Mutated lamin A modulates stiffness in muscle cells

The cytoskeleton is a complex network interlinking filaments that extend throughout the cytoplasm from the nucleus to the plasma membrane. Three major types of filaments are found in the cytoskeleton: actin filaments, microtubules, and intermediate filaments. They play a key role in the ability of cells to both resist mechanical stress and generate force. However, the precise involvement of intermediate filament proteins in these processes remains unclear. Here, we focused on nuclear A-type lamins, which are connected to the cytoskeleton via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Using micro-constriction rheology, we investigated the impact of A-type lamins (p.H222P) mutation on the mechanical properties of muscle cells. We demonstrate that the expression of point mutation of lamin A in muscle cells increases cellular stiffness compared with cells expressing wild type lamin A and that the chemical agent selumetinib, an inhibitor of the ERK1/2 signaling, reversed the mechanical alterations in mutated cells. These results highlight the interplay between A-type lamins and mechano-signaling, which are supported by cell biology measurements

S6K2-mediated regulation of TRBP as a determinant of miRNA expression in human primary lymphatic endothelial cells

MicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs. They are generated following transcription and cleavage by the DROSHA/DGCR8 and DICER/TRBP/PACT complexes. Although it is known that components of the miRNA biogenesis machinery can be phosphorylated, it remains poorly understood how these events become engaged during physiological cellular activation. We demonstrate that S6 kinases can phosphorylate the extended C-terminal domain of TRBP and interact with TRBP in situ in primary cells. TRBP serines 283/286 are essential for S6K-mediated TRBP phosphorylation, optimal expression of TRBP, and the S6K-TRBP interaction in human primary cells. We demonstrate the functional relevance of this interaction in primary human dermal lymphatic endothelial cells (HDLECs). Angiopoietin-1 (ANG1) can augment miRNA biogenesis in HDLECs through enhancing TRBP phosphorylation and expression in an S6K2-dependent manner. We propose that the S6K2/TRBP node controls miRNA biogenesis in HDLECs and provides a molecular link between the mTOR pathway and the miRNA biogenesis machinery

Actin-microtubule cytoskeletal interplay mediated by MRTF-A/SRF signaling promotes dilated cardiomyopathy caused by LMNA mutations

Publisher Copyright: © 2022, The Author(s).Mutations in the lamin A/C gene (LMNA) cause dilated cardiomyopathy associated with increased activity of ERK1/2 in the heart. We recently showed that ERK1/2 phosphorylates cofilin-1 on threonine 25 (phospho(T25)-cofilin-1) that in turn disassembles the actin cytoskeleton. Here, we show that in muscle cells carrying a cardiomyopathy-causing LMNA mutation, phospho(T25)-cofilin-1 binds to myocardin-related transcription factor A (MRTF-A) in the cytoplasm, thus preventing the stimulation of serum response factor (SRF) in the nucleus. Inhibiting the MRTF-A/SRF axis leads to decreased α-tubulin acetylation by reducing the expression of ATAT1 gene encoding α-tubulin acetyltransferase 1. Hence, tubulin acetylation is decreased in cardiomyocytes derived from male patients with LMNA mutations and in heart and isolated cardiomyocytes from Lmnap.H222P/H222P male mice. In Atat1 knockout mice, deficient for acetylated α-tubulin, we observe left ventricular dilation and mislocalization of Connexin 43 (Cx43) in heart. Increasing α-tubulin acetylation levels in Lmnap.H222P/H222P mice with tubastatin A treatment restores the proper localization of Cx43 and improves cardiac function. In summary, we show for the first time an actin-microtubule cytoskeletal interplay mediated by cofilin-1 and MRTF-A/SRF, promoting the dilated cardiomyopathy caused by LMNA mutations. Our findings suggest that modulating α-tubulin acetylation levels is a feasible strategy for improving cardiac function.Peer reviewe

Die Rollen der ERK1/2 Signalisierung in der Pathophysiologie der LMNA- Kardiomyopathie

Dilated cardiomyopathy is one of the leading causes of heart failure in Europe. Despite of the conventional medical care, there is no definitive and satisfactory treatment for the progressive cardiac dilatation and loss of contractility in LMNA cardiomyopathy often leading to sudden death or heart transplantation. LMNA gene encodes nuclear A-type lamins, which are the main constituents of the nuclear lamina. To explain how mutations in proteins of the nuclear envelope can cause a disease of the heart, it has been proposed that nuclear envelope abnormalities bring about cellular fragility and a decrease in the mechanical resistance to stress, which could partially explain the cardiac muscle disease, considering that the heart muscle is constantly subjected to mechanical force. In previous work, it has been showed that there is an abnormal activation of stress-activated ERK1/2 signaling in hearts that carry LMNA mutations. Administration of drugs inhibiting ERK1/2 signaling improves cardiac ejection fraction in mice, and blunts further increase in left ventricular dilatation. These studies clearly show that the abnormal ERK1/2 activation is involved in the pathophysiology of LMNA dilated cardiomyopathy. However, its role in the development of cardiac dysfunction remains unclear. Inhibition of ERK1/2 signaling also slows progression of myocardial fibrosis, which is prominent in humans with dilated cardiomyopathy. I suggested that aberrant TGF-β signaling activity could participate to the abnormal ERK1/2 activation and be involved is the pathophysiology of left- ventricular contractile dysfunction in LMNA cardiomyopathy. My work led us to describe the TGF-β/ERK1/2/CTGF axis as a key player for the onset of myocardial fibrosis, which impairs left ventricular function, a major symptom of LMNA cardiomyopathy. Given that the understanding of molecular and cellular mechanisms underlying the modulation of ERK1/2 signaling in the heart caused by LMNA mutation remains totally unclear, I tested the hypothesis that ERK1/2 abnormal modulation leads to alteration of cytosolic targets and alter cardiac cytoskeleton network. This may lead to LMNA cardiomyopathy. My work highlighted a novel partner of activated (phosphorylated) ERK1/2, ADF/cofilin-1. Cofilin promotes debranching of actin filaments. I showed that disrupted actin dynamics leads to abnormal destructuration of sarcomere and ADF/cofilin accumulation in the heart from a mouse model of LMNA cardiomyopathy, suggesting a defect in actin depolymerization. This project unraveled an unexpected role played by ERK1/2 signaling in actin dynamics and in the development of left-ventricular dysfunction in LMNA cardiomyopathy.Dilatative Kardiomyopathie ist eine der häufigsten Ursachen der Herzinsuffizienz in Europa. Neben einer konventionellen medizinischen Versorgung gibt es, außer der Herztransplantation, keine kurative Behandlung gegen die progressive Herzdilatation und den Verlust der Kontraktilität, die in der LMNA Kardiomyopathie auftreten. Das LMNA Gen kodiert die nuklearen A-typ Lamine, die den Hauptbestandteil der nuklearen Lamina bilden. Um zu erklären wie Mutationen in Proteinen der nuklearen Lamina zu Krankheiten des Herzens führen können, wird vermutet, dass die Anomalien der Kernhülle zu Fragilität und einer Abnahme der mechanischen Widerstandsfähigkeit der Zelle gegen mechanischen Stress führt. In der Erwägung, dass der Herzmuskel ständig mechanischen Kräften ausgesetzt ist, könnte dies zur Entstehung der Herzmuskelerkrankung beitragen. In früheren Arbeiten wurde bereits eine abnormale Aktivierung des stress-aktivierten ERK1/2 Signalwegs im Herzmuskelzellen, die eine LMNA Mutationen tragen, gezeigt. Die Verabreichung von Arzneimitteln, die die ERK1/2 Aktivierung hemmen, verbessert die Herzauswurffraktion und verhindert das weitere Fortschreiten der Dilatation des linken Ventrikels im Mausexperiment. Diese Studien zeigen deutlich, dass die abnormale ERK1/2 Aktivierung in der Pathophysiologie der dilatativen Kardiomyopathie beteiligt ist. Die genaue Rolle von ERK1/2 bei der Entstehung der Herzdysfunktion bleibt jedoch unklar. Die Hemmung der ERK1/2 Aktivität verlangsamt außerdem das Fortschreiten der myokardialen Fibrose, die häufig bei Menschen mit dilatativer Kardiomyopathie beobachtet wird. Ich habe vermutet, dass eine anomale TGF-Beta Signalaktivität zu der abnormalen ERK1/2 Aktivität beiträgt und daher in die Pathophysiologie der linksventrikulären kontraktilen Dysfunktion der LMNA Kardiomyopathie involviert ist. Meine Arbeit zeigt den TGF-Beta/ERK1/2/CTGF Signalweg als einen wichtigen Akteur in der Entstehung der myokardialen Fibrose, welche die linksventrikuläre Funktion beeinträchtigt und ein Hauptsymptom der Kardiomyopathie darstellt. Da der molekulare und zelluläre Mechanismus der ERK1/2 Signaltransduktion in Herzen, versucht durch die LMNA mutation, völlig ungeklärt ist, habe ich die Hypothese getestet, dass die abnormale ERK1/2 Modulation zu Veränderungen der zytosolischen Interaktionspartner sowie zu einer Veränderung des Herz- Zytoskelettes führt. Diese Veränderungen wiederum könnten zur Kardiomyopathie führen. Meine Arbeit zeigt einen neuen Interaktionspartner von aktiviertem ERK1/2, Cofilin1. Cofilin1 reguliert das Entzweigen von Aktin-filamenten. Ich konnte zeigen, dass eine gestörte Aktindynamik zu einer abnormalen Desorganisation des Sarkomers und einer Akkumulation von ADF/Cofilin im Herzen des Mausmodells der LMNA Kardiomyopathie führt. Dies weist auf einen Defekt in der Aktinpolymerisierung hin. Dieses Projekt deckt eine unerwartete Rolle der ERK1/2 Signaltransduktion in der Regulierung der Aktindynamik und in der Entstehung der linksventrikulären Dysfunktion in der LMNA Kardiomyopathie auf

