation at the start of force development, accompanied by transient disordering of motors in the regions of the myosin filament near its midpoint, suggesting that filament zonal dynamics also play a role in its activation. Key points Activation of myosin filaments in extensor digitorum longus muscles of the mouse is delayed by imposing rapid shortening from the start of stimulation.Stress is the major mechanism of myosin filament activation in these muscles, but there is a small component of filament activation during electrical stimulation at zero stress.Myosin motors switch rapidly from the folded inhibited conformation to the actin‐attached force‐generating conformation early in force development.

Small Molecule RPI-194 Stabilizes Activated Troponin to Increase the Calcium Sensitivity of Striated Muscle Contraction.

Small molecule cardiac troponin activators could potentially enhance cardiac muscle contraction in the treatment of systolic heart failure. We designed a small molecule, RPI-194, to bind cardiac/slow skeletal muscle troponin (Cardiac muscle and slow skeletal muscle share a common isoform of the troponin C subunit.) Using solution NMR and stopped flow fluorescence spectroscopy, we determined that RPI-194 binds to cardiac troponin with a dissociation constant KD of 6–24 μM, stabilizing the activated complex between troponin C and the switch region of troponin I. The interaction between RPI-194 and troponin C is weak (KD 311 μM) in the absence of the switch region. RPI-194 acts as a calcium sensitizer, shifting the pCa50 of isometric contraction from 6.28 to 6.99 in mouse slow skeletal muscle fibers and from 5.68 to 5.96 in skinned cardiac trabeculae at 100 μM concentration. There is also some cross-reactivity with fast skeletal muscle fibers (pCa50 increases from 6.27 to 6.52). In the slack test performed on the same skinned skeletal muscle fibers, RPI-194 slowed the velocity of unloaded shortening at saturating calcium concentrations, suggesting that it slows the rate of actin-myosin cross-bridge cycling under these conditions. However, RPI-194 had no effect on the ATPase activity of purified actin-myosin. In isolated unloaded mouse cardiomyocytes, RPI-194 markedly decreased the velocity and amplitude of contractions. In contrast, cardiac function was preserved in mouse isolated perfused working hearts. In summary, the novel troponin activator RPI-194 acts as a calcium sensitizer in all striated muscle types. Surprisingly, it also slows the velocity of unloaded contraction, but the cause and significance of this is uncertain at this time. RPI-194 represents a new class of non-specific troponin activator that could potentially be used either to enhance cardiac muscle contractility in the setting of systolic heart failure or to enhance skeletal muscle contraction in neuromuscular disorders.

Analysis of Cardiac Contractile Dysfunction and Ca&lt;sup&gt;2+&lt;/sup&gt; Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes.

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

RPI‐194 is a Novel Troponin Activator that Increases the Calcium Sensitivity of Striated Muscle Contraction

IntroductionSystolic heart failure is a cardiac disease in which the contractile function of heart muscle becomes weakened and unable to pump blood with adequate force. As of yet, no existing positive inotrope is able to lower the mortality rate in systolic heart failure. Cardiac troponin complex (cTn) is a trimeric complex consisting of troponin C (cTnC), troponin I (cTnI) and troponin T (cTnT). cTnC regulates blood supply to the heart by turning muscle contraction on and off in a calcium‐dependent manner. More precisely, the regulatory N‐domain of cTnC (cNTnC) binds the switch region of cTnI to activate contraction. Drug‐like molecules need to bind to this cNTnC‐cTnI switch region interface and modulate the pumping activity of the heart. Our objective was to identify a small molecule that has the ability to stabilize the activated conformation of cTn by binding to this interface and thereby enhance cardiac muscle contraction.HypothesisA cardiac troponin activator could potentially compensate the impaired contractile function of the heart in the treatment of systolic heart failureMethodsA total of 47 new compounds were designed using existing knowledge of calcium sensitizers. The compounds were screened against a recombinantly purified cNTnC‐cTnI chimeric construct, named gChimera, using nuclear magnetic resonance and fluorescence spectroscopy. We identified a novel small molecule cardiac troponin activator, RPI‐194, and measured its binding affinity to recombinantly purified cNTnC and gChimera. We also investigated the activity of RPI‐194 in rat skinned cardiac muscle trabeculae, isolated mouse cardiomyocytes, and isolated working mouse hearts. RPI‐194 activity was also tested in skinned skeletal muscle fibers from rats as both cardiac and slow skeletal muscle share the same isoform of cTnC.ResultsWe demonstrated that a small molecule troponin activator RPI‐194 binds to gChimera with a KD of 12‐24 µM and is able to stabilize activated cTn. It also slowed down the rate of calcium release from reconstituted cTn. RPI‐194 showed increasing calcium sensitivity of isometric contraction in skinned cardiac muscle trabeculae, as well as in slow and fast skeletal muscle fibers, suggesting it to be a calcium sensitizer, with cross‐reactivity within striated muscle. Contrarily, RPI‐194 was unable to slow down calcium release from isolated cNTnC. It also reduced the velocity of unloaded shortening in skeletal muscle fibers, suggesting that it slows the rate of actin‐myosin cross‐bridging, although no effect on myosin ATPase activity was found. RPI‐194 decreased the velocity and amplitude of contractions in isolated cardiomyocytes but contractility was preserved in isolated working hearts.ConclusionsRPI‐194 is a small molecule troponin activator that acts as a calcium sensitizer in striated muscle. Because of the isoform sharing of cTnC in cardiac and slow skeletal muscle, it is nearly impossible to develop a cardiac‐specific troponin activator. Whole animal model studies are needed to determine the impact of RPI‐194 on the different striated muscle types and whether this would be acceptable for therapeutic intervention.

