Structure determination and analysis of titin A-band fibronectin type III domains provides insights for disease-linked variants and protein oligomerisation.

Titin is the largest protein found in nature and spans half a sarcomere in vertebrate striated muscle. The protein has multiple functions, including in the organisation of the thick filament and acting as a molecular spring during the muscle contraction cycle. Missense variants in titin have been linked to both cardiac and skeletal myopathies. Titin is primarily composed of tandem repeats of immunoglobulin and fibronectin type III (Fn3) domains in a variety of repeat patterns; however, the vast majority of these domains have not had their high-resolution structure determined experimentally. Here, we present the crystal structures of seven wild type titin Fn3 domains and two harbouring rare missense variants reported in hypertrophic cardiomyopathy (HCM) patients. All domains present the typical Fn3 fold, with the domains harbouring variants reported in HCM patients retaining the wild-type conformation. The effect on domain folding and stability were assessed for five rare missense variants found in HCM patients: four caused thermal destabilization of between 7 and 13 °C and one prevented the folding of its domain. The structures also allowed us to locate the positions of residues whose mutations have been linked to congenital myopathies and rationalise how they convey their deleterious effects. We find no evidence of physiological homodimer formation, excluding one hypothesised mechanism as to how titin variants could exert pathological effects.

Obscurin Rho GEF domains are phosphorylated by MST-family kinases but do not exhibit nucleotide exchange factor activity towards Rho GTPases in vitro.

Obscurin is a giant muscle protein (>800 kDa) featuring multiple signalling domains, including an SH3-DH-PH domain triplet from the Trio-subfamily of guanosine nucleotide exchange factors (GEFs). While previous research suggests that these domains can activate the small GTPases RhoA and RhoQ in cells, in vitro characterization of these interactions using biophysical techniques has been hampered by the intrinsic instability of obscurin GEF domains. To study substrate specificity, mechanism and regulation of obscurin GEF function by individual domains, we successfully optimized recombinant production of obscurin GEF domains and found that MST-family kinases phosphorylate the obscurin DH domain at Thr5798. Despite extensive testing of multiple GEF domain fragments, we did not detect any nucleotide exchange activity in vitro against 9 representative small GTPases. Bioinformatic analyses show that obscurin differs from other Trio-subfamily GEFs in several important aspects. While further research is necessary to evaluate obscurin GEF activity in vivo, our results indicate that obscurin has atypical GEF domains that, if catalytically active at all, are subject to complex regulation.

Molecular noise filtering in the ß-adrenergic signaling network by phospholamban pentamers.

Phospholamban (PLN) is an important regulator of cardiac calcium handling due to its ability to inhibit the calcium ATPase SERCA. ß-Adrenergic stimulation reverses SERCA inhibition via PLN phosphorylation and facilitates fast calcium reuptake. PLN also forms pentamers whose physiological significance has remained elusive. Using mathematical modeling combined with biochemical and cell biological experiments, we show that pentamers regulate both the dynamics and steady-state levels of monomer phosphorylation. Substrate competition by pentamers and a feed-forward loop involving inhibitor-1 can delay monomer phosphorylation by protein kinase A (PKA), whereas cooperative pentamer dephosphorylation enables bistable PLN steady-state phosphorylation. Simulations show that phosphorylation delay and bistability act as complementary filters that reduce the effect of random fluctuations in PKA activity, thereby ensuring consistent monomer phosphorylation and SERCA activity despite noisy upstream signals. Preliminary analyses suggest that the PLN mutation R14del could impair noise filtering, offering a new perspective on how this mutation causes cardiac arrhythmias.

High Throughput Screen Identifies Small Molecule Effectors That Modulate Thin Filament Activation in Cardiac Muscle.

