RESUMO
BACKGROUND: Cardiac contractile function requires high energy from mitochondria, and Ca2+ from the sarcoplasmic reticulum (SR). Via local Ca2+ transfer at close mitochondria-SR contacts, cardiac excitation feedforward regulates mitochondrial ATP production to match surges in demand (excitation-bioenergetics coupling). However, pathological stresses may cause mitochondrial Ca2+ overload, excessive reactive oxygen species production and permeability transition, risking homeostatic collapse and myocyte loss. Excitation-bioenergetics coupling involves mitochondria-SR tethers but the role of tethering in cardiac physiology/pathology is debated. Endogenous tether proteins are multifunctional; therefore, nonselective targets to scrutinize interorganelle linkage. Here, we assessed the physiological/pathological relevance of selective chronic enhancement of cardiac mitochondria-SR tethering. METHODS: We introduced to mice a cardiac muscle-specific engineered tether (linker) transgene with a fluorescent protein core and deployed 2D/3D electron microscopy, biochemical approaches, fluorescence imaging, in vivo and ex vivo cardiac performance monitoring and stress challenges to characterize the linker phenotype. RESULTS: Expressed in the mature cardiomyocytes, the linker expanded and tightened individual mitochondria-junctional SR contacts; but also evoked a marked remodeling with large dense mitochondrial clusters that excluded dyads. Yet, excitation-bioenergetics coupling remained well-preserved, likely due to more longitudinal mitochondria-dyad contacts and nanotunnelling between mitochondria exposed to junctional SR and those sealed away from junctional SR. Remarkably, the linker decreased female vulnerability to acute massive ß-adrenergic stress. It also reduced myocyte death and mitochondrial calcium-overload-associated myocardial impairment in ex vivo ischemia/reperfusion injury. CONCLUSIONS: We propose that mitochondria-SR/endoplasmic reticulum contacts operate at a structural optimum. Although acute changes in tethering may cause dysfunction, upon chronic enhancement of contacts from early life, adaptive remodeling of the organelles shifts the system to a new, stable structural optimum. This remodeling balances the individually enhanced mitochondrion-junctional SR crosstalk and excitation-bioenergetics coupling, by increasing the connected mitochondrial pool and, presumably, Ca2+/reactive oxygen species capacity, which then improves the resilience to stresses associated with dysregulated hyperactive Ca2+ signaling.
Assuntos
Sinalização do Cálcio , Retículo Sarcoplasmático , Feminino , Camundongos , Animais , Retículo Sarcoplasmático/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Cálcio/metabolismoRESUMO
Cardiovascular diseases cause a high mortality rate worldwide and represent a major burden for health care systems. Experimental rodent models play a central role in cardiovascular disease research by effectively simulating human cardiovascular diseases. Using mice, the International Mouse Phenotyping Consortium (IMPC) aims to target each protein-coding gene and phenotype multiple organ systems in single-gene knockout models by a global network of mouse clinics. In this review, we summarize the current advances of the IMPC in cardiac research and describe in detail the diagnostic requirements of high-throughput electrocardiography and transthoracic echocardiography capable of detecting cardiac arrhythmias and cardiomyopathies in mice. Beyond that, we are linking metabolism to the heart and describing phenotypes that emerge in a set of known genes, when knocked out in mice, such as the leptin receptor (Lepr), leptin (Lep), and Bardet-Biedl syndrome 5 (Bbs5). Furthermore, we are presenting not yet associated loss-of-function genes affecting both, metabolism and the cardiovascular system, such as the RING finger protein 10 (Rfn10), F-box protein 38 (Fbxo38), and Dipeptidyl peptidase 8 (Dpp8). These extensive high-throughput data from IMPC mice provide a promising opportunity to explore genetics causing metabolic heart disease with an important translational approach.
Assuntos
Doenças Cardiovasculares , Sistema Cardiovascular , Camundongos , Animais , Humanos , Camundongos Knockout , Doenças Cardiovasculares/genética , Técnicas de Inativação de Genes , FenótipoRESUMO
Reference ranges provide a powerful tool for diagnostic decision-making in clinical medicine and are enormously valuable for understanding normality in pre-clinical scientific research that uses in vivo models. As yet, there are no published reference ranges for electrocardiography (ECG) in the laboratory mouse. The first mouse-specific reference ranges for the assessment of electrical conduction are reported herein generated from an ECG dataset of unprecedented scale. International Mouse Phenotyping Consortium data from over 26,000 conscious or anesthetized C57BL/6N wildtype control mice were stratified by sex and age to develop robust ECG reference ranges. Interesting findings include that heart rate and key elements from the ECG waveform (RR-, PR-, ST-, QT-interval, QT corrected, and QRS complex) demonstrate minimal sexual dimorphism. As expected, anesthesia induces a decrease in heart rate and was shown for both inhalation (isoflurane) and injectable (tribromoethanol) anesthesia. In the absence of pharmacological, environmental, or genetic challenges, we did not observe major age-related ECG changes in C57BL/6N-inbred mice as the differences in the reference ranges of 12-week-old compared to 62-week-old mice were negligible. The generalizability of the C57BL/6N substrain reference ranges was demonstrated by comparison with ECG data from a wide range of non-IMPC studies. The close overlap in data from a wide range of mouse strains suggests that the C57BL/6N-based reference ranges can be used as a robust and comprehensive indicator of normality. We report a unique ECG reference resource of fundamental importance for any experimental study of cardiac function in mice.
Assuntos
Eletrocardiografia , Técnicas Eletrofisiológicas Cardíacas , Camundongos , Animais , Camundongos Endogâmicos C57BL , Camundongos EndogâmicosRESUMO
Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with ß-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.
Assuntos
Miócitos Cardíacos , Retículo Sarcoplasmático , Ratos , Masculino , Feminino , Animais , Humanos , Idoso , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Caracteres Sexuais , Mitocôndrias/metabolismo , Sinalização do Cálcio , Cálcio/metabolismoRESUMO
The use of fixed fibroblasts from familial and sporadic Alzheimer's disease patients has previously indicated an upregulation of mitochondria-ER contacts (MERCs) as a hallmark of Alzheimer's disease. Despite its potential significance, the relevance of these results is limited because they were not extended to live neurons. Here we performed a dynamic in vivo analysis of MERCs in hippocampal neurons from McGill-R-Thy1-APP transgenic rats, a model of Alzheimer's disease-like amyloid pathology. Live FRET imaging of neurons from transgenic rats revealed perturbed 'lipid-MERCs' (gap width <10â nm), while 'Ca2+-MERCs' (10-20â nm gap width) were unchanged. In situ TEM showed no significant differences in the lipid-MERCs:total MERCs or lipid-MERCs:mitochondria ratios; however, the average length of lipid-MERCs was significantly decreased in neurons from transgenic rats as compared to controls. In accordance with FRET results, untargeted lipidomics showed significant decreases in levels of 12 lipids and bioenergetic analysis revealed respiratory dysfunction of mitochondria from transgenic rats. Thus, our results reveal changes in MERC structures coupled with impaired mitochondrial functions in Alzheimer's disease-related neurons.This article has an associated First Person interview with the first author of the paper.
Assuntos
Doença de Alzheimer , Retículo Endoplasmático , Mitocôndrias , Neurônios , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos TransgênicosRESUMO
BACKGROUND: Endothelial progenitor cells (EPCs) were indicated in vascular repair, angiogenesis of ischemic organs, and inhibition of formation of initial hyperplasia. Differentiation of endothelial cells (ECs) from human induced pluripotent stem cells (hiPSC)-derived endothelial cells (hiPSC-ECs) provides an unlimited supply for clinical application. Furthermore, magnetic cell labelling offers an effective way of targeting and visualization of hiPSC-ECs and is the next step towards in vivo studies. METHODS: ECs were differentiated from hiPSCs and labelled with uncoated superparamagnetic iron-oxide nanoparticles (uSPIONs). uSPION uptake was compared between hiPSC-ECs and mature ECs isolated from patients by software analysis of microscopy pictures after Prussian blue cell staining. The acute and long-term cytotoxic effects of uSPIONs were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) and Annexin assay. RESULTS: We showed, for the first time, uptake of uncoated SPIONs (uSPIONs) by hiPSC-ECs. In comparison with mature ECs of identical genetic background hiPSC-ECs showed lower uSPION uptake. However, all the studied endothelial cells were effectively labelled and showed magnetic properties even with low labelling concentration of uSPIONs. uSPIONs prepared by microwave plasma synthesis did not show any cytotoxicity nor impair endothelial properties. CONCLUSION: We show that hiPSC-ECs labelling with low concentration of uSPIONs is feasible and does not show any toxic effects in vitro, which is an important step towards animal studies.
