Sarcomere Length Measurements Research Articles

Calmodulin kinase II inhibition suppresses atrioventricular conduction by regulating intracellular Ca2+ homeostasis

BackgroundCa2+/calmodulin-dependent protein kinase II (CaMKII) inhibition decelerates atrioventricular node (AVN) conduction, providing a potential treatment of tachycardia. However, the effectiveness of CaMKII inhibition on tachycardia and its underlying mechanism remains unclear. ObjectiveWe aimed to assess the effectiveness of CaMKII inhibition in reducing ventricular rates during atrial fibrillation and to elucidate the underlying mechanism in affecting AVN electrophysiology. MethodsCardiac CaMKII inhibition (AC3-I) mice were used. Transesophageal atrial pacing was performed to evaluate AVN conduction function and to induce atrial fibrillation. Patch-clamp techniques were employed to record action potentials and ionic currents in AVN cells. Intracellular Ca2+ transients and sarcomere length measurements were obtained with the IonOptix system. Masson trichrome stain was used to evaluate fibrosis in the AVN region. Western blotting and immunofluorescence techniques were employed to detect connexin expression and localization. ResultsCaMKII inhibition decreased the ventricular rate during atrial fibrillation and isoproterenol-induced tachycardia. Esophageal electrocardiogram results from AC3-I mice showed longer AVN conduction than in wild-type mice. AN- and N-type AVN cells from AC3-I mice exhibited slower action potential frequencies and diastolic depolarization rates than those of wild-type mice. The study revealed that CaMKII inhibition reduced AVN cell sarcoplasmic reticulum (SR) Ca2+ content, Ca2+ release rate from the SR during diastole, Ca2+ transient amplitude, and SR Ca2+ uptake rate. In addition, CaMKII inhibition prolonged the sarcomere diastole duration and enhanced the sensitivity of sarcomeres to Ca2+. ConclusionCaMKII inhibition effectively decreases the ventricular rate during atrial fibrillation and tachycardia by slowing down AVN conduction through suppressing Ca2+ overload in AVN cells.

Senolysis ameliorates increased susceptibility to ventricular arrhythmias in prediabetes

Abstract Background Obesity and associated prediabetes are major risk factors for cardiovascular disease that promote cellular senescence and predispose to ventricular arrhythmias (VA). In consequence, targeting cellular senescence may represent a feasible therapeutical approach that can decrease VA susceptibility in aged hearts. The aim of the present study was to characterize the impact of senolysis on glucose control, left ventricular function (LVF) and VA susceptibility in a prediabetic mouse model. Methods Ten – 12 week-old C57Bl/6J mice were fed a high-fat diet over a period of 12 weeks to generate diet-induced-obesity (DIO). Afterwards, mice were treated with dasatinib (5 mg/kg) and quercetin (50 mg/kg) (DQ) or vehicle (10 % DMSO) by oral gavage three times in 7 days. Cardiac senescence was assessed by measuring Cdkn1a mRNA expression levels. Glucose control was monitored by glucose tolerance testing. LVF was evaluated by echocardiography and myocardial contractility was assessed by sarcomere length measurements in paced single cardiomyocytes. VA susceptibility testing was performed by programmed ventricle stimulation combined with an epicardial fluorescence imaging of the LV anterior wall in isolated hearts. VA susceptibility was quantified by an established scoring system. Results Cardiac senescence, as assessed by expression of Cdkn1a mRNA (encoding p21), was increased in DIO mice (P=0.0004, Figure 1A), whereas DQ reduced cardiac senescence (P<0.0001, Figure 1A). Glucose control was impaired in DIO mice (P<0.0001) and could be partially restored by DQ (P=0.003). LVF was ameliorated by DQ in DIO mice (P=0.018). In agreement, contractility in single cardiomyocytes was improved (Amplitude of sarcomere contraction: P=0.0003, figure 1B; speed of sarcomere contraction: P=0.046). VA susceptibility was increased in DIO mice (number of inducible VAs: P=0.001, figure 1C; VA score: P=0.0002; VT inducibility: P=0.001) whereas DQ limited VA susceptibility (number of induced VAs: P=0.004, Figure 1C; VA score: P=0.004; VT inducibility: P=0.025) in isolated hearts. Conclusions Our findings demonstrate that senolysis ameliorates glucose tolerance and LVF while reducing VA susceptibility in obesity and prediabetes. In consequence, senolysis may offer a perspective therapeutical approach for obese patients at risk for VAs. Further experiments will address underlying molecular mechanisms to identify potential therapeutical targets.