Current therapeutic interventions for both heart disease and heart failure are largely insufficient and associated with undesired side effects. Biomedical research has emphasized the role of sarcomeric protein function for the normal performance and energy efficiency of the heart, suggesting that directly targeting the contractile myofilaments themselves using small molecule effectors has therapeutic potential and will likely result in greater drug efficacy and selectivity. In this study, we developed a robust and highly reproducible fluorescence polarization-based high throughput screening (HTS) assay that directly targets the calcium-dependent interaction between cardiac troponin C (cTnC) and the switch region of cardiac troponin I (cTnISP), with the aim of identifying small molecule effectors of the cardiac thin filament activation pathway. We screened a commercially available small molecule library and identified several hit compounds with both inhibitory and activating effects. We used a range of biophysical and biochemical methods to characterize hit compounds and identified fingolimod, a sphingosin-1-phosphate receptor modulator, as a new troponin-based small molecule effector. Fingolimod decreased the ATPase activity and calcium sensitivity of demembranated cardiac muscle fibers in a dose-dependent manner, suggesting that the compound acts as a calcium desensitizer. We investigated fingolimod's mechanism of action using a combination of computational studies, biophysical methods, and synthetic chemistry, showing that fingolimod bound to cTnC repels cTnISP via mainly electrostatic repulsion of its positively charged tail. These results suggest that fingolimod is a potential new lead compound/scaffold for the development of troponin-directed heart failure therapeutics.

Nerve growth factor plays a role in the neurotherapeutic effect of a CD45+ pan-hematopoietic subpopulation derived from human umbilical cord blood in a traumatic brain injury model.

BACKGROUND AIMS: Human umbilical cord blood (HUCB) is an important source of stem cells for therapy of hematopoietic disorders and is a potential therapy for various neurological disorders, including traumatic brain injury (TBI). The expression of nerve growth factor (NGF) and its receptors TrkA, p75NTR and α9ß1 integrin on an HUCB CD45+ pan-hematopoietic subpopulation was investigated in the context of its neurotherapeutic potential after TBI. METHODS: NGF and its receptors were detected on CD45+ cells by reverse transcriptase polymerase chain reaction, flow cytometry analysis and confocal microscopy. CD45+ cells were stimulated by TBI brain extracts, and NGF levels were measured by enzyme-linked immunosorbent assay. TBI mice were divided into six groups for xenogeneic intravenous transplantation, 1 day post-trauma, with 1 × 106 CD45+ cells untreated or treated with the anti-NGF neutralizing antibody K252a, a TrkA antagonist; VLO5, an α9ß1 disintegrin; or negative (vehicle) and positive (NGF) controls. RESULTS: The HUCB CD45+ subpopulation constitutively expresses NGF and its receptors, mainly TrkA and p75NTR and minor levels of α9ß1. In vitro experiments provided evidence that trauma-related mediators from brain extracts of TBI mice induced release of NGF from HUCB CD45+ cell cultures. HUCB CD45+ cells induced a neurotherapeutic effect in TBI mice, abrogated by cell treatment with either anti-NGF antibody or K252a, but not VLO5. CONCLUSIONS: These findings strengthen the role of NGF and its TrkA receptor in the HUCB CD45+ subpopulation's neurotherapeutic effect. The presence of neurotrophin receptors in the HUCB CD45+ pan-hematopoietic subpopulation may explain the neuroprotective effect of cord blood in therapy of a variety of neurological disorders.

Increasing evidence of mechanical force as a functional regulator in smooth muscle myosin light chain kinase.

Mechanosensitive proteins are key players in cytoskeletal remodeling, muscle contraction, cell migration and differentiation processes. Smooth muscle myosin light chain kinase (smMLCK) is a member of a diverse group of serine/threonine kinases that feature cytoskeletal association. Its catalytic activity is triggered by a conformational change upon Ca2+/calmodulin (Ca2+/CaM) binding. Due to its significant homology with the force-activated titin kinase, smMLCK is suspected to be also regulatable by mechanical stress. In this study, a CaM-independent activation mechanism for smMLCK by mechanical release of the inhibitory elements is investigated via high throughput AFM single-molecule force spectroscopy. The characteristic pattern of transitions between different smMLCK states and their variations in the presence of different substrates and ligands are presented. Interaction between kinase domain and regulatory light chain (RLC) substrate is identified in the absence of CaM, indicating restored substrate-binding capability due to mechanically induced removal of the auto-inhibitory regulatory region.

Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy.

Strep-Tactin, an engineered form of streptavidin, binds avidly to the genetically encoded peptide Strep-tag II in a manner comparable to streptavidin binding to biotin. These interactions have been used in protein purification and detection applications. However, in single-molecule studies, for example using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), the tetravalency of these systems impedes the measurement of monodispersed data. Here, we introduce a monovalent form of Strep-Tactin that harbours a unique binding site for Strep-tag II and a single cysteine that allows Strep-Tactin to specifically attach to the atomic force microscope cantilever and form a consistent pulling geometry to obtain homogeneous rupture data. Using AFM-SMFS, the mechanical properties of the interaction between Strep-tag II and monovalent Strep-Tactin were characterized. Rupture forces comparable to biotin:streptavidin unbinding were observed. Using titin kinase and green fluorescent protein, we show that monovalent Strep-Tactin is generally applicable to protein unfolding experiments. We expect monovalent Strep-Tactin to be a reliable anchoring tool for a range of single-molecule studies.