A high-whey-protein diet does not enhance mechanical and structural remodeling of cardiac muscle in response to aerobic exercise in rats.

[Purpose]Aerobic exercise training results in distinct structural and mechanical myocardial adaptations. In skeletal muscle, whey protein supplementation is effective in enhancing muscle adaptation following resistance exercise. However, it is unclear whether cardiac adaptation to aerobic exercise can be enhanced by systematic protein supplementation.[Methods]Twelve-week-old rats were assigned to 12 weeks of either sedentary or aerobic exercise with either a standard (Sed+Standard, Ex+Standard) or high-protein (Sed+Pro, Ex+Pro) diet. Echocardiography was used to measure cardiac structural remodeling and performance. Skinned cardiac fiber bundles were used to determine the active and passive stress properties, maximum shortening velocity, and calcium sensitivity.[Results]Aerobic training was characterized structurally by increases in ventricle volume (Ex+Standard, 19%; Ex+Pro, 29%) and myocardial thickness (Ex+Standard, 26%; Ex+-Pro, 12%) compared to that of baseline. Skinned trabecula r fiber bundles also had a greater unloaded shortening velocity (Sed+Standard, 1.04±0.05; Sed+Pro, 1.07±0.03; Ex-+Standard, 1.16±0.04; Ex+Pro, 1.18±0.05 FL/s) and calcium sensitivity (pCa50: Sed+Standard, 6.04±0.17; Sed+Pro, 6.08±0.19; Ex+Standard, 6.30±0.09; Ex+Pro, 6.36±0.12) in trained hearts compared to that of hearts from sedentary animals. However, the addition of a high-protein diet did not provide additional benefits to either the structural or mechanical adaptations of the myocardium.[Conclusion]Therefore, it seems that a high-whey-protein diet does not significantly enhance adaptations of the heart to aerobic exercise in comparison to that of a standard diet.

Investigating the active contractile function of the rat paraspinal muscles reveals unique cross-bridge kinetics in the multifidus

Various aspects of paraspinal muscle anatomy, biology, and histology have been studied; however, information on paraspinal muscle contractile function is almost nonexistent, thus hindering functional interpretation of these muscles in healthy individuals and those with low back disorders. The aim of this study was to measure and compare the contractile function and force-sarcomere length properties of muscle fibers from the multifidus (MULT) and erector spinae (ES) as well as a commonly studied lower limb muscle (Extensor digitorum longus (EDL)) in the rat. Single muscle fibers (n = 77 total from 6 animals) were isolated from each of the muscles and tested to determine their active contractile function; all fibers used in the analyses were type IIB. There were no significant differences between muscles for specific force (sFo) (p = 0.11), active modulus (p = 0.63), average optimal sarcomere length (p = 0.27) or unloaded shortening velocity (Vo) (p = 0.69). However, there was a significant difference in the rate of force redevelopment (ktr) between muscles (p = < 0.0001), with MULT being significantly faster than both the EDL (p = < 0.0001) and ES (p = 0.0001) and no difference between the EDL and ES (p = 0.41). This finding suggests that multifidus has faster cross-bridge turnover kinetics when compared to other muscles (ES and EDL) when matched for fiber type. Whether the faster cross-bridge kinetics translate to a functionally significant difference in whole muscle performance needs to be studied further.

