Cardiomyopathies Associated with Myofibrillar Myopathies (Pathophysiology and Genetics of Cardiomyopathies) Part 2

The smallest contractile unit: The sarcomere

The myocardium is composed of an assembly of a number of interconnecting, branching fibers, or short cells, separated at their end by the intercalated disk. The fibers contain numerous fibrils, composed of a regular repeating structure termed the "sarcomere" (Figure 2A) (Sonnenblick, 1968). The sarcomere is the basic and fundamental unit of striated muscles. Understanding the structure-function relationship linking the structure of the sarcomere to the physiology of normal or pathologic heart is therefore essential to understand myofibrillar myopathies development. Sarcomeres are distinguished by the striated distribution of their proteins, visible in light microscopy as three major bands, called A, I and Z (Figure 2B). A bands contain thick filaments of myosin and proteins that bind to myosin. The I band comprise thin actin filaments and proteins that bind actin. In the middle of the A is the "M band" also called "M line". The middle part of the I band is the "Z band", also called the "Z line" or "Z disk". The basic contractile system is the well known actin-myosin tandem. Two heavy myosin chains are associated to two light chains and form a globular part. Actin filaments, the thin structure, are composed of a double helix of G-actin (a globular molecule of 46 kDa) polymerized into a chain (Lehninger et al., 2005). aB-crystallin as chaperone molecule, myotilin and filamin C as scaffolding molecules, are known to interact with actin.

Force transmission

The first important role of Z-discs is passive transmission of tension through the Z-disc structural assembly. When a mutation occurs, like in MFM, the mechanism that maintains fixed Z-disc may go awry. In addition, Z-disc proteins allow to transmit force and ensure mechanical coupling between sarcomeres and the sarcolemma via the costameres. Three of four filaments systems of the sarcomere, filamentous F-actin, titin and nebulin/nebulette, interact with the Z-disc structure. Two proteins participate to the cardiac sacomeric cytoskeleton: titin and nebulette (Figure 2B).

Titin is a giant 3 MDa elastic protein that spans half sarcomeres from Z-disc to M-band, thus forming a continuous structure from one end of the sarcomere to the other, with consecutive titins. Titin can be considered as a giant bidirectional spring responsible for the generation of passive retraction force in mechanically stretched cardiac myocytes (Granzier & Labeit, 2004). Stiffness of titin can be adjusted during development and diseases through a shift in the expression ratio of the two main titin isoforms in cardiac sarcomeres (Lahmers et al., 2004; Opitz et al., 2004; Warren et al., 2004). Titin binds to more than 20 structural, contractile or signaling molecules, and therefore plays a role as major integrating component in the mechanosensory complexes associated to the sarcomeres. Nebulette is a 107 kDa nebulin homologue present in the cardiac muscle Z-disc. It is composed of only 22 nebulin motifs (compared to up to 185 in nebulin), and contains a nebulin-like C terminus, mediating Z-disc localization (Moncman & Wang, 1995). At present, however, its molecular function in cardiac myocytes is still unclear. The backbone of the Z-disc consists of layers of a-actinin aligned in an antiparallel fashion. In muscle, it cross-links actin filaments of opposite polarity originating from adjacent sarcomeres (Stromer & Goll, 1972) and provides anchors for the binding of actin thin filaments, as well as titin and nebulin/nebulette (Otey & Carpen, 2004). Myotilin and ZASP interact with a-actinin, and myotilin is linked to filamin C, thus creating a network of proteins at the Z-disc.

Schematic representation of the general organization of muscular fibers (A), sarcomere (B) and schematic localization of the major proteins involved in the cardiac Z-disc structure (C).

Fig. 2. Schematic representation of the general organization of muscular fibers (A), sarcomere (B) and schematic localization of the major proteins involved in the cardiac Z-disc structure (C).

Figure 2B represents the enlarged dotted rectangle in figure 2A, and Figure 2C the enlarged representation of the dotted rectangle in Figure 2B. Names in red and bold correspond to proteins involved in myofibrillar myopathies, excepted for Bag3 which is not represented. Not all proteins participating to the Z-disc structure or signaling are represented, due to the complexity of this structure. For more details, see text. Figure C is adapted from Frank et al., 2006.

Another essential component is CapZ, a heterodimer composed of a and P subunits, which caps the barbed ends of actin filaments. CapZ is proposed to regulate actin dynamics at the barbed end, thereby anchoring the thin filament system to the Z-disc (Schafer et al., 1996).

The costameres are multiproteic complexes which link the marginal Z-discs at their circumferences to the sarcolemma, the specialized membrane of the individual myofibers. Costameres have been described as transmitters of contractile force to the sarcolemma and extracellular matrix. This lateral force transmission ensures identical sarcomere length, thereby minimizing shear stress. However, desmin, filamin C, dystrophin, sarcoglycans, integrins, melusin and focal adhesion kinases have been involved in its structure (reviewed in Bloch et al., 2002).