Assuntos
Diferenciação Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Compostos Férricos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Nanopartículas de Magnetita , Biomarcadores , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/ultraestrutura , Compostos Férricos/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Nanopartículas de Magnetita/químicaRESUMO
Mutations in the LMNA gene encoding lamins A/C cause an array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the perturbations emanating from LMNA mutations, an integrative understanding of the pathogenesis underlying cardiac dysfunction remains elusive. Using a novel conditional deletion model capable of translatome profiling, we observed that cardiomyocyte-specific Lmna deletion in adult mice led to rapid cardiomyopathy with pathological remodeling. Before cardiac dysfunction, Lmna-deleted cardiomyocytes displayed nuclear abnormalities, Golgi dilation/fragmentation, and CREB3-mediated stress activation. Translatome profiling identified MED25 activation, a transcriptional cofactor that regulates Golgi stress. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the Golgi. Systemic administration of modulators of autophagy or ER stress significantly delayed cardiac dysfunction and prolonged survival. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the LMNA cardiomyopathy development.
Assuntos
Cardiomiopatias , Lamina Tipo A , Miócitos Cardíacos , Membrana Nuclear , Animais , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Camundongos , Membrana Nuclear/metabolismo , Cardiomiopatias/metabolismo , Cardiomiopatias/etiologia , Cardiomiopatias/patologia , Cardiomiopatias/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Autofagia , Estresse Fisiológico , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Complexo de Golgi/metabolismo , Camundongos KnockoutRESUMO
Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation.
Assuntos
Ciclo-Oxigenase 1 , Complexo IV da Cadeia de Transporte de Elétrons , Inflamação , Fígado , Fosforilação Oxidativa , Espécies Reativas de Oxigênio , Animais , Feminino , Humanos , Masculino , Camundongos , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Inflamação/metabolismo , Inflamação/genética , Inflamação/patologia , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Mutação , Biossíntese de Proteínas , Espécies Reativas de Oxigênio/metabolismo , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismoRESUMO
Mutations in the LMNA gene encoding nuclear lamins A/C cause a diverse array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the molecular perturbations emanating from LMNA mutations, an integrative understanding of the pathogenesis leading to cardiac dysfunction remains elusive. Using a novel cell-type specific Lmna deletion mouse model capable of translatome profiling, we found that cardiomyocyte-specific Lmna deletion in adult mice led to rapid cardiomyopathy with pathological remodeling. Prior to the onset of cardiac dysfunction, lamin A/C-depleted cardiomyocytes displayed nuclear envelope deterioration, golgi dilation/fragmentation, and CREB3-mediated golgi stress activation. Translatome profiling identified upregulation of Med25, a transcriptional co-factor that can selectively dampen UPR axes. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the golgi or inducing nuclear damage by increased matrix stiffness. Systemic administration of pharmacological modulators of autophagy or ER stress significantly improved the cardiac function. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the development of LMNA cardiomyopathy. Teaser: Interplay of stress responses underlying the development of LMNA cardiomyopathy.
RESUMO
Mitochondria are central for cellular metabolism and energy supply. Barth syndrome (BTHS) is a severe disorder, due to dysfunction of the mitochondrial cardiolipin acyl transferase tafazzin. Altered cardiolipin remodeling affects mitochondrial inner membrane organization and function of membrane proteins such as transporters and the oxidative phosphorylation (OXPHOS) system. Here, we describe a mouse model that carries a G197V exchange in tafazzin, corresponding to BTHS patients. TAZG197V mice recapitulate disease-specific pathology including cardiac dysfunction and reduced oxidative phosphorylation. We show that mutant mitochondria display defective fatty acid-driven oxidative phosphorylation due to reduced levels of carnitine palmitoyl transferases. A metabolic switch in ATP production from OXPHOS to glycolysis is apparent in mouse heart and patient iPSC cell-derived cardiomyocytes. An increase in glycolytic ATP production inactivates AMPK causing altered metabolic signaling in TAZG197V . Treatment of mutant cells with AMPK activator reestablishes fatty acid-driven OXPHOS and protects mice against cardiac dysfunction.