Generative adversarial networks for construction of virtual populations of mechanistic models: simulations to study Omecamtiv Mecarbil action

Biophysical models are increasingly used to gain mechanistic insights by fitting and reproducing experimental and clinical data. The inherent variability in the recorded datasets, however, presents a key challenge. In this study, we present a novel approach, which integrates mechanistic modeling and machine learning to analyze in vitro cardiac mechanics data and solve the inverse problem of model parameter inference. We designed a novel generative adversarial network (GAN) and employed it to construct virtual populations of cardiac ventricular myocyte models in order to study the action of Omecamtiv Mecarbil (OM), a positive cardiac inotrope. Populations of models were calibrated from mechanically unloaded myocyte shortening recordings obtained in experiments on rat myocytes in the presence and absence of OM. The GAN was able to infer model parameters while incorporating prior information about which model parameters OM targets. The generated populations of models reproduced variations in myocyte contraction recorded during in vitro experiments and provided improved understanding of OM’s mechanism of action. Inverse mapping of the experimental data using our approach suggests a novel action of OM, whereby it modifies interactions between myosin and tropomyosin proteins. To validate our approach, the inferred model parameters were used to replicate other in vitro experimental protocols, such as skinned preparations demonstrating an increase in calcium sensitivity and a decrease in the Hill coefficient of the force–calcium (F–Ca) curve under OM action. Our approach thereby facilitated the identification of the mechanistic underpinnings of experimental observations and the exploration of different hypotheses regarding variability in this complex biological system.

Combinational Therapy of Cardiac Atrial Appendage Stem Cells and Pyridoxamine: The Road to Cardiac Repair?

Myocardial infarction (MI) occurs when the coronary blood supply is interrupted. As a consequence, cardiomyocytes are irreversibly damaged and lost. Unfortunately, current therapies for MI are unable to prevent progression towards heart failure. As the renewal rate of cardiomyocytes is minimal, the optimal treatment should achieve effective cardiac regeneration, possibly with stem cells transplantation. In that context, our research group identified the cardiac atrial appendage stem cells (CASCs) as a new cellular therapy. However, CASCs are transplanted into a hostile environment, with elevated levels of advanced glycation end products (AGEs), which may affect their regenerative potential. In this study, we hypothesize that pyridoxamine (PM), a vitamin B6 derivative, could further enhance the regenerative capacities of CASCs transplanted after MI by reducing AGEs’ formation. Methods and Results: MI was induced in rats by ligation of the left anterior descending artery. Animals were assigned to either no therapy (MI), CASCs transplantation (MI + CASCs), or CASCs transplantation supplemented with PM treatment (MI + CASCs + PM). Four weeks post-surgery, global cardiac function and infarct size were improved upon CASCs transplantation. Interstitial collagen deposition, evaluated on cryosections, was decreased in the MI animals transplanted with CASCs. Contractile properties of resident left ventricular cardiomyocytes were assessed by unloaded cell shortening. CASCs transplantation prevented cardiomyocyte shortening deterioration. Even if PM significantly reduced cardiac levels of AGEs, cardiac outcome was not further improved. Conclusion: Limiting AGEs’ formation with PM during an ischemic injury in vivo did not further enhance the improved cardiac phenotype obtained with CASCs transplantation. Whether AGEs play an important deleterious role in the setting of stem cell therapy after MI warrants further examination.

Botox Injections in Paraspinal Muscles Result in Low Maximal Specific Force and Shortening Velocity in Fast but Not Slow Skinned Muscle Fibers.

