Re: 근수축 과정에서 티틴(titin)의 역할 탐구!!

[문형철]

Biophys Rev

. 2018 Jan 20;10(4):1187–1199. doi: 10.1007/s12551-017-0395-y

The multiple roles of titin in muscle contraction and force production

Walter Herzog 1,✉

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PMCID: PMC6082311  PMID: 29353351

Abstract

Titin is a filamentous protein spanning the half-sarcomere, with spring-like properties in the I-band region. Various structural, signaling, and mechanical functions have been associated with titin, but not all of these are fully elucidated and accepted in the scientific community. Here, I discuss the primary mechanical functions of titin, including its accepted role in passive force production, stabilization of half-sarcomeres and sarcomeres, and its controversial contribution to residual force enhancement, passive force enhancement, energetics, and work production in shortening muscle. Finally, I provide evidence that titin is a molecular spring whose stiffness changes with muscle activation and actin–myosin-based force production, suggesting a novel model of force production that, aside from actin and myosin, includes titin as a “third contractile” filament. Using this three-filament model of sarcomeres, the stability of (half-) sarcomeres, passive force enhancement, residual force enhancement, and the decrease in metabolic energy during and following eccentric contractions can be explained readily.

타이틴은

반근절 half sarcomere에 걸쳐 있는 필라멘트형 단백질로,

I-밴드 영역에 스프링과 같은 특성을 가지고 있습니다.

다양한 구조적, 신호 전달 및 기계적 기능이 타이틴과 연관되어 있지만,

과학계에서 이 모든 기능이 완전히 밝혀지고 받아들여지고 있는 것은 아닙니다.

여기에서는

수동적 힘 생성,

반근절 및 근절의 안정화,

그리고 논란이 되고 있는 잔류 힘 강화,

수동적 힘 강화,

에너지 및 단축 근육의 일 생산에 대한 기여를 포함하여

타이틴의 주요 기계적 기능에 대해 논의합니다.

마지막으로, 저는

타이틴이

근육 활성화와 액틴-미오신 기반 힘 생성에 따라

강성이 변화하는 분자 스프링이라는 증거를 제시하여

액틴과 미오신 외에 타이틴을 “제3의 수축성” 필라멘트로 포함하는 새로운 힘 생성 모델을 제안합니다.

이 세 가지 필라멘트 근절 모델을 사용하면

(반) 근절의 안정성,

수동적 힘 강화,

잔류 힘 강화 및 편심 수축 중 및 이후의 대사 에너지 감소를

쉽게 설명 할 수 있습니다.

Keywords: Titin, Molecular spring, Mechanical functions, Active/passive force regulation, Muscle shortening, Force production, Cross-bridge theory, Three filament sarcomere model, Mechanisms of muscle contraction, Muscle energetics

Background

Since the 1950s, muscle contraction and force production have been associated with the relative sliding of the two contractile filaments, actin and myosin (referred to as the sliding filament theory) (Huxley and Hanson 1954; Huxley and Niedergerke 1954), and the cyclic interaction of myosin-based cross-bridges with specialized attachment sites on the actin filaments (referred to as the cross-bridge theory) (Huxley 1957a). However, from the very onset, the cross-bridge theory could not predict well some of the experimentally observed properties in skeletal muscles (Huxley 1957a). For example, the well-recognized and generally accepted residual force enhancement and residual force depression properties of muscles, observed well before the development of the cross-bridge theory (Abbott and Aubert 1952), cannot be predicted without making fundamental changes to the cross-bridge theory (Walcott and Herzog 2008). Furthermore, the stability of myosin filaments in the center of sarcomeres (Iwazumi 1979; Horowits and Podolsky 1987) and that of sarcomeres on the so-called descending limb of the force–length relationship (Zahalak 1997; Novak and Truskinovsky 2014) cannot be predicted with the cross-bridge theory (Iwazumi and Noble 1989; Zahalak 1997), and the forces and metabolic cost predicted by the original cross-bridge theory were much too high for eccentric contractions (Huxley 1957a).

Andrew Huxley, the father of the cross-bridge theory (Huxley 1957a), recognized the shortcomings of his approach. For example, in order to account for the excessive metabolic cost of eccentric contractions, he proposed that there might be multiple cross-bridge cycles for each energy unit hydrolyzed [adenosine triphosphate (ATP)], while for concentric and isometric contractions, the hydrolysis of one ATP molecule was tightly linked to one cross-bridge cycle (Huxley 1957a, 1969; Huxley and Simmons 1971; Rayment et al. 1993). Also, the excessive eccentric forces could be reduced to experimentally observed values by assuming that attached cross-bridges are torn from actin prior to the full completion of the cross-bridge cycle (Huxley 1957a). However, for the residual force enhancement properties of skeletal muscle, Huxley had no solution to offer. In his 1980 book, “Reflections on Muscle,” Huxley acknowledged the insufficiencies of current cross-bridge thinking in eccentric muscle contraction (Huxley 1980). Specifically, he mentions that special features must have evolved that allow the elongation of active muscles to take place without damaging muscles, that these special features allow for explanations of the mechanics and energetics of eccentric contractions, and that force regulation in eccentric contractions bears little relation to what happens in concentric muscle action (Huxley 1980).

In the mid- and late 1970s, just prior to the publication of Huxley’s book on muscle contraction, titin (initially also referred to as connection) was discovered (Maruyama 1976; Maruyama et al. 1977; Wang et al. 1979). Titin is a filamentous protein spanning the half-sarcomere from the M-band to Z-band (Fig. 1). While thought to be essentially rigidly attached to myosin in the A-band region (except possibly for extreme sarcomere excursions beyond the normal range encountered in typical everyday movements), titin’s I-band structure allows for large elongations and passive force production, and thus has been termed a “spring-like” molecule. Just prior to inserting into the Z-band, titin binds to actin along its most proximal 50 nm, thereby establishing a “permanent” bridge between actin and myosin (Trombitas and Pollack 1993; Linke et al. 1997; Trombitas and Granzier 1997): a bridge that is in parallel with attached cross-bridges and in series with the myosin filament in the passive muscle. With an estimated six titin filaments per half myosin (Granzier and Irving 1995; Cazorla et al. 2000; Liversage et al. 2001; Granzier and Labeit 2007), there is one titin for each actin filament in vertebrate skeletal muscles where actin filaments surround myosin in a hexagonal array (Huxley 1953b, 1957).

배경

1950년대 이후 근육 수축과 힘 생성은

액틴과 미오신이라는 두 수축성 필라멘트(슬라이딩 필라멘트 이론이라고 함)의

상대적 미끄러짐(슬라이딩 필라멘트 이론이라고 함)과

미오신 기반 교차교량과 액틴 필라멘트의 특수 부착 부위(교차교량 이론이라고 함)의

주기적 상호작용(교차교량 이론이라고 함)과 연관되어 왔습니다(Huxley 1957a).

그러나

처음부터 크로스 브리지 이론은

골격근에서 실험적으로 관찰된 일부 특성을 잘 예측하지 못했습니다(Huxley 1957a).

예를 들어,

크로스 브리지 이론이 개발되기 훨씬 전에 관찰되어 잘 알려져 있고

일반적으로 인정되는 근육의 잔류력 향상 및 잔류력 저하 특성은

크로스 브리지 이론을 근본적으로 변경하지 않고는 예측할 수 없습니다 (Walcott and Herzog 2008).

또한, 근절의 중심에있는 미오신 필라멘트의 안정성 (이와즈미 1979; 호로위츠와 포돌스키 1987) 및 힘-길이 관계의 소위 하강 사지 (Zahalak 1997; Novak and Truskinovsky 2014)는 크로스 브리지 이론 (Iwazumi and Noble 1989; Zahalak 1997)으로 예측할 수 없으며, 원래 크로스 브리지 이론에서 예측 한 힘과 대사 비용은 편심 수축 (Huxley 1957a)에 비해 너무 높았습니다.

크로스 브리지 이론의 아버지인 앤드류 헉슬리(Andrew Huxley)는

자신의 접근법의 단점을 인식했습니다(Huxley 1957a).

예를 들어,

편심 수축의 과도한 대사 비용을 설명하기 위해

그는 가수 분해된 각 에너지 단위[아데노신 삼인산(ATP)]에 대해

여러 교차 다리 주기가 있을 수 있다고 제안한 반면,

동심 및 등척성 수축의 경우 한 ATP 분자의 가수 분해가

하나의 교차 다리 주기에 밀접하게 연결되어 있다고 설명했습니다

(Huxley 1957a, 1969; Huxley and Simmons 1971; Rayment et al. 1993).

