Sarcomere Force Research Articles

What Can We Learn from Single Sarcomere and Myofibril Preparations?

Sarcomeres are the smallest functional contractile unit of muscle, and myofibrils are striated muscle organelles that are comprised of sarcomeres that are strictly aligned in series. Furthermore, passive forces in sarcomeres and myofibrils are almost exclusively produced by the structural protein titin, and all contractile, regulatory, and structural proteins are in their natural configuration. For these mechanical and structural reasons single sarcomere and myofibril preparations are arguably the most powerful to answer questions on the mechanisms of striated muscle contraction. We developed and optimized single myofibril research over the past 20 years and were the first to mechanically isolate and test single sarcomeres. The results from this research led to the uncovering of the crucial role of titin in muscle contraction, first molecular explanations for the origins of the passive and the residual force enhancement properties of skeletal and cardiac muscles, the discovery of sarcomere length stability on the descending limb of the force-length relationship, and culminating in the formulation of the three-filament theory of muscle contraction that, aside from actin and myosin, proposes a crucial role of titin in active force production. Aside from all the advantages and possibilities that single sarcomere and myofibril preparations offer, there are also disadvantages. These include the fragility of the preparation, the time-consuming training to master these preparations, the limited spatial resolution for length and force measurements, and the unavailability of commercial systems for single sarcomere/myofibril research. Ignoring the mechanics that govern serially linked systems, not considering the spatial resolution and associated accuracies of myofibril systems, and neglecting the fragility of myofibril preparations, has led to erroneous interpretations of results and misleading conclusions. Here, we will attempt to describe the methods and possible applications of single sarcomere/myofibril research and discuss the advantages and disadvantages by focusing on specific applications. It is hoped that this discussion may contribute to identifying the enormous potential of single sarcomere/myofibril research in discovering the secrets of muscle contraction.

Muscle LIM Protein Force-Sensing Mediates Sarcomeric Biomechanical Signaling in Human Familial Hypertrophic Cardiomyopathy.

Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics. Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell-derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations. Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain (MYH7) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin-nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein-W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations. Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex.

2-Deoxy-ATP improves ventricular function via combined effects on myosin recruitment from the super-relaxed state, crossbridge cycling and calcium dynamics in healthy and failing conditions

2-deoxy-ATP (dATP) has been shown to enhance cardiac contractility by acting on myosin to increase the rate of crossbridge binding and cycling. An increased rate of Ca2+ transient decay has also been observed with elevated dATP. However, it is unclear how dATP affects feedback between sarcomere force and Ca2+ dynamics, and to what degree these cellular level effects can explain experimentally observed improvements in ventricular function with elevated dATP. Further, low concentrations of dATP have been shown to significantly improve cardiomyocyte and ventricular function, but the mechanisms behind this are not well understood.

Effect of ACTN3 Genotype on Sports Performance, Exercise-Induced Muscle Damage, and Injury Epidemiology.

Genetic factors play a significant role in athletic performance and its related phenotypes such as power, strength and aerobic capacity. In this regard, the lack of a muscle protein due to a genetic polymorphism has been found to affect sport performance in a wide variety of ways. α-actinin-3 is a protein located within the skeletal muscle with a key role in the production of sarcomeric force. A common stop-codon polymorphism (rs1815739; R577X) in the gene that codes for α-actinin-3 (ACTN3) produces individuals with the XX genotype that lack expression of a functional α-actinin-3. In contrast, individuals with the R-allele (i.e., RX vs. RR genotypes) in this polymorphism can express α-actinin-3. Interestingly, around ~18% of the world population have the XX genotype and much has been debated about why a polymorphism that produces a lack of a muscle protein has endured natural selection. Several investigations have found that α-actinin-3 deficiency due to XX homozygosity in the ACTN3 R577X polymorphism can negatively affect sports performance through several structural, metabolic, or signaling changes. In addition, new evidence suggests that α-actinin-3 deficiency may also impact sports performance through indirect factors such a higher risk for injury or lower resistance to muscle-damaging exercise. The purpose of this discussion is to provide a clear explanation of the effect of α-actinin-3 deficiency due to the ACTN3 XX genotype on sport. Key focus has been provided about the effect of α-actinin-3 deficiency on morphologic changes in skeletal muscle, on the low frequency of XX athletes in some athletic disciplines, and on injury epidemiology.

