Missense mutations in the central domains of cardiac myosin binding protein-C and their potential contribution to hypertrophic cardiomyopathy.

Myosin binding protein-C (MyBP-C) is a multidomain protein that regulates muscle contraction. Mutations in MYBPC3, the gene encoding for the cardiac variant (henceforth called cMyBP-C), are amongst the most frequent causes of hypertrophic cardiomyopathy. Most mutations lead to a truncated version of cMyBP-C, which is most likely unstable. However, missense mutations have also been reported, which tend to cluster in the central domains of the cMyBP-C molecule. This suggests that these central domains are more than just a passive spacer between the better characterized N- and C-terminal domains. Here, we investigated the potential impact of four different missense mutations, E542Q, G596R, N755K, and R820Q, which are spread over the domains C3 to C6, on the function of MyBP-C on both the isolated protein level and in cardiomyocytes in vitro. Effect on domain stability, interaction with thin filaments, binding to myosin, and subcellular localization behavior were assessed. Our studies show that these missense mutations result in slightly different phenotypes at the molecular level, which are mutation specific. The expected functional readout of each mutation provides a valid explanation for why cMyBP-C fails to work as a brake in the regulation of muscle contraction, which eventually results in a hypertrophic cardiomyopathy phenotype. We conclude that missense mutations in cMyBP-C must be evaluated in context of their domain localization, their effect on interaction with thin filaments and myosin, and their effect on protein stability to explain how they lead to disease.

Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, are able to invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis and here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the ß1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. Spatially, sEV were secreted via filopodia-like cellular protrusions at the leading edge of migrating cells. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI. In vitro assays identified collagen VI as playing the key role in cell adhesion and invasion. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate migration and invasion by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction forces to enable VSMC movement within the complex vascular ECM network.

AMPK is a mechano-metabolic sensor linking cell adhesion and mitochondrial dynamics to Myosin-dependent cell migration.

Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton.

Making sense of missense variants in TTN-related congenital myopathies.

Mutations in the sarcomeric protein titin, encoded by TTN, are emerging as a common cause of myopathies. The diagnosis of a TTN-related myopathy is, however, often not straightforward due to clinico-pathological overlap with other myopathies and the prevalence of TTN variants in control populations. Here, we present a combined clinico-pathological, genetic and biophysical approach to the diagnosis of TTN-related myopathies and the pathogenicity ascertainment of TTN missense variants. We identified 30 patients with a primary TTN-related congenital myopathy (CM) and two truncating variants, or one truncating and one missense TTN variant, or homozygous for one TTN missense variant. We found that TTN-related myopathies show considerable overlap with other myopathies but are strongly suggested by a combination of certain clinico-pathological features. Presentation was typically at birth with the clinical course characterized by variable progression of weakness, contractures, scoliosis and respiratory symptoms but sparing of extraocular muscles. Cardiac involvement depended on the variant position. Our biophysical analyses demonstrated that missense mutations associated with CMs are strongly destabilizing and exert their effect when expressed on a truncating background or in homozygosity. We hypothesise that destabilizing TTN missense mutations phenocopy truncating variants and are a key pathogenic feature of recessive titinopathies that might be amenable to therapeutic intervention.

Electrical stimulation applied during differentiation drives the hiPSC-CMs towards a mature cardiac conduction-like cells.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) resemble fetal cardiomyocytes and electrical stimulation (ES) has been explored to mature the differentiated cells. Here, we hypothesize that ES applied at the beginning of the differentiation process, triggers both differentiation of the hiPSC-CMs into a specialized conduction system (CS) phenotype and cell maturation. We applied ES for 15 days starting on day 0 of the differentiation process and found an increased expression of transcription factors and proteins associated with the development and function of CS including Irx3, Nkx2.5 and contactin 2, Hcn4 and Scn5a, respectively. We also found activation of intercalated disc proteins (Nrap and ß-catenin). We detected ES-induced CM maturation as indicated by increased Tnni1 and Tnni3 expression. Confocal micrographs showed a shift towards expression of the gap junction protein connexin 40 in ES hiPSC-CM compared to the more dominant expression of connexin 43 in controls. Finally, analysis of functional parameters revealed that ES hiPSC-CMs exhibited faster action potential (AP) depolarization, longer intracellular Ca2+ transients, and slower AP duration at 90% of repolarization, resembling fast conducting fibers. Altogether, we provided evidence that ES during the differentiation of hiPSC to cardiomyocytes lead to development of cardiac conduction-like cells with more mature cytoarchitecture. Thus, hiPSC-CMs exposed to ES during differentiation can be instrumental to develop CS cells for cardiac disease modelling, screening individual drugs on a precison medicine type platform and support the development of novel therapeutics for arrhythmias.

