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1.
Circulation ; 143(16): 1614-1628, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33682422

RESUMO

BACKGROUND: Despite in-depth knowledge of the molecular mechanisms controlling embryonic heart development, little is known about the signals governing postnatal maturation of the human heart. METHODS: Single-nucleus RNA sequencing of 54 140 nuclei from 9 human donors was used to profile transcriptional changes in diverse cardiac cell types during maturation from fetal stages to adulthood. Bulk RNA sequencing and the Assay for Transposase-Accessible Chromatin using sequencing were used to further validate transcriptional changes and to profile alterations in the chromatin accessibility landscape in purified cardiomyocyte nuclei from 21 human donors. Functional validation studies of sex steroids implicated in cardiac maturation were performed in human pluripotent stem cell-derived cardiac organoids and mice. RESULTS: Our data identify the progesterone receptor as a key mediator of sex-dependent transcriptional programs during cardiomyocyte maturation. Functional validation studies in human cardiac organoids and mice demonstrate that the progesterone receptor drives sex-specific metabolic programs and maturation of cardiac contractile properties. CONCLUSIONS: These data provide a blueprint for understanding human heart maturation in both sexes and reveal an important role for the progesterone receptor in human heart development.


Assuntos
Coração/fisiopatologia , Receptores de Progesterona/metabolismo , Feminino , Humanos , Masculino , Fatores Sexuais
2.
Am J Physiol Heart Circ Physiol ; 321(4): H807-H817, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34533400

RESUMO

Multiple mouse lines lacking the orphan G protein-coupled receptor, GPR37L1, have elicited disparate cardiovascular phenotypes. The first Gpr37l1 knockout mice study to be published reported a marked elevation in systolic blood pressure (SBP; ∼60 mmHg), revealing a potential therapeutic opportunity. The phenotype differed from our own independently generated knockout line, where male mice exhibited equivalent baseline blood pressure to wild type. Here, we attempted to reproduce the first study by characterizing the cardiovascular phenotype of both the original knockout and transgenic lines alongside a C57BL/6J control line, using the same method of blood pressure measurement. The present study supports the findings from our independently developed Gpr37l1 knockout line, finding that SBP and diastolic blood pressure (DBP) are not different in the original Gpr37l1 knockout male mice (SBP: 130.9 ± 5.3 mmHg; DBP: 90.7 ± 3.0 mmHg) compared with C57BL/6J mice (SBP: 123.1 ± 4.1 mmHg; DBP: 87.0 ± 2.7 mmHg). Instead, we attribute the apparent hypertension of the knockout line originally described to comparison with a seemingly hypotensive transgenic line (SBP 103.7 ± 5.0 mmHg; DBP 71.9 ± 3.7 mmHg). Additionally, we quantified myocardial GPR37L1 transcript in humans, which was suggested to be downregulated in cardiovascular disease. We found that GPR37L1 has very low native transcript levels in human myocardium and that expression is not different in tissue samples from patients with heart failure compared with sex-matched healthy control tissue. These findings indicate that cardiac GPR37L1 expression is unlikely to contribute to the pathophysiology of human heart failure.NEW & NOTEWORTHY This study characterizes systolic blood pressure (SBP) in a Gpr37l1 knockout mouse line, which was previously reported to have ∼60 mmHg higher SBP compared with a transgenic line. We observed only a ∼27 mmHg SBP difference between the lines. However, when compared with C57BL/6J mice, knockout mice showed no difference in SBP. We also investigated GPR37L1 mRNA abundance in human hearts and observed no difference between healthy and failing heart samples.


Assuntos
Pressão Sanguínea , Insuficiência Cardíaca/metabolismo , Hipertensão/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Animais , Estudos de Casos e Controles , Feminino , Genótipo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertensão/genética , Hipertensão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fenótipo , Receptores Acoplados a Proteínas G/genética , Especificidade da Espécie
3.
J Mol Cell Cardiol ; 128: 212-226, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30742812

RESUMO

The protein kinase C (PKC) and closely related protein kinase N (PKN) families of serine/threonine protein kinases play crucial cellular roles. Both kinases belong to the AGC subfamily of protein kinases that also include the cAMP dependent protein kinase (PKA), protein kinase B (PKB/AKT), protein kinase G (PKG) and the ribosomal protein S6 kinase (S6K). Involvement of PKC family members in heart disease has been well documented over the years, as their activity and levels are mis-regulated in several pathological heart conditions, such as ischemia, diabetic cardiomyopathy, as well as hypertrophic or dilated cardiomyopathy. This review focuses on the regulation of PKCs and PKNs in different pathological heart conditions and on the influences that PKC/PKN activation has on several physiological processes. In addition, we discuss mechanisms by which PKCs and the closely related PKNs are activated and turned-off in hearts, how they regulate cardiac specific downstream targets and pathways, and how their inhibition by small molecules is explored as new therapeutic target to treat cardiomyopathies and heart failure.


