티틴(sarcomere 끝 부분 부착 단백질)의 기능.. 장력 센서...

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참고) 이러한 티틴이 산화 스트레스를 받아서 stiffness 현상이 나타나면.. 심근 탄력 기능 저하...

질문 근절 단백질 티틴을 암호화하는 유전자 티틴(TTN)의 유전적 변이와 조기 발병 심방세동 사이에 연관성이 있을까요? 결과 조기 발병 심방세동 환자 2781명과 대조군 4959명을 대상으로 한 이 사례-대조군 연구에서 TTN의 기능 상실 변이와 심방세동 사이에는 통계적으로 유의미한 연관성이 있었으며(오즈비, 1.76 [95% CI, 1.04-2.97]), 사례 참가자의 2.1%와 대조군의 1.1%에서 변이가 나타났습니다(오즈비, 1.76 [95% CI, 1.04-2.97]). 의미 TTN 유전자의 기능 상실 돌연변이는 일부 환자에서 조기 발병 심방세동과 관련이 있었지만, 인과 관계를 파악하기 위해서는 추가 연구가 필요합니다.

ReviewPassive Properties of Muscle

Titin as a force-generating muscle protein under regulatory control

Johanna K. Freundt and 

Wolfgang A. Linke

16 May 2019https://doi.org/10.1152/japplphysiol.00865.2018

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Abstract

Titin has long been recognized as a mechanical protein in muscle cells that has a main function as a molecular spring in the contractile units, the sarcomeres. Recent work suggests that the titin spring contributes to muscle contraction in a more active manner than previously thought. In this review, we highlight this property, specifically the ability of the immunoglobulin-like (Ig) domains of titin to undergo unfolding-refolding transitions when isolated titin molecules or skeletal myofibrils are held at physiological force levels. Folding of titin Ig domains under force is a hitherto unappreciated, putative source of work production in muscle cells, which could work in synergy with the actomyosin system to maximize the energy delivered by a stretched, actively contracting muscle.

This review also focuses on the mechanisms shown to modulate titin-based viscoelastic forces in skeletal muscle cells, including chaperone binding, titin oxidation, phosphorylation, Ca2+ binding, and interaction with actin filaments. Along the way, we discuss which of these modulatory mechanisms might contribute to the phenomenon of residual force enhancement relevant for eccentric muscle contractions. Finally, a brief perspective is added on the potential for the alterations in titin-based force to dynamically alter mechano-chemical signaling pathways in the muscle cell. We conclude that titin from skeletal muscle is a determinant of both passive and active tension and a bona fide mechanosensor, whose stiffness is tuned by various independent mechanisms.

초록

타이틴(titin)은 

근육 세포에서 수축 단위인 sarcomere에서 분자 스프링의 주요 기능을 하는 

기계적 단백질로 오랫동안 인식되어 왔습니다. 

최근 연구에 따르면 

타이틴 스프링이 이전에 생각했던 것보다 

더 적극적인 방식으로 근육 수축에 기여하는 것으로 나타났습니다. 

이 리뷰에서는 이 특성, 

특히 분리된 타이틴 분자 또는 골격 근섬유가 생리적 힘 수준에서 유지될 때 

타이틴의 면역글로불린 유사(Ig) 도메인이 

접힘-재접힘 전환을 겪는 능력을 강조합니다. 

unfolding-refolding transitions

힘을 받는 타이틴 Ig 도메인의 접힘은 

지금까지 잘 알려지지 않은 근육 세포의 일 생산 원천으로 추정되며,

이는 액토미오신 시스템과 시너지 효과를 발휘하여 늘어나고

활발하게 수축하는 근육이 전달하는 에너지를 최대화할 수 있습니다.

이 리뷰에서는 또한

샤페론 결합,

티틴 산화,

인산화,

Ca2+ 결합,

액틴 필라멘트와의 상호작용 등

골격근 세포에서 티틴 기반 점탄성 힘을 조절하는 메커니즘에 초점을 맞춥니다.

This review also focuses on the mechanisms shown to modulate

titin-based viscoelastic forces in skeletal muscle cells,

including

chaperone binding,

titin oxidation,

phosphorylation,

Ca2+ binding, and

interaction with actin filaments

티틴 산화는 산화 스트레스로 인해 거대 근육 단백질인 티틴이 변형되는 것을 말합니다. 이 과정은 심근(심장 근육) 경직의 주요 원인인 티틴의 강성을 변화시킵니다. 심장 질환에서 티틴 강성 증가가 종종 관찰됩니다 [1][2]. 티틴에는 특히 산화에 취약한 시스테인 잔기가 포함되어 있어 기계적 특성에 영향을 미치고 잠재적으로 심혈관 질환의 원인이 될 수 있습니다[4][5]. 티틴 산화를 이해하면 심근 탄력과 심혈관 건강을 목표로 하는 치료법에 대한 통찰력을 얻을 수 있습니다[6].

그 과정에서

이러한 조절 메커니즘 중 어떤 것이

편심성 근육 수축과 관련된 잔류력 강화 현상에 기여할 수 있는지에 대해 논의합니다.

마지막으로,

티틴 기반 힘의 변화가

근육 세포의 기계 화학적 신호 경로를 동적으로 변화시킬 가능성에 대한

간략한 관점을 추가합니다.

결론적으로

골격근의 티틴은

수동 및 능동 장력의 결정 요인이며,

다양한 독립적인 메커니즘에 의해 강성이 조절되는 진정한 기계 센서라는 결론을 내립니다.

INTRODUCTION

In muscle mechanics, the terms “passive” and “active” are used to distinguish the force of a muscle that is stretched but not activated by electrical stimuli/action potentials (and the ensuing rise in intracellular free Ca2+) from that developed because of excitation-contraction coupling. However, “passive” in this context is not to be confused with “less important.” Various lines of research performed over the last years have suggested that passive and active muscle forces are more closely interconnected than previously thought and that, e.g., the level of passive force regulates active contractile properties (1, 32, 48). This connection results, in part, from the unique arrangement of the major myofilaments in the contractile units, the sarcomeres, which consist of myosin-based thick filaments, actin-based thin filaments, and titin-based elastic filaments (Fig. 1, top). Despite the obvious structural and functional links between those myofilaments, the field most often still separates titin-based “passive” from actomyosin-based “active” forces. We have advocated a view in which the interdependence between titin and actomyosin mechanical properties in the sarcomere serves to optimize muscle contraction (14, 48, 78).