Individual Limb Muscle Bundles Are Formed through Progressive Steps Orchestrated by Adjacent Connective Tissue Cells during Primary Myogenesis.

Although the factors regulating muscle cell differentiation are well described, we know very little about how differentiating muscle fibers are organized into individual muscle tissue bundles. Disruption of these processes leads to muscle hypoplasia or dysplasia, and replicating these events is vital in tissue engineering approaches. We describe the progressive cellular events that orchestrate the formation of individual limb muscle bundles and directly demonstrate the role of the connective tissue cells that surround muscle precursors in controlling these events. We show how disruption of gene activity within or genetic ablation of connective tissue cells impacts muscle precursors causing disruption of muscle bundle formation and subsequent muscle dysplasia and hypoplasia. We identify several markers of the populations of connective tissue cells that surround muscle precursors and provide a model for how matrix-modifying proteoglycans secreted by these cells may influence muscle bundle formation by effects on the local extracellular matrix (ECM) environment.

SUN1/2 Are Essential for RhoA/ROCK-Regulated Actomyosin Activity in Isolated Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the blood vessel wall. Changes in VSMC actomyosin activity and morphology are prevalent in cardiovascular disease. The actin cytoskeleton actively defines cellular shape and the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, comprised of nesprin and the Sad1p, UNC-84 (SUN)-domain family members SUN1/2, has emerged as a key regulator of actin cytoskeletal organisation. Although SUN1 and SUN2 function is partially redundant, they possess specific functions and LINC complex composition is tailored for cell-type-specific functions. We investigated the importance of SUN1 and SUN2 in regulating actomyosin activity and cell morphology in VSMCs. We demonstrate that siRNA-mediated depletion of either SUN1 or SUN2 altered VSMC spreading and impaired actomyosin activity and RhoA activity. Importantly, these findings were recapitulated using aortic VSMCs isolated from wild-type and SUN2 knockout (SUN2 KO) mice. Inhibition of actomyosin activity, using the rho-associated, coiled-coil-containing protein kinase1/2 (ROCK1/2) inhibitor Y27632 or blebbistatin, reduced SUN2 mobility in the nuclear envelope and decreased the association between SUN2 and lamin A, confirming that SUN2 dynamics and interactions are influenced by actomyosin activity. We propose that the LINC complex exists in a mechanical feedback circuit with RhoA to regulate VSMC actomyosin activity and morphology.

Impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy.

Nemaline myopathy (NM) is a skeletal muscle disorder caused by mutations in genes that are generally involved in muscle contraction, in particular those related to the structure and/or regulation of the thin filament. Many pathogenic aspects of this disease remain largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mouse models and patients with NM: irregular spacing and morphology of nuclei; disrupted nuclear envelope; altered chromatin arrangement; and disorganisation of the cortical cytoskeleton. Impairments in contractility are the primary cause of these nuclear defects. We also establish the role of microtubule organisation in determining nuclear morphology, a phenomenon which is likely to contribute to nuclear alterations in this disease. Our results overlap with findings in diseases caused directly by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to explain some of the hallmarks of NM, including contractile filament disarray, altered mechanical properties and broad transcriptional alterations.

Trauma and Orthopedic Surgery Curriculum Concordance: An Operative Learning Curve Trajectory Perspective.