Many other proteins (myopalladin, obscurin, Enigma, telethonin, zyxin, …) participate to the Z-disc structure (Table 1), but their study is beyond the scope of this review (reviewed in Frank et al., 2006). The six genes held responsible for MFM are involved at various degrees in Z-disc structure and force transmission (Figure 2C). Desmin forms a continuous network that maintains a spatial relationship between the contractile apparatus and other structural elements of the cells, and is believed to provide maintenance of structural integrity, force transmission, mechanosignaling, and resistance to external mechanical stress. Myotilin constitutes the core of a network of proteins, including actin, a-actinin and filamin C, that are part of the force transmission mechanism. In turn, filamin C provides a scaffold for signaling proteins at the Z-disc, and may be part of a mechanosensing device.

Energy metabolism

In this part, we will focus on specific effects of the alteration of genes causing MFMs on energy in muscle. The main effects have been studied on the localization and function of mitochondria. Structural studies of intracellular arrangements of mitochondria into functional complexes with myofibrils and sarcoplasmic reticulum demonstrate their importance in mitochondrial oxydative activity and membrane permeability (Andrienko et al., 2003; Appaix et al., 2003). There are findings with pathological respiratory chain enzyme activities in patients with MFM (Reimann et al., 2003). Desmin intermediate filaments (IFs) might participate in mitochondrial positioning to areas of high energy demand, respiratory function, and calcium cycling in cardiac and skeletal muscle (Capetanaki, 2002).

Dynamic view of the sarcomere

While often described as a static structure, the sarcomere is actually dynamic and undergoes constant turnover, allowing to adapt to physiological changes while still maintaining its function. New factors have been identified that play a role in the regulation of protein quality control in the sarcomere, including chaperones that mediate the assembly of sarcomere components and ubiquitin ligases that control their specific degradation. The Z-disc has additional important roles as it houses or anchors many additional proteins, which have various roles, including stretch sensing and signaling or protein quality control. The Z-disc can therefore be considered as a nodal point in signaling and disease (reviewed in Frank et al., 2006). In MFMs, the Z-discs are abnormal, the arrangements of myofibrils are in disarray, or aggregates of proteins are present, often near the Z-disc. The highly ordered arrangements of proteins in the sarcomere, which persists even as contractile force is generated, suggest that binding interactions between Z-disc proteins are strong and very stable (Sanger & Sanger, 2008). Thus, the continual remodelling of the cardiac sarcomere allows efficient adaptation to physiological stresses, including exercice or metabolic variations, but also initial efficient adaptation to starting pathologies such as ischemic heart disease and myofibrillar myopathies. Since this dynamic turnover is the basis of homeostatic mechanism of sarcomere maintenance, it is essential to better understand it.


Size (kDa)







calmodulin, titin

Rho-GEF domain signaling

Giant protein



Nebulette, a-actinin cadiac ankyrin repeat proteins (CARP)

Intra-Z-disc meshwork binds nebulette (directly) and titin (via a-actinin) to actin, CARP signaling ?

Palladin familly



Spectrin repeats of a-actinin

enhances cross-linking actin to a-actinin

PDZ and LIM domain protein 3 (PDLIM3)



Ret kinase, actin, insulin receptor P tropomyosin

anchor for LIM proteins




a-actinin 2, PKCs (brain)

hypertrophic program




a-actinin 2, Clik1 kinase

stress fibers control, FA




binds actin to MLP a-actinin

close to Z-disc and FAK signalling / antiapoptotic

LIM protein family

Nucleoplasmic shuttling



a-actinin, telethonin calcineurin, P spectrin

cardiac stretch receptor and mechanosignalling, negative regulator of cardiac hypertrophy

Nucleoplasmic shuttling



Titin N-terminus K+ channels, calsarcin

cardiomyocytes passive tension

negative regulator of myostatin = T-Cap


27 – 32 *

Calcineurin, a-actinin telethonin, Filamin C, a-actinin

crosslinks Z-disc and Ca2+/ calcineurin signalling, inhibits hypertrophic genes sensor for

biomechanical stress

= synaptopodin-2


118 – 136 *

a-actinin colocalization

multiadaptator protein ?

nucleoplasmic shuttling

Table 1. Proteins involved in the Z-disc structure as well as in Z-disc signaling, and not detailed in the text.

Other structural and signaling molecules are described paragraphs 3.1, 3.2 and paragraph 3.4.6, respectively. ALP: Actinin-associated LIM Protein. PDZ: PSD-95 / SAP90 / ZO-1 proteins common domain. LIM domains: LIN-11 / Isl1m / MEC-3 proteins common domain. ENH: Enigma Homologue protein. MLP: Muscle LIM Protein. FA: Focal Adhesion. FAK: Focal Adhesion Kinase. PKC: Protein Kinase C.

Main contractile protein turnover

The following half-lives have been estimated in myocytes: Actin: 7 to 10 days (Zak, 1977). Myosin: 5 to 8 days (Martin et al., 1977). Tropomyosin: 7 to 10 days (Zak, 1977). Troponin: 3 to 5 days (Michele et al., 1999).