Age-related blunting of serial sarcomerogenesis and mechanical adaptations following 4 weeks of maximal eccentric resistance training

During natural aging, muscles atrophy, which is partly accounted for by a loss of sarcomeres in series. Serial sarcomere number (SSN) is associated with aspects of muscle mechanical function including the force-length and force-velocity-power relationships; hence, the age-related loss of SSN contributes to declining performance. Training emphasizing muscle lengthening (eccentric) contractions increases SSN in young healthy rodents. However, the ability for eccentric training to increase SSN and improve mechanical function in old age is unknown. Older individuals often exhibit a blunted ability for training-induced hypertrophic adaptations. Therefore, the purpose of this study was to investigate the sarcomerogenic response to eccentric training in old rats. We hypothesized that old rats would experience a smaller magnitude of serial sarcomerogenesis than young rats, which would correspond to smaller beneficial adaptations in mechanical function as compared with young. Ten young (9 months) and 11 old (33 months) Fisher344/BN F1 rats completed 4 weeks of unilateral isokinetic eccentric plantar flexion training 3 days/week. Pre- and post-training, the plantar flexors were assessed for maximum tetanic torque (ankle angles of 70° and 90°), the torque-frequency relationship (stimulation frequencies of 1-100 Hz), the passive torque-angle relationship (ankle angles of 110-70°), and the torque-angular velocity-power relationship (isotonic loads of 10%-80% maximum). Following post-training testing, rats were sacrificed, and the soleus, lateral gastrocnemius (LG), and medial gastrocnemius (MG) were harvested for SSN assessment by measuring sarcomere lengths with laser diffraction, with the untrained leg used as a control. In the untrained leg/pre-training, old rats had lower SSN in the soleus (−9%), LG (−7%), and MG (−14%), lower maximum torque (−27 to −42%), power (−63%), and shortening velocity (−35%), and greater passive torque (+62 to +191%) than young. Young rats showed 6% increased SSN from the untrained to the trained soleus and MG. In contrast, old rats had no change in soleus SSN, only a 2% increase in MG SSN, and 4% SSN loss in the LG. Young rats saw modest improvements in isometric mechanical function, including a 13% increase in maximum torque at 90° and 4-11% increases in 10-60 Hz torque. Old rats, however, had reductions in maximum torque (−35%), shortening velocity (−46%), and power (−63%), and increased passive torque (+24 to +51%). Altogether, eccentric training induced serial sarcomerogenesis and improved mechanical function in young rats, while old rats exhibited dysfunctional remodelling that led to impaired muscle mechanical performance. Supported by NSERC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Surgically Repairing Achilles Tendon Ruptures Restores Plantar Flexor Function Better Than Non- Surgical Treatment in a Rat Model

