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Sigma 1 receptor (Sigmar1) is a widely expressed, multitasking molecular chaperone protein that plays functional roles in several cellular processes. Mutations in the Sigmar1 gene are associated with several distal neuropathies with strong manifestation in skeletal muscle dysfunction with phenotypes like muscle wasting and atrophy. However, the physiological function of Sigmar1 in skeletal muscle remains unknown. Herein, the physiological role of Sigmar1 in skeletal muscle structure and function in gastrocnemius, quadriceps, soleus, extensor digitorum longus, and tibialis anterior muscles was determined. Quantification of myofiber cross-sectional area showed altered myofiber size distribution and changes in myofiber type in the skeletal muscle of the Sigmar1-/- mice. Interestingly, ultrastructural analysis by transmission electron microscopy showed the presence of abnormal mitochondria, and immunostaining showed derangements in dystrophin localization in skeletal muscles from Sigmar1-/- mice. In addition, myopathy in Sigmar1-/- mice was associated with an increased number of central nuclei, increased collagen deposition, and fibrosis. Functional studies also showed reduced endurance and exercise capacity in the Sigmar1-/- mice without any changes in voluntary locomotion, markers for muscle denervation, and muscle atrophy. Overall, this study shows, for the first time, a potential physiological function of Sigmar1 in maintaining healthy skeletal muscle structure and function.
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Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Receptores sigma/deficiencia , Animales , Colágeno/metabolismo , Distrofina/metabolismo , Fibrosis , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/ultraestructura , Condicionamiento Físico Animal , Transporte de Proteínas , Receptores sigma/metabolismo , Receptor Sigma-1RESUMEN
The molecular architecture of pH-responsive amphiphilic block copolymers, their self-assembly behavior to form nanoparticles (NPs), and doxorubicin (DOX)-loading technique govern the extent of DOX-induced cardiotoxicity. We observed that the choice of pH-sensitive tertiary amines, surface charge, and DOX-loading techniques within the self-assembled NPs strongly influence the release and stimulation of DOX-induced cardiotoxicity in primary cardiomyocytes. However, covalent conjugation of DOX to a pH-sensitive nanocarrier through a "conditionally unstable amide" linkage (PCPY-cDOX; PC = polycarbonate and PY = 2-pyrrolidine-1-yl-ethyl-amine) significantly reduced the cardiotoxicity of DOX in cardiomyocytes as compared to noncovalently encapsulated DOX NPs (PCPY-eDOX). When these formulations were tested for drug release in serum-containing media, the PCPY-cDOX systems showed prolonged control over drug release (for â¼72 h) at acidic pH compared to DOX-encapsulated nanocarriers, as expected. We found that DOX-encapsulated nanoformulations triggered cardiotoxicity in primary cardiomyocytes more acutely, while conjugated systems such as PCPY-cDOX prevented cardiotoxicity by disabling the nuclear entry of the drug. Using 2D and 3D (spheroid) cultures of an ER + breast cancer cell line (MCF-7) and a triple-negative breast cancer cell line (MDA-MB-231), we unravel that, similar to encapsulated systems (PCPY-eDOX-type) as reported earlier, the PCPY-cDOX system suppresses cellular proliferation in both cell lines and enhances trafficking through 3D spheroids of MDA-MB-231 cells. Collectively, our studies indicate that PCPY-cDOX is less cardiotoxic as compared to noncovalently encapsulated variants without compromising the chemotherapeutic properties of the drug. Thus, our studies suggest that the appropriate selection of the nanocarrier for DOX delivery may prove fruitful in shifting the balance between low cardiotoxicity and triggering the chemotherapeutic potency of DOX.
