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1.
Sci Rep ; 9(1): 6801, 2019 05 01.
Article in English | MEDLINE | ID: mdl-31043634

ABSTRACT

Heart failure (HF) is characterized by poor survival, a loss of catecholamine reserve and cellular structural remodeling in the form of disorganization and loss of the transverse tubule network. Indeed, survival rates for HF are worse than many common cancers and have not improved over time. Tadalafil is a clinically relevant drug that blocks phosphodiesterase 5 with high specificity and is used to treat erectile dysfunction. Using a sheep model of advanced HF, we show that tadalafil treatment improves contractile function, reverses transverse tubule loss, restores calcium transient amplitude and the heart's response to catecholamines. Accompanying these effects, tadalafil treatment normalized BNP mRNA and prevented development of subjective signs of HF. These effects were independent of changes in myocardial cGMP content and were associated with upregulation of both monomeric and dimerized forms of protein kinase G and of the cGMP hydrolyzing phosphodiesterases 2 and 3. We propose that the molecular switch for the loss of transverse tubules in HF and their restoration following tadalafil treatment involves the BAR domain protein Amphiphysin II (BIN1) and the restoration of catecholamine sensitivity is through reductions in G-protein receptor kinase 2, protein phosphatase 1 and protein phosphatase 2 A abundance following phosphodiesterase 5 inhibition.


Subject(s)
Catecholamines/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5/chemistry , Heart Failure/drug therapy , Myocardial Contraction/drug effects , Myocytes, Cardiac/drug effects , Phosphodiesterase 5 Inhibitors/pharmacology , Ventricular Remodeling/drug effects , Animals , Disease Models, Animal , Female , Heart Failure/metabolism , Heart Failure/pathology , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Sheep , Tadalafil/pharmacology
2.
PLoS One ; 13(4): e0196232, 2018.
Article in English | MEDLINE | ID: mdl-29689070

ABSTRACT

BACKGROUND: Vascular calcification is associated with increased cardiovascular morbidity and mortality in patients with atherosclerosis, diabetes and chronic kidney disease. However, no viable treatments for this condition have been identified. This study aimed to determine whether farnesyl transferase inhibitors (FTIs) can reduce vascular calcification and the mechanism by which this reduction occurs. RESULTS: We demonstrate that FTI-277 significantly inhibits phosphate-induced mineral deposition by vascular smooth muscle cells (VSMC) in vitro, prevents VSMC osteogenic differentiation, and increases mRNA expression of matrix Gla protein (MGP), an inhibitor of mineralization. FTI-277 increases Akt signaling in VSMC in short-term serum-stimulation assays and in long-term mineralization assays. In contrast, manumycin A has no effect on Akt signaling or mineralization. Co-incubation of VSMC with FTI-277 and SH6 (an Akt inhibitor) significantly reduces the inhibitory effect of FTI-277 on mineralization, demonstrating that FTI-277 inhibits calcification by activating Akt signaling. Over-expression of the constitutively active p110 sub-unit of PI3K in VSMC using adenovirus activates Akt, inhibits mineralization, suppresses VSMC differentiation and significantly enhances MGP mRNA expression. FTI-277 also inhibits phosphate-induced activation of caspase 3 and apoptosis of VSMC, and these effects are negated by co-incubation with SH6. Finally, using an ex vivo model of vascular calcification, we demonstrate that FTI-277 inhibits high phosphate-induced mineralization in aortic rings derived from rats with end-stage renal failure. CONCLUSIONS: Together, these results demonstrate that FTI-277 inhibits VSMC mineral deposition by up-regulating PI3K/Akt signaling and preventing apoptosis, suggesting that targeting farnesylation, or Akt specifically, may have therapeutic potential for the prevention of vascular calcification.


Subject(s)
Methionine/analogs & derivatives , Muscle, Smooth, Vascular/cytology , Renal Insufficiency, Chronic/complications , Signal Transduction/drug effects , Vascular Calcification/metabolism , Animals , Apoptosis/drug effects , Cattle , Cell Differentiation/drug effects , Cells, Cultured , Disease Models, Animal , Humans , Male , Methionine/pharmacology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Osteogenesis/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rats , Renal Insufficiency, Chronic/genetics , Renal Insufficiency, Chronic/metabolism , Vascular Calcification/drug therapy , Vascular Calcification/genetics , alpha-Galactosidase
3.
Circ Res ; 115(12): 986-96, 2014 Dec 05.
Article in English | MEDLINE | ID: mdl-25332206

ABSTRACT

RATIONALE: Transverse tubules (t-tubules) regulate cardiac excitation-contraction coupling and exhibit interchamber and interspecies differences in expression. In cardiac disease, t-tubule loss occurs and affects the systolic calcium transient. However, the mechanisms controlling t-tubule maintenance and whether these factors differ between species, cardiac chambers, and in a disease setting remain unclear. OBJECTIVE: To determine the role of the Bin/Amphiphysin/Rvs domain protein amphiphysin II (AmpII) in regulating t-tubule maintenance and the systolic calcium transient. METHODS AND RESULTS: T-tubule density was assessed by di-4-ANEPPS, FM4-64 or WGA staining using confocal microscopy. In rat, ferret, and sheep hearts t-tubule density and AmpII protein levels were lower in the atrium than in the ventricle. Heart failure (HF) was induced in sheep using right ventricular tachypacing and ferrets by ascending aortic coarctation. In both HF models, AmpII protein and t-tubule density were decreased in the ventricles. In the sheep, atrial t-tubules were also lost in HF and AmpII levels decreased. Conversely, junctophilin 2 levels did not show interchamber differences in the rat and ferret nor did they change in HF in the sheep or ferret. In addition, in rat atrial and sheep HF atrial cells where t-tubules were absent, junctophilin 2 had sarcomeric intracellular distribution. Small interfering RNA-induced knockdown of AmpII protein reduced t-tubule density, calcium transient amplitude, and the synchrony of the systolic calcium transient. CONCLUSIONS: AmpII is intricately involved in t-tubule maintenance. Reducing AmpII protein decreases t-tubule density, reduces the amplitude, and increases the heterogeneity of the systolic calcium transient.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Calcium/metabolism , Excitation Contraction Coupling , Heart Failure/metabolism , Myocardial Contraction , Myocytes, Cardiac/metabolism , Nerve Tissue Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cells, Cultured , Disease Models, Animal , Ferrets , Heart Atria/metabolism , Heart Atria/pathology , Heart Atria/physiopathology , Heart Failure/genetics , Heart Failure/pathology , Heart Failure/physiopathology , Heart Ventricles/metabolism , Heart Ventricles/pathology , Heart Ventricles/physiopathology , Membrane Proteins/metabolism , Microscopy, Confocal , Myocytes, Cardiac/pathology , Nerve Tissue Proteins/genetics , RNA Interference , Rats , Sarcoplasmic Reticulum/metabolism , Sheep , Transfection , Tumor Suppressor Proteins/genetics
4.
PLoS One ; 9(7): e102096, 2014.
Article in English | MEDLINE | ID: mdl-25019319

ABSTRACT

Chronic kidney disease (CKD) is defined as the progressive loss of renal function often involving glomerular, tubulo-interstitial and vascular pathology. CKD is associated with vascular calcification; the extent of which predicts morbidity and mortality. However, the molecular regulation of these events and the progression of chronic kidney disease are not fully elucidated. To investigate the function of Axl receptor tyrosine kinase in CKD we performed a sub-total nephrectomy and fed high phosphate (1%) diet to Axl+/+ and Axl-/- mice. Plasma Gas6 (Axl' ligand), renal Axl expression and downstream Akt signalling were all significantly up-regulated in Axl+/+ mice following renal mass reduction and high phosphate diet, compared to age-matched controls. Axl-/- mice had significantly enhanced uraemia, reduced bodyweight and significantly reduced survival following sub-total nephrectomy and high phosphate diet compared to Axl+/+ mice; only 45% of Axl-/- mice survived to 14 weeks post-surgery compared to 87% of Axl+/+ mice. Histological analysis of kidney remnants revealed no effect of loss of Axl on glomerular hypertrophy, calcification or renal sclerosis but identified significantly increased tubulo-interstitial apoptosis in Axl-/- mice. Vascular calcification was not induced in Axl+/+ or Axl-/- mice in the time frame we were able to examine. In conclusion, we identify the up-regulation of Gas6/Axl signalling as a protective mechanism which reduces tubulo-interstitial apoptosis and slows progression to end-stage renal failure in the murine nephrectomy and high phosphate diet model of CKD.


Subject(s)
Apoptosis/physiology , Gene Expression Regulation/physiology , Hyperphosphatemia/physiopathology , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Renal Insufficiency, Chronic/physiopathology , Analysis of Variance , Animals , Blotting, Western , DNA Primers/genetics , Hyperphosphatemia/enzymology , In Situ Nick-End Labeling , Intercellular Signaling Peptides and Proteins/blood , Kidney/metabolism , Mice , Mice, Knockout , Nephrectomy , Phosphates/administration & dosage , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Renal Insufficiency, Chronic/enzymology , Signal Transduction/physiology , Axl Receptor Tyrosine Kinase
5.
PLoS One ; 8(4): e60658, 2013.
Article in English | MEDLINE | ID: mdl-23577141

ABSTRACT

Recent studies support a role for FGF23 and its co-receptor Klotho in cardiovascular pathology, yet the underlying mechanisms remain largely elusive. Herein, we analyzed the expression of Klotho in mouse arteries and generated a novel mouse model harboring a vascular smooth muscle cell specific deletion of Klotho (Sm22-KL(-/-) ). Arterial Klotho expression was detected at very low levels with quantitative real-time PCR; Klotho protein levels were undetectable by immunohistochemistry and Western blot. There was no difference in arterial Klotho between Sm22-KL(-/-) and wild-type mice, as well as no changes in serum markers of mineral metabolism. Intravenous delivery of FGF23 elicited a rise in renal (0.005; p<0.01) but not arterial Egr-1 expression, a marker of Klotho-dependent FGF23 signaling. Further, the impact of FGF23 on vascular calcification and endothelial response was evaluated in bovine vascular smooth muscle cells (bVSMC) and in a murine ex vivo model of endothelial function, respectively. FGF23 treatment (0.125-2 ng/mL) did not modify calcification in bVSMCs or dilatory, contractile and structural properties in mice arterial specimen ex vivo. Collectively, these results demonstrate that FGF23-Klotho signaling is absent in mouse arteries and that the vascular response was unaffected by FGF23 treatment. Thus, our data do not support Klotho-mediated FGF23 effects in the vasculature although confirmative studies in humans are warranted.


Subject(s)
Arteries/drug effects , Arteries/metabolism , Calcinosis/metabolism , Fibroblast Growth Factors/pharmacology , Gene Expression Regulation/drug effects , Glucuronidase/metabolism , Animals , Arteries/pathology , Arteries/physiopathology , Calcinosis/genetics , Cattle , Early Growth Response Protein 1/genetics , Female , Fibroblast Growth Factor-23 , Gene Deletion , Glucuronidase/deficiency , Glucuronidase/genetics , Klotho Proteins , Male , Mice , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Phenotype , Time Factors
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