소개

근육 역학에서 “수동적”과 “능동적”이라는 용어는 전기 자극/활동 전위(및 그에 따른 세포 내 유리 Ca2+의 상승)에 의해 신장되었지만 활성화되지 않은 근육의 힘과 여기-수축 결합으로 인해 발생하는 힘을 구별하는 데 사용됩니다. 그러나 이 맥락에서 “수동적”을 “덜 중요”과 혼동해서는 안 됩니다.

지난 몇 년 동안 수행된 다양한 연구에 따르면

수동 및 능동 근육의 힘이 이전에 생각했던 것보다

더 밀접하게 연결되어 있으며,

예를 들어 수동 힘의 수준이 능동 수축 특성을 조절한다고 합니다(1, 32, 48).

이러한 연결은 부분적으로는

미오신 기반의 굵은 필라멘트,

액틴 기반의 얇은 필라멘트 및

티틴 기반의 탄성 필라멘트로 구성된 수축 단위의 주요 근섬유,

즉  근절의 독특한 배열에서 비롯됩니다(그림 1, 상단).

이러한 근섬유 사이의 명백한 구조적 및 기능적 연관성에도 불구하고,

현장에서는 여전히 티틴 기반의 '수동적' 힘과 액토미오신 기반의 '능동적' 힘을

구분하는 경우가 많습니다.

우리는

근섬유의 타이틴과 액토미오신 기계적 특성 간의 상호 의존성이

근육 수축을 최적화하는 역할을 한다는 견해를 지지해 왔습니다(14, 48, 78).

Fig. 1.Layout and domain architecture of titin filaments in the skeletal muscle sarcomere. Top: schematic of the 3-filament sarcomere highlighting myosin, actin, and titin filaments. Middle: protein domains expressed in the N2A isoform of skeletal muscle titin (consensus sequence). Differentially spliced titin segments are indicated. FnIII, fibronectin type-III-like; Ig, immunoglobulin-like; PEVK, protein domain rich in proline, glutamic acid, valine, and lysine residues; TK, titin kinase. Bottom: layout of the titin spring (consensus sequence of N2A isoform) in the half-sarcomere. Color coding of titin domains is as in middle panel. Note that there is a break inserted in the A-band region close to the M band, which indicates that the A-band segment is much longer than actually shown.

그림 1:골격근 근절에서 타이틴 필라멘트의 레이아웃과 도메인 구조. 위: 미오신, 액틴, 타이틴 필라멘트가 강조된 3-필라멘트 sarcomere의 모식도. 가운데: 골격근 티틴의 N2A 이소형에서 발현되는 단백질 도메인(합의 서열). 차등적으로 접합된 티틴 세그먼트가 표시되어 있습니다.

FnIII, 피브로넥틴 타입 III 유사체; Ig, 면역글로불린 유사체; PEVK, 프롤린, 글루탐산, 발린 및 라이신 잔기가 풍부한 단백질 도메인; TK, 타이틴 키나아제. 아래: 반육종에서 타이틴 스프링(N2A 이소폼의 합의 서열)의 레이아웃. 타이틴 도메인의 색상 코딩은 가운데 패널과 같습니다. M 밴드에 가까운 A 밴드 영역에 단절이 삽입되어 있는데, 이는 A 밴드 세그먼트가 실제보다 훨씬 길다는 것을 나타냅니다.

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Much of a muscle’s passive force upon stretching is generated by titin, a giant protein with a molecular mass of 3–4 MDa (Fig. 1). Titin-based force directly or indirectly contributes to and modulates active tension (48). Moreover, conformational changes attendant with the stretch of the titin spring can trigger mechanical signaling events in the myocytes leading to, e.g., enhanced muscle protein degradation and activation of protein synthesis (55). In this minireview, we provide an update on the molecular mechanisms of titin extensibility in the sarcomere and point out how new insights into these properties suggest a more active role for the protein in force development. We then highlight the mechanisms shown to acutely modulate the stiffness of the titin spring in skeletal muscle, including chaperone binding, posttranslational modification, Ca2+ binding, and actin-titin interaction. We discuss which of these mechanisms could contribute to enhanced force production in active lengthening (eccentric) muscle contractions, which is a phenomenon that fails to be explained by actomyosin properties (27). The work reviewed here leads us to conclude that titin is a determinant of muscle stress, whose mechanical properties are highly regulated. These characteristics place titin in a unique position to act as a mechanosensor in skeletal muscle cells.

스트레칭 시

근육의 수동적 힘의 대부분은

분자량이 3~4MDa인 거대 단백질인 타이틴에 의해 생성됩니다(그림 1).

타이틴 기반 힘은

직간접적으로 활성 장력에 기여하고 이를 조절합니다(48).

또한, 타이틴 스프링의 신장에 수반되는 형태 변화는

근육 단백질 분해 및 단백질 합성 활성화로 이어지는

근세포의 기계적 신호 이벤트를 유발할 수 있습니다(55).

이 미니리뷰에서는

sarcomere에서 타이틴 확장성의 분자적 메커니즘에 대한 최신 정보를 제공하고

이러한 특성에 대한 새로운 통찰력이 어떻게 힘 발달에서

단백질의 보다 적극적인 역할을 시사하는지 설명합니다.

그런 다음

샤페론 결합,

번역 후 변형,

Ca2+ 결합,

액틴-티틴 상호 작용 등 골격근에서

티틴 스프링의 강성을 급격하게 조절하는 것으로 보이는 메커니즘을 강조합니다.