Category: Basic Sciences/Biologics; Sports Introduction/Purpose: Achilles tendon ruptures cause long-term functional deficits in nearly 2 out of 3 patients. Our group has established a complex muscle-tendon pathology following isolated Achilles tendon ruptures. Our long-term goal is to determine how rehabilitation loads can simultaneously accomplish 2 competing objectives: (1) prevent muscle remodeling while (2) not overloading the healing tendon that causes permanent tendon elongation - unloading the muscle and stimulating muscle remodeling. To do this, we developed a novel experimental framework to test the potential therapeutic effectiveness of different loading paradigms on the healing muscle-tendon following surgically induced Achilles tendon ruptures in an established rat model. We hypothesized that overloading the healing tendon would lead to worse functional outcomes compared to unloaded immobilization. Methods: Achilles tendon rupture, repair, and immobilization. Male Sprague-Dawley rats underwent a surgically induced Achilles tendon rupture by blunt dissection followed by a modified Kessler repair in this IACUC approved study. The repaired limb was immobilized in either unloaded plantar flexion (n=7) to simulate the clinical standard of rehabilitative care or loaded dorsiflexion (n=5) to simulate clinically damaging rehabilitation. Structural analyses. Following 2 weeks of immobilization and 2 weeks of cage recovery, we sacrificed our study animals and measured tendon elongation and serial sarcomere count. Plantar flexor functional assessment. We developed an ankle dynamometer to quantify plantar flexor function that mirrored our clinical assessments in patients. To demonstrate the clinical- relevance of this device, we tested 1 animal that was immobilized in unloaded plantar flexion for 1 week and 1 animal that was immobilized in loaded dorsiflexion for 1 week - both then completed 2 weeks of unrestricted cage activity. Results: Muscle-tendon structural changes. Achilles tendon ruptures caused the tendon to heal in an elongated position with fewer sarcomeres in series (Fig. A). Tendon length increased by 73% following 2-weeks of loaded immobilization in dorsiflexion and 23% in unloaded immobilization in plantar flexion (p<.001). We measured sarcomere length in a subset of these animals but found similar trends in serial sarcomere reductions, where unloaded immobilization in plantar flexion caused 14% reduced serial sarcomeres (p<.01, n=4) and loaded immobilization in dorsiflexion caused 32% reduced serial sarcomeres (n=2). Plantar flexor function. Plantar flexor work was adversely impacted by the Achilles tendon ruptures (Fig. B). We found that unloaded immobilization in plantar flexion caused a 13% decrease in plantar flexor work with the detectable deficits occurring in ankle plantar flexion. Loaded immobilization in dorsiflexion caused greater plantar flexor work deficits (28%) with greater torque deficits in dorsiflexion and continuing through plantar flexion angles. Conclusion: Our exciting experimental findings are the first of their kind to directly link muscle-tendon structural deficits with plantar flexor functional deficits in an animal model of Achilles tendon rupture using a clinically relevant ankle dynamometer. We established preliminary evidence that supports our overarching hypothesis that joint immobilization, whether unloaded or loaded, causes detrimental muscle-tendon structural changes that impose functional constraints on plantar flexor work. Our preliminary findings suggest that rehabilitation loading is critical to structural and functional outcomes following surgically repairing the Achilles tendon. Too little loading promotes muscle remodeling while too much loading elongates the healing tendon.

Brachial plexus birth injury and cerebral palsy lead to a common contracture phenotype characterized by reduced functional muscle length and strength.

IntroductionBrachial plexus birth injury (BPBI) and cerebral palsy (CP) both cause disabling contractures for which no curative treatments exist, largely because contracture pathophysiology is incompletely understood. The distinct neurologic nature of BPBI and CP suggest different potential contracture etiologies, although imbalanced muscle strength and insufficient muscle length have been variably implicated. The current study directly compares the muscle phenotype of elbow flexion contractures in human subjects with BPBI and CP to test the hypothesis that both conditions cause contractures characterized by a deficit in muscle length rather than an excess in muscle strength.MethodsSubjects over 6 years of age with unilateral BPBI or hemiplegic CP, and with elbow flexion contractures greater than 10 degrees on the affected side, underwent bilateral elbow flexion isokinetic strength testing to identify peak torque and impulse, or area under the torque-angle curve. Subjects then underwent needle microendoscopic sarcomere length measurement of bilateral biceps brachii muscles at symmetric joint angles.ResultsIn five subjects with unilateral BPBI and five with hemiplegic CP, peak torque and impulse were significantly lower on the affected versus unaffected sides, with no differences between BPBI and CP subjects in the percent reduction of either strength measurement. In both BPBI and CP, the percent reduction of impulse was significantly greater than that of peak torque, consistent with functionally shorter muscles. Similarly, in both conditions, affected muscles had significantly longer sarcomeres than unaffected muscles at symmetric joint angles, indicating fewer sarcomeres in series, with no differences between BPBI and CP subjects in relative sarcomere overstretch.DiscussionThe current study reveals a common phenotype of muscle contracture in BPBI and CP, with contractures in both conditions characterized by a similar deficit in muscle length rather than an excess in muscle strength. These findings support contracture treatments that lengthen rather than weaken affected muscles. Moreover, the discovery of a common contracture phenotype between CP and BPBI challenges the presumed dichotomy between upper and lower motor neuron lesions in contracture pathogenesis, instead revealing the broader concept of “myobrevopathy”, or disorder of short muscle, warranting increased investigation into the poorly understood mechanisms regulating muscle length.

