The contractile units of striated muscle, the sarcomeres, comprise the thick (myosin) and thin (actin) filaments mediating active contraction and the titin filaments determining "passive" elasticity. We hypothesized that titin may be more active in muscle contraction by directly modulating thick-filament properties. We used single-myofibril mechanical measurements and atomic force microscopy of individual sarcomeres to quantify the effects of sarcomere strain and titin spring length on both the inter-filament lattice spacing and the lateral stiffness of the actin-myosin overlap zone (A-band). We found that strain reduced the lattice spacing similarly in sarcomeres with stiff (rabbit psoas) or compliant titin (rabbit diaphragm), but increased A-band lateral stiffness much more in psoas than in diaphragm. The strain-induced alterations in A-band stiffness that occur independently of lattice spacing effects may be due to titin stiffness-sensing by A-band proteins. This mechanosensitivity could play a role in the physiologically important phenomenon of length-dependent activation of striated muscle.

In myocytes, small heat shock proteins (sHSPs) are preferentially translocated under stress to the sarcomeres. The functional implications of this translocation are poorly understood. We show here that HSP27 and αB-crystallin associated with immunoglobulin-like (Ig) domain-containing regions, but not the disordered PEVK domain (titin region rich in proline, glutamate, valine, and lysine), of the titin springs. In sarcomeres, sHSP binding to titin was actin filament independent and promoted by factors that increased titin Ig unfolding, including sarcomere stretch and the expression of stiff titin isoforms. Titin spring elements behaved predominantly as monomers in vitro. However, unfolded Ig segments aggregated, preferentially under acidic conditions, and αB-crystallin prevented this aggregation. Disordered regions did not aggregate. Promoting titin Ig unfolding in cardiomyocytes caused elevated stiffness under acidic stress, but HSP27 or αB-crystallin suppressed this stiffening. In diseased human muscle and heart, both sHSPs associated with the titin springs, in contrast to the cytosolic/Z-disk localization seen in healthy muscle/heart. We conclude that aggregation of unfolded titin Ig domains stiffens myocytes and that sHSPs translocate to these domains to prevent this aggregation.

The titin springs of muscle sarcomeres largely determine tensile muscle stress. However, titin is incorporated into sarcomeric I-bands not strictly in parallel with the sarcomere axis, being anchored to actin at Z-disks and myosin at A-bands. On tensile sarcomere strain, which stretches the titin springs, a force component arises longitudinally, but somewhat also laterally. The latter component may contribute to increased sarcomeric transversal stiffness, decreased lateral myofilament spacing, and length-dependent activation of stretched muscle. We aimed to directly test by atomic force microscopy (AFM) force mapping whether the titin springs contribute to sarcomeric transversal stiffness and how a titin-based lateral force component would compare with other possible sources of lateral stiffness, such as osmotic forces. Single myofibrils were isolated from rabbit psoas (stiff titin-isoform) or diaphragm (compliant titin-isoform) muscles and placed in physiological buffer under the MFP-3D-BIO AFM (Asylum Research). Force curves (50x50) were performed over a region-of-interest encompassing a whole sarcomere. Force mapping revealed distinct transversal stiffness patterns along psoas and diaphragm sarcomeres, reflecting different Z-disk, I-, A-, and M-band stiffness. A-band transversal stiffness at 25nm indentation was higher in rigor (4.2pN/nm) than in relaxed sarcomeres (0.4pN/nm). Titin digestion by low-dose trypsin decreased rigor but not relaxed stiffness. A-band lateral stiffness did not differ between relaxed psoas and diaphragm sarcomeres at slack length (∼2.2µm) but increased significantly after stretch to ∼3.2µm, albeit more highly in psoas than in diaphragm. Following osmotic compression by 5% dextran, A-band lateral stiffness rose 5-fold, similarly in psoas and diaphragm. We conclude that stiff titin contributes more to transversal stiffness than compliant titin, confirming a role for titin in lateral force generation. However, osmotic forces alike those present in-vivo may laterally compress the sarcomeric lattice to a degree that the titin contribution becomes negligible.

Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. HCM is caused by mutations in sarcomeric genes, but in >40% of patients, the mutation is not yet identified. We hypothesized that FHL1, encoding four-and-a-half-LIM domains 1, could be another disease gene since it has been shown to cause distinct myopathies, sometimes associated with cardiomyopathy. We evaluated 121 HCM patients, devoid of a mutation in known disease genes. We identified three novel variants in FHL1 (c.134delA/K45Sfs, c.459C>A/C153X and c.827G>C/C276S). Whereas the c.459C>A variant was associated with muscle weakness in some patients, the c.134delA and c.827G>C variants were associated with isolated HCM. Gene transfer of the latter variants in C2C12 myoblasts and cardiac myocytes revealed reduced levels of FHL1 mutant proteins, which could be rescued by proteasome inhibition. Contractility measurements after adeno-associated virus transduction in rat-engineered heart tissue (EHT) showed: (i) higher and lower forces of contraction with K45Sfs and C276S, respectively, and (ii) prolonged contraction and relaxation with both mutants. All mutants except one activated the fetal hypertrophic gene program in EHT. In conclusion, this study provides evidence for FHL1 to be a novel gene for isolated HCM. These data, together with previous findings of proteasome impairment in HCM, suggest that FHL1 mutant proteins may act as poison peptides, leading to hypertrophy, diastolic dysfunction and/or altered contractility, all features of HCM.

