Individual Sarcomere Lengths Research Articles

Gaining new understanding of sarcomere length non-uniformities in skeletal muscles.

Sarcomere lengths are non-uniform on all structural levels of mammalian skeletal muscle. These non-uniformities have been associated with a variety of mechanical properties, including residual force enhancement and depression, creep, increased force capacity, and extension of the plateau of the force-length relationship. However, the nature of sarcomere length non-uniformities has not been explored systematically. The purpose of this study was to determine the properties of sarcomere length non-uniformities in active and passive muscle. Single myofibrils of rabbit psoas (n = 20; with 412 individual sarcomeres) were subjected to three activation/deactivation cycles and individual sarcomere lengths were measured at 4 passive and 3 active points during the activation/deactivation cycles. The myofibrils were divided into three groups based on their initial average sarcomere lengths: short, intermediate, and long average sarcomere lengths of 2.7, 3.2, and 3.6µm. The primary results were that sarcomere length non-uniformities did not occur randomly but were governed by some structural and/or contractile properties of the sarcomeres and that sarcomere length non-uniformities increased when myofibrils went from the passive to the active state. We propose that the mechanisms that govern the systematic sarcomere lengths non-uniformities observed in active and passive myofibrils may be associated with the variable number of contractile proteins and the variable number and the adjustable stiffness of titin filaments in individual sarcomeres.

An Examination of Sarcomere Length Non-Uniformities in Actively Stretched Muscle Myofibrils

Residual force enhancement (RFE) is a characteristic of skeletal muscle describing the increase in force exhibited following an active stretch on the descending limb of the force-length relationship, compared to the force of an isometric contraction at the final length. It has previously been argued that RFE is a result of instable sarcomeres on the descending limb, causing longer, weaker sarcomeres to lengthen to a greater extent than shorter, stronger sarcomeres, when a myofibril is actively stretched. If this were the mechanism of RFE, then sarcomeres should become more non-uniform in length after an active stretch. The purpose of this study was to investigate length non-uniformities between sarcomeres within a myofibril in isometric contractions pre- and post-active stretch. We hypothesized that sarcomere lengths would be less uniform in the post-stretch condition. Rabbit psoas muscle myofibrils were stretched passively to an average sarcomere length of 3.2 μm. The myofibrils were then activated. Five seconds after full activation, myofibrils were rapidly shortened to an average sarcomere length of 2.4 μm, held at that length for ten seconds and then stretched back to 3.2 μm. Individual sarcomere lengths were then determined during the initial isometric contraction and again following the active stretch. Standard deviations of sarcomere lengths were compared to analyze non-uniformity. Preliminary results gave normalized sarcomere length standard deviations of 5.7 % and 10.2 % for the initial isometric contraction and following active stretch respectively (103 % RFE). This supports the hypothesis that sarcomere lengths might be less uniform after active stretch; however, further testing will increase the sample size to 20. This will allow for a more general idea of the development of sarcomere length non-uniformities following active stretch and might provide additional insight into the mechanism of RFE.

Mathematical modeling of the dynamic mechanical behavior of neighboring sarcomeres in actin stress fibers.

Actin stress fibers (SFs) in live cells consist of series of dynamic individual sarcomeric units. Within a group of consecutive SF sarcomeres, individual sarcomeres can spontaneously shorten or lengthen without changing the overall length of this group, but the underlying mechanism is unclear. We used a computational model to test our hypothesis that this dynamic behavior is inherent to the heterogeneous mechanical properties of the sarcomeres and the cytoplasmic viscosity. Each sarcomere was modeled as a discrete element consisting of an elastic spring, a viscous dashpot and an active contractile unit all connected in parallel, and experiences forces as a result of actin filament elastic stiffness, myosin II contractility, internal viscoelasticity, or cytoplasmic drag. When all four types of forces are considered, the simulated dynamic behavior closely resembles the experimental observations, which include a low-frequency fluctuation in individual sarcomere length and compensatory lengthening and shortening of adjacent sarcomeres. Our results suggest that heterogeneous stiffness and viscoelasticity of actin fibers, heterogeneous myosin II contractility, and the cytoplasmic drag are sufficient to cause spontaneous fluctuations in SF sarcomere length. Our results shed new light to the dynamic behavior of SF and help design experiments to further our understanding of SF dynamics.

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).

