Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A.

PKA-mediated phosphorylation of sarcomeric proteins enhances heart muscle performance in response to ß-adrenergic stimulation and is associated with accelerated relaxation and increased cardiac output for a given preload. At the cellular level, the latter translates to a greater dependence of Ca2+ sensitivity and maximum force on sarcomere length (SL), that is, enhanced length-dependent activation. However, the mechanisms by which PKA phosphorylation of the most notable sarcomeric PKA targets, troponin I (cTnI) and myosin-binding protein C (cMyBP-C), lead to these effects remain elusive. Here, we specifically altered the phosphorylation level of cTnI in heart muscle cells and characterized the structural and functional effects at different levels of background phosphorylation of cMyBP-C and with two different SLs. We found Ser22/23 bisphosphorylation of cTnI was indispensable for the enhancement of length-dependent activation by PKA, as was cMyBP-C phosphorylation. This high level of coordination between cTnI and cMyBP-C may suggest coupling between their regulatory mechanisms. Further evidence for this was provided by our finding that cardiac troponin (cTn) can directly interact with cMyBP-C in vitro, in a phosphorylation- and Ca2+-dependent manner. In addition, bisphosphorylation at Ser22/Ser23 increased Ca2+ sensitivity at long SL in the presence of endogenously phosphorylated cMyBP-C. When cMyBP-C was dephosphorylated, bisphosphorylation of cTnI increased Ca2+ sensitivity and decreased cooperativity at both SLs, which may translate to deleterious effects in physiological settings. Our results could have clinical relevance for disease pathways, where PKA phosphorylation of cTnI may be functionally uncoupled from cMyBP-C phosphorylation due to mutations or haploinsufficiency.

Cardiac myosin regulatory light chain kinase modulates cardiac contractility by phosphorylating both myosin regulatory light chain and troponin I.

Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been studied extensively in vitro and in vivo, the precise role of cardiac myosin light chain kinase (cMLCK), the primary kinase acting upon RLC, in the regulation of cardiomyocyte contractility remains poorly understood. In this study, using recombinantly expressed and purified proteins, various analytical methods, in vitro and in situ kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions. We show that cMLCK specifically monophosphorylates Ser23 of human cardiac troponin I (cTnI) in isolation and in the trimeric troponin complex in vitro and in situ in the native environment of the muscle myofilament lattice. Moreover, we observed that human cMLCK phosphorylates rodent cTnI to a much smaller extent in vitro and in situ, suggesting species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with rat or human troponin increased their cross-bridge kinetics, the increase in sensitivity of myofilaments to calcium was significantly blunted by human TnI, suggesting that human cTnI phosphorylation by cMLCK modifies the functional consequences of RLC phosphorylation. We propose that cMLCK-mediated phosphorylation of TnI is functionally significant and represents a critical signaling pathway that coordinates the regulatory states of thick and thin filaments in both physiological and potentially pathophysiological conditions of the heart.

Site-specific phosphorylation of myosin binding protein-C coordinates thin and thick filament activation in cardiac muscle.

The heart's response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and Cε (PKCε), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C's multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites. PKA phosphorylation of cMyBP-C's N terminus on 3 conserved serine residues is hierarchical and antagonizes phosphorylation by PKCε, and vice versa. In contrast, RSK2 phosphorylation of cMyBP-C accelerates PKA phosphorylation. We used cMyBP-C's regulatory N-terminal domains in defined phosphorylation states for protein-protein interaction studies with isolated cardiac native thin filaments and the S2 domain of cardiac myosin to show that site-specific phosphorylation of this region of cMyBP-C controls its interaction with both the actin-containing thin and myosin-containing thick filaments. We also used fluorescence probes on the myosin-associated regulatory light chain in the thick filaments and on troponin C in the thin filaments to monitor structural changes in the myofilaments of intact heart muscle cells associated with activation of myocardial contraction by the N-terminal region of cMyBP-C in its different phosphorylation states. Our results suggest that cMyBP-C acts as a sarcomeric integrator of multiple signaling pathways that determines downstream physiological function.

