RESUMO
A hallmark of human and experimental heart failure is deficient sarcoplasmic reticulum (SR) Ca-uptake reflecting impaired contractile function. This is at least partially attributed to dephosphorylation of phospholamban by increased protein phosphatase 1 (PP1) activity. Indeed inhibition of PP1 by transgenic overexpression or gene-transfer of constitutively active inhibitor-1 improved Ca-cycling, preserved function and decreased fibrosis in small and large animal models of heart failure, suggesting that inhibitor-1 may represent a potential therapeutic target. We recently identified a novel human polymorphism (G109E) in the inhibitor-1 gene with a frequency of 7% in either normal or heart failure patients. Transgenic mice, harboring cardiac-specific expression of G109E inhibitor-1, exhibited decreases in contractility, Ca-kinetics and SR Ca-load. These depressive effects were relieved by isoproterenol stimulation. Furthermore, stress conditions (2Hz +/- Iso) induced increases in Ca-sparks, Ca-waves (60% of G109E versus 20% in wild types) and after-contractions (76% of G109E versus 23% of wild types) in mutant cardiomyocytes. Similar findings were obtained by acute expression of the G109E variant in adult cardiomyocytes in the absence or presence of endogenous inhibitor-1. The underlying mechanisms included reduced binding of mutant inhibitor-1 to PP1, increased PP1 activity, and dephosphorylation of phospholamban at Ser16 and Thr17. However, phosphorylation of the ryanodine receptor at Ser2808 was not altered while phosphorylation at Ser2814 was increased, consistent with increased activation of Ca/calmodulin-dependent protein kinase II (CaMKII), promoting aberrant SR Ca-release. Parallel in vivo studies revealed that mutant mice developed ventricular ectopy and complex ventricular arrhythmias (including bigeminy, trigeminy and ventricular tachycardia), when challenged with isoproterenol. Inhibition of CaMKII activity by KN-93 prevented the increased propensity to arrhythmias. These findings suggest that the human G109E inhibitor-1 variant impairs SR Ca-cycling and promotes arrhythmogenesis under stress conditions, which may present an additional insult in the compromised function of heart failure carriers.
Assuntos
Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Polimorfismo de Nucleotídeo Único/genética , Proteínas/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Catecolaminas/farmacologia , Diástole/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiopatologia , Humanos , Isoproterenol/farmacologia , Cinética , Camundongos Transgênicos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas/metabolismo , Ratos , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismoRESUMO
Arterial myocytes express α1-catalytic subunit isoform Na(+) pumps (75-80% of total), which are ouabain resistant in rodents, and high ouabain affinity α2-Na(+) pumps. Mice with globally reduced α2-pumps (but not α1-pumps), mice with mutant ouabain-resistant α2-pumps, and mice with a smooth muscle (SM)-specific α2-transgene (α2 (SM-Tg)) that induces overexpression all have altered blood pressure (BP) phenotypes. We generated α2 (SM-DN) mice with SM-specific α2 (not α1) reduction (>50%) using nonfunctional dominant negative (DN) α2. We compared α2 (SM-DN) and α2 (SM-Tg) mice to controls to determine how arterial SM α2-pumps affect vasoconstriction and BP. α2 (SM-DN) mice had elevated basal mean BP (mean BP by telemetry: 117 ± 4 vs. 106 ± 1 mmHg, n = 7/7, P < 0.01) and enhanced BP responses to chronic ANG II infusion (240 ng·kg(-1)·min(-1)) and high (6%) NaCl. Several arterial Ca(2+) transporters, including Na(+)/Ca(2+) exchanger 1 (NCX1) and sarcoplasmic reticulum and plasma membrane Ca(2+) pumps [sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 (SERCA2) and plasma membrane Ca(2+)-ATPase 1 (PMCA1)], were also reduced (>50%). α2 (SM-DN) mouse isolated small arteries had reduced myogenic reactivity, perhaps because of reduced Ca(2+) transporter expression. In contrast, α2 (SM-Tg) mouse aortas overexpressed α2 (>2-fold), NCX1, SERCA2, and PMCA1 (43). α2 (SM-Tg) mice had reduced basal mean BP (104 ± 1 vs. 109 ± 2 mmHg, n = 15/9, P < 0.02) and attenuated BP responses to chronic ANG II (300-400 ng·kg(-1)·min(-1)) with or without 2% NaCl but normal myogenic reactivity. NCX1 expression was inversely related to basal BP in SM-α2 engineered mice but was directly related in SM-NCX1 engineered mice. NCX1, which usually mediates arterial Ca(2+) entry, and α2-Na(+) pumps colocalize at plasma membrane-sarcoplasmic reticulum junctions and functionally couple via the local Na(+) gradient to help regulate cell Ca(2+). Altered Ca(2+) transporter expression in SM-α2 engineered mice apparently compensates to minimize Ca(2+) overload (α2 (SM-DN)) or depletion (α2 (SM-Tg)) and attenuate BP changes. In contrast, Ca(2+) transporter upregulation, observed in many rodent hypertension models, should enhance Ca(2+) entry and signaling and contribute significantly to BP elevation.