Palmitoyl Serine: An Endogenous Neuroprotective Endocannabinoid-Like Entity After Traumatic Brain Injury.

The endocannabinoid (eCB) system helps recovery following traumatic brain injury (TBI). Treatment with 2-arachidonoylglycerol (2-AG), a cerebral eCB ligand, was found to ameliorate the secondary damage. Interestingly, the fatty acid amino acid amide (FAAA) N-arachidonoyl-L-serine (AraS) exerts similar eCB dependent neuroprotective. The present study aimed to investigate the effects of the FAAA palmitoyl-serine (PalmS) following TBI. We utilized the TBI model in mice to examine the therapeutic potential of PalmS, injected 1 h following closed head injury (CHI). We followed the functional recovery of the injured mice for 28 days post-CHI, and evaluated cognitive and motor function, lesion volume, cytokines levels, molecular signaling, and infarct volume at different time points after CHI. PalmS treatment led to a significant improvement of the neurobehavioral outcome of the treated mice, compared with vehicle. This effect was attenuated in the presence of eCBR antagonists and in CB2-/- mice, compared to controls. Unexpectedly, treatment with PalmS did not affect edema and lesion volume, TNFα and IL1ß levels, anti-apoptotic mechanisms, nor did it exert improvement in cognitive and motor function. Finally, co-administration of PalmS, AraS and 2-AG, did not enhance the effect of the individual drugs. We suggest that the neuroprotective action of PalmS is mediated by indirect activation of the eCB receptors following TBI. One such mechanism may involve receptor palmitoylation which has been reported to result in structural stabilization of the receptors and to an increase in their activity. Further research is required in order to establish this assumption.

Angiotensin receptor type 2 activation induces neuroprotection and neurogenesis after traumatic brain injury.

Angiotensin II receptor type 2 (AT(2)) agonists have been shown to limit brain ischemic insult and to improve its outcome. The activation of AT(2) was also linked to induced neuronal proliferation and differentiation in vitro. In this study, we examined the therapeutic potential of AT(2) activation following traumatic brain injury (TBI) in mice, a brain pathology that displays ischemia-like secondary damages. The AT(2) agonist CGP42112A was continuously infused immediately after closed head injury (CHI) for 3 days. We have followed the functional recovery of the injured mice for 35 days post-CHI, and evaluated cognitive function, lesion volume, molecular signaling, and neurogenesis at different time points after the impact. We found dose-dependent improvement in functional recovery and cognitive performance after CGP42112A treatment that was accompanied by reduced lesion volume and induced neurogenesis in the neurogenic niches of the brain and also in the injury region. At the cellular/molecular level, CGP42112A induced early activation of neuroprotective kinases protein kinase B (Akt) and extracellular-regulated kinases ½ (ERK½), and the neurotrophins nerve growth factor and brain-derived neurotrophic factor; all were blocked by treatment with the AT(2) antagonist PD123319. Our results suggest that AT(2) activation after TBI promotes neuroprotection and neurogenesis, and may be a novel approach for the development of new drugs to treat victims of TBI.

Neuroprotection after traumatic brain injury in heat-acclimated mice involves induced neurogenesis and activation of angiotensin receptor type 2 signaling.

Long-term exposure of mice to mild heat (34°C±1°C) confers neuroprotection against traumatic brain injury (TBI); however, the underling mechanisms are not fully understood. Heat acclimation (HA) increases hypothalamic angiotensin II receptor type 2 (AT2) expression and hypothalamic neurogenesis. Accumulating data suggest that activation of the brain AT2 receptor confers protection against several types of brain pathologies, including ischemia, a hallmark of the secondary injury occurring following TBI. As AT2 activates the same pro-survival pathways involved in HA-mediated neuroprotection (e.g., Akt phosphorylation, hypoxia-inducible factor 1α (HIF-1α), and brain-derived neurotrophic factor (BDNF)), we examined the role of AT2 in HA-mediated neuroprotection after TBI. Using an AT2-specific antagonist PD123319, we found that the improvements in motor and cognitive recovery as well as reduced lesion volume and neurogenesis seen in HA mice were all diminished by AT2 inhibition, whereas no significant alternations were observed in control mice. We also found that nerve growth factor/tropomyosin-related kinase receptor A (TrkA), BDNF/TrkB, and HIF-1α pathways are upregulated by HA and inhibited on PD123319 administration, suggesting that these pathways play a role in AT2 signaling in HA mice. In conclusion, AT2 is involved in HA-mediated neuroprotection, and AT2 activation may be protective and should be considered a novel drug target in the treatment of TBI patients.