Overextended sarcomeres regain filament overlap following stretch

Sarcomere overextension has been widely implicated in stretch-induced muscle injury. Yet, sarcomere overextensions are typically inferred based on indirect evidence obtained in muscle and fibre preparations, where individual sarcomeres cannot be observed during dynamic contractions. Therefore, it remains unclear whether sarcomere overextensions are permanent following injury-inducing stretch-shortening cycles, and thus, if they can explain stretch-induced force loss. We tested the hypothesis that overextended sarcomeres can regain filament overlap in isolated myofibrils from rabbit psoas muscles. Maximally activated myofibrils (n=13) were stretched from an average sarcomere length of 2.6±0.04μm by 0.9μm sarcomere−1 at a speed of 0.1μm sarcomere−1s−1 and immediately returned to the starting lengths at the same speed (sarcomere strain=34.1±2.3%). Myofibrils were then allowed to contract isometrically at the starting lengths (2.6μm) for ∼30s before relaxing. Force and individual sarcomere lengths were measured continuously. Out of the 182 sarcomeres, 35 sarcomeres were overextended at the peak of stretch, out of which 26 regained filament overlap in the shortening phase while 9 (∼5%) remained overextended. About 35% of the sarcomeres with initial lengths on the descending limb of the force-length relationship and ∼2% of the sarcomeres with shorter initial lengths were overextended. These findings provide first ever direct evidence that overextended sarcomeres can regain filament overlap in the shortening phase following stretch, and that the likelihood of overextension is higher for sarcomeres residing initially on the descending limb.

Individual Sarcomere Lengths in Whole Muscle Fibers and Optimal Fiber Length Computation

Estimation of muscle fiber optimum length is typically accomplished using either laser diffraction or by counting the number of sarcomeres in a portion of the muscle fiber, measuring the distance that encompasses those sarcomeres and dividing by the number of sarcomeres to obtain an average sarcomere length. If the sarcomeres are not uniformly distributed, either of these techniques could produce errors when estimating optimum lengths. The purposes of this study were: to describe new software that automatically analyzes digital images of skeletal muscle fibers to measure individual sarcomere lengths; and to use this software to measure individual sarcomere lengths along complete muscle fibers to examine the influence of computing whole muscle fiber properties from portions of the fiber. Six complete muscle fibers were imaged using a digital camera attached to a microscope. The images were then processed to achieve the best resolution possible, individual sarcomeres along the image were detected, and each individual sarcomere length was measured. The software accuracy was compared with that of manual measurement and was found to be as accurate. In addition, the time to measure individual sarcomere lengths was greatly reduced using the software compared with manual measurement. The arrangement of individual sarcomere lengths demonstrated long-range correlations, which indicates problems in assuming only a portion of a fiber can be used to determine whole fiber properties. This study has provided evidence on the number of sarcomeres which must be analyzed to infer the properties of whole muscles.

Passive Force Augmentation in Actively Stretched Myofibrils and Sarcomeres

Single skeletal muscle myofibrils were stretched actively and passively (pCa2+ = 3.5 and 8 respectively) from optimal length to beyond myofilament overlap (4.0μm) and individual sarcomere lengths and the associated stretch forces were measured. Actively stretched myofibrils produced much more force than passively stretched myofibrils at all sarcomere lengths, including when all sarcomeres were beyond myofilament overlap (Figure 1). In order to confirm that cross bridge based acto-myosin forces were not present at sarcomere lengths beyond myofilament overlap, passively and actively stretched myofibrils were deactivated and activated, respectively at 5μm. In both cases, no change in force was observed, indicating that active cross bridge forces were not present at long (5μm) length. Based on these results, we suggest that there is a dramatic passive force augmentation in passively stretched myofibrils and the individual sarcomeres.

Force depression in single myofibrils

Force depression after active shortening has been observed in different muscle preparations. It has been assumed that force depression is caused by the development of sarcomere length nonuniformities after shortening. However, this hypothesis has never been investigated in a preparation where individual sarcomere lengths could be directly measured. Here, we investigated force depression in single myofibrils (n = 11) and tracked simultaneously the changes in individual sarcomere lengths (n = 60) before, during, and after shortening and after a purely isometric contraction performed at the final length. Shortening produced force depression in all myofibrils (mean +/- SE; 30.9 +/- 3.9%). During shortening, all sarcomeres shortened, but not by the same amount. Sarcomere lengths were nonuniform, with the same mean SD before (0.11 +/- 0.06 microm) and after shortening (0.11 +/- 0.06 microm) and after a purely isometric contraction at the final length (0.10 +/- 0.05 microm). Furthermore, greater shortening magnitudes were found for sarcomeres that were long in the initial isometric configuration. Nonuniformities of half-sarcomere lengths were also the same before (SD = 0.13 microm) and after (SD = 0.14 microm) shortening. We conclude from these results that the development of sarcomere (or half-sarcomere) length nonuniformities does not play a major role in force depression. Rather, force depression seems an intrinsic property of individual (half-) sarcomeres and muscle contraction.

Sarcomere dynamics in skeletal muscle myofibrils during isometric contractions

The main goal of this study was to evaluate the dynamics of sarcomeres during isometric activation of skeletal muscle myofibrils. Rabbit psoas myofibrils ( n=14) were attached between a pair of cantilevers for force measurements at one side and a rigid glass needle at the other side, and their images were used for measurements of individual sarcomere lengths (SL) during contractions. Myofibrils were set at average SL between 2.13 and 3.06 μm, and were activated and held isometric for 20–35 s during which SL and force were continuously measured. SL dispersion increased from the rest state to activation, but it remained mostly constant during the activation period. Even with the length non-uniformity developed during myofibril activation, most sarcomeres stabilized their length changes during the isometric contraction. As a result, sarcomeres contracted at different degrees of filament overlap while producing similar forces. When the myofibrils were separated in two groups that produced force at averaged short (≤2.5 μm) or long (≥2.5 μm) SL, the initial non-uniformity was greater in long lengths, but changes observed in sarcomeres during the activation period were similar, suggesting that sarcomere stability is not length-dependent.