Assuntos
Síndrome de Barth , Camundongos , Animais , Síndrome de Barth/metabolismo , Síndrome de Barth/patologia , Cardiolipinas/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Glicólise , Ácidos Graxos/metabolismo , Trifosfato de AdenosinaRESUMO
The animal models of myocardial injury induced by systemic ß-adrenergic receptor agonist administration represent an experimental approach of persisting interest. These models were found useful especially for studies of structural and functional adaptation of myocardium during the progression of cardiac adaptive response towards maladaptive hypertrophy and insufficiency. The pathological alterations induced by isoproterenol (ISO) do not develop evenly. The ISO models may contribute effectively to understanding of pathologies in signal transduction, energetics, excitability and contractility that may contribute concomitantly to cardiac dysfunction and heart failure. In this minireview we focused on the alterations in general characteristics and heart function as well as on the morphological changes of cardiomyocytes developed during ISO administration. The morphological alterations within the cellular macro- and microdomains correspond to the electrical remodelling and contractile dysfunction of ventricular myocardium that could be used to identify pathological changes ranging from hypertrophy to failing heart.
Assuntos
Modelos Animais de Doenças , Sistema de Condução Cardíaco/fisiopatologia , Isoproterenol , Contração Miocárdica/efeitos dos fármacos , Miocárdio/patologia , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/fisiopatologia , Animais , Sistema de Condução Cardíaco/efeitos dos fármacos , Humanos , Disfunção Ventricular Esquerda/patologiaRESUMO
Endoplasmic reticulum-mitochondria contacts (ERMCs) are restructured in response to changes in cell state. While this restructuring has been implicated as a cause or consequence of pathology in numerous systems, the underlying molecular dynamics are poorly understood. Here, we show means to visualize the capture of motile IP3 receptors (IP3Rs) at ERMCs and document the immediate consequences for calcium signaling and metabolism. IP3Rs are of particular interest because their presence provides a scaffold for ERMCs that mediate local calcium signaling, and their function outside of ERMCs depends on their motility. Unexpectedly, in a cell model with little ERMC Ca2+ coupling, IP3Rs captured at mitochondria promptly mediate Ca2+ transfer, stimulating mitochondrial oxidative metabolism. The Ca2+ transfer does not require linkage with a pore-forming protein in the outer mitochondrial membrane. Thus, motile IP3Rs can traffic in and out of ERMCs, and, when 'parked', mediate calcium signal propagation to the mitochondria, creating a dynamic arrangement that supports local communication.
Assuntos
Sinalização do Cálcio , Mitocôndrias , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Sinalização do Cálcio/fisiologia , Mitocôndrias/metabolismo , Respiração Celular , Cálcio/metabolismo , Estresse OxidativoRESUMO
Friedreich's ataxia (FRDA) is an inherited disorder caused by reduced levels of frataxin (FXN), which is required for iron-sulfur cluster biogenesis. Neurological and cardiac comorbidities are prominent and have been a major focus of study. Skeletal muscle has received less attention despite indications that FXN loss affects it. Here, we show that lean mass is lower, whereas body mass index is unaltered, in separate cohorts of adults and children with FRDA. In adults, lower lean mass correlated with disease severity. To further investigate FXN loss in skeletal muscle, we used a transgenic mouse model of whole-body inducible and progressive FXN depletion. There was little impact of FXN loss when FXN was approximately 20% of control levels. When residual FXN was approximately 5% of control levels, muscle mass was lower along with absolute grip strength. When we examined mechanisms that can affect muscle mass, only global protein translation was lower, accompanied by integrated stress response (ISR) activation. Also in mice, aerobic exercise training, initiated prior to the muscle mass difference, improved running capacity, yet, muscle mass and the ISR remained as in untrained mice. Thus, FXN loss can lead to lower lean mass, with ISR activation, both of which are insensitive to exercise training.
Assuntos
Ataxia de Friedreich , Proteínas de Ligação ao Ferro , Animais , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , FrataxinaRESUMO
Aged cardiomyocytes develop a mismatch between energy demand and supply, the severity of which determines the onset of heart failure, and become prone to undergo cell death. The FoF1-ATP synthase is the molecular machine that provides >90% of the ATP consumed by healthy cardiomyocytes and is proposed to form the mitochondrial permeability transition pore (mPTP), an energy-dissipating channel involved in cell death. We investigated whether aging alters FoF1-ATP synthase self-assembly, a fundamental biological process involved in mitochondrial cristae morphology and energy efficiency, and the functional consequences this may have. Purified heart mitochondria and cardiomyocytes from aging mice displayed an impaired dimerization of FoF1-ATP synthase (blue native and proximity ligation assay), associated with abnormal mitochondrial cristae tip curvature (TEM). Defective dimerization did not modify the in vitro hydrolase activity of FoF1-ATP synthase but reduced the efficiency of oxidative phosphorylation in intact mitochondria (in which membrane architecture plays a fundamental role) and increased cardiomyocytes' susceptibility to undergo energy collapse by mPTP. High throughput proteomics and fluorescence immunolabeling identified glycation of 5 subunits of FoF1-ATP synthase as the causative mechanism of the altered dimerization. In vitro induction of FoF1-ATP synthase glycation in H9c2 myoblasts recapitulated the age-related defective FoF1-ATP synthase assembly, reduced the relative contribution of oxidative phosphorylation to cell energy metabolism, and increased mPTP susceptibility. These results identify altered dimerization of FoF1-ATP synthase secondary to enzyme glycation as a novel pathophysiological mechanism involved in mitochondrial cristae remodeling, energy deficiency, and increased vulnerability of cardiomyocytes to undergo mitochondrial failure during aging.