Basic science, experimental animal study. To determine the effects of Botulinum toxin type A (BTX-A) injections on the mechanical properties of skinned muscle fibers (cells) of rabbit paraspinal muscles. BTX-A has been widely used in the treatment of disorders of muscle hyperactivity, such as spasticity, dystonia, and back pain. However, BTX-A injection has been shown to cause muscle atrophy, fat infiltration, and decreased force output in target muscles, but its potential effects on the contractile machinery and force production on the cellular level remain unknown. Nineteen-month-old, male New Zealand White Rabbits received either saline or BTX-A injections into the paraspinal muscles, equally distributed along the left and right sides of the spine at T12, L1, and L2 at 0, 8, 12, 16, 20, and 24 weeks. Magnetic resonance imaging was used to quantify muscle crosssectional area and structural changes before and at 28 weeks following the initial injection. Skinned fibers isolated from the paraspinal muscles were tested for their active and passive force-length relationships, unloaded shortening velocity, and myosin heavy chain isoforms. BTX-A injections led to significant fat infiltration within the injected muscles and a greater proportion of IIa to IIx fibers. Isolated fast fibers from BTX-A injected animals had lower active force and unloaded shortening velocity compared with fibers from saline-injected control animals. Force and velocity properties were not different between groups for the slow fibers. Injection of BTX-A into the paraspinal rabbit muscles leads to significant alterations in the contractile properties of fast, but not slow, fibers.Level of Evidence: N/A.

Consumption of a high-fat-high-sucrose diet partly diminishes mechanical and structural adaptations of cardiac muscle following resistance training.

[Purpose]The purpose of this study was to investigate the effects of a high-fat high-sucrose (HFHS) diet on previously reported adaptations of cardiac morphological and contractile properties to resistance training.[Methods]Twelve-week-old rats participated in 12-weeks of resistance exercise training and consumed an HFHS diet. Echocardiography and skinned cardiac muscle fiber bundle testing were performed to determine the structural and mechanical adaptations.[Results]Compared to chow-fed sedentary animals, both HFHS- and chow-fed resistance-trained animals had thicker left ventricular walls. Isolated trabecular fiber bundles from chow-fed resistance-trained animals had greater force output, shortening velocities, and calcium sensitivities than those of chow-fed sedentary controls. However, trabeculae from the HFHS resistance-trained animals had greater force output but no change in unloaded shortening velocity or calcium sensitivity than those of the chow-fed sedentary group animals.[Conclusion]Resistance exercise training led to positive structural and mechanical adaptations of the heart, which were partly offset by the HFHS diet.

Contractility of permeabilized rat vastus intermedius muscle fibres following high-fat, high-sucrose diet consumption.

Obesity is a worldwide health concern associated with impaired physical function. It is not clear if contractile protein dysfunction contributes to the impairment of muscle function observed with obesity. The purpose of this study was to examine if diet-induced obesity affects contractile function of chemically permeabilized vastus intermedius fibres of male Sprague-Dawley rats expressing fast myosin heavy chain (MHC) IIa or slow MHC I. Rats consumed either a high-fat, high sucrose (HFHS) diet or a standard (CHOW) diet beginning as either weanlings (7-week duration: WEAN7 cohort, or 14-week duration: WEAN14 cohort) or young adults (12-week duration: ADULT12 cohort, 24-week duration: ADULT24 cohort). HFHS-fed rats had higher (P < 0.05) whole-body adiposity (derived from dual-energy X-ray absorptiometry) than CHOW-fed rats in all cohorts. Relative to CHOW diet groups, the HFHS diet was associated with impaired force production in (a) MHC I fibres in the ADULT24 cohort; and (b) MHC IIa fibres in the ADULT12 and ADULT24 cohorts combined. However, the HFHS diet did not significantly affect the Ca2+-sensitivity of force production, unloaded shortening velocity, or ratio of active force to active stiffness in any cohort. We conclude that diet-induced obesity can impair force output of permeabilized muscle fibres of adult rats. Novelty: We assessed contractile function of permeabilized skeletal muscle fibres in a rat model of diet-induced obesity. The high-fat, high-sucrose diet was associated with impaired force output of fibres expressing MHC I or MHC IIa in some cohorts of rats. Other measures of contractile function were not significantly affected by diet.

Mandibular muscle troponin of the Florida carpenter ant Camponotus floridanus: extending our insights into invertebrate Ca2+ regulation.