또한

크로스 브리지 사이클이 완전히 완료되기 전에

부착된 크로스 브리지가 액틴에서 찢어진다고 가정함으로써

과도한 편심력을 실험적으로 관찰된 값으로 줄일 수 있었습니다(Huxley 1957a).

그러나

골격근의 잔류 힘 강화 특성에 대해서는

헉슬리가 제시할 수 있는 해결책이 없었습니다.

1980년 저서 “근육에 대한 고찰”에서 헉슬리는

편심 근육 수축에 대한 현재의 크로스 브리지 사고의 불충분함을 인정했습니다(Huxley 1980).

구체적으로 그는

근육을 손상시키지 않고

활동성 근육의 신장을 가능하게 하는 특수 기능이 진화했을 것이며,

이러한 특수 기능이 편심성 수축의 역학과 에너지학을 설명할 수 있고,

편심성 수축에서의 힘 조절은 동심성 근육 작용에서 일어나는 것과는 거의 관계가 없다고 언급했습니다(Huxley 1980).

헉슬리의 근육 수축에 관한 책이 출판되기 직전인

1970년대 중후반에 타이틴(처음에는 연결이라고도 불림)이

발견되었습니다(마루야마 1976; 마루야마 외. 1977; 왕 외. 1979).

타이틴은

M-밴드에서 Z-밴드까지

반근절에 걸쳐 있는 필라멘트 단백질입니다(그림 1).

기본적으로

A-밴드 영역의 미오신에 단단히 부착되어 있는 것으로 생각되지만

(일반적인 일상적인 움직임에서 발생하는 정상 범위를 벗어난 극단적인 근절의 이탈을 제외하고),

타이틴의 I-밴드 구조는

큰 신장과 수동적인 힘 생성을 허용하기 때문에

“스프링 같은” 분자로 불려왔다.

타이틴은

Z-밴드에 삽입되기 직전에 가장 근위부 50nm를 따라

액틴에 결합하여 액틴과 미오신 사이에 “영구적인” 다리를 형성합니다

(Trombitas and Pollack 1993; Linke et al. 1997; Trombitas and Granzier 1997):

이 다리는

부착된 교차 다리와 평행하고

수동 근육의 미오신 필라멘트와 직렬로 연결됩니다.

절반의 미오신당 약 6개의 티틴 필라멘트가 있는 것으로 추정되며

(Granzier and Irving 1995; Cazorla 외. 2000; Liversage 외. 2001; Granzier and Labeit 2007),

척추 골격근에는 액틴 필라멘트가

육각형 배열로 미오신을 둘러싼 각 액틴 필라멘트당 1개의 티틴이 있습니다(Huxley 1953b, 1957).

Fig. 1.

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Schematic two-dimensional illustration of a sarcomere bordered by Z-bands at either end. Thick, myosin-based filaments are in the center of the sarcomere (green), thin, actin-based filaments insert into the Z-band at either end of the sarcomere (red), and titin filaments (blue) run from the M-line in the middle of the sarcomere to the Z-band. Adapted from Granzier and Labeit (2007) with permission

양쪽 끝이 Z-밴드로 둘러싸인 근절의 개략적인 2차원 그림.

두꺼운 미오신 기반 필라멘트가 육종의 중앙에 있고(녹색), 얇은 액틴 기반 필라멘트가 육종의 양쪽 끝에 있는 Z-밴드에 삽입되어 있으며(빨간색), 티틴 필라멘트(파란색)가 육종 중앙의 M-라인에서 Z-밴드까지 이어져 있습니다.

Granzier와 Labeit(2007)에서 허가를 받아 수정함.

Ever since its discovery, titin’s functions have been questioned, and titin’s recently proposed roles in active force regulation and mechanical work in muscle shortening are current topics of intense debate in the scientific community. At the recent European Muscle Conference (September 2017), debates on the functional role of titin ended inconclusively. Here, I will attempt to summarize both the acknowledged and the controversial aspects of titin’s mechanical functions, with an emphasis on titin’s proposed role in active force regulation and mechanical work.

타이틴의 발견 이후 

타이틴의 기능에 대한 의문이 제기되어 왔으며, 

최근 타이틴이 근육 단축에서 활동력 조절과 기계적 작용에 미치는 역할이 

과학계에서 격렬한 논쟁의 대상이 되고 있습니다. 

최근 유럽 근육 학회(2017년 9월)에서도 타이틴의 기능적 역할에 대한 논쟁은 결론을 내리지 못하고 끝났습니다. 여기에서는 타이틴의 기계적 기능에 대해 인정된 측면과 논란의 여지가 있는 측면을 모두 요약하고, 활성 힘 조절 및 기계적 작업에서 타이틴의 제안된 역할에 중점을 두어 설명하려고 합니다.

Titin’s proposed functions

Titin has been associated with a variety of functions, including mechanical roles in active and passive force regulation in cardiac and skeletal muscles (e.g., Linke et al. 1994, 1996; Granzier and Labeit 2002, 2007; Linke and Fernandez 2002; Herzog et al. 2006; LeWinter and Granzier 2010; Herzog 2014a), structural and developmental roles in sarcomere organization (e.g., Linke and Fernandez 2002; Granzier and Labeit 2007), and functions associated with mechano-sensing and signaling (Schwarz et al. 2008; Kruger and Linke 2009; Linke and Kruger 2010; Granzier et al. 2014). Here, I primarily focus on the proposed mechanical functions of titin in skeletal muscles, although some comparisons with cardiac muscles will be made. However, many extensive reviews on titin’s properties in cardiac muscle have been published recently (Granzier and Labeit 2002, 2007; Granzier et al. 2002), whereas comparatively little has been said on titin’s mechanical role in skeletal muscle.

티틴의 제안된 기능

티틴은

심장 및 골격근의 능동적 및 수동적 힘 조절에서의 기계적 역할

(예: Linke 외. 1994, 1996; Granzier와 Labeit 2002, 2007; Linke와 Fernandez 2002; Herzog 외. 2006; LeWinter와 Granzier 2010; Herzog 2014a),

근조직에서의 구조적 및 발달적 역할 등 다양한 기능과 연관되어 왔다(예:, Linke와 Fernandez 2002; Granzier와 Labeit 2007), 기계 감지 및 신호와 관련된 기능 (Schwarz 외. 2008; Kruger와 Linke 2009; Linke와 Kruger 2010; Granzier 외. 2014).

여기서는 주로 골격근에서 타이틴의 기계적 기능에 초점을 맞추지만 심장 근육과의 비교도 일부 이루어질 것입니다. 그러나 최근 심장 근육에서 타이틴의 특성에 대한 광범위한 리뷰가 많이 발표되었지만(Granzier and Labeit 2002, 2007; Granzier 등. 2002), 골격근에서 타이틴의 기계적 역할에 대해서는 상대적으로 거의 언급되지 않았습니다.

Passive force contributions of titin

It is generally accepted that titin contributes to the passive forces in skeletal and cardiac muscles. Passive force is defined here as any force that arises from structural elements of muscle, is not associated with metabolic energy consumption, and is not part of the actin–myosin-based cross-bridge forces. The primary passive force contributors in cardiac and skeletal muscles are collagen filaments embedded in the various connective tissue layers of muscles, and the sarcomeric filament titin. In isolated myofibril preparations, titin is the primary passive force contributor (Maruyama 1976; Funatsu et al. 1990; Bartoo et al. 1997; Colomo et al. 1997; Linke and Fernandez 2002; Joumaa et al. 2008b; Leonard and Herzog 2010; Herzog et al. 2012), and the elimination of titin abolishes virtually all passive force (e.g., Leonard and Herzog 2010) .

In skinned single fibers and myofibrils, passive force and titin isoforms are tightly related. Increasing molecular weight, and thus increasing titin subunits and length, are associated with decreasing passive forces. Prado et al. (2005) determined the molecular weights of titin in 37 rabbit skeletal muscles and compared the molecular weights to the passive forces in myofibrils, skinned fibers, and intact and skinned fiber bundles. These authors found a strong inverse relationship between the size of titin and passive force in myofibrils and skinned fibers, i.e., the greater the molecular weight of titin, the smaller the corresponding passive force for a given sarcomere length. However, titin size was not associated in any systematic manner with the passive force in intact fiber bundles (and thus the entire muscle), and the contribution of titin to the total passive force in fiber bundles varied considerably between muscles, ranging from a high of 57% in rabbit psoas (Granzier et al. 2002) to a low of 24% for soleus in the range of 2.0 to 3.2 μm/sarcomere (Prado et al. 2005).