Les rôles de la signalisation ERK1/2 dans la cardiomyopathie liée aux mutations du gène LMNA

Dilated cardiomyopathy is one of the leading causes of heart failure in Europe. Despite of the conventional medical care, there is no definitive treatment for the progressive cardiac dilatation and loss of contractility in LMNA cardiomyopathy often leading to sudden death or heart transplantation. LMNA gene encodes nuclear A-type lamins, which are the main constituents of the nuclear lamina. Previous studies clearly show that the abnormal ERK1/2 activation is involved in the pathophysiology of LMNA dilated cardiomyopathy. However, its role in the development of cardiac dysfunction remains unclear. Inhibition of ERK1/2 signaling also slows progression of myocardial fibrosis, which is prominent in humans with dilated cardiomyopathy. I suggested that aberrant TGF-β signaling activity could participate to the abnormal ERK1/2 activation and be involved is the pathophysiology of left-ventricular contractile dysfunction in LMNA cardiomyopathy. Given that the understanding of molecular and cellular mechanisms underlying the modulation of ERK1/2 signaling in the heart caused by LMNA mutation remains totally unclear, I tested the hypothesis that ERK1/2 abnormal modulation leads to alteration of cytosolic targets and alter cardiac cytoskeleton network. My work highlighted a novel partner of activated (phosphorylated) ERK1/2, ADF/cofilin-1. Cofilin promotes debranching of actin filaments. I showed that disrupted actin dynamics leads to abnormal sarcomere structure. This project unraveled an unexpected role played by ERK1/2 signaling in actin dynamics and in the development of left-ventricular dysfunction in LMNA cardiomyopathy.La cardiomyopathie dilatée est l'une des principales causes d'insuffisance cardiaque en Europe. Dans le cadre de la cardiomyopathie liée aux mutations du gène LMNA, en dépit des soins médicaux conventionnels, aucun traitement satisfaisant ne permet de pallier à la dilatation cardiaque progressive et à la perte de la contractilité. Le gène LMNA code pour les lamines nucléaires de type A, qui sont les principaux constituants de la lamina nucléaire. De précédents travaux démontrent que l'activation anormale de ERK 1/2 est impliquée dans la pathophysiologie de la cardiomyopathie dilatée liée aux mutations du gène LMNA. L'inhibition de la voie ERK 1/2 ralentit également la progression de la fibrose myocardique, relativement développée chez l'homme, en cas de cardiomyopathie dilatée. Dans le cadre de ma thèse, j’ai suggéré que l'activité aberrante de la voie TGF-β pourrait participer à l’activation anormale de ERK 1/2 et être impliquée dans la physiopathologie de dysfonction contractile ventriculaire gauche dans la cardiomyopathie liée aux mutations du gène LMNA. Les mécanismes moléculaires sous-jacents à la modulation de la signalisation ERK1/2, causée dans le cœur, par les mutations du gène LMNA, restent totalement incertains. De ce fait, j'ai testé l'hypothèse selon laquelle la modulation anormale de ERK1/2 induirait l'altération des cibles cytosoliques et modifierait le réseau du cytosquelette cardiaque. Mon travail a mis en évidence un nouveau partenaire de la forme active (phosphorylée) d’ERK1/2 : cofiline-1. La cofiline induit la déramification des filaments d'actine. Ce projet met en lumière un rôle inattendu joué par la signalisation ERK1/2 dans la dynamique de l'actine et dans le développement de la dysfonction ventriculaire gauche de la cardiomyopathie liée aux mutations du gène LMNA

When Hippo meets actin in the nucleus

Nuclear actin is exported from the nucleus via the Exportin-6 (XPO6)/RAN GTPase complex. We recently identified that RASSF1A and the hippo pathway kinase Mammalian STE20-like protein kinase 2 (MST2) play a pivotal role in nucleocytoplasmic shuttling of actin by regulating the association of XPO6 with RAN GTPase. Importantly, loss of Ras association domain family 1A (RASSF1A) and hippo signaling in cancer cells highlights a key mechanism by which nuclear actin promotes tumorigenesis

Rescue of biosynthesis of nicotinamide adenine dinucleotide protects the heart in cardiomyopathy caused by lamin A/C gene mutation

International audienceCardiomyopathy caused by lamin A/C gene (LMNA) mutations (hereafter referred as LMNA cardiomyopathy) is an anatomic and pathologic condition associated with muscle and electrical dysfunction of the heart, often leading to heart failure-related disability. There is currently no specific therapy available for patients that target the molecular pathophysiology of LMNA cardiomyopathy. Recent studies suggested that nicotinamide adenine dinucleotide (NAD+) cellular content could be a critical determinant for heart function. Biosynthesis of NAD+ from vitamin B3 (known as salvage pathways) is the primary source of NAD+. We showed here that NAD+ salvage pathway was altered in the heart of mouse and human carrying LMNA mutation, leading to an alteration of one of NAD+ co-substrate enzymes, PARP-1. Oral administration of nicotinamide riboside, a natural NAD+ precursor and a pyridine-nucleoside form of vitamin B3, leads to a marked improvement of the NAD+ cellular content, an increase of PARylation of cardiac proteins and an improvement of left ventricular structure and function in a model of LMNA cardiomyopathy. Collectively, our results provide mechanistic and therapeutic insights into dilated cardiomyopathy caused by LMNA mutations

Cardioprotective effects of α‐cardiac actin on oxidative stress in a dilated cardiomyopathy mouse model