Neurotherapeutic effect of cord blood derived CD45+ hematopoietic cells in mice after traumatic brain injury.

Treatment of traumatic brain injury (TBI) is still an unmet need. Cell therapy by human umbilical cord blood (HUCB) has shown promising results in animal models of TBI and is under evaluation in clinical trials. HUCB contains different cell populations but to date, only mesenchymal stem cells have been evaluated for therapy of TBI. Here we present the neurotherapeutic effect, as evaluated by neurological score, using a single dose of HUCB-derived mononuclear cells (MNCs) upon intravenous (IV) administration one day post-trauma in a mouse model of closed head injury (CHI). Delayed (eight days post-trauma) intracerebroventricular administration of MNCs showed improved neurobehavioral deficits thereby extending the therapeutic window for treating TBI. Further, we demonstrated for the first time that HUCB-derived pan-hematopoietic CD45 positive (CD45(+)) cells, isolated by magnetic sorting and characterized by expression of CD45 and CD11b markers (96-99%), improved the neurobehavioral deficits upon IV administration, which persisted for 35 days. The therapeutic effect was in a direct correlation to a reduction in the lesion volume and decreased by pre-treatment of the cells with anti-human-CD45 antibody. At the site of brain injury, 1.5-2 h after transplantation, HUCB-derived cells were identified by near infrared scanning and immunohistochemistry using anti-human-CD45 and anti-human-nuclei antibodies. Nerve growth factor and vascular endothelial growth factor levels were differentially expressed in both ipsilateral and contralateral brain hemispheres, thirty-five days after CHI, measured by enzyme-linked immunosorbent assay. These findings indicate the neurotherapeutic potential of HUCB-derived CD45(+) cell population in a mouse model of TBI and propose their use in the clinical setting of human TBI.

Inosine improves functional recovery after experimental traumatic brain injury.

Despite years of research, no effective therapy is yet available for the treatment of traumatic brain injury (TBI). The most prevalent and debilitating features in survivors of TBI are cognitive deficits and motor dysfunction. A potential therapeutic method for improving the function of patients following TBI would be to restore, at least in part, plasticity to the CNS in a controlled way that would allow for the formation of compensatory circuits. Inosine, a naturally occurring purine nucleoside, has been shown to promote axon collateral growth in the corticospinal tract (CST) following stroke and focal TBI. In the present study, we investigated the effects of inosine on motor and cognitive deficits, CST sprouting, and expression of synaptic proteins in an experimental model of closed head injury (CHI). Treatment with inosine (100 mg/kg i.p. at 1, 24 and 48 h following CHI) improved outcome after TBI, significantly decreasing the neurological severity score (NSS, p<0.04 vs. saline), an aggregate measure of performance on several tasks. It improved non-spatial cognitive performance (object recognition, p<0.016 vs. saline) but had little effect on sensorimotor coordination (rotarod) and spatial cognitive functions (Y-maze). Inosine did not affect CST sprouting in the lumbar spinal cord but did restore levels of the growth-associated protein GAP-43 in the hippocampus, though not in the cerebral cortex. Our results suggest that inosine may improve functional outcome after TBI.

Recessive TTN truncating mutations define novel forms of core myopathy with heart disease.

