Gene Remodeling in Type 2 Diabetic Cardiomyopathy and Its Phenotypic Rescue with SERCA2a

Background/Aim: Diabetes-associated myocardial dysfunction results in altered gene expression in the heart. We aimed to investigate the changes in gene expression profiles accompanying diabetes-induced cardiomyopathy and its phenotypic rescue by restoration of SERCA2a expression. Methods/Results: Using the Otsuka Long-Evans Tokushima Fatty rat model of type 2 diabetes and the Agilent rat microarray chip, we analyzed gene expression by comparing differential transcriptional changes in age-matched control versus diabetic hearts and diabetic hearts that received gene transfer of SERCA2a. Microarray expression profiles of selected genes were verified with real-time qPCR and immunoblotting. Our analysis indicates that diabetic cardiomyopathy is associated with a downregulation of transcripts. Diabetic cardiomyopathic hearts have reduced levels of SERCA2a. SERCA2a gene transfer in these hearts reduced diabetes-associated hypertrophy, and differentially modulated the expression of 76 genes and reversed the transcriptional profile induced by diabetes. In isolated cardiomyocytes in vitro, SERCA2a overexpression significantly modified the expression of a number of transcripts known to be involved in insulin signaling, glucose metabolism and cardiac remodeling. Conclusion: This investigation provided insight into the pathophysiology of cardiac remodeling and the potential role o

Bone Morphogenetic Protein‐2 Decreases MicroRNA‐30b and MicroRNA‐30c to Promote Vascular Smooth Muscle Cell Calcification

Background: Vascular calcification resembles bone formation and involves vascular smooth muscle cell (SMC) transition to an osteoblast‐like phenotype to express Runx2, a master osteoblast transcription factor. One possible mechanism by which Runx2 protein expression is induced is downregulation of inhibitory microRNAs (miR). Methods and Results: Human coronary artery SMCs (CASMCs) treated with bone morphogenetic protein‐2 (BMP‐2; 100 ng/mL) demonstrated a 1.7‐fold (P<0.02) increase in Runx2 protein expression at 24 hours. A miR microarray and target prediction database analysis independently identified miR‐30b and miR‐30c (miR‐30b‐c) as miRs that regulate Runx2 expression. Real‐time–polymerase chain reaction confirmed that BMP‐2 decreased miR‐30b and miR‐30c expression. A luciferase reporter assay verified that both miR‐30b and miR‐30c bind to the 3′‐untranslated region of Runx2 mRNA to regulate its expression. CASMCs transfected with antagomirs to downregulate miR‐30b‐c demonstrated significantly increased Runx2, intracellular calcium deposition, and mineralization. Conversely, forced expression of miR‐30b‐c by transfection with pre–miR‐30b‐c prevented the increase in Runx2 expression and mineralization of SMCs. Calcified human coronary arteries demonstrated higher levels of BMP‐2 and lower levels of miR‐30b than did noncalcified donor coronary arteries. Conclusions: BMP‐2 downregulates miR‐30b and miR‐30c to increase Runx2 expression in CASMCs and promote mineralization. Strategies that modulate expression of miR‐30b and miR‐30c may influence vascular calcification

Chronic treatment with Carvedilol improves ventricular function and reduces myocyte apoptosis in an animal model of heart failure

BACKGROUND: β-blocker treatment has emerged as an effective treatment modality for heart failure. Interestingly, β-blockers can activate both pro-apoptotic and anti-apoptotic pathways. Nevertheless, the mechanism for improved cardiac function seen with β-blocker treatment remains largely unknown. Carvedilol is a non-selective β-blocker with α-receptor blockade and antioxidant properties. We therefore studied the impact of the effects of carvedilol in an animal model of end-stage heart failure. RESULTS: To test whether chronic treatment with β-blockade decreases apoptosis, we treated myopathic turkeys with two dosages of carvedilol, 1 mg/kg (DCM(1)) and 20 mg/kg (DCM(20)), for four weeks and compared them to non-treated DCM animals (DCM(0)) and to control turkeys (CON). Echocardiographic measurements showed that non-treated DCM animals had a significantly lower fractional shortening (FS) when compared to CON (68.73 ± 1.37 vs. 18.76 ± 0.59%, p < 0.001). Both doses of carvedilol significantly improved FS (33.83 ± 10.11 and 27.73 ± 6.18% vs. 18.76 ± 0.59 % for untreated DCM, p < 0.001). DCM left ventricles were characterized by a higher percentage of apoptotic nuclei when compared to CON (5.64 ± 0.49 vs. 1.72 ± 0.12%, respectively p < 0.001). Both doses of carvedilol significantly reduced the number of apoptotic nuclei (2.32 ± 0.23% and 2.36 ± 0.26% 1 mg and 20 mg/kg respectively). CONCLUSIONS: Carvedilol improves ventricular function. Furthermore, treatment with carvedilol decreased the incidence of apoptosis in cardiac myocytes from failing hearts at both doses. These data suggest that the inhibition of apoptosis with carvedilol may lead to improvement in ventricular function and may underlie a beneficial effect of β-blockade independent of heart rate lowering effects