International audienceThe expression of α‐cardiac actin, a major constituent of the cytoskeleton of cardiomyocytes, is dramatically decreased in a mouse model of dilated cardiomyopathy triggered by inducible cardiac‐specific serum response factor (Srf) gene disruption that could mimic some forms of human dilated cardiomyopathy. To investigate the consequences of the maintenance of α‐cardiac actin expression in this model, we developed a new transgenic mouse based on Cre/LoxP strategy, allowing together the induction of SRF loss and a compensatory expression of α‐cardiac actin. Here, we report that maintenance of α‐cardiac actin within cardiomyocytes temporally preserved cytoarchitecture from adverse cardiac remodeling through a positive impact on both structural and transcriptional levels. These protective effects were accompanied in vivo by the decrease of ROS generation and protein carbonylation and the downregulation of NADPH oxidases NOX2 and NOX4. We also show that ectopic expression of α‐cardiac actin protects HEK293 cells against oxidative stress induced by H2O2. Oxidative stress plays an important role in the development of cardiac remodeling and contributes also to the pathogenesis of heart failure. Taken together, these findings indicate that α‐cardiac actin could be involved in the regulation of oxidative stress that is a leading cause of adverse remodeling during dilated cardiomyopathy development

Microtubule cytoskeleton regulates Connexin 43 localization and cardiac conduction in cardiomyopathy caused by mutation in A-type lamins gene

International audienceMutations in the lamin A/C gene (LMNA) cause an autosomal dominant inherited form of dilated cardiomyopathy associated with cardiac conduction disease (hereafter referred to as LMNA cardiomyopathy). Compared with other forms of dilated cardiomyopathy, mutations in LMNA are responsible for a more aggressive clinical course owing to a high rate of malignant ventricular arrhythmias. Gap junctions are intercellular channels that allow direct communication between neighboring cells, which are involved in electrical impulse propagation and coordinated contraction of the heart. For gap junctions to properly control electrical synchronization in the heart, connexin-based hemichannels must be correctly targeted to intercalated discs, Cx43 being the major connexin in the working myocytes. We here showed an altered distribution of Cx43 in a mouse model of LMNA cardiomyopathy. However, little is known on the molecular mechanisms of Cx43 remodeling in pathological context. We now show that microtubule cytoskeleton alteration and decreased acetylation of α-tubulin lead to remodeling of Cx43 in LMNA cardiomyopathy, which alters the correct communication between cardiomyocytes, ultimately leading to electrical conduction disturbances. Preventing or reversing this process could offer a strategy to repair damaged heart. Stabilization of microtubule cytoskeleton using Paclitaxel improved intraventricular conduction defects. These results indicate that microtubule cytoskeleton contributes to the pathogenesis of LMNA cardiomyopathy and that drugs stabilizing the microtubule may be beneficial for patients

ERK1/2 directly acts on CTGF/CCN2 expression to mediate myocardial fibrosis in cardiomyopathy caused by mutations in the lamin A/C gene

International audienceCardiomyopathy caused by lamin A/C gene mutations (LMNA cardiomyopathy) is characterized by increased myocardial fibrosis, which impairs left ventricular relaxation and predisposes to heart failure, and cardiac conduction abnormalities. While we previously discovered abnormally elevated extracellular signal-regulated kinase 1/2 (ERK1/2) activities in heart in LMNA cardiomyopathy, its role on the development of myocardial fibrosis remains unclear. We now showed that transforming growth factor (TGF)-β/Smad signaling participates in the activation of ERK1/2 signaling in LMNA cardiomyopathy. ERK1/2 acts on connective tissue growth factor (CTGF/CCN2) expression to mediate the myocardial fibrosis and left ventricular dysfunction. Studies in vivo demonstrate that inhibiting CTGF/CCN2 using a specific antibody decreases myocardial fibrosis and improves the left ventricular dysfunction. Together, these findings show that cardiac ERK1/2 activity is modulated in part by TGF-β/Smad signaling, leading to altered activation of CTGF/CCN2 to mediate fibrosis and alter cardiac function. This identifies a novel mechanism in the development of LMNA cardiomyopathy