Development of an Integrated Human Model of Electrophysiology and Actin-Myosin Binding to Describe Sarcomere Force Generation in Cardiomyocytes

We have developed an integrated human model which links detailed sub-models of sarcomere electrophysiology and actin-myosin kinetics to describe sarcomere force generation. While many published quantitative models connect cardiac electrophysiology to force generation or full heart mechanics, these models typically include a lumped representation of actin-myosin mechanics without sufficient granularity. In order to mechanistically represent both heart failure conditions and treatment strategies which operate at the actin-myosin level, we have developed an integrated multiscale model of sarcomere force generation. Our platform connects published electrophysiology and lumped force generation models to an actin-myosin (AM) model developed based on published data on cardiac myosin modulators. Specifically, the AM model was calibrated to published in vitro data of phosphate release and ATP turnover, and can successfully reproduce the molecular effect of cardiac myosin modulation. We connected this in vitro model with healthy and heart failure phenotypes to describe cardiac force generation before and after therapy. With this multiscale approach we can also simulate sarcomere length-dependent activation, calcium-force relationships, and determine how these simulations are affected in failing myocardium after treatment with cardiac modulators. We demonstrate that models calibrated to data at one scale often need to be adjusted in the context of a multiscale model. For example, the calcium transient produced from a published heart failure electrophysiology model generates little force in the integrated multiscale model, underscoring the continuing challenge of linking in vitro experimental efforts to clinical outputs. We discuss our methods to connect information across scales into one coherent mathematical framework. The final model describes how specific therapeutic interventions at different stages of the actin-myosin cycle will alter sarcomere force generation, which will propagate to changes in clinical outcomes like ejection fraction.

Force\u2013length relation of skeletal muscles: from sarcomeres to myofibril

Sarcomeres are building blocks of skeletal muscles. Given force–length relations of sarcomeres serially connected in a myofibril, the myofibril force–length relation can be uniquely determined. Necessary and sufficient conditions are derived for capability of fully lengthening or completely shortening a myofibril under isometric, eccentric or concentric contraction, and for the myofibril force–length relation to be a continuous single-valued function. Intriguing phenomena such as sarcomere force–length hysteresis and myofibril regularity are investigated and their important roles in determining myofibril force–length relations are explored. The theoretical analysis leads to experimentally verifiable predictions on myofibril force–length relations. For illustration, simulated force–length relations of a myofibril portion consisting of a sarcomere pair are presented.

A small-molecule modulator of cardiac myosin acts on multiple stages of the myosin chemomechanical cycle

Mavacamten, formerly known as MYK-461 is a recently discovered novel small-molecule modulator of cardiac myosin that targets the underlying sarcomere hypercontractility of hypertrophic cardiomyopathy, one of the most prevalent heritable cardiovascular disorders. Studies on isolated cells and muscle fibers as well as intact animals have shown that mavacamten inhibits sarcomere force production, thereby reducing cardiac contractility. Initial mechanistic studies have suggested that mavacamten primarily reduces the steady-state ATPase activity by inhibiting the rate of phosphate release of β-cardiac myosin-S1, but the molecular mechanism of action of mavacamten has not been described. Here we used steady-state and presteady-state kinetic analyses to investigate the mechanism of action of mavacamten. Transient kinetic analyses revealed that mavacamten modulates multiple steps of the myosin chemomechanical cycle. In addition to decreasing the rate-limiting step of the cycle (phosphate release), mavacamten reduced the number of myosin-S1 heads that can interact with the actin thin filament during transition from the weakly to the strongly bound state without affecting the intrinsic rate. Mavacamten also decreased the rate of myosin binding to actin in the ADP-bound state and the ADP-release rate from myosin-S1 alone. We, therefore, conclude that mavacamten acts on multiple stages of the myosin chemomechanical cycle. Although the primary mechanism of mavacamten-mediated inhibition of cardiac myosin is the decrease of phosphate release from β-cardiac myosin-S1, a secondary mechanism decreases the number of actin-binding heads transitioning from the weakly to the strongly bound state, which occurs before phosphate release and may provide an additional method to modulate myosin function.

A chemo-mechanical model for the single myofibril in striated muscle contraction

Based on the framework of sliding-filament theory and on the cross-bridges dynamics, a mathematical model for the simulation of the force response and length change of individual myofibril is presented. The myofibril is modeled as a group of segments placed in series, each segment represents a half-sarcomere with active and elastic properties. A multiple-state cross-bridge formalism relates the half Sarcomere force to the chemical kinetics of ATP hydrolysis. The corresponding system of nonlinear nonlocal partial differential equations of the model is analyzed. A numerical approach is introduced and some numerical tests are performed. The proposed in-silico model enables the study of biologically relevant process in the muscle contraction process, also in the case of muscular diseases, with reasonable computational effort.