Ants use their mandibles for a variety of functions and behaviors. We investigated mandibular muscle structure and function from major workers of the Florida carpenter ant Camponotusfloridanus: force-pCa relation and velocity of unloaded shortening of single, permeabilized fibres, primary sequences of troponin subunits (TnC, TnI and TnT) from a mandibular muscle cDNA library, and muscle fibre ultrastructure. From the mechanical measurements, we found Ca2+-sensitivity of isometric force was markedly shifted rightward compared with vertebrate striated muscle. From the troponin sequence results, we identified features that could explain the rightward shift of Ca2+-activation: the N-helix of TnC is effectively absent and three of the four EF-hands of TnC (sites I, II and III) do not adhere to canonical sequence rules for divalent cation binding; two alternatively spliced isoforms of TnI were identified with the alternatively spliced exon occurring in the region of the IT-arm α-helical coiled-coil, and the N-terminal extension of TnI may be involved in modulation of regulation, as in mammalian cardiac muscle; and TnT has a Glu-rich C-terminus. In addition, a structural homology model was built of C.floridanus troponin on the thin filament. From analysis of electron micrographs, we found thick filaments are almost as long as the 6.8μm sarcomeres, have diameter of ~ 16nm, and typical center-to-centerspacing of ~ 46nm. These results have implications for the mechanisms by which mandibular muscle fibres perform such a variety of functions, and how the structure of the troponin complex aids in these tasks.

CMyBP-C phosphorylation modulates the time-dependent slowing of unloaded shortening in murine skinned myocardium.

In myocardium, phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) is thought to modulate the cooperative activation of the thin filament by binding to myosin and/or actin, thereby regulating the probability of cross-bridge binding to actin. At low levels of Ca2+ activation, unloaded shortening velocity (Vo) in permeabilized cardiac muscle is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The velocities in these phases scale with the level of activation, culminating in a single high-velocity phase (Vmax) at saturating Ca2+. To test the idea that cMyBP-C phosphorylation contributes to the activation dependence of Vo, we measured Vo before and following treatment with protein kinase A (PKA) in skinned trabecula isolated from mice expressing either wild-type cMyBP-C (tWT), nonphosphorylatable cMyBP-C (t3SA), or phosphomimetic cMyBP-C (t3SD). During maximal Ca2+ activation, Vmax was monophasic and not significantly different between the three groups. Although biphasic shortening was observed in all three groups at half-maximal activation under control conditions, the high- and low-velocity phases were faster in the t3SD myocardium compared with values obtained in either tWT or t3SA myocardium. Treatment with PKA significantly accelerated both the high- and low-velocity phases in tWT myocardium but had no effect on Vo in either the t3SD or t3SA myocardium. These results can be explained in terms of a model in which the level of cMyBP-C phosphorylation modulates the extent and rate of cooperative spread of myosin binding to actin.

Paraspinal Muscle Contractile Function is Impaired in the ENT1-deficient Mouse Model of Progressive Spine Pathology.

Basic science study of the relationship between spine pathology and the contractile ability of the surrounding muscles. The aim of this study was to investigate single muscle fiber contractile function in a model of progressive spine mineralization (ENT1-/- mice). Altered muscle structure and function have been associated with various spine pathologies; however, studies to date have provided limited insight into the fundamental ability of spine muscles to actively contract and generate force, and how this may change in response to spine pathology. Experiments were performed on two groups (ENT1-/- [KO] and ENT1+/+ [WT]) of mice at 8 months of age (n = 12 mice/group). Single muscle fibers were isolated from lumbar multifidus and erector spinae, as well as tibialis anterior (a non-spine-related control) and tested to determine their active contractile characteristics. The multifidus demonstrated decreases in specific force (type IIax fibers: 36% decrease; type IIb fibers: 29% decrease), active modulus (type IIax: 35% decrease; type IIb: 30% decrease), and unloaded shortening velocity (Vo) (type IIax: 31% decrease) in the ENT1-/- group when compared to WT controls. The erector spinae specific force was reduced in the ENT1-/- mice when compared to WT (type IIax: 29% decrease), but active modulus and Vo were unchanged. There were no differences in any of the active contractile properties of the lower limb TA muscle, validating that impairments observed in the spine muscles were specific to the underlying spine pathology and not the global loss of ENT1. These results provide the first direct evidence of cellular level impairments in the active contractile force generating properties of spine muscles in response to chronic spine pathology.Level of Evidence: N/A.