Effects of Length Changes on Force Produced by Ca2+ and ADP-Activated Myofibrils along the Ascending Limb of the Force-Length Relation

Isometric forces produced by skeletal muscles are higher after stretch and smaller after shortening. A few studies investigating these length-dependent changes in force were conducted on the ascending limb of the force-length (FL) relation, showing conflicting results with elusive mechanisms. The purposes of this study were: (i) to evaluate the effects of muscle stretching and shortening on forces along the ascending limb of the FL relation, (ii) to evaluate if sarcomere length dispersion changes after the imposed length changes, and (iii) to assess if cross-bridges play a role in the length-induced force changes. Rabbit psoas myofibrils were attached between two pre-calibrated micro-needles, and their images were projected into a photodiode array for measurements of individual sarcomere length (SL). Myofibrils were activated by Ca2+ or ADP - the later induces cross-bridge attachment to actin independently of Ca2+. After activation myofibrils were subjected to three stretches or shortenings (∼4%SL), with isometric periods allowed between length changes so that force would achieve a steady-state. Forces of ADP-activated myofibrils were greater (7-8%) than those of Ca2+-activated myofibrils at corresponding SLs (range: 2.2-2.4μm) after shortening, but forces were similar after stretch. Forces were greater (26% with ADP and 15% with Ca2+, SL: 2.2μm) after stretch than after shortening. Sarcomere dispersion was similar after stretch or shortening in Ca2+ and ADP-activated myofibrils. The results suggest that stretching and shortening affects isometric forces on the ascending limb of the FL relation through different mechanisms, and are not associated with SL dispersion. While cross-bridges seem to be involved in force depression, they are likely not involved in force enhancement.

Intracellular Ca 2+ dynamics and sarcomere length in single ventricular myocytes

The measurements of the sarcomere length in dissociated cardiac ventricular myocytes are discussed using mainly our own experimental data. The striation periodicity of these unloaded cells was found to be that which is to be expected of a myocyte free of the ultrastrucural constraints imposed upon it by the normal syncytial matrix of the ventricular wall. The sarcomere length and [Ca 2+] relationship was consistent as expected from the intact tissue, when it was measured soon after partial rupturing the cell membrane. Miniature fluctuations of individual sarcomere length were demonstrated during rest, which was augmented by the Ca 2+ overload. The [Ca 2+] could be estimated from the measurements of sarcomere length during the positive staircase of contraction. The usefulness of the optical measurement of sarcomere pattern was indicated.

Sarcomere lengths of thick skeletal muscle specimens measured under an epi-illumination-type polarization microscope.

The purpose of this study was to measure sarcomere lengths of thick muscle fiber bundles at resting and at isometric tetanic contractions. We developed a novel measurement system using an epi-illumination-type polarization microscope and an image processing algorithm using an ellipse-type Gabor filter. Images with striation patterns of frog skeletal muscle were obtained by the microscope and the image processing algorithm. Individual lengths of 10 consecutive sarcomeres of a single muscle fiber were measured by gauging each width of the striation pattern, which was proved to be derived from striation structures of the single fiber by performing experiments using different polarization lights and different focus depths. At the resting state, each sarcomere length was identical at the fixed muscle length and in proportion to the length ranging over 91-123% of the natural length. Each sarcomere length was unchanged at the steady state during isometric tetanic contractions. Individual sarcomere lengths in the central part of the skeletal muscle were identical at resting and at isometric tetanic contractions even in the thick muscle fiber specimen.

Individual sarcomere length determination from isolated cardiac cells using high-resolution optical microscopy and digital image processing

Discrete sarcomere lengths have been determined from dynamically contracting isolated cardiac cells with a high-speed, high-resolution direct optical imaging system. Calcium-tolerant cardiac cells from the rat are isolated by perfusion with collagenase and hyaluronidase. Individual sarcomere lengths can be determined by directly imaging the cell's striation pattern onto a solid-state charge-coupled device (CCD) detector interfaced with a digital computer. The precision of detection in a real light microscopic optical system is discussed in relation to the type of image detector, optical contract enhancement techniques, and digital image processing. The optical performance of the direct striation pattern image apparatus has been determined empirically with test grids under standard bright-field and Nomarski-differential interference contrast (DIC) conditions for application to real muscle imaging. Discrete striation positions of isolated cells have been detected and followed with high precision during phasic contraction-relaxation cycles down to average sarcomere lengths as short as 1.43 +/- 0.053 microns. The maximum rates of contraction and relaxation are rapid and synchronous in time course along the length of the cell. These results indicate that direct optical imaging can provide an accurate means to monitor discrete striations and sarcomere lengths along the length of Ca2+-tolerant heart cells.