Nano-imaging of the beating mouse heart in vivo: Importance of sarcomere dynamics,as opposed to sarcomere length per se,in the regulation of cardiac function

Sarcomeric contraction in cardiomyocytes serves as the basis for the heart’s pump functions in mammals. Although it plays a critical role in the circulatory system, myocardial sarcomere length (SL) change has not been directly measured in vivo under physiological conditions because of technical difficulties. In this study, we developed a high speed (100–frames per second), high resolution (20-nm) imaging system for myocardial sarcomeres in living mice. Using this system, we conducted three-dimensional analysis of sarcomere dynamics in left ventricular myocytes during the cardiac cycle, simultaneously with electrocardiogram and left ventricular pressure measurements. We found that (a) the working range of SL was on the shorter end of the resting distribution, and (b) the left ventricular–developed pressure was positively correlated with the SL change between diastole and systole. The present findings provide the first direct evidence for the tight coupling of sarcomere dynamics and ventricular pump functions in the physiology of the heart.     

Intracellular Ca 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 [Ca²⁺] 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 Ca²⁺ overload. The [Ca²⁺] 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.     

Lymphatic muscle: a review of contractile function

A recent surge in lymphangiogenesis research has led to a greater understanding of lymphatic endothelial cell biology. However, a general understanding of lymphatic muscle cell biology lags far behind its endothelial counterpart. Lymphatics at the level of the collecting vessels and higher contain muscular walls capable of both tonic and phasic contractions, which both generate and regulate lymph flow. Because lymphatic contraction is crucial to lymphatic function, a solid understanding of lymphatic muscle development and function is necessary to understand lymphatic biology. This review summarizes the current body of lymphatic muscle research and addresses important questions that are currently unanswered.     

Three-dimensional structure of the Z band in a normal mammalian skeletal muscle

The three-dimensional structure of the vertebrate skeletal muscle Z band reflects its function as the muscle component essential for tension transmission between successive sarcomeres. We have investigated this structure as well as that of the nearby I band in a normal, unstimulated mammalian skeletal muscle by tomographic three- dimensional reconstruction from electron micrograph tilt series of sectioned tissue. The three-dimensional Z band structure consists of interdigitating axial filaments from opposite sarcomeres connected every 18 +/- 12 nm (mean +/- SD) to one to four cross-connecting Z- filaments are observed to meet the axial filaments in a fourfold symmetric arrangement. The substantial variation in the spacing between cross-connecting Z-filament to axial filament connection points suggests that the structure of the Z band is not determined solely by the arrangement of alpha-actinin to actin-binding sites along the axial filament. The cross-connecting filaments bind to or form a "relaxed interconnecting body" halfway between the axial filaments. This filamentous body is parallel to the Z band axial filaments and is observed to play an essential role in generating the small square lattice pattern seen in electron micrographs of unstimulated muscle cross sections. This structure is absent in cross section of the Z band from muscles fixed in rigor or in tetanus, suggesting that the Z band lattice must undergo dynamic rearrangement concomitant with crossbridge binding in the A band.     

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 ultrastructural 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.     

Contraction dynamics and function of the muscle-tendon complex depend on the muscle fibre-tendon length ratio: a simulation study