Core myopathies (CM), the main non-dystrophic myopathies in childhood, remain genetically unexplained in many cases. Heart disease is not considered part of the typical CM spectrum. No congenital heart defect has been reported, and childhood-onset cardiomyopathy has been documented in only two CM families with homozygous mutations of the TTN gene. TTN encodes titin, a giant protein of striated muscles. Recently, heterozygous TTN truncating mutations have also been reported as a major cause of dominant dilated cardiomyopathy. However, relatively few TTN mutations and phenotypes are known, and titin pathophysiological role in cardiac and skeletal muscle conditions is incompletely understood. We analyzed a series of 23 families with congenital CM and primary heart disease using TTN M-line-targeted sequencing followed in selected patients by whole-exome sequencing and functional studies. We identified seven novel homozygous or compound heterozygous TTN mutations (five in the M-line, five truncating) in 17% patients. Heterozygous parents were healthy. Phenotype analysis identified four novel titinopathies, including cardiac septal defects, left ventricular non-compaction, Emery-Dreifuss muscular dystrophy or arthrogryposis. Additionally, in vitro studies documented the first-reported absence of a functional titin kinase domain in humans, leading to a severe antenatal phenotype. We establish that CM are associated with a large range of heart conditions of which TTN mutations are a major cause, thereby expanding the TTN mutational and phenotypic spectrum. Additionally, our results suggest titin kinase implication in cardiac morphogenesis and demonstrate that heterozygous TTN truncating mutations may not manifest unless associated with a second mutation, reassessing the paradigm of their dominant expression.

The role and dynamics of ß-catenin in precondition induced neuroprotection after traumatic brain injury.

Preconditioning via heat acclimation (34°C 30 d) results in neuroprotection from traumatic brain injury due to constitutive as well as dynamic changes triggered by the trauma. Among these changes is Akt phosphorylation, which decreases apoptosis and induces HIF1α. In the present study we investigated the Akt downstream GSK3ß/ß-catenin pathway and focused on post injury alternations of ß catenin and its impact on the cellular response in preconditioned heat acclimated mice. We found that the reduction in motor disability is accompanied with attenuation of depressive like behavior in heat acclimated mice that correlates with the GSK3ß phosphorylation state. Concomitantly, a robust ß catenin phosphorylation is not followed by its degradation, or by reduced nuclear accumulation. Enhanced tyrosine phosphorylation of ß catenin in the injured area weakens the ß catenin-N cadherin complex. Membrane ß catenin is transiently reduced in heat acclimated mice and its recovery 7 days post TBI is accompanied by induction of the synaptic marker synaptophysin. We suggest a set of cellular events following traumatic brain injury in heat acclimated mice that causes ß catenin to participate in cell-cell adhesion alternations rather than in Wnt signaling. These events may contribute to synaptogenesis and the improved motor and cognitive abilities seen heat acclimated mice after traumatic brain injury.

N-arachidonoyl-L-serine (AraS) possesses proneurogenic properties in vitro and in vivo after traumatic brain injury.

N-arachidonoyl-L-serine (AraS) is a novel neuroprotective endocannabinoid. We aimed to test the effects of exogenous AraS on neurogenesis after traumatic brain injury (TBI). The effects of AraS on neural progenitor cells (NPC) proliferation, survival, and differentiation were examined in vitro. Next, mice underwent TBI and were treated with AraS or vehicle. Lesion volumes and clinical outcome were evaluated and the effects on neurogenesis were tested using immunohistochemistry. Treatment with AraS led to a dose-dependent increase in neurosphere size without affecting cell survival. These effects were partially reversed by CB1, CB2, or TRPV1 antagonists. AraS significantly reduced the differentiation of NPC in vitro to astrocytes or neurons and led to a 2.5-fold increase in expression of the NPC marker nestin. Similar effects were observed in vivo in mice treated with AraS 7 days after TBI. These effects were accompanied by a reduction in lesion volume and an improvement in neurobehavioral function compared with controls. AraS increases proliferation of NPCs in vitro in cannabinoid-receptor-mediated mechanisms and maintains NPC in an undifferentiated state in vitro and in vivo. Moreover, although given at 7 days post injury, these effects are associated with significant neuroprotective effects leading to an improvement in neurobehavioral functions.

Hypoxia-inducible factor 1 is essential for spontaneous recovery from traumatic brain injury and is a key mediator of heat acclimation induced neuroprotection.

Heat acclimation (HA), a well-established preconditioning model, confers neuroprotection in rodent models of traumatic brain injury (TBI). It increases neuroprotective factors, among them is hypoxia-inducible factor 1α (HIF-1α), which is important in the response to postinjury ischemia. However, little is known about the role of HIF-1α in TBI and its contribution to the establishment of the HA protecting phenotype. Therefore, we aimed to explore HIF-1α role in TBI defense mechanisms as well as in HA-induced neuroprotection. Acriflavine was used to inhibit HIF-1 in injured normothermic (NT) or HA mice. After TBI, we evaluated motor function recovery, lesion volume, edema formation, and body temperature as well as HIF-1 downstream transcription targets, such as glucose transporter 1 (GLUT1), vascular endothelial growth factor, and aquaporin 4. We found that HIF-1 inhibition resulted in deterioration of motor function, increased lesion volume, hypothermia, and reduced edema formation. All these parameters were significantly different in the HA mice. Western blot analysis and enzyme-linked immunosorbent assay showed reduced levels of all HIF-1 downstream targets in HA mice, however, only GLUT1 was downregulated in NT mice. We conclude that HIF-1 is a key mediator in both spontaneous recovery and HA-induced neuroprotection after TBI.