Differences in titin segmental elongation between passive and active stretch in skeletal muscle.

Since the 1950s, muscle contraction has been explained using a two-filament system in which actin and myosin exclusively dictate active force in muscle sarcomeres. Decades later, a third filament called titin was discovered. This titin filament has recently been identified as an important regulator of active force, but has yet to be incorporated into contemporary theories of muscle contraction. When sarcomeres are actively stretched, a substantial and rapid increase in force occurs, which has been suggested to arise in part from titin-actin binding that is absent in passively stretched sarcomeres. However, there is currently no direct evidence for such binding within muscle sarcomeres. Therefore, we aimed to determine whether titin binds to actin in actively but not in passively stretched sarcomeres by observing length changes of proximal and distal titin segments in the presence and absence of calcium. We labeled I-band titin with fluorescent F146 antibody in rabbit psoas myofibrils and tracked segmental elongations during passive (no calcium) and active (high calcium) stretch. Without calcium, proximal and distal segments of titin elongated as expected based on their free spring properties. In contrast, active stretch differed statistically from passive stretch, demonstrating that calcium activation increases titin segment stiffness, but not in an actin-dependent manner. The consistent elongation of the proximal segment was contrary to what was expected if titin's proximal segment was attached to actin. This rapid calcium-dependent change in titin stiffness likely contributes to active muscle force regulation in addition to actin and myosin.

Human soleus sarcomere lengths measured using in vivo microendoscopy at two ankle flexion angles

The forces generated by the soleus muscle play an important role in standing and locomotion. The lengths of the sarcomeres of the soleus affect its force-generating capacity, yet it is unknown how sarcomere lengths in the soleus change as a function of ankle flexion angle. In this study, we used microendoscopy to measure resting sarcomere lengths at 10° plantarflexion and 20° dorsiflexion in 7 healthy individuals. Mean sarcomere lengths at 10° plantarflexion were 2.84±0.09µm (mean±S.E.M.), near the optimal length for sarcomere force generation. Sarcomere lengths were 3.43±0.09µm at 20° dorsiflexion, indicating that they were longer than optimal length when the ankle was in dorsiflexion and the muscle was inactive. Our results indicate a smaller sarcomere length difference between two ankle flexion angles compared to estimates from musculoskeletal models and suggest why these models frequently underestimate the force-generating capacity of the soleus.

Computing Average Passive Forces in Sarcomeres in Length-Ramp Simulations.

Passive forces in sarcomeres are mainly related to the giant protein titin. Titin’s extensible region consists of spring-like elements acting in series. In skeletal muscles these elements are the PEVK segment, two distinct immunoglobulin (Ig) domain regions (proximal and distal), and a N2A portion. While distal Ig domains are thought to form inextensible end filaments in intact sarcomeres, proximal Ig domains unfold in a force- and time-dependent manner. In length-ramp experiments of single titin strands, sequential unfolding of Ig domains leads to a typical saw-tooth pattern in force-elongation curves which can be simulated by Monte Carlo simulations. In sarcomeres, where more than a thousand titin strands are arranged in parallel, numerous Monte Carlo simulations are required to estimate the resultant force of all titin filaments based on the non-uniform titin elongations. To simplify calculations, the stochastic model of passive forces is often replaced by linear or non-linear deterministic and phenomenological functions. However, new theories of muscle contraction are based on the hypothesized binding of titin to the actin filament upon activation, and thereby on a prominent role of the structural properties of titin. Therefore, these theories necessitate a detailed analysis of titin forces in length-ramp experiments. In our study we present a simple and efficient alternative to Monte Carlo simulations. Based on a structural titin model, we calculate the exact probability distributions of unfolded Ig domains under length-ramp conditions needed for rigorous analysis of expected forces, distribution of unfolding forces, etc. Due to the generality of our model, the approach is applicable to a wide range of stochastic protein unfolding problems.