The Sex-Dependent Role of Myostatin Signaling in Contractures Following Neonatal Brachial Plexus Injury

Purpose: Neonatal brachial plexus injury (NBPI) causes disabling and incurable muscle contractures, arising from impaired longitudinal growth of denervated muscles. This growth impairment is caused by dysregulation of muscle proteostasis characterized by increased proteasome-mediated protein degradation. Myostatin (MSTN), a secreted growth differentiation factor and prominent muscle-specific negative regulator of proteostasis, drives muscle protein degradation. MSTN signaling is thus a possible pathway through which denervation impairs muscle growth and a potential muscle-specific molecular target for preventing contractures. This study uses pharmacologic inhibition of MSTN signaling in a mouse model of NBPI to test the hypothesis that NBPI induces contractures through MSTN-dependent impairment of longitudinal muscle growth. Methods: Unilateral NBPI was generated in postnatal day (P)5 mice. Mice were treated weekly with Dulbecco’s phosphate-buffered saline (DPBS) or ACVR2B-Fc, a soluble inhibitory decoy receptor for MSTN, starting at P4. Mice were sacrificed at P33, whereupon elbow and shoulder range of motion were measured to assess contractures. Brachialis muscles were prepared for microCT assessment of cross-sectional area (CSA) and volume, then processed for sarcomere length measurement as a readout of longitudinal muscle growth. Total protein content in biceps was assessed via Bradford assay. Protein degradation in triceps was quantified via 20S proteasome β1 subunit activity assay. Results: In female mice, myostatin inhibition with ACVR2B-Fc enhanced muscle weight and total protein content of denervated biceps (Figure 1), increased cross-sectional and volumetric growth of denervated brachialis (Figure 2), preserved longitudinal growth in denervated brachialis (Figure 3), and reduced elbow and shoulder contracture severity in the denervated limbs (Figure 4). Analysis of protein dynamics demonstrates the improvements in female denervated muscles were associated with a reduction in proteasome β1 subunit activity (Figure 5). However, no such improvements in denervated muscles occurred in male mice with ACVR2B-Fc. Conversely, myostatin inhibition increased muscle weight, CSA, volume, and total protein content of the normally innervated, contralateral control muscles in both male and female mice (Figures 1 and 2). Conclusion: Our findings reveal MSTN as a driver of impaired muscle growth and contracture formation in female but not male mice. This sex dimorphism lies in the pathophysiology of denervation-induced contractures rather than drug pharmacodynamics, given the sex-independent effects of MSTN inhibition on normally innervated muscle. Future studies must rigorously interrogate the contributions of sex in contracture pathophysiology. Significance: Our discovery identifies MSTN as a signaling target for modulating contractures in females and highlights the overlooked role of sex in the pathophysiology of acquired neuromuscular disorders. Level of Evidence: Not Applicable FDA: The FDA has not cleared the following pharmaceuticals and/or medical devices for the use described in this presentation.

Shortening the thick filament by partial deletion of titin's C-zone alters cardiac function by reducing the operating sarcomere length range

Titin's C-zone is an inextensible segment in titin, comprised of 11 super-repeats and located in the cMyBP-C-containing region of the thick filament. Previously we showed that deletion of titin's super-repeats C1 and C2 (TtnΔC1–2 model) results in shorter thick filaments and contractile dysfunction of the left ventricular (LV) chamber but that unexpectedly LV diastolic stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the LV and intact cardiomyocyte, cellular work loops of intact cardiomyocytes, Ca2+ transients, cross-bridge kinetics, and myofilament Ca2+ sensitivity. Intact cardiomyocytes of TtnΔC1–2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance at both LV and single-cell levels showed that activation kinetics are normal in TtnΔC1–2 mice, but that relaxation is slower. The slowed relaxation is, in part, attributable to an increased myofilament Ca2+ sensitivity and slower early Ca2+ reuptake. Cross-bridge dynamics showed that cross-bridge kinetics are normal but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements revealed that in TtnΔC1–2 mice the operating SL range of the LV is shifted towards shorter lengths. This normalizes the apparent cell and LV diastolic stiffness but further reduces systolic force as systole occurs further down on the ascending limb of the force-SL relation. We propose that the reduced working SLs reflect titin's role in regulating diastolic stiffness by altering the number of sarcomeres in series. Overall, our study reveals that thick filament length regulation by titin's C-zone is critical for normal cardiac function.