Experimental studies show different muscle-tendon complex (MTC) functions (e.g. motor or spring) depending on the muscle fibre-tendon length ratio. Comparing different MTC of different animals examined experimentally, the extracted MTC functions are biased by, for example, MTC-specific pennation angle and fibre-type distribution or divergent experimental protocols (e.g. influence of temperature or stimulation on MTC force). Thus, a thorough understanding of variation of these inner muscle fibre-tendon length ratios on MTC function is difficult. In this study, we used a hill-type muscle model to simulate MTC. The model consists of a contractile element (CE) simulating muscle fibres, a serial element (SE) as a model for tendon, and a parallel elastic element (PEE) modelling tissue in parallel to the muscle fibres. The simulation examines the impact of length variations of these components on contraction dynamics and MTC function. Ensuring a constant overall length of the MTC by \(L_\mathrm{MTC} = L_\mathrm{SE} + L_\mathrm{CE}\), the SE rest length was varied over a broad physiological range from 0.1 to 0.9 MTC length. Five different MTC functions were investigated by simulating typical physiological experiments: the stabilising function with isometric contractions, the motor function with contractions against a weight, the capability of acceleration with contractions against a small inertial mass, the braking function by decelerating a mass, and the spring function with stretch-shortening cycles. The ratio of SE and CE mainly determines the MTC function. MTC with comparably short tendon generates high force and maximal shortening velocity and is able to produce maximal work and power. MTC with long tendon is suitable to store and release a maximum amount of energy. Variation of muscle fibre-tendon ratio yielded two peaks for MTC’s force response for short and long SE lengths. Further, maximum work storage capacity of the SE is at long \(\mathrm{rel}L_\mathrm{SE,0}\). Impact of fibre-tendon length ratio on MTC functions will be discussed. Considering a constant set of MTC parameters, quantitative changes in MTC performance (work, stiffness, force, energy storage, dissipation) depending on varying muscle fibre-tendon length ratio were provided, which enables classification and grading of different MTC designs.     

Magnitude of sarcomere extension correlates with initial sarcomere length during lengthening of activated single fibers from soleus muscle of rats

A laser-diffraction technique was developed that rapidly reports the lengths of sarcomeres (L_s) in serially connected sectors of permeabilized single fibers. The apparatus translates a laser beam along the entire length of a fiber segment within 2 ms, with brief stops at each of 20 contiguous sectors. We tested the hypothesis that during lengthening contractions, when maximally activated fibers are stretched, sectors that contain the longer sarcomeres undergo greater increases in L_s than those containing shorter sarcomeres. Fibers (n = 16) were obtained from the soleus muscles of adult male rats and the middle portions (length = 1.05 ± 0.11 mm; mean ± SD) were investigated. Single stretches of strain 27% and a strain rate of 54% s⁻¹ were initiated at maximum isometric stress and resulted in a 19 ± 9% loss in isometric stress. The data on L_s revealed that 1), the stretch was not distributed uniformly among the sectors, and 2), during the stretch, sectors at long L_s before the stretch elongated more than those at short lengths. The findings support the hypothesis that during stretches of maximally activated skeletal muscles, sarcomeres at longer lengths are more susceptible to damage by excessive strain.     

Light diffraction studies of sarcomere dynamics in single skeletal muscle fibers.

A position-sensitive optical diffractometer has been used to examine the diffraction spectra produced by single skeletal muscle fibers during twitch and tetanic contraction. First-order diffraction lines were computer-analyzed for mean sarcomere length, line intensity, and percent dispersion in sarcomere length. Line intensity was observed to decrease rapidly by about 60 percent during a twitch, with an exponential recovery to resting intensity persisting well beyond cessation of sarcomere shortening; recovery was particularly prolonged at zero myofilament overlap. A number of single fibers at initial lengths from 2.5 to 3.5 MICRON EXHIBITED a splitting of the first-order line into two or more components during relaxation, with components merging back into a single peak by 200 ms after stimulation. This splitting reflects the asynchronous nature of myofibrillar relaxation within a single fiber. During tetanus, the dispersion decreased by more than 10 percent from onset to plateau, implying a gradual stabilization of sarcomeres.     