KChIP2 attenuates cardiac hypertrophy through regulation of Ito and intracellular calcium signaling

Recent evidence shows that the auxiliary subunit KChIP2, which assembles with pore-forming Kv4-subunits, represents a new potential regulator of the cardiac calcium-independent transient outward potassium current ( I to ) density. In hypertrophy and heart failure, KChIP2 expression has been found to be significantly decreased. Our aim was to examine the role of KChIP2 in cardiac hypertrophy and the effect of restoring its expression on electrical remodeling and cardiac mechanical function using a combination of molecular, biochemical and gene targeting approaches. KChIP2 overexpression through gene transfer of Ad.KChIP2 in neonatal cardiomyocytes resulted in a significant increase in I to -channel forming Kv4.2 and Kv4.3 protein levels. In vivo gene transfer of KChIP2 in aortic banded adult rats showed that, compared to sham-operated or Ad.β-gal-transduced hearts, KChIP2 significantly attenuated the developed left ventricular hypertrophy, robustly increased I to densities, shortened action potential duration, and significantly altered myocyte mechanics by shortening contraction amplitudes and maximal rates of contraction and relaxation velocities and decreasing Ca 2+ transients. Interestingly, blocking I to with 4-aminopyridine in KChIP2-overexpressing adult cardiomyocytes significantly increased the Ca 2+ transients to control levels. One-day-old rat pups intracardially transduced with KChIP2 for two months then subjected to aortic banding for 6–8 weeks (to induce hypertrophy) showed similar echocardiographic, electrical and mechanical remodeling parameters. In addition, in cultured adult cardiomyocytes, KChIP2 overexpression increased the expression of Ca 2+ -ATPase (SERCA2a) and sodium calcium exchanger but had no effect on ryanodine receptor 2 or phospholamban expression. In neonatal myocytes, KChIP2 notably reversed Ang II-induced hypertrophic changes in protein synthesis and MAP-kinase activation. It also significantly decreased calcineurin expression, NFATc1 expression and nuclear translocation and its downstream target, MCiP1.4. Altogether, these data show that KChIP2 can attenuate cardiac hypertrophy possibly through modulation of intracellular calcium concentration and calcineurin/NFAT pathway.Centro de Investigaciones Cardiovasculare

Reversal of Cardiac Dysfunction After Long-Term Expression of SERCA2a by Gene Transfer in a Pre-Clinical Model of Heart Failure

ObjectivesThe aim of this study was to examine the effects of sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) gene transfer in a swine heart failure (HF) model.BackgroundReduced expression and activity of SERCA2a have been documented in HF. Prior studies have reported the beneficial effects of short-term SERCA2a overexpression in rodent models. However, the effects of long-term expression of SERCA2a in pre-clinical large animal models are not known.MethodsYorkshire-Landrace pigs were used (n = 16) to create volume overload by percutaneously severing chordae tendinae of the mitral apparatus with a bioptome to induce mitral regurgitation. At 2 months, pigs underwent intracoronary delivery of either recombinant adeno-associated virus type 1 (rAAV1) carrying SERCA2a under a cytomegalovirus promoter (rAAV1.SERCA2a) (n = 10; group 1) or saline (n = 6; group 2).ResultsAt 2 months, study animals were found to be in a compensated state of volume-overload HF (increased left ventricular internal diastolic and systolic diameters [LVIDd and LVIDs]). At 4 months, gene transfer resulted in: 1) positive left ventricular (LV) inotropic effects (adjusted peak left ventricular pressure rate of rise (dP/dt)max/P, 21.2 ± 3.2 s−1 group 1 vs. 15.5 ± 3.0 s−1 group 2; p < 0.01); 2) improvement in LV remodeling (% change in LVIDs −3.0 ± 10% vs. +15 ± 11%, respectively; p < 0.01). At follow-up, brain natriuretic peptide levels remained stable in group 1 after gene transfer, in contrast to rising levels in group 2. Further, cardiac SERCA2a expression was significantly decreased in group 2 whereas in group 1 it was restored to normal levels. There was no histopathological evidence of acute myocardial inflammation or necrosis.ConclusionsUsing a large-animal, volume-overload model of HF, we report that long-term overexpression of SERCA2a by in vivo rAAV1-mediated intracoronary gene transfer preserved systolic function, potentially prevented diastolic dysfunction, and improved ventricular remodeling