Prado et al. (2005) also found that the contribution of titin to passive muscle force depends on the length of the muscle (i.e., the average sarcomere length). This result agrees with observations in cardiac muscle where titin is thought to contribute more substantially to the passive forces at short (average sarcomere length 2.0–2.2 μm) compared to long (> 2.3 μm/sarcomere) sarcomere length (Cazorla et al. 2000; Freiburg et al. 2000; Granzier et al. 2002). Since the physiologic cardiac cycle occurs between sarcomere lengths that range from approximately 1.9 to 2.3 μm (Ter Keurs et al. 1980), titin plays a significant role in the beating heart.

Whether titin plays an equally important role within the functional range of skeletal muscles has not been determined systematically. It has been observed that in rabbit psoas myofibrils, passive, titin-based forces start to emerge at average sarcomere lengths of approximately 2.6–2.7 μm (Linke et al. 1996; Bartoo et al. 1997; Joumaa et al. 2007; Leonard and Herzog 2010). However, our group measured the shortest (hip fully flexed) and longest (hip fully extended) sarcomere length for rabbit psoas muscles as 1.9 and 2.6 μm, respectively. Knowing that rabbits never fully extend their hip, the maximal sarcomere lengths are probably never reached in the live animal. Furthermore, our measurements were performed on the passive muscle in the anesthetized animal, while in the active muscle, a substantial fiber and sarcomere length shortening would be expected with force production, as elastic elements are stretched and the contractile machinery shortens (Fukunaga et al. 1997; Ichinose et al. 1997; Vaz et al. 2012; de Brito Fontana and Herzog 2016). Therefore, maximal sarcomere lengths in the rabbit psoas likely never exceed about 2.3–2.4 μm, and thus are below the sarcomere lengths where passive titin forces have been first observed. A similar argument could be made for the rabbit soleus and medial gastrocnemius muscles. Thus, it appears that titin passive force does not play a functional role in many skeletal muscles. Whether this statement can be generalized is not yet known, as the functional sarcomere length of most animal muscles are not known. However, should skeletal muscle functional sarcomere length reach values in excess of 2.6 μm, then titin would likely contribute to the passive force of intact muscles (Prado et al. 2005). Also, in the following text I discuss titin’s possible role in shifting its slack length upon muscle activation to shorter sarcomere lengths than those observed in the passive muscle, which could potentially change the argument made here, with titin possibly emerging as a powerful passive force contributor in active skeletal muscles after all (Herzog 2014b; Herzog et al. 2015, 2016).

Titin’s stiffness, and thus passive force at a given sarcomere length, can be modulated in a variety of ways, such as calcium binding to titin upon muscle activation (Labeit et al. 2003; Joumaa et al. 2008b; DuVall et al. 2013), titin phosphorylation (Yamasaki et al. 2002; Borbely et al. 2009; Anderson et al. 2010; Perkin et al. 2015), and titin interactions with actin and other sarcomeric proteins (Li et al. 1995; Linke et al. 1997, 2002; Trombitas and Granzier 1997; Astier et al. 1998; Kulke et al. 2001; Yamasaki et al. 2001; Nagy et al. 2004; Bianco et al. 2007; Leonard and Herzog 2010; Chung et al. 2011), to just name the most common mechanisms. Some of these mechanisms will be discussed below in the section on residual force enhancement properties of skeletal muscles in general (Abbott and Aubert 1952; Edman et al. 1982; Herzog et al. 2006) and the passive residual force enhancement specifically (Herzog and Leonard 2002). Excellent reviews on the regulation of titin’s stiffness in cardiac muscle are abundant (e.g., Granzier and Labeit 2002; Granzier et al. 2002; Linke and Fernandez 2002; LeWinter and Granzier 2010), and these results will not be repeated here, except for comparative purposes.

티틴의 수동적 힘 기여

일반적으로 티틴은 골격근과 심장 근육의 수동적 힘에 기여하는 것으로 알려져 있습니다. 여기서 수동적 힘은 근육의 구조적 요소에서 발생하고 대사 에너지 소비와 관련이 없으며 액틴-미오신 기반 교차 교량 힘의 일부가 아닌 모든 힘으로 정의됩니다. 심장 및 골격근의 주요 수동적 힘 기여자는 근육의 다양한 결합 조직 층에 내장된 콜라겐 필라멘트와 근절 필라멘트 티틴입니다. 분리된 근섬유 제제에서 타이틴은 수동적 힘의 주요 기여자이며(Maruyama 1976; Funatsu 외. 1990; Bartoo 외. 1997; Colomo 외. 1997; Linke and Fernandez 2002; Joumaa 외. 2008b; Leonard and Herzog 2010; Herzog 외. 2012), 타이틴을 제거하면 사실상 모든 수동적 힘이 사라집니다(예: Leonard and Herzog 2010) .

피부가 벗겨진 단일 섬유와 근섬유에서 수동 힘과 타이틴 이소폼은 밀접한 관련이 있습니다. 분자량이 증가하여 티틴 서브유닛과 길이가 증가하면 수동력이 감소하는 것과 관련이 있습니다. Prado 등(2005)은 토끼 골격근 37개에서 타이틴의 분자량을 측정하고 이 분자량을 근섬유, 피막 섬유, 손상되지 않은 섬유 다발 및 피막 섬유 다발의 수동력과 비교했습니다. 그 결과, 티틴의 크기와 근섬유 및 피부 섬유의 수동력 사이에 강한 반비례 관계, 즉 티틴의 분자량이 클수록 주어진 근절 길이에 상응하는 수동력이 작아지는 것을 발견했습니다. 그러나 티틴 크기는 온전한 섬유 다발 (따라서 전체 근육)의 수동력과 체계적인 방식으로 연관되지 않았으며 섬유 다발의 총 수동력에 대한 티틴의 기여도는 토끼 요근에서 최고 57 % (Granzier et al. 2002)에서 2.0 ~ 3.2 μm / sarcomere 범위의 가자미의 경우 24 %의 최저에 이르기까지 근육간에 상당히 다양했습니다 (Prado et al. 2005).

Prado 등. (2005) 또한 수동 근력에 대한 티틴의 기여도는 근육의 길이 (즉, 평균 근절 길이)에 따라 달라진다는 것을 발견했습니다. 이 결과는 심장 근육에서 티틴이 긴 (> 2.3 μm / 근절) 근절 길이에 비해 짧은 (평균 근절 길이 2.0-2.2 μm) 수동력에 더 크게 기여한다고 생각되는 관찰과 일치합니다 (Cazorla 외. 2000; Freiburg 외. 2000; Granzier 외. 2002). 생리적 심장 주기는 약 1.9~2.3 μm 범위의 근절 길이 사이에서 발생하기 때문에(Ter Keurs 등. 1980), 티틴은 심장 박동에서 중요한 역할을 합니다.