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Cardiotoxicidad/prevención & control , Doxorrubicina/administración & dosificación , Portadores de Fármacos/química , Neoplasias/tratamiento farmacológico , Polímeros/química , Animales , Animales Recién Nacidos , Cardiotoxicidad/etiología , Línea Celular Tumoral , Doxorrubicina/farmacocinética , Doxorrubicina/toxicidad , Composición de Medicamentos/métodos , Liberación de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración de Iones de Hidrógeno , Miocitos Cardíacos , Nanopartículas/química , Neoplasias/patología , Cemento de Policarboxilato , Cultivo Primario de Células , Pirrolidinas/química , Ratas , Esferoides Celulares , Pruebas de Toxicidad AgudaRESUMEN
Background: Peripheral artery disease (PAD) is on the rise worldwide, ranking as the third leading cause of atherosclerosis-related morbidity; much less is known about its trends in hospitalizations among methamphetamine and cocaine users. Objectives: We aim to evaluate the overall trend in the prevalence of hospital admission for PAD with or without the use of stimulant abuse (methamphetamine and cocaine) across the United States. Additionally, we evaluated the PAD-related hospitalizations trend stratified by age, race, sex, and geographic location. Methods: We used the National Inpatient Sample (NIS) database from 2008 to 2020. The Cochran Armitage trend test was used to compare the trend between groups. Multivariate logistic regression was used to examine adjusted odds for PAD and CLI hospitalizations among methamphetamine and cocaine users. Results: Between 2008 and 2020, PAD-related hospitalizations showed an increasing trend in Hispanics, African Americans, and western states, while a decreasing trend in southern and Midwestern states (p-trend <0.05). Among methamphetamine users, an overall increasing trend was observed in men, women, western, southern, and midwestern states (p-trend <0.05). However, among cocaine users, PAD-related hospitalization increased significantly for White, African American, age group >64 years, southern and western states (p-trend <0.05). Overall, CLI-related hospitalizations showed an encouraging decreasing trend in men and women, age group >64 years, and CLI-related amputations declined for women, White patient population, age group >40, and all regions (p-trend <0.05). However, among methamphetamine users, a significantly increasing trend in CLI-related hospitalization was seen in men, women, White & Hispanic population, age group 26-45, western, southern, and midwestern regions. Conclusions: There was an increasing trend in PAD-related hospitalizations among methamphetamine and cocaine users for both males and females. Although an overall decreasing trend in CLI-related hospitalization was observed for both genders, an up-trend in CLI was seen among methamphetamine users. The upward trends were more prominent for White, Hispanic & African Americans, and southern and western states, highlighting racial and geographic variations over the study period.
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Alzheimer's Disease (AD) is a progressive neurodegenerative disease caused by the deposition of Aß aggregates or neurofibrillary tangles. AD patients are primarily diagnosed with the concurrent development of several cardiovascular dysfunctions. While few studies have indicated the presence of intramyocardial Aß aggregates, none of the studies have performed detailed analyses for pathomechanism of cardiac dysfunction in AD patients. This manuscript used aged APPSWE/PS1 Tg and littermate age-matched wildtype (Wt) mice to characterize cardiac dysfunction and analyze associated pathophysiology. Detailed assessment of cardiac functional parameters demonstrated the development of diastolic dysfunction in APPSWE/PS1 Tg hearts compared to Wt hearts. Muscle function evaluation showed functional impairment (decreased exercise tolerance and muscle strength) in APPSWE/PS1 Tg mice. Biochemical and histochemical analysis revealed Aß aggregate accumulation in APPSWE/PS1 Tg mice myocardium. APPSWE/PS1 Tg mice hearts also demonstrated histopathological remodeling (increased collagen deposition and myocyte cross-sectional area). Additionally, APPSWE/PS1 Tg hearts showed altered mitochondrial dynamics, reduced antioxidant protein levels, and impaired mitochondrial proteostasis compared to Wt mice. APPSWE/PS1 Tg hearts also developed mitochondrial dysfunction with decreased OXPHOS and PDH protein complex expressions, altered ETC complex dynamics, decreased complex activities, and reduced mitochondrial respiration. Our results indicated that Aß aggregates in APPSWE/PS1 Tg hearts are associated with defects in mitochondrial respiration and complex activities, which may collectively lead to cardiac diastolic dysfunction and myocardial pathological remodeling.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Modelos Animales de Enfermedad , Ratones Transgénicos , Animales , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Ratones , Péptidos beta-Amiloides/metabolismo , Miocardio/metabolismo , Miocardio/patología , Mitocondrias/metabolismo , Diástole , Humanos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , MasculinoRESUMEN
Sigmar1 is a ubiquitously expressed, multifunctional protein known for its cardioprotective roles in cardiovascular diseases. While accumulating evidence indicate a critical role of Sigmar1 in cardiac biology, its physiological function in the vasculature remains unknown. In this study, we characterized the expression of Sigmar1 in the vascular wall and assessed its physiological function in the vascular system using global Sigmar1 knockout (Sigmar1-/-) mice. We determined the expression of Sigmar1 in the vascular tissue using immunostaining and biochemical experiments in both human and mouse blood vessels. Deletion of Sigmar1 globally in mice (Sigmar1-/-) led to blood vessel wall reorganizations characterized by nuclei disarray of vascular smooth muscle cells, altered organizations of elastic lamina, and higher collagen fibers deposition in and around the arteries compared to wildtype littermate controls (Wt). Vascular function was assessed in mice using non-invasive time-transit method of aortic stiffness measurement and flow-mediated dilation (FMD) of the left femoral artery. Sigmar1-/- mice showed a notable increase in arterial stiffness in the abdominal aorta and failed to increase the vessel diameter in response to reactive-hyperemia compared to Wt. This was consistent with reduced plasma and tissue nitric-oxide bioavailability (NOx) and decreased phosphorylation of endothelial nitric oxide synthase (eNOS) in the aorta of Sigmar1-/- mice. Ultrastructural analysis by transmission electron microscopy (TEM) of aorta sections showed accumulation of elongated shaped mitochondria in both vascular smooth muscle and endothelial cells of Sigmar1-/- mice. In accordance, decreased mitochondrial respirometry parameters were found in ex-vivo aortic rings from Sigmar1 deficient mice compared to Wt controls. These data indicate a potential role of Sigmar1 in maintaining vascular homeostasis.