15-deoxy-Δ12,14-Prostaglandin J2 inhibits human soluble epoxide hydrolase by a dual orthosteric and allosteric mechanism.

Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.

Structural and functional effects of myosin-binding protein-C phosphorylation in heart muscle are not mimicked by serine-to-aspartate substitutions.

Myosin-binding protein-C (cMyBP-C) is a key regulator of contractility in heart muscle, and its regulatory function is controlled in turn by phosphorylation of multiple serines in its m-domain. The structural and functional effects of m-domain phosphorylation have often been inferred from those of the corresponding serine-to-aspartate (Ser-Asp) substitutions, in both in vivo and in vitro studies. Here, using a combination of in vitro binding assays and in situ structural and functional assays in ventricular trabeculae of rat heart and the expressed C1mC2 region of cMyBP-C, containing the m-domain flanked by domains C1 and C2, we tested whether these substitutions do in fact mimic the effects of phosphorylation. In situ changes in thin and thick filament structure were determined from changes in polarized fluorescence from bifunctional probes attached to troponin C or myosin regulatory light chain, respectively. We show that both the action of exogenous C1mC2 to activate contraction in the absence of calcium and the accompanying change in thin filament structure are abolished by tris-phosphorylation of the m-domain, but unaffected by the corresponding Ser-Asp substitutions. The latter produced an intermediate change in thick filament structure. Both tris-phosphorylation and Ser-Asp substitutions abolished the interaction between C1mC2 and myosin sub-fragment 2 (myosin S2) in vitro, but yielded different effects on thin filament binding. These results suggest that some previous inferences from the effects of Ser-Asp substitutions in cMyBP-C should be reconsidered and that the distinct effects of tris-phosphorylation and Ser-Asp substitutions on cMyBP-C may provide a useful basis for future studies.

Reversible Covalent Reaction of Levosimendan with Cardiac Troponin C in Vitro and in Situ.

The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties, including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target because cardiac troponin C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan. We have recently shown that when a covalent cTnC-levosimendan analogue is exchanged into cardiac muscle cells, they become constitutively active, demonstrating the potency of a covalent complex. We have also demonstrated that levosimendan reacts in vitro to form a reversible covalent thioimidate bond specifically with cysteine 84, unique to cTnC. In this study, we use mass spectrometry to show that the in vitro mechanism of action of levosimendan is consistent with an allosteric, reversible covalent inhibitor; to determine whether the presence of the cTnI switch peptide or changes in either Ca2+ concentration or pH modify the reaction kinetics; and to determine whether the reaction can occur with cTnC in situ in cardiac myofibrils. Using the derived kinetic rate constants, we predict the degree of covalently modified cTnC in vivo under the conditions studied. We observe that covalent bond formation would be highest under the acidotic conditions resulting from ischemia and discuss whether the predicted level could be sufficient to have therapeutic value. Irrespective of the in vivo mechanism of action for levosimendan, our results provide a rationale and basis for the development of reversible covalent drugs to target the failing heart.

Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament.

KEY POINTS: Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle. Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament. Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto-myosin ATPase cycle. Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto-myosin ATPase. Thick filament regulation is a promising target for novel therapeutics in heart disease. ABSTRACT: Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin-containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules - omecamtiv mecarbil (OM) and blebbistatin (BS) - that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small-molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin-myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin-myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co-operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments.

Distinct contributions of the thin and thick filaments to length-dependent activation in heart muscle.