티틴이 골격근의 기능 범위 내에서 똑같이 중요한 역할을 하는지는 체계적으로 밝혀지지 않았습니다. 토끼 요근 근섬유에서 수동적 인 티틴 기반 힘이 약 2.6-2.7 μm의 평균 근절 길이에서 나타나기 시작하는 것으로 관찰되었습니다 (Linke 외. 1996; Bartoo 외. 1997; Joumaa 외. 2007; Leonard and Herzog 2010). 그러나 우리 그룹은 토끼 요근의 가장 짧은 (고관절이 완전히 구부러진 상태) 및 가장 긴 (고관절이 완전히 펴진 상태) 근절 길이를 각각 1.9 및 2.6 μm로 측정했습니다. 토끼가 엉덩이를 완전히 펴지 않는다는 점을 고려할 때, 살아있는 동물에서는 최대 근절 길이에 도달하지 못할 것입니다. 또한, 우리의 측정은 마취 된 동물의 수동 근육에서 수행 된 반면, 활성 근육에서는 탄성 요소가 늘어나고 수축 기계가 짧아지기 때문에 힘 생성으로 상당한 섬유 및 육종 길이 단축이 예상됩니다 (Fukunaga 외 1997; Ichinose 외 1997; Vaz 외 2012; de Brito Fontana 및 Herzog 2016). 따라서 토끼 요근의 최대 근절 길이는 약 2.3-2.4 μm를 초과하지 않을 가능성이 높으며, 따라서 수동적 인 티틴 힘이 처음 관찰 된 근절 길이보다 낮습니다. 토끼 가자미근과 내측 비복근에 대해서도 비슷한 주장이 제기될 수 있습니다. 따라서 타이틴 수동력은 많은 골격근에서 기능적인 역할을 하지 않는 것으로 보입니다. 대부분의 동물 근육의 기능적 근절 길이가 알려지지 않았기 때문에이 진술을 일반화 할 수 있는지 여부는 아직 알려지지 않았습니다. 그러나 골격근의 기능적 근절 길이가 2.6 μm를 초과하는 값에 도달하면 티틴이 온전한 근육의 수동적 힘에 기여할 가능성이 높습니다 (Prado et al. 2005). 또한 다음 본문에서는 근육 활성화시 이완 길이를 수동 근육에서 관찰 된 것보다 더 짧은 근절 길이로 이동시키는 타이틴의 가능한 역할에 대해 논의하며, 이는 결국 타이틴이 활성 골격근에서 강력한 수동 힘 기여자로 부상 할 가능성이있는 잠재적으로 여기에서 제기 된 주장을 바꿀 수 있습니다 (Herzog 2014b; Herzog et al. 2015, 2016).

타이틴의 강성, 따라서 주어진 근절 길이에서의 수동적 힘은 근육 활성화시 타이틴에 대한 칼슘 결합 (Labeit et al. 2003; Joumaa et al. 2008b; DuVall et al. 2013), 타이틴 인산화 (Yamasaki et al. 2002; Borbely et al. 2009; Anderson et al. 2010; Perkin et al. 2015), 액틴 및 기타 근절 단백질과의 타이틴 상호 작용 (Li et al. 1995; Linke et al. 1997, 2002; Trombitas and Granzier 1997; Astier et al. 1998; Kulke et al. 2001; Yamasaki et al. 2001; Nagy et al. 2004; Bianco et al. 2007; Leonard and Herzog 2010; Chung et al. 2011), 가장 일반적인 메커니즘의 이름을 지정합니다. 이러한 기전 중 일부는 아래에서 골격근의 잔류력 향상 특성(Abbott and Aubert 1952; Edman 등 1982; Herzog 등 2006)과 수동적 잔류력 향상(Herzog and Leonard 2002) 섹션에서 구체적으로 논의될 것입니다. 심장 근육에서 타이틴의 강성 조절에 대한 훌륭한 리뷰는 풍부하며(예: Granzier and Labeit 2002; Granzier et al. 2002; Linke and Fernandez 2002; LeWinter and Granzier 2010), 이러한 결과는 비교 목적을 제외하고는 여기서 반복하지 않겠습니다.

Titin as a stabilizer of sarcomeres and half-sarcomeres

In the two-filament (actin and myosin) cross-bridge model of muscle contraction, half-sarcomeres and sarcomeres are unstable (Morgan 1990, 1994; Allinger et al. 1996; Zahalak 1997; Epstein and Herzog 1998; Morgan et al. 2000; Novak and Truskinovsky 2014). The half-sarcomere is unstable because small differences in half-sarcomere forces, caused by the stochastic interaction of cross-bridges with actin, will cause an initially centered myosin filament to be displaced from its mid-point position in the sarcomere. This perturbation is unstable as the overlap between actin and myosin will increase in the half-sarcomere of initial myosin drift and thus make this half-sarcomere increasingly stronger, while in the other half-sarcomere, the actin–myosin filament overlap is lost, and force continuously decreases (Iwazumi 1979). An analogous argument can be made for the instability of serially arranged sarcomeres (for example, as they occur in a myofibril; Gordon et al. 1966; Campbell 2009; Stoecker et al. 2009) that are located on the descending limb of the force–length relationship (Hill 1953).

A-band shifts to one end of the sarcomere were observed after prolonged activation in rabbit psoas skinned fibers with an average sarcomere length of 2.6 μm and zero passive force (Horowits and Podolsky 1987, 1988). These shifts, as well as non-uniformities in associated half-sarcomere lengths, were abolished at average sarcomere lengths of about 3.0 μm and a passive stress of approximately 2 N/cm2, corresponding to approximately 20% of the maximum, active, isometric force of these fibers at optimal sarcomere length and 7 °C. The A-band shifts observed in these studies were interpreted as indicating myosin instability in the center of the sarcomere when titin forces were zero or small, while myosin was stabilized in the center of the sarcomere once titin forces had reached a certain passive force level corresponding to approximately 2 N/cm2 (e.g., Horowits 1992). The same observations were made for rabbit soleus fibers, with the notable difference that A-band shifts were greater in soleus than in psoas fibers at corresponding sarcomere lengths and that half-sarcomere uniformity and stability (absence of A-band shifts) were only observed at average sarcomere lengths of about 3.6 μm when passive, titin-based tension had reached 2 N/cm2. The explanation for this result was based on the smaller size of the titin isoforms for rabbit psoas (3.3 and 3.4 MDa in a 70:30% ratio) compared to that of the single titin isoform observed in rabbit soleus (3.6 MDa) (Prado et al. 2005), resulting in reduced titin-based passive forces at a given sarcomere length for rabbit soleus compared to psoas.

Instability of muscles and sarcomeres on the descending limb of the force–length relationship has been used to explain specific muscle properties for more than half a century (Hill 1953; Gordon et al. 1966; Julian et al. 1978; Julian and Morgan 1979). For example, the so-called “creep” property, which is a slow change in isometric force for muscles/fibers on the descending limb of the force–length relationship (Hill 1953; Gordon et al. 1966), and the residual force enhancement and residual force depression properties (Morgan 1990, 1994) have been thought to be caused by instabilities in sarcomere length and the associated development of sarcomere length non-uniformities in active muscles, particularly if the muscles were stretched onto the descending limb of the force–length relationship. Indeed, vast sarcomere length non-uniformities have been observed in entire muscle preparations (Llewellyn et al. 2008; Moo et al. 2016), single fibers (Huxley and Peachey 1961), and isolated myofibrils (Rassier et al. 2003a; Joumaa et al. 2008a; Johnston et al. 2016). However, these sarcomere length non-uniformities occur at all lengths (ascending, plateau, and descending portions of the force–length relationship; Moo et al. 2016) and similarly in isometric, shortening, and stretched muscles (Johnston et al. 2016); thus, they do not seem to be associated with the proposed instability of the negative slope of the descending limb of the force–length relationship.

Surprisingly, Rassier et al. (2003a, b) observed that strictly serially arranged sarcomeres in single, active myofibrils stretched onto the descending limb of the force–length relationship are highly non-uniform, but perfectly stable. Sarcomeres half-way down the descending limb of the force–length relationship, and thus at half of the maximal actin–myosin filament overlap, were perfectly stable and remained at a constant length in the presence of sarcomeres on the plateau of the force–length relationship with maximal overlap between actin and myosin (Fig. 2). It would appear, therefore, that sarcomere length, and thus actin–myosin filament overlap, alone does not predict the isometric force of a sarcomere for a given level of activation within a myofibril. Rather, since the force in serially arranged sarcomeres must be identical (neglecting any inertial effects, which can safely be done), there must be another way other than just actin–myosin filament overlap to determine the isometric, steady-state force of a sarcomere within its natural environment of a myofibril. Active or passive stabilizing mechanisms must ensure that the weakening behavior of sarcomeres on the descending limb of the force–length relationship is compensated for in some (as of yet unknown) manner to ensure stability of the muscle.

