Biological responses to mechanical stress require strain-sensing molecules, whose mechanically induced conformational changes are relayed to signaling cascades mediating changes in cell and tissue properties. In vertebrate muscle, the giant elastic protein titin is involved in strain sensing via its C-terminal kinase domain (TK) at the sarcomeric M-band and contributes to the adaptation of muscle in response to changes in mechanical strain. TK is regulated in a unique dual autoinhibition mechanism by a C-terminal regulatory tail, blocking the ATP binding site, and tyrosine autoinhibition of the catalytic base. For access to the ATP binding site and phosphorylation of the autoinhibitory tyrosine, the C-terminal autoinhibitory tail needs to be removed. Here, we use AFM-based single-molecule force spectroscopy, molecular dynamics simulations, and enzymatics to study the conformational changes during strain-induced activation of human TK. We show that mechanical strain activates ATP binding before unfolding of the structural titin domains, and that TK can thus act as a biological force sensor. Furthermore, we identify the steps in which the autoinhibition of TK is mechanically relieved at low forces, leading to binding of the cosubstrate ATP and priming the enzyme for subsequent autophosphorylation and substrate turnover.

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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.

Traumatic brain injury is a leading cause of mortality and morbidity among young people. For the last couple of decades, it was believed that excess stimulation of brain receptors for the excitatory neurotransmitter glutamate was a major cause of delayed neuronal death after head injury, and several major clinical trials in severely head injured patients used blockers of the glutamate N-methyl-D-aspartate (NMDA) receptor. All of these trials failed to show efficacy. Using a mouse model of traumatic brain injury and quantitative autoradiography of the activity-dependent NMDA receptor antagonist MK801, we show that hyperactivation of glutamate NMDA receptors after injury is short-lived (<1 h) and is followed by a profound and long-lasting (>7 days) loss of function. Furthermore, stimulation of NMDA receptors by NMDA 24 and 48 h postinjury produced a significant attenuation of neurological deficits (blocked by coadministration of MK801) and restored cognitive performance 14 days postinjury. These results provide the underlying mechanism for the well known but heretofore unexplained short therapeutic window of glutamate antagonists after brain injury and support a pharmacological intervention with a relatively long (>24 h) time window easily attainable for treatment of human accidental head injury.H ead trauma is a leading cause of mortality and morbidity among young people in the western world (1). Traumatic and ischemic brain injury triggers a large, transient increase in excitatory amino acid transmitter efflux in the brain of experimental animals and human subjects (2-6). Glutamate activation of the N-methyl-D-aspartate (NMDA) receptor (NMDAR), which is a ligand-gated ion (calcium and sodium) channel, results in channel opening and ion influx into the cell. It has been suggested that this process mediates delayed excitotoxic neuronal death after brain ischemia and trauma (7,8), although the concept is not universally accepted (9, 10). Support for the involvement of NMDAR activation in neuronal death after brain injury has come from numerous studies showing that NMDAR antagonists reduce cell death and improve outcome in animal models of traumatic brain injury (TBI) and stroke. NMDAR antagonists appear to be most efficacious when given before or immediately after the insult and lose efficacy if administered Ͼ30-60 min postinjury (11)(12)(13)(14)(15).Microdialysis studies of extracellular glutamate in human TBI and stroke patients suggested that the increase in glutamate in humans is more sustained [6 h to several days (7, 8)] than in rodents, where it only lasts minutes (2-6). This result may have contributed to a decision to administer NMDAR antagonists in clinical trials of head injury for several days rather than once after severe nonpenetrating injury. All clinical trials of NMDAR antagonists to date failed to show efficacy. Furthermore, some of these trials had to be stopped prematurely because of increases in mortality and morbidity in the drug arm of the stroke trials (16-18), suggesting that prolonged bloc...