The Frank-Starling relation is a fundamental auto-regulatory property of the heart that ensures the volume of blood ejected in each heartbeat is matched to the extent of venous filling. At the cellular level, heart muscle cells generate higher force when stretched, but despite intense efforts the underlying molecular mechanism remains unknown. We applied a fluorescence-based method, which reports structural changes separately in the thick and thin filaments of rat cardiac muscle, to elucidate that mechanism. The distinct structural changes of troponin C in the thin filaments and myosin regulatory light chain in the thick filaments allowed us to identify two aspects of the Frank-Starling relation. Our results show that the enhanced force observed when heart muscle cells are maximally activated by calcium is due to a change in thick filament structure, but the increase in calcium sensitivity at lower calcium levels is due to a change in thin filament structure.

Reversible Covalent Binding to Cardiac Troponin C by the Ca2+-Sensitizer Levosimendan.

The binding of Ca2+ to cardiac troponin C (cTnC) triggers contraction in heart muscle. In the diseased heart, the myocardium is often desensitized to Ca2+, which leads to impaired contractility. Therefore, compounds that sensitize cardiac muscle to Ca2+ (Ca2+-sensitizers) have therapeutic promise. The only Ca2+-sensitizer used regularly in clinical settings is levosimendan. While the primary target of levosimendan is thought to be cTnC, the molecular details of this interaction are not well understood. In this study, we used mass spectrometry, computational chemistry, and nuclear magnetic resonance spectroscopy to demonstrate that levosimendan reacts specifically with cysteine 84 of cTnC to form a reversible thioimidate bond. We also showed that levosimendan only reacts with the active, Ca2+-bound conformation of cTnC. Finally, we propose a structural model of levosimendan bound to cTnC, which suggests that the Ca2+-sensitizing function of levosimendan is due to stabilization of the Ca2+-bound conformation of cTnC.

Novel myosin-based therapies for congenital cardiac and skeletal myopathies.

The dysfunction in a number of inherited cardiac and skeletal myopathies is primarily due to an altered ability of myofilaments to generate force and motion. Despite this crucial knowledge, there are, currently, no effective therapeutic interventions for these diseases. In this short review, we discuss recent findings giving strong evidence that genetically or pharmacologically modulating one of the myofilament proteins, myosin, could alleviate the muscle pathology. This should constitute a research and clinical priority.

Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments.

Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease.

Probing the mechanism of cardiovascular drugs using a covalent levosimendan analog.

One approach to improve contraction in the failing heart is the administration of calcium (Ca(2+)) sensitizers. Although it is known that levosimendan and other sensitizers bind to troponin C (cTnC), their in vivo mechanism is not fully understood. Based on levosimendan, we designed a covalent Ca(2+) sensitizer (i9) that targets C84 of cTnC and exchanged this complex into cardiac muscle. The NMR structure of the covalent complex showed that i9 binds deep in the hydrophobic pocket of cTnC. Despite slightly reducing troponin I affinity, i9 enhanced the Ca(2+) sensitivity of cardiac muscle. We conclude that i9 enhances Ca(2+) sensitivity by stabilizing the open conformation of cTnC. These findings provide new insights into the in vivo mechanism of Ca(2+) sensitization and demonstrate that directly targeting cTnC has significant potential in cardiovascular therapy.

The structural and functional effects of the familial hypertrophic cardiomyopathy-linked cardiac troponin C mutation, L29Q.

Familial hypertrophic cardiomyopathy (FHC) is characterized by severe abnormal cardiac muscle growth. The traditional view of disease progression in FHC is that an increase in the Ca(2+)-sensitivity of cardiac muscle contraction ultimately leads to pathogenic myocardial remodeling, though recent studies suggest this may be an oversimplification. For example, FHC may be developed through altered signaling that prevents downstream regulation of contraction. The mutation L29Q, found in the Ca(2+)-binding regulatory protein in heart muscle, cardiac troponin C (cTnC), has been linked to cardiac hypertrophy. However, reports on the functional effects of this mutation are conflicting, and our goal was to combine in vitro and in situ structural and functional data to elucidate its mechanism of action. We used nuclear magnetic resonance and circular dichroism to solve the structure and characterize the backbone dynamics and stability of the regulatory domain of cTnC with the L29Q mutation. The overall structure and dynamics of cTnC were unperturbed, although a slight rearrangement of site 1, an increase in backbone flexibility, and a small decrease in protein stability were observed. The structure and function of cTnC was also assessed in demembranated ventricular trabeculae using fluorescence for in situ structure. L29Q reduced the cooperativity of the Ca(2+)-dependent structural change in cTnC in trabeculae under basal conditions and abolished the effect of force-generating myosin cross-bridges on this structural change. These effects could contribute to the pathogenesis of this mutation.

