Regulation of the actin–myosin interaction by titin

Abstract

Titin is known to interact with actin thin filaments within the I-band region of striated muscle sarcomeres. In this study, we have used a titin fragment of 800 kDa (T800) purified from striated skeletal muscle to measure the effect of this interaction on the functional properties of the actinmyosin complex. MALDI-TOF MS revealed that T800 contains the entire titin PEVK (Pro, Glu, Val, Lys-rich) 1 domain. In the presence of tropomyosin-troponin, T800 increased the sliding velocity (both average and maximum values) of actin filaments on heavy-meromyosin (HMM)-coated surfaces and dramatically decreased the number of stationary filaments. These results were correlated with a 30% reduction in actin-activated HMM ATPase activity and with an inhibition of HMM binding to actin N-terminal residues as shown by chemical cross-linking. At the same time, T800 did not affect the efficiency of the Ca 2+ -controlled on/off switch, nor did it alter the overall binding energetics of HMM to actin, as revealed by cosedimentation experiments. These data are consistent with a competitive effect of PEVK domain-containing T800 on the electrostatic contacts at the actin-HMM interface. They also suggest that titin may participate in the regulation of the active tension generated by the actin-myosin complex.Keywords: ATPase; chemical cross-linking; mass spectrometry; motility assay; muscle contraction.Titin is the largest known protein, containing more than 38 000 residues in its longest human striated muscle isoform. It represents the third most abundant component of vertebrate striated muscle, after myosin and actin, and is also present in smooth muscle and nonmuscle cells (recently reviewed in [1,2]). The importance of intact titin for normal muscle function has been demonstrated in vitro [3][4][5], as well as in vivo through its implication in muscular dystrophies such as dilated cardiomyopathies and Udd's tibial muscular dystrophy (reviewed in [6]).In striated muscle, titin is involved in several fundamental processes, including sarcomere assembly, possibly in thick filament length control [4,[7][8][9], maintenance of the sarcomeric structure, muscle elasticity and passive tension development [10][11][12]. These functions are related to three main structural properties of the protein: titin spans half a sarcomere, from the Z disks to the M line (connecting the Z disks to myosin thick filaments), it contains subdomains that confer unusual elastic properties, and it interacts with several protein partners such as myosin, actin, M protein, C protein, MURF-1, calpain 3, myomesin, a-actinin, nebulin, telethonin and obscurin.The elastic domains are made of tandemly arranged immunoglobulin (Ig)-like domains and a unique PEVK domain (Pro, Glu, Val, Lys-rich) whose size depends on the muscle fibre isotype. Specific structural properties and mechanical force/extension measurements made on muscle fibres or at the single molecule level suggest that the tandem Ig-and PEVK-domains are two elements of differential stiffness that function as a two-...

Is a parallel elastic element responsible for the enhancement of steady-state muscle force following active stretch?

“…Titin is known to bind to actin (Jin, 1995;Soteriou et al, 1993) and to modify in vitro motility of actin moving on myosin (Li et al, 1995;Niederländer et al, 2004). However it is not known whether these properties have an influence on active muscle mechanics in vivo.…”

Force enhancement and stability of finite element muscle models

“…This phenomenon is called force enhancement and is frequently attributed to a semi-active behaviour of the titin filament, which connects the thick filament to the Z-discs within the sarcomeres. In detail, it is assumed that in the presence of calcium [3][4][5], i. e., during activation, the PEVK (rich in proline (P), glutamic acid (E), valine (V), and lysine (K)) region of titin's molecular spring region can attach to actin at available myosin binding sites on the thin filaments [6]. Through this attachment, titin's free molecular spring length is dramatically reduced, which leads to vastly increased titin forces during and after active stretch.…”

Restriktive Kardiomyopathie

“…Myosin S1 gibt (Muhle-Goll et al, 2001 . Nachfolgend beschrieb dieselbe Arbeitsgruppe eine Inhibierung der Aktin-Myosin Interaktion durch dieses 800 kDa Titinfragment, welche nur in Anwesenheit von Tropomyosin und Troponin stattfand (Niederländer et al, 2004). Dies führte einerseits zu einer erhöhten Gleitgeschwindigkeit dünner Filamente auf einer myosinbeschichteten Oberfläche, andererseits aber zu einer niedrigeren steady-state Aktomyosin-ATPase Aktivität.…”

“…Niederländers (Niederländer et al, 2004) -Sensitivität des dünnen Filamentes in den Aktomyosin-S1-ATPase Assays beobachtet werden konnte.…”

“…Raynaud und Niederländer Niederländer et al, 2004) gingen, basierend auf massenspektrometrischen Daten und Überlegungen bezüglich der Struktur des Titins, bei ihrem 800 kDa Titinfragment von einem reinen I-Banden Fragment aus, welches die PEVK-Domäne enthält. Da die Sequenzabdeckung des Riesenproteins Titin in massenspektrometrischen Messungen bekanntermaßen gering ist und in den zitierten Arbeiten nur wenige Peptide untersucht und noch weniger tatsächlich zugeordnet werden konnten, kann das Vorhandensein von Teilen des A-Banden Titins im 800 kDa Fragment nicht eindeutig ausgeschlossen werden, zumal die Abschätzung der tatsächlichen Größe des Fragmentes auf Grund fehlender geeigneter Größenstandards schwierig ist.…”