근절 및 반근절의 안정제로서의 티틴

근육 수축의 두 필라멘트 (액틴과 미오신) 교차 다리 모델에서 반육종과 근절은 불안정합니다 (Morgan 1990, 1994; Allinger 외 1996; Zahalak 1997; Epstein과 Herzog 1998; Morgan 외 2000; Novak과 Truskinovsky 2014). 교차 다리와 액틴의 확률 적 상호 작용으로 인한 반 육종체의 작은 차이로 인해 반 육종체 힘이 불안정 해지면 처음에 중심이 된 미오신 필라멘트가 근절의 중간 지점 위치에서 변위 될 수 있기 때문에 반 육종체가 불안정합니다. 이 섭동은 불안정한데, 초기 미오신 드리프트의 반 육종체에서는 액틴과 미오신 사이의 중첩이 증가하여 이 반 육종체가 점점 더 강해지는 반면, 다른 반 육종체에서는 액틴-미오신 필라멘트 중첩이 사라지고 힘이 지속적으로 감소하기 때문입니다 (Iwazumi 1979). 힘-길이 관계의 하강 사지에 위치한 직렬로 배열된 근절의 불안정성에 대해서도 유사한 주장을 할 수 있습니다(예: 근섬유에서 발생하는 것처럼; Gordon 외. 1966; Campbell 2009; Stoecker 외. 2009) (Hill 1953).

평균 근절 길이가 2.6 μm이고 수동 힘이 0 인 토끼 요근 피부 섬유에서 장기간 활성화 된 후 근절의 한쪽 끝으로의 A- 밴드 이동이 관찰되었습니다 (Horowits and Podolsky 1987, 1988). 이러한 이동과 관련 반육종 길이의 불균일성은 평균 육종 길이가 약 3.0 μm이고 수동 응력이 약 2 N / cm2 인 경우 폐지되었으며, 이는 최적의 육종 길이와 7 ° C에서 이러한 섬유의 최대 활성 등척 힘의 약 20 %에 해당합니다. 이 연구에서 관찰된 A-밴드 이동은 티틴 힘이 0이거나 작을 때 근절 중심부의 미오신 불안정성을 나타내는 것으로 해석되었으며, 티틴 힘이 약 2 N/cm2에 해당하는 특정 수동력 수준에 도달하면 근절 중심부에서 미오신이 안정화되었습니다 (예: Horowits 1992). 토끼 가자미근 섬유에 대해서도 동일한 관찰이 이루어졌지만, 해당 육종 길이에서 요근 섬유보다 가자미근에서 A- 밴드 이동이 더 컸고 수동적 인 티틴 기반 장력이 2 N / cm2에 도달했을 때 약 3.6 μm의 평균 육종 길이에서만 반 육종 균일 성 및 안정성 (A- 밴드 이동 없음)이 관찰되었다는 주목할만한 차이점이 있습니다. 이 결과에 대한 설명은 토끼 가자미근에서 관찰 된 단일 티틴 이소형 (3.6 MDa)에 비해 토끼 가자미근의 티틴 이소형 (70 : 30 비율로 3.3 및 3.4 MDa)의 크기가 더 작아서 (Prado et al. 2005) 가자미근에 비해 주어진 근절 길이에서 티틴 기반 수동 힘이 감소한 결과입니다.

힘-길이 관계의 하강 사지에있는 근육과 근절의 불안정성은 반세기 이상 동안 특정 근육 특성을 설명하는 데 사용되었습니다 (Hill 1953; Gordon 외. 1966; Julian 외. 1978; Julian and Morgan 1979). 예를 들어, 힘-길이 관계의 하강 사지에있는 근육 / 섬유의 등척성 힘의 느린 변화 인 소위 “크리프”속성 (Hill 1953; Gordon 외 1966) 및 잔류력 향상 및 잔류력 저하 속성 (Morgan 1990, 1994)은 특히 근육이 힘-길이 관계의 하강 사지로 늘어나는 경우 활동 근육에서 근절 길이의 불안정과 관련 근절 길이 불균일의 발달로 인한 것으로 생각되어 왔습니다. 실제로, 전체 근육 준비 (Llewellyn 외. 2008; Moo 외. 2016), 단일 섬유 (헉슬리와 피치 1961) 및 분리 된 근섬유 (라시에 외. 2003a; 주마 외. 2008a; 존스턴 외. 2016)에서 방대한 근절 길이 불균일성이 관찰되었습니다. 그러나 이러한 근절 길이 불균일성은 모든 길이 (힘-길이 관계의 상승, 고원 및 하강 부분; 무 등 2016) 및 유사하게 등척성, 단축 및 신장 근육에서 발생합니다 (존스턴 등 2016); 따라서 힘-길이 관계의 하강 사지의 음의 기울기의 제안 된 불안정성과 관련이없는 것으로 보입니다.

놀랍게도 Rassier 등 (2003a, b) 힘-길이 관계의 하강 사지에 뻗은 단일 활성 근섬유에서 엄격하게 연속적으로 배열 된 근절은 매우 비 균일하지만 완벽하게 안정적이라는 것을 관찰했습니다. 힘-길이 관계의 하강 사지 중간, 즉 최대 액틴-미오신 필라멘트 중첩의 절반에 있는 근절은 완벽하게 안정적이었으며, 액틴과 미오신 사이의 최대 중첩이 있는 힘-길이 관계의 고원에 있는 근절의 존재에서도 일정한 길이를 유지했습니다 (그림 2). 따라서 근섬유 길이, 따라서 액틴-미오신 필라멘트 중첩만으로는 근섬유 내에서 주어진 활성화 수준에 대한 근섬유의 등척성 힘을 예측할 수 없는 것으로 보입니다. 오히려 직렬로 배열된 근절의 힘은 동일해야 하므로(관성 효과를 무시하고 안전하게 수행 가능), 근섬유라는 자연 환경 내에서 근절의 등척성 정상 상태 힘을 결정하려면 액틴-미오신 필라멘트 중첩 이외의 다른 방법이 있어야 합니다. 능동적 또는 수동적 안정화 메커니즘은 힘-길이 관계의 하강 사지에 있는 근절의 약화 동작이 근육의 안정성을 보장하기 위해 어떤 (아직 알려지지 않은) 방식으로 보상되도록 보장해야 합니다.

Fig. 2.

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Rabbit psoas myofibril comprised of six sarcomeres that is stretched while activated from an average sarcomere length of about 2.4 μm to about 3.0 μm. After active stretching, all individual sarcomeres are on the descending limb of the force–length relationship, but there is no apparent overstretching or popping (quick sarcomere elongations beyond actin–myosin filament overlap: 3.9 μm) as has been proposed by proponents of the sarcomere length non-uniformity theory. Rather, sarcomeres seem to remain at an essentially constant length following the active stretch

토끼 요근 근섬유는 6개의 근절로 구성되어 있으며, 평균 근절 길이가 약 2.4 μm에서 약 3.0 μm로 활성화되는 동안 늘어납니다. 활성 스트레칭 후 모든 개별 육종체는 힘-길이 관계의 하강 지점에 있지만, 육종체 길이 비균일성 이론의 지지자들이 제안한 것처럼 과도한 스트레칭이나 터짐(액틴-미오신 필라멘트 중첩을 넘어서는 빠른 육종체 신장: 3.9μm)은 나타나지 않습니다. 오히려, 근절은 활성 신장 후에도 본질적으로 일정한 길이를 유지하는 것으로 보입니다.

Titin has been implicated as a stretch sensor that activates so-called “super-relaxed” cross-bridges, thereby providing a mechanism by which a disadvantage caused by reduced actin–myosin filament overlap (long sarcomere length) can be compensated for by an increased proportion of attached cross-bridges per unit length of myofilament overlap (Fusi et al. 2016). Titin would be an ideal candidate for regulating cross-bridge kinetics in sarcomeres of different lengths, thereby guaranteeing stability. Also, since titin in adjacent half-sarcomeres overlap in the M-band and the Z-band regions (Granzier and Labeit 2007), it is easy to imagine that force transmission across the M-band and Z-band is continuous, thus providing possibilities for force transfer within half-sarcomeres and between sarcomeres. Such a passive, structural force transmission between half-sarcomeres would also provide sarcomere length/force stability and would eliminate the rather odd notion that sarcomeres are the smallest “independent” unit of force production. Muscle mechanics would be simplified greatly if serial sarcomeres in a myofibril were indeed not “independent” but rather mutually dependent force producers, such that forces can be transmitted and “re-distributed” along serially arranged sarcomeres. Aside from structural evidence for such mutual dependence among sarcomeres, there is also functional support for this notion (e.g., Yasuda et al. 1996).