Orientation of the N- and C-terminal lobes of the myosin regulatory light chain in cardiac muscle.

The orientations of the N- and C-terminal lobes of the cardiac isoform of the myosin regulatory light chain (cRLC) in the fully dephosphorylated state in ventricular trabeculae from rat heart were determined using polarized fluorescence from bifunctional sulforhodamine probes. cRLC mutants with one of eight pairs of surface-accessible cysteines were expressed, labeled with bifunctional sulforhodamine, and exchanged into demembranated trabeculae to replace some of the native cRLC. Polarized fluorescence data from the probes in each lobe were combined with RLC crystal structures to calculate the lobe orientation distribution with respect to the filament axis. The orientation distribution of the N-lobe had three distinct peaks (N1-N3) at similar angles in relaxation, isometric contraction, and rigor. The orientation distribution of the C-lobe had four peaks (C1-C4) in relaxation and isometric contraction, but only two of these (C2 and C4) remained in rigor. The N3 and C4 orientations are close to those of the corresponding RLC lobes in myosin head fragments bound to isolated actin filaments in the absence of ATP (in rigor), but also close to those of the pair of heads folded back against the filament surface in isolated thick filaments in the so-called J-motif conformation. The N1 and C1 orientations are close to those expected for actin-bound myosin heads with their light chain domains in a pre-powerstroke conformation. The N2 and C3 orientations have not been observed previously. The results show that the average change in orientation of the RLC region of the myosin heads on activation of cardiac muscle is small; the RLC regions of most heads remain in the same conformation as in relaxation. This suggests that the orientation of the dephosphorylated RLC region of myosin heads in cardiac muscle is primarily determined by an interaction with the thick filament surface.

Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells.

Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.

Regulatory domain of troponin moves dynamically during activation of cardiac muscle.

Heart muscle is activated by Ca(2+) to generate force and shortening, and the signaling pathway involves allosteric mechanisms in the thin filament. Knowledge about the structure-function relationship among proteins in the thin filament is critical in understanding the physiology and pathology of the cardiac function, but remains obscure. We investigate the conformation of the cardiac troponin (Tn) on the thin filament and its response to Ca(2+) activation and propose a molecular mechanism for the regulation of cardiac muscle contraction by Tn based uniquely on information from in situ protein domain orientation. Polarized fluorescence from bifunctional rhodamine is used to determine the orientation of the major component of Tn core domain on the thin filaments of cardiac muscle. We show that the C-terminal lobe of TnC (CTnC) does not move during activation, suggesting that CTnC, together with the coiled coil formed by the TnI and TnT chains (IT arm), acts as a scaffold that holds N-terminal lobe of TnC (NTnC) and the actin binding regions of troponin I. The NTnC, on the other hand, exhibits multiple orientations during both diastole and systole. By combining the in situ orientation data with published in vitro measurements of intermolecular distances, we construct a model for the in situ structure of the thin filament. The conformational dynamics of NTnC plays an important role in the regulation of cardiac muscle contraction by moving the C-terminal region of TnI from its actin-binding inhibitory location and enhancing the movement of tropomyosin away from its inhibitory position.

Structural dynamics of troponin during activation of skeletal muscle.

Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s(-1)) matches the rate of Ca(2+) binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s(-1)) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s(-1)) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament-myosin head complex under physiological conditions.

Conformation of the troponin core complex in the thin filaments of skeletal muscle during relaxation and active contraction.