Mechanisms governing protective pregnancy-induced adaptations of the pelvic floor muscles in the rat preclinical model

The intrinsic properties of pelvic soft tissues in women who do and do not sustain birth injuries are likely divergent. However, little is known about this. Rat pelvic floor muscles undergo protective pregnancy-induced structural adaptations-sarcomerogenesis and increase in intramuscular collagen content-that protect against birth injury. We aimed to test the following hypotheses: (1) the increased mechanical load of a gravid uterus drives antepartum adaptations; (2) load-induced changes are sufficient to protect pelvic muscles from birth injury. The independent effects of load uncoupled from the hormonal milieu of pregnancy were tested in 3- to 4-month-old Sprague-Dawley rats randomly divided into the following 4 groups, with N of 5 to 14 per group: (1) load-/pregnancy hormones- (controls), (2) load+/pregnancy hormones-, (3) reduced load/pregnancy hormones+, and (4) load+/pregnancy hormones+. Mechanical load of a gravid uterus was simulated by weighing uterine horns with beads similar to fetal rat size and weight. A reduced load was achieved by unilateral pregnancy after unilateral uterine horn ligation. To assess the acute and chronic phases required for sarcomerogenesis, the rats were sacrificed at 4 hours or 21 days after bead loading. The coccygeus, iliocaudalis, pubocaudalis, and nonpelvic tibialis anterior musles were harvested for myofiber and sarcomere length measurements. The intramuscular collagen content was assessed using a hydroxyproline assay. An additional 20 load+/pregnancy hormones- rats underwent vaginal distention to determine whether the load-induced changes are sufficient to protect from mechanical muscle injury in response to parturition-associated strains of various magnitude. The data, compared using 2-way repeated measures analysis of variance followed by pairwise comparisons, are presented as mean±standard error of mean. An acute increase in load resulted in significant pelvic floor muscle stretch, accompanied by an acute increase in sarcomere length compared with nonloaded control muscles (coccygeus: 2.69±0.03 vs 2.30±0.06 μm, respectively, P<.001; pubocaudalis: 2.71±0.04 vs 2.25±0.03 μm, respectively, P<.0001; and iliocaudalis: 2.80±0.06 vs 2.35±0.04 μm, respectively, P<.0001). After 21 days of sustained load, the sarcomeres returned to operational length in all pelvic muscles (P>.05). However, the myofibers remained significantly longer in the load+/pregnancy hormones- than the load-/pregnancy hormones- in coccygeus (13.33±0.94 vs 9.97±0.26 mm, respectively, P<.0001) and pubocaudalis (21.20±0.52 vs 19.52±0.34 mm, respectively, P<.04) and not different from load+/pregnancy hormones+ (12.82±0.30 and 22.53±0.32 mm, respectively, P>.1), indicating that sustained load-induced sarcomerogenesis in these muscles. The intramuscular collagen content in the load+/pregnancy hormones- group was significantly greater relative to the controls in coccygeus (6.55±0.85 vs 3.11±0.47 μg/mg, respectively, P<.001) and pubocaudalis (5.93±0.79 vs 3.46±0.52 μg/mg, respectively, P<.05) and not different from load+/pregnancy hormones+ (7.45±0.65 and 6.05±0.62 μg/mg, respectively, P>.5). The iliocaudalis required both mechanical and endocrine cues for sarcomerogenesis. The tibialis anterior was not affected by mechanical or endocrine alterations. Despite an equivalent extent of adaptations, load-induced changes were only partially protective against sarcomere hyperelongation. Load induces plasticity of the intrinsic pelvic floor muscle components, which renders protection against mechanical birth injury. The protective effect, which varies between the individual muscles and strain magnitudes, is further augmented by the presence of pregnancy hormones. Maximizing the impact of mechanical load on the pelvic floormuscles during pregnancy, such as with specialized pelvic floor muscle stretching regimens, is a potentially actionable target for augmenting pregnancy-induced adaptations to decrease birth injury in women who may otherwise have incomplete antepartum muscle adaptations.

Abstract 13931: Upregulation of the AKT/mTOR Pathway in Premature Ventricular Contractions (PVC)-Induced Cardiomyopathy