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ABSTRACT: Venous thromboembolic events are significant contributors to morbidity and mortality in patients with stroke. Neutrophils are among the first cells in the blood to respond to stroke and are known to promote deep vein thrombosis (DVT). Integrin α9 is a transmembrane glycoprotein highly expressed on neutrophils and stabilizes neutrophil adhesion to activated endothelium via vascular cell adhesion molecule 1 (VCAM-1). Nevertheless, the causative role of neutrophil integrin α9 in poststroke DVT remains unknown. Here, we found higher neutrophil integrin α9 and plasma VCAM-1 levels in humans and mice with stroke. Using mice with embolic stroke, we observed enhanced DVT severity in a novel model of poststroke DVT. Neutrophil-specific integrin α9-deficient mice (α9fl/flMrp8Cre+/-) exhibited a significant reduction in poststroke DVT severity along with decreased neutrophils and citrullinated histone H3 in thrombi. Unbiased transcriptomics indicated that α9/VCAM-1 interactions induced pathways related to neutrophil inflammation, exocytosis, NF-κB signaling, and chemotaxis. Mechanistic studies revealed that integrin α9/VCAM-1 interactions mediate neutrophil adhesion at the venous shear rate, promote neutrophil hyperactivation, increase phosphorylation of extracellular signal-regulated kinase, and induce endothelial cell apoptosis. Using pharmacogenomic profiling, virtual screening, and in vitro assays, we identified macitentan as a potent inhibitor of integrin α9/VCAM-1 interactions and neutrophil adhesion to activated endothelial cells. Macitentan reduced DVT severity in control mice with and without stroke, but not in α9fl/flMrp8Cre+/- mice, suggesting that macitentan improves DVT outcomes by inhibiting neutrophil integrin α9. Collectively, we uncovered a previously unrecognized and critical pathway involving the α9/VCAM-1 axis in neutrophil hyperactivation and DVT.
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Integrinas , Neutrófilos , Accidente Cerebrovascular , Molécula 1 de Adhesión Celular Vascular , Trombosis de la Vena , Animales , Humanos , Masculino , Ratones , Adhesión Celular , Modelos Animales de Enfermedad , Integrinas/metabolismo , Ratones Noqueados , Activación Neutrófila , Neutrófilos/metabolismo , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/etiología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Trombosis de la Vena/metabolismo , Trombosis de la Vena/etiologíaRESUMEN
Sigma1 receptor protein (Sigmar1) is a small, multifunctional molecular chaperone protein ubiquitously expressed in almost all body tissues. This protein has previously shown its cardioprotective roles in rodent models of cardiac hypertrophy, heart failure, and ischemia-reperfusion injury. Extensive literature also suggested its protective functions in several central nervous system disorders. Sigmar1's molecular functions in the pulmonary system remained unknown. Therefore, we aimed to determine the expression of Sigmar1 in the lungs. We also examined whether Sigmar1 ablation results in histological, ultrastructural, and biochemical changes associated with lung pathology over aging in mice. In the current study, we first confirmed the presence of Sigmar1 protein in human and mouse lungs using immunohistochemistry and immunostaining. We used the Sigmar1 global knockout mouse (Sigmar1-/-) to determine the pathophysiological role of Sigmar1 in lungs over aging. The histological staining of lung sections showed altered alveolar structures, higher immune cells infiltration, and upregulation of inflammatory markers (such as pNFκB) in Sigmar1-/- mice compared to wildtype (Wt) littermate control mice (Wt). This indicates higher pulmonary inflammation resulting from Sigmar1 deficiency in mice, which was associated with increased pulmonary fibrosis. The protein levels of some fibrotic markers, fibronectin, and pSMAD2 Ser 245/250/255 and Ser 465/467, were also elevated in mice lungs in the absence of Sigmar1 compared to Wt. The ultrastructural analysis of lungs in Wt mice showed numerous multilamellar bodies of different sizes with densely packed lipid lamellae and mitochondria with a dark matrix and dense cristae. In contrast, the Sigmar1-/- mice lung tissues showed altered multilamellar body structures in alveolar epithelial type-II pneumocytes with partial loss of lipid lamellae structures in the lamellar bodies. This was further associated with higher protein levels of all four surfactant proteins, SFTP-A, SFTP-B, SFTP-C, and SFTP-D, in the Sigmar1-/- mice lungs. This is the first study showing Sigmar1's expression pattern in human and mouse lungs and its association with lung pathophysiology. Our findings suggest that Sigmar1 deficiency leads to increased pulmonary inflammation, higher pulmonary fibrosis, alterations of the multilamellar body stuructures, and elevated levels of lung surfactant proteins.