Contraction of skeletal and cardiac muscles is regulated by Ca(2+) binding to troponin in the actin-containing thin filaments, leading to an azimuthal movement of tropomyosin around the filament that uncovers the myosin binding sites on actin. Here, we use polarized fluorescence to determine the orientation of the C-terminal lobe of troponin C (TnC) in skeletal muscle cells as a step toward elucidating the molecular mechanism of troponin-mediated regulation. Assuming, as shown by X-ray crystallography, that this lobe of TnC is part of a well-defined troponin domain called the IT arm, we show that the coiled coil formed by troponin components I and T makes an angle of about 55° with the thin filament axis in relaxed muscle, in contrast with previous models based on electron microscopy in which this angle is close to 0°. The E helix of TnC makes an angle of about 45° with the thin filament axis. Both the IT coiled coil and the TnC E helix tilt by about 10° on muscle activation. By combining in situ measurements of the orientation of the IT arm and regulatory domain of troponin, which together form the troponin core complex, with published intermolecular distances between thin filament components, we derive models of thin filament structure in which the IT arm of troponin holds its regulatory domain close to the actin surface. Although the structure and function of troponin regions outside the core complex remain to be characterized, the present results provide useful constraints for molecular models of the mechanism of muscle regulation.

Orientation of the N-terminal lobe of the myosin regulatory light chain in skeletal muscle fibers.

The orientation of the N-terminal lobe of the myosin regulatory light chain (RLC) in demembranated fibers of rabbit psoas muscle was determined by polarized fluorescence. The native RLC was replaced by a smooth muscle RLC with a bifunctional rhodamine probe attached to its A, B, C, or D helix. Fiber fluorescence data were interpreted using the crystal structure of the head domain of chicken skeletal myosin in the nucleotide-free state. The peak angle between the lever axis of the myosin head and the fiber or actin filament axis was 100-110° in relaxation, isometric contraction, and rigor. In each state the hook helix was at an angle of ∼40° to the lever/filament plane. The in situ orientation of the RLC D and E helices, and by implication of its N- and C-lobes, was similar in smooth and skeletal RLC isoforms. The angle between these two RLC lobes in rigor fibers was different from that in the crystal structure. These results extend previous crystallographic evidence for bending between the two lobes of the RLC to actin-attached myosin heads in muscle fibers, and suggest that such bending may have functional significance in contraction and regulation of vertebrate striated muscle.

Uncoordinated transcription and compromised muscle function in the lmna-null mouse model of Emery- Emery-Dreyfuss muscular dystrophy.

LMNA encodes both lamin A and C: major components of the nuclear lamina. Mutations in LMNA underlie a range of tissue-specific degenerative diseases, including those that affect skeletal muscle, such as autosomal-Emery-Dreifuss muscular dystrophy (A-EDMD) and limb girdle muscular dystrophy 1B. Here, we examine the morphology and transcriptional activity of myonuclei, the structure of the myotendinous junction and the muscle contraction dynamics in the lmna-null mouse model of A-EDMD. We found that there were fewer myonuclei in lmna-null mice, of which ∼50% had morphological abnormalities. Assaying transcriptional activity by examining acetylated histone H3 and PABPN1 levels indicated that there was a lack of coordinated transcription between myonuclei lacking lamin A/C. Myonuclei with abnormal morphology and transcriptional activity were distributed along the length of the myofibre, but accumulated at the myotendinous junction. Indeed, in addition to the presence of abnormal myonuclei, the structure of the myotendinous junction was perturbed, with disorganised sarcomeres and reduced interdigitation with the tendon, together with lipid and collagen deposition. Functionally, muscle contraction became severely affected within weeks of birth, with specific force generation dropping as low as ∼65% and ∼27% of control values in the extensor digitorum longus and soleus muscles respectively. These observations illustrate the importance of lamin A/C for correct myonuclear function, which likely acts synergistically with myotendinous junction disorganisation in the development of A-EDMD, and the consequential reduction in force generation and muscle wasting.