Assuntos
Cardiopatias/genética , Miocárdio/enzimologia , Proteína Quinase C/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Cardiopatias/enzimologia , Cardiopatias/patologia , Humanos , Miocárdio/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética
4.
J Biol Chem ; 293(22): 8588-8599, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29669813

RESUMO

The acceleration of myocardial relaxation produced by ß-adrenoreceptor stimulation is mediated in part by protein kinase A (PKA)-mediated phosphorylation of cardiac troponin-I (cTnI), which decreases myofibrillar Ca2+ sensitivity. Previous evidence suggests that phosphorylation of both Ser-23 and Ser-24 in cTnI is required for this Ca2+ desensitization. PKA-mediated phosphorylation also partially protects cTnI from proteolysis by calpain. Here we report that protein kinase D (PKD) phosphorylates only one serine of cTnI Ser-23/24. To explore the functional consequences of this monophosphorylation, we examined the Ca2+ sensitivity of force production and susceptibility of cTnI to calpain-mediated proteolysis when Ser-23/24 of cTnI in mouse cardiac myofibrils was nonphosphorylated, mono-phosphorylated, or bisphosphorylated (using sequential incubations in λ-phosphatase, PKD, and PKA, respectively). Phos-tag gels, Western blotting, and high-resolution MS revealed that PKD produced >90% monophosphorylation of cTnI, primarily at Ser-24, whereas PKA led to cTnI bisphosphorylation exclusively. PKD markedly decreased the Ca2+ sensitivity of force production in detergent-permeabilized ventricular trabeculae, whereas subsequent incubation with PKA produced only a small further fall of Ca2+ sensitivity. Unlike PKD, PKA also substantially phosphorylated myosin-binding protein-C and significantly accelerated cross-bridge kinetics (ktr). After phosphorylation by PKD or PKA, cTnI in isolated myofibrils was partially protected from calpain-mediated degradation. We conclude that cTnI monophosphorylation at Ser-23/24 decreases myofibrillar Ca2+ sensitivity and partially protects cTnI from calpain-induced proteolysis. In healthy cardiomyocytes, the basal monophosphorylation of cTnI may help tonically regulate myofibrillar Ca2+ sensitivity.


Assuntos
Cálcio/metabolismo , Calpaína/farmacologia , Miócitos Cardíacos/fisiologia , Miofibrilas/fisiologia , Proteólise/efeitos dos fármacos , Serina/metabolismo , Troponina I/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Serina/química
5.
Am J Pathol ; 187(12): 2645-2658, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28935576

RESUMO

Peripartum cardiomyopathy (PPCM) and dilated cardiomyopathy (DCM) show similarities in clinical presentation. However, although DCM patients do not recover and slowly deteriorate further, PPCM patients show either a fast cardiac deterioration or complete recovery. The aim of this study was to assess if underlying cellular changes can explain the clinical similarities and differences in the two diseases. We, therefore, assessed sarcomeric protein expression, modification, titin isoform shift, and contractile behavior of cardiomyocytes in heart tissue of PPCM and DCM patients and compared these with nonfailing controls. Heart samples from ischemic heart disease (ISHD) patients served as heart failure control samples. Passive force was only increased in PPCM samples compared with controls, whereas PPCM, DCM, and ISHD samples all showed increased myofilament Ca2+ sensitivity. Length-dependent activation was significantly impaired in PPCM compared with controls, no impairment was observed in ISHD samples, and DCM samples showed an intermediate response. Contractile impairments were caused by impaired protein kinase A (PKA)-mediated phosphorylation because exogenous PKA restored all parameters to control levels. Although DCM samples showed reexpression of EH-myomesin, an isoform usually only expressed in the heart before birth, PPCM and ISHD did not. The lack of EH-myomesin, combined with low PKA-mediated phosphorylation of myofilament proteins and increased compliant titin isoform, may explain the increase in passive force and blunted length-dependent activation of myofilaments in PPCM samples.


Assuntos
Cardiomiopatias/fisiopatologia , Cardiomiopatia Dilatada/fisiopatologia , Miócitos Cardíacos/patologia , Miofibrilas/patologia , Período Periparto , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/fisiopatologia , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Gravidez
6.
Proc Natl Acad Sci U S A ; 112(50): E7003-12, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26621701

RESUMO

Diastolic dysfunction is general to all idiopathic dilated (IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from increased actin-myosin formation during diastole due to altered tropomyosin position, which blocks myosin binding to actin in the absence of Ca(2+). We investigated whether ADP-stimulated force development (without Ca(2+)) can be used to reveal changes in actin-myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7mut, MYBPC3mut) and thin-filament (TNNT2mut, TNNI3mut) mutations, and IDCM were compared with sarcomere mutation-negative HCM (HCMsmn) and nonfailing donors. Myofilament ADP sensitivity was higher in IDCM and HCM compared with donors, whereas it was lower for MYBPC3. Increased ADP sensitivity in IDCM, HCMsmn, and MYH7mut was caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment. Troponin exchange experiments in a TNNT2mut sample corrected the abnormal actin-myosin blockade. In MYBPC3trunc samples, ADP sensitivity highly correlated with cardiac myosin-binding protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with cMyBP-C antibody against the actin-binding N-terminal region reduced ADP sensitivity, indicative of cMyBP-C's role in actin-myosin regulation. In the presence of Ca(2+), ADP increased myofilament force development and sarcomere stiffness. Enhanced sarcomere stiffness in sarcomere mutation-positive HCM samples was irrespective of the phosphorylation background. In conclusion, ADP-stimulated contraction can be used as a tool to study how protein phosphorylation and mutant proteins alter accessibility of myosin binding on actin. In the presence of Ca(2+), pathologic [ADP] and low PKA-phosphorylation, high actin-myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies.


Assuntos
Difosfato de Adenosina/farmacologia , Cardiopatias/metabolismo , Contração Miocárdica/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Fosforilação
7.
J Mol Cell Cardiol ; 106: 68-83, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28392437

RESUMO

Experimental data from human cardiac myocytes at body temperature is crucial for a quantitative understanding of clinically relevant cardiac function and development of whole-organ computational models. However, such experimental data is currently very limited. Specifically, important measurements to characterize changes in tension development in human cardiomyocytes that occur with perturbations in cell length are not available. To address this deficiency, in this study we present an experimental data set collected from skinned human cardiac myocytes, including the passive and viscoelastic properties of isolated myocytes, the steady-state force calcium relationship at different sarcomere lengths, and changes in tension following a rapid increase or decrease in length, and after constant velocity shortening. This data set is, to our knowledge, the first characterization of length and velocity-dependence of tension generation in human skinned cardiac myocytes at body temperature. We use this data to develop a computational model of contraction and passive viscoelasticity in human myocytes. Our model includes troponin C kinetics, tropomyosin kinetics, a three-state crossbridge model that accounts for the distortion of crossbridges, and the cellular viscoelastic response. Each component is parametrized using our experimental data collected in human cardiomyocytes at body temperature. Furthermore we are able to confirm that properties of length-dependent activation at 37°C are similar to other species, with a shift in calcium sensitivity and increase in maximum tension. We revise our model of tension generation in the skinned isolated myocyte to replicate reported tension traces generated in intact muscle during isometric tension, to provide a model of human tension generation for multi-scale simulations. This process requires changes to calcium sensitivity, cooperativity, and crossbridge transition rates. We apply this model within multi-scale simulations of biventricular cardiac function and further refine the parametrization within the whole organ context, based on obtaining a healthy ejection fraction. This process reveals that crossbridge cycling rates differ between skinned myocytes and intact myocytes.


Assuntos
Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Troponina C/química , Humanos , Contração Isométrica/fisiologia , Cinética , Contração Muscular/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/patologia , Sarcômeros/química , Sarcômeros/metabolismo , Troponina C/metabolismo
8.
Proc Natl Acad Sci U S A ; 110(4): 1446-51, 2013 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-23302686

RESUMO

The human heart is believed to grow by enlargement but not proliferation of cardiomyocytes (heart muscle cells) during postnatal development. However, recent studies have shown that cardiomyocyte proliferation is a mechanism of cardiac growth and regeneration in animals. Combined with evidence for cardiomyocyte turnover in adult humans, this suggests that cardiomyocyte proliferation may play an unrecognized role during the period of developmental heart growth between birth and adolescence. We tested this hypothesis by examining the cellular growth mechanisms of the left ventricle on a set of healthy hearts from humans aged 0-59 y (n = 36). The percentages of cardiomyocytes in mitosis and cytokinesis were highest in infants, decreasing to low levels by 20 y. Although cardiomyocyte mitosis was detectable throughout life, cardiomyocyte cytokinesis was not evident after 20 y. Between the first year and 20 y of life, the number of cardiomyocytes in the left ventricle increased 3.4-fold, which was consistent with our predictions based on measured cardiomyocyte cell cycle activity. Our findings show that cardiomyocyte proliferation contributes to developmental heart growth in young humans. This suggests that children and adolescents may be able to regenerate myocardium, that abnormal cardiomyocyte proliferation may be involved in myocardial diseases that affect this population, and that these diseases might be treatable through stimulation of cardiomyocyte proliferation.


Assuntos
Coração/crescimento & desenvolvimento , Miócitos Cardíacos/citologia , Adolescente , Adulto , Ciclo Celular , Crescimento Celular , Proliferação de Células , Criança , Pré-Escolar , Feminino , Fibrose , Coração/fisiologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Miocárdio/citologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Ploidias , Regeneração , Adulto Jovem
9.
J Mol Cell Cardiol ; 85: 262-72, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26100051

RESUMO

Decreased expression of cardiac myosin binding protein-C (cMyBP-C) in the myocardium is thought to be a contributing factor to hypertrophic cardiomyopathy in humans, and the initial molecular defect is likely abnormal cross-bridge (XB) function which leads to impaired force generation, decreased contractile performance, and hypertrophy in vivo. The myosin activator omecamtiv mecarbil (OM) is a pharmacological drug that specifically targets the myosin XB and recent evidence suggests that OM induces a significant decrease in the in vivo motility velocity and an increase in the XB duty cycle. Thus, the molecular effects of OM maybe beneficial in improving contractile function in skinned myocardium lacking cMyBP-C because absence of cMyBP-C in the sarcomere accelerates XB kinetics and enhances XB turnover rate, which presumably reduces contractile efficiency. Therefore, parameters of XB function were measured in skinned myocardium lacking cMyBP-C prior to and following OM incubation. We measured ktr, the rate of force redevelopment as an index of XB transition from both the weakly- to strongly-bound state and from the strongly- to weakly-bound states and performed stretch activation experiments to measure the rates of XB detachment (krel) and XB recruitment (kdf) in detergent-skinned ventricular preparations isolated from hearts of wild-type (WT) and cMyBP-C knockout (KO) mice. Samples from donor human hearts were also used to assess the effects of OM in cardiac muscle expressing a slow ß-myosin heavy chain (ß-MHC). Incubation of skinned myocardium with OM produced large enhancements in steady-state force generation which were most pronounced at low levels of [Ca(2+)] activations, suggesting that OM cooperatively recruits additional XB's into force generating states. Despite a large increase in steady-state force generation following OM incubation, parallel accelerations in XB kinetics as measured by ktr were not observed, and there was a significant OM-induced decrease in krel which was more pronounced in the KO skinned myocardium compared to WT skinned myocardium (58% in WT vs. 76% in KO at pCa 6.1), such that baseline differences in krel between KO and WT skinned myocardium were no longer apparent following OM-incubation. A significant decrease in the kdf was also observed following OM incubation in all groups, which may be related to the increase in the number of cooperatively recruited XB's at low Ca(2+)-activations which slows the overall rate of force generation. Our results indicate that OM may be a useful pharmacological approach to normalize hypercontractile XB kinetics in myocardium with decreased cMyBP-C expression due to its molecular effects on XB behavior.


Assuntos
Proteínas de Transporte/metabolismo , Ativadores de Enzimas/farmacologia , Contração Miocárdica/efeitos dos fármacos , Ureia/análogos & derivados , Animais , Cálcio/fisiologia , Proteínas de Transporte/genética , Feminino , Humanos , Cinética , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Miocárdio/metabolismo , Miosinas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Ureia/farmacologia
10.
Circ Res ; 112(4): 664-74, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23283722

RESUMO

RATIONALE: Myocardial diastolic stiffness and cardiomyocyte passive force (F(passive)) depend in part on titin isoform composition and phosphorylation. Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca(2+)-handling proteins, and chromatin-modifying enzymes in the heart, but has not been known to target titin. OBJECTIVE: To elucidate whether CaMKII phosphorylates titin and modulates F(passive) in normal and failing myocardium. METHODS AND RESULTS: Titin phosphorylation was assessed in CaMKIIδ/γ double-knockout (DKO) mouse, transgenic CaMKIIδC-overexpressing mouse, and human hearts, by Pro-Q-Diamond/Sypro-Ruby staining, autoradiography, and immunoblotting using phosphoserine-specific titin-antibodies. CaMKII-dependent site-specific titin phosphorylation was quantified in vivo by mass spectrometry using stable isotope labeling by amino acids in cell culture mouse heart mixed with wild-type (WT) or DKO heart. F(passive) of single permeabilized cardiomyocytes was recorded before and after CaMKII-administration. All-titin phosphorylation was reduced by >50% in DKO but increased by up to ≈100% in transgenic versus WT hearts. Conserved CaMKII-dependent phosphosites were identified within the PEVK-domain of titin by quantitative mass spectrometry and confirmed in recombinant human PEVK-fragments. CaMKII also phosphorylated the cardiac titin N2B-unique sequence. Phosphorylation at specific PEVK/titin N2B-unique sequence sites was decreased in DKO and amplified in transgenic versus WT hearts. F(passive) was elevated in DKO and reduced in transgenic compared with WT cardiomyocytes. CaMKII-administration lowered F(passive) of WT and DKO cardiomyocytes, an effect blunted by titin antibody pretreatment. Human end-stage failing hearts revealed higher CaMKII expression/activity and phosphorylation at PEVK/titin N2B-unique sequence sites than nonfailing donor hearts. CONCLUSIONS: CaMKII phosphorylates the titin springs at conserved serines/threonines, thereby lowering F(passive). Deranged CaMKII-dependent titin phosphorylation occurs in heart failure and contributes to altered diastolic stress.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Insuficiência Cardíaca/enzimologia , Proteínas Musculares/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biomecânicos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Complacência (Medida de Distensibilidade) , Conectina , Diástole/fisiologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/fisiologia , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/fisiologia
11.
Circ Res ; 112(4): 633-9, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23277198

RESUMO

RATIONALE: Cardiac myosin-binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C. OBJECTIVE: To identify and characterize additional phosphorylation sites in human cMyBP-C. METHODS AND RESULTS: Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine-rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3ß (GSK3ß) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3ß showed phosphorylation on Ser133. In addition, GSK3ß phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A-induced phosphorylation at Ser304. GSK3ß treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment. CONCLUSIONS: GSK3ß phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine-rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3ß at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.


Assuntos
Proteínas de Transporte/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Contração Miocárdica/fisiologia , Sequência de Aminoácidos , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Proteínas de Transporte/química , Glicogênio Sintase Quinase 3 beta , Ventrículos do Coração/química , Humanos , Dados de Sequência Molecular , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Fragmentos de Peptídeos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Sarcômeros/fisiologia , Espectrometria de Massas em Tandem
12.
Am J Physiol Heart Circ Physiol ; 306(8): H1171-81, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24585778

RESUMO

Frank-Starling's law reflects the ability of the heart to adjust the force of its contraction to changes in ventricular filling, a property based on length-dependent myofilament activation (LDA). The threonine at amino acid 143 of cardiac troponin I (cTnI) is prerequisite for the length-dependent increase in Ca(2+) sensitivity. Thr143 is a known target of protein kinase C (PKC) whose activity is increased in cardiac disease. Thr143 phosphorylation may modulate length-dependent myofilament activation in failing hearts. Therefore, we investigated if pseudo-phosphorylation at Thr143 modulates length dependence of force using troponin exchange experiments in human cardiomyocytes. In addition, we studied effects of protein kinase A (PKA)-mediated cTnI phosphorylation at Ser23/24, which has been reported to modulate LDA. Isometric force was measured at various Ca(2+) concentrations in membrane-permeabilized cardiomyocytes exchanged with recombinant wild-type (WT) troponin or troponin mutated at the PKC site Thr143 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after incubation with exogenous PKA. Pseudo-phosphorylation of Thr143 increased myofilament Ca(2+) sensitivity compared with WT without affecting LDA in failing and donor cardiomyocytes. Subsequent PKA treatment enhanced the length-dependent shift in Ca(2+) sensitivity after WT and 143D exchange. Exchange with Ser23/24 variants demonstrated that pseudo-phosphorylation of both Ser23 and Ser24 is needed to enhance the length-dependent increase in Ca(2+) sensitivity. cTnI pseudo-phosphorylation did not alter length-dependent changes in maximal force. Thus phosphorylation at Thr143 enhances myofilament Ca(2+) sensitivity without affecting LDA, while Ser23/24 bisphosphorylation is needed to enhance the length-dependent increase in myofilament Ca(2+) sensitivity.


Assuntos
Miócitos Cardíacos/metabolismo , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Troponina I/metabolismo , Cálcio/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Contração Miocárdica/fisiologia , Miofibrilas/efeitos dos fármacos , Miofibrilas/fisiologia , Fosforilação , Proteína Quinase C/metabolismo , Sarcômeros/fisiologia
13.
Arch Biochem Biophys ; 554: 11-21, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24814372

RESUMO

Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal force. We investigated whether Ser42/44 pseudo-phosphorylation affects force development and ATPase activity using troponin exchange in human myocardium. Additionally, we studied if pseudo-phosphorylated Ser42/44 modulates length-dependent activation of force, which is regulated by protein kinase A (PKA)-mediated cTnI-Ser23/24 phosphorylation. Isometric force was measured in membrane-permeabilized cardiomyocytes exchanged with human recombinant wild-type troponin or troponin mutated at Ser42/44 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after PKA incubation. ATPase activity was measured in troponin-exchanged cardiac muscle strips. Compared to wild-type, 42D/44D decreased Ca(2+)-sensitivity without affecting maximal force in failing and donor cardiomyocytes. In donor myocardium, 42D/44D did not affect maximal ATPase activity or tension cost. Interestingly, 42D/44D blunted the length-dependent increase in Ca(2+)-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca(2+)-sensitivity at physiological Ca(2+)-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Quinase C/metabolismo , Troponina I/química , Troponina I/metabolismo , Adenosina Trifosfatases/metabolismo , Substituição de Aminoácidos , Cálcio/metabolismo , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Feminino , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Técnicas In Vitro , Contração Isométrica/fisiologia , Masculino , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Contração Miocárdica/fisiologia , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Troponina I/genética
14.
Proc Natl Acad Sci U S A ; 108(35): 14405-10, 2011 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-21844370

RESUMO

Immobilizing a protein, that is fully compatible with the patient, on the surface of a biomedical device should make it possible to avoid adverse responses such as inflammation, rejection, or excessive fibrosis. A surface that strongly binds and does not denature the compatible protein is required. Hydrophilic surfaces do not induce denaturation of immobilized protein but exhibit a low binding affinity for protein. Here, we describe an energetic ion-assisted plasma process that can make any surface hydrophilic and at the same time enable it to covalently immobilize functional biological molecules. We show that the modification creates free radicals that migrate to the surface from a reservoir beneath. When they reach the surface, the radicals form covalent bonds with biomolecules. The kinetics and number densities of protein molecules in solution and free radicals in the reservoir control the time required to form a full protein monolayer that is covalently bound. The shelf life of the covalent binding capability is governed by the initial density of free radicals and the depth of the reservoir. We show that the high reactivity of the radicals renders the binding universal across all biological macromolecules. Because the free radical reservoir can be created on any solid material, this approach can be used in medical applications ranging from cardiovascular stents to heart-lung machines.


Assuntos
Equipamentos e Provisões/efeitos adversos , Radicais Livres , Máquina Coração-Pulmão/efeitos adversos , Proteínas/química , Stents/efeitos adversos , Humanos , Conformação Proteica , Propriedades de Superfície
15.
Biosens Bioelectron ; 246: 115879, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38056344

RESUMO

Motor proteins, such as myosin and kinesin, are biological molecular motors involved in force generation and intracellular transport within living cells. The characteristics of molecular motors, i.e., their motility over long distances, their capacity of transporting cargoes, and their very efficient energy consumption, recommend them as potential operational elements of a new class of dynamic nano-devices, with potential applications in biosensing, analyte concentrators, and biocomputation. A possible design of a biosensor based on protein molecular motor comprises a surface with immobilized motors propelling cytoskeletal filaments, which are decorated with antibodies, presented as side-branches. Upon biomolecular recognition of these branches by secondary antibodies, the 'extensions' on the cytoskeletal filaments can achieve considerable lengths (longer than several diameters of the cytoskeletal filament carrier), thus geometrically impairing or halting motility. Because the filaments are several micrometers long, this sensing mechanism converts an event in the nanometer range, i.e., antibody-antigen sizes, into an event in the micrometer range: the visualization of the halting of motility of microns-long cytoskeletal filaments. Here we demonstrate the proof of concept of a sensing system comprising heavy-mero-myosin immobilized on surfaces propelling actin filaments decorated with actin antibodies, whose movement is halted upon the recognition with secondary anti-actin antibodies. Because antibodies to the actin-myosin system are involved in several rare diseases, the first possible application for such a device may be their prognosis and diagnosis. The results also provide insights into guidelines for designing highly sensitive and very fast biosensors powered by motor proteins.


Assuntos
Actinas , Técnicas Biossensoriais , Citoesqueleto de Actina/metabolismo , Miosinas/metabolismo , Citoesqueleto/metabolismo , Anticorpos/metabolismo , Cinesinas/metabolismo
16.
Circ Genom Precis Med ; 17(3): e004369, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38853772

RESUMO

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by sarcomere gene mutations (genotype-positive HCM) in ≈50% of patients and occurs in the absence of mutations (genotype-negative HCM) in the other half of patients. We explored how alterations in the metabolomic and lipidomic landscape are involved in cardiac remodeling in both patient groups. METHODS: We performed proteomics, metabolomics, and lipidomics on myectomy samples (genotype-positive N=19; genotype-negative N=22; and genotype unknown N=6) from clinically well-phenotyped patients with HCM and on cardiac tissue samples from sex- and age-matched and body mass index-matched nonfailing donors (N=20). These data sets were integrated to comprehensively map changes in lipid-handling and energy metabolism pathways. By linking metabolomic and lipidomic data to variability in clinical data, we explored patient group-specific associations between cardiac and metabolic remodeling. RESULTS: HCM myectomy samples exhibited (1) increased glucose and glycogen metabolism, (2) downregulation of fatty acid oxidation, and (3) reduced ceramide formation and lipid storage. In genotype-negative patients, septal hypertrophy and diastolic dysfunction correlated with lowering of acylcarnitines, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines. In contrast, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines were positively associated with septal hypertrophy and diastolic impairment in genotype-positive patients. CONCLUSIONS: We provide novel insights into both general and genotype-specific metabolic changes in HCM. Distinct metabolic alterations underlie cardiac disease progression in genotype-negative and genotype-positive patients with HCM.


Assuntos
Cardiomiopatia Hipertrófica , Genótipo , Fenótipo , Humanos , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Miocárdio/metabolismo , Miocárdio/patologia , Metabolômica , Proteômica , Lipidômica , Metabolismo dos Lipídeos/genética , Sarcômeros/metabolismo , Sarcômeros/genética , Metabolismo Energético/genética , Idoso , Multiômica
17.
J Mol Cell Cardiol ; 65: 59-66, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24083979

RESUMO

Hypertrophic cardiomyopathy (HCM) is predominantly caused by mutations in genes encoding sarcomeric proteins. One of the most frequent affected genes is MYBPC3, which encodes the thick filament protein cardiac myosin binding protein C. Despite the prevalence of HCM, disease pathology and clinical outcome of sarcomeric mutations are largely unknown. We hypothesized that microRNAs (miRNAs) could play a role in the disease process. To determine which miRNAs were changed in expression, miRNA arrays were performed on heart tissue from HCM patients with a MYBPC3 mutation (n=6) and compared with hearts of non-failing donors (n=6). 532 out of 664 analyzed miRNAs were expressed in at least one heart sample. 13 miRNAs were differentially expressed in HCM compared with donors (at p<0.01, fold change ≥ 2). The genomic context of these differentially expressed miRNAs revealed that miR-204 (fold change 2.4 in HCM vs. donor) was located in an intron of the TRPM3 gene, encoding an aspecific cation channel involved in calcium entry. RT-PCR analysis revealed a trend towards TRPM3 upregulation in HCM compared with donor myocardium (fold change 2.3, p=0.078). In silico identification of mRNA targets of differentially expressed miRNAs showed a large proportion of genes involved in cardiac hypertrophy and cardiac beta-adrenergic receptor signaling and we showed reduced phosphorylation of cardiac troponin I in the HCM myocardium when compared with donor. HCM patients with MYBPC3 mutations have a specific miRNA expression profile. Downstream mRNA targets reveal possible involvement in cardiac signaling pathways.


Assuntos
Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , Mutação/genética , Miocárdio/metabolismo , Transcriptoma/genética , Adulto , Idoso , Cardiomiopatia Hipertrófica/patologia , Simulação por Computador , Feminino , Genoma Humano/genética , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Miocárdio/patologia , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Troponina I/metabolismo , Adulto Jovem
18.
Circulation ; 126(15): 1828-37, 2012 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-22972900

RESUMO

BACKGROUND: Human cardiac troponin I is known to be phosphorylated at multiple amino acid residues by several kinases. Advances in mass spectrometry allow sensitive detection of known and novel phosphorylation sites and measurement of the level of phosphorylation simultaneously at each site in myocardial samples. METHODS AND RESULTS: On the basis of in silico prediction and liquid chromatography/mass spectrometry data, 14 phosphorylation sites on cardiac troponin I, including 6 novel residues (S4, S5, Y25, T50, T180, S198), were assessed in explanted hearts from end-stage heart failure transplantation patients with ischemic heart disease or idiopathic dilated cardiomyopathy and compared with samples obtained from nonfailing donor hearts (n=10 per group). Thirty mass spectrometry-based multiple reaction monitoring quantitative tryptic peptide assays were developed for each phosphorylatable and corresponding nonphosphorylated site. The results show that in heart failure there is a decrease in the extent of phosphorylation of the known protein kinase A sites (S22, S23) and other newly discovered phosphorylation sites located in the N-terminal extension of cardiac troponin I (S4, S5, Y25), an increase in phosphorylation of the protein kinase C sites (S41, S43, T142), and an increase in phosphorylation of the IT-arm domain residues (S76, T77) and C-terminal domain novel phosphorylation sites of cardiac troponin I (S165, T180, S198). In a canine dyssynchronous heart failure model, enhanced phosphorylation at 3 novel sites was found to decline toward control after resynchronization therapy. CONCLUSIONS: Selective, functionally significant phosphorylation alterations occurred on individual residues of cardiac troponin I in heart failure, likely reflecting an imbalance in kinase/phosphatase activity. Such changes can be reversed by cardiac resynchronization.


Assuntos
Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Troponina I/metabolismo , Aminoácidos/metabolismo , Animais , Terapia de Ressincronização Cardíaca , Cães , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/terapia , Transplante de Coração , Humanos , Espectrometria de Massas , Fosforilação
19.
Am J Physiol Heart Circ Physiol ; 304(2): H260-8, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23144315

RESUMO

PKA-mediated phosphorylation of contractile proteins upon ß-adrenergic stimulation plays an important role in the regulation of cardiac performance. Phosphorylation of the PKA sites (Ser(23)/Ser(24)) of cardiac troponin (cTn)I results in a decrease in myofilament Ca(2+) sensitivity and an increase in the rate of relaxation. However, the relation between the level of phosphorylation of the sites and the functional effects in the human myocardium is unknown. Therefore, site-directed mutagenesis was used to study the effects of phosphorylation at Ser(23) and Ser(24) of cTnI on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnI-DD mimics both sites phosphorylated, cTnI-AD mimics Ser(23) unphosphorylated and Ser(24) phosphorylated, cTnI-DA mimics Ser(23) phosphorylated and Ser(24) unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca(2+) concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca(2+) sensitivity (pCa(50)) was significantly lower in cTnI-DD (pCa(50): 5.39 ± 0.01) compared with cTnI-AA (pCa(50): 5.50 ± 0.01), cTnI-AD (pCa(50): 5.48 ± 0.01), and cTnI-DA (pCa(50): 5.51 ± 0.01) at ~70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa(50) with cTnI-DD content was observed, saturating at ~55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca(2+) sensitivity, which is maximal at ~55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the DISCUSSION in this article.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Contração Miocárdica , Miócitos Cardíacos/enzimologia , Troponina/metabolismo , Cálcio/metabolismo , Humanos , Força Muscular , Mutagênese Sítio-Dirigida , Mutação , Miofibrilas/metabolismo , Fosforilação , Proteínas Recombinantes/metabolismo , Serina , Troponina/química , Troponina/genética
20.
Int J Mol Sci ; 14(6): 11034-60, 2013 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-23708103

RESUMO

Prostate cancer (PCa) is a leading cause of cancer-related death of men globally. Since its introduction, there has been intense debate as to the effectiveness of the prostate specific antigen (PSA) test as a screening tool for PCa. It is now evident that the PSA test produces unacceptably high rates of false positive results and is not prognostic. Here we review the current status of molecular biomarkers that promise to be prognostic and that might inform individual patient management. It highlights current efforts to identify biomarkers obtained by minimally invasive methods and discusses current knowledge with regard to gene fusions, mRNA and microRNAs, immunology, and cancer-associated microparticles.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Próstata/diagnóstico , Fosfatase Ácida , Humanos , Masculino , Antígeno Prostático Específico/metabolismo , Proteínas Tirosina Fosfatases/metabolismo
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