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Amyotrophic lateral sclerosis (ALS) is a complex systemic disease that primarily involves motor neuron dysfunction and skeletal muscle atrophy. One commonly used mouse model to study ALS was generated by transgenic expression of a mutant form of human superoxide dismutase 1 (SOD1) gene harboring a single amino acid substitution of glycine to alanine at codon 93 (G93A*SOD1). Although mutant-SOD1 is ubiquitously expressed in G93A*SOD1 mice, a detailed analysis of the skeletal muscle expression pattern of the mutant protein and the resultant muscle pathology were never performed. Using different skeletal muscles isolated from G93A*SOD1 mice, we extensively characterized the pathological sequelae of histological, molecular, ultrastructural, and biochemical alterations. Muscle atrophy in G93A*SOD1 mice was associated with increased and differential expression of mutant-SOD1 across myofibers and increased MuRF1 protein level. In addition, high collagen deposition and myopathic changes sections accompanied the reduced muscle strength in the G93A*SOD1 mice. Furthermore, all the muscles in G93A*SOD1 mice showed altered protein levels associated with different signaling pathways, including inflammation, mitochondrial membrane transport, mitochondrial lipid uptake, and antioxidant enzymes. In addition, the mutant-SOD1 protein was found in the mitochondrial fraction in the muscles from G93A*SOD1 mice, which was accompanied by vacuolized and abnormal mitochondria, altered OXPHOS and PDH complex protein levels, and defects in mitochondrial respiration. Overall, we reported the pathological sequelae observed in the skeletal muscles of G93A*SOD1 mice resulting from the whole-body mutant-SOD1 protein expression.
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The subcellular localization is critical to delineating proper function and determining the molecular mechanisms of a particular protein. Several qualitative and quantitative techniques are used to determine the subcellular localization of proteins. One of the emerging techniques in determining the subcellular localization of a protein is quantum dots (QD)-mediated immunolabeling of a protein followed by imaging them with transmission electron microscopy (TEM). QD is a semiconductor nanocrystal with a dual property of crystalline structure and high electron density, which makes them applicable to electron microscopy. This current method visualized the subcellular localization of Sigma 1 receptor (Sigmar1) protein using QD-TEM in the heart tissue at ultrastructural level. Small cubes of the heart tissue sections from a wild-type mouse were fixed in 3% glutaraldehyde, subsequently osmicated, stained with uranyl acetate, followed by sequential dehydration with ethanol and acetone. These dehydrated heart tissue sections were embedded in low-viscosity epoxy resins, cut into thin sections of 500 nm thickness, put on the grid, and subsequently subjected to antigen unmasking with 5% sodium metaperiodate, followed by quenching of the residual aldehydes with glycine. The tissues were blocked, followed by sequential incubation in primary antibody, biotinylated secondary antibody, and streptavidin-conjugated QD. These stained sections were blot dried and imaged at high magnification using TEM. The QD-TEM technique allowed the visualization of Sigmar1 protein's subcellular localization at the ultrastructural level in the heart. These techniques can be used to visualize the presence of any protein and subcellular localization in any organ system.
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Puntos Cuánticos , Acetona , Animales , Resinas Epoxi , Etanol , Glutaral , Glicina , Ratones , Microscopía Electrónica de Transmisión , EstreptavidinaRESUMEN
Doxorubicin (DOX)-induced cardiomyopathy constitutes dose-dependent cardiac toxicity, culminating in fatal heart failure progression. Cardiac toxicity limits effective and subsequent use of DOX in chemotherapy regimens in pediatric, adult, and recurrent cancer patients. DOX-induced profound alterations in mitochondrial morphology, dynamics, and defects in mitochondrial energy metabolism in the heart comprise key stressors in DOX-induced cardiotoxicity. Hence, the discovery of novel molecular targets and therapeutics to mitigate DOX-induced mitochondrial dysfunctions are imperative. Herein, we provided two laboratory protocols to monitor DOX-induced alterations in mitochondrial morphology and respiration in isolated primary neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes are extensively used to monitor signaling mechanisms regulating cardiomyopathy in vitro. Therefore, these protocols will help researchers study the effects of novel pharmacological and genetic manipulations against DOX-induced alterations in mitochondrial morphology and energy metabolism in cardiomyocytes.
