Plasticity of cardiac titin/connectin in heart development

Many sarcomeric proteins in the myocardium alter their isoform pattern during perinatal development to adjust to the intensified pump function of the postnatal heart. These changes also involve the giant protein titin/connectin. Here we show by low-percentage polyacrylamide-gel electrophoresis that developmentally regulated switching of cardiac titin/connectin size occurs in the hearts of mouse, rat, pig, and chicken. Mammalian hearts express, well before birth, large foetal (∼3.7 MDa) N2BA-titin/connectin isoform but no N2B-isoform (3.0 MDa). During perinatal heart development the 3.7-MDa N2BA-isoform is replaced by a mix of smaller isoforms. At birth a plethora of intermediate-size N2BA-isoforms appears together with the N2B-isoform. In postnatal heart development the larger-size N2BA-isoforms disappear and smaller-size N2BA-isoforms are upregulated, whereas the proportion of N2B-titin/connectin increases to species-specific adult levels. The time courses of isoform switching are faster in small than in large mammals. Titin/connectin isoform switching also takes place in developing chicken hearts, but the largest embryonic isoform found here was less than 3.4 MDa. At hatching, various smaller-size isoforms appeared and within a week the adult expression pattern was established representing a major 3.0-MDa isoform and a minor 3.15-MDa isoform. The ratio between the two adult isoforms differed between the left ventricle and the right atrium. The perinatal changes toward smaller cardiac titin/connectin isoforms in mammals and chicken greatly increase the myofibrillar passive tension of postnatal hearts. Plasticity of titin/connectin at approximately the time of birth thus affects myocardial mechanics but could also be an important factor in developmentally regulated assembly and signalling processes.Proceedings of the International Symposium on Muscle Elastic Proteins:Koscak Maruyama Memorial Meeting, Chiba, Japan, November 2004     

Calcium binding to an elastic portion of connectin/titin filaments

-Connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the -connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on -connectin. Purified -connectin was digested by trypsin into 1700- and 400-kDa fragments, which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 × 10⁷ M^–1 and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 M. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of -connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of -connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl₂ and 70 M leupeptin to split connectin filaments into -connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200 ± 33 kDa (mean ± SD), while - and -connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 m at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N₂ line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction–relaxation cycle of skeletal muscle.     

Restoring force development by titin/connectin and assessment of Ig domain unfolding

Titin/connectin is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is composed of serially-linked immunoglobulin (Ig)-like domains and several unique sequence elements. Here we address the role of titin/connectin in sarcomeres shortened to below the slack length (length attained by an un-activated cell in absence of external forces). Such shortened cells develop so-called restoring forces that re-extend the cells upon relaxation. The experiments that we present are based on a high throughput method with a rapid solution switching system which allows unattached single cardiac myocytes to be activated (resulting in shortening below the slack length) and then to be rapidly relaxed while their maximal re-lengthening velocity is measured at the sarcomere level (dSL/dt _max), with high-resolution imaging techniques. Experiments were carried out on myocytes that express different isoforms of titin/connectin. We measured the relation between dSL/dt _max and the minimal SL during contraction (SL_min) and determined the slope of this relation as a measure of ‘restoring stiffness.’ We found that the restoring stiffness correlates with the isoform expression profile with myocytes that express high levels of the stiff isoform (N2B) having the highest restoring stiffness. These results support the notion that titin/connectin is a bi-directional spring that develops passive force when stretched above the slack length and restoring force when shortened to below this length. We also discuss in detail the mechanisms that underlie titin/connectin’s restoring force development and focus on whether or not unfolding of Ig domains plays a role.     