Multi-scale modeling of chemo-mechanical coupling in muscle contraction and applications to cardiac modeling

We propose a muscle chemo-mechanical model by which myosin heads - that can chemically bind to actin, thus creating so-called cross-bridges producing contraction forces in sarcomeres at the subcellular level - are considered as special chemical entities having internal mechanical variables pertaining to the actual geometric configuration. This provides a thermodynamical basis for modeling the complex interplay of chemical and mechanical phenomena at the sarcomere level. The resulting model is in the form of stochastic equations governing the dynamics of these microscopic mechanical variables in a Langevin framework. Equivalently, Fokker-Planck equations can be derived to describe the evolution of the associated probability densities. Under certain assumptions the corresponding moment equations can be closed, thus directly providing access to macroscopic quantities that can be incorporated in the overall constitutive equations of the muscle tissue. The underlying thermodynamical framework also enables the derivation of compatible numerical schemes, in particular in terms of energy balances. These modeling and discretization ingredients can be integrated in a global model of the cardiac system, to represent physiological and pathological phenomena in various medical applications.

Titin (visco-) elasticity in skeletal muscle myofibrils.

Titin is the third most abundant protein in sarcomeres and fulfills a number of mechanical and signaling functions. Specifically, titin is responsible for most of the passive forces in sarcomeres and the passive visco-elastic behaviour of myofibrils and muscles. It has been suggested, based on mechanical testing of isolated titin molecules, that titin is an essentially elastic spring if Ig domain un/refolding is prevented either by working at short titin lengths, prior to any un- folding of Ig domains, or at long sarcomere (and titin) lengths when Ig domain un/refolding is effectively prevented. However, these properties of titin, and by extension of muscles, have not been tested with titin in its natural structural environment within a sarcomere. The purpose of this study was to gain insight into the Ig domain un/refolding kinetics and test the idea that titin could behave essentially elastically at any sarcomere length by preventing Ig domain un/refolding during passive stretch-shortening cycles. Although not completely successful, we demonstrate here that titin's visco-elastic properties appear to depend on the Ig do- main un/refolding kinetics and that indeed, titin (and thus myofibrils) can become virtually elastic when Ig domain un/refolding is prevented.

Residual force enhancement: the neglected property of striated muscle contraction

As pointed out by Dr Edman in his thoughtful review (Edman, 2012), residual force enhancement in striated muscles has been observed consistently over the past half-century, its properties have been well described, and its lack of explanation within the framework of the cross-bridge theory has been acknowledged. However, and this is the topic of Dr Edman's review, the primary mechanism producing force enhancement remains a matter of scientific debate. On the one hand, there is the classic explanation of the development of non-uniformities that develop upon muscle stretch; on the other hand, there is the idea of the ‘engagement’ of a passive structural element upon activation that produces the enhanced force following active muscle stretching. In his review, Dr Edman favours the idea that residual force enhancement is primarily a consequence of (half-) sarcomere length non-uniformities (e.g. [residual force enhancement]…‘is ultimately based on non-uniform sarcomere behaviour with the result that populations of sarcomeres, or half-sarcomeres, actually acquire a greater amount of filament overlap than expected from the overall sarcomere length recorded after stretch’; Edman, 2012). Here, we would like to make the argument that ‘the role of a passive structural element’, the second idea mentioned above, should not be dismissed prematurely. First, Dr Edman mentions the advantages of single fibre preparations over myofibril preparations, and all the points he makes are perfectly valid. However, the great advantage of myofibrils over single fibre preparations is that single myofibrils consist of serially arranged sarcomeres. Therefore, the forces measured at the ends of a myofibril reflect directly the force in each sarcomere. Furthermore, the instantaneous lengths of each and all sarcomeres of a myofibril can easily be quantified. In contrast, in a single fibre, sarcomeres are connected partly in parallel and partly in series forming a highly redundant network of force transmission and individual sarcomere lengths cannot be measured, thus making any inferences to sarcomere dynamics highly suspect. Regarding the involvement of a passive structural element in force enhancement, Dr Edman makes several statements that we do not agree with. For example, he states that: ‘It seems quite clear, however, that the phenomenon “force enhancement after stretch” is entirely limited to the active period of the fibre. It disappears completely as the fibre relaxes after the stimulation period.’ (Edman, 2012). This statement ignores the well-described passive force enhancement observed following active stretches of muscles (e.g. Herzog & Leonard, 2002), fibres (e.g. Lee et al. 2007) and myofibrils (e.g. Joumaa et al. 2007, 2008) which persists long after muscle stimulation has ceased. Dr Edman also mentions that: ‘Another strong indication that strain of elastic elements does not by itself determine the measured force is given by the fact that the force recorded during stretch is quite independent of the amplitude of the stretch ramp.’ (Edman, 2012). Again, we disagree; there is ample evidence that the forces during stretch increase with increasing stretch magnitudes, and that the forces at the end-length following stretches of different magnitudes are well correlated with the residual force enhancement (e.g. Bullimore et al. 2007). The sarcomere length non-uniformity theory, favoured in Dr Edman's review, also provides testable predictions. Arguably the most important of these is that a muscle cannot produce enhanced force that exceeds the maximal isometric force at optimal length (Edman et al. 1982). However, there are numerous studies showing peak force enhancements well in excess of 10% above the plateau forces (Lee & Herzog, 2008), and most importantly, force enhancement in single sarcomeres has been shown to exceed 35% on average, with peak values exceeding 50%, above the plateau of the force–length relationship (Leonard et al. 2010). These values cannot be attributed to random fluctuations or artifacts. In summary, we agree with Dr Edman that the mechanisms underlying residual force enhancement remain unknown. However, inferring single sarcomere mechanics from fibre experiments appears impossible. Myofibrils offer an elegant preparation to measure the mechanics of isolated sarcomeres directly and accurately, and have revealed enhanced forces exceeding 50% of the maximal isometric forces in single sarcomeres (Leonard et al. 2010). These results cannot be explained with increased myofilament overlap due to (half-) sarcomere length non-uniformities, but are consistent with the idea of the ‘engagement’ of a passive structural element upon activation. In agreement with Dr Edman, we speculate that titin might play a crucial role in this ‘passive’ force regulation (Herzog et al. 2012).