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Cardiomiopatías , Cardiotoxicidad , Animales , Antibióticos Antineoplásicos/efectos adversos , Apoptosis , Cardiomiopatías/inducido químicamente , Cardiomiopatías/metabolismo , Doxorrubicina/efectos adversos , Humanos , Miocitos Cardíacos/metabolismo , Ratas , RespiraciónRESUMEN
The recent rise in illicit use of methamphetamine (METH), a highly addictive psychostimulant, is a huge health care burden due to its central and peripheral toxic effects. Mounting clinical studies have noted that METH use in humans is associated with the development of cardiomyopathy; however, preclinical studies and animal models to dissect detailed molecular mechanisms of METH-associated cardiomyopathy development are scarce. The present study utilized a unique very long-access binge and crash procedure of METH self-administration to characterize the sequelae of pathological alterations that occur with METH-associated cardiomyopathy. Rats were allowed to intravenously self-administer METH for 96 h continuous weekly sessions over 8 weeks. Cardiac function, histochemistry, ultrastructure, and biochemical experiments were performed 24 h after the cessation of drug administration. Voluntary METH self-administration induced pathological cardiac remodeling as indicated by cardiomyocyte hypertrophy, myocyte disarray, interstitial and perivascular fibrosis accompanied by compromised cardiac systolic function. Ultrastructural examination and native gel electrophoresis revealed altered mitochondrial morphology and reduced mitochondrial oxidative phosphorylation (OXPHOS) supercomplexes (SCs) stability and assembly in METH exposed hearts. Redox-sensitive assays revealed significantly attenuated mitochondrial respiratory complex activities with a compensatory increase in pyruvate dehydrogenase (PDH) activity reminiscent of metabolic remodeling. Increased autophagy flux and increased mitochondrial antioxidant protein level was observed in METH exposed heart. Treatment with mitoTEMPO reduced the autophagy level indicating the involvement of mitochondrial dysfunction in the adaptive activation of autophagy in METH exposed hearts. Altogether, we have reported a novel METH-associated cardiomyopathy model using voluntary drug seeking behavior. Our studies indicated that METH self-administration profoundly affects mitochondrial ultrastructure, OXPHOS SCs assembly and redox activity accompanied by increased PDH activity that may underlie observed cardiac dysfunction.
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Cardiomiopatías , Estimulantes del Sistema Nervioso Central , Metanfetamina , Humanos , Ratas , Animales , Metanfetamina/toxicidad , Estimulantes del Sistema Nervioso Central/farmacología , Autofagia , MitocondriasRESUMEN
Sigmar1 is a widely expressed molecular chaperone protein in mammalian cell systems. Accumulating research demonstrated the cardioprotective roles of pharmacologic Sigmar1 activation by ligands in preclinical rodent models of cardiac injury. Extensive biochemical and immuno-electron microscopic research demonstrated Sigmar1's sub-cellular localization largely depends on cell and organ types. Despite comprehensive studies, Sigmar1's direct molecular role in cardiomyocytes remains elusive. In the present study, we determined Sigmar1's subcellular localization, transmembrane topology, and function using complementary microscopy, biochemical, and functional assays in cardiomyocytes. Quantum dots in transmission electron microscopy showed Sigmar1 labeled quantum dots on the mitochondrial membranes, lysosomes, and sarcoplasmic reticulum-mitochondrial interface. Subcellular fractionation of heart cell lysates confirmed Sigmar1's localization in purified mitochondria fraction and lysosome fraction. Immunocytochemistry confirmed Sigmar1 colocalization with mitochondrial proteins in isolated adult mouse cardiomyocytes. Sigmar1's mitochondrial localization was further confirmed by Sigmar1 colocalization with Mito-Tracker in isolated mouse heart mitochondria. A series of biochemical experiments, including alkaline extraction and proteinase K treatment of purified heart mitochondria, demonstrated Sigmar1 as an integral mitochondrial membrane protein. Sigmar1's structural requirement for mitochondrial localization was determined by expressing FLAG-tagged Sigmar1 fragments in cells. Full-length Sigmar1 and Sigmar1's C terminal-deletion fragments were able to localize to the mitochondrial membrane, whereas N-terminal deletion fragment was unable to incorporate into the mitochondria. Finally, functional assays using extracellular flux analyzer and high-resolution respirometry showed Sigmar1 siRNA knockdown significantly altered mitochondrial respiration in cardiomyocytes. Overall, we found that Sigmar1 localizes to mitochondrial membranes and is indispensable for maintaining mitochondrial respiratory homeostasis in cardiomyocytes.