타이틴은 소위 “초이완” 교차교량을 활성화하는 스트레치 센서로서, 액틴-미오신 필라멘트 겹침(긴 근절 길이) 감소로 인한 단점을 근섬유 겹침의 단위 길이당 부착된 교차교량 비율 증가로 보상할 수 있는 메커니즘을 제공합니다(푸시 외. 2016). 티틴은 길이가 다른 근절에서 교차 다리 동역학을 조절하여 안정성을 보장하는 데 이상적인 후보가 될 것입니다. 또한 인접한 반육종의 타이틴은 M- 밴드와 Z- 밴드 영역에서 겹치기 때문에 (Granzier and Labeit 2007), M- 밴드와 Z- 밴드를 통한 힘 전달이 연속적이어서 반육종 내 및 육종 간 힘 전달 가능성을 제공한다고 쉽게 상상할 수 있습니다. 반육종체 사이의 이러한 수동적이고 구조적인 힘 전달은 또한 근절 길이/힘 안정성을 제공하고 근절이 힘 생성의 가장 작은 “독립적인” 단위라는 다소 이상한 개념을 제거할 수 있습니다. 근섬유에서 일련의 근절이 실제로 “독립적”이 아니라 상호 의존적인 힘 생산자라면 근육 역학은 크게 단순화될 것이며, 따라서 힘이 연속적으로 배열된 근절을 따라 전달되고 “재분배”될 수 있습니다. 근절 간의 상호 의존성에 대한 구조적 증거 외에도 이 개념을 뒷받침하는 기능적 증거도 있습니다(예: 야스다 외. 1996).

Titin’s role in the residual force enhancement property of skeletal muscle

When an active muscle is stretched, its isometric, steady-state force following the stretch is greater than the corresponding (same length, same activation) purely isometric contraction. This increase in force caused by active stretching has been termed residual force enhancement (RFE) (Edman et al. 1982). RFE can produce forces twice as high as purely isometric contractions (Edman et al. 1982; Leonard and Herzog 2010; Leonard et al. 2010). Force in the enhanced state can easily exceed the isometric force at the plateau of the force–length relationship (Abbott and Aubert 1952; Rassier et al. 2003c; Peterson et al. 2004; Leonard et al. 2010). RFE increases with increasing stretch magnitude (Bullimore et al. 2007; Hisey et al. 2009) but is independent of stretch speed (Edman et al. 1982), is associated with a substantial decrease in the metabolic cost of force production (Joumaa and Herzog 2013), is long-lasting (Abbott and Aubert 1952; Herzog and Leonard 2002; Leonard et al. 2010), and has a passive component that contributes substantially to the enhanced force (Fig. 3a, b) (Herzog and Leonard 2002, 2005; Herzog et al. 2003; Joumaa et al. 2007, 2008b).

골격근의 잔류력 향상 특성에서 티틴의 역할

활동성 근육을 스트레칭하면 스트레칭 후 등척성 정상 상태의 힘은 해당(동일한 길이, 동일한 활성화) 순수 등척성 수축보다 더 커집니다. 이러한 활성 스트레칭으로 인한 힘의 증가를 잔류 힘 향상(RFE)이라고 합니다(Edman 외. 1982). RFE는 순수 등척성 수축보다 두 배 높은 힘을 생성할 수 있습니다(Edman 외. 1982; Leonard and Herzog 2010; Leonard 외. 2010). 강화된 상태의 힘은 힘-길이 관계의 정점에서 등척성 힘을 쉽게 초과할 수 있습니다(Abbott and Aubert 1952; Rassier 외 2003c; Peterson 외 2004; Leonard 외 2010). RFE는 스트레치 크기가 증가함에 따라 증가하지만 (Bullimore et al. 2007; Hisey et al. 2009) 스트레치 속도와는 독립적이며 (Edman et al. 1982), 힘 생성의 대사 비용의 상당한 감소와 관련이 있으며 (Joumaa and Herzog 2013), 오래 지속되며 (Abbott and Aubert 1952; Herzog and Leonard 2002; Leonard et al. 2010), 강화된 힘에 실질적으로 기여하는 수동적 요소를 가지고 있습니다(그림 3a, b)(Herzog and Leonard 2002, 2005; Herzog et al. 2003; Joumaa et al. 2007, 2008b).

Fig. 3.

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Residual force enhancement observed in whole muscle (cat soleus; a), single myofibrils (rabbit psoas; b), and single sarcomeres (rabbit psoas; c). Note the increase in force enhancement (FE; a) with increasing stretch magnitude, and the increased passive force [passive force enhancement (PFE)] following deactivation of the muscles after an active stretch (a, b). Note also the vast FE in a single sarcomere (c) and the substantially greater force after active stretching compared to the isometric, steady-state force prior to stretching which occurred at the plateau of the force–length relationship (2.4 μm)

RFE was first studied and described systematically in 1952 (Abbott and Aubert 1952), and it has subsequently been observed consistently across all structural levels of muscle, from entire muscles (Fig. 3a) (Abbott and Aubert 1952; Lee and Herzog 2002; Oskouei and Herzog 2005; Hahn et al. 2010; Seiberl et al. 2012; Fortuna et al. 2016), to single fibers (Edman et al. 1982; Sugi and Tsuchiya 1988; Peterson et al. 2004; Lee and Herzog 2008) and myofibrils (Fig. 3b) (Rassier et al. 2003a, b; Leonard and Herzog 2010; Leonard et al. 2010; Pun et al. 2010; Rassier and Pavlov 2012), and even in single sarcomeres (Fig. 3c) (Leonard et al. 2010).

For most of the twentieth century, RFE was explained by the instability of sarcomere lengths and the associated development of sarcomere length non-uniformities when muscles are stretched onto the descending limb of the force–length relationship (Morgan 1990, 1994; Edman and Tsuchiya 1996; Morgan et al. 2000; Morgan and Proske 2004, 2006). The concepts of the so-called sarcomere length non-uniformity theory have been well discussed (for review, see Morgan 1994; Herzog et al. 2016) and will not be repeated here, with the exception of experimental results that demonstrate that this theory cannot explain most of the fundamental RFE properties of skeletal muscles.

The most basic predictions of the sarcomere length non-uniformity theory that have been shown to be violated are that:

1.  
2.  
3.  
4.  
5.  

Fig. 4.

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Steady-state isometric forces obtained in single, mechanically isolated sarcomeres (rabbit psoas) at sarcomere lengths of 2.4 μm [optimal length = 100% force (filled brown circle)] and 3.4 μm [approximately 50% of maximal isometric force at the plateau length (filled blue diamonds and filled black square = mean force). Also shown are the isometric steady-state forces of these isolated sarcomeres following a stretch from 2.4 to 3.4 μm (filled green triangles and filled black circle). FE Mean force enhancement observed in these sarcomeres, OFE the mean force above the maximal, isometric plateau forces for these sarcomeres. Note the enormous force enhancement and the consistently greater forces in the enhanced state compared to the plateau force. Adapted from Leonard et al. (2010) with permission

For these reasons, it would appear that the sarcomere length non-uniformity theory has little direct support. It will not be discussed further in this review, but interested readers may want to consult the following references for a more in depth treatment of this theory (Morgan 1990, 1994; Herzog 2014a, b; Herzog et al. 2015).

RFE increases with increasing stretch magnitude (Edman et al. 1982; Bullimore et al. 2007; Hisey et al. 2009), is independent of stretch speed (Edman et al. 1982), and is long lasting (Abbott and Aubert 1952; Leonard and Herzog 2010); however, it can be abolished instantaneously if activation of the muscle is interrupted for long enough for the force to drop to zero (Morgan et al. 2000). These properties gave early rise to the notion that RFE might be caused by the engagement of an elastic structural element upon muscle activation (Forcinito et al. 1998). Because of its location, attachment, and mechanical properties, titin was an early favorite for this role (Noble 1992). Simple modeling of passive structural element engagements upon activation allowed for excellent qualitative predictions of the RFE properties (Forcinito et al. 1998). However, direct proof of a passive element playing a role in RFE was missing for a long time.

In 2002, our research group discovered in experiments with cat soleus muscle that RFE was associated with a distinct increase in passive force (Herzog and Leonard 2002). Specifically, we demonstrated that an active stretch resulted in a substantial increase in the passive force (following the active stretch and following deactivation of the muscle) compared to the passive forces measured when the muscle was activated isometrically at the corresponding length or stretched passively to the final length (Fig. 5) (Herzog and Leonard 2002). We termed this increase in passive force following active muscle stretching “passive force enhancement” (PFE) and showed that PFE was long-lasting and increased with stretch magnitude and with final muscle length, but that it could be abolished instantaneously by a quick release of the muscle to a short (prior to stretch) length (Herzog et al. 2003). These studies provided the first direct evidence that a passive component was likely contributing to the RFE property of skeletal muscles.