Sarcomeric dysfunction contributes to muscle weakness in facioscapulohumeral muscular dystrophy

To investigate whether sarcomeric dysfunction contributes to muscle weakness in facioscapulohumeral muscular dystrophy (FSHD). Sarcomeric function was evaluated by contractile studies on demembranated single muscle fibers obtained from quadriceps muscle biopsies of 4 patients with FSHD and 4 healthy controls. The sarcomere length dependency of force was determined together with measurements of thin filament length using immunofluorescence confocal scanning laser microscopy. X-ray diffraction techniques were used to study myofilament lattice spacing. FSHD muscle fibers produced only 70% of active force compared to healthy controls, a reduction which was exclusive to type II muscle fibers. Changes in force were not due to changes in thin filament length or sarcomere length. Passive force was increased 5- to 12-fold in both fiber types, with increased calcium sensitivity of force generation and decreased myofilament lattice spacing, indicating compensation by the sarcomeric protein titin. We have demonstrated a reduction in sarcomeric force in type II FSHD muscle fibers, and suggest compensatory mechanisms through titin stiffening. Based on these findings, we propose that sarcomeric dysfunction plays a critical role in the development of muscle weakness in FSHD.

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Force enhancement following stretch in a single sarcomere

It has been accepted for half a century that, for a given level of activation, the steady-state isometric force of a muscle sarcomere depends exclusively on the amount of overlap between the contractile filaments actin and myosin, or equivalently sarcomere length (Gordon AM et al., J Physiol 184: 170-192, 1966). Moreover, according to the generally accepted paradigm of muscle contraction, the cross-bridge theory (Huxley AF, Prog Biophys Biophys Chem 7: 255-318, 1957), this steady-state isometric sarcomere force is independent of the muscle's contractile history (Huxley AF, Prog Biophys Biophys Chem 7: 255-318, 1957; Walcott S and Herzog W, Math Biosci 216: 172-186, 2008); i.e., it is independent of whether a muscle is held at a constant length before and during the contraction or whether the muscle is shortened or lengthened to the same constant length. This, however, is not the case, as muscles and single fibers that are stretched show greatly increased steady-state isometric forces compared with preparations that are held at a constant length (Abbott BC and Aubert XM, J Physiol 117: 77-86, 1952; De Ruiter CJ et al., J Physiol 526.3: 671-681, 2000; Edman KAP et al., J Physiol 281: 139-155, 1978; Edman KAP et al., J Gen Physiol 80: 769-784, 1982; Edman KAP and Tsuchiya T, J Physiol 490.1: 191-205, 1996). This so-called "residual force enhancement" (Edman KAP et al., J Gen Physiol 80: 769-784, 1982) offers a perplexing puzzle for muscle physiologists. Many theories have been advanced to address the discrepancy between prediction and observation with the most popular and accepted being the sarcomere length nonuniformity theory (Morgan DL, Biophys J 57: 209-221, 1990), which explains the residual force enhancement with the development of large nonuniformities in sarcomere lengths during muscle stretching. Here, we performed experiments in mechanically isolated sarcomeres and observed that the residual force enhancement following active stretching is preserved. Since our preparation utilizes a single sarcomere, a redistribution of the length of neighboring sarcomeres to produce the higher force following stretch is, by design, precluded. Furthermore, the enhanced forces in the single sarcomeres always exceed the isometric forces on the plateau of the force-length relationship, thereby eliminating the possibility that our result might have been obtained because of a redistribution of half-sarcomere lengths. Since force enhancement in single myofibrils has been associated with actin-titin interactions (Kulke M et al., Circ Res 89: 874-881, 2001; Li Q et al., Biophys J 69: 1508-1518, 1995) and calcium binding to titin (Joumaa V et al., Am J Physiol Cell Physiol 294: C74-C78, 2008; Labeit D et al., Proc Natl Acad Sci USA 100: 13716-13721, 2003), titin may regulate the sarcomeric force enhancement observed here.