Assuntos
Artérias/metabolismo , Pressão Sanguínea , Músculo Liso Vascular/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Angiotensina II/farmacologia , Animais , Artérias/fisiologia , Camundongos , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
BACKGROUND: Heat shock proteins (Hsp) are known to enhance cell survival under various stress conditions. In the heart, the small Hsp20 has emerged as a key mediator of protection against apoptosis, remodeling, and ischemia/reperfusion injury. Moreover, Hsp20 has been implicated in modulation of cardiac contractility ex vivo. The objective of this study was to determine the in vivo role of Hsp20 in the heart and the mechanisms underlying its regulatory effects in calcium (Ca) cycling. METHODS AND RESULTS: Hsp20 overexpression in intact animals resulted in significant enhancement of cardiac function, coupled with augmented Ca cycling and sarcoplasmic reticulum Ca load in isolated cardiomyocytes. This was associated with specific increases in phosphorylation of phospholamban (PLN) at both Ser16 and Thr17, relieving its inhibition of the apparent Ca affinity of SERCA2a. Accordingly, the inotropic effects of Hsp20 were abrogated in cardiomyocytes expressing nonphosphorylatable PLN (S16A/T17A). Interestingly, the activity of type 1 protein phosphatase (PP1), a known regulator of PLN signaling, was significantly reduced by Hsp20 overexpression, suggesting that the Hsp20 stimulatory effects are partially mediated through the PP1-PLN axis. This hypothesis was supported by cell fractionation, coimmunoprecipitation, and coimmunolocalization studies, which revealed an association between Hsp20, PP1, and PLN. Furthermore, recombinant protein studies confirmed a physical interaction between AA 73 to 160 in Hsp20 and AA 163 to 330 in PP1. CONCLUSIONS: Hsp20 is a novel regulator of sarcoplasmic reticulum Ca cycling by targeting the PP1-PLN axis. These findings, coupled with the well-recognized cardioprotective role of Hsp20, suggest a dual benefit of targeting Hsp20 in heart disease.
Assuntos
Cálcio/metabolismo , Proteínas de Choque Térmico HSP20/biossíntese , Contração Miocárdica , Miocárdio/metabolismo , Proteína Fosfatase 1/metabolismo , Retículo Sarcoplasmático/metabolismo , Substituição de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Choque Térmico HSP20/genética , Cardiopatias/genética , Cardiopatias/metabolismo , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Fosforilação/genética , Proteína Fosfatase 1/genética , Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
BACKGROUND: Recent studies have identified critical roles for microRNAs (miRNAs) in a variety of cellular processes, including regulation of cardiomyocyte death. However, the signature of miRNA expression and possible roles of miRNA in the ischemic heart have been less well studied. METHODS AND RESULTS: We performed miRNA arrays to detect the expression pattern of miRNAs in murine hearts subjected to ischemia/reperfusion (I/R) in vivo and ex vivo. Surprisingly, we found that only miR-320 expression was significantly decreased in the hearts on I/R in vivo and ex vivo. This was further confirmed by TaqMan real-time polymerase chain reaction. Gain-of-function and loss-of-function approaches were employed in cultured adult rat cardiomyocytes to investigate the functional roles of miR-320. Overexpression of miR-320 enhanced cardiomyocyte death and apoptosis, whereas knockdown was cytoprotective, on simulated I/R. Furthermore, transgenic mice with cardiac-specific overexpression of miR-320 revealed an increased extent of apoptosis and infarction size in the hearts on I/R in vivo and ex vivo relative to the wild-type controls. Conversely, in vivo treatment with antagomir-320 reduced infarction size relative to the administration of mutant antagomir-320 and saline controls. Using TargetScan software and proteomic analysis, we identified heat-shock protein 20 (Hsp20), a known cardioprotective protein, as an important candidate target for miR-320. This was validated experimentally by utilizing a luciferase/GFP reporter activity assay and examining the expression of Hsp20 on miR-320 overexpression and knockdown in cardiomyocytes. CONCLUSIONS: Our data demonstrate that miR-320 is involved in the regulation of I/R-induced cardiac injury and dysfunction via antithetical regulation of Hsp20. Thus, miR-320 may constitute a new therapeutic target for ischemic heart diseases.
Assuntos
Proteínas de Choque Térmico HSP20/genética , MicroRNAs/fisiologia , Traumatismo por Reperfusão Miocárdica/genética , Animais , Cardiotônicos , Morte Celular , Células Cultivadas , Perfilação da Expressão Gênica , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Miócitos CardíacosRESUMO
The Na(+)-K(+)-ATPase (NKA) can affect intracellular Ca(2+) concentration regulation via coupling to the Na(+)-Ca(2+) exchanger and may be important in myogenic tone. We previously reported that in mice carrying a transgene for the NKA alpha(2)-isoform in smooth muscle (alpha(2sm+)), the alpha(2)-isoform protein as well as the alpha(1)-isoform (not contained in the transgene) increased to similar degrees (2-7-fold). Aortas from alpha(2sm+) mice relaxed faster from a KCl-induced contraction, hypothesized to be related to more rapid Ca(2+) clearance. To elucidate the mechanisms underlying this faster relaxation, we therefore measured the expression and distribution of proteins involved in Ca(2+) clearance. Na(+)-Ca(2+) exchanger, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), and plasma membrane Ca(2+)-ATPase (PMCA) proteins were all elevated up to approximately fivefold, whereas actin, myosin light chain, and calponin proteins were not changed in smooth muscle from alpha(2sm+) mice. Interestingly, the corresponding Ca(2+) clearance mRNA levels were unchanged. Immunocytochemical data indicate that the Ca(2+) clearance proteins are distributed similarly in wild-type and alpha(2sm+) aorta cells. In studies measuring relaxation half-times from a KCl-induced contraction in the presence of pharmacological inhibitors of SERCA and PMCA, we estimated that together these proteins were responsible for approximately 60-70% of relaxation in aorta. Moreover, the percent contribution of SERCA and PMCA to relaxation rates in alpha(2sm+) aorta was not significantly different from that in wild-type aorta. The coordinate expressions of NKA and Ca(2+) clearance proteins without change in the relative contributions of each individual protein to smooth muscle function suggest that NKA may be but one component of a larger functional Ca(2+) clearance system.
Assuntos
Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Transporte Biológico , Pressão Sanguínea , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/genética , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , RNA Mensageiro/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Fatores de Tempo , Vasoconstrição , VasodilataçãoRESUMO
Contractile dysfunction and ventricular arrhythmias associated with heart failure have been attributed to aberrant sarcoplasmic reticulum (SR) Ca(2+) cycling. The study of junctin (JCN) and histidine-rich Ca(2+) binding protein (HRC) becomes of particular importance since these proteins have been shown to be critical regulators of Ca(2+) cycling. Specifically, JCN is a SR membrane protein, which is part of the SR Ca(2+) release quaternary structure that also includes the ryanodine receptor, triadin and calsequestrin. Functionally, JCN serves as a bridge between calsequestrin and the Ca(2+) release channel, ryanodine receptor. HRC is a SR luminal Ca(2+) binding protein known to associate with both triadin and the sarcoplasmic reticulum Ca(2+)-ATPase, and may thus mediate the crosstalk between SR Ca(2+) uptake and release. Indeed, evidence from genetic models of JCN and HRC indicate that they are important in cardiophysiology as alterations in these proteins affect SR Ca(2+) handling and cardiac function. In addition, downregulation of JCN and HRC may contribute to Ca(2+) cycling perturbations manifest in the failing heart, where their protein levels are significantly reduced. This review examines the roles of JCN and HRC in SR Ca(2+) cycling and their potential significance in heart failure.
Assuntos
Arritmias Cardíacas/etiologia , Proteínas de Ligação ao Cálcio/fisiologia , Insuficiência Cardíaca/etiologia , Proteínas de Membrana/fisiologia , Oxigenases de Função Mista/fisiologia , Proteínas Musculares/fisiologia , Animais , Arritmias Cardíacas/fisiopatologia , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Insuficiência Cardíaca/fisiopatologia , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Oxigenases de Função Mista/química , Oxigenases de Função Mista/deficiência , Oxigenases de Função Mista/genética , Modelos Cardiovasculares , Proteínas Musculares/química , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Ratos , Retículo Sarcoplasmático/fisiologiaRESUMO
BACKGROUND: Summer bridges facilitate the transition from high school to college. Although many schools employ summer bridges, few have published outcomes. This article's purpose is to share preconceptions of college by underrepresented and disadvantaged nursing students and describe important elements and long-term impact of a summer bridge, a component of the Leadership 2.0 program. METHOD: A longitudinal study design was used to collect baseline, short-term, and long-term post-summer bridge data. Methods included pre- and postsurveys, interviews, and focus groups. RESULTS: After bridge completion, students felt more prepared for the nursing program. Students ranked socialization components as most important. The summer bridge had lasting impact through the first year, where grade point average and retention of underrepresented and disadvantaged bridge students was comparable to the majority first-year students. CONCLUSION: The summer bridge was effective in preparing nursing students for the first year of college. Through holistic evaluation, unique aspects of socialization critical to student success were uncovered.
Assuntos
Grupos Minoritários/psicologia , Estudantes de Enfermagem/psicologia , Populações Vulneráveis/psicologia , Diversidade Cultural , Bacharelado em Enfermagem , Feminino , Humanos , Liderança , Estudos Longitudinais , Masculino , Grupos Minoritários/estatística & dados numéricos , Pesquisa em Educação em Enfermagem , Pesquisa em Avaliação de Enfermagem , Ohio , Pesquisa Qualitativa , Estudantes de Enfermagem/estatística & dados numéricos , Populações Vulneráveis/estatística & dados numéricosRESUMO
BACKGROUND: Innovative strategies may support the Institute of Medicine's recommendation to increase the number of doctorally prepared nurses by 50% by 2020. Moreover, strategies implemented may increase the number and diversity of Doctor of Philosophy (PhD)-prepared nurses in particular. METHOD: The purpose of this article is to describe the approaches used by one college of nursing to enact a research-doctorate pipeline initiative to inspire a diverse pool of undergraduate students to consider pursuing a PhD degree. Principles that served as the foundation for this pipeline initiative are identified. RESULTS: Sixteen undergraduate students, with varying degrees of research interest, participated in this initiative. Students contributed an average of 35 hours as full members of research teams, and 94% completed the experience. Students with initial low interest in research reported enhanced interest after participation. Overall student experiences were positive and influenced future career plans. CONCLUSION: Lessons learned and future steps for the pipeline initiative are presented.
Assuntos
Bacharelado em Enfermagem , Educação de Pós-Graduação em Enfermagem , Pesquisa em Enfermagem , Escolha da ProfissãoRESUMO
Impaired sarcoplasmic reticulum calcium cycling and depressed contractility are key characteristics in heart failure. Defects in sarcoplasmic reticulum function are characterized by decreased SERCA2a Ca-transport that is partially attributable to dephosphorylation of its regulator phospholamban by increased protein phosphatase 1 activity. Inhibition of protein phosphatase 1 through activation of its endogenous inhibitor-1 has been shown to enhance cardiac Ca-handling and contractility as well as protect from pathological stress remodeling in young mice. In this study, we assessed the long-term effects of inducible expression of constitutively active inhibitor-1 in the adult heart and followed function and remodeling through the aging process, up to 20 months. Mice with inhibitor-1 had normal survival and similar function to WTs. There was no overt remodeling as evidenced by measures of left ventricular end-systolic and diastolic diameters and posterior wall dimensions, heart weight to tibia length ratio, and histology. Higher phosphorylation of phospholamban at both Ser16 and Thr17 was maintained in aged hearts with active inhibitor-1, potentially offsetting the effects of elevated Ser2815-phosphorylation in ryanodine receptor, as there were no increases in arrhythmias under stress conditions in 20-month old mice. Furthermore, long-term expression of active inhibitor-1 via recombinant adeno-associated virus type 9 gene transfer in rats with pressure-overload induced heart failure improved function and prevented remodeling, associated with increased phosphorylation of phospholamban at Ser16 and Thr17. Thus, chronic inhibition of protein phosphatase 1, through increases in active inhibitor-1, does not accelerate age-related cardiomyopathy and gene transfer of this molecule in vivo improves function and halts remodeling in the long term.
Assuntos
Envelhecimento/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Proteínas/metabolismo , Remodelação Ventricular , Envelhecimento/genética , Envelhecimento/patologia , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Fosforilação/genética , Proteínas/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
The Na(+)-K(+)-ATPase (NKA) is a transmembrane protein that sets and maintains the electrochemical gradient by extruding three Na(+) in exchange for two K(+). An important physiological role proposed for vascular smooth muscle NKA is the regulation of blood pressure via modulation of vascular smooth muscle contractility (5). To investigate the relations between the level of NKA in smooth muscle and blood pressure, we developed mice carrying a transgene for either the NKA alpha(1)- or alpha(2)-isoform (alpha(1 sm+) or alpha(2 sm+) mice) driven by the smooth muscle-specific alpha-actin promoter SMP8. Interestingly, both alpha-isoforms, the one contained in the transgene and the one not contained, were increased to a similar degree at both protein and mRNA levels. The total alpha-isoform protein was increased from 1.5-fold (alpha(1 sm+) mice) to 7-fold (alpha(2 sm+) mice). The increase in total NKA alpha-isoform protein was accompanied by a 2.5-fold increase in NKA activity in alpha(2 sm+) gastric antrum. Immunocytochemistry of the alpha(1)- and alpha(2)-isoforms in alpha(2 sm+) aortic smooth muscle cells indicated that alpha-isoform distributions were similar to those shown in wild-type cells. alpha(2 sm+) Mice (high expression) were hypotensive (109.9 +/- 1.6 vs. 121.3 +/- 1.4 mmHg; n = 13 and 11, respectively), whereas alpha(1 sm+) mice (low expression) were normotensive (122.7 +/- 2.5 vs. 117.4 +/- 2.3; n = 11 or 12). alpha(2 sm+) Aorta, but not alpha(1 sm+) aorta, relaxed faster from a KCl-induced contraction than wild-type aorta. Our results show that smooth muscle displays unique coordinate expression of the alpha-isoforms. Increasing smooth muscle NKA decreases blood pressure and is dependent on the degree of increased alpha-isoform expression.