Assuntos
Envelhecimento , Mitocôndrias Cardíacas , ATPases Mitocondriais Próton-Translocadoras , Miócitos Cardíacos , Trifosfato de Adenosina/metabolismo , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Animais , Cálcio/metabolismo , Dimerização , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Camundongos , Mitocôndrias Cardíacas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Cardiac excitation-contraction coupling relies on dyads, the intracellular calcium synapses of cardiac myocytes, where the plasma membrane contacts sarcoplasmic reticulum and where electrical excitation triggers calcium release. The morphology of dyads and dynamics of local calcium release vary substantially. To better understand the correspondence between the structure and the functionality of dyads, we estimated incidences of structurally different dyads and of kinetically different calcium release sites and tested their responsiveness to experimental myocardial injury in left ventricular myocytes of rats. According to the structure of dyads estimated in random electron microscopic images of myocardial tissue, the dyads were sorted into 'compact' or 'loose' types. The calcium release fluxes, triggered at local calcium release sites in patch-clamped ventricular myocytes and recorded by laser scanning confocal fluorescence microscopy, were decomposed into 'early' and 'late' components. ANOVA tests revealed very high correlation between the relative amplitudes of early and late calcium release flux components and the relative occurrences of compact and loose dyads in the control and in the injured myocardium. This finding ascertained the relationship between the structure of dyads and the functionality of calcium release sites and the responsiveness of calcium release sites to physical load in cardiac myocytes.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Ventrículos do Coração/fisiopatologia , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Animais , Acoplamento Excitação-Contração , Masculino , Miócitos Cardíacos/citologia , Ratos , Ratos Wistar , Retículo Sarcoplasmático/metabolismoRESUMO
Aims: Endoplasmic reticulum (ER) stress has recently emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases. However, the molecular mechanisms by which ER stress leads to cardiac dysfunction remain poorly understood. Methods and results: In this study, we evaluated the early cardiac effects of ER stress induced by tunicamycin (TN) in mice. Echocardiographic analysis indicated that TN-induced ER stress led to a significant impairment of the cardiac function. Electron microscopic observations revealed that ultrastructural changes of cardiomyocytes in response to ER stress manifested extensively at the level of the reticular membrane system. Smooth tubules of sarcoplasmic reticulum in connection with short sections of rough ER were observed. The presence of rough instead of smooth reticulum was increased at the interfibrillar space, at the level of dyads, and in the vicinity of mitochondria. At the transcriptional level, ER stress resulted in a substantial decrease in the expression of the major regulator of mitochondrial biogenesis PGC-1α and of its targets NRF1, Tfam, CS, and COXIV. At the functional level, ER stress also induced an impairment of mitochondrial Ca2+ uptake, an alteration of mitochondrial oxidative phosphorylation, and a metabolic remodelling characterized by a shift from fatty acid to glycolytic substrate consumption. Conclusions: Our findings show that ER stress induces cytoarchitectural and metabolic alterations in cardiomyocytes and provide evidences that ER stress could represent a primary mechanism that contributes to the impairment of energy metabolism reported in most cardiac diseases.
Assuntos
Estresse do Retículo Endoplasmático , Cardiopatias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ácidos Graxos/metabolismo , Glicólise , Cardiopatias/induzido quimicamente , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Camundongos , Mitocôndrias Cardíacas/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Fator 1 Relacionado a NF-E2/genética , Fator 1 Relacionado a NF-E2/metabolismo , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais , TunicamicinaRESUMO
Contact sites of endoplasmic reticulum (ER) and mitochondria locally convey calcium signals between the IP3 receptors (IP3R) and the mitochondrial calcium uniporter, and are central to cell survival. It remains unclear whether IP3Rs also have a structural role in contact formation and whether the different IP3R isoforms have redundant functions. Using an IP3R-deficient cell model rescued with each of the three IP3R isoforms and an array of super-resolution and ultrastructural approaches we demonstrate that IP3Rs are required for maintaining ER-mitochondrial contacts. This role is independent of calcium fluxes. We also show that, while each isoform can support contacts, type 2 IP3R is the most effective in delivering calcium to the mitochondria. Thus, these studies reveal a non-canonical, structural role for the IP3Rs and direct attention towards the type 2 IP3R that was previously neglected in the context of ER-mitochondrial calcium signaling.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Retículo Endoplasmático/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocôndrias/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Galinhas , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Isoformas de Proteínas/genéticaRESUMO
Though congenital hydrocephalus is heritable, it has been linked only to eight genes, one of which is MPDZ Humans and mice that carry a truncated version of MPDZ incur severe hydrocephalus resulting in acute morbidity and lethality. We show by magnetic resonance imaging that contrast medium penetrates into the brain ventricles of mice carrying a Mpdz loss-of-function mutation, whereas none is detected in the ventricles of normal mice, implying that the permeability of the choroid plexus epithelial cell monolayer is abnormally high. Comparative proteomic analysis of the cerebrospinal fluid of normal and hydrocephalic mice revealed up to a 53-fold increase in protein concentration, suggesting that transcytosis through the choroid plexus epithelial cells of Mpdz KO mice is substantially higher than in normal mice. These conclusions are supported by ultrastructural evidence, and by immunohistochemistry and cytology data. Our results provide a straightforward and concise explanation for the pathophysiology of Mpdz-linked hydrocephalus.
Assuntos
Permeabilidade Capilar , Proteínas de Transporte/genética , Plexo Corióideo/patologia , Plexo Corióideo/fisiopatologia , Hidrocefalia/patologia , Hidrocefalia/fisiopatologia , Animais , Meios de Contraste/análise , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Imageamento por Ressonância Magnética , Proteínas de Membrana , CamundongosRESUMO
Mitochondrial Ca2+ elevations enhance ATP production, but uptake must be balanced by efflux to avoid overload. Uptake is mediated by the mitochondrial Ca2+ uniporter channel complex (MCUC), and extrusion is controlled largely by the Na+/Ca2+ exchanger (NCLX), both driven electrogenically by the inner membrane potential (ΔΨm). MCUC forms hotspots at the cardiac mitochondria-junctional SR (jSR) association to locally receive Ca2+ signals; however, the distribution of NCLX is unknown. Our fractionation-based assays reveal that extensively jSR-associated mitochondrial segments contain a minor portion of NCLX and lack Na+-dependent Ca2+ extrusion. This pattern is retained upon in vivo NCLX overexpression, suggesting extensive targeting to non-jSR-associated submitochondrial domains and functional relevance. In cells with non-polarized MCUC distribution, upon NCLX overexpression the same given increase in matrix Ca2+ expends more ΔΨm. Thus, cardiac mitochondrial Ca2+ uptake and extrusion are reciprocally polarized, likely to optimize the energy efficiency of local calcium signaling in the beating heart.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , Animais , Linhagem Celular , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Sódio/metabolismoRESUMO
Glioblastoma multiforme are considered to be aggressive high-grade tumors with poor prognosis for patient survival. Photodynamic therapy is one of the adjuvant therapies which has been used for glioblastoma multiforme during last decade. Hypericin, a photosensitizer, can be employed in this treatment. We have studied the effect of hypericin on PKCδ phosphorylation in U87 MG cells before and after light application. Hypericin increased PKCδ phosphorylation at tyrosine 155 in the regulatory domain and serine 645 in the catalytic domain. However, use of the light resulted in apoptosis, decreased phosphorylation of tyrosine 155 and enhanced serine 645. The PKCδ localization and phosphorylation of regulatory and catalytic domains were shown to play a distinct role in the anti-apoptotic response of glioma cells. We hypothesized that PKCδ phosphorylated at the regulatory domain is primarily present in the cytoplasm and in mitochondria before irradiation, and it may participate in Bcl-2 phosphorylation. After hypericin and light application, PKCδ phosphorylated at a regulatory domain which is in the nucleus. In contrast, PKCδ phosphorylated at the catalytic domain may be mostly active in the nucleus before irradiation, but active in the cytoplasm after the irradiation. In summary, light-induced oxidative stress significantly regulates PKCδ pro-survival and pro-apoptotic activity in glioma cells by its phosphorylation at serine 645 and tyrosine 155.