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Mitocondrias Cardíacas/fisiología , Miocitos Cardíacos/metabolismo , Transporte de Proteínas/fisiología , Receptores sigma/metabolismo , Animales , Metabolismo Energético/fisiología , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Masculino , Ratones , ARN Interferente Pequeño , Ratas , Receptores sigma/genética , Receptor Sigma-1RESUMEN
The Sigma 1 receptor (Sigmar1) is a ubiquitously expressed multifunctional inter-organelle signaling chaperone protein playing a diverse role in cellular survival. Recessive mutation in Sigmar1 have been identified as a causative gene for neuronal and neuromuscular disorder. Since the discovery over 40 years ago, Sigmar1 has been shown to contribute to numerous cellular functions, including ion channel regulation, protein quality control, endoplasmic reticulum-mitochondrial communication, lipid metabolism, mitochondrial function, autophagy activation, and involved in cellular survival. Alterations in Sigmar1's subcellular localization, expression, and signaling has been implicated in the progression of a wide range of diseases, such as neurodegenerative diseases, ischemic brain injury, cardiovascular diseases, diabetic retinopathy, cancer, and drug addiction. The goal of this review is to summarize the current knowledge of Sigmar1 biology focusing the recent discoveries on Sigmar1's molecular, cellular, pathophysiological, and biological functions.
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Mitochondria are the key to properly functioning energy generation in the metabolically demanding cardiomyocytes and thus essential to healthy heart contractility on a beat-to-beat basis. Mitochondria being the central organelle for cellular metabolism and signaling in the heart, its dysfunction leads to cardiovascular disease. The healthy mitochondrial functioning critical to maintaining cardiomyocyte viability and contractility is accomplished by adaptive changes in the dynamics, biogenesis, and degradation of the mitochondria to ensure cellular proteostasis. Recent compelling evidence suggests that the classical protein quality control system in cardiomyocytes is also under constant mitochondrial control, either directly or indirectly. Impairment of cytosolic protein quality control may affect the position of the mitochondria in relation to other organelles, as well as mitochondrial morphology and function, and could also activate mitochondrial proteostasis. Despite a growing interest in the mitochondrial quality control system, very little information is available about the molecular function of mitochondria in cardiac proteostasis. In this review, we bring together current understanding of the adaptations and role of the mitochondria in cardiac proteostasis and describe the adaptive/maladaptive changes observed in the mitochondrial network required to maintain proteomic integrity. We also highlight the key mitochondrial signaling pathways activated in response to proteotoxic stress as a cellular mechanism to protect the heart from proteotoxicity. A deeper understanding of the molecular mechanisms of mitochondrial adaptations and their role in cardiac proteostasis will help to develop future therapeutics to protect the heart from cardiovascular diseases.
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Background The mutated α-B-Crystallin (CryABR120G) mouse model of desmin-related myopathy (DRM) shows an age-dependent onset of pathologic cardiac remodeling and progression of heart failure. CryABR120G expression in cardiomyocytes affects the mitochondrial spatial organization within the myofibrils, but the molecular perturbation within the mitochondria in the relation of the overall course of the proteotoxic disease remains unclear. Methods and Results CryABR120G mice show an accumulation of electron-dense aggregates and myofibrillar degeneration associated with the development of cardiac dysfunction. Though extensive studies demonstrated that these altered ultrastructural changes cause cardiac contractility impairment, the molecular mechanism of cardiomyocyte death remains elusive. Here, we explore early pathological processes within the mitochondria contributing to the contractile dysfunction and determine the pathogenic basis for the heart failure observed in the CryABR120G mice. In the present study, we report that the CryABR120G mice transgenic hearts undergo altered mitochondrial dynamics associated with increased level of dynamin-related protein 1 and decreased level of optic atrophy type 1 as well as mitofusin 1 over the disease process. In association with these changes, an altered level of the components of mitochondrial oxidative phosphorylation and pyruvate dehydrogenase complex regulatory proteins occurs before the manifestation of pathologic adverse remodeling in the CryABR120G hearts. Mitochondria isolated from CryABR120G transgenic hearts without visible pathology show decreased electron transport chain complex activities and mitochondrial respiration. Taken together, we demonstrated the involvement of mitochondria in the pathologic remodeling and progression of DRM-associated cellular dysfunction. Conclusions Mitochondrial dysfunction in the form of altered mitochondrial dynamics, oxidative phosphorylation and pyruvate dehydrogenase complex proteins level, abnormal electron transport chain complex activities, and mitochondrial respiration are evident on the CryABR120G hearts before the onset of detectable pathologies and development of cardiac contractile dysfunction.