The I-band portion of the giant muscle-protein titin is elastic but detailed knowledge of titin arrangement and interactions in this region is lacking. We determined whether or not select domains from this region can associate with one another, as observed for titin's "distal" immunoglobulin domain-like (Ig-) region. Using sedimentation-velocity centrifugation and size-exclusion chromatography of purified human titin fragments, we detected a monomeric state of the titin N2B-domain or the N2B-unique sequence contained therein. The constitutively expressed PEVK-domain and the Ig-only fragment I9-I12 also behaved as monomers in-vitro, whereas the N2A-segment showed both dimeric and (to a lesser degree) monomeric behavior. Yeast-2-hybrid and GST-pulldown interaction tests demonstrated that both the N2B and the N2A domain, but not the PEVK-fragment, bind to the small heat-shock proteins (sHSPs), alphaB-crystallin and HSP27. These interactions were confirmed on isolated human cardiac or rabbit psoas myofibrils incubated ex-vivo with either sHSP type. Stretching myofibrils to promote titin-domain unfolding increased the propensity of sHSPs to bind I-band titin. Protein unfolding in-vitro by 8M urea caused aggregation of the N2A-segment under acidic conditions (pH6.7), but not at pH7.2. Importantly, alphaB-crystallin prevented the aggregation of the N2A-segment at pH6.7 partially (molar ratio of N2A:alphaB-crystallin, 1:5) or fully (ratio, 1:10). In cultured neonatal rat cardiomyocytes, both sHSPs translocated from the cytosol to the sarcomeric Z-disk/I-band region on inhibition of the proteasome. In adult rat cardiomyocytes both sHSPs associated with I-band titin already under normal culture conditions, suggesting an age-related increase in chaperoning activity. We conclude that titin filaments may run through the elastic segment mainly as monomers; sHSPs associate with elastic titin domains under various stress conditions; and sHSPs are able to protect I-band titin regions from aggregation under adverse intracellular circumstances.

Titin's elastic I-band region is a hotspot for protein-protein interactions and important for myocyte extensibility and passive stiffness. The titin springs can be phosphorylated at the N2B-unique sequence (N2Bus) by protein kinases (PK)A or PKG and at the PEVK-domain by PKCalpha, which affects passive stiffness. We searched for protein phosphatase(s) acting on the N2Bus. A yeast-2-hybrid (Y2H) screen with the human N2Bus ("bait") and a human heart cDNA library ("prey") detected the catalytic domain of the serine/threonine protein phosphatase-5 (PP5) as a binding partner of the cardiac N2Bus. The interaction was confirmed in forced Y2H screens with the N2Bus and full-length PP5 or PP5 catalytic subunit (PP5c), and also in GST-pulldown assays. In cardiomyocytes, PP5 was mainly in the cytosol but also in the nucleus and at the sarcomeric I-bands. Recombinant PP5 was found by autoradiography to dephosphorylate recombinant, PKG-phosphorylated, N2Bus, and PP5 bound more strongly to (PKA-/PKG-)phosphorylated N2Bus than to non-phosphorylated N2Bus. Phosphorylation of titin could be reduced in human heart tissue treated ex vivo with recombinant PP5c, an effect detected with phospho-N2Bus (S469) specific antibodies. A transgenic mouse model with PP5 overexpression revealed reduced cardiac titin phosphorylation levels compared to wildtype mouse hearts. PP5 expression was elevated in human heart failure, while titin phosphorylation was depressed. PP5c treatment of enzymatically skinned single human cardiomyocytes significantly reduced passive stiffness, which can potentially be explained by dephosphorylation of the PEVK-domain adjacent to the N2Bus. In conclusion, PP5 is a novel binding partner of cardiac titin at the N2Bus and acts to reduce passive myocyte stiffness by dephosphorylating the titin springs. PP5 may participate in mechanical signaling pathways converging on the titin springs.

Rationale: Telethonin (also known as titin-cap or t-cap ) is a 19-kDa Z-disk protein with a unique β-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin's in vivo function. Objective: Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation. Methods and Results: By using a variety of different genetically altered animal models and biophysical experiments we show that contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin–titin cross-links via α-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the proapoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis (“mechanoptosis”). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect that may contribute to enhanced rates of apoptosis found in these hearts. Conclusions: Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect that may also play a role in human heart failure.

The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by PKG activation. We used back-phosphorylation assays to determine whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. Using site-directed mutagenesis, serine residue S469 near the COOH terminus of the cardiac N2-B-unique sequence (N2-Bus) was identified as a PKG and PKA phosphorylation site. To address the mechanism of the PKG effect on titin stiffness, single-molecule atomic force microscopy force-extension experiments were performed on engineered N2-Bus-containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus; phosphorylation of other titin sites could affect protein-protein interactions. The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure.