RESUMO
Sarco(endo)plasmic reticulum calcium (Ca2+) ATPase (SERCA) transports Ca2+ in muscle. Impaired SERCA activity may contribute to diabetic myopathy. Sirtuin (SIRT) 3 regulates muscle metabolism and function; however, it is unknown if SIRT3 regulates muscle SERCA activity or acetylation. We determined if SIRT3 overexpression enhances SERCA activity in mouse gastrocnemius muscle and if SIRT3 overexpression preserves gastrocnemius SERCA activity in a model of type 2 diabetes, induced by high fat - high sucrose (HFHS) feeding. We also determined if the acetylation status of SERCA proteins in mouse gastrocnemius is altered by SIRT3 overexpression or HFHS feeding. Wild-type (WT) and SIRT3 transgenic (SIRT3TG) mice, overexpressing SIRT3 in skeletal muscle, were fed a standard or HFHS diet for 4 months. SIRT3TG and WT mice developed obesity and glucose intolerance after 4 months of HFHS feeding. SERCA Vmax was higher in gastrocnemius of SIRT3TG mice compared with WT mice. HFHS-fed mice had lower SERCA1a protein levels and lower SERCA Vmax in their gastrocnemius than control-fed mice. The decrease in SERCA Vmax in gastrocnemius muscle due to HFHS feeding was attenuated by SIRT3 overexpression in HFHS-fed SIRT3TG mice. SERCA1a and SERCA2a acetylation in mouse gastrocnemius was not altered by genotype or diet. These findings suggest SIRT3 overexpression improves SERCA function in mouse skeletal muscle.
Assuntos
Diabetes Mellitus Tipo 2 , Músculo Esquelético/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Sirtuína 3 , Animais , Cálcio/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Estresse do Retículo Endoplasmático , Camundongos , Retículo Sarcoplasmático/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo , Sacarose/metabolismoRESUMO
Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting "undruggable" regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand-membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.
Assuntos
Proteínas de Membrana/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Ativação Enzimática/efeitos dos fármacos , Células Gigantes/enzimologia , Humanos , Células-Tronco Pluripotentes Induzidas/enzimologia , Microssomos/enzimologia , Simulação de Dinâmica Molecular , Estrutura Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Fosfatidilcolinas , Domínios Proteicos/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologia , Bibliotecas de Moléculas Pequenas/efeitos adversos , Bibliotecas de Moléculas Pequenas/farmacologia , Suínos , ÁguaRESUMO
The sarco-plasmic reticulum calcium pump (SERCA) plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Peptídeos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/enzimologia , Proteínas de Ligação ao Cálcio/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/genética , Conformação Proteica , Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Diabetic retinopathy is a multifactorial microvascular complication, and its pathogenesis hasn't been fully elucidated. The irreversible oxidation of cysteine 674 (C674) in the sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) was increased in the type 1 diabetic retinal vasculature. SERCA2 C674S knock-in (SKI) mouse line that half of C674 was replaced by serine 674 (S674) was used to study the effect of C674 inactivation on retinopathy. Compared with wild type (WT) mice, SKI mice had increased number of acellular capillaries and pericyte loss similar to those in type 1 diabetic WT mice. In the retina of SKI mice, pro-apoptotic proteins and intracellular Ca2+-dependent signaling pathways increased, while anti-apoptotic proteins and vessel density decreased. In endothelial cells, C674 inactivation increased the expression of pro-apoptotic proteins, damaged mitochondria, and induced cell apoptosis. These results suggest that a possible mechanism of retinopathy induced by type 1 diabetes is the interruption of calcium homeostasis in the retina by oxidation of C674. C674 is a key to maintain retinal health. Its inactivation can cause retinopathy similar to type 1 diabetes by promoting apoptosis. SERCA2 might be a potential target for the prevention and treatment of diabetic retinopathy.
Assuntos
Cisteína/genética , Retinopatia Diabética/enzimologia , Retículo Endoplasmático/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/enzimologia , Adenoviridae , Animais , Apoptose , Western Blotting , Calcineurina/metabolismo , Capilares/enzimologia , Capilares/patologia , Cisteína/metabolismo , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/genética , Retinopatia Diabética/patologia , Técnica Indireta de Fluorescência para Anticorpo , Técnicas de Introdução de Genes , Inativação Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Imuno-Histoquímica , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/metabolismo , Oxirredução , Reação em Cadeia da Polimerase em Tempo Real , Vasos Retinianos/enzimologia , Vasos Retinianos/patologia , Transdução de Sinais , EstreptozocinaRESUMO
Calcium homeostasis in the cardiomyocyte is critical to the regulation of normal cardiac function. Abnormal calcium dynamics such as altered uptake by the sarcoplasmic reticulum (SR) Ca2+-ATPase and increased diastolic SR calcium leak are involved in the development of maladaptive cardiac remodeling under pathological conditions. Ca2+/calmodulin-dependent protein kinase II-δ (CaMKIIδ) is a well-recognized key molecule in calcium dysregulation in cardiomyocytes. Elevated cellular stress is known as a common feature during pathological remodeling, and c-jun N-terminal kinase (JNK) is an important stress kinase that is activated in response to intrinsic and extrinsic stress stimuli. Our lab recently identified specific actions of JNK isoform 2 (JNK2) in CaMKIIδ expression, activation, and CaMKIIδ-dependent SR Ca2+ mishandling in the stressed heart. This review focuses on the current understanding of cardiac SR calcium handling under physiological and pathological conditions as well as the newly identified contribution of the stress kinase JNK2 in CaMKIIδ-dependent SR Ca2+ abnormal mishandling. The new findings identifying dual roles of JNK2 in CaMKIIδ expression and activation are also discussed in this review.
Assuntos
Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/enzimologia , Retículo Sarcoplasmático/enzimologia , Animais , HumanosRESUMO
Inositol trisphosphate (IP3) is a Ca2+-mobilizing second messenger shown to modulate atrial muscle contraction and is thought to contribute to atrial fibrillation. Cellular pathways underlying IP3 actions in cardiac tissue remain poorly understood, and the work presented here addresses the question whether IP3-mediated Ca2+ release from the sarcoplasmic reticulum is linked to adenylyl cyclase activity including Ca2+-stimulated adenylyl cyclases (AC1 and AC8) that are selectively expressed in atria and sinoatrial node (SAN). Immunocytochemistry in guinea pig atrial myocytes identified colocalization of type 2 IP3 receptors with AC8, while AC1 was located in close vicinity. Intracellular photorelease of IP3 by UV light significantly enhanced the amplitude of the Ca2+ transient (CaT) evoked by electrical stimulation of atrial myocytes (31 ± 6% increase 60 s after photorelease, n = 16). The increase in CaT amplitude was abolished by inhibitors of adenylyl cyclases (MDL-12,330) or protein kinase A (H89), showing that cAMP signaling is required for this effect of photoreleased IP3. In mouse, spontaneously beating right atrial preparations, phenylephrine, an α-adrenoceptor agonist with effects that depend on IP3-mediated Ca2+ release, increased the maximum beating rate by 14.7 ± 0.5%, n = 10. This effect was substantially reduced by 2.5 µmol/L 2-aminoethyl diphenylborinate and abolished by a low dose of MDL-12,330, observations which are again consistent with a functional interaction between IP3 and cAMP signaling involving Ca2+ stimulation of adenylyl cyclases in the SAN pacemaker. Understanding the interaction between IP3 receptor pathways and Ca2+-stimulated adenylyl cyclases provides important insights concerning acute mechanisms for initiation of atrial arrhythmias.NEW & NOTEWORTHY This study provides evidence supporting the proposal that IP3 signaling in cardiac atria and sinoatrial node involves stimulation of Ca2+-activated adenylyl cyclases (AC1 and AC8) by IP3-evoked Ca2+ release from junctional sarcoplasmic reticulum. AC8 and IP3 receptors are shown to be located close together, while AC1 is nearby. Greater understanding of these novel aspects of the IP3 signal transduction mechanism is important for future study in atrial physiology and pathophysiology, particularly atrial fibrillation.
Assuntos
Adenilil Ciclases/metabolismo , Relógios Biológicos , Sinalização do Cálcio , Átrios do Coração/enzimologia , Frequência Cardíaca , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Miócitos Cardíacos/enzimologia , Nó Sinoatrial/enzimologia , Potenciais de Ação , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cobaias , Átrios do Coração/citologia , Isoenzimas , Masculino , Camundongos , Retículo Sarcoplasmático/enzimologia , Fatores de TempoRESUMO
AIMS: Ventricular myocytes (VM) depolarization activates L-type Ca2+ channels (LCC) allowing Ca2+ influx (ICa) to synchronize sarcoplasmic reticulum (SR) Ca2+ release, via Ca2+-release channels (RyR2). The resulting whole-cell Ca2+ transient triggers contraction, while cytosolic Ca2+ removal by SR Ca2+ pump (SERCA2) and sarcolemmal Na+/Ca2+ exchanger (NCX) allows relaxation. In diseased hearts, extensive VM remodeling causes heterogeneous, blunted and slow Ca2+ transients. Among remodeling changes are: A) T-tubules disorganization. B) Diminished SERCA2 and low SR Ca2+. However, those often overlap, hindering their relative contribution to contractile dysfunction (CD). Furthermore, few studies have assessed their specific impact on the spatiotemporal Ca2+ transient properties and contractile dynamics simultaneously. Therefore, we sought to perform a quantitative comparison of how heterogeneous and slow Ca2+ transients, with different underlying determinants, affect contractile performance. METHODS: We used two experimental models: A) formamide-induced acute "detubulation", where VM retain functional RyR2 and SERCA2, but lack T-tubules-associated LCC and NCX. B) Intact VM from hypothyroid rats, presenting decreased SERCA2 and SR Ca2+, but maintained T-tubules. By confocal imaging of Fluo-4-loaded VM, under field-stimulation, simultaneously acquired Ca2+ transients and shortening, allowing direct correlations. KEY FINDINGS: We found near-linear correlations among key parameters of altered Ca2+ transients, caused independently by T-tubules disruption or decreased SR Ca2+, and shortening and relaxation, SIGNIFICANCE: Unrelated structural and molecular alterations converge in similarly abnormal Ca2+ transients and CD, highlighting the importance of independently reproduce disease-specific alterations, to quantitatively assess their impact on Ca2+ signaling and contractility, which would be valuable to determine potential disease-specific therapeutic targets.
Assuntos
Ventrículos do Coração/citologia , Contração Miocárdica , Miócitos Cardíacos/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/enzimologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Citosol/metabolismo , Formamidas , Hipotireoidismo/patologia , Masculino , Ratos Wistar , Fatores de TempoRESUMO
The effects of the newly synthesized covalent conjugates of water-soluble fullerene derivatives (WSFD) with xanthene dyes: polyanionic WSFD-fluorescein (1), polycationic WSFD-fluorescein (2), polyanionic WSFD-eosin (3), and polyanionic WSFD (4), polycationic WSFD (5), fluorescein (6) and eosin (7), on activity of the membrane-bound Ca2+-ATPase of the sarcoplasmic reticulum (SR Ca2+-ATPase) were studied. Compounds 1, 3, 4, 6, and 7 inhibit the hydrolytic function of the enzyme, the inhibition constants for these compounds are Ki=1.3×10-5 M (1), Ki=4.7×10-6 M (3), Ki=2.5×10-6 M (4), Ki=6.1×10-5 M (6), and Ki=5.8×10-6 M (7). The effects of compounds 3, 6, and 7 on the hydrolytic function of the enzyme is competitive; compounds 1 and 4 are noncompetitive. Polycationic WSFD fluorescein (2) and polycationic WSFD (5) do not affect ATP hydrolysis, but inhibit active Ca2+ transport in a concentration of 0.01 mM by 100±10 and 40±4%, respectively. Conjugates 1 and 3 completely inhibit the hydrolytic and transport functions of the enzyme in a concentration of 0.01 mM, and in a concentration of 0.001 mM inhibit active Ca2+ transport by 60±6 and 55±6% uncoupling the hydrolytic and transport functions of SR Ca2+-ATPases. The obtained results demonstrate a significant effect of the studied compounds on the active transmembrane transfer of Ca2+ and make it possible to predict the presence of antimetastatic and antiaggregatory activities of the studied compounds.
Assuntos
ATPases Transportadoras de Cálcio/efeitos dos fármacos , Fulerenos/farmacologia , Retículo Sarcoplasmático/enzimologia , Xantenos/farmacologia , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Corantes/química , Corantes/farmacologia , Fulerenos/química , Humanos , Cinética , Ligação Proteica/efeitos dos fármacos , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Xantenos/químicaRESUMO
RATIONALE: Diabetes mellitus is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII (Ca2+/calmodulin-dependent kinase II) activation may occur in diabetes mellitus and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality. OBJECTIVE: To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII. METHODS AND RESULTS: We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using DCF (2',7'-dichlorodihydrofluorescein diacetate) and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation-an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac-specific CaMKIIδ KO (knockout) mice. CaMKII KO or inhibition also prevented Hi-Glu-induced sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks). Thus, CaMKII activation is required for Hi-Glu-induced ROS generation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes. To test the involvement of O-GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (ThmG), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1 [(αR)-α-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide]) prevented ROS induction in response to either Hi-Glu or ThmG. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIδ was ablated (S280A), neither Hi-Glu nor ThmG induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX (NADPH oxidase) 2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (MitoTempo), and NOS (NO synthase) pathway inhibitors (L-NAME, L-NIO, and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/ThmG-induced ROS generation. CONCLUSIONS: Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires O-GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatias Diabéticas/etiologia , Glucose/toxicidade , Hiperglicemia/complicações , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Animais , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Cardiomiopatias Diabéticas/enzimologia , Cardiomiopatias Diabéticas/fisiopatologia , Ativação Enzimática , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glicosilação , Humanos , Hiperglicemia/enzimologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/enzimologia , NADPH Oxidase 2/deficiência , NADPH Oxidase 2/genética , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologiaRESUMO
Plasmodium falciparum sarcoplasmic reticulum Ca2+ ATPase (PfSERCA) is sarcoplasmic reticulum membrane bound transporter to regulate cytosol Ca2+ ions. Ca2+ act as secondary messenger and play important role in differentiation of parasite during its life cycle. Present study is epidemiological surveillance of PfSERCA (Pf3D7_0106300) gene fragment harboring 263, 402, 431 codon to look for its single nucleotide polymorphism which is well documented to be associated with Artemisinin tolerance. Filter paper with finger pricked blood samples for Plasmodium falciparum infected uncomplicated malaria patients were obtained for region as diverse as down the longitude from east to west of India i.e. Mizoram, Tripura, Meghalaya, Jharkhand, Odhisa. There observed no mutation for codon 263 at all study sites. Mizoram showed highest PfSERCA diversity with well known SNPs of L402â¯V, E431â¯K, A438â¯V and novel mutations as well i.e. A338â¯V, S357Y, S379Y. Tripura reported highest proportion of Plasmodium isolates (18.5%) with E431â¯K single nucleotide polymorphism. Moving towards the west i.e. Meghalaya, Jharkhand, Odhisa showed no occurrence of most prevalent PfSERCA 431, 402 polymorphism worldwide but some novel mutations and its haplotypes. In present study, significantly increased proportion of novel PfSERCA polymorphism among children suggests the susceptibility of these Plasmodium falciparum strains to acquired immunity. Mizoram, sharing open international border with south east asia, demonstrated highest PfSERCA diversity. Spatial PfSERCA diversity from far north east India to moving towards west implies its association with antimalarial susceptibility.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Genótipo , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Retículo Sarcoplasmático/enzimologia , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Artemisininas , ATPases Transportadoras de Cálcio/genética , Criança , Resistência a Medicamentos/genética , Haplótipos , Humanos , Índia/epidemiologia , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , MutaçãoRESUMO
We are currently facing an "obesity epidemic" worldwide. Promoting inefficient metabolism in muscle represents a potential treatment for obesity and its complications. Sarco(endo)plasmic reticulum (SR) Ca2+-ATPase (SERCA) pumps in muscle are responsible for maintaining low cytosolic Ca2+ concentration through the ATP-dependent pumping of Ca2+ from the cytosol into the SR lumen. SERCA activity has the potential to be a critical regulator of body mass and adiposity given that it is estimated to contribute upwards of 20% of daily energy expenditure. More interestingly, this fraction can be modified physiologically in the face of stressors, such as ambient temperature and diet, through its physical interaction with several regulators known to inhibit Ca2+ uptake and muscle function. In this review, we discuss advances in our understanding of Ca2+-cycling thermogenesis within skeletal muscle, focusing on SERCA and its protein regulators, which were thought previously to only modulate muscular contractility. Novelty ATP consumption by SERCA pumps comprises a large proportion of resting energy expenditure in muscle and is dynamically regulated through interactions with small SERCA regulatory proteins. SERCA efficiency correlates significantly with resting metabolism, such that individuals with a higher resting metabolic rate have less energetically efficient SERCA Ca2+ pumping in muscle (i.e., lower coupling ratio). Futile Ca2+ cycling is a versatile heat generating mechanism utilized by both skeletal muscle and beige fat.
Assuntos
ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Retículo Sarcoplasmático , Termogênese/fisiologia , Animais , Humanos , Camundongos , Modelos Biológicos , Músculo Esquelético/fisiologia , Retículo Sarcoplasmático/enzimologia , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/fisiologiaRESUMO
AIMS: Matrix metalloproteinase-2 (MMP-2) is a zinc-dependent protease which contributes to cardiac contractile dysfunction when activated during myocardial ischaemia-reperfusion (IR) injury. MMP-2 is localized to several subcellular sites inside cardiac myocytes; however, its role in the sarcoplasmic reticulum (SR) is unknown. The Ca2+ ATPase SERCA2a, which pumps cytosolic Ca2+ into the SR to facilitate muscle relaxation, is degraded in cardiac IR injury; however, the protease responsible for this is unclear. We hypothesized that MMP-2 contributes to cardiac contractile dysfunction by proteolyzing SERCA2a, thereby impairing its activity in IR injury. METHODS AND RESULTS: Isolated rat hearts were subjected to IR injury in the presence or absence of the selective MMP inhibitor ARP-100, or perfused aerobically as a control. Inhibition of MMP activity with ARP-100 significantly improved the recovery of cardiac mechanical function and prevented the increase of a 70 kDa SERCA2a degradation fragment following IR injury, although 110 kDa SERCA2a and phospholamban levels appeared unchanged. Electrophoresis of IR heart samples followed by LC-MS/MS confirmed the presence of a SERCA2a fragment of â¼70 kDa. MMP-2 activity co-purified with SR-enriched microsomes prepared from the isolated rat hearts. Endogenous SERCA2a in SR-enriched microsomes was proteolyzed to â¼70 kDa products when incubated in vitro with exogenous MMP-2. MMP-2 also cleaved purified porcine SERCA2a in vitro. SERCA activity in SR-enriched microsomes was decreased by IR injury; however, this was not prevented with ARP-100. CONCLUSION: This study shows that MMP-2 activity is found in SR-enriched microsomes from heart muscle and that SERCA2a is proteolyzed by MMP-2. The cardioprotective actions of MMP inhibition in myocardial IR injury may include the prevention of SERCA2a degradation.
Assuntos
Metaloproteinase 2 da Matriz/metabolismo , Contração Miocárdica , Traumatismo por Reperfusão Miocárdica/enzimologia , Miocárdio/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/enzimologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Ácidos Hidroxâmicos/farmacologia , Preparação de Coração Isolado , Masculino , Inibidores de Metaloproteinases de Matriz/farmacologia , Contração Miocárdica/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/patologia , Proteólise , Ratos Sprague-Dawley , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/patologia , Sulfonas/farmacologiaRESUMO
Impaired sarcoplasmic reticulum (SR) calcium transport ATPase (SERCA) gives rise to Ca2+ alternans and changes of the Ca2+release amount. These changes in Ca2+ release amount can reveal the mechanism underlying how the interaction between Ca2+ release and Ca2+ uptake induces Ca2+ alternans. This study of alternans by calculating the values of Ca2+ release properties with impaired SERCA has not been explored before. Here, we induced Ca2+ alternans by using an impaired SERCA pump under ischemic conditions. The results showed that the recruitment and refractoriness of the Ca2+ release increased as Ca2+ alternans occurred. This indicates triggering Ca waves. As the propagation of Ca waves is linked to the occurrence of Ca2+ alternans, the "threshold" for Ca waves reflects the key factor in Ca2+ alternans development, and it is still controversial nowadays. We proposed the ratio between the diastolic network SR (NSR) Ca content (Cansr) and the cytoplasmic Ca content (Ca i ) (Cansr/Ca i ) as the "threshold" of Ca waves and Ca2+ alternans. Diastolic Cansr, Ca i , and their ratio were recorded at the onset of Ca2+ alternans. Compared with certain Cansr and Ca i , the "threshold" of the ratio can better explain the comprehensive effects of the Ca2+ release and the Ca2+ uptake on Ca2+ alternans onset. In addition, these ratios are related with the function of SERCA pumps, which vary with different ischemic conditions. Thus, values of these ratios could be used to differentiate Ca2+ alternans from different ischemic cases. This agrees with some experimental results. Therefore, the certain value of diastolic Cansr/Ca i can be the better "threshold" for Ca waves and Ca2+ alternans.
Assuntos
Sinalização do Cálcio , Isquemia Miocárdica/fisiopatologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/enzimologia , Potenciais de Ação , Animais , Arritmias Cardíacas/fisiopatologia , Cálcio/metabolismo , Citoplasma/metabolismo , Diástole , Humanos , Concentração de Íons de Hidrogênio , Modelos Cardiovasculares , Isquemia Miocárdica/enzimologia , Pericárdio/metabolismo , Fosforilação , Coelhos , TermodinâmicaRESUMO
Thapsigargin (SERCA ATPase inhibitor) inhibited the S100A4 metastatic marker expression in MDA-MB231 breast cancer cells. We found that S100A4 gene transcription is regulated by Ca2+ signaling pathways. We found that the synthesis of S100A4 mRNA and S100A4 protein in MDA-MB231 cells was effectively suppressed by thapsigargin at a concentration of 0.4-4 µM with retaining cell viability. We assume that the change in the gene transcription in response to disturbance of Ca2+ homeostasis is directly involved in the remodeling of Ca2+ signaling pathways.
Assuntos
Neoplasias da Mama/patologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Retículo Sarcoplasmático/enzimologia , Tapsigargina/farmacologia , Linhagem Celular Tumoral , Humanos , Proteína A4 de Ligação a Cálcio da Família S100/genética , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
Changes of intracellular Ca2+ concentration regulate many aspects of cardiac myocyte function. About 99% of the cytoplasmic calcium in cardiac myocytes is bound to buffers, and their properties will therefore have a major influence on Ca2+ signaling. This article considers the fundamental properties and identities of the buffers and how to measure them. It reviews the effects of buffering on the systolic Ca2+ transient and how this may change physiologically, and in heart failure and both atrial and ventricular arrhythmias, as well. It is concluded that the consequences of this strong buffering may be more significant than currently appreciated, and a fuller understanding is needed for proper understanding of cardiac calcium cycling and contractility.
Assuntos
Sinalização do Cálcio/fisiologia , Miócitos Cardíacos/metabolismo , Animais , Fibrilação Atrial/metabolismo , Sítios de Ligação , Soluções Tampão , Proteínas de Ligação ao Cálcio/metabolismo , ATPases Transportadoras de Cálcio/fisiologia , Cardiomiopatia Hipertrófica/metabolismo , Citoplasma/metabolismo , Insuficiência Cardíaca/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Líquido Intracelular/metabolismo , Ligantes , Contração Miocárdica , Retículo Sarcoplasmático/enzimologia , Troponina C/metabolismoRESUMO
Schefflera kwangsiensis Merr. ex H.L. Li (Araliaceae) is a widely used traditional Chinese medicine for pain management in the clinic. In the present study, we isolated a previously undescribed lupane saponin, designated as schekwanglupaside C (Sch C) from the ethanolic extract of S. kwangsiensis. The structure of Sch C was determined by comprehensive spectroscopic and spectrometric analyses and chemical degradation. In primary cultured cortical neurons, Sch C altered the pattern of spontaneous Ca2+ oscillation (SCO) with a slight increase in the frequency of SCO right after addition and a gradual decrease in the frequency and amplitude of SCO, that dynamic change mimicked by an activator of sarcoplasmic reticulum Ca2+-ATPase (SERCA). The IC50 values for Sch C suppression of the frequency and amplitude of SCO were 1.75 and 2.51⯵M, respectively. Furthermore, we demonstrated that Sch C is a potent SERCA activator (EC50â¯=â¯1.20⯵M). Given the pivotal role of SERCA in the progression of neuropathic pain and neurodegenerative diseases, Sch C represents a new drug lead compound to develop the treatment of neuropathic pain and Alzheimer's disease.
Assuntos
Araliaceae/química , Neurônios/efeitos dos fármacos , Saponinas/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Triterpenos/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , China , Feminino , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Componentes Aéreos da Planta/química , Saponinas/isolamento & purificação , Retículo Sarcoplasmático/enzimologia , Triterpenos/isolamento & purificaçãoRESUMO
The type 2a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a key role in Ca2+ regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca2+ sensor R-CEPIA1er was used to measure the concentration of Ca2+ in the lumen of the endoplasmic reticulum (ER) ([Ca2+]ER) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca2+ release channel ryanodine receptor (RyR2) created a Ca2+ release/reuptake system that mimicked aspects of cardiac myocyte Ca2+ handling. SERCA2a function was quantified from the rate of [Ca2+]ER refilling after ER Ca2+ depletion; then, ER Ca2+ leak was measured after SERCA inhibition. ER Ca2+ uptake and leak were analyzed as a function of [Ca2+]ER to determine maximum ER Ca2+ uptake rate and maximum ER Ca2+ load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/[ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function. NEW & NOTEWORTHY The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) function in the cellular environment. The newly developed Ca2+ sensor R-CEPIA1er was used to successfully analyze Ca2+ uptake mediated by recombinant and native cardiac SERCA. These results demonstrate that this new approach can be used as a powerful tool to study new mechanisms of Ca2+ pump regulation.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Miócitos Cardíacos/enzimologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico , Técnicas Biossensoriais , Proteínas de Ligação ao Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Fatores de TempoRESUMO
RATIONALE: SPEG (Striated muscle preferentially expressed protein kinase) has 2 kinase-domains and is critical for cardiac development and function. However, it is not clear how these 2 kinase-domains function to maintain cardiac performance. OBJECTIVE: To determine the molecular functions of the 2 kinase-domains of SPEG. METHODS AND RESULTS: A proteomics approach identified SERCA2a (sarcoplasmic/endoplasmic reticulum calcium ATPase 2a) as a protein interacting with the second kinase-domain but not the first kinase-domain of SPEG. Furthermore, the second kinase-domain of SPEG could phosphorylate Thr484 on SERCA2a, promote its oligomerization and increase calcium reuptake into the sarcoplasmic/endoplasmic reticulum in culture cells and primary neonatal rat cardiomyocytes. Phosphorylation of SERCA2a by SPEG enhanced its calcium-transporting activity without affecting its ATPase activity. Depletion of Speg in neonatal rat cardiomyocytes inhibited SERCA2a-Thr484 phosphorylation and sarcoplasmic reticulum calcium reuptake. Moreover, overexpression of SERCA2aThr484Ala mutant protein also slowed sarcoplasmic reticulum calcium reuptake in neonatal rat cardiomyocytes. In contrast, domain mapping and phosphorylation analysis revealed that the first kinase-domain of SPEG interacted and phosphorylated its recently identified substrate JPH2 (junctophilin-2). An inducible heart-specific Speg knockout mouse model was generated to further study this SPEG-SERCA2a signal nexus in vivo. Inducible deletion of Speg decreased SERCA2a-Thr484 phosphorylation and its oligomerization in the heart. Importantly, inducible deletion of Speg inhibited SERCA2a calcium-transporting activity and impaired calcium reuptake into the sarcoplasmic reticulum in cardiomyocytes, which preceded morphological and functional alterations of the heart and eventually led to heart failure in adult mice. CONCLUSIONS: Our data demonstrate that the 2 kinase-domains of SPEG may play distinct roles to regulate cardiac function. The second kinase-domain of SPEG is a critical regulator for SERCA2a. Our findings suggest that SPEG may serve as a new target to modulate SERCA2a activation for treatment of heart diseases with impaired calcium homeostasis.
Assuntos
Sinalização do Cálcio , Cardiomiopatia Dilatada/enzimologia , Insuficiência Cardíaca/enzimologia , Proteínas Musculares/metabolismo , Miócitos Cardíacos/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/enzimologia , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Modelos Animais de Doenças , Células HEK293 , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/genética , Miócitos Cardíacos/patologia , Quinase de Cadeia Leve de Miosina/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases , Ratos , Retículo Sarcoplasmático/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genéticaRESUMO
AIMS: Abnormal Ca2+ release from the sarcoplasmic reticulum (SR), associated with Ca2+-calmodulin kinase II (CaMKII)-dependent phosphorylation of RyR2 at Ser2814, has consistently been linked to arrhythmogenesis and ischaemia/reperfusion (I/R)-induced cell death. In contrast, the role played by SR Ca2+ uptake under these stress conditions remains controversial. We tested the hypothesis that an increase in SR Ca2+ uptake is able to attenuate reperfusion arrhythmias and cardiac injury elicited by increased RyR2-Ser2814 phosphorylation. METHODS AND RESULTS: We used WT mice, which have been previously shown to exhibit a transient increase in RyR2-Ser2814 phosphorylation at the onset of reperfusion; mice with constitutive pseudo-phosphorylation of RyR2 at Ser2814 (S2814D) to exacerbate CaMKII-dependent reperfusion arrhythmias and cardiac damage, and phospholamban (PLN)-deficient-S2814D knock-in (SDKO) mice resulting from crossbreeding S2814D with phospholamban knockout deficient (PLNKO) mice. At baseline, S2814D and SDKO mice had structurally normal hearts. Moreover none of the strains were arrhythmic before ischaemia. Upon cardiac I/R, WT, and S2814D hearts exhibited abundant arrhythmias that were prevented by PLN ablation. In contrast, PLN ablation increased infarct size compared with WT and S2814D hearts. Mechanistically, the enhanced SR Ca2+ sequestration evoked by PLN ablation in SDKO hearts prevented arrhythmogenic events upon reperfusion by fragmenting SR Ca2+ waves into non-propagated and non-arrhythmogenic events (mini-waves). Conversely, the increase in SR Ca2+ sequestration did not reduce but rather exacerbated I/R-induced SR Ca2+ leak, as well as mitochondrial alterations, which were greatly avoided by inhibition of RyR2. These results indicate that the increase in SR Ca2+ uptake is ineffective in preventing the enhanced SR Ca2+ leak of PLN ablated myocytes from either entering into nearby mitochondria and/or activating additional CaMKII pathways, contributing to cardiac damage. CONCLUSION: Our results demonstrate that increasing SR Ca2+ uptake by PLN ablation can prevent the arrhythmic events triggered by CaMKII-dependent phosphorylation of RyR2-induced SR Ca2+ leak. These findings underscore the benefits of increasing SERCA2a activity in the face of SR Ca2+ triggered arrhythmias. However, enhanced SERCA2a cannot prevent but rather exacerbates I/R cardiac injury.
Assuntos
Arritmias Cardíacas/enzimologia , Proteínas de Ligação ao Cálcio/deficiência , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Mitocôndrias Cardíacas/enzimologia , Infarto do Miocárdio/enzimologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Miócitos Cardíacos/enzimologia , Potenciais de Ação , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/genética , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Frequência Cardíaca , Preparação de Coração Isolado , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/patologia , Fosforilação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
The effect of iron nitrosyl complexes, NO donors, of a general formula [Fe2(L)2(NO)4] with functional sulfur-containing ligands (L-3-nitro-phenol-2-yl, 4-nitro-phenol-2-yl, or 1-methyl-tetrazol-5-yl) on the activity of sarcoplasmic reticulum Ca2+-ATPase and cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) was studied. The test complexes uncoupled the hydrolytic and transport functions of Ca2+- ATPase, thus disturbing the balance of Ca2+ ions in cells, which may affect the formation of thrombi and adhesion of metastatic cells to the endothelium of capillaries. They also inhibited the activity of cGMP PDE, thereby contributing to the accumulation of the second messenger cGMP. The studied iron nitrosyl complexes can be considered as potential drugs.