Branch Mode Selection during Early Lung Development

Many organs of higher organisms, such as the vascular system, lung, kidney, pancreas, liver and glands, are heavily branched structures. The branching process during lung development has been studied in great detail and is remarkably stereotyped. The branched tree is generated by the sequential, non-random use of three geometrically simple modes of branching (domain branching, planar and orthogonal bifurcation). While many regulatory components and local interactions have been defined an integrated understanding of the regulatory network that controls the branching process is lacking. We have developed a deterministic, spatio-temporal differential-equation based model of the core signaling network that governs lung branching morphogenesis. The model focuses on the two key signaling factors that have been identified in experiments, fibroblast growth factor (FGF10) and sonic hedgehog (SHH) as well as the SHH receptor patched (Ptc). We show that the reported biochemical interactions give rise to a Schnakenberg-type Turing patterning mechanisms that allows us to reproduce experimental observations in wildtype and mutant mice. The kinetic parameters as well as the domain shape are based on experimental data where available. The developed model is robust to small absolute and large relative changes in the parameter values. At the same time there is a strong regulatory potential in that the switching between branching modes can be achieved by targeted changes in the parameter values. We note that the sequence of different branching events may also be the result of different growth speeds: fast growth triggers lateral branching while slow growth favours bifurcations in our model. We conclude that the FGF10-SHH-Ptc1 module is sufficient to generate pattern that correspond to the observed branching modesComment: Initially published at PLoS Comput Bio

FGF10 maintains distal lung bud epithelium and excessive signaling leads to progenitor state arrest, distalization, and goblet cell metaplasia

<p>Abstract</p> <p>Background</p> <p>Interaction with the surrounding mesenchyme is necessary for development of endodermal organs, and Fibroblast growth factors have recently emerged as mesenchymal-expressed morphogens that direct endodermal morphogenesis. The fibroblast growth factor 10 (<it>Fgf10</it>) null mouse is characterized by the absence of lung bud development. Previous studies have shown that this requirement for <it>Fgf10 </it>is due in part to its role as a chemotactic factor during branching morphogenesis. In other endodermal organs <it>Fgf10 </it>also plays a role in regulating differentiation.</p> <p>Results</p> <p>Through gain-of-function analysis, we here find that FGF10 inhibits differentiation of the lung epithelium and promotes distalization of the embryonic lung. Ectopic expression of FGF10 in the lung epithelium caused impaired lung development and perinatal lethality in a transgenic mouse model. Lung lobes were enlarged due to increased interlobular distance and hyperplasia of the airway epithelium. Differentiation of bronchial and alveolar cell lineages was inhibited. The transgenic epithelium consisted predominantly of proliferating progenitor-like cells expressing Pro-surfactant protein C, TTF1, PEA3 and Clusterin similarly to immature distal tip cells. Strikingly, goblet cells developed within this arrested epithelium leading to goblet cell hyperplasia.</p> <p>Conclusion</p> <p>We conclude that FGF10 inhibits terminal differentiation in the embryonic lung and maintains the distal epithelium, and that excessive levels of FGF10 leads to metaplastic differentiation of goblet cells similar to that seen in chronic inflammatory diseases.</p

Diabetes Alters Intracellular Calcium Transients in Cardiac Endothelial Cells

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca2+]i) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca2+]i homeostasis due to altered sarcoplasmic reticulum Ca2+ ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca2+ regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca2+]i homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca2+]i transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca2+ ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca2+]i sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment

Cellular magnesium acquisition : an anomaly in embryonic cation homeostasis

Author Posting. © The Author(s), 2007. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Experimental and Molecular Pathology 83 (2007): 224-240, doi:10.1016/j.yexmp.2007.03.007.The intracellular dominance of magnesium ion makes clinical assessment difficult despite the critical role of Mg++ in many key functions of cells and enzymes. There is general consensus that serum Mg++ levels are not representative of the growing number of conditions for which magnesium is known to be important. There is no consensus method or sample source for testing for clinical purposes. High intracellular Mg++ in vertebrate embryos results in part from interactions of cations which influence cell membrane transport systems. These are functionally competent from the earliest stages, at least transiently held over from the unfertilized ovum. Kinetic studies with radiotracer cations, osmolar variations, media lacking one or more of the four biological cations, Na+, Mg++, K+, and Ca++, and metabolic poison 0.05 mEq/L NaF, demonstrated: (1) all four cations influence the behavior of the others, and (2) energy is required for uptake and efflux on different time scales, some against gradient. Na+ uptake is energy dependent against an efflux gradient. The rate of K+ loss is equal with or without fluoride, suggesting a lack of an energy requirement at these stages. Ca++ efflux took twice as long in the presence of fluoride, likely due in part to intracellular binding. Mg++ is anomalous in that early teleost vertebrate embryos have an intracellular content exceeding the surrounding sea water, an isolated unaffected yolk compartment, and a clear requirement for energy for both uptake and efflux. The physiological, pathological, and therapeutic roles of magnesium are poorly understood. This will change: (1) when 28Mg is once again generally available at a reasonable cost for both basic research and clinical assessment, and (2) when serum or plasma levels are determined simultaneously with intracellular values, preferably as part of complete four cation profiles. Atomic absorption spectrophotometry, energy-dispersive x-ray analysis, and inductively coupled plasma emission spectroscopy on sublingual mucosal and peripheral blood samples are potential methods of value for coordinated assessments.AEC Grant No. 134