Functional analysis of novel KCNQ2 mutations found in patients with Benign Familial Neonatal Convulsions

Benign Familial Neonatal Convulsions (BFNC) are a rare epilepsy disorder with an autosomal-dominant inheritance. It is linked to mutations in the potassium channel genes KCNQ2 and KCNQ3. These encode for Kv7.2 and Kv7.3 potassium ion channels, which produce an M-current that regulates the potential firing action in neurons through modulation of the membrane potential. We report on the biophysical and biochemical properties of V589X, T359K and P410fs12X mutant-KCNQ2 ion channels that were detected in three BFNC families. Mutant KCNQ2 cDNAs were co-expressed with WT-KCNQ2 and KCNQ3 cDNAs in HEK293 cells to mimic heterozygous expression of the KCNQ2 mutations in BFNC patients. The resulting potassium currents were measured using patch-clamp techniques and showed an approximately 75% reduction in current and a depolarized shift in the voltage dependence of activation. Furthermore, the time-constant of activation of M-currents in cells expressing T359K and P410fs12X was slower compared to cells expressing only wild-type proteins. Immunofluorescent labeling of HEK293 cells stably expressing GFP-tagged KCNQ2-WT or mutant α-subunits indicated cell surface expression of WT, V589X and T359K mutants, suggesting a loss-of-function, while P410fs12X was predominantly retained in the ER and sub-cellular compartments outside the ER suggesting an effectively haplo-insufficient effect.     

The organization of titin (connectin) and nebulin in the sarcomeres: an immunocytolocalization study

Summary Monospecific polyclonal antibodies against two exceptionally large proteins, titin (a-T) and nebulin (a-N) isolated from rabbit skeletal muscles, were raised in guinea pig. Using an immuno-pre-embedding method, we have localized at the ultrastructural level of resolution the reactivity sites in skinned muscle fibres. At resting length a-T and a-N antibodies recognize epitopes which only partially overlap. a-T antibodies decorate mostly the A band with at least four clearly distinguished lines of reaction and one line in the I band, all near the A/I limit; a-N antibodies bind to the same region, but with wider areas of reaction in both A and I bands. To study whether the localization of these reaction sites varies according to the sarcomere length, skinned rabbit psoas fibres were incubated at sarcomere lengths ranging from maximum shortening to overstretching. The results indicate that lines decorated by a-T move away from the Z disc when the sarcomere is lengthened. With respect to the M line, the behaviour was biphasic. When the sarcomere was stretched up to about 2.8 m, the decorated lines maintain almost the same distance from the M line. When the sarcomere is stretched beyond 2.8 m, all a-T epitopes move away from the M line and the molecule behaves elastically. At resting length the a-N decoration appears to be localized on three large adjacent bands at the I, A/I and A level. The a-N line of reaction at the edge of the A band moves away from the Z discs as the sarcomere lengthens, while a second line which seems to be localized at the tip of the thin filament moves away from M line when the sarcomere lengthens. In non-overlapping sarcomeres a-N antibodies decorate only the tip of the thin filaments. Our results indicate that titin forms a polar filament connecting the M line to the Z line. In short sarcomeres, the filament seems to have some connections with structures of the A band, since titin epitopes do not move during stretching. These connections are lost at longer sarcomere lengths. On the other hand, our results suggest that nebulin is probably not a constituent of the titin filament.     

Developmental changes in rat cardiac titin/connectin: transitions in normal animals and in mutants with a delayed pattern of isoform transition

Rat cardiac titin undergoes developmental changes in isoform expression during the period from late embryonic through the first 20–25 days of life. At least five size classes of titin isoforms have been identified using SDS agarose gel electrophoresis. The longest normal isoform is expressed in the embryonic stages, and it is progressively replaced with increasingly smaller versions. The isoform switching is consistent with changes in resting tension from lower values in one-day neonates to higher levels in adult myocytes. Considerable micro-heterogeneity in alternative splicing patterns also was found, particularly in the N2BA PEVK region of human, rat, and dog ventricle. A rat mutation has been identified in which the embryonic-neonatal titin isoform transitions are markedly delayed. These mutant animals may prove useful for examining the role of titin in stretch-activated signal transduction and in the Frank–Starling relationship.     