INTRODUCTION: Certification of completion of training in Trauma and Orthopedic (T&O) surgery in the UK requires the demonstration of operative competence in 12 index procedures, achieved through attaining a level 4 consultant-validated procedure-based assessment (PBA). The aim of this study was to evaluate the trajectory of operative learning curves related to PBA performance with respect to operative caseload and training time. DESIGN: Logbook data from consecutive 24 higher T&O trainees were compared with PBA evaluations to determine the relationship between PBA level, operative experience, training time, and indicative numbers. Learning curve gradients were calculated using trigonometry related to operative experience and training time. SETTING: A higher surgical orthopedic training program serving a single UK (Wales) Deanery. PARTICIPANTS: Twenty-four consecutive higher T&O surgery trainees. RESULTS: Median caseload to achieve level 4 competences ranged from 9 (interquartile range 6-12) for tension band wiring (olecranon or patella) to 101 (61-127) for arthroscopy, with significant variation between all 12 procedures (p < 0.001). Median number of PBAs to reach level 4 competences was 4 (2-6) with significant variation between procedures (p < 0.001). Median learning curve gradients to achieve level 4 competence for tension band wiring were 68.2° and 33.7° by caseload and training time respectively, compared with 12.2° and 45° for arthroscopy, with significant learning curve variation for all procedures related to caseload between first level 3 and first level 4 PBA (p < 0.001). Competence ratios were <1 (median 0.99, range 0.70-2.53) for 6 of the 12 indicative procedures. CONCLUSIONS: Significant learning curve trajectory variance was observed, with discrepancies between indicative operative numbers and the point at which competence was judged achieved. Numbers of index operations to achieve certification of completion of training warrant further examination.

FRCS first pass variance: deanery and specialty contrariety.

BACKGROUND: FRCS exit examination success may be interpreted as a surrogate marker for UK Deanery-related training quality. The aim of this study was to evaluate relative FRCS examination pass rates related to Deanery and Surgical Specialty. METHODS: Joint Committee on Surgical Training-published examination first attempt pass rates were scrutinised for type I higher surgical trainees and outcomes compared related to Deanery and Surgical Specialty. RESULTS: Of 9363 FRCS first attempts, 3974 were successful (42.4%). Median and mean pass rates related to Deanery were 42.1% and 30.7%, respectively, and ranged from 26.7% to 45.6%. Median (range) pass rates by specialty were urology 76.3% (60%-100%), trauma and orthopaedic surgery 74.7% (58.2%-100%), general surgery 70.0% (63.1%-86%), ENT 62.5% (50%-100%), cardiothoracic surgery 50.0% (25%-100%), oral and maxillofacial surgery 50% (40.0%-100%), neurosurgery 50% (22.7%-100%), plastic surgery 47.6% (30.0%-100%) and paediatric surgery 25% (16.7%-100%). Significant variance was observed across all specialties and deaneries (p=0.001). CONCLUSION: As much as threefold variance exists related to FRCS examination first attempt success, trainees should be aware of this spectrum when preferencing deaneries during national selection.

Rural Rotations at Core: Rarefied Exposure or Real Experience?

OBJECTIVE: Surgical rotations involving rural General Hospitals (rGH) are frequently associated with recruitment challenges, partly because of adverse perceptions regarding distances from social support networks and training opportunities. The aim of this study was to determine the outcomes of core surgical training rotations involving rGHs when compared with urban hospitals in a single UK Deanery. DESIGN: Online Intercollegiate Surgical Curriculum Programme portfolios from 163 core surgical trainees (CST) were examined related to postlocation, operative experience, workplace-based assessments, and academic achievement. Of the 163 CSTs, 27 had completed at least 50% of their 2-year training posts at rGHs and were compared with 136 control CSTs completing rotations in urban general and teaching hospitals (uGH). The primary outcome measures were MRCS pass rate and success at national ST3 selection. SETTING: A core surgical training program serving a single UK Deanery. PARTICIPANTS: Consecutive 177 CSTs appointed to a single UK Deanery between 2010 and 2016. RESULTS: Success at MRCS and national ST3 selection were similar for CSTs from rGH vs uGH rotations-MRCS success: 70.4 vs 72.8% (p = 0.816), and ST3 success: 22.2% vs 27.0% (p = 0.811). Median rGH vs uGH curriculum-based outcomes were operative case load: 378 vs 422 (p = 0.300); workplace-based assessments completed: 79 vs 94 (p = 0.499); audits performed: 4 vs 4 (p = 0.966); learned society communications: 1 vs 2 (p = 0.020); and scientific publications: 0 vs 0 (p = 0.478). CONCLUSION: CST rotations including rGHs produced a different spectrum of training experience compared with uGH rotations but overall primary outcomes were similar.