Fig. 5.

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Force–time histories of cat soleus muscle stretched passively (lowest trace at 6 s), stretched actively (highest trace at 6 s), and activated isometrically at the final stretch length (middle trace at 6 s). Note the increased passive force following muscle deactivation (at 12 s) for the actively stretched muscle, compared to the passively stretched muscle and the muscle activated isometrically at the final stretch length. The increase in passive force following active muscle stretching (here seen at 12 s) was termed passive force enhancement (PFE). Adapted from Herzog and Leonard (2002) with permission

Subsequently, PFE was also observed in isolated myofibrils and sarcomeres (Fig. 3b) (Joumaa et al. 2007, 2008b). Since titin is the primary passive force producing structure in myofibrils (it produces in excess of 95% of the passive force), titin became directly implicated in the mechanisms causing PFE and contributing substantially to the total RFE (Herzog et al. 2006; Leonard and Herzog 2010; Powers et al. 2014).

How might titin contribute to the residual force enhancement?

If titin were to contribute to the PFE and the RFE, its force for a given muscle/sarcomere length would need to be greater when a muscle is stretched actively compared to when it is stretched passively. Such an increase in force could be achieved if titin was stiffer in an active muscle than in a passive muscle. There are two basic mechanisms by which titin (or any molecular spring) can increase its stiffness: (1) it can increase its inherent stiffness (a change in material property) or (2) it can shorten its spring length (resting length) while its material property remains unaltered.

Changes in the inherent stiffness of titin upon activation may occur if calcium binds to titin, thereby increasing its stiffness and force upon stretching. Labeit et al. (2003) showed that there was an approximately 20% increase in non-cross-bridge-based force in skinned mouse soleus fibers activated with calcium ([PCa 4.0]) whose cross-bridge forces were inhibited by 2,3-butanedione monoxime (BDM) compared to fibers stretched passively ([PCa 9.0]). Single molecule experiments with PEVK fragments of titin suggested that specific E-rich motifs in PEVK can bind calcium, thereby becoming stiffer. Our research group showed, using fluorescence spectroscopy, that I27 cardiac immunoglobulin domains also bind calcium in a dose-dependent and reversible manner and demonstrated, with atomic force microscopy, that unfolding of these domains required 20–25% more force in the presence of physiologically relevant concentrations of calcium compared to the passive state with low calcium concentration [PCa 8] (Fig. 6) (DuVall et al. 2013). Further experiments in which single myofibrils were activated with calcium but whose cross-bridge forces were inhibited (with BDM and/or troponin C deletion) also showed an increase in titin-based force (Joumaa et al. 2008b; Leonard and Herzog 2010) compared to low calcium (passive) conditions, as did experiments in which myofibrils were stretched actively and passively beyond the actin–myosin filament overlap (Leonard and Herzog 2010; Powers et al. 2014). These experiments all led to the conclusion that titin is indeed a spring whose stiffnessis changed by calcium, and thus, muscle activation. Aside from calcium activation, titin’s spring stiffness can also be changed with phosphorylation (Yamasaki et al. 2002; Borbely et al. 2009; Anderson et al. 2010; Hudson et al. 2010; Perkin et al. 2015) and disulfide bridging (Scott et al. 2002), among others. In summary, titin is a molecular spring whose inherent stiffness can be modulated by muscle activation. However, changes in the inherent stiffness of titin only seem to account for up to about 20% of the maximal RFE observed experimentally under optimal conditions (Granzier 2010; Leonard and Herzog 2010). Therefore, there must be other mechanisms to explain the remainder of the RFE.

Fig. 6.

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Unfolding force of the first five (out of 8 identical) cardiac I27 immunoglobulin (Ig) domains of titin. Note that unfolding of the I27 Ig domains in the absence of calcium (Control) required about 20% less force than in the presence of calcium (Calcium). Adapted from DuVall et al. (2013) with permission

Another way of increasing titin’s spring stiffness that may potentially account for the full RFE observed experimentally, is a change in titin’s resting or free spring length. A decrease in free spring length could be achieved theoretically if some of titin’s extensible domains were to become inextensible—for example, by binding to a more rigid structure. In vitro, titin fragments have been found to bind to actin (Linke et al. 1997, 2002; Yamasaki et al. 2001; Nagy et al. 2004; Li et al. 1995; Trombitas and Granzier 1997; Astier et al. 1998; Kulke et al. 2001; Bianco et al. 2007; Chung et al. 2011;), thereby slowing the progress of actin–myosin filament sliding in motility assays. However, functionally relevant binding seems to be restricted to cardiac titin’s PEVK domain, which has been found to interact with actin in a calcium-dependent manner (Kulke et al. 2001; Yamasaki et al. 2001). Specifically, calcium seems to release actin from titin, thereby reducing titin-based passive force and stiffness and facilitating the cardiac cycle (Yamasaki et al. 2001). Permanent titin–actin binding has been shown to occur in the most proximal titin segments near the Z-band (Trombitas and Pollack 1993; Trombitas and Granzier 1997). Together with the relatively rigid association of titin with myosin, this titin link to actin results in a passive molecular spring that is arranged in parallel with attached cross-bridges, while it is in series with the myosin filament and the actin filament near its insertion into the Z-band in the relaxed muscle. However, in vitro assays have largely excluded strong binding of skeletal titin sub-fragments to actin (Kulke et al. 2001; Yamasaki et al. 2001).

Antibody labeling of titin demonstrates that titin is extensible along its entire I-band length (except for titin’s most proximal 50–100 nm) in passive muscle (Horowits et al. 1989; Trombitas et al. 2000; Minajeva et al. 2001; Linke and Fernandez 2002). Horowits et al. (1989) used monoclonal antibodies that bind in the I-band region of titin to observe distal and proximal (relative to the antibody) titin segment elongation in passive and active rabbit psoas fibers. Passive fibers were analyzed for sarcomere lengths ranging from 2.1 to 3.8 μm, while activated fibers were kept at a constant sarcomere length of 2.6 μm (fibers were activated for 5 min to produce A-band shifts to one side of the sarcomere, thus compressing one-half of the sacromere and extending the other half). These authors found that in both passive and active fibers, the lengths of the proximal and distal titin segments depended only on the length of the half-sarcomere, leading them to conclude that activation did not change the mechanical properties of titin (Horowits et al. 1989).

In contrast, DuVall et al. (2017), using a variety of I-band-specific titin antibodies, found that stretching of rabbit psoas myofibrils resulted in different segmental elongations in the active and passive conditions. While these authors replicated previous results for segmental titin elongations for passive myofibril stretching (Horowits et al. 1989), they observed that proximal segments of titin stopped elongating after a short stretch amplitude in active conditions (Fig. 7). They interpreted their results with an activation- and stretch-induced entanglement of titin with myosin or cross-bridges that rendered some of the extensible distal regions of titin inextensible. However, an equally valid explanation would be that titin’s proximal segments bind to actin after a short stretch, thereby rendering the proximal segments inextensible. Since elongations of proximal titin segments stop occurring at sarcomere lengths as short as 2.3 μm, which is a sarcomere length at which titin is known to be unstrained in rabbit psoas myofibrils, this latter explanation seems the more feasible of the two. If correct, such titin–actin interactions in actively stretched muscles change titin’s free spring length, thereby increasing titin’s stiffness and consequently its force upon sarcomere elongation (Herzog 2014a). Theoretical modeling of titin–actin interactions, as observed experimentally (DuVall et al. 2017), demonstrate that even the largest RFE observed experimentally (e.g., Leonard et al. 2010) can be explained using titin binding to actin in actively stretched muscle (Schappacher-Tilp et al. 2015).

Fig. 7.