Functional analysis of neurofibromatosis 2 (NF2) missense mutations.

Neurofibromatosis 2 (NF2) is a tumor predisposition syndrome in which affected individuals develop nervous system tumors at an increased frequency. The most common tumor in individuals with NF2 is the schwannoma, which is composed of neoplastic Schwann cells lacking NF2 gene expression. Moreover, inactivation of the NF2 gene is observed in nearly all sporadic schwannomas, suggesting that the NF2 gene is a critical growth regulator for Schwann cells. In an effort to gain insights into the function of the NF2 gene product, merlin or schwannomin, we performed a detailed functional analysis of eight naturally occurring non-conservative missense mutations in the NF2 gene. Using a regulatable expression system in rat schwannoma cells, we analyzed proliferation, actin cytoskeleton-mediated events and merlin folding. In this report, we demonstrate that mutations clustered in the predicted alpha-helical region did not impair the function of merlin whereas those in either the N- or C-terminus of the protein rendered merlin inactive as a negative growth regulator. These results suggest that the key functional domains of merlin lie within the highly conserved FERM domain and the unique C-terminus of the protein.     

Functional studies of yeast actin mutants corresponding to human cardiomyopathy mutations

The molecular mechanisms by which different mutations in actin lead to distinct cardiomyopathies are unknown. Here, actin mutants corresponding to α-cardiac actin mutations causing hypertrophic cardiomyopathy [(HCM) P164A and A331P] and dilated cardiomyopathy [(DCM) R312H and E361G] were expressed in yeast and purified for in vitro functional studies. While P164A appeared unaltered compared to wild-type (WT) actin, A331P function was impaired. A331P showed reduced stability in circular dichroism melting experiments; its monomer unfolding transition was 10°C lower compared to WT actin. Additionally, in vitro filament formation was hampered, and yeast cell cultures were temperature sensitive, implying perturbations in actin–actin interactions. Filament instability of the A331P mutant actin could lead to actomyosin dysfunction observed in HCM. Yeast strains harboring the R312H mutation did not grow well in culture, suggesting that cell viability is compromised. The E361G substitution is located at an α-actinin binding region where the actin filament is anchored. The mutant actin, though unaltered in the in vitro motility and standard actomyosin functions, had a threefold reduction in α-actinin binding. This could result in impairment of force-transduction in muscle fibers, and a DCM phenotype. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of eccentric exercise on joint stiffness and muscle connectin (titin) isoform in the rat hindlimb

We investigated the effects of repeated eccentric exercise for rat medial gastrocnemius muscle on ankle joint stiffness and muscle connectin (titin) isoform composition (longer form, alpha-connectin; shorter form, beta-connectin). Male Wistar rats were trained on a custom-made, isokinetic dynamometer (eccentric-exercise group, n = 6; sham-operated group, n = 6). The exercise session consisted of 20 eccentric contractions elicited by submaximal electric stimulations under anesthesia. The contracting muscle was forcibly lengthened by an isokinetic dorsi-flexion of the ankle joint (velocity, 30 degrees/s; range of motion, 45 degrees). Rats in the eccentric-exercise group were trained every two days for 20 days (10 sessions in total). The static passive resistive torque (PRT) of 45 degrees at the ankle joint was used as a measure of the joint stiffness, and was determined before and after the experimental period. After 10 sessions of eccentric exercise, the wet weight of medial gastrocnemius muscle significantly increased (P < 0.05), whereas the static PRT significantly decreased (P < 0.05) in the eccentric-exercise group, when compared to the sham-operated group. Myosin-ATPase staining showed a decrease in the number of type IIb/IId fibers (P < 0.001) and an increase in the number of type IIa fibers (P < 0.05). However, no significant difference was seen in the connectin (titin) isoform composition between the eccentric-exercise group and the sham-operated group, suggesting that the reduction in PRT was not due to change in resting mechanical properties of muscle fibers.     