Introduction: Premature ventricular contractions (PVCs) are early ventricular depolarizations that, if frequent, can be associated with PVC-induced cardiomyopathy (PVC-CM). PVC-CM in our translational model is characterized by LV systolic dysfunction and eccentric hypertrophy. However, the mechanisms involved in this LV remodeling remain unknown. Objectives: Evaluate changes in cardiomyocyte size and sarcomere shortening, along with classical molecular pathways involved in pathological or physiological hypertrophy in the PVC-CM model. Methods: Pacemakers were implanted in healthy canines to reproduce 3 months of bigeminal PVCs (50% burden at 200-220 ms coupling interval). Isolated ventricular cardiomyocytes were electrically paced to measure sarcomere length. Formaldehyde-fixed LV sections were stained with WGA-AF633. Expression of β-MHC, ACTA1, ANP, calcineurin, AKT, mTOR, S6, 4EBP1, NFAT and their phosphorylated forms were measured by Western blot. Calcineurin activity was determined by colorimetry. Results: Cardiomyocyte area was larger in PVC group vs. sham (357.4 ± 12.9 μm 2 n= 121 vs. 271.8 + 7 μm 2 n = 161, p&lt;0.001). Time to peak sarcomere contraction was longer in the PVC group (124.9 + 25.2 ms, n=101 vs 116.2 + 27.4 ms, n=61, p&lt;0.05), whereas maximal cell shortening was similar between groups. Western blot analyses (n=6) showed significant increases in AKT expression (1 + 0.18 vs 1.6 + 0.43, p&lt;0.001), and in its downstream effectors, S6 (1 + 0.26 vs 1.46 + 0.4, p&lt;0.05) and 4EBP1 (1 + 0.3 vs 1.57 + 0.38, p&lt;0.001) in the PVC group, without changes in mTOR. On the contrary, PVC group displayed a reduction in phosphorylated AKT (Ser473, 1 + 0.14 vs 0.77 + 0.26, p&lt;0.05) and 4EBP1 (Thr37/46, 1 + 0.15 vs 0.75 + 0.1, p&lt;0.001) without changes in S6 (Ser235/236) or mTOR (Ser2448) phosphorylation. The activity and expression of calcineurin, total and phospho- NFAT, and the expression of β-MHC, ACTA1, and ANP were not different. Conclusion: The pathological calcineurin/NFAT/fetal genes pathway was unchanged in the PVC-induced eccentric hypertrophy. Thus, this hypertrophy is “atypical” characterized by an upregulation of the AKT/mTOR pathway and cellular hypertrophy, together with a paradoxical decrease in contractility and LV systolic dysfunction.

Abstract P315: Shortening The Thick Filament By Partial Deletion Of Titin’S C-zone Alters Cardiac Function By Reducing The Operating Sarcomere Length Range Of The Heart.

Titin’s C-zone is the inextensible part of titin that binds along the thick filament at its cMyBP-C -containing region. Previously it was shown that deletion of titin’s super-repeats C1 and C2 ( Ttn ΔC1-2 mouse model) results in shorter thick filaments and contractile dysfunction, but LV chamber stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the left ventricle (LV) and from cellular work loops of intact loaded cardiac myocytes. Ca 2+ transients were also measured as well as crossbridge cycling kinetics and Ca 2+ sensitivity of force. It was found that intact cardiomyocytes of Ttn ΔC1-2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance of the LV chamber showed that the kinetics of LV activation are normal but that relaxation is slower in Ttn ΔC1-2 mice. The slowed relaxation was, in part, attributable to an increased myofilament Ca 2+ sensitivity and slower early Ca 2+ reuptake. Dynamic stiffness at the myofilament level showed that cross-bridge kinetics are normal, but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements in the mid-wall region of the LV revealed that the operating SL range is shifted in Ttn ΔC1-2 mice towards shorter lengths. This normalizes the apparent cell and LV chamber stiffness but reduces the number of force generating cross-bridges due to suboptimal thin and thick filament overlap. Thus the contractile dysfunction in Ttn ΔC1-2 mice is not only due to shorter thick filaments but also to a reduced operating sarcomere length range. Overall these results reveal that for normal cardiac function, thick filament length regulation by titin’s C-zone is critical.

The cibarial pump of the xylem-feeding froghopper Philaenus spumarius produces negative pressures exceeding 1 MPa.

The xylem sap of vascular plants is an unlikely source of nutrition, being both nutrient poor and held under tensions (negative pressures) that can exceed 1 MPa. But some insects feed on xylem sap exclusively, extracting copious quantities using a muscular cibarial pump. However, neither the strength of the insect's suction, nor the direct energetic cost of xylem ingestion, have ever been quantified. Philaenus spumarius froghoppers were used to address these gaps in our knowledge. Micro-CT scans of its cibarium and measurements of cibarial muscle sarcomere length revealed that P. spumarius can generate a maximum tension of 1.3 ± 0.2 MPa within its cibarium. The energetic cost of xylem extraction was quantified using respirometry to measure the metabolic rate (MR) of P. spumarius while they fed on hydroponically grown legumes, while xylem sap excretion rate and cibarial pumping frequency were simultaneously recorded. Increasing the plants' xylem tensions up to 1.1 MPa by exposing their roots to polyethylene glycol did not reduce the insects' rate of xylem excretion, but significantly increased both MR and pumping frequency. We conclude that P. spumarius can gain energy feeding on xylem sap containing previously reported energy densities and at xylem tensions up to their maximum suction capacity.

Sarcomere length measurement reliability in single myofibrils

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

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.

Quantitative Evaluation of the Sarcomere Network of Human hiPSC-Derived Cardiomyocytes Using Single-Molecule Localization Microscopy.

The maturation of iPSC-derived cardiomyocytes is still a critical point for their application in cardiovascular research as well as for their clinical use. Although multiple differentiation protocols have been established, researchers failed to generate fully mature cardiomyocytes in vitro possessing identical phenotype-related and functional properties as their native adult counterparts. Besides electrophysiological and metabolic changes, the establishment of a well structured sarcomere network is important for the development of a mature cardiac phenotype. Here, we present a super resolution-based approach to quantitatively evaluate the structural maturation of iPSC-derived cardiomyocytes. Fluorescence labelling of the α-actinin cytoskeleton and subsequent visualization by photoactivated localization microscopy allows the acquisition of highly resolved images for measuring sarcomere length and z-disc thickness. Our image analysis revealed that iPSC and neonatal cardiomyocyte share high similarity with respect to their sarcomere organization, however, contraction capacity was inferior in iPSC-derived cardiac cells, indicating an early maturation level. Moreover, we demonstrate that this imaging approach can be used as a tool to monitor cardiomyocyte integrity, helping to optimize iPSC differentiation as well as somatic cell direct-reprogramming strategies.

Tissue-engineered human embryonic stem cell-containing cardiac patches: evaluating recellularization of decellularized matrix

Decellularized cardiac extracellular matrix scaffolds with preserved composition and architecture can be used in tissue engineering to reproduce the complex cardiac extracellular matrix. However, evaluating the extent of cardiomyocyte repopulation of decellularized cardiac extracellular matrix scaffolds after recellularization attempts is challenging. Here, we describe a unique combination of biochemical, biomechanical, histological, and physiological parameters for quantifying recellularization efficiency of tissue-engineered cardiac patches compared with native cardiac tissue. Human embryonic stem cell-derived cardiomyocytes were seeded into rat heart atrial and ventricular decellularized cardiac extracellular matrix patches. Confocal and atomic force microscopy showed cell integration within the extracellular matrix basement membrane that was accompanied by restoration of native cardiac tissue passive mechanical properties. Multi-electrode array and immunostaining (connexin 43) were used to determine synchronous field potentials with electrical coupling. Myoglobin content (~60%) and sarcomere length measurement (>45% vs 2D culture) were used to evaluate cardiomyocyte maturation of integrated cells. The combination of these techniques allowed us to demonstrate that as cellularization efficiency improves, cardiomyocytes mature and synchronize electrical activity, and tissue mechanical/biochemical properties improve toward those of native tissue.

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.

Chronic uphill and downhill exercise protocols do not lead to sarcomerogenesis in mouse skeletal muscle

It has been suggested that eccentric contraction (EC) is associated with increases in serially arranged sarcomeres (sarcomerogenesis), while concentric contraction (CC) has been associated with serial sarcomeres decrease. Sarcomerogenesis following EC is thought to be a protective muscle adaptation, preventing muscle injury in future eccentric exercise bouts (repeated bout effect). However, the mechanisms underlying sarcomerogenesis in EC remain unknown, and the sarcomerogenic responses observed in response to EC and CC are contradictory. We measured sarcomere length, sarcomere length uniformity, serial sarcomere number, and fascicle length in gastrocnemius medialis, tibialis anterior, vastus medialis and vastus lateralis in sedentary (SED) mice, and in mice following protocols of moderate uphill (TRU) and downhill (TRD) training and uphill (OTU) and downhill (OTD) overtraining. We found pain sensitivity after the first bout of EC exercise on TRD and OTD followed by a normalized sensory response after four weeks of training, indicating a repeated bout effect. However, these findings were not associated with sarcomerogenesis, as serial sarcomere numbers did not increase in TRD and OTD skeletal muscle samples compared to controls (SED). However, we found a decrease in serial sarcomere number in VL and TA in OTU group mice, which was associated with a decrease in fascicle length and no change of sarcomere length at the tested joint configuration. We conclude that excessive concentric muscle contraction (OTU group mice), leads to a decrease in serial sarcomere number, while moderate or excessive eccentric training, did not result in sarcomerogenesis, as reported in the literature.