우리는 이러한 메커니즘 중

어떤 것이 액토미오신 특성으로는 설명되지 않는 현상인

활성 신장(편심) 근육 수축에서 향상된 힘 생성에 기여할 수 있는지에 대해 논의합니다(27).

여기서 검토한 연구를 통해

티틴은 근육 스트레스의 결정 요인이며,

그 기계적 특성이 고도로 조절된다는 결론을 내릴 수 있습니다.

이러한 특성으로 인해

티틴은 골격근 세포에서

기계 센서 역할을 하는 독특한 위치에 놓이게 됩니다.

MECHANICALLY ACTIVE TITIN SPRING ELEMENTS

The titin molecule (Fig. 1) is capped at the NH2 terminus by telethonin (35, 97) and anchored within the Z disk through interactions with α-actinin and actin (51, 82, 96). The elastic segment of titin is in the sarcomeric I band and exists in many different length isoforms (17) generated by differential splicing of “I-band” exons in the titin gene (TTN; higher vertebrates have a single titin gene). Thus, the titin spring segment varies in size by ~400 kDa in different skeletal muscles from the same species, which gives rise to highly variable titin-based stiffness (74). This variability is even larger in cardiac muscle, where I-band titin size varies by ~700 kDa (17). The titin springs can be subdivided into a proximal, a differentially spliced, and a distal region consisting of immunoglobulin-like (Ig) domains arranged in tandem (Fig. 1). Two additional spring elements are present between the differentially spliced and distal Ig regions: the N2A element consisting of several Ig domains interspersed with unique sequences and the PEVK element, which is rich in proline (P), glutamate (E), valine (V), and lysine (K) residues and is considered to be an intrinsically disordered protein region. The PEVK element comprises repetitive, mostly 28-residue-long motifs, each encoded by a single TTN exon (21, 43). The PEVK repeats are exposed to intense differential splicing, such that the number of motifs (and thus PEVK residues) included in a given muscle varies greatly (17, 21, 43). Both the PEVK segment and all Ig-domain regions represent important spring elements in titin (53). The remainder of the titin molecule is part of the sarcomeric A-band and M-band regions (Fig. 1) and is rendered largely inextensible via binding to myosin, myosin-binding protein-C (MyBPC), and myomesin. However, this part of titin may be centrally involved in A-band mechanosensory functions by translating a stretch signal into conformational changes of proteins within the actin-myosin filament overlap zone (2, 20, 45, 48, 89).

기계적으로 활성인 타이틴 스프링 요소

타이틴 분자(그림 1)는 텔레토닌(35, 97)에 의해 NH2 말단에서 캡핑되고 α-액티닌 및 액틴(51, 82, 96)과의 상호작용을 통해 Z 디스크 내에 고정됩니다. 타이틴의 탄성 세그먼트는 육종성 I 밴드에 있으며, 타이틴 유전자에서 “I-밴드” 엑손의 차등 접합에 의해 생성된 다양한 길이의 이소형(17)으로 존재합니다(TTN; 고등 척추동물은 단일 타이틴 유전자를 가짐). 따라서 타이틴 스프링 세그먼트는 같은 종의 다른 골격근에서 크기가 ~400kDa까지 다양하며, 이로 인해 타이틴 기반 강성이 매우 가변적입니다(74). 이러한 변동성은 심장 근육에서 훨씬 더 커서, I-밴드 티틴 크기는 ~700 kDa까지 다양합니다(17). 타이틴 스프링은 근위부, 차동 접합부, 면역글로불린 유사(Ig) 도메인이 나란히 배열된 원위부로 세분화할 수 있습니다(그림 1). 차동 스플라이스된 원위 Ig 영역 사이에는 두 가지 스프링 요소가 추가로 존재하는데, 고유 서열이 산재한 여러 Ig 도메인으로 구성된 N2A 요소와 프롤린(P), 글루타메이트(E), 발린(V), 라이신(K) 잔기가 풍부하고 본질적으로 무질서한 단백질 영역으로 간주되는 PEVK 요소가 그것입니다. PEVK 요소는 대부분 28개의 잔기 길이로 반복되는 모티프로 구성되며, 각각 단일 TTN 엑손(21, 43)으로 암호화됩니다. PEVK 반복은 강렬한 차등 스플라이싱에 노출되어 특정 근육에 포함된 모티프(따라서 PEVK 잔기)의 수가 크게 달라집니다(17, 21, 43). PEVK 세그먼트와 모든 Ig 도메인 영역은 모두 티틴에서 중요한 스프링 요소를 나타냅니다(53). 나머지 타이틴 분자는 육종성 A-밴드 및 M-밴드 영역의 일부이며(그림 1) 미오신, 미오신 결합 단백질-C(MyBPC) 및 미오메신과의 결합을 통해 대부분 확장되지 않게 됩니다. 그러나 티틴의 이 부분은 스트레치 신호를 액틴-미오신 필라멘트 중첩 영역 내 단백질의 형태 변화로 변환함으로써 A-밴드 기계 감각 기능에 중심적으로 관여할 수 있습니다(2, 20, 45, 48, 89).

SARCOMERE STRETCHING CAUSES TITIN SEGMENT EXTENSION AND IG DOMAIN UNFOLDING

When the sarcomeres of a skeletal muscle are “passively” stretched, e.g., by the antagonist muscle or gravitational forces, the titin Ig-domain segments and the PEVK region extend sequentially (Fig. 2). In the absence of external force, at slack length, the Ig domains are in their native, folded state and the linker regions between the domains adopt a bent configuration [analogy of a “carpenter’s ruler” (91)]. The PEVK domain is not extended. When low forces are applied to stretch the sarcomere, the linker regions connecting the Ig domains are straightened out; the domains are still folded (53). This provides initial extensibility at low force (Fig. 2) (53, 56). When the stretch force increases further, the PEVK domain begins to extend and provides ample extensibility (52, 53, 56, 84). The spring force of these titin segments arises predominantly from their entropic elasticity (34, 77, 86), although additional factors, such as electrostatic interactions, may be important for PEVK elasticity (52, 65). Models of entropic elasticity theory were successfully applied to quantitatively describe the stepwise extension mechanism of the titin spring in the sarcomere (44, 52, 56, 84).

sarcomere 스트레칭은 타이틴 세그먼트 확장과 IG 도메인 전개를 유발합니다.