Assembly of the cardiac I-band region of titin/connectin: expression of the cardiac-specific regions and their structural relation to the elastic segments

Summary The giant molecule titin (also called connectin) provides an elastic connection in the I-band between the Z-disk and A-band of striated muscle. This region is assembled in a tissue-specific way by extensive differential splicing events. We have raised monoclonal antibodies against the two N2-line isoforms of titin and demonstrate that both forms of cardiac I-band titin are constitutively co-expressed in atrial and ventricular muscle. In developing mouse embryos, the expression of the cardiac N2-B isoform remains strictly cardiac-specific and is linked to the expression of the ubiquitous N2-A isoform. The mechanical function of the cardiac N2-line region was investigated ultrastructurally. Immunoelectron microscopy reveals that the N2-B region separates two mechanically distinct sections of titin with a hyperextensible segment spanning the distance to the Z-disk. The formation of a plateau in the extension of cardiac titin rules out that Ig-domains can be unfolded as a mechanism of elasticity.     

Functional analysis of mutations in TGIF associated with holoprosencephaly

Holoprosencephaly (HPE) is the most common structural malformation of the forebrain and face in humans. Our current understanding of the pathogenesis of HPE attempts to integrate genetic susceptibility, evidenced by mutations in the known HPE genes, with the epigenetic influence of environmental factors. Mutations or deletions of the human TGIF gene have been associated with HPE in multiple population cohorts. Here we examine the functional effects of all previously reported mutations, and describe four additional variants. Of the eleven sequence variations in TGIF, all but four can be demonstrated to be functionally abnormal. In contrast, no potentially pathogenic sequence alterations were detected in the related gene TGIF2. These results provide further evidence of a role for TGIF in HPE and demonstrate the importance of functional analysis of putative disease-associated alleles.     

Ten novel mutations found in aniridia

Aniridia (AN) is a sight-threatening congenital ocular disorder characterized by iris hypoplasia, corneal pannus, foveal and optic nerve hypoplasia, cataract formation, and glaucoma. In two-thirds of the patients, AN is inherited in an autosomal dominant fashion with almost complete penetrance but variable expression. The remaining cases are sporadic. Aniridia has been shown to be associated with mutations in the PAX6 gene, located on chromosome 11p13, telomeric to the Wilms' tumor predisposition gene (WT1). This paper describes 14 mutations in the PAX6 gene in patients with AN. Among these 14 mutations, 10 have been unpublished until now. They result most probably in haploinsufficiency and consequently in a reduced protein level of functional PAX6 protein. The mutations reported here are scattered all over the gene, including the paired-box, the glycine-rich region, the homeobox, and the proline–serine–threonine (PST)-rich region. Hum Mutat 12:304–313, 1998.© 1998 Wiley-Liss, Inc     

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments

Summary The localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 m remained at about 0.8 m from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 m. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 m of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.     

Behaviour of connectin (titin) and nebulin in skinned muscle fibres released after extreme stretch as revealed by immunoelectron microscopy

Summary Stretching of skinned fibres of frog skeletal muscle beyond the overlap of the thin and thick filaments followed by release to resting length results in disorganization of the thin filaments at the A-I junction of a sarcomere (Higuchiet al. (1988) J. Muscl. Res. Cell. Motility 9, 491–8). Immunoelectron microscopic observations showed that the binding sites of antibodies against connectin (titin) returned to the original position after extreme stretch and release but those of anti-nebulin antibodies were largely disorganized. The binding sites of anti-connectin antibodies moved within an I band with the change in sarcomere length, but those of anti-nebulin antibodies did not. Nebulin remained in the I band at extreme stretch. Thus connectin filaments appear to be responsible for maintaining mechanical continuity of a sarcomere and appear to behave independently of thin filaments. It is suggested that nebulin is localized in the I band but not in the A band and is associated with thin filaments but not with the elastic structure of myofibrils.