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Cardiomiopatías/etiología , Cardiomiopatías/patología , Dinámicas Mitocondriales/fisiología , Fosforilación Oxidativa , Animales , Cardiomiopatías/metabolismo , Desmina , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Cadena B de alfa-CristalinaRESUMEN
Mitochondria are highly dynamic organelles that constantly undergo fission and fusion events to adapt to changes in the cellular environment. Aberrant mitochondrial fission has been associated with several types of cardiovascular dysfunction; inhibition of pathologically aberrant mitochondrial fission has been shown to be cardioprotective. Pathological fission is mediated by the excessive activation of GTPase dynamin-related protein 1 (Drp1), making it an attractive therapeutic target in numerous cardiovascular diseases. Mitochondrial division inhibitor (mdivi-1) is widely used small molecule reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate injury. The purpose of our study was to understand the pleiotropic effects of mdivi-1 on mitochondrial dynamics, mitochondrial respiration, electron transport activities, and macro-autophagy. In this study, we found that mdivi-1 treatment decreased Drp1 expression, proteolytically cleaved L-OPA1, and altered the expression of OXPHOS complex proteins, resulting in increased superoxide production. The altered expression of OXPHOS complex proteins may be directly associated with decreased Drp1 expression, as Drp1 siRNA knockdown in cardiomyocytes showed similar effects. Results from an autophagy flux assay showed that mdivi-1 induced impaired autophagy flux that could be restored by Atg7 overexpression, suggesting that mdivi-1 mediated inhibition of macro-autophagy in cardiomyocytes. Treatment with mdivi-1 resulted in increased expression of p62, which is required for Atg7 overexpression-induced rescue of mdivi-1-mediated impaired autophagy flux. In addition, mdivi-1-dependent proteolytic processing of L-OPA1 was associated with increased mitochondrial superoxide production and altered expression of mitochondrial serine/proteases. Overall, the novel pleiotropic effect of mdivi-1 in cardiomyocytes included proteolytically cleaved L-OPA1, altered expression of OXPHOS complex proteins, and increased superoxide production, which together resulted in defects in mitochondrial respiration and inhibition of macro-autophagy.
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Dinámicas Mitocondriales , Miocitos Cardíacos , Autofagia , Dinaminas/genética , Proteínas Mitocondriales/genética , Quinazolinonas/farmacología , RespiraciónRESUMEN
Methamphetamine-associated cardiomyopathy is the leading cause of death linked with illicit drug use. Here we show that Sigmar1 is a therapeutic target for methamphetamine-associated cardiomyopathy and defined the molecular mechanisms using autopsy samples of human hearts, and a mouse model of "binge and crash" methamphetamine administration. Sigmar1 expression is significantly decreased in the hearts of human methamphetamine users and those of "binge and crash" methamphetamine-treated mice. The hearts of methamphetamine users also show signs of cardiomyopathy, including cellular injury, fibrosis, and enlargement of the heart. In addition, mice expose to "binge and crash" methamphetamine develop cardiac hypertrophy, fibrotic remodeling, and mitochondrial dysfunction leading to contractile dysfunction. Methamphetamine treatment inhibits Sigmar1, resulting in inactivation of the cAMP response element-binding protein (CREB), decreased expression of mitochondrial fission 1 protein (FIS1), and ultimately alteration of mitochondrial dynamics and function. Therefore, Sigmar1 is a viable therapeutic agent for protection against methamphetamine-associated cardiomyopathy.
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Cardiomiopatías/inducido químicamente , Metanfetamina/toxicidad , Mitocondrias/efectos de los fármacos , Receptores sigma/metabolismo , Animales , Cardiomiopatías/prevención & control , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Esquema de Medicación , Regulación de la Expresión Génica/efectos de los fármacos , Corazón/efectos de los fármacos , Humanos , Metanfetamina/administración & dosificación , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Receptores sigma/genética , Receptor Sigma-1RESUMEN
Doxorubicin (Dox) is a highly effective anticancer drug but cause acute ventricular dysfunction, and also induce late-onset cardiomyopathy and heart failure. Despite extensive studies, the pathogenic sequelae leading to the progression of Dox-associated cardiomyopathy remains unknown. We assessed temporal changes in autophagy, mitochondrial dynamics, and bioenergetics in mouse models of acute and chronic Dox-cardiomyopathy. Time course study of acute Dox-treatment showed accumulation of LC3B II in heart lysates. Autophagy flux assays confirmed that the Dox-induced accumulation of autophagosomes occurs due to blockage of the lysosomal degradation process. Dox-induced autophagosomes and autolysosome accumulation were confirmed in vivo by using GFP-LC3 and mRFP-GFP-LC3 transgenic (Tg) mice. Mitochondria isolated from acute Dox-treated hearts showed significant suppression of oxygen consumption rate (OCR). Chronic Dox-cardiotoxicity also exhibited time-dependent accumulation of LC3B II levels and increased accumulation of green puncta in GFP-LC3 Tg hearts. Mitochondria isolated from chronic Dox-treated hearts also showed significant suppression of mitochondrial OCR. The in vivo impairment of autophagic degradation process and mitochondrial dysfunction data were confirmed in vitro using cultured neonatal cardiomyocytes. Both acute and chronic Dox-associated cardiomyopathy involves a multifocal disease process resulting from autophagosomes and autolysosomes accumulation, altered expression of mitochondrial dynamics and oxidative phosphorylation regulatory proteins, and mitochondrial respiratory dysfunction.