A model of semitendinosus muscle sarcomere length, knee and hip joint interaction in the frog hindlimb

The interaction between the semitendinosus muscle and both hip and knee joint angles was examined in the frog (Rana pipiens) hindlimb. Sarcomere length was measured by laser diffraction in passive muscle during hip and knee rotation. A model was then developed to predict semitendinosus sarcomere length as a function of both hip and knee flexion angle. Based on published frog muscle fiber length-tension [Gordon, A. M. et al., J. Physiol. 184, 170-192 (1966)] and force-velocity [Edman, K. A. P., J. Physiol. 291, 143-159 (1979)] properties, and published joint angles during hopping [Calow, L. J. and Alexander, R. McN., J. Zool. (Lond.) 171, 293-321 (1973)], muscle sarcomere length, force and hip and knee torque during a hop were predicted. The semitendinosus muscle generally operated on the descending limb of the length-tension curve at normal joint angle combinations. The model predicted that, during a single coordinated movement, a period of sarcomere shortening (concentric) was followed by a period of sarcomere lengthening (eccentric). Based on calculated torque profiles at the hip and knee joints, this study suggested that the semitendinosus muscle probably functions more as a hip extensor than a knee flexor. In addition, based on the nature of the shortening-lengthening cycle, the semitendinosus may act to mechanically link the force of knee extension to hip extension.     

Heterogeneity of Z-band structure within a single muscle sarcomere: implications for sarcomere assembly

The vertebrate striated muscle Z-band connects actin filaments of opposite polarity from adjacent sarcomeres and allows tension to be transmitted along a myofibril during contraction. Z-bands in different muscles have a modular structure formed by layers of alpha-actinin molecules cross-linking actin filaments. Successive layers occur at 19 nm intervals and have 90 degrees rotations between them. 3D reconstruction from electron micrographs show a two-layer "simple" Z-band in fish body fast muscle, a three-layer Z-band in fish fin fast muscle, and a six-layer Z-band in mammalian slow muscle. Related to the number of these layers, longitudinal sections of the Z-band show a number of zigzag connections between the oppositely oriented actin filaments. The number of layers also determines the axial width of the Z-band, which is a useful indicator of fibre type; fast fibres have narrow (approximately 30-50 nm) Z-bands; slow and cardiac fibres have wide (approximately 100-140 nm) Z-bands. Here, we report the first observation of two different Z-band widths within a single sarcomere. By comparison with previous studies, the narrower Z-band comprises three layers. Since the increase in width of the wider Z-band is about 19 nm, we conclude that it comprises four layers. This finding is consistent with a Z-band assembly model involving molecular control mechanisms that can add additional layers of 19 nm periodicity. These multiple Z-band structures suggest that different isoforms of nebulin and titin with a variable number of Z-repeats could be present within a single sarcomere.     

The active state of cross-striated muscle cell sarcomere (an imitation model)]

An index of the sarcomere active state is introduced. It reflects the most important processes of sarcomere contraction, such as calcium diffusion taking into account permeability of calcium channels and its binding with troponin, formation of energy supplies and interaction of sarcomere contractile proteins. The effect of changes of the values of diffusion coefficients and chemical reactions rates was studied theoretically.     

Measurement of sarcomere length during fast contraction of muscle fibers by digital image analysis.

A low-cost, high-resolution (spatial and temporal) image analysis system was developed to measure sarcomere length (Sl) during fast twitch of isolated striated muscle fibers at different temperatures. Fiber images were examined during twitch with an imaging rate of 220 Hz. To increase temporal resolution beyond 220 Hz, consecutive temporally shifted image sequences (N sequences) were acquired. Individual or average Sl was directly measured from a horizontal profile without spatial-frequency assessment. Measurement precision (E) was determined and expressed as: E(%) = 100xPs/(IsxSl), where Ps is the pixel size and Is the involved sarcomere number. At 18 degrees C during isometric twitch, Sls were measured with 220 Hz temporal and 0.2% spatial resolutions. Sl shortened in the central region (0.21+/-0.12 microm) as tension developed, reaching a maximal shortening of 8.09 + 2.05% (at rest, Sl = 2.59+/-0.05 microm, n = 4) in 32.5+/-1.96 ms. At 30 degrees C, Sl variations were examined with 880 Hz temporal resolution, in which case maximal S1 shortening was reached in 15.74+/-1.99 ms, and then decreased to 5.19+/-1.97% (at rest, S1 = 2.6+/-0.06 microm). The twitch tension developed by the whole fiber was recorded and compared with sarcomere length behavior. Sarcomere length variations in the central region were representative of overall developed tensions at 18 and 30 degrees C.     