골격근의 근절이 길항근이나 중력에 의해 “수동적으로” 늘어나면,

티틴 Ig 도메인 세그먼트와 PEVK 영역이 순차적으로 확장됩니다(그림 2).

외력이 없을 때, 느슨한 길이에서 Ig 도메인은 원래의 접힌 상태에 있고

도메인 사이의 링커 영역은 구부러진 구성을 채택합니다 [“목수의 자”의 비유 (91)].

PEVK 도메인은 확장되지 않습니다.

근절을 늘리기 위해 낮은 힘을 가하면 Ig 도메인을 연결하는 링커 영역이 곧게 펴지고 도메인은 여전히 접혀 있습니다(53). 이것은 낮은 힘에서 초기 확장성을 제공합니다 (그림 2) (53, 56). 스트레치 힘이 더 증가하면 PEVK 도메인이 확장되기 시작하여 충분한 확장성을 제공합니다(52, 53, 56, 84).

이러한 티틴 세그먼트의 스프링 힘은

주로 엔트로피 탄성(34, 77, 86)에서 발생하지만

정전기적 상호 작용과 같은 추가 요인이 PEVK 탄성에 중요할 수 있습니다(52, 65).

엔트로피 탄성 이론의 모델은

근절에서 타이틴 스프링의 단계적 확장 메커니즘을

정량적으로 설명하는 데 성공적으로 적용되었습니다(44, 52, 56, 84).

Fig. 2.State-of-the-art model of differential titin spring extension and contraction on sarcomere stretching and release, respectively, and levels of force/titin molecule encountered. The main schematic depicts the initial straightening of the immunoglobulin-like (Ig) domain segment(s) from a buckled appearance at low stretch force (F), followed by unraveling of the protein domain rich in proline, glutamic acid, valine, and lysine residues (PEVK) at intermediate and high stretch forces. The unfolding probability of individual Ig domains increases with the stretch force and becomes physiologically significant at forces of ≥4 pN/titin. Upon release, the unfolded Ig domains perform a folding contraction under force, which generates work during the transition to a molten globule/collapsed state (which is spared during unfolding). The native, folded state needs relatively more time to be reached. Inset, relationship between force/titin molecule and relative sarcomere extension. The contributions of the Ig domain segments (straightening; unfolding of individual modules) and the PEVK region at the respective lengths are indicated.

근절의 신축과 이완에 따른 차동 타이틴 스프링의 신축과 수축, 그리고 발생하는 힘/타이틴 분자의 수준에 대한 최첨단 모델입니다. 주요 모식도는 낮은 신장력(F)에서 면역글로불린 유사(Ig) 도메인 세그먼트가 좌굴된 모습에서 곧게 펴진 후 프롤린, 글루탐산, 발린, 라이신 잔기(PEVK)가 풍부한 단백질 도메인이 중간 및 높은 신장력에서 풀리는 모습을 보여줍니다. 개별 Ig 도메인의 펼쳐질 확률은 스트레치 힘에 따라 증가하며 ≥4pN/titin의 힘에서 생리적으로 유의미해집니다. 풀리면 펼쳐진 Ig 도메인은 힘을 받아 접힘 수축을 수행하여 용융 소구/접힌 상태로 전환하는 동안(펼쳐지는 동안에는 여유가 있음) 일을 생성합니다. 원래의 접힌 상태에 도달하는 데는 상대적으로 더 많은 시간이 필요합니다. 삽입, 힘/티틴 분자와 상대적 육종 확장 사이의 관계. 각 길이에서 Ig 도메인 세그먼트(곧게 펴짐, 개별 모듈의 펼침)와 PEVK 영역의 기여도가 표시되어 있습니다.

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Contrary to an early proposal (15), unfolding of titin Ig domains was long considered to be unlikely in the sarcomere, or at best to occur only beyond physiological muscle lengths (50, 85). This view was supported by the results of single-molecule mechanical measurements using the atomic force microscope (AFM), which recorded force in response to quick stretching at constant velocity and suggested that these domains unfold at nonphysiological forces above 100–200 pN (44, 77). Since the unfolding probability of the Ig domains increases with the stretch force (Fig. 2, inset), Ig unfolding was still suspected as a source of viscoelastic force decay in more highly stretched sarcomeres (61) (Fig. 2, left). However, only recently was unfolding at low (physiological) force levels demonstrated convincingly at the single-molecule level, and an effort was made to confirm‎ this unfolding in isolated skeletal myofibrils (78). The study used magnetic tweezers, which are more stable than the AFM in the low force range. When recombinant titin fragments from the proximal Ig region were held at a constant force of 6 pN (range 4–10 pN) for up to several hours, many unfolding and refolding length steps were observed. A force of ~6 pN/titin corresponds to a sarcomere length (SL) of ~3 µm in m. psoas myofibrils (56), which is near the high end of the physiological SL range (57). Therefore, single isolated myofibrils were labeled by antibody-functionalized quantum dots (Qdots) positioned within the differentially spliced titin Ig segment and stretched quickly to ~3 µm SL. Then, the distance separating pairs of individual Qdots (measured across the Z disk) was followed over time (78). Many stepwise alterations in the Qdot separation distance were observed, suggesting unfolding-refolding transitions of proximal titin Ig domains in stretched sarcomeres. Stepwise force/length transitions under low constant stretch/force levels were also detected in isolated native titin molecules held by optical tweezers and were interpreted as Ig domain unfolding-refolding events (60). Collectively, these experiments established that Ig domain unfolding and refolding are part of the titin extension-relaxation mechanism and occur at low forces (Fig. 2). Although there can be no doubt that Ig-domain unfolding takes place in isolated titin molecules and probably in stretched isolated myofibrils at “physiological” levels of force/titin (<10 pN), the concept warrants support from experiments on living muscle to become universally accepted.

초기 제안(15)과는 달리, 티틴 Ig 도메인의 전개는 오랫동안 근절에서 일어날 가능성이 낮거나 기껏해야 생리적 근육 길이를 넘어서서만 일어나는 것으로 여겨졌습니다(50, 85). 이러한 견해는 원자힘 현미경(AFM)을 사용한 단일 분자 기계적 측정 결과에 의해 뒷받침되었는데, 이 현미경은 일정한 속도에서 빠른 신장에 대한 힘을 기록하여 이러한 도메인이 100-200 pN 이상의 비생리적 힘에서 펼쳐진다고 제안했습니다(44, 77). Ig 도메인의 전개 확률은 신장력에 따라 증가하기 때문에(그림 2, 삽입), 더 많이 신장된 육종에서 점탄성 힘 붕괴의 원인으로 여전히 Ig 전개가 의심되었습니다(61)(그림 2, 왼쪽). 그러나 최근에야 낮은 (생리적) 힘 수준에서의 전개가 단일 분자 수준에서 설득력있게 입증되었으며, 분리 된 골격 근섬유에서 이러한 전개를 확인하기위한 노력이 이루어졌습니다 (78). 이 연구에서는 낮은 힘 범위에서 AFM보다 더 안정적인 자기 핀셋을 사용했습니다. 근위 Ig 영역의 재조합 티틴 조각을 최대 몇 시간 동안 6pN(범위 4-10pN)의 일정한 힘으로 유지했을 때, 많은 접힘과 재접힘 길이 단계가 관찰되었습니다. 6 pN/티틴의 힘은 생리적 SL 범위(57)의 최상단에 가까운 m. 요근 근섬유(56)에서 ~3 µm의 육종 길이(SL)에 해당합니다. 따라서 분리된 단일 근섬유를 차동 접합된 티틴 Ig 세그먼트 내에 위치한 항체 기능화 양자점(Qdot)으로 표지하고 ~3 µm SL까지 빠르게 늘렸습니다. 그런 다음 시간이 지남에 따라 개별 큐닷 쌍을 분리하는 거리(Z 디스크 전체에서 측정)를 추적했습니다(78). Qdot 분리 거리의 많은 단계적 변화가 관찰되었으며, 이는 늘어난 육종에서 근위 티틴 Ig 도메인의 펼쳐짐-접힘 전이를 시사합니다. 낮은 일정한 신축/힘 수준에서 단계적 힘/길이 전이는 광학 핀셋으로 잡은 분리된 네이티브 타이틴 분자에서도 검출되었으며, 이는 Ig 도메인 전개-접힘 사건으로 해석되었습니다(60). 이러한 실험을 종합해 볼 때, Ig 도메인 전개 및 재접힘은 타이틴 확장-이완 메커니즘의 일부이며 낮은 힘에서 발생한다는 사실이 밝혀졌습니다(그림 2). 분리된 티틴 분자와 아마도 “생리적” 수준의 힘/티틴(<10 pN)에서 늘어난 분리된 근섬유에서 Ig 도메인 전개가 일어난다는 것은 의심의 여지가 없지만, 이 개념이 보편적으로 수용되기 위해서는 살아있는 근육에 대한 실험의 뒷받침이 필요합니다.

TITIN IG DOMAIN REFOLDING UNDER FORCE: A SOURCE OF WORK PRODUCTION IN MUSCLE

The refolding of titin Ig domains in the sarcomere has far-reaching implications for myocyte function, because shortening against a force produces work. The mechanical work is generated before the titin domains attain the native, folded state, specifically, upon transition from the unfolded state to a collapsed/molten globule state (Fig. 2) (14, 60, 78). [In comparison, the unfolding mechanism includes only very short-lived unfolding intermediates, if any (59).] On the basis of these findings, it has been suggested that titin generates a certain amount of the total work produced by a contracting muscle (78). Theoretical considerations reveal that titin recovers the energy stored upon a stretch only to a minor degree via entropic recoil, whereas the majority is returned via the titin folding contraction (14). The importance of the titin folding contraction for mechanical muscle function has been extensively reviewed recently (14, 48). Whether titin folding-unfolding gives intact muscle the extra “edge” is not yet known and will be an interesting topic for future exploration.

Various issues in the above scenario require further study. For instance, it should be ascertained that the SL/force levels in passively stretched muscle cells are high and variable enough for substantial titin Ig unfolding-refolding to take place. Live in situ measurements on rodent and human skeletal muscles did suggest relatively long physiological SLs and SL excursions reaching up to >3 µm (57). Nevertheless, it remains unknown how many Ig domains per titin molecule unfold-refold at which physiological SL. Another point is that Ig domain refolding must occur synchronously in the >1,000 parallel titin molecules present in a sarcomere to make an impact on work production. The trigger for synchronized domain refolding was suggested to be the onset of myosin motor activity at the start of an active contraction (14), which is a possibility not yet tested experimentally. Furthermore, the time course of titin domain refolding is important but difficult to measure in living sarcomeres. Arguments have been put forth suggesting that the extremely fast entropic recoil of the titin spring elements plus the slower folding contractions of the Ig domains can work in synergy with the fast myosin motor to enhance the power output of the sarcomere (14). Importantly, the titin folding mechanism is effective at SLs comprising the higher end of the physiological length range in skeletal muscles, where the overlap between actin and myosin filaments is reduced (14, 48). Thus, the mechanism could explain, in part, why forces on the descending limb of the active length-tension curve can significantly deviate from those predicted based only on thick and thin filament overlap. In conclusion, titin domain folding against a force represents a previously unappreciated potential source of work production in muscle, which presumably acts synchronously with the actomyosin contractile mechanism. The sum of the energy delivered by the titin-based folding contractions is less than that generated by the actomyosin system, but the two mechanisms are inextricably coupled in the sarcomere. This way, titin is an active component in the sarcomere that helps to maximize muscle work output. Whereas the above suggestions are a logical consequence of the measured single-molecule properties of titin, there is more research to be done to unequivocally demonstrate their relevance for real muscle.