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Autofagia/efectos de los fármacos , Cardiomiopatías/inducido químicamente , Cardiomiopatías/patología , Respiración de la Célula/efectos de los fármacos , Doxorrubicina/efectos adversos , Mitocondrias/efectos de los fármacos , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Femenino , Masculino , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Fosforilación Oxidativa/efectos de los fármacosRESUMEN
BACKGROUND: Desmin filament proteins interlink the contractile myofibrillar apparatus with mitochondria, nuclei and the sarcolemma. Mutations in the human desmin gene cause cardiac disease, remodeling, and heart failure but the pathophysiological mechanisms remain unknown. METHODS AND RESULTS: Cardiomyocyte-specific overexpression of mutated desmin (a 7 amino acid deletion R172-E178, D7-Des Tg) causes accumulations of electron-dense aggregates and myofibrillar degeneration associated with cardiac dysfunction. Though extensive studies demonstrated that these altered ultrastructural changes cause impairment of cardiac contractility, the molecular mechanism of cardiomyocyte death remains elusive. In the present study, we report that the D7-Des Tg mouse hearts undergo aberrant mitochondrial fission associated with increased expression of mitochondrial fission regulatory proteins. Mitochondria isolated from D7-Des Tg hearts showed decreased mitochondrial respiration and increased apoptotic cell death. Overexpression of mutant desmin by adenoviral infection in cultured cardiomyocytes led to increased mitochondrial fission, inhibition of mitochondrial respiration, and activation of cellular toxicity. Inhibition of mitochondrial fission by mitochondrial division inhibitor mdivi-1 significantly improved mitochondrial respiration and inhibited cellular toxicity associated with D7-Des overexpression in cardiomyocytes. CONCLUSIONS: Aberrant mitochondrial fission results in mitochondrial respiratory defects and apoptotic cell death in D7-Des Tg hearts. Inhibition of aberrant mitochondrial fission using mitochondrial division inhibitor significantly preserved mitochondrial function and decreased apoptotic cell death. Taken together, our study shows that maladaptive aberrant mitochondrial fission causes desminopathy-associated cellular dysfunction.
Asunto(s)
Cardiomiopatías/genética , ADN/genética , Desmina/genética , Mitocondrias Cardíacas/metabolismo , Mutación , Miocitos Cardíacos/metabolismo , Animales , Animales Recién Nacidos , Western Blotting , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Células Cultivadas , Análisis Mutacional de ADN , Desmina/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/patología , Ratas Sprague-DawleyRESUMEN
Background The Sigma 1 receptor (Sigmar1) functions as an interorganelle signaling molecule and elicits cytoprotective functions. The presence of Sigmar1 in the heart was first reported on the basis of a ligand-binding assay, and all studies to date have been limited to pharmacological approaches using less-selective ligands for Sigmar1. However, the physiological function of cardiac Sigmar1 remains unknown. We investigated the physiological function of Sigmar1 in regulating cardiac hemodynamics using the Sigmar1 knockout mouse (Sigmar1-/-). Methods and Results Sigmar1-/- hearts at 3 to 4 months of age showed significantly increased contractility as assessed by left ventricular catheterization with stimulation by increasing doses of a ß1-adrenoceptor agonist. Noninvasive echocardiographic measurements were also used to measure cardiac function over time, and the data showed the development of cardiac contractile dysfunction in Sigmar1 -/- hearts as the animals aged. Histochemistry demonstrated significant cardiac fibrosis, collagen deposition, and increased periostin in the Sigmar1 -/- hearts compared with wild-type hearts. Ultrastructural analysis of Sigmar1-/- cardiomyocytes revealed an irregularly shaped, highly fused mitochondrial network with abnormal cristae. Mitochondrial size was larger in Sigmar1-/- hearts, resulting in decreased numbers of mitochondria per microscopic field. In addition, Sigmar1-/- hearts showed altered expression of mitochondrial dynamics regulatory proteins. Real-time oxygen consumption rates in isolated mitochondria showed reduced respiratory function in Sigmar1-/- hearts compared with wild-type hearts. Conclusions We demonstrate a potential function of Sigmar1 in regulating normal mitochondrial organization and size in the heart. Sigmar1 loss of function led to mitochondrial dysfunction, abnormal mitochondrial architecture, and adverse cardiac remodeling, culminating in cardiac contractile dysfunction.