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Z-discs are core ultrastructural organizers of cardiomyocytes that modulate many facets of cardiac pathogenesis. Yet a comprehensive proteomic atlas of Z-disc-associated components remain incomplete. Here, we established an adeno-associated virus (AAV)-delivered, cardiomyocyte-specific, proximity-labeling approach to characterize the Z-disc proteome in vivo. We found palmdelphin (PALMD) as a novel Z-disc-associated protein in both adult murine cardiomyocytes and human pluripotent stem cell-derived cardiomyocytes. Germline and cardiomyocyte-specific Palmd knockout mice were grossly normal at baseline but exhibited compromised cardiac hypertrophy and aggravated cardiac injury upon long-term isoproterenol treatment. By contrast, cardiomyocyte-specific PALMD overexpression was sufficient to mitigate isoproterenol-induced cardiac injury. PALMD ablation perturbed the transverse tubule (T-tubule)-sarcoplasmic reticulum (SR) ultrastructures, which formed the Z-disc-associated junctional membrane complex (JMC) essential for calcium handling and cardiac function. These phenotypes were associated with the reduction of nexilin (NEXN), a crucial Z-disc-associated protein that is essential for both Z-disc and JMC structures and functions. PALMD interacted with NEXN and enhanced its protein stability while the Nexn mRNA level was not affected. AAV-based NEXN addback rescued the exacerbated cardiac injury in isoproterenol-treated PALMD-depleted mice. Together, this study discovered PALMD as a potential target for myocardial protection and highlighted in vivo proximity proteomics as a powerful approach to nominate novel players regulating cardiac pathogenesis.
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BACKGROUND: Mutations in tafazzin (TAZ), a gene required for biogenesis of cardiolipin, the signature phospholipid of the inner mitochondrial membrane, causes Barth syndrome (BTHS). Cardiomyopathy and risk of sudden cardiac death are prominent features of BTHS, but the mechanisms by which impaired cardiolipin biogenesis causes cardiac muscle weakness and arrhythmia are poorly understood. METHODS: We performed in vivo electrophysiology to define arrhythmia vulnerability in cardiac-specific TAZ knockout mice. Using cardiomyocytes derived from human induced pluripotent stem cells and cardiac-specific TAZ knockout mice as model systems, we investigated the effect of TAZ inactivation on Ca2+ handling. Through genome editing and pharmacology, we defined a molecular link between TAZ mutation and abnormal Ca2+ handling and contractility. RESULTS: A subset of mice with cardiac-specific TAZ inactivation developed arrhythmias, including bidirectional ventricular tachycardia, atrial tachycardia, and complete atrioventricular block. Compared with wild-type controls, BTHS-induced pluripotent stem cell-derived cardiomyocytes had increased diastolic Ca2+ and decreased Ca2+ transient amplitude. BTHS-induced pluripotent stem cell-derived cardiomyocytes had higher levels of mitochondrial and cellular reactive oxygen species than wild-type controls, which activated CaMKII (Ca2+/calmodulin-dependent protein kinase II). Activated CaMKII phosphorylated the RYR2 (ryanodine receptor 2) on serine 2814, increasing Ca2+ leak through RYR2. Inhibition of this reactive oxygen species-CaMKII-RYR2 pathway through pharmacological inhibitors or genome editing normalized aberrant Ca2+ handling in BTHS-induced pluripotent stem cell-derived cardiomyocytes and improved their contractile function. Murine Taz knockout cardiomyocytes also exhibited elevated diastolic Ca2+ and decreased Ca2+ transient amplitude. These abnormalities were ameliorated by Ca2+/calmodulin-dependent protein kinase II or reactive oxygen species inhibition. CONCLUSIONS: This study identified a molecular pathway that links TAZ mutation with abnormal Ca2+ handling and decreased cardiomyocyte contractility. This pathway may offer therapeutic opportunities to treat BTHS and potentially other diseases with elevated mitochondrial reactive oxygen species production.
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Síndrome de Barth/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Síndrome de Barth/fisiopatologia , Humanos , Camundongos , Camundongos KnockoutRESUMO
STIM1- and Orai1-mediated store-operated Ca2+ entry (SOCE) constitutes the major Ca2+ influx in almost all electrically non-excitable cells. However, little is known about the spatiotemporal organization at the elementary level. Here, we developed Orai1-tethered or palmitoylated biosensor GCaMP6f to report subplasmalemmal Ca2+ signals. We visualized spontaneous discrete and long-lasting transients ('Ca2+ glows') arising from STIM1-Orai1 in invading melanoma cells. Ca2+ glows occurred preferentially in single invadopodia and at sites near the cell periphery under resting conditions. Re-addition of external Ca2+ after store depletion elicited spatially synchronous Ca2+ glows, followed by high-rate discharge of asynchronous local events. Knockout of STIM1 or expression of the dominant-negative Orai1-E106A mutant markedly decreased Ca2+ glow frequency, diminished global SOCE and attenuated invadopodial formation. Functionally, invadopodial Ca2+ glows provided high Ca2+ microdomains to locally activate Ca2+/calmodulin-dependent Pyk2 (also known as PTK2B), which initiates the SOCE-Pyk2-Src signaling cascade required for invasion. Overall, the discovery of elemental Ca2+ signals of SOCE not only unveils a previously unappreciated gating mode of STIM1-Orai1 channels in situ, but also underscores a critical role of the spatiotemporal dynamics of SOCE in orchestrating complex cell behaviors such as invasion. This article has an associated First Person interview with the first author of the paper.
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Sinalização do Cálcio/fisiologia , Quinase 2 de Adesão Focal/metabolismo , Proteínas de Neoplasias/metabolismo , Células Neoplásicas Circulantes/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Técnicas Biossensoriais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células HEK293 , Humanos , Microscopia Confocal , Imagem Molecular/métodosRESUMO
The store-operated calcium (Ca2+) entry (SOCE) is the Ca2+ entry mechanism used by cells to replenish depleted Ca2+ store. The dysregulation of SOCE has been reported in metastatic cancer. It is believed that SOCE promotes migration and invasion by remodeling the actin cytoskeleton and cell adhesion dynamics. There is recent evidence supporting that SOCE is critical for the spatial and the temporal coding of Ca2+ signals in the cell. In this review, we critically examined the spatiotemporal control of SOCE signaling and its implication in the specificity and robustness of signaling events downstream of SOCE, with a focus on the spatiotemporal SOCE signaling during cancer cell migration, invasion and metastasis. We further discuss the limitation of our current understanding of SOCE in cancer metastasis and potential approaches to overcome such limitation.
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Cálcio/metabolismo , Metástase Neoplásica , Neoplasias/metabolismo , Sinalização do Cálcio , Humanos , Transporte de Íons , Neoplasias/patologiaRESUMO
BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited cardiac arrhythmia characterized by adrenergically triggered arrhythmias, is inadequately treated by current standard of care. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an adrenergically activated kinase that contributes to arrhythmogenesis in heart disease models, is a candidate therapeutic target in CPVT. However, translation of CaMKII inhibition has been limited by the need for selective CaMKII inhibition in cardiomyocytes. Here, we tested the hypothesis that CaMKII inhibition with a cardiomyocyte-targeted gene therapy strategy would suppress arrhythmia in CPVT mouse models. METHODS: We developed AAV9-GFP-AIP, an adeno-associated viral vector in which a potent CaMKII inhibitory peptide, autocamtide-2-related inhibitory peptide [AIP], is fused to green fluorescent protein (GFP) and expressed from a cardiomyocyte selective promoter. The vector was delivered systemically. Arrhythmia burden was evaluated with invasive electrophysiology testing in adult mice. AIP was also tested on induced pluripotent stem cells derived from patients with CPVT with different disease-causing mutations to determine the effectiveness of our proposed therapy on human induced pluripotent stem cell-derived cardiomyocytes and different pathogenic genotypes. RESULTS: AAV9-GFP-AIP was robustly expressed in the heart without significant expression in extracardiac tissues, including the brain. Administration of AAV9-GFP-AIP to neonatal mice with a known CPVT mutation (RYR2R176Q/+) effectively suppressed ventricular arrhythmias induced by either ß-adrenergic stimulation or programmed ventricular pacing, without significant proarrhythmic effect. Intravascular delivery of AAV9-GFP-AIP to adolescent mice transduced ≈50% of cardiomyocytes and was effective in suppressing arrhythmia in CPVT mice. Induced pluripotent stem cell-derived cardiomyocytes derived from 2 different patients with CPVT with different pathogenic mutations demonstrated increased frequency of abnormal calcium release events, which was suppressed by a cell-permeable form of AIP. CONCLUSIONS: This proof-of-concept study showed that AAV-mediated delivery of a CaMKII peptide inhibitor to the heart was effective in suppressing arrhythmias in a murine model of CPVT. CaMKII inhibition also reversed the arrhythmia phenotype in human CPVT induced pluripotent stem cell-derived cardiomyocyte models with different pathogenic mutations.
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Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Terapia Genética/métodos , Taquicardia Ventricular/genética , Taquicardia Ventricular/terapia , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Camundongos , Camundongos Transgênicos , Taquicardia Ventricular/enzimologiaRESUMO
BACKGROUND: Modeling of human arrhythmias with induced pluripotent stem cell-derived cardiomyocytes has focused on single-cell phenotypes. However, arrhythmias are the emergent properties of cells assembled into tissues, and the impact of inherited arrhythmia mutations on tissue-level properties of human heart tissue has not been reported. METHODS: Here, we report an optogenetically based, human engineered tissue model of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia caused by mutation of the cardiac ryanodine channel and triggered by exercise. We developed a human induced pluripotent stem cell-derived cardiomyocyte-based platform to study the tissue-level properties of engineered human myocardium. We investigated pathogenic mechanisms in CPVT by combining this novel platform with genome editing. RESULTS: In our model, CPVT tissues were vulnerable to developing reentrant rhythms when stimulated by rapid pacing and catecholamine, recapitulating hallmark features of the disease. These conditions elevated diastolic Ca2+ levels and increased temporal and spatial dispersion of Ca2+ wave speed, creating a vulnerable arrhythmia substrate. Using Cas9 genome editing, we pinpointed a single catecholamine-driven phosphorylation event, ryanodine receptor-serine 2814 phosphorylation by Ca2+/calmodulin-dependent protein kinase II, that is required to unmask the arrhythmic potential of CPVT tissues. CONCLUSIONS: Our study illuminates the molecular and cellular pathogenesis of CPVT and reveals a critical role of calmodulin-dependent protein kinase II-dependent reentry in the tissue-scale mechanism of this disease. We anticipate that this approach will be useful for modeling other inherited and acquired cardiac arrhythmias.
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Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Taquicardia Ventricular/patologia , Taquicardia Ventricular/fisiopatologia , Engenharia Tecidual/métodos , Potenciais de Ação/fisiologia , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/química , Miócitos Cardíacos/química , Optogenética/métodosRESUMO
Cyclophilin D (CyP-D) is the mitochondrial-specific member of the evolutionally conserved cyclophilin family, and plays an important role in the regulation of mitochondrial permeability transition (MPT) under stress. Recently we have demonstrated that respiratory mitochondria undergo mitochondrial flash ("mitoflash") activity which is coupled with transient MPT under physiological conditions. However, whether and how CyP-D regulates mitoflashes remain incompletely understood. By using both loss- and gain-of-function approaches in isolated cardiomyocytes, beating hearts, and skeletal muscles in living mice, we revisited the role of CyP-D in the regulation of mitoflashes. Overexpression of CyP-D increased, and knockout of it halved, cardiac mitoflash frequency, while mitoflash amplitude and kinetics remained unaffected. However, CyP-D ablation did not alter mitoflash frequency, with mitoflash amplitude increased, in gastrocnemius muscles. This disparity was accompanied by 4-fold higher CyP-D expression in mouse cardiac than skeletal muscles. The mitochondrial maximal respiration rate and reserved capacity were reduced in CyP-D-null cardiomyocytes. These data indicate that CyP-D is a significant regulator of mitoflash ignition and mitochondrial metabolism in heart. In addition, tissue-specific CyP-D expression may partly explain the differential regulation of mitoflashes in the two types of striated muscles.
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Ciclofilinas/metabolismo , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Feminino , Regulação da Expressão Gênica , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Músculo Estriado/metabolismo , Músculo Estriado/ultraestrutura , Miocárdio/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Técnicas de Cultura de Órgãos , Especificidade de Órgãos , Cultura Primária de Células , Transdução de SinaisRESUMO
RATIONALE: In cardiac dyads, junctional Ca2+ directly controls the gating of the ryanodine receptors (RyRs), and is itself dominated by RyR-mediated Ca2+ release from the sarcoplasmic reticulum. Existing probes do not report such local Ca2+ signals because of probe diffusion, so a junction-targeted Ca2+ sensor should reveal new information on cardiac excitation-contraction coupling and its modification in disease states. OBJECTIVE: To investigate Ca2+ signaling in the nanoscopic space of cardiac dyads by targeting a new sensitive Ca2+ biosensor (GCaMP6f) to the junctional space. METHODS AND RESULTS: By fusing GCaMP6f to the N terminus of triadin 1 or junctin, GCaMP6f-triadin 1/junctin was targeted to dyadic junctions, where it colocalized with t-tubules and RyRs after adenovirus-mediated gene transfer. This membrane protein-tagged biosensor displayed ≈4× faster kinetics than native GCaMP6f. Confocal imaging revealed junctional Ca2+ transients (Ca2+ nanosparks) that were ≈50× smaller in volume than conventional Ca2+ sparks (measured with diffusible indicators). The presence of the biosensor did not disrupt normal Ca2+ signaling. Because no indicator diffusion occurred, the amplitude and timing of release measurements were improved, despite the small recording volume. We could also visualize coactivation of subclusters of RyRs within a single junctional region, as well as quarky Ca2+ release events. CONCLUSIONS: This new, targeted biosensor allows selective visualization and measurement of nanodomain Ca2+ dynamics in intact cells and can be used to give mechanistic insights into dyad RyR operation in health and in disease states such as when RyRs become orphaned.
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Técnicas Biossensoriais/tendências , Sinalização do Cálcio/genética , Cálcio/fisiologia , Simulação de Dinâmica Molecular/tendências , Miócitos Cardíacos/fisiologia , Nanotecnologia/tendências , Animais , Técnicas Biossensoriais/métodos , Células Cultivadas , Coração , Masculino , Miócitos Cardíacos/química , Nanotecnologia/métodos , Ratos , Ratos Sprague-DawleyRESUMO
Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and regeneration. Challenges with quality, inter-batch consistency, cryopreservation and scale remain, reducing experimental reproducibility and clinical translation. Here, we report a robust stirred suspension cardiac differentiation protocol, and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines, the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs, bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks, which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible, scalable, and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications, and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols.
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Reatores Biológicos , Técnicas de Cultura de Células , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Organoides , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Organoides/citologia , Técnicas de Cultura de Células/métodos , Reprodutibilidade dos Testes , Células Cultivadas , Criopreservação/métodosRESUMO
AIMS: Calcium-handling capacity is a major gauge of cardiomyocyte maturity. Ryanodine receptor 2 (RYR2) is the pre-dominant calcium channel that releases calcium from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) to activate cardiomyocyte contraction. Although RYR2 was previously implied as a key regulator of cardiomyocyte maturation, the mechanisms remain unclear. The aim of this study is to solve this problem. METHODS AND RESULTS: We performed Cas9/AAV9-mediated somatic mutagenesis to knockout RYR2 specifically in cardiomyocytes in mice. We conducted a genetic mosaic analysis to dissect the cell-autonomous function of RYR2 during cardiomyocyte maturation. We found that RYR2 depletion triggered ultrastructural and transcriptomic defects relevant to cardiomyocyte maturation. These phenotypes were associated with the drastic activation of ER stress pathways. The ER stress alleviator tauroursodeoxycholic acid partially rescued the defects in RYR2-depleted cardiomyocytes. Overexpression of ATF4, a key ER stress transcription factor, recapitulated defects in RYR2-depleted cells. Integrative analysis of RNA-Seq and bioChIP-Seq data revealed that protein biosynthesis-related genes are the major direct downstream targets of ATF4. CONCLUSION: RYR2-regulated ER homeostasis is essential for cardiomyocyte maturation. Severe ER stress perturbs cardiomyocyte maturation primarily through ATF4 activation. The major downstream effector genes of ATF4 are related to protein biosynthesis.
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Miócitos Cardíacos , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Camundongos , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Cálcio/metabolismo , Retículo Sarcoplasmático/metabolismo , Resposta a Proteínas não Dobradas , Sinalização do CálcioRESUMO
Z-lines are core ultrastructural organizers of cardiomyocytes that modulate many facets of cardiac pathogenesis. Yet a comprehensive proteomic atlas of Z-line-associated components remain incomplete. Here, we established an adeno-associated virus (AAV)-delivered, cardiomyocyte-specific, proximity-labeling approach to characterize the Z-line proteome in vivo. We found palmdelphin (PALMD) as a novel Z-line-associated protein in both adult murine cardiomyocytes and human pluripotent stem cell-derived cardiomyocytes. Germline and cardiomyocyte-specific palmd knockout mice were grossly normal at baseline but exhibited compromised cardiac hypertrophy and aggravated cardiac injury upon long-term isoproterenol treatment. By contrast, cardiomyocyte-specific PALMD overexpression was sufficient to mitigate isoproterenol-induced cardiac injury. PALMD ablation perturbed transverse tubules (T-tubules) and their association with sarcoplasmic reticulum, which formed the Z-line-associated junctional membrane complex (JMC) essential for calcium handling and cardiac function. These phenotypes were associated with disrupted localization of T-tubule markers caveolin-3 (CAV3) and junctophilin-2 (JPH2) and the reduction of nexilin (NEXN) protein, a crucial Z-line-associated protein that is essential for both Z-line and JMC structures and functions. PALMD was found to interact with NEXN and enhance its protein stability while the Nexn mRNA level was not affected. Together, this study discovered PALMD as a potential target for myocardial protection and highlighted in vivo proximity proteomics as a powerful approach to nominate novel players regulating cardiac pathogenesis. Highlights: In vivo proximity proteomics uncover novel Z-line components that are undetected in in vitro proximity proteomics in cardiomyocytes.PALMD is a novel Z-line-associated protein that is dispensable for baseline cardiomyocyte function in vivo.PALMD mitigates cardiac dysfunction and myocardial injury after repeated isoproterenol insults.PALMD stabilizes NEXN, an essential Z-line-associated regulator of the junctional membrane complex and cardiac systolic function.
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Understanding how the atrial and ventricular chambers of the heart maintain their distinct identity is a prerequisite for treating chamber-specific diseases. Here, we selectively inactivated the transcription factor Tbx5 in the atrial working myocardium of the neonatal mouse heart to show that it is required to maintain atrial identity. Atrial Tbx5 inactivation downregulated highly chamber specific genes such as Myl7 and Nppa , and conversely, increased the expression of ventricular identity genes including Myl2 . Using combined single nucleus transcriptome and open chromatin profiling, we assessed genomic accessibility changes underlying the altered atrial identity expression program, identifying 1846 genomic loci with greater accessibility in control atrial cardiomyocytes compared to KO aCMs. 69% of the control-enriched ATAC regions were bound by TBX5, demonstrating a role for TBX5 in maintaining atrial genomic accessibility. These regions were associated with genes that had higher expression in control aCMs compared to KO aCMs, suggesting they act as TBX5-dependent enhancers. We tested this hypothesis by analyzing enhancer chromatin looping using HiChIP and found 510 chromatin loops that were sensitive to TBX5 dosage. Of the loops enriched in control aCMs, 73.7% contained anchors in control-enriched ATAC regions. Together, these data demonstrate a genomic role for TBX5 in maintaining the atrial gene expression program by binding to atrial enhancers and preserving tissue-specific chromatin architecture of atrial enhancers.
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Understanding how the atrial and ventricular heart chambers maintain distinct identities is a prerequisite for treating chamber-specific diseases. Here, we selectively knocked out (KO) the transcription factor Tbx5 in the atrial working myocardium to evaluate its requirement for atrial identity. Atrial Tbx5 inactivation downregulated atrial cardiomyocyte (aCM) selective gene expression. Using concurrent single nucleus transcriptome and open chromatin profiling, genomic accessibility differences were identified between control and Tbx5 KO aCMs, revealing that 69% of the control-enriched ATAC regions were bound by TBX5. Genes associated with these regions were downregulated in KO aCMs, suggesting they function as TBX5-dependent enhancers. Comparing enhancer chromatin looping using H3K27ac HiChIP identified 510 chromatin loops sensitive to TBX5 dosage, and 74.8% of control-enriched loops contained anchors in control-enriched ATAC regions. Together, these data demonstrate TBX5 maintains the atrial gene expression program by binding to and preserving the tissue-specific chromatin architecture of atrial enhancers.
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Objectives: Heparan sulfate (HS) forms heparan sulfate proteoglycans (HSPGs), such as syndecans (SDCs) and glypicans (GPCs), to perform biological processes in the mammals. This study aimed to explore the role of HS in dilated cardiomyopathy (DCM). Methods: Two high throughput RNA sequencing, two microarrays, and one single-cell RNA sequencing dataset of DCM hearts were downloaded from the Gene Expression Omnibus (GEO) database and integrated for bioinformatics analyses. Differential analysis, pathway enrichment, immunocytes infiltration, subtype identification, and single-cell RNA sequencing analysis were used in this study. Results: The expression level of most HSPGs was significantly upregulated in DCM and was closely associated with immune activation, cardiac fibrosis, and heart failure. Syndecan2 (SDC2) was highly associated with collagen I and collagen III in cardiac fibroblasts of DCM hearts. HS biosynthetic pathway was activated, while the only enzyme to hydrolyze HS was downregulated. Based on the expression of HSPGs, patients with DCM were classified into three molecular subtypes, i.e., C1, C2, and C3. Cardiac fibrosis and heart failure were more severe in the C1 subtype. Conclusion: Heparan sulfate is closely associated with immune activation, cardiac fibrosis, and heart failure in DCM. A novel molecular classification of patients with DCM is established based on HSPGs.
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Cardiac excitation-contraction coupling requires dyads, the nanoscopic microdomains formed adjacent to Z-lines by apposition of transverse tubules and junctional sarcoplasmic reticulum. Disruption of dyad architecture and function are common features of diseased cardiomyocytes. However, little is known about the mechanisms that modulate dyad organization during cardiac development, homeostasis, and disease. Here, we use proximity proteomics in intact, living hearts to identify proteins enriched near dyads. Among these proteins is CMYA5, an under-studied striated muscle protein that co-localizes with Z-lines, junctional sarcoplasmic reticulum proteins, and transverse tubules in mature cardiomyocytes. During cardiac development, CMYA5 positioning adjacent to Z-lines precedes junctional sarcoplasmic reticulum positioning or transverse tubule formation. CMYA5 ablation disrupts dyad architecture, dyad positioning at Z-lines, and junctional sarcoplasmic reticulum Ca2+ release, leading to cardiac dysfunction and inability to tolerate pressure overload. These data provide mechanistic insights into cardiomyopathy pathogenesis by demonstrating that CMYA5 anchors junctional sarcoplasmic reticulum to Z-lines, establishes dyad architecture, and regulates dyad Ca2+ release.
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Acoplamento Excitação-Contração , Retículo Sarcoplasmático , Cálcio/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Sarcolema/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) has been shown to possess pro-hypertrophic properties in the heart, but the detailed molecular mechanism that underlies the pathological process is rarely explored. In the present study, we aim to explore the role of S1P-mediated intracellular Ca2+ signaling, with a focus on sarcoplasmic reticulum (SR)-mitochondria communication, in cardiomyocyte hypertrophy. Cultured neonatal rat ventricular myocytes (NRVMs) displayed significantly hypertrophic growth after treatment with 1 µmol/L S1P for 48 h, as indicated by the cell surface area or mRNA expressions of hypertrophic marker genes (ANP, BNP and ß-MHC). Importantly, mitochondrial Ca2+ and reactive oxygen species (ROS) levels were dramatically elevated upon S1P stimulation, and pharmacological blockage of which abolished NRVM hypertrophy. 0.5 Hz electrical pacing induced similar cytosolic Ca2+ kinetics to S1P stimulation, but unaffected the peak of mitochondrial [Ca2+]. With interference of the expression of type 2 inositol 1,4,5-trisphosphate receptors (IP3R2), which are unemployed in electrical paced Ca2+ activity but may be activated by S1P, alteration in mitochondrial Ca2+ as well as the hypertrophic effect in NRVMs under S1P stimulation were attenuated. The hypertrophic effect of S1P can also be abolished by pharmacological block of S1PR1 or Gi signaling. Collectively, our study highlights the mechanistic role of IP3R2-mediated excess SR-mitochondria Ca2+ transport in S1P-induced cardiomyocyte hypertrophy.
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Sinalização do Cálcio/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/patologia , Retículo Sarcoplasmático/metabolismo , Esfingosina/análogos & derivados , Animais , Animais Recém-Nascidos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertrofia , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Esfingosina/farmacologiaRESUMO
Cardiac excitation-contraction (EC) coupling, which links plasma membrane depolarization to activation of cardiomyocyte contraction, occurs at dyads, the nanoscopic microdomains formed by apposition of transverse (T)-tubules and junctional sarcoplasmic reticulum (jSR). In a dyadic junction, EC coupling occurs through Ca2+-induced Ca2+ release. Membrane depolarization opens voltage-gated L-type Ca2+ channels (LTCCs) in the T-tubule. The resulting influx of extracellular Ca2+ into the dyadic cleft opens Ca2+ release channels known as ryanodine receptors (RYRs) in the jSR, leading to the rapid increase in cytosolic Ca2+ that triggers sarcomere contraction. The efficacy of LTCC-RYR communication greatly affects a myriad of downstream intracellular signaling events, and it is controlled by many factors, including T-tubule and jSR structure, spatial distribution of ion channels, and regulatory proteins that closely regulate the activities of channels within dyads. Alterations in dyad architecture and/or channel activity are seen in many types of heart disease. This review will focus on the current knowledge regarding cardiac dyad structure and function, their alterations in heart failure, and new approaches to study the composition and function of dyads.
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Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell-derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas dos Microfilamentos/metabolismo , Oxigenases de Função Mista/metabolismo , Mutação de Sentido Incorreto , Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Taquicardia Ventricular/enzimologia , Substituição de Aminoácidos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Oxigenases de Função Mista/genética , Miocárdio/patologia , Miócitos Cardíacos/patologia , Oxirredução , Taquicardia Ventricular/genética , Taquicardia Ventricular/patologiaRESUMO
Mitochondrial calcium ([Ca2+]mito) dynamics plays vital roles in regulating fundamental cellular and organellar functions including bioenergetics. However, neuronal [Ca2+]mito dynamics in vivo and its regulation by brain activity are largely unknown. By performing two-photon Ca2+ imaging in the primary motor (M1) and visual cortexes (V1) of awake behaving mice, we find that discrete [Ca2+]mito transients occur synchronously over somatic and dendritic mitochondrial network, and couple with cytosolic calcium ([Ca2+]cyto) transients in a probabilistic, rather than deterministic manner. The amplitude, duration, and frequency of [Ca2+]cyto transients constitute important determinants of the coupling, and the coupling fidelity is greatly increased during treadmill running (in M1 neurons) and visual stimulation (in V1 neurons). Moreover, Ca2+/calmodulin kinase II is mechanistically involved in modulating the dynamic coupling process. Thus, activity-dependent dynamic [Ca2+]mito-to-[Ca2+]cyto coupling affords an important mechanism whereby [Ca2+]mito decodes brain activity for the regulation of mitochondrial bioenergetics to meet fluctuating neuronal energy demands as well as for neuronal information processing.