Loss of Protein Kinase Novel 1 (PKN1) is associated with mild systolic and diastolic contractile dysfunction, increased phospholamban Thr17 phosphorylation, and exacerbated ischaemia-reperfusion injury.

Aims: PKN1 is a stress-responsive protein kinase acting downstream of small GTP-binding proteins of the Rho/Rac family. The aim was to determine its role in endogenous cardioprotection. Methods and results: Hearts from PKN1 knockout (KO) or wild type (WT) littermate control mice were perfused in Langendorff mode and subjected to global ischaemia and reperfusion (I/R). Myocardial infarct size was doubled in PKN1 KO hearts compared to WT hearts. PKN1 was basally phosphorylated on the activation loop Thr778 PDK1 target site which was unchanged during I/R. However, phosphorylation of p42/p44-MAPK was decreased in KO hearts at baseline and during I/R. In cultured neonatal rat ventricular cardiomyocytes (NRVM) and NRVM transduced with kinase dead (KD) PKN1 K644R mutant subjected to simulated ischaemia/reperfusion (sI/R), PhosTag® gel analysis showed net dephosphorylation of PKN1 during sI and early R despite Thr778 phosphorylation. siRNA knockdown of PKN1 in NRVM significantly decreased cell survival and increased cell injury by sI/R which was reversed by WT- or KD-PKN1 expression. Confocal immunofluorescence analysis of PKN1 in NRVM showed increased localization to the sarcoplasmic reticulum (SR) during sI. GC-MS/MS and immunoblot analysis of PKN1 immunoprecipitates following sI/R confirmed interaction with CamKIIδ. Co-translocation of PKN1 and CamKIIδ to the SR/membrane fraction during sI correlated with phospholamban (PLB) Thr17 phosphorylation. siRNA knockdown of PKN1 in NRVM resulted in increased basal CamKIIδ activation and increased PLB Thr17 phosphorylation only during sI. In vivo PLB Thr17 phosphorylation, Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA2) expression and Junctophilin-2 (Jph2) expression were also basally increased in PKN1 KO hearts. Furthermore, in vivo P-V loop analysis of the beat-to-beat relationship between rate of LV pressure development or relaxation and end diastolic P (EDP) showed mild but significant systolic and diastolic dysfunction with preserved ejection fraction in PKN1 KO hearts. Conclusion: Loss of PKN1 in vivo significantly reduces endogenous cardioprotection and increases myocardial infarct size following I/R injury. Cardioprotection by PKN1 is associated with reduced CamKIIδ-dependent PLB Thr17 phosphorylation at the SR and therefore may stabilize the coupling of SR Ca2+ handling and contractile function, independent of its kinase activity.

Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection.

The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signaling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1):myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of ∼135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.

Prelamin A Accumulation Attenuates Rac1 Activity and Increases the Intrinsic Migrational Persistence of Aged Vascular Smooth Muscle Cells.

Vascular smooth muscle cell (VSMC) motility is essential during both physiological and pathological vessel remodeling. Although ageing has emerged as a major risk factor in the development of cardiovascular disease, our understanding of the impact of ageing on VSMC motility remains limited. Prelamin A accumulation is known to drive VSMC ageing and we show that presenescent VSMCs, that have accumulated prelamin A, display increased focal adhesion dynamics, augmented migrational velocity/persistence and attenuated Rac1 activity. Importantly, prelamin A accumulation in proliferative VSMCs, induced by depletion of the prelamin A processing enzyme FACE1, recapitulated the focal adhesion, migrational persistence and Rac1 phenotypes observed in presenescent VSMCs. Moreover, lamin A/C-depleted VSMCs also display reduced Rac1 activity, suggesting that prelamin A influences Rac1 activity by interfering with lamin A/C function at the nuclear envelope. Taken together, these data demonstrate that lamin A/C maintains Rac1 activity in VSMCs and prelamin A disrupts lamin A/C function to reduce Rac1 activity and induce migrational persistence during VSMC ageing.

A novel role for atypical MAPK kinase ERK3 in regulating breast cancer cell morphology and migration.

ERK3 is an atypical Mitogen-activated protein kinase (MAPK6). Despite the fact that the Erk3 gene was originally identified in 1991, its function is still unknown. MK5 (MAP kinase- activated protein kinase 5) also called PRAK is the only known substrate for ERK3. Recently, it was found that group I p21 protein activated kinases (PAKs) are critical effectors of ERK3. PAKs link Rho family of GTPases to actin cytoskeletal dynamics and are known to be involved in the regulation of cell adhesion and migration. In this study we demonstrate that ERK3 protein levels are elevated as MDA-MB-231 breast cancer cells adhere to collagen I which is concomitant with changes in cellular morphology where cells become less well spread following nascent adhesion formation. During this early cellular adhesion event we observe that the cells retain protrusive activity while reducing overall cellular area. Interestingly exogenous expression of ERK3 delivers a comparable reduction in cell spread area, while depletion of ERK3 expression increases cell spread area. Importantly, we have detected a novel specific endogenous ERK3 localization at the cell periphery. Furthermore we find that ERK3 overexpressing cells exhibit a rounded morphology and increased cell migration speed. Surprisingly, exogenous expression of a kinase inactive mutant of ERK3 phenocopies ERK3 overexpression, suggesting a novel kinase independent function for ERK3. Taken together our data suggest that as cells initiate adhesion to matrix increasing levels of ERK3 at the cell periphery are required to orchestrate cell morphology changes which can then drive migratory behavior.

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro.

L-selectin is a cell adhesion molecule that tethers free-flowing leukocytes from the blood to luminal vessel walls, facilitating the initial stages of their emigration from the circulation toward an extravascular inflammatory insult. Following shear-resistant adhesion to the vessel wall, L-selectin has frequently been reported to be rapidly cleaved from the plasma membrane (known as ectodomain shedding), with little knowledge of the timing or functional consequence of this event. Using advanced imaging techniques, we observe L-selectin shedding occurring exclusively as primary human monocytes actively engage in transendothelial migration (TEM). Moreover, the shedding was localized to transmigrating pseudopods within the subendothelial space. By capturing monocytes in midtransmigration, we could monitor the subcellular distribution of L-selectin and better understand how ectodomain shedding might contribute to TEM. Mechanistically, L-selectin loses association with calmodulin (CaM; a negative regulator of shedding) specifically within transmigrating pseudopods. In contrast, L-selectin/CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphorylation of L-selectin at Ser 364 is critical for CaM dissociation, which is also restricted to the transmigrating pseudopod. Pharmacological or genetic inhibition of L-selectin shedding significantly increased pseudopodial extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the establishment of front/back polarity for directional migration persistence once TEM was complete. We conclude that L-selectin shedding directly regulates polarity in transmigrated monocytes, which affirms an active role for this molecule in driving later stages of the multistep adhesion cascade.

The crystal structure of the human titin:obscurin complex reveals a conserved yet specific muscle M-band zipper module.

M10 is the most C-terminal immunoglobulin (Ig) domain of the giant protein titin and a frequent target of disease-linked mutations. Currently, it is the only known muscle Ig domain able to interact with two alternative ligands-obscurin and obscurin-like-1 (Obsl1)-in different sarcomeric subregions. Obscurin and Obsl1 use their homologous N-terminal Ig domain (O1 in obscurin and OL1 in Obsl1) to bind M10 in a mutually exclusive manner. We present here the X-ray structure of the human titin:obscurin M10:O1 complex extending our previous work on the M10:OL1 interaction. Similar to M10:OL1, the M10:O1 complex displays a chevron-shaped antiparallel Ig-Ig architecture held together by a conserved molecular interface, which we validated by isothermal titration calorimetry and sorting experiments in neonatal rat cardiomyocytes. O1, although structurally related to OL1 and M10, both members of the intermediate set (I-set) Ig family, presents an intriguing switch of its ßA' strand. This leads to structural differences between the complexes, particularly for the "open side" of the chevron-shaped assembly. A bioinformatics analysis reveals that the ßA'-switch observed for O1 is rare and that it is involved in mediating protein-protein interactions. Molecular dynamics simulations also suggest that this topological alteration substantially increases local flexibility compared to the conventional I-set Ig domains. The O1/OL1 Ig domains are candidate discriminatory structural modules potentially directing the binding of specific additional partners at the M-band. Cellular sorting experiments in neonatal rat cardiomyocytes are consistent with the view that the titin:obscurin/Obsl1 complexes might be a platform for higher-order interactions.

The structure and regulation of human muscle α-actinin.

The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins.