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BACKGROUND: How the sarcomeric complex is continuously turned over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres. METHODS: We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands. RESULTS: We disprove the prevailing protein pool model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age and that proteolytic extraction is a rate-limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells. CONCLUSIONS: Our findings establish a unidirectional replacement model for cardiac sarcomeres subunit replacement and identify their turnover principles.
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Conectina , Miócitos Cardíacos , Sarcômeros , Sarcômeros/metabolismo , Animais , Miócitos Cardíacos/metabolismo , Conectina/metabolismo , Células Cultivadas , Proteólise , Camundongos , Biossíntese de Proteínas , Proteínas Musculares/metabolismo , Ratos , Masculino , Camundongos Endogâmicos C57BLRESUMO
Cardiovascular disease (CVD) is the leading cause of morbimortality in Europe and worldwide. CVD imposes a heterogeneous spectrum of cardiac remodelling, depending on the insult nature, that is, pressure or volume overload, ischaemia, arrhythmias, infection, pathogenic gene variant, or cardiotoxicity. Moreover, the progression of CVD-induced remodelling is influenced by sex, age, genetic background and comorbidities, impacting patients' outcomes and prognosis. Cardiac reverse remodelling (RR) is defined as any normative improvement in cardiac geometry and function, driven by therapeutic interventions and rarely occurring spontaneously. While RR is the outcome desired for most CVD treatments, they often only slow/halt its progression or modify risk factors, calling for novel and more timely RR approaches. Interventions triggering RR depend on the myocardial insult and include drugs (renin-angiotensin-aldosterone system inhibitors, beta-blockers, diuretics and sodium-glucose cotransporter 2 inhibitors), devices (cardiac resynchronization therapy, ventricular assist devices), surgeries (valve replacement, coronary artery bypass graft), or physiological responses (deconditioning, postpartum). Subsequently, cardiac RR is inferred from the degree of normalization of left ventricular mass, ejection fraction and end-diastolic/end-systolic volumes, whose extent often correlates with patients' prognosis. However, strategies aimed at achieving sustained cardiac improvement, predictive models assessing the extent of RR, or even clinical endpoints that allow for distinguishing complete from incomplete RR or adverse remodelling objectively, remain limited and controversial. This scientific statement aims to define RR, clarify its underlying (patho)physiologic mechanisms and address (non)pharmacological options and promising strategies to promote RR, focusing on the left heart. We highlight the predictors of the extent of RR and review the prognostic significance/impact of incomplete RR/adverse remodelling. Lastly, we present an overview of RR animal models and potential future strategies under pre-clinical evaluation.
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Remodelação Ventricular , Humanos , Remodelação Ventricular/fisiologia , Prognóstico , Doenças Cardiovasculares/terapia , Doenças Cardiovasculares/fisiopatologia , Europa (Continente) , Relevância ClínicaRESUMO
BACKGROUND: Alterations in the buffering of intracellular Ca2+, for which myofilament proteins play a key role, have been shown to promote cardiac arrhythmia. It is interesting that although studies report atrial myofibrillar degradation in patients with persistent atrial fibrillation (persAF), the intracellular Ca2+ buffering profile in persAF remains obscure. Therefore, we aimed to investigate the intracellular buffering of Ca2+ and its potential arrhythmogenic role in persAF. METHODS: Transmembrane Ca2+ fluxes (patch-clamp) and intracellular Ca2+ signaling (fluo-3-acetoxymethyl ester) were recorded simultaneously in myocytes from right atrial biopsies of sinus rhythm (Ctrl) and patients with persAF, alongside human atrial subtype induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs). Protein levels were quantified by immunoblotting of human atrial tissue and induced pluripotent stem cell-derived cardiac myocytes. Mouse whole heart and atrial electrophysiology were measured on a Langendorff system. RESULTS: Cytosolic Ca2+ buffering was decreased in atrial myocytes of patients with persAF because of a depleted amount of Ca2+ buffers. In agreement, protein levels of selected Ca2+ binding myofilament proteins, including cTnC (cardiac troponin C), a major cytosolic Ca2+ buffer, were significantly lower in patients with persAF. Small interfering RNA (siRNA)-mediated knockdown of cTnC (si-cTNC) in atrial iPSC-CM phenocopied the reduced cytosolic Ca2+ buffering observed in persAF. Si-cTnC treated atrial iPSC-CM exhibited a higher predisposition to spontaneous Ca2+ release events and developed action potential alternans at low stimulation frequencies. Last, indirect reduction of cytosolic Ca2+ buffering using blebbistatin in an ex vivo mouse whole heart model increased vulnerability to tachypacing-induced atrial arrhythmia, validating the direct mechanistic link between impaired cytosolic Ca2+ buffering and atrial arrhythmogenesis. CONCLUSIONS: Our findings suggest that loss of myofilament proteins, particularly reduced cTnC protein levels, causes diminished cytosolic Ca2+ buffering in persAF, thereby potentiating the occurrence of spontaneous Ca2+ release events and atrial fibrillation susceptibility. Strategies targeting intracellular buffering may represent a promising therapeutic lead in persAF management.
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Fibrilação Atrial , Cálcio , Átrios do Coração , Miócitos Cardíacos , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Animais , Cálcio/metabolismo , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Camundongos , Masculino , Células-Tronco Pluripotentes Induzidas/metabolismo , Feminino , Sinalização do Cálcio , Pessoa de Meia-Idade , Idoso , Potenciais de AçãoRESUMO
In striated muscle, the sarcomeric protein myosin-binding protein-C (MyBP-C) is bound to the myosin thick filament and is predicted to stabilize myosin heads in a docked position against the thick filament, which limits crossbridge formation. Here, we use the homozygous Mybpc2 knockout (C2-/-) mouse line to remove the fast-isoform MyBP-C from fast skeletal muscle and then conduct mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers present deficits in force production and calcium sensitivity. Structurally, passive C2-/- fibers present altered sarcomere length-independent and -dependent regulation of myosin head conformations, with a shift of myosin heads towards actin. At shorter sarcomere lengths, the thin filament is axially extended in C2-/-, which we hypothesize is due to increased numbers of low-level crossbridges. These findings provide testable mechanisms to explain the etiology of debilitating diseases associated with MyBP-C.
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Proteínas de Transporte , Camundongos Knockout , Animais , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Camundongos , Sarcômeros/metabolismo , Miofibrilas/metabolismo , Miofibrilas/genética , Músculo Esquelético/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/genética , Masculino , Miosinas/metabolismo , Miosinas/genéticaRESUMO
Background: The global rise of obesity and its association with cardiovascular risk factors (CVRF) have highlighted its connection to chronic heart failure (CHF). Paradoxically, obese CHF patients often experience better outcomes, a phenomenon known as the 'obesity paradox'. This study evaluated the 'obesity paradox' within a large cohort in Germany and explored how varying degrees of obesity affect HF outcome. Methods: Anonymized health claims data from the largest German insurer (AOK) for the years 2014-2015 were utilized to analyze 88,247 patients hospitalized for myocardial infarction. This analysis encompassed baseline characteristics, comorbidities, interventions, complications, and long-term outcomes, including overall survival, freedom from CHF, and CHF-related rehospitalization. Patients were categorized based on body mass index. Results: Obese patients encompassed 21.3% of our cohort (median age 68.69 years); they exhibited a higher prevalence of CVRF (p < 0.001) and comorbidities than non-obese patients (median age 70.69 years). Short-term outcomes revealed lower complication rates and mortality (p < 0.001) in obese compared to non-obese patients. Kaplan-Meier estimations for long-term analysis illustrated increased incidences of CHF and rehospitalization rates among the obese, yet with lower overall mortality. Multivariable Cox regression analysis indicated that obese individuals faced a higher risk of developing CHF and being rehospitalized due to CHF but demonstrated better overall survival for those classified as having low-level obesity (p < 0.001). Conclusions: This study underscores favorable short-term outcomes among obese individuals. The 'obesity paradox' was confirmed, with more frequent CHF cases and rehospitalizations in the long term, alongside better overall survival for certain degrees of obesity.
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Damage of the endothelial glycocalyx (eGC) plays a central role in the development of vascular hyperpermeability and organ damage during systemic inflammation. However, the specific signalling pathways for eGC damage remain poorly defined. Aim of this study was to combine sublingual video-microscopy, plasma proteomics and live cell imaging to uncover further pathways of eGC damage in patients with coronavirus disease 2019 (COVID-19) or bacterial sepsis. This secondary analysis of the prospective multicenter MICROCODE study included 22 patients with COVID-19 and 43 patients with bacterial sepsis admitted to intermediate or intensive care units and 10 healthy controls. Interleukin-6 (IL-6) was strongly associated with damaged eGC and correlated both with eGC dimensions (rs=0.36, p = 0.0015) and circulating eGC biomarkers. In vitro, IL-6 reduced eGC height and coverage, which was inhibited by blocking IL-6 signalling with the anti-IL-6 receptor antibody tocilizumab or the Janus kinase inhibitor tofacitinib. Exposure of endothelial cells to 5% serum from COVID-19 or sepsis patients resulted in a significant decrease in eGC height, which was attenuated by co-incubation with tocilizumab. In an external COVID-19 cohort of 219 patients from Massachusetts General Hospital, a previously identified proteomic eGC signature correlated with IL-6 (rs=-0.58, p < 0.0001) and predicted the combined endpoint of 28-day mortality and/or intubation (ROC-AUC: 0.86 [95% CI: 0.81-0.91], p < 0.001). The data suggest that IL-6 may significantly drive eGC damage in COVID-19 and bacterial sepsis. Our findings provide valuable insights into pathomechanisms of vascular dysfunction during systemic inflammation and highlight the need for further in vivo studies.
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COVID-19 , Glicocálix , Interleucina-6 , Sepse , Humanos , COVID-19/patologia , COVID-19/metabolismo , COVID-19/complicações , Glicocálix/metabolismo , Glicocálix/patologia , Interleucina-6/metabolismo , Interleucina-6/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Sepse/patologia , Sepse/metabolismo , Sepse/complicações , Idoso , Estudos Prospectivos , SARS-CoV-2/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Anticorpos Monoclonais HumanizadosRESUMO
Muscle contraction is produced via the interaction of myofilaments and is regulated so that muscle performance matches demand. Myosin-binding protein C (MyBP-C) is a long and flexible protein that is tightly bound to the thick filament at its C-terminal end (MyBP-CC8C10), but may be loosely bound at its middle- and N-terminal end (MyBP-CC1C7) to myosin heads and/or the thin filament. MyBP-C is thought to control muscle contraction via the regulation of myosin motors, as mutations lead to debilitating disease. We use a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-CC1C7 domains of fast MyBP-C in permeabilized skeletal muscle. We show that cleavage leads to alterations in crossbridge kinetics and passive structural signatures of myofilaments that are indicative of a shift of myosin heads towards the ON state, highlighting the importance of MyBP-CC1C7 to myofilament force production and regulation.
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Proteínas de Transporte , Sarcômeros , Sarcômeros/metabolismo , Proteínas de Transporte/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Miosinas/metabolismoRESUMO
Both diastolic filling and systolic pumping of the heart are dependent on the passive stiffness characteristics of various mechanical elements of myocardium. However, the specific contribution from each element, including the extracellular matrix, actin filaments, microtubules, desmin intermediate filaments, and sarcomeric titin springs, remains challenging to assess. Recently, a mouse model allowing for precise and acute cleavage of the titin springs was used to remove one mechanical element after the other from cardiac fibers and record the effect on passive stiffness. It became clear that the stiffness contribution from each element is context-dependent and varies depending on strain level and the force component considered (elastic or viscous); elements do not act in isolation but in a tensegral relationship. Titin is a substantial contributor under all conditions and dominates the elastic forces at both low and high strains. The contribution to viscous forces is more equally shared between microtubules, titin, and actin. However, the extracellular matrix substantially contributes to both force components at higher strain levels. Desmin filaments may bear low stiffness. These insights enhance our understanding of how different filament networks contribute to passive stiffness in the heart and offer new perspectives for targeting this stiffness in heart failure treatment.
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Proteínas Musculares , Miocárdio , Animais , Camundongos , Conectina , Desmina , CoraçãoRESUMO
In striated muscle, some sarcomere proteins regulate crossbridge cycling by varying the propensity of myosin heads to interact with actin. Myosin-binding protein C (MyBP-C) is bound to the myosin thick filament and is predicted to interact and stabilize myosin heads in a docked position against the thick filament and limit crossbridge formation, the so-called OFF state. Via an unknown mechanism, MyBP-C is thought to release heads into the so-called ON state, where they are more likely to form crossbridges. To study this proposed mechanism, we used the C2-/- mouse line to knock down fast-isoform MyBP-C completely and total MyBP-C by ~24%, and conducted mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers presented deficits in force production and reduced calcium sensitivity. Structurally, passive C2-/- fibers presented altered SL-independent and SL-dependent regulation of myosin head ON/OFF states, with a shift of myosin heads towards the ON state. Unexpectedly, at shorter sarcomere lengths, the thin filament was axially extended in C2-/- vs. non-transgenic controls, which we postulate is due to increased low-level crossbridge formation arising from relatively more ON myosins in the passive muscle that elongates the thin filament. The downstream effect of increasing crossbridge formation in a passive muscle on contraction performance is not known. Such widespread structural changes to sarcomere proteins provide testable mechanisms to explain the etiology of debilitating MyBP-C-associated diseases.
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While chronic heart failure (CHF) treatment has considerably improved patient prognosis and survival, the therapeutic management of acute heart failure (AHF) has remained virtually unchanged in the last decades. This is partly due to the scarcity of pre-clinical models for the pathophysiological assessment and, consequently, the limited knowledge of molecular mechanisms involved in the different AHF phenotypes. This scientific statement outlines the different trajectories from acute to CHF originating from the interaction between aetiology, genetic and environmental factors, and comorbidities. Furthermore, we discuss the potential molecular targets capable of unveiling new therapeutic perspectives to improve the outcome of the acute phase and counteracting the evolution towards CHF.
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Insuficiência Cardíaca , Humanos , Doença Aguda , Prognóstico , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Doença Crônica , Fatores de RiscoRESUMO
The Frank-Starling law states that the heart's stroke volume increases with greater preload due to increased venous return, allowing the heart to adapt to varying circulatory demands. Molecularly, increasing preload increases sarcomere length (SL), which alters sarcomere structures that are correlated to increased calcium sensitivity upon activation. The titin protein, spanning the half-sarcomere, acts as a spring in the I-band, applying a SL-dependent force suggested to pull against and alter myofilaments in a way that supports the Frank-Starling effect. To evaluate this, we employed the titin cleavage (TC) model, where a tobacco-etch virus protease recognition site is inserted into distal I-band titin and allows for rapid, specific cleavage of titin in an otherwise-healthy sarcomere. Here, we evaluated the atomic-level structures of amyopathic cardiac myofilaments following 50% titin cleavage under passive stretch conditions using small-angle X-ray diffraction, which measures these structures under near-physiological (functional) conditions. We report that titin-based forces in permeabilized papillary muscle regulate both thick and thin myofilament structures clearly supporting titin's role in the Frank-Starling mechanism.
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BACKGROUND: Anthracycline-related cardiac toxicity is a recognized consequence of cancer therapies. We assess resting cardiac and skeletal muscle energetics and myocyte, sarcomere, and mitochondrial integrity in patients with breast cancer receiving epirubicin. METHODS: In a prospective, mechanistic, observational, longitudinal study, we investigated chemotherapy-naive patients with breast cancer receiving epirubicin versus sex- and age-matched healthy controls. Resting energetic status of cardiac and skeletal muscle (phosphocreatine/gamma ATP and inorganic phosphate [Pi]/phosphocreatine, respectively) was assessed with 31P-magnetic resonance spectroscopy. Cardiac function and tissue characterization (magnetic resonance imaging and 2D-echocardiography), cardiac biomarkers (serum NT-pro-BNP and high-sensitivity troponin I), and structural assessments of skeletal muscle biopsies were obtained. All study assessments were performed before and after chemotherapy. RESULTS: Twenty-five female patients with breast cancer (median age, 53 years) received a mean epirubicin dose of 304 mg/m2, and 25 age/sex-matched controls were recruited. Despite comparable baseline cardiac and skeletal muscle energetics with the healthy controls, after chemotherapy, patients with breast cancer showed a reduction in cardiac phosphocreatine/gamma ATP ratio (2.0±0.7 versus 1.1±0.5; P=0.001) and an increase in skeletal muscle Pi/phosphocreatine ratio (0.1±0.1 versus 0.2±0.1; P=0.022). This occurred in the context of increases in left ventricular end-systolic and end-diastolic volumes (P=0.009 and P=0.008, respectively), T1 and T2 mapping (P=0.001 and P=0.028, respectively) but with preserved left ventricular ejection fraction, mass and global longitudinal strain, and no change in cardiac biomarkers. There was preservation of the mitochondrial copy number in skeletal muscle biopsies but a significant increase in areas of skeletal muscle degradation (P=0.001) in patients with breast cancer following chemotherapy. Patients with breast cancer demonstrated a reduction in skeletal muscle sarcomere number from the prechemotherapy stage compared with healthy controls (P=0.013). CONCLUSIONS: Contemporary doses of epirubicin for breast cancer treatment result in a significant reduction of cardiac and skeletal muscle high-energy 31P-metabolism alongside structural skeletal muscle changes. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT04467411.
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Antraciclinas , Antibióticos Antineoplásicos , Neoplasias da Mama , Epirubicina , Feminino , Humanos , Pessoa de Meia-Idade , Trifosfato de Adenosina , Antraciclinas/efeitos adversos , Antibióticos Antineoplásicos/efeitos adversos , Biomarcadores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Epirubicina/efeitos adversos , Estudos Longitudinais , Músculo Esquelético/diagnóstico por imagem , Fosfocreatina , Estudos Prospectivos , Volume Sistólico , Função Ventricular EsquerdaRESUMO
Contraction force in muscle is produced by the interaction of myosin motors in the thick filaments and actin in the thin filaments and is fine-tuned by other proteins such as myosin-binding protein C (MyBP-C). One form of control is through the regulation of myosin heads between an ON and OFF state in passive sarcomeres, which leads to their ability or inability to interact with the thin filaments during contraction, respectively. MyBP-C is a flexible and long protein that is tightly bound to the thick filament at its C-terminal end but may be loosely bound at its middle- and N-terminal end (MyBP-CC1C7). Under considerable debate is whether the MyBP-CC1C7 domains directly regulate myosin head ON/OFF states, and/or link thin filaments ("C-links"). Here, we used a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-CC1C7 domains of fast MyBP-C in permeabilized skeletal muscle. After cleavage, the thin filaments were significantly shorter, a result consistent with direct interactions of MyBP-C with thin filaments thus confirming C-links. Ca2+ sensitivity was reduced at shorter sarcomere lengths, and crossbridge kinetics were increased across sarcomere lengths at submaximal activation levels, demonstrating a role in crossbridge kinetics. Structural signatures of the thick filaments suggest that cleavage also shifted myosin heads towards the ON state - a marker that typically indicates increased Ca2+ sensitivity but that may account for increased crossbridge kinetics at submaximal Ca2+ and/or a change in the force transmission pathway. Taken together, we conclude that MyBP-CC1C7 domains play an important role in contractile performance which helps explain why mutations in these domains often lead to debilitating diseases.
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BACKGROUND: Increasing cardiomyocyte contraction during myocardial stretch serves as the basis for the Frank-Starling mechanism in the heart. However, it remains unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres. We investigated sarcomere contractile synchrony and how intersarcomere dynamics contribute to increasing contractility during cell lengthening. METHODS: Sarcomere strain and Ca2+ were simultaneously recorded in isolated left ventricular cardiomyocytes during 1 Hz field stimulation at 37 °C, at resting length and following stepwise stretch. RESULTS: We observed that in unstretched rat cardiomyocytes, differential sarcomere deformation occurred during each beat. Specifically, while most sarcomeres shortened during the stimulus, ≈10% to 20% of sarcomeres were stretched or remained stationary. This nonuniform strain was not traced to regional Ca2+ disparities but rather shorter resting lengths and lower force production in systolically stretched sarcomeres. Lengthening of the cell recruited additional shortening sarcomeres, which increased contractile efficiency as less negative, wasted work was performed by stretched sarcomeres. Given the known role of titin in setting sarcomere dimensions, we next hypothesized that modulating titin expression would alter intersarcomere dynamics. Indeed, in cardiomyocytes from mice with titin haploinsufficiency, we observed greater variability in resting sarcomere length, lower recruitment of shortening sarcomeres, and impaired work performance during cell lengthening. CONCLUSIONS: Graded sarcomere recruitment directs cardiomyocyte work performance, and harmonization of sarcomere strain increases contractility during cell stretch. By setting sarcomere dimensions, titin controls sarcomere recruitment, and its lowered expression in haploinsufficiency mutations impairs cardiomyocyte contractility.
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Miócitos Cardíacos , Sarcômeros , Ratos , Camundongos , Animais , Sarcômeros/metabolismo , Conectina/genética , Conectina/metabolismo , Miócitos Cardíacos/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/metabolismoRESUMO
Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking.
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Transdução de Sinais , Linfócitos T , Humanos , Conectina/metabolismo , Adesão Celular/fisiologia , Isoformas de Proteínas/metabolismo , Ativação LinfocitáriaRESUMO
Myofibrillar myopathies (MFM) are a group of chronic muscle diseases pathophysiologically characterized by accumulation of protein aggregates and structural failure of muscle fibers. A subtype of MFM is caused by heterozygous mutations in the filamin C (FLNC) gene, exhibiting progressive muscle weakness, muscle structural alterations and intracellular protein accumulations. Here, we characterize in depth the pathogenicity of two novel truncating FLNc variants (p.Q1662X and p.Y2704X) and assess their distinct effect on FLNc stability and distribution as well as their impact on protein quality system (PQS) pathways. Both variants cause a slowly progressive myopathy with disease onset in adulthood, chronic myopathic alterations in muscle biopsy including the presence of intracellular protein aggregates. Our analyses revealed that p.Q1662X results in FLNc haploinsufficiency and p.Y2704X in a dominant-negative FLNc accumulation. Moreover, both protein-truncating variants cause different PQS alterations: p.Q1662X leads to an increase in expression of several genes involved in the ubiquitin-proteasome system (UPS) and the chaperone-assisted selective autophagy (CASA) system, whereas p.Y2704X results in increased abundance of proteins involved in UPS activation and autophagic buildup. We conclude that truncating FLNC variants might have different pathogenetic consequences and impair PQS function by diverse mechanisms and to varying extents. Further studies on a larger number of patients are necessary to confirm our observations.
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Miopatias Congênitas Estruturais , Agregados Proteicos , Humanos , Filaminas/genética , Filaminas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismoRESUMO
Palladin (PALLD) belongs to the PALLD/myopalladin (MYPN)/myotilin family of actin-associated immunoglobulin-containing proteins in the sarcomeric Z-line. PALLD is ubiquitously expressed in several isoforms, and its longest 200 kDa isoform, predominantly expressed in striated muscle, shows high structural homology to MYPN. MYPN gene mutations are associated with human cardiomyopathies, whereas the role of PALLD in the heart has remained unknown, partly due to embryonic lethality of PALLD knockout mice. In a yeast two-hybrid screening, CARP/Ankrd1 and FHOD1 were identified as novel interaction partners of PALLD's N-terminal region. To study the role of PALLD in the heart, we generated conditional (cPKO) and inducible (cPKOi) cardiomyocyte-specific PALLD knockout mice. While cPKO mice exhibited no pathological phenotype, ablation of PALLD in adult cPKOi mice caused progressive cardiac dilation and systolic dysfunction, associated with reduced cardiomyocyte contractility, intercalated disc abnormalities, and fibrosis, demonstrating that PALLD is essential for normal cardiac function. Double cPKO and MYPN knockout (MKO) mice exhibited a similar phenotype as MKO mice, suggesting that MYPN does not compensate for the loss of PALLD in cPKO mice. Altered transcript levels of MYPN and PALLD isoforms were found in myocardial tissue from human dilated and ischemic cardiomyopathy patients, whereas their protein expression levels were unaltered.
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Cardiomiopatias , Cardiomiopatia Dilatada , Proteínas do Citoesqueleto , Animais , Humanos , Camundongos , Cardiomiopatias/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Camundongos Knockout , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Isoformas de Proteínas/genéticaRESUMO
In muscle, titin proteins connect myofilaments together and are thought to be critical for contraction, especially during residual force enhancement (RFE) when force is elevated after an active stretch. We investigated titin's function during contraction using small-angle X-ray diffraction to track structural changes before and after 50% titin cleavage and in the RFE-deficient, mdm titin mutant. We report that the RFE state is structurally distinct from pure isometric contractions, with increased thick filament strain and decreased lattice spacing, most likely caused by elevated titin-based forces. Furthermore, no RFE structural state was detected in mdm muscle. We posit that decreased lattice spacing, increased thick filament stiffness, and increased non-crossbridge forces are the major contributors to RFE. We conclude that titin directly contributes to RFE.