Conformational dynamics of unfolded peptides as a function of chain length: a frequency domain fluorescence approach

The fluorescence emission decays of single-tryptophan-containing peptides of different chain lengths in their unfolded state were investigated in the frequency domain. The data were analyzed using different functions, i.e., exponential fit and probability-density functions of different shape. We found that unimodal Lorentzian distributions best describe the fluorescence decays. This finding agrees with the point of view, now broadly accepted, that rapid motions exist in polypeptides. As a consequence of this flexibility, a large variety of conformations, with an unequal perturbation of tryptophan in its excited state, is generated. The lifetime distribution center was independent of the length of the polypeptide chain but strongly related to the nature of the amino acid residues located in the proximity of the tryptophan in the primary structure. The full width at half maximum, W, of the lifetime distribution was found to be related to the length of unfolded polypeptide by the empirical logarithmic relationship W = 0.83 log n, where n indicates the number of residues. For short peptides, a single lifetime or a narrow range of lifetimes is observed because of the fast relaxation of the tryptophanyl environment. On peptide lengthening, the spectrum of conformations, which the peptide can assume, increases; this causes a complex fluorescence decay represented by a lifetime distribution. For long polypeptide chains, the motions of the regions far from tryptophan do not significantly perturb the chromophore environment.     

Spectral analysis of muscle fiber images as a means of assessing sarcomere heterogeneity.

A new image-analysis-based method is described for assessing sarcomere heterogeneity in skinned rabbit psoas muscle fiber segments. This method consists of off-line, two-dimensional Fourier spectral analysis of video-taped muscle images. Local sarcomere length is assessed by partitioning the muscle images into half and quarter images spanning the original image and analyzing the associated spectra. The spectra are analyzed in two different ways, yielding two measures of sarcomere length. The first measure is obtained by calculating and inverting the centroid frequency of the first-order peak associated with each two-dimensional Fourier spectrum. The second measure is obtained in a similar manner, the only difference being that the two-dimensional spectra are first collapsed into one-dimensional line spectra by summing the pixels perpendicular to the fiber axis. Comparison of the two measures provides a measure of striation skewness that cannot be obtained by other image analysis based methods that determine sarcomere length by analyzing selected line luminance profiles.     

Soluble miniagrin enhances contractile function of engineered skeletal muscle

Neural agrin plays a pleiotropic role in skeletal muscle innervation and maturation, but its specific effects on the contractile function of aneural engineered muscle remain unknown. In this study, neonatal rat skeletal myoblasts cultured within 3-dimensional engineered muscle tissue constructs were treated with 10 nM soluble recombinant miniagrin and assessed using histological, biochemical, and functional assays. Depending on the treatment duration and onset time relative to the stage of myogenic differentiation, miniagrin was found to induce up to 1.7-fold increase in twitch and tetanus force amplitude. This effect was associated with the 2.3-fold up-regulation of dystrophin gene expression at 6 d after agrin removal and enhanced ACh receptor (AChR) cluster formation, but no change in cell number, expression of muscle myosin, or important aspects of intracellular Ca(2+) handling. In muscle constructs with endogenous ACh levels suppressed by the application of α-NETA, miniagrin increased AChR clustering and twitch force amplitude but failed to improve intracellular Ca(2+) handling and increase tetanus-to-twitch ratio. Overall, our studies suggest that besides its synaptogenic function that could promote integration of engineered muscle constructs in vivo, neural agrin can directly promote the contractile function of aneural engineered muscle via mechanisms distinct from those involving endogenous ACh.