Abstract Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower ...forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n = 96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n = 19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16–18 days at 0.5 Hz developed 2.2 × higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca 2 + -response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2 Hz in the first week and 1.5 Hz thereafter developed 1.5 × higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT.
The Z-disc, appearing as a fine dense line forming sarcomere boundaries in striated muscles, when studied in detail reveals crosslinked filament arrays that transmit tension and house myriads of ...proteins with diverse functions. At the Z-disc the barbed ends of the antiparallel actin filaments from adjoining sarcomeres interdigitate and are crosslinked primarily by layers of α-actinin. The Z-disc is therefore the site of polarity reversal of the actin filaments, as needed to interact with the bipolar myosin filaments in successive sarcomeres. The layers of α-actinin determine the Z-disc width: fast fibres have narrow (~30-50 nm) Z-discs and slow and cardiac fibres have wide (~100 nm) Z-discs. Comprehensive reviews on the roles of the numerous proteins located at the Z-disc in signalling and disease have been published; the aim here is different, namely to review the advances in structural aspects of the Z-disc.
Muscle contraction is orchestrated by the well-understood thin filaments and the markedly complex thick filaments. Studies by Dutta et al. and Tamborrini et al., discussed here, have unravelled the ...structure of the mammalian heart thick filament in exquisite near-atomic detail and pave the way for understanding physiological modulation pathways and mutation-induced dysfunction and for designing potential drugs to modify defects.
Muscle contraction is orchestrated by the well-understood thin filaments and the markedly complex thick filaments. Studies by Dutta et al. and Tamborrini et al., discussed here, have unravelled the structure of the mammalian heart thick filament in exquisite near-atomic detail and pave the way for understanding physiological modulation pathways and mutation-induced dysfunction and for designing potential drugs to modify defects.
Early x-ray diffraction studies of muscle revealed spacings larger than the basic thick filament lattice spacing and led to a number of speculations on the mutual rotations of the filaments in the ...myosin lattice. The nature of the arrangements of the filaments was resolved by John Squire and Pradeep Luther using careful electron microscopy and image analysis. The intriguing disorder in the rotations, that they termed the myosin superlattice, remained a curiosity, until work with Rick Millane and colleagues showed a connection to “geometric frustration,” a well-known phenomenon in statistical and condensed matter physics. In this review, we describe how this connection gives a satisfying physical basis for the myosin superlattice, and how recent work has shown relationships to muscle mechanical behaviour.
Myosin-binding protein C (MyBP-C) is a thick filament protein playing an essential role in muscle contraction, and MyBP-C mutations cause heart and skeletal muscle disease in millions worldwide. ...Despite its discovery 40 y ago, the mechanism of MyBP-C function remains unknown. In vitro studies suggest that MyBP-C could regulate contraction in a unique way--by bridging thick and thin filaments--but there has been no evidence for this in vivo. Here we use electron tomography of exceptionally well preserved muscle to demonstrate that MyBP-C does indeed bind to actin in intact muscle. This binding implies a physical mechanism for communicating the relative sliding between thick and thin filaments that does not involve myosin and which could modulate the contractile process.
Obituary: Professor John Michael Squire Morris, Edward P.; Knupp, Carlo; Luther, Pradeep K.
Journal of muscle research and cell motility,
09/2023, Letnik:
44, Številka:
3
Journal Article
The Z-band in vertebrate striated muscle crosslinks actin filaments of opposite polarity from adjoining sarcomeres and transmits tension along myofibrils during muscular contraction. It is also the ...location of a number of proteins involved in signalling and myofibrillogenesis; mutations in these proteins lead to myopathies. Understanding the high-resolution structure of the Z-band will help us understand its role in muscle contraction and the role of these proteins in the function of muscle. The appearance of the Z-band in transverse-section electron micrographs typically resembles a small-square lattice or a basketweave appearance. In longitudinal sections, the Z-band width varies more with muscle type than species: slow skeletal and cardiac muscles have wider Z-bands than fast skeletal muscles. As the Z-band is periodic, Fourier methods have previously been used for three-dimensional structural analysis. To cope with variations in the periodic structure of the Z-band, we have used subtomogram averaging of tomograms of rat cardiac muscle in which subtomograms are extracted and compared and similar ones are averaged. We show that the Z-band comprises four to six layers of links, presumably α-actinin, linking antiparallel overlapping ends of the actin filaments from the adjoining sarcomeres. The reconstruction shows that the terminal 5–7nm of the actin filaments within the Z-band is devoid of any α-actinin links and is likely to be the location of capping protein CapZ.
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•The vertebrate muscle Z-band connects antiparallel actin filaments from two adjacent sarcomeres.•Contractile tension is transmitted via α-actinin linking the antiparallel actin filaments.•We have used electron tomography and subtomogram averaging to investigate the three-dimensional structure of the Z-band in rat cardiac muscle.•The Z-band is composed of overlapping ends of actin filaments crosslinked by four to six layers of α-actinin.•The terminal 5–7nm of the actin filament within the Z-band is devoid of links and may be due to capping protein CapZ.
Striated muscle enables movement in all animals by the contraction of myriads of sarcomeres joined end to end by the Z-bands. The contraction is due to tension generated in each sarcomere between ...overlapping arrays of actin and myosin filaments. At the Z-band, actin filaments from adjoining sarcomeres overlap and are cross-linked in a regular pattern mainly by the protein α-actinin. The Z-band is dynamic, reflected by the 2 regular patterns seen in transverse section electron micrographs; the so-called small-square and basketweave forms. Although these forms are attributed, respectively, to relaxed and actively contracting muscles, the basketweave form occurs in certain relaxed muscles as in the muscle studied here. We used electron tomography and subtomogram averaging to derive the 3D structure of the Z-band in the swimbladder sonic muscle of type I male plainfin midshipman fish (Porichthys notatus), into which we docked the crystallographic structures of actin and α-actinin. The α-actinin links run diagonally between connected pairs of antiparallel actin filaments and are oriented at an angle of about 25° away from the actin filament axes. The slightly curved and flattened structure of the α-actinin rod has a distinct fit into the map. The Z-band model provides a detailed understanding of the role of α-actinin in transmitting tension between actin filaments in adjoining sarcomeres.
Background
Myopalladin (MYPN) is a striated muscle‐specific, immunoglobulin‐containing protein located in the Z‐line and I‐band of the sarcomere as well as the nucleus. Heterozygous MYPN gene ...mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss‐of‐function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe.
Methods
Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro.
Results
MKO mice were 13% smaller compared with wild‐type controls and exhibited a 48% reduction in myofibre cross‐sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild‐type mice, MKO mice showed progressively decreased exercise capability, Z‐line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z‐line widening starting from 8 months of age. RNA‐sequencing analysis revealed down‐regulation of serum response factor (SRF)‐target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF‐cofactor myocardin‐related transcription factor A (MRTF‐A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF‐A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF‐A‐mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway.
Conclusions
Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z‐line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.
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