Carvacrol together with TRPC1 elimination improve functional recovery after traumatic brain injury in mice.

Death of Central Nervous System (CNS) neurons following traumatic brain injury (TBI) is a complex process arising from a combination of factors, many of which are still unknown. It has been found that inhibition of transient receptor potential (TRP) channels constitutes an effective strategy for preventing death of CNS neurons following TBI. TRP channels are classified into seven related subfamilies, most of which are Ca(2+) permeable and involved in many cellular functions, including neuronal cell death. We hypothesized that TRP channels of the TRPC subfamily may be involved in post-TBI pathophysiology and that the compound 5-isopropyl-2-methylphenol (carvacrol), by inhibition of TRP channels, may exert neuroprotective effect after TBI. To test these suppositions, carvacrol was given to mice after TBI and its effect on their functional recovery was followed for several weeks. Our results show that neurological recovery after TBI was significantly enhanced by application of carvacrol. To better define the type of the specific channel involved, the effect of carvacrol on the extent and speed of recovery after TBI was compared among mice lacking TRPC1, TRPC3, or TRPC5, relative to wild type controls. We found that neurological recovery after TBI was significantly enhanced by combining carvacrol with TRPC1 elimination, but not by the absence of TRPC3 or TRPC5, showing a synergistic effect between carvacrol application and TRPC1 elimination. We conclude that TRPC1-sensitive mechanisms are involved in TBI pathology, and that inhibition of this channel by carvacrol enhances recovery and should be considered for further studies in animal models and humans.

The function of the M-line protein obscurin in controlling the symmetry of the sarcomere in the flight muscle of Drosophila.

Obscurin (also known as Unc-89 in Drosophila) is a large modular protein in the M-line of Drosophila muscles. Drosophila obscurin is similar to the nematode protein UNC-89. Four isoforms are found in the muscles of adult flies: two in the indirect flight muscle (IFM) and two in other muscles. A fifth isoform is found in the larva. The larger IFM isoform has all the domains that were predicted from the gene sequence. Obscurin is in the M-line throughout development of the embryo, larva and pupa. Using P-element mutant flies and RNAi knockdown flies, we have investigated the effect of decreased obscurin expression on the structure of the sarcomere. Embryos, larvae and pupae developed normally. In the pupa, however, the IFM was affected. Although the Z-disc was normal, the H-zone was misaligned. Adults were unable to fly and the structure of the IFM was irregular: M-lines were missing and H-zones misplaced or absent. Isolated thick filaments were asymmetrical, with bare zones that were shifted away from the middle of the filaments. In the sarcomere, the length and polarity of thin filaments depends on the symmetry of adjacent thick filaments; shifted bare zones resulted in abnormally long or short thin filaments. We conclude that obscurin in the IFM is necessary for the development of a symmetrical sarcomere in Drosophila IFM.

The sarcomeric cytoskeleton as a target for pharmacological intervention.

Many diseases of heart and skeletal muscle, from heart failure to muscle atrophy, pose unmet needs for specific and effective treatments. Recent advances suggest that sarcomeres, the smallest contractile units of heart and skeletal muscles, can be viable pharmacological targets. In sarcomeres, the contractile actin and myosin filaments are organised by a network of proteins combining structural and signalling functions, forming the sarcomeric cytoskeleton. This includes the giant proteins titin, obscurin and nebulin, which contain protein-binding sites along with signalling domains such as protein kinase, Rho activator, and Src-homology domains. These signalling domains have recently been implicated in sarcomere assembly, and the regulation of muscle contractile and metabolic adaptation. Although many functions of sarcomeric proteins remain to be discovered, their potential as pharmacological targets is now emerging. Here, we will review recent insight into the physiological and pathological signalling functions of sarcomeric cytoskeletal proteins and discuss new aspects and strategies in skeletal muscle signalling, pathomechanisms and therapy.