TITIN SPRING STIFFNESS CAN BE TUNED BY SEVERAL INDEPENDENT MECHANISMS

The elasticity and stretch-dependent “passive” force of titin are highly variable. There are different mechanisms by which titin stiffness can be modified, including the increase in inherent spring stiffness and/or the shortening/lengthening of spring length. The latter is done by differential splicing of I-band exons causing highly variable “hardware” stiffness among different skeletal muscles (74). Much evidence has been assembled demonstrating that titin-based stiffness is modulated beyond I-band titin splicing in cardiomyocytes, e.g., via phosphorylation-dephosphorylation (25, 40). However, the mechanical properties of the titin spring can also be fine-tuned in skeletal muscle. Mechanisms include chaperone binding, oxidation, Ca2+ binding, actin-titin interaction, and phosphorylation.

Heat Shock Protein Binding and Titin-Based Passive Tension

The unfolding of protein domains in the titin spring bears the risk of causing misfolding and aggregation and thus loss of protein function. Indeed, misfolding events have been reported for recombinantly expressed titin Ig domains unfolded by stretching in single-molecule mechanical studies, although most of these domains refold correctly (7, 67, 68). However, when recombinant titin Ig domains are chemically unfolded in bulk experiments, they aggregate (37, 93). Unlike the Ig segments of titin, the PEVK region has the typical properties of an intrinsically disordered region (24), which avoids aggregation: it is enriched in structure-breaking (proline) and charged (glutamate, lysine) residues, devoid of cysteine and asparagine, and altogether has a high net charge and low hydrophobicity, as well as no aggregating sequences (37). Therefore, the titin Ig segments, but not the PEVK region, require protection against aggregation when they become extended/unfolded by mechanical strain in muscle cells.

This protection is provided by a specific class of chaperones, the small heat shock proteins (sHSPs), notably HSP27 (HSPB1) and αB-crystallin (CryaB; HSPB5) (Fig. 3), which were shown to bind to I-band titin Ig domains but not to PEVK (37). These (ATP independent) chaperones are very abundant in the cytosol of muscle cells. They are further induced and frequently translocated to the sarcomeres under diverse stress conditions, including eccentric exercise (72), myocyte strain (37), inherited muscle disease (16, 88) [where binding to the sarcomeric I band can be massive (88)], aging (10), and oxidative or acidic stress (63). CryaB prevented the aggregation of chemically unfolded Ig domains from titin’s proximal Ig or N2A regions in in vitro assays, in which both the aggregation tendency and the protective effect by CryaB were enhanced by modest acidosis (37). The presence of recombinant sHSPs in skinned myocytes that were overstretched under mildly acidic conditions protected the cells from pathological stiffening possibly following from intrasarcomeric titin aggregation (37). In summary, binding of sHSPs to the unfolded titin spring regions may be a crucial protective mechanism in mechanically stressed myocytes. The interaction is likely to prevent titin aggregation, promote correct refolding of unfolded Ig domains, and thus act as an early protective measure, and it could also provide a signal for the eventual targeted degradation of titin.

Fig. 3.Overview of the mechanisms known to modulate titin-based force in the skeletal muscle sarcomere. For detailed explanation, see text. Additionally, various titin-interacting proteins are depicted at their respective binding sites on titin. Some of these molecules may undergo a conformational change when titin is stretched, triggering strain-induced mechano-chemical signaling pathways that promote muscle protein turnover. These mechanosensory events are reviewed in detail elsewhere (47). ADP, adenosine diphosphate; ATP, adenosine triphosphate; Ca2+, concentration of free calcium ions; FHL2, four-and-a-half-LIM domains protein 2; G, glutathione; H, hydrogen atom; HSP27, heat shock protein 27 (HSPB1); HSP90, heat shock protein 90; Ig, immunoglobulin-like; MARPs, muscle ankyrin repeat proteins; MuRF1/2, muscle-specific RING finger-1 and -2 proteins; MyBPC, myosin-binding protein-C; Nbr1, neighbor of BRCA1 gene-1 protein; P, inorganic phosphate; ROS, reactive oxygen species; S, sulfur atom in cysteine; sHSP, small heat shock protein; Smyd2, SET and MYND domain containing 2 protein (a lysine methyltransferase); TK, titin kinase domain.

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Binding of sHSPs to titin Ig domains in stretched myocytes appears to be triggered by the exposure of previously buried hydrophobic residues in the titin protein chain upon Ig domain unfolding (37). However, sHSPs such as CryaB may be able to also bind to folded titin Ig domains (8, 37). The physiological function of such an interaction remains obscure, but a side effect seems to be mildly elevated titin-based passive tension (Fig. 3, inset, top left) (88). This stiffening effect can be explained, in theory, by an increase in the unfolding forces of titin Ig domains in the presence of CryaB, resulting in mechanical stabilization of the titin spring (8), and/or by an effect of the sHSP binding on the entropic elasticity of titin. In any case, HSP binding to the titin spring is a cause of altered titin-based stiffness.

Modulation of Titin Stiffness by Oxidative Modifications

Oxidative stress is associated with intense muscle activity and can be increased further in myopathic muscles. Oxidative modifications affect, among others, the mechanical properties of muscle cells via alterations in sarcomere protein function (5). The titin spring appears to be a main target for oxidation (3, 19, 23), which serves to tune its stiffness (Fig. 3) Two types of oxidative modification have the potential to alter titin elasticity in skeletal myocytes, and both of them require the unfolding of I-band titin Ig domains. The first is S-glutathiolation of cysteines that become exposed when an Ig domain unfolds (“cryptic” cysteines) (3). Glutathione forms a mixed disulfide with the exposed titin site, which hinders the refolding of the domain. Since the prior unfolding of the Ig domain increased the contour length of the titin spring by ~30 nm, lowering titin’s elastic force, the overall effect of preventing titin Ig domain refolding by S-glutathiolation is a long-lasting (but reversible) reduction in titin-based passive force (Fig. 3) (3).

The second mechanism involves the oxidant-induced formation of a disulfide bridge within the mechanically unfolded titin Ig domain (19). This intramolecular bonding reduces the length of the unfolded Ig region and is therefore predicted to increase titin-based spring stiffness (Fig. 3). Interestingly, many differentially spliced Ig domains in I-band titin contain at least three (evolutionarily well conserved) cysteines, which allow for S-S isomerization (19). Depending on which S-S isomer is formed within the domain, the free length of the unfolded segment is shorter or longer, implicating an additional mode of titin elasticity regulation via oxidation. The role of Ig domain oxidation in titin elasticity modulation in vivo is still unknown. An important test will be to study whether muscle stretching under oxidative stress really increases Ig domain oxidation in I-band titin.

Stiffness Increase Through Ca2+ Binding to Titin Spring Elements

Calcium binds to glutamate-rich segments within titin’s differentially spliced portion of the PEVK region (42). Because this PEVK subsegment is negatively charged (unlike the constitutively expressed bit of PEVK, which has a net positive charge), Ca2+ binding will increase intramolecular attractions within this subdomain, thus elevating titin-based force in response to stretching (Fig. 3). An increase in mechanical stability was also reported for a distal titin Ig domain when it was unfolded in single-molecule mechanical measurements in the presence of free Ca2+, which could also translate into some stiffening of titin in sarcomeres (12). A large stiffness increase was observed in mechanically overstretched (to beyond overlap of actin and myosin filaments) single skeletal myofibrils in high-Ca2+ versus low-Ca2+ activation buffer and was speculated to be due somewhat to Ca2+ binding to titin (15%) but mostly to Ca2+-promoted interactions of titin with the actin filaments (85%) (28, 73). However, since the overstretch of sarcomeres causes loss of ordered sarcomere structure, e.g., the breaking loose of A-band titin from myosin (49, 53, 92), the large magnitude of the stiffness increase seen in the presence of high Ca2+ could have been an experimental artifact. Nevertheless, a small increase in titin-based stiffness is expected from the Ca2+ binding to PEVK (42) and Ig domains (12), which seems to be a consensus interpretation in the field. This titin-stiffening mechanism from Ca2+ binding could explain the increased tension and stiffness of a non-cross-bridge structure during Ca2+ influx in active skeletal muscle (4, 9, 66, 76). The mechanism could be responsible for a small proportion of the residual force enhancement of actively stretched muscle (48), which is a characteristic of eccentric muscle contractions but unexplained by the sliding filament and cross bridge theories (27).

Actin-Titin Binding and Viscoelastic Force in the Sarcomere: Regulation by Ca2+?

Titin binds at several sites along its length to the sarcomeric actin filaments, notably at the periphery of the Z disk, which serves an anchoring function (51, 83). Additionally, both the differentially spliced and the constitutively expressed PEVK subsegments, but not the Ig domains in I-band titin or some Ig/FnIII domains in A-band titin, interact with the actin filaments (6, 41, 51, 54, 64, 94). Conflicting results have been obtained as regards the propensity of the N2A element to bind actin filaments. Earlier work found no evidence for this binding in “competition assays,” in which recombinant N2A titin fragment was added to single isolated myofibrils and a possible effect on passive stiffness measured (51). In contrast, recent work demonstrated interaction between actin and recombinant N2A fragment by cosedimentation assay, rupture force measurements using single-molecule force spectroscopy, and actin-myosin in vitro motility assay (11). No matter where exactly the main interactions of I-band titin with actin filaments take place, this (relatively weak) association is a source of viscous drag force in the sarcomere (Fig. 3) (41) and could shorten the effective length of the titin spring in relaxed sarcomeres (64), which would also increase viscoelasticity (6). In support of these suggestions, there is reduced internal viscous resistance to passive shortening in sarcomeres after cleavage of the actin filaments (69).

Interestingly, the actin-titin interactions depend, to variable degrees, on the concentration of free Ca2+ ions (11, 33, 41, 54). The earliest study testing the functional effect of interaction between actin filaments and native isolated titin (by means of observing the actin filament sliding in the actin-myosin in vitro motility assay) suggested that this interaction was increased in the presence of physiological levels of Ca2+ (33). Since then, various studies have measured the impact of Ca2+ on the interaction between actin and recombinant fragments from the elastic titin spring segment or from A-band titin, with different results. Some studies found that physiological levels of Ca2+ had no effect on the interaction (51, 64, 94). Others found that the presence of Ca2+ made the interaction between actin and PEVK titin (especially the constitutive subfragment) weaker (41, 54). This reduction is consistent with the results of a newer study investigating the segmental extension of titin spring elements with stretching under passive and Ca2+-activated conditions, revealing a more stretchable PEVK region in the presence of Ca2+ (13). A novel result is the increase in actin-binding strength of titin’s N2A element when Ca2+ is present (11). The idea of a stronger interaction between actin and titin spring regions that takes place when Ca2+ is raised to higher physiological levels has been around for some time (28, 46), but without strong evidence supporting it. The Ca2+-dependent increase in N2A-actin interaction thus is a first hint that a stiffening of the titin spring may occur in actively contracting versus nonactivated muscle through increased interaction with actin. However, the issue remains highly controversial: another recent study concluded that the PEVK region does not interact with actin during active stretch (13). As it stands, then, there is a weak foundation for the proposal (28, 46) that Ca2+-triggered actin-titin binding contributes substantially to force enhancement in actively stretched muscle, i.e., in eccentric contractions. Ca2+ could still affect the interaction between actin and titin in regions not yet tested, e.g., in A-band segments.

In summary, there is good evidence that actin and titin interact in the sarcomere, including in the I band. Functionally, this interaction increases titin-based viscoelasticity in the sarcomere. It is less sure whether physiologically relevant Ca2+ levels alter the binding strength between titin spring elements and actin filaments. This remains an interesting area for future research.

Modulation of Titin Stiffness by Phosphorylation

Arguably, the best-studied regulatory mechanism modulating titin-based force in cardiac muscle is phosphorylation (26, 30, 38–40, 75, 95). A focus of these studies was the only cardiac-specific segment in titin, known as the N2B element (encoded by TTN exon 49), which is spliced out in the skeletal muscle titin isoforms. Thus, the phosphorylation-dependent stiffness regulation of this titin region does not apply to skeletal myocytes. However, skeletal muscle titin has long been known to be phosphorylatable in vivo (81), and there are hundreds of potential phosphosites in titin regions that are included in skeletal muscle (25).

Measurements of total titin phosphorylation in skeletal muscle demonstrated variable degrees of change under diverse stress conditions, including unloading during 30-day spaceflight [hyperphosphorylation in m. gastrocnemius of mice (87)], hibernation [constant phosphorylation in hibernating brown bears (80)], 6-mo chronic alcohol intake [hyperphosphorylation in m. gastrocnemius and m. soleus of rats (22)], or inherited forms of myopathy [hypophosphorylation in m. vastus lateralis of human patients vs. healthy subjects (88)]. Some of these changes were suggested to trigger increased proteolytic degradation of titin, which then contributes to the development of skeletal muscle atrophy (79). Various other studies proposed that altered phosphorylation state of I-band titin modifies titin-based spring force in skeletal myocytes (Fig. 3), as it does in cardiomyocytes (31, 58, 62, 71, 88, 90).

A number of evolutionarily conserved phosphosites were identified in titin’s constitutively expressed PEVK subsegment, confirmed by phosphoantibody-based detection and implicated in the modulation of titin spring force (26, 29, 30, 88). Phosphosites were also detected in the N2A element (38, 58). Several studies showed changes in the phosphorylation level of two serines in the PEVK domain, S11878 and S12022, which are known substrates of protein kinase Cα and calcium/calmodulin-dependent kinase IIδ, respectively (26, 30). Increased phosphorylation at these sites within the (positively charged) PEVK subsegment raised titin-based stiffness (Fig. 3) (30). Elevated phosphorylation at least at one of these sites can explain an increased myofiber stiffness found under diverse physiological and pathological conditions, such as in muscles of patients with Ehlers-Danlos syndrome due to deficiency in the extracellular matrix protein tenascin-X (71), in mouse diaphragm muscles after exercise training (31), and in m. vastus lateralis of rats after acute exercise (62). Conversely, reduced phosphorylation of these PEVK sites observed in vastus lateralis muscles of chronically ill myopathy patients versus healthy subjects (88) could be a compensatory mechanism attempting to reduce the increased myofiber stiffness resulting from massive chaperone binding to I-band titin. In conclusion, phosphorylation of PEVK sites modulates titin-based force in skeletal muscle. Altered phosphorylation status of the PEVK domain is observed after acute exercise and in muscle disease and is associated with altered muscle stiffness.

ALTERED TITIN-BASED STIFFNESS CAN TRIGGER MECHANO-CHEMICAL SIGNALING PATHWAYS

Acute or chronic change in the elastic force level of titin, by any one of the mechanisms discussed above, has the potential to initiate mechano-chemical signaling pathways in skeletal myocytes. Through manifold interactions with diverse molecules (Fig. 3), titin can thus act as a bona fide mechanosensor. Suggested mechanosensory mechanisms triggered by titin have been reviewed in detail elsewhere (18, 36, 47, 48, 55, 58, 70) and are not covered here. These pathways are involved in the regulation of muscle protein turnover and encompass prohypertrophic and antihypertrophic signaling cascades, as well as the ubiquitin-proteasome system and the autophagy-lysosomal protein degradation machinery. Additionally, titin seems to be an integral part of a novel mechanosensory system that functions in the sarcomeric A band, which could be responsible for the length-dependent activation of muscle (48). This way, titin-based “passive” force levels may translate more directly into actomyosin-based “active” force levels.

CONCLUSIONS

The work reviewed above leaves no doubt that the giant protein titin is a main determinant of muscle force in both the nonactivated and activated states. Titin’s mechanical role in the sarcomere, formerly described as “passive,” reaches far beyond that of a rubberlike elastomer, since the protein is both a “passive” spring and a generator of “active” contractile work via Ig domain folding contractions. The mechanical properties of the titin spring can be tuned acutely by various mechanisms, which include posttranslational modifications and binding of chaperones, Ca2+, or sarcomeric actin. These alterations, along with the high connectivity with other proteins in the myocyte, give rise to a mechanosensor function of titin that is probably activated in a dynamic manner, depending on the level of change in titin’s spring force. A challenge for future research will be to elucidate the molecular mechanisms of these titin force-triggered mechanosensing pathways, as well as the role of titin and its interactions in eccentric muscle contractions.

GRANTS

We acknowledge financial support from the European Union and Federal Ministry for Education and Research (ERA-Net, MINOTAUR) and from the German Research Foundation (SFB 1002, TP A08).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

W.A.L. conceived and designed research; J.K.F. and W.A.L. prepared figures; J.K.F. and W.A.L. drafted manuscript; W.A.L. edited and revised manuscript; J.K.F. and W.A.L. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank the many past and present laboratory members, whose work has contributed to the insight reviewed here.

AUTHOR NOTES

• Address for reprint requests and other correspondence: W. A. Linke, Inst. of Physiology II, Univ. of Muenster, Robert-Koch-Str. 27b, D-48149 Muenster, Germany (e-mail: wlinke@uni-muenster.de).
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  • Address for reprint requests and other correspondence: W. A. Linke, Inst. of Physiology II, Univ. of Muenster, Robert-Koch-Str. 27b, D-48149 Muenster, Germany (e-mail: wlinke@uni-muenster.de).
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    • Address for reprint requests and other correspondence: W. A. Linke, Inst. of Physiology II, Univ. of Muenster, Robert-Koch-Str. 27b, D-48149 Muenster, Germany (e-mail: wlinke@uni-muenster.de).

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