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Passive (a) and active (b) stretching of proximal titin segments labeled using an antibody [F146 that binds to the PEVK region (diamond symbols)] region that allows for measurements of proximal and distal titin segment elongations during passive and active stretching of single rabbit psoas myofibrils. Figures on the left show elongation of the half-sarcomere [top traces (circular symbols) using an M-line label) and elongations of the proximal titin segment (bottom traces: from X-band to F146 label) for two representative sarcomeres from two different myofibrils. Note in a (passive stretching) that the two proximal titin segments elongate continuously with half-sarcomere elongations, reaching final lengths of approximately 0.95 μm (at a sarcomere length of 4.0 μm) and about 0.6 μm (at a sarcomere length of about 3.5 μm). In contrast, when the myofibrils are stretched while activated, the proximal segments elongate similarly to the elongations observed in the passive condition, but then stop elongating and remain substantially shorter than in the passive case (i.e., with a length of about 0.6 and 0.35 μm, respectively). The panels on the right illustrate schematically what we believe might be happening. In the passive stretch (a), the proximal and distal titin segments elongate in accordance with their stiffness properties. In the active stretch (b), titin is thought to attach to actin at some point, thereby shortening titin’s free spring length, increasing its stiffness, eliminating elongation of proximal titin, and increasing titin-based force

The results by DuVall et al. (2017) do not agree with those observed by Horowits et al. (1989). However, in the DuVall study (DuVall et al. 2017), some sarcomere stretching was required prior to the loss of proximal titin segment elongation, while in the Horowits study (Horowits et al. 1989), sarcomeres were kept at a constant isometric length. Also, in the DuVall study, segmental elongations were measured continuously during the active and passive stretching, while in the Horowits study, the active and passive fibers were fixed at specific lengths, and thus measurements of titin segment lengths were not continuous, but were made at a single (average) sarcomere length following fixation. It is not known how fixation might affect possible titin-to-actin binding in active fibers, and if indeed some sarcomere stretching is required to initiate titin–actin interaction; both these aspects might have been missed in the Horowits experiments.

Linke et al. (1996) used epitope tracking to determine the elongations of specific titin segments in passive rabbit cardiac myofibrils and in rabbit soleus and psoas myofibrils. Their results using an N2A-based epitope do not mimic those by DuVall et al. (2017) for passive stretching of titin segments in rabbit psoas. While they found that proximal titin segment elongation stopped at average sarcomere lengths of about 2.5 μm with little if any further elongation, DuVall et al. (2017) found continuous elongations of proximal titin segments up to a sarcomere length of 4.0 μm (Fig. 7). Leonard and Herzog (2010) showed that titin-based forces were much greater in psoas myofibrils stretched actively than in those stretched passively from optimal lengths to lengths beyond the actin–myosin filament overlap (Fig. 8). When cross-bridge interaction in the active [pCa 3.5] myofibrils was inhibited by BDM or troponin C deletion (Joumaa et al. 2008b), titin-based forces were still greater than in myofibrils under passive [pCa 8.5] conditions but much smaller than those when cross-bridge interactions with actin were allowed to occur normally (Leonard and Herzog 2010; Leonard et al. 2010). Starting active stretching from a longer length (3.4 μm) than optimal (2.3–2.5 μm) also decreased the titin-based forces compared to when stretches were initiated at optimal length (Leonard and Herzog 2010). Although the molecular mechanisms associated with segment-specific changes in titin’s mechanical properties need further elucidation, it appears that changes in the titin’s mechanical properties that occur with muscle activation and stretching contribute substantially to the experimentally observed RFE and PFE in skeletal muscles. Furthermore, the results by Leonard and Herzog (2010) suggest that it is not calcium activation that produces these substantial changes in passive/titin-based forces; rather, active cross-bridge binding is required to observe substantial force enhancement. A possible explanation could be that strong cross-bridge binding causes a movement of the regulatory proteins (troponin and tropomyosin) that may expose titin binding sites on actin that cannot be accessed by titin in the passive muscle or in calcium-activated muscle in which strong cross-bridge binding to actin is inhibited.

Fig. 8.

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Stress (force/cross-sectional area) versus sarcomere length relationship for single rabbit psoas myofibrils stretched from an average sarcomere length of 2.0 μm to 6.0 μm. Myofibrils were stretched passively (Passive), actively (Active), actively from an average sarcomere length of 3.4 μm (Half-Force), and after deletion of titin (No Titin). Actin myosin filament overlap is lost at an average sarcomere length of about 4.0 μm (indicated by the shaded area). According to the cross-bridge theory, one would expect the forces beyond actin myosin filament overlap (non-shaded area) to be purely passive and the same for all conditions with intact titin filaments. However, the forces in the actively stretched myofibrils were substantially greater than those for the passively stretched myofibrils in the area where myofilament overlap was lost. Deactivation of selected myofibrils at an average sarcomere length of 5.0 μm did not result in a loss of force (results not shown), indicating that there was no remnant cross-bridge-based force at these lengths. Elimination of titin from the myofibrils abolished all passive and all active force in myofibrils, indicating that titin is not only an essential protein for passive force production but is absolutely essential for active force transmission from the cross-bridges to the Z-bands and for centering the myosin filaments in the sarcomere. Adapted from Leonard and Herzog (2010) with permission

Stretching psoas sarcomeres to very long lengths, i.e., in excess of 5.0 μm, has been criticized for its potential to damage sarcomeres permanently by dislodging titin from its attachment to the Z-band or the myosin filament. However, Herzog et al. (2012) found that stretching of myofibrils to an average sarcomere length of > 5 μm (and individual sarcomeres within these myofibrils up to 6 μm) was not associated with permanent damage (i.e., loss of force). Rather, given sufficient time (about 10 min) and recovery at a very short average sarcomere length (1.8–2.0 μm), full recovery of titin-based passive force was observed, indicating that for stretches of sarcomeres up to 5.0 μm, there does not appear to be permanent damage to the structural network of (rabbit psoas) sarcomeres that would prevent full force recovery (Fig. 9). The fact that force recovery required several minutes and needed to be done at short sarcomere lengths (recovery for < 10 min or at sarcomere lengths averaging ≥ 2.6 μm was always incomplete) was interpreted as indicating that titin immunoglobulin unfolding, which would have taken place at these long sarcomere lengths (Kellermayer et al. 1997; Herzog et al. 2012), is slow and only occurs when forces acting on titin are essentially zero (Kellermayer et al. 1997).

Fig. 9.

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Examples of two separate rabbit psoas myofibrils that were repeatedly stretched to sarcomere lengths beyond actin myosin filament overlap. Note that in both cases repeat stretches did not result in a decrease in peak force or loading energy, thus indicating that even stretching to lengths up to 5.0 μm did not result in permanent damage and loss of force. a Myofibril stretched to an average sarcomere length of approximately 5.2 μm (stretch 1), then shortened and rested for 10 min at an average sarcomere length of 2.6 μm and re-stretched (stretch 2). Two sets of three stretch-shortening cycles were performed and the third stretch of the first set (stretch 1), and the first stretch of the second set (stretch 2) are shown b Myofibril stretched to an average sarcomere length of approximately 4.2 μm, then shortened and rested for 10 min at an average sarcomere length of approximately 1.8 μm. Two sets of three stretch-shortening cycles were performed (with a 10-min rest in between) and the first cycles (stretch 1) of the first and second set (stretch 2) are shown. Adapted from Herzog et al. (2014) with permission

This interpretation has been challenged recently where immunoglobulin refolding has been thought to occur at force levels acting on titin of about 8 pN, a relatively large force corresponding to the force exerted by about two to four attached cross-bridges (Rivas-Pardo et al. 2016). If these latest results receive further support, then the notion of unfolding and subsequent refolding of titin’s immunoglobulin domain having physiological relevance may have to be revisited.

Conclusion

Titin is a multi-purpose spring with many acknowledged mechanical functions, including the provision of passive force, stability of the myosin filaments, and stability of sarcomeres on the descending limb of the force–length relationship (Granzier et al. 1996, 2002; Linke et al. 1998; Granzier and Labeit 2002, 2007; Anderson et al. 2010; Linke and Kruger 2010). Less clear are its possible functions in explaining the RFE property of skeletal and cardiac muscle and the molecular details of its function as an adaptable molecular spring (Herzog et al. 2006, 2016; Herzog 2014a). Specifically, many unexplained phenomena in eccentric muscle action (increased force, RFE, decreased energetic requirements) can easily be accounted for within the framework of the cross-bridge theory, assuming that titin is an adaptable spring (Schappacher-Tilp et al. 2015). It will be an exciting challenge in this upcoming decade to elucidate the detailed molecular properties of titin in actively elongating muscle.

Compliance with ethical standards

Walter Herzog declares that he has no conflict of interest.

Ethics approvals for all experiments described in this study were obtained by the Life Sciences and Animal Research Ethics Commitee of the University of Calgary.

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