Nonuniform activation and the mechanics of myocardial trabeculae with fast or slow myosin

Left ventricular (LV) wall motion abnormalities reflect regional nonuniform contraction which may be arrhythmogenic. We studied sarcomere mechanics and force development (F) in uniform and nonuniform trabeculae using a model in which half of the muscle can be rendered weak by exposure to low [Ca2+]o. Stretch allowed the weak muscle segment to generate a force that was four-fold higher than force when the whole muscle was exposed to low [Ca2+]o. The sarcomere force-velocity relationships (FSVR) and the force-sarcomere-length relationships (FSLR) explained the force increase in the weak segment and the decrease of force in the strong segment such that both carried the same force. Correction for muscle stiffness converted the FSVR into a [Ca2+]o-independent linear FVRXB for "the single cross-bridge (XB)." Stretch increased XB force<10% above FXB-max, but recruited more XBs by feedback of V to the rate of XB, weakening (g=g0+g1V). The g1 here was indistinguishable from g1 of XBs in slow myosin of aged animals. The mechanics of nonuniform muscle can be explained by a linear FVRXB combined with the effect of V on the XB weakening rate.

Extensible Behavior of Titin in the Miniswine Left Ventricle

The sarcomeric protein titin is a molecular spring responsible for passive tension and restoring forces of cardiomyocytes. Extension of titin as a function of sarcomere length (SL) has been studied in rodents, which predominantly express the smaller, stiffer N2B titin isoform. Large mammals coexpress roughly equal proportions of N2B and N2BA titin, the larger, more compliant isoform. We hypothesized that extension of titin in relation to SL differs in large mammals and that this difference is functionally important. We characterized the filling pressure-SL relation in diastolic-arrested miniswine left ventricles. SL was 2.15 to 2.25 mum at a filling pressure of approximately 0 mm Hg and reached a maximum of approximately 2.50 mum with overfilling. In the normal filling pressure range, SL ranged from approximately 2.32 to approximately 2.40 mum. We assessed titin extension as a function of SL using immunoelectron microscopy, which allowed delineation of the behavior of specific spring segments. The major isoform difference was that the N2B-Us segment extended approximately 4-fold more as a function of SL in N2B compared with N2BA titin. Using this segment, we estimated sarcomeric force development with a worm-like chain model and found that N2B develops markedly greater force than N2BA titin. The resulting force with coexpression of N2B and N2BA titin is intermediate. In light of murine studies showing that operating SLs are shorter than in miniswine, our results indicate that coexpression of the 2 titin isoforms in large mammals allows longer SLs without the development of excessive diastolic tension.

Sarcomere Mechanics in Capillary Endothelial Cells

Tension generation in endothelial cells of the aorta, spleen, and eye occurs in actin stress fibers, and is necessary for normal cell function. Sarcomeres are the tension-generating units of actin stress fibers in endothelial cells. How sarcomeres generate and maintain tension in stress fibers is not well understood. Using femtosecond laser ablation, we severed living stress fibers and measured sarcomere contraction under zero tension. The length of the sarcomere decreased in two phases: an instantaneous initial response, followed by a slower change in length attributed to myosin activity. The latter phase ceased abruptly after a minimum sarcomere length was reached, suggesting a rigid resistance that prevents further contraction. Furthermore, severed, contracted stress fibers did not relax when treated with myosin inhibitors, indicating that contracted stress fibers do not store elastic potential energy. These novel measurements combined with modeling suggest that myosin-generated forces in adjacent sarcomeres are directly in balance, and argue against sarcomere models with springlike elements in parallel with myosin contractile elements. We propose a new model for tension generation in the sarcomere, which provides a mechanistic interpretation for our observations and previous observations of inhomogeneous sarcomere contraction and apparent stress fiber viscoelastic behavior.