Iodine Staining Results in Significant Shrinkage of Sarcomere Lengths in Macaque Biceps Brachii Muscle

Sarcomere lengths are widely used to calculate scaled sarcomere models of whole muscle length‐tension curves. Previous studies have shown that scaled sarcomere models of fresh tissues accurately predict experimental whole muscle length‐tension curves. Diffusible iodine‐based contrast enhanced CT (diceCT) imaging employs iodine staining with Lugol's solution (I2KI) to improve visualization of muscle tissue to the level of individual fascicles, which holds great promise to allow length‐tension modeling of multiple muscles. However, staining skeletal muscles with I2KI causes significant volumetric shrinkage of the muscle, even at levels as low as 2% I2/4% KI, which may affect the validity of length‐tension curves calculated from these tissues. Here we test the hypothesis that iodine staining distorts sarcomere length measurements. We evaluated the impact of I2KI on sarcomere lengths measured from muscle samples of the right biceps brachii from one formalin‐fixed rhesus macaque (Macaca mulatta). We sectioned the muscle into a primary control (no I2KI) and two experimental groups: one treated with 20% (w/v) I2KI and one pretreated with a sucrose prewash (20% w/v) prior to iodine staining. The muscle sections then were chemically digested in 70% nitric acid in saline and mounted on slides. A minimum of three sarcomere length measurements was taken per fiber using laser diffraction and the averages used for ANOVA and post hoc t‐tests. The results show that iodine staining significantly shrinks sarcomere lengths (2.08 ± 0.22 μm) compared with primary controls (2.45 ± 0.25 μm) (p &lt; 0.001). The sucrose prewash group also experienced significant shrinkage (2.13 ± 0.23 μm). These data demonstrate the need for methods which correct for this pronounced sarcomere shrinkage before iodine stained skeletal muscle samples can be used to calculate whole muscle length‐tension curves.Support or Funding InformationNational Science Foundation Grant, Division of Behavioral and Cognitive Science, DDRI Bio Anthro, Award #1732175This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Diverse relaxation rates exist among rat cardiomyocytes isolated from a single myocardial region.

Prior studies have shown variation in the functional properties of cardiomyocytes isolated from different regions of the left ventricular myocardium. We found that these region-dependent variations vanish below a tissue volume of ∼7 mm3 in the adult rat myocardium, revealing a fixed level of intrinsic relaxation rate heterogeneity that is independent of tissue volume. Within these microscopically varying cell populations, fast-relaxing cells were shown to have elevated phosphorylated troponin I compared to slow-relaxing cells. Relaxation rate was also correlated with cardiomyocyte length, in that slow-relaxing cells were longer than fast-relaxing cells. These results show a new relationship between cardiomyocyte morphology and myofilament relaxation, and suggest that functional diversity among individual myocytes at the microscale may contribute to bulk relaxation of the myocardium. The mean contractility and calcium handling properties of cardiomyocytes isolated from different regions of the ventricular myocardium are known to vary significantly. We designed experiments to quantify the variance in contractile properties among cells within the same myocardial region. Longitudinal strips of myocardial tissue were excised from the epicardial left ventricular free walls of adult Sprague-Dawley rats and then treated with collagenase to isolate individual myocytes. Cardiomyocytes were characterized by measuring sarcomere length changes and calcium transients during electrical pacing. Variance of the time from peak sarcomere shortening to 50% re-lengthening (RT50 ) was assessed in each cell population. Isolating cells from progressively shorter strips allowed an estimate of the myocardial volume below which regional variation vanished and only microscale heterogeneity remained (∼7 mm3 ). The SD of RT50 within this myocardial volume was 28% of the mean. In a series of follow-up experiments, RT50 was shown to correlate significantly with resting myocyte length, suggesting a connection between cell morphology and intrinsic relaxation behaviour. To explore the mechanistic basis of varying RT50 , a novel single-cell aspirator was employed to collect small batches of cardiomyocytes grouped according to their relaxation rates (fast or slow). Western blot analysis of the two groups revealed significantly elevated troponin I phosphorylation in fast-relaxing cells. Our observations suggest that cell-to-cell heterogeneity of active contractile properties is substantial, with implications for how we understand myocardial relaxation and design drug therapies intended to alter relaxation rate.