Exercise training reverses myocardial dysfunction induced by CaMKIIδC overexpression by restoring Ca2+-homeostasis

Several conditions of heart disease, including heart failure and diabetic cardiomyopathy, are associated with upregulation of cytosolic Ca2+/calmodulin-dependent protein kinase II (CaMKIIδC) activity. In the heart, CaMKIIδC isoform targets several proteins involved in intracellular Ca2+ homeostasis. We hypothesized that high-intensity endurance training activates mechanisms that enable a rescue of dysfunctional cardiomyocyte Ca2+ handling and thereby ameliorate cardiac dysfunction despite continuous and chronic elevated levels of CaMKIIδC. CaMKIIδC transgenic (TG) and wild-type (WT) mice performed aerobic interval exercise training over 6 wk. Cardiac function was measured by echocardiography in vivo, and cardiomyocyte shortening and intracellular Ca2+ handling were measured in vitro. TG mice had reduced global cardiac function, cardiomyocyte shortening (47% reduced compared with WT, P < 0.01), and impaired Ca2+ homeostasis. Despite no change in the chronic elevated levels of CaMKIIδC, exercise improved global cardiac function, restored cardiomyocyte shortening, and reestablished Ca2+ homeostasis to values not different from WT. The key features to explain restored Ca2+ homeostasis after exercise training were increased L-type Ca2+ current density and flux by 79 and 85%, respectively (P < 0.01), increased sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) function by 50% (P < 0.01), and reduced diastolic SR Ca2+ leak by 73% (P < 0.01), compared with sedentary TG mice. In conclusion, exercise training improves global cardiac function as well as cardiomyocyte function in the presence of a maintained high CaMKII activity. The main mechanisms of exercise-induced improvements in TG CaMKIIδC mice are mediated via increased L-type Ca2+ channel currents and improved SR Ca2+ handling by restoration of SERCA2a function in addition to reduced diastolic SR Ca2+ leak

The Mechanism of Flexible Controlling as an Innovative Method in Management of Corporate Structures

У статті представлений принцип дії механізму гнучкого контролінгу як інноваційного методу управління корпоративними структурами. Розглянуто принципи застосування даного методу як інструменту ефективного управління корпоративними структурами, що дозволяють забезпечити різноманітність і гнучкість процесів управління для досягнення поставлених цілей. Розглянуто особливості контролінгу в управлінні корпоративними структурами в умовах нестабільного економічного середовища. Подано концепцію механізму контролінгу в управлінні корпоративними структурами, яка дозволяє забезпечити необхідну різноманітність процесів управління для досягнення динамічного комплексу цілей. Розглянуто особливості функціонування корпоративних структур у нестабільному ринковому оточенні. На прикладі механізму гнучкого контролінгу як способу адаптації до реалій сформованої нестабільності в економіці України представлена його здатність оперативно і тонко спрямовувати управління корпоративними структурами для прийняття правильних рішень та погодження роботи всіх підсистем підприємства.В статье представлен принцип действия механизма гибкого контроллинга как инновационного метода управления корпоративными структурами. Рассмотрены принципы применения данного метода как инструмента эффективного управления корпоративными структурами, позволяющие обеспечить разнообразие и гибкость процессов управления для достижения поставленных целей. Рассмотрены особенности контроллинга в управлении корпоративными структурами в условиях нестабильной экономической среды. Представлена концепция механизма контроллинга в управлении корпоративными структурами, которая позволяет обеспечить необходимое разнообразие процессов управления для достижения динамического комплекса целей. Рассмотрены особенности функционирования корпоративных структур в нестабильном рыночном окружении. На примере механизма гибкого контроллинга как способа адаптации к реалиям сложившейся нестабильности в экономике Украины представлена его способность оперативно и тонко направлять управление корпоративными структурами для принятия правильных решений и согласования работы всех подсистем предприятия.This article presents the principle of the mechanism controlling the flexible as an innovative method of managing corporate structures. The principles of this method as a tool for the effective management of corporate structures, allowing for variety and flexibility of management processes to achieve their goals. Features of controlling in management of corporate structures in the conditions of the unstable economic environment are considered. The concept of the mechanism of controlling in management of corporate structures which allows to provide a necessary variety of management processes for achievement of a dynamic complex of the purposes is presented. Features of functioning of corporate structures in an unstable market environment are considered. On the example of controlling a flexible mechanism as a way of adapting to the realities of the current instability in the economy of Ukraine, represented by its ability to quickly and subtly direct the management of the corporate structure to make the right decisions and coordination of all sub-systems of the enterprise

G12 Requirement for Thrombin-stimulated Gene Expression and DNA Synthesis in 1321N1 Astrocytoma Cells

Thrombin stimulation of 1321N1 astrocytoma cells leads to Ras-dependent AP-1-mediated transcriptional activation and to DNA replication. In contrast to what has been observed in most cell systems, in 1321N1 cells these responses are pertussis toxin-insensitive. The pertussis toxin-insensitive G-protein G12 has been implicated in cell growth and transformation in different cell systems. We have examined the potential role of this protein in AP-1-mediated transcriptional activation and DNA synthesis in 1321N1 cells. Transient expression of an activated (GTPase-deficient) mutant of Gα12 increased AP-1-dependent gene expression. This response was inhibited by co-expression of a dominant negative Ala-15 Ras protein. To determine whether the pertussis toxin-insensitive G12 protein is involved in the thrombin-stimulated DNA synthesis, an inhibitory antibody against the C-terminal sequence of Gα12 subunit was microinjected into 1321N1 cells. Microinjection of the anti-Gα12 resulted in a concentration-dependent inhibition of thrombin-stimulated DNA synthesis. In contrast, microinjection of nonimmune IgG or an antibody directed against the C terminus of Gα(o) did not reduce the mitogenic response to thrombin. Furthermore, microinjection of the anti-Gα12 antibody had no effect on fibroblast growth factor-stimulated DNA synthesis. These results demonstrate a specific role for Gα12 in the mitogenic response to thrombin in human astroglial cells

ATPase Inhibitory Factor-1 Disrupts Mitochondrial Ca2+ Handling and Promotes Pathological Cardiac Hypertrophy through CaMKIIδ

ATPase inhibitory factor-1 (IF1) preserves cellular ATP under conditions of respiratory collapse, yet the function of IF1 under normal respiring conditions is unresolved. We tested the hypothesis that IF1 promotes mitochondrial dysfunction and pathological cardiomyocyte hypertrophy in the context of heart failure (HF). Methods and results: Cardiac expression of IF1 was increased in mice and in humans with HF, downstream of neurohumoral signaling pathways and in patterns that resembled the fetal-like gene program. Adenoviral expression of wild-type IF1 in primary cardiomyocytes resulted in pathological hypertrophy and metabolic remodeling as evidenced by enhanced mitochondrial oxidative stress, reduced mitochondrial respiratory capacity, and the augmentation of extramitochondrial glycolysis. Similar perturbations were observed with an IF1 mutant incapable of binding to ATP synthase (E55A mutation), an indication that these effects occurred independent of binding to ATP synthase. Instead, IF1 promoted mitochondrial fragmentation and compromised mitochondrial Ca2+ handling, which resulted in sarcoplasmic reticulum Ca2+ overloading. The effects of IF1 on Ca2+ handling were associated with the cytosolic activation of calcium-calmodulin kinase II (CaMKII) and inhibition of CaMKII or co-expression of catalytically dead CaMKIIδC was sufficient to prevent IF1 induced pathological hypertrophy. Conclusions: IF1 represents a novel member of the fetal-like gene program that contributes to mitochondrial dysfunction and pathological cardiac remodeling in HF. Furthermore, we present evidence for a novel, ATP-synthase-independent, role for IF1 in mitochondrial Ca2+ handling and mitochondrial-to-nuclear crosstalk involving CaMKII

CaMKII delta C Drives Early Adaptive Ca(2+)Change and Late Eccentric Cardiac Hypertrophy

Rationale: CaMKII (Ca2+-Calmodulin dependent protein kinase) delta C activation is implicated in pathological progression of heart failure (HF) and CaMKII delta C transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca(2+)handling and CaMKII activation in hypertrophy and HF. Objective: To measure time- and location-dependent activation of CaMKII delta C signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKII delta C transgenic mice. Methods and Results: We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKII delta-knockout), CaMKII delta C transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKII delta C activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKII delta activation caused a compensatory increase in sarcoplasmic reticulum Ca(2+)content, Ca(2+)transient amplitude, and [Ca2+] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKII delta TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca(2+)transient effects reversed, but further increases in nuclear and time-averaged [Ca2+] and CaMKII activation occurred. CaMKII delta TG mice lacking delta B exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKII delta expression, especially for CaMKII delta C in nuclear fractions. Conclusions: We conclude that in early TAC perinuclear CaMKII delta C activation promotes adaptive increases in myocyte Ca(2+)transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca2+-CaMKII delta C axis contributes to eccentric hypertrophy and HF

Akt regulates L-type Ca2+ channel activity by modulating Cavα1 protein stability

The insulin IGF-1–PI3K–Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca2+ handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca2+ channel (LTCC) protein density. The pore-forming channel subunit Cavα1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Cavα1 protein levels. Our findings show that Akt-dependent phosphorylation of Cavβ2, the LTCC chaperone for Cavα1, antagonizes Cavα1 protein degradation by preventing Cavα1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca2+ channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca2+ entry, Ca2+ handling, and contractility

Novel Allosteric Sites on Ras for Lead Generation

Aberrant Ras activity is a hallmark of diverse cancers and developmental diseases. Unfortunately, conventional efforts to develop effective small molecule Ras inhibitors have met with limited success. We have developed a novel multi-level computational approach to discover potential inhibitors of previously uncharacterized allosteric sites. Our approach couples bioinformatics analysis, advanced molecular simulations, ensemble docking and initial experimental testing of potential inhibitors. Molecular dynamics simulation highlighted conserved allosteric coupling of the nucleotide-binding switch region with distal regions, including loop 7 and helix 5. Bioinformatics methods identified novel transient small molecule binding pockets close to these regions and in the vicinity of the conformationally responsive switch region. Candidate binders for these pockets were selected through ensemble docking of ZINC and NCI compound libraries. Finally, cell-based assays confirmed our hypothesis that the chosen binders can inhibit the downstream signaling activity of Ras. We thus propose that the predicted allosteric sites are viable targets for the development and optimization of new drugs