Titin is subjected to a "rapid" turnover, with a half-life estimated to be 3 days in myocytes (Fong et al., 1996).

Protein quality control: Role of chaperone molecules

Multiple endogenous pathways are engaged in restoring cellular homeostasis, among which one of the best characterized mechanism involves protein folding by the heat-shock family of stress proteins (HSP), also termed chaperones. There are several families of molecular chaperones present in the cytoplasm of mammalian cells, including Hsp90, Hsp70, TCP1 (CCT, TriC) and small HSP (sHSPs). Members of the Hsp90 family are the most abundant chaperones located in the cytosol in non-stressed cells which are inducible with stress.

Hsp70 and Hsc70 (Hsp70 cognate protein) are major players in cardiomyocyte protection: the induction of HSP70 by ischemia, and conversely, overexpression of Hsp70 or Hsc70 promotes substantial cardioprotective benefits (Donnelly et al., 1992; Hutter et al., 1994).

Chaperonin TRiC requires additional components, such as Hsp40, to stimulate the Hsc70 ATPase for protein folding, and is required for folding of actin and tubulin in vivo. Unlike Hsp70, small HSPs, including aB-crystallin, Hsp27, Hsp22 and Hsp20 cannot bind nor hydrolyze ATP, and are not able to refold proteins, but can buffer them against aggregation (Merck et al., 1993). aB-crystallin represents a substantial fraction of adult heart total soluble proteins (1 to 3%) (Kato et al., 1991). The highest level of aB-crystallin expression in muscles has been found in the cardiac conduction fibers (Leach et al., 1994). Previous studies indicate that aB-crystallin is highly soluble and localized in the cytosolic fraction in unstimulated cardiac myocytes. Heat or ischemia triggers rapid translocation of aB-crystallin into the cytoskeletal and nuclear fraction and specific interactions at the Z-disc (Neufer et al., 1998). HspB8 (Hsp22) and HspB6 (Hsp20) are also expressed in striated myogenic lineages with high oxidative capacity, such as the heart and type I skeletal muscle fibers (Depre et al., 2002).

HSP molecules are assisted by co-chaperones which perform a variety of tasks, including modulation of ATPase activity (DNAJ), substrate protein binding and release (BAG : Bcl-2 -Associated athanoGene), protein folding (Hsp40 family), assembly, and translocation or degradation (CHIP : Carboxyterminus of Hsp70 Interacting Protein). Co-chaperones also bind substrate proteins to modulate folding in a substrate-specific manner (reviewed in Willis & Patterson, 2010). Among many proteins (more than 40 Co-chaperones), only DnaJ, BAG-1, Hop and CHIP have been described in the heart. pDJA1 (DnaJ-like molecule) expression is restricted to cardiomyocytes. Its levels increase four-fold after reperfusion (Depre et al., 2003). BAG-1 is able to inhibit apoptosis and to induce autophagy by interacting with Hsc70, stimulated after ischemia / reperfusion injury (Townsend et al., 2004). The BAG-3 isoform participates in the induction of macroautophagy in association with HspB8 (Gurusamy et al., 2009). Another protein,CHIP, plays a role as cochaperone of Hsp70 and in the ubiquitin-proteasome system as an ubiquitin ligase. CHIP may exert a critical function in shuttling damaged and oxydized proteins into autophagic pathways after ischemia / reperfusion injury (Zhang et al., 2005). Increased cochaperone expression in the heart has been found to be cardioprotective in ischemia (Benjamin & McMillan, 1998).

During assembling of the sarcomere, molecular chaperones are needed for the correct folding, assembly and prevention of aggregation. Two molecular chaperone GimC (prefoldin) and TriC (TCP-1 Ring Complex) have been found to play a synergistic role during synthesis and incorporation of actin filaments into the sarcomere. In contrast to actin, myosin cannot self-assemble without additional factors, including chaperones such as Unc45, Hsp90 and Hsp70. The assembly of desmin requires the chaperone aB-crystallin, to prevent its misfolding and aggregation (Bennardini et al., 1992). aB-crystallin also interacts with titin and actin. These data suggest a highly cooperative relationship between various chaperones during the assembly of the actin filaments in the sarcomere.

In animal models with targeted deletion of muscle-specific chaperone proteins, there is a clear evidence of sarcomere disorganization. Cardiac chaperones such as Hsp70, aB-crystallin and HspB8 levels are increased during the development of cardiac hypertrophy. Hsp27 and aB-crystallin can protect cardiomyocytes against ischemic damages (Martin et al., 1997). Increase in HspB8 expression also results in the reexpression of the foetal gene program characteristic of cardiac hypertrophy (Depre et al., 2002). The protective role of Hsp22 in ischemia / reperfusion injury appears to be due to its function in activating autophagy, which is critical during the course of this type of injury (Carra et al., 2008b).

Next post:

Previous post: