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1.
Proc Natl Acad Sci U S A ; 121(22): e2402890121, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38771868

ABSTRACT

Maintaining the structure of cardiac membranes and membrane organelles is essential for heart function. A critical cardiac membrane organelle is the transverse tubule system (called the t-tubule system) which is an invagination of the surface membrane. A unique structural characteristic of the cardiac muscle t-tubule system is the extension of the extracellular matrix (ECM) from the surface membrane into the t-tubule lumen. However, the importance of the ECM extending into the cardiac t-tubule lumen is not well understood. Dystroglycan (DG) is an ECM receptor in the surface membrane of many cells, and it is also expressed in t-tubules in cardiac muscle. Extensive posttranslational processing and O-glycosylation are required for DG to bind ECM proteins and the binding is mediated by a glycan structure known as matriglycan. Genetic disruption resulting in defective O-glycosylation of DG results in muscular dystrophy with cardiorespiratory pathophysiology. Here, we show that DG is essential for maintaining cardiac t-tubule structural integrity. Mice with defects in O-glycosylation of DG developed normal t-tubules but were susceptible to stress-induced t-tubule loss or severing that contributed to cardiac dysfunction and disease progression. Finally, we observed similar stress-induced cardiac t-tubule disruption in a cohort of mice that solely lacked matriglycan. Collectively, our data indicate that DG in t-tubules anchors the luminal ECM to the t-tubule membrane via the polysaccharide matriglycan, which is critical to transmitting structural strength of the ECM to the t-tubules and provides resistance to mechanical stress, ultimately preventing disruptions in cardiac t-tubule integrity.


Subject(s)
Dystroglycans , Myocardium , Animals , Mice , Myocardium/metabolism , Myocardium/pathology , Glycosylation , Dystroglycans/metabolism , Extracellular Matrix/metabolism , Mice, Knockout
2.
Circulation ; 2024 Sep 18.
Article in English | MEDLINE | ID: mdl-39291390

ABSTRACT

BACKGROUND: Excitation-contraction (E-C) coupling processes become disrupted in heart failure (HF), resulting in abnormal Ca2+ homeostasis, maladaptive structural and transcriptional remodeling, and cardiac dysfunction. Junctophilin-2 (JP2) is an essential component of the E-C coupling apparatus but becomes site-specifically cleaved by calpain, leading to disruption of E-C coupling, plasmalemmal transverse tubule degeneration, abnormal Ca2+ homeostasis, and HF. However, it is not clear whether preventing site-specific calpain cleavage of JP2 is sufficient to protect the heart against stress-induced pathological cardiac remodeling in vivo. METHODS: Calpain-resistant JP2 knock-in mice (JP2CR) were generated by deleting the primary JP2 calpain cleavage site. Stress-dependent JP2 cleavage was assessed through in vitro cleavage assays and in isolated cardiomyocytes treated with 1 Āµmol/L isoproterenol by immunofluorescence. Cardiac outcomes were assessed in wild-type and JP2CR mice 5 weeks after transverse aortic constriction compared with sham surgery using echocardiography, histology, and RNA-sequencing methods. E-C coupling efficiency was measured by in situ confocal microscopy. E-C coupling proteins were evaluated by calpain assays and Western blotting. The effectiveness of adeno-associated virus gene therapy with JP2CR, JP2, or green fluorescent protein to slow HF progression was evaluated in mice with established cardiac dysfunction. RESULTS: JP2 proteolysis by calpain and in response to transverse aortic constriction and isoproterenol was blocked in JP2CR cardiomyocytes. JP2CR hearts are more resistant to pressure-overload stress, having significantly improved Ca2+ homeostasis and transverse tubule organization with significantly attenuated cardiac dysfunction, hypertrophy, lung edema, fibrosis, and gene expression changes relative to wild-type mice. JP2CR preserves the integrity of calpain-sensitive E-C coupling-related proteins, including ryanodine receptor 2, CaV1.2, and sarcoplasmic reticulum calcium ATPase 2a, by attenuating transverse aortic constriction-induced increases in calpain activity. Furthermore, JP2CR gene therapy after the onset of cardiac dysfunction was found to be effective at slowing the progression of HF and superior to wild-type JP2. CONCLUSIONS: The data presented here demonstrate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain cleavage of JP2 offers multifaceted beneficial effects, conferring cardiac protection against stress-induced proteolysis, hypertrophy, and HF. Our data also indicate that specifically targeting the primary calpain cleavage site of JP2 by gene therapy approaches holds great therapeutic potential as a novel precision medicine for treating HF.

3.
Eur J Nucl Med Mol Imaging ; 51(5): 1395-1408, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38095674

ABSTRACT

PURPOSE: Cancer treatment with alpha-emitter-based radioligand therapies (α-RLTs) demonstrates promising tumor responses. Radiolabeled peptides are filtered through glomeruli, followed by potential reabsorption of a fraction by proximal tubules, which may cause acute kidney injury (AKI) and chronic kidney disease (CKD). Because tubular cells are considered the primary site of radiopeptides' renal reabsorption and potential injury, the current use of kidney biomarkers of glomerular functional loss limits the evaluation of possible nephrotoxicity and its early detection. This study aimed to investigate whether urinary secretion of tubular injury biomarkers could be used as an additional non-invasive sensitive diagnostic tool to identify unrecognizable tubular damage and risk of long-term α-RLT nephrotoxicity. METHODS: A bifunctional cyclic peptide, melanocortin 1 ligand (MC1L), labeled with [203Pb]Pb-MC1L, was used for [212Pb]Pb-MC1L biodistribution and absorbed dose measurements in CD-1 Elite mice. Mice were treated with [212Pb]Pb-MC1L in a dose-escalation study up to levels of radioactivity intended to induce kidney injury. The approach enabled prospective kidney functional and injury biomarker evaluation and late kidney histological analysis to validate these biomarkers. RESULTS: Biodistribution analysis identified [212Pb]Pb-MC1L reabsorption in kidneys with a dose deposition of 2.8, 8.9, and 20 Gy for 0.9, 3.0, and 6.7 MBq injected [212Pb]Pb-MC1L doses, respectively. As expected, mice receiving 6.7 MBq had significant weight loss and CKD evidence based on serum creatinine, cystatin C, and kidney histological alterations 28 weeks after treatment. A dose-dependent urinary neutrophil gelatinase-associated lipocalin (NGAL, tubular injury biomarker) urinary excretion the day after [212Pb]Pb-MC1L treatment highly correlated with the severity of late tubulointerstitial injury and histological findings. CONCLUSION: Urine NGAL secretion could be a potential early diagnostic tool to identify unrecognized tubular damage and predict long-term α-RLT-related nephrotoxicity.


Subject(s)
Lead , Renal Insufficiency, Chronic , Mice , Animals , Lipocalin-2/urine , Tissue Distribution , Early Detection of Cancer , Biomarkers , Creatinine
4.
Circ Res ; 130(9): 1306-1317, 2022 04 29.
Article in English | MEDLINE | ID: mdl-35317607

ABSTRACT

BACKGROUND: Transcriptional remodeling is known to contribute to heart failure (HF). Targeting stress-dependent gene expression mechanisms may represent a clinically relevant gene therapy option. We recently uncovered a salutary mechanism in the heart whereby JP2 (junctophilin-2), an essential component of the excitation-contraction coupling apparatus, is site-specifically cleaved and releases an N-terminal fragment (JP2NT [N-terminal fragment of JP2]) that translocates into the nucleus and functions as a transcriptional repressor of HF-related genes. This study aims to determine whether JP2NT can be leveraged by gene therapy techniques for attenuating HF progression in a preclinical pressure overload model. METHODS: We intraventricularly injected adeno-associated virus (AAV) (2/9) vectors expressing eGFP (enhanced green fluorescent protein), JP2NT, or DNA-binding deficient JP2NT (JP2NTΔbNLS/ARR) into neonatal mice and induced cardiac stress by transaortic constriction (TAC) 9 weeks later. We also treated mice with established moderate HF from TAC stress with either AAV-JP2NT or AAV-eGFP. RNA-sequencing analysis was used to reveal changes in hypertrophic and HF-related gene transcription by JP2NT gene therapy after TAC. Echocardiography, confocal imaging, and histology were performed to evaluate heart function and pathological myocardial remodeling following stress. RESULTS: Mice preinjected with AAV-JP2NT exhibited ameliorated cardiac remodeling following TAC. The JP2NT DNA-binding domain is required for cardioprotection as its deletion within the AAV-JP2NT vector prevented improvement in TAC-induced cardiac dysfunction. Functional and histological data suggest that JP2NT gene therapy after the onset of cardiac dysfunction is effective at slowing the progression of HF. RNA-sequencing analysis further revealed a broad reversal of hypertrophic and HF-related gene transcription by JP2NT overexpression after TAC. CONCLUSIONS: Our prevention- and intervention-based approaches here demonstrated that AAV-mediated delivery of JP2NT into the myocardium can attenuate stress-induced transcriptional remodeling and the development of HF when administered either before or after cardiac stress initiation. Our data indicate that JP2NT gene therapy holds great potential as a novel therapeutic for treating hypertrophy and HF.


Subject(s)
Heart Failure , Animals , DNA , Dependovirus , Disease Models, Animal , Genetic Therapy , Heart Failure/genetics , Heart Failure/metabolism , Heart Failure/therapy , Membrane Proteins , Mice , Mice, Inbred C57BL , RNA , Ventricular Remodeling
5.
Circulation ; 145(7): 513-530, 2022 02 15.
Article in English | MEDLINE | ID: mdl-35000411

ABSTRACT

BACKGROUND: Aortic valve stenosis is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain underexplored. METHODS: Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA sequencing was used to define pathways involved in driving sex-dependent activation. Interventions with small molecule inhibitors and siRNA transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. RESULTS: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation through Rho-associated protein kinase signaling. CONCLUSIONS: Together, in vivo and in vitro results confirm sex dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.


Subject(s)
Aortic Valve/cytology , Gene Expression , Genes, X-Linked , Myofibroblasts/metabolism , X Chromosome Inactivation , Actins/genetics , Actins/metabolism , Animals , Aortic Valve Stenosis/etiology , Aortic Valve Stenosis/metabolism , Aortic Valve Stenosis/pathology , Biomarkers , Cells, Cultured , Disease Models, Animal , Female , Gene Expression Regulation/drug effects , Humans , Immunohistochemistry , Male , Myofibroblasts/drug effects , Sex Factors , Signal Transduction , Swine , Transcriptome
6.
FASEB J ; 36(5): e22306, 2022 05.
Article in English | MEDLINE | ID: mdl-35385164

ABSTRACT

As aortic valve stenosis develops, valve tissue becomes stiffer. In response to this change in environmental mechanical stiffness, valvular interstitial cells (VICs) activate into myofibroblasts. We aimed to investigate the role of mechanosensitive calcium channel Transient Receptor Potential Vanilloid type 4 (TRPV4) in stiffness induced myofibroblast activation. We verified TRPV4 functionality in VICs using live calcium imaging during application of small molecule modulators of TRPV4 activity. We designed hydrogel biomaterials that mimic mechanical features of healthy or diseased valve tissue microenvironments, respectively, to investigate the role of TRPV4 in myofibroblast activation and proliferation. Our results show that TRPV4 regulates VIC proliferation in a microenvironment stiffness-independent manner. While there was a trend toward inhibiting myofibroblast activation on soft microenvironments during TRPV4 inhibition, we observed near complete deactivation of myofibroblasts on stiff microenvironments. We further identified Yes-activated protein (YAP) as a downstream target for TRPV4 activity on stiff microenvironments. Mechanosensitive TRPV4 channels regulate VIC myofibroblast activation, whereas proliferation regulation is independent of the microenvironmental stiffness. Collectively, the data suggests differential regulation of stiffness-induced proliferation and myofibroblast activation. Our data further suggest a regulatory role for TRPV4 regarding YAP nuclear localization. TRPV4 is an important regulator for VIC myofibroblast activation, which is linked to the initiation of valve fibrosis. Although more validation studies are necessary, we suggest TRPV4 as a promising pharmaceutical target to slow aortic valve stenosis progression.


Subject(s)
Aortic Valve Stenosis , Calcinosis , Myofibroblasts , Animals , Aortic Valve/metabolism , Aortic Valve Stenosis/metabolism , Calcinosis/metabolism , Cell Proliferation , Cells, Cultured , Hydrogels , Myofibroblasts/metabolism , Swine , TRPV Cation Channels/metabolism
7.
Cell ; 133(3): 462-74, 2008 May 02.
Article in English | MEDLINE | ID: mdl-18455987

ABSTRACT

Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Heart Diseases/metabolism , Methionine/metabolism , Myocytes, Cardiac/metabolism , Signal Transduction , Angiotensin II , Animals , Apoptosis , Calcium , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calmodulin/metabolism , Methionine Sulfoxide Reductases , Mice , Mutagenesis, Site-Directed , Myocytes, Cardiac/cytology , Oxidation-Reduction , Oxidoreductases/genetics , Rats , Reactive Oxygen Species/metabolism
8.
FASEB J ; 35(3): e21382, 2021 03.
Article in English | MEDLINE | ID: mdl-33554387

ABSTRACT

Pro-inflammatory cytokines play critical roles in regulating valvular interstitial cell (VIC) phenotypic changes that can cause heart valve fibrosis and calcification. Tumor necrosis factor alpha (TNF-α) is a cytokine known to influence VIC behavior and has been reported at high levels in calcified valves ex vivo. We sought to understand the specific effects of TNF-α on VIC phenotypes (eg, fibroblast, profibrotic activated myofibroblasts) and its link with heart valve disorders. We characterize human aortic valve tissue from patients with valve disorders and identify a high variability of fibrotic and calcific markers between tissues. These results motivated in vitro studies to explore the effects of TNF-α on defined VIC fibroblasts and profibrotic activated myofibroblasts, induced via FGF-2 and TGF-Ɵ1 treatment. Using 3D hydrogels to culture VICs, we measure the effect of TNF-α (0.1-10Ā ng/mL) on key markers of fibrosis (eg, αSMA, COL1A1) and calcification (eg, RUNX2, BMP2, and calcium deposits). We observe calcification in TNF-α-treated VIC activated myofibroblasts and identify the MAPK/ERK signaling cascade as a potential pathway for TNF-α mediated calcification. Conversely, VIC fibroblasts respond to TNF-α with decreased calcification. Treatment of VIC profibrotic activated myofibroblast populations with TNF-α leads to increased calcification. Our in vitro findings correlate with findings in diseased human valves and highlight the importance of understanding the effect of cytokines and signaling pathways on specific VIC phenotypes. Finally, we reveal MAPK/ERK as a potential pathway involved in VIC-mediated matrix calcification with TNF-α treatment, suggesting this pathway as a potential pharmaceutical target for aortic valve disease.


Subject(s)
Aortic Valve Stenosis/etiology , Aortic Valve/pathology , Calcinosis/etiology , Myofibroblasts/pathology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Aortic Valve Stenosis/pathology , Fibrosis , Humans , MAP Kinase Signaling System/physiology , Male , Swine
9.
J Cardiovasc Pharmacol ; 80(2): 261-269, 2022 08 01.
Article in English | MEDLINE | ID: mdl-35904815

ABSTRACT

ABSTRACT: Selective serotonin reuptake inhibitors (SSRIs) are antidepressants prescribed in 10% of pregnancies in the United States. Maternal use of SSRIs has been linked to an elevated rate of congenital heart defects, but the exact mechanism of pathogenesis is unknown. Previously, we have shown a decrease in cardiomyocyte proliferation, left ventricle size, and reduced cardiac expression of the serotonin receptor 5-HT 2B in offspring of mice exposed to the SSRI sertraline during pregnancy, relative to offspring of untreated mice. These results suggest that disruption of serotonin signaling leads to heart defects. Supporting this conclusion, we show here that zebrafish embryos exposed to sertraline develop with a smaller ventricle, reduced cardiomyocyte number, and lower cardiac expression of htr2b relative to untreated embryos. Moreover, zebrafish embryos homozygous for a nonsense mutation of htr2b ( htr2bsa16649 ) were sensitized to sertraline treatment relative to wild-type embryos. Specifically, the ventricle area was reduced in the homozygous htr2b mutants treated with sertraline compared with wild-type embryos treated with sertraline and homozygous htr2b mutants treated with vehicle control. Whereas long-term effects on left ventricle shortening fraction and stroke volume were observed by echocardiography in adult mice exposed to sertraline in utero, echocardiograms of adult zebrafish exposed to sertraline as embryos were normal. These results implicate the 5-HT 2B receptor functions in heart development and suggest zebrafish are a relevant animal model that can be used to investigate the connection between maternal SSRI use and elevated risk of congenital heart defects.


Subject(s)
Heart Defects, Congenital , Sertraline , Animals , Female , Heart Defects, Congenital/chemically induced , Heart Defects, Congenital/genetics , Mice , Myocytes, Cardiac/metabolism , Pregnancy , Serotonin/metabolism , Selective Serotonin Reuptake Inhibitors/toxicity , Sertraline/toxicity , Zebrafish/genetics
10.
Nanomedicine ; 40: 102477, 2022 02.
Article in English | MEDLINE | ID: mdl-34740868

ABSTRACT

Magnetic resonance is a key imaging tool for the detection of prostate cancer; however, better tools focusing on cancer specificity are required to distinguish benign from cancerous regions. We found higher expression of claudin-3 (CLDN-3) and -4 (CLDN-4) in higher grade than lower-grade human prostate cancer biopsies (nĆ¢Ā€ĀÆ=Ć¢Ā€ĀÆ174), leading to the design of functionalized nanoparticles (NPs) with a non-toxic truncated version of the natural ligand Clostridium perfringens enterotoxin (C-CPE) that has a strong binding affinity to Cldn-3 and Cldn-4 receptors. We developed a first-of-its-type, C-CPE-NP-based MRI detection tool in a prostate tumor-bearing mouse model. NPs with an average diameter of 152.9Ć¢Ā€ĀÆĀ±Ć¢Ā€ĀÆ15.7Ć¢Ā€ĀÆnm (RS1) had a 2-fold enhancement of tumor specificity compared to larger (421.2Ć¢Ā€ĀÆĀ±Ć¢Ā€ĀÆ33.8Ć¢Ā€ĀÆnm) NPs (RS4). There was a 1.8-fold (PĆ¢Ā€ĀÆ<Ć¢Ā€ĀÆ0.01) and 1.6-fold (PĆ¢Ā€ĀÆ<Ć¢Ā€ĀÆ0.01) upregulation of the tumor-to-liver signal intensities of C-RS1 and C-RS4 (functionalized NPs) compared to controls, respectively. Also, tumor specificity was 3.1-fold higher (PĆ¢Ā€ĀÆ<Ć¢Ā€ĀÆ0.001) when comparing C-RS1 to C-RS4. This detection tool improved tumor localization of contrast-enhanced MRI, supporting potential clinical applicability.


Subject(s)
Nanoparticles , Prostatic Neoplasms , Animals , Enterotoxins/metabolism , Humans , Magnetic Resonance Imaging , Male , Mice , Prostatic Neoplasms/diagnostic imaging , Prostatic Neoplasms/metabolism
11.
Am J Physiol Heart Circ Physiol ; 321(5): H850-H864, 2021 11 01.
Article in English | MEDLINE | ID: mdl-34477461

ABSTRACT

Molecular mechanisms underlying cardiac dysfunction and subsequent heart failure in diabetic cardiomyopathy are incompletely understood. Initially we intended to test the role of G protein-coupled receptor kinase 2 (GRK2), a potential mediator of cardiac dysfunction in diabetic cardiomyopathy, but found that control animals on HFD did not develop cardiomyopathy. Cardiac function was preserved in both wild-type and GRK2 knockout animals fed high-fat diet as indicated by preserved left ventricular ejection fraction (LVEF) although heart mass was increased. The absence of cardiac dysfunction led us to rigorously evaluate the utility of diet-induced obesity to model diabetic cardiomyopathy in mice. Using pure C57BL/6J animals and various diets formulated with different sources of fat-lard (32% saturated fat, 68% unsaturated fat) or hydrogenated coconut oil (95% saturated fat), we consistently observed left ventricular hypertrophy, preserved LVEF, and preserved contractility measured by invasive hemodynamics in animals fed high-fat diet. Gene expression patterns that characterize pathological hypertrophy were not induced, but a modest induction of various collagen isoforms and matrix metalloproteinases was observed in heart with high-fat diet feeding. PPARα-target genes that enhance lipid utilization such as Pdk4, CD36, AcadL, and Cpt1b were induced, but mitochondrial energetics was not impaired. These results suggest that although long-term fat feeding in mice induces cardiac hypertrophy and increases cardiac fatty acid metabolism, it may not be sufficient to activate pathological hypertrophic mechanisms that impair cardiac function or induce cardiac fibrosis. Thus, additional factors that are currently not understood may contribute to the cardiac abnormalities previously reported by many groups.NEW & NOTEWORTHY Dietary fat overload (DFO) is widely used to model diabetic cardiomyopathy but the utility of this model is controversial. We comprehensively characterized cardiac contractile and mitochondrial function in C57BL6/J mice fed with lard-based or saturated fat-enriched diets initiated at two ages. Despite cardiac hypertrophy, contractile and mitochondrial function is preserved, and molecular adaptations likely limit lipotoxicity. The resilience of these hearts to DFO underscores the need to develop robust alternative models of diabetic cardiomyopathy.


Subject(s)
Diabetic Cardiomyopathies/etiology , Diet, High-Fat , Hypertrophy, Left Ventricular/etiology , Obesity/complications , Stroke Volume , Ventricular Dysfunction, Left/etiology , Ventricular Function, Left , Age Factors , Animals , Diabetic Cardiomyopathies/enzymology , Diabetic Cardiomyopathies/pathology , Diabetic Cardiomyopathies/physiopathology , Disease Models, Animal , Energy Metabolism , Female , Fibrosis , G-Protein-Coupled Receptor Kinase 2/genetics , G-Protein-Coupled Receptor Kinase 2/metabolism , Hypertrophy, Left Ventricular/enzymology , Hypertrophy, Left Ventricular/pathology , Hypertrophy, Left Ventricular/physiopathology , Male , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Heart/enzymology , Mitochondria, Heart/pathology , Myocardium/enzymology , Myocardium/pathology , Ventricular Dysfunction, Left/enzymology , Ventricular Dysfunction, Left/pathology , Ventricular Dysfunction, Left/physiopathology , Ventricular Remodeling
12.
Arterioscler Thromb Vasc Biol ; 40(11): e296-e308, 2020 11.
Article in English | MEDLINE | ID: mdl-32938214

ABSTRACT

OBJECTIVE: Resident valvular interstitial cells (VICs) activate to myofibroblasts during aortic valve stenosis progression, which further promotes fibrosis or even differentiate into osteoblast-like cells that can lead to calcification of valve tissue. Inflammation is a hallmark of aortic valve stenosis, so we aimed to determine proinflammatory cytokines secreted from M1 macrophages that give rise to a transient VIC phenotype that leads to calcification of valve tissue. Approach and Results: We designed hydrogel biomaterials as valve extracellular matrix mimics enabling the culture of VICs in either their quiescent fibroblast or activated myofibroblast phenotype in response to the local matrix stiffness. When VIC fibroblasts and myofibroblasts were treated with conditioned media from THP-1-derived M1 macrophages, we observed robust reduction of αSMA (alpha smooth muscle actin) expression, reduced stress fiber formation, and increased proliferation, suggesting a potent antifibrotic effect. We further identified TNF (tumor necrosis factor)-α and IL (interleukin)-1Ɵ as 2 cytokines in M1 media that cause the observed antifibrotic effect. After 7 days of culture in M1 conditioned media, VICs began differentiating into osteoblast-like cells, as measured by increased expression of RUNX2 (runt-related transcription factor 2) and osteopontin. We also identified and validated IL-6 as a critical mediator of the observed pro-osteogenic effect. CONCLUSIONS: Proinflammatory cytokines in M1 conditioned media inhibit myofibroblast activation in VICs (eg, TNF-α and IL-1Ɵ) and promote their osteogenic differentiation (eg, IL-6). Together, our work suggests inflammatory M1 macrophages may drive a myofibroblast-to-osteogenic intermediate VIC phenotype, which may mediate the switch from fibrosis to calcification during aortic valve stenosis progression.


Subject(s)
Aortic Valve Stenosis/metabolism , Aortic Valve/metabolism , Aortic Valve/pathology , Calcinosis/metabolism , Cytokines/metabolism , Inflammation Mediators/metabolism , Macrophages/metabolism , Myofibroblasts/metabolism , Osteoblasts/metabolism , Osteogenesis , Paracrine Communication , Animals , Aortic Valve Stenosis/pathology , Calcinosis/pathology , Cell Proliferation , Extracellular Matrix/metabolism , Fibrosis , Humans , Male , Myofibroblasts/pathology , Osteoblasts/pathology , Phenotype , Secretory Pathway , Signal Transduction , Sus scrofa , THP-1 Cells
13.
Mol Pharm ; 17(9): 3643-3648, 2020 09 08.
Article in English | MEDLINE | ID: mdl-32786958

ABSTRACT

Sympathetic excitation contributes to clinical deterioration in systolic heart failure (HF). Significant inhibition of hypothalamic paraventricular nucleus (PVN) ERK1/2 signaling and a subsequent reduction of plasma norepinephrine (NE) levels in HF rats were achieved 2 weeks after a single subcutaneous injection of PD98059-loaded polymeric microparticles, without apparent adverse events, while blank microparticles had no effect. Similar reductions in plasma NE, a general indicator of sympathetic excitation, were previously achieved in HF rats by intracerebroventricular infusion of PD98059 or genetic knockdown of PVN ERK1/2 expression. This study presents a clinically feasible therapeutic approach to the central abnormalities contributing to HF progression.


Subject(s)
Heart Failure/drug therapy , MAP Kinase Signaling System/drug effects , Paraventricular Hypothalamic Nucleus/drug effects , Pharmaceutical Preparations/administration & dosage , Animals , Chemistry, Pharmaceutical/methods , Disease Models, Animal , Norepinephrine/metabolism , Paraventricular Hypothalamic Nucleus/metabolism , Rats , Signal Transduction/drug effects
14.
Am J Physiol Heart Circ Physiol ; 316(1): H70-H79, 2019 01 01.
Article in English | MEDLINE | ID: mdl-30289294

ABSTRACT

Sex differences in the presentation, outcome, and responses to treatment of systolic heart failure (HF) have been reported. In the present study, we examined the effect of sex on central neural mechanisms contributing to neurohumoral excitation and its peripheral manifestations in rats with HF. Male and female Sprague-Dawley rats underwent coronary artery ligation (CL) to induce HF. Age-matched rats served as controls. Ischemic zone and left ventricular function were similar 24 h and 4 wk after CL. Female rats with HF had a lower mortality rate and less hemodynamic compromise, pulmonary congestion, and right ventricular remodeling 4 wk after CL. Plasma angiotensin II (ANG II), arginine vasopressin (AVP), and norepinephrine levels were increased in HF rats in both sexes, but AVP and norepinephrine levels increased less in female rats. In the hypothalamic paraventricular nucleus, a key cardiovascular-related nucleus contributing to neurohumoral excitation in HF, mRNA levels for the proinflammatory cytokines tumor necrosis factor-α and interleukin-1Ɵ as well as cyclooxygenase-2 and the ANG II type 1a receptor were increased in HF rats of both sexes, but less so in female rats. Angiotensin-converting enzyme 2 protein levels increased in female HF rats but decreased in male HF rats. mRNA levels of AVP were lower in female rats in both control and HF groups compared with the respective male groups. Activation of extracellular signal-regulated protein kinases 1 and 2 increased similarly in both sexes in HF. The results suggest that female HF rats have less central neural excitation and less associated hemodynamic compromise than male HF rats with the same degree of initial ischemic cardiac injury. NEW & NOTEWORTHY Sex differences in the presentation and responses to treatment of heart failure (HF) are widely recognized, but the underlying mechanisms are poorly understood. The present study describes sex differences in the central nervous system mechanisms that drive neurohumoral excitation in ischemia-induced HF. Female rats had a less intense central neurochemical response to HF and experienced less hemodynamic compromise. Sex hormones may contribute to these differences in the central and peripheral adaptations to HF.


Subject(s)
Heart Failure/physiopathology , Hemodynamics , Hypothalamus/metabolism , Myocardial Ischemia/physiopathology , Animals , Arginine Vasopressin/blood , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Cytokines/genetics , Cytokines/metabolism , Female , Heart Failure/etiology , Heart Failure/metabolism , Male , Myocardial Ischemia/complications , Myocardial Ischemia/metabolism , Norepinephrine/blood , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Angiotensin/genetics , Receptors, Angiotensin/metabolism , Sex Factors , Ventricular Function
15.
Circulation ; 135(24): e1115-e1134, 2017 Jun 13.
Article in English | MEDLINE | ID: mdl-28533303

ABSTRACT

Cardiac arrest in patients on mechanical support is a new phenomenon brought about by the increased use of this therapy in patients with end-stage heart failure. This American Heart Association scientific statement highlights the recognition and treatment of cardiovascular collapse or cardiopulmonary arrest in an adult or pediatric patient who has a ventricular assist device or total artificial heart. Specific, expert consensus recommendations are provided for the role of external chest compressions in such patients.


Subject(s)
American Heart Association , Cardiopulmonary Resuscitation/standards , Heart Arrest/epidemiology , Heart Arrest/therapy , Heart-Assist Devices/standards , Adult , Cardiopulmonary Resuscitation/trends , Child , Emergency Medical Services/standards , Emergency Medical Services/trends , Extracorporeal Circulation/standards , Extracorporeal Circulation/trends , Heart-Assist Devices/trends , Humans , United States/epidemiology
16.
Proc Natl Acad Sci U S A ; 112(29): 9129-34, 2015 Jul 21.
Article in English | MEDLINE | ID: mdl-26153425

ABSTRACT

Myocardial mitochondrial Ca(2+) entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca(2+) are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca(2+) uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca(2+) entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU-permeabilized myocardial fibers or isolated mitochondria in low Ca(2+) were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca(2+) homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca(2+)] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca(2+) homeostasis. Mitochondrial Ca(2+) overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca(2+) homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.


Subject(s)
Adaptation, Physiological , Calcium Channels/metabolism , Heart/physiopathology , Mitochondria, Heart/metabolism , Stress, Physiological , Animals , Blood Pressure , Calcium/metabolism , Cardiac Pacing, Artificial , Cellular Reprogramming , Cytosol/drug effects , Cytosol/metabolism , Diastole , Electrocardiography , Genes, Dominant , Glucose/metabolism , Heart Ventricles/pathology , Heart Ventricles/physiopathology , Mice , Mitochondria, Heart/drug effects , Myocardial Reperfusion , Myocardium/metabolism , Myocardium/pathology , Oxygen Consumption , Prostaglandin-Endoperoxide Synthases/metabolism , Sarcoplasmic Reticulum/metabolism , Transcription, Genetic
17.
J Mol Cell Cardiol ; 112: 123-130, 2017 11.
Article in English | MEDLINE | ID: mdl-28822805

ABSTRACT

The cardiac transverse (T)-tubule membrane system is the safeguard for cardiac function and undergoes dramatic remodeling in response to cardiac stress. However, the mechanism by which cardiomyocytes repair damaged T-tubule network remains unclear. In the present study, we tested the hypothesis that MG53, a muscle-specific membrane repair protein, antagonizes T-tubule damage to protect against maladaptive remodeling and thereby loss of excitation-contraction coupling and cardiac function. Using MG53-knockout (MG53-KO) mice, we first established that deficiency of MG53 had no impact on maturation of the T-tubule network in developing hearts. Additionally, MG53 ablation did not influence T-tubule integrity in unstressed adult hearts as late as 10months of age. Following left ventricular pressure overload-induced cardiac stress, MG53 protein levels were increased by approximately three-fold in wild-type mice, indicating that pathological stress induces a significant upregulation of MG53. MG53-deficient mice had worsened T-tubule disruption and pronounced dysregulation of Ca2+ handling properties, including decreased Ca2+ transient amplitude and prolonged time to peak and decay. Moreover, MG53 deficiency exacerbated cardiac hypertrophy and dysfunction and decreased survival following cardiac stress. Our data suggest MG53 is not required for T-tubule development and maintenance in normal physiology. However, MG53 is essential to preserve T-tubule integrity and thereby Ca2+ handling properties and cardiac function under pathological cardiac stress.


Subject(s)
Carrier Proteins/metabolism , Myocardium/metabolism , Myocardium/pathology , Sarcolemma/metabolism , Animals , Calcium Signaling , Down-Regulation , Excitation Contraction Coupling , Heart/embryology , Male , Membrane Proteins , Mice, Inbred C57BL , Mice, Knockout , Myocytes, Cardiac/metabolism , Sarcolemma/ultrastructure , Sodium-Calcium Exchanger/metabolism
18.
Am J Physiol Heart Circ Physiol ; 313(4): H744-H756, 2017 Oct 01.
Article in English | MEDLINE | ID: mdl-28710070

ABSTRACT

In systolic heart failure (HF), circulating proinflammatory cytokines upregulate inflammation and renin-angiotensin system (RAS) activity in cardiovascular regions of the brain, contributing to sympathetic excitation and cardiac dysfunction. Important among these is the subfornical organ (SFO), a forebrain circumventricular organ that lacks an effective blood-brain barrier and senses circulating humors. We hypothesized that the tumor necrosis factor-α (TNF-α) receptor 1 (TNFR1) in the SFO contributes to sympathetic excitation and cardiac dysfunction in HF rats. Rats received SFO microinjections of a TNFR1 shRNA or a scrambled shRNA lentiviral vector carrying green fluorescent protein, or vehicle. One week later, some rats were euthanized to confirm the accuracy of the SFO microinjections and the transfection potential of the lentiviral vector. Other rats underwent coronary artery ligation (CL) to induce HF or a sham operation. Four weeks after CL, vehicle- and scrambled shRNA-treated HF rats had significant increases in TNFR1 mRNA and protein, NF-κB activity, and mRNA for inflammatory mediators, RAS components and c-Fos protein in the SFO and downstream in the hypothalamic paraventricular nucleus, along with increased plasma norepinephrine levels and impaired cardiac function, compared with vehicle-treated sham-operated rats. In HF rats treated with TNFR1 shRNA, TNFR1 was reduced in the SFO but not paraventricular nucleus, and the central and peripheral manifestations of HF were ameliorated. In sham-operated rats treated with TNFR1 shRNA, TNFR1 expression was also reduced in the SFO but there were no other effects. These results suggest a key role for TNFR1 in the SFO in the pathophysiology of systolic HF.NEW & NOTEWORTHY Activation of TNF-α receptor 1 in the subfornical organ (SFO) contributes to sympathetic excitation in heart failure rats by increasing inflammation and renin-angiotensin system activity in the SFO and downstream in the hypothalamic paraventricular nucleus. Cytokine receptors in the SFO may be a target for central intervention in cardiovascular conditions characterized by peripheral inflammation.


Subject(s)
Coronary Circulation/genetics , Heart Failure/genetics , Heart Failure/physiopathology , Receptors, Tumor Necrosis Factor, Type I/genetics , Subfornical Organ/physiopathology , Sympathetic Nervous System/physiopathology , Animals , Electrocardiography , Gene Knockdown Techniques , Hemodynamics/drug effects , Male , NF-kappa B/metabolism , Norepinephrine/blood , Proto-Oncogene Proteins c-fos/biosynthesis , RNA, Small Interfering/genetics , Rats , Rats, Sprague-Dawley , Receptors, Tumor Necrosis Factor, Type I/biosynthesis , Renin-Angiotensin System , Tumor Necrosis Factor-alpha/pharmacology
19.
Am J Physiol Heart Circ Physiol ; 313(6): H1168-H1179, 2017 Dec 01.
Article in English | MEDLINE | ID: mdl-28971841

ABSTRACT

Oxidative stress plays a fundamental role in abdominal aortic aneurysm (AAA) formation. Activated polymorphonuclear leukocytes (or neutrophils) are associated with AAA and express myeloperoxidase (MPO), which promotes inflammation, matrix degradation, and other pathological features of AAA, including enhanced oxidative stress through generation of reactive oxygen species. Both plasma and aortic MPO levels are elevated in patients with AAA, but the role of MPO in AAA pathogenesis has, heretofore, never been investigated. Here, we show that MPO gene deletion attenuates AAA formation in two animal models: ANG II infusion in apolipoprotein E-deficient mice and elastase perfusion in C57BL/6 mice. Oral administration of taurine [1% or 4% (wt/vol) in drinking water], an amino acid known to react rapidly with MPO-generated oxidants like hypochlorous acid, also prevented AAA formation in the ANG II and elastase models as well as the CaCl2 application model of AAA formation while reducing aortic peroxidase activity and aortic protein-bound dityrosine levels, an oxidative cross link formed by MPO. Both MPO gene deletion and taurine supplementation blunted aortic macrophage accumulation, elastin fragmentation, and matrix metalloproteinase activation, key features of AAA pathogenesis. Moreover, MPO gene deletion and taurine administration significantly attenuated the induction of serum amyloid A, which promotes ANG II-induced AAAs. These data implicate MPO in AAA pathogenesis and suggest that studies exploring whether taurine can serve as a potential therapeutic for the prevention or treatment of AAA in patients merit consideration.NEW & NOTEWORTHY Neutrophils are abundant in abdominal aortic aneurysm (AAA), and myeloperoxidase (MPO), prominently expressed in neutrophils, is associated with AAA in humans. This study demonstrates that MPO gene deletion or supplementation with the natural product taurine, which can scavenge MPO-generated oxidants, can prevent AAA formation, suggesting an attractive potential therapeutic strategy for AAA.


Subject(s)
Antioxidants/pharmacology , Aorta, Abdominal/drug effects , Aortic Aneurysm, Abdominal/prevention & control , Neutrophils/drug effects , Oxidative Stress/drug effects , Peroxidase/metabolism , Taurine/pharmacology , Angiotensin II , Animals , Aorta, Abdominal/enzymology , Aorta, Abdominal/pathology , Aortic Aneurysm, Abdominal/chemically induced , Aortic Aneurysm, Abdominal/enzymology , Aortic Aneurysm, Abdominal/genetics , Calcium Chloride , Disease Models, Animal , Gene Deletion , Male , Mice, Inbred C57BL , Mice, Knockout, ApoE , Neutrophils/enzymology , Pancreatic Elastase , Peroxidase/deficiency , Peroxidase/genetics , Reactive Oxygen Species/metabolism , Serum Amyloid A Protein/metabolism
20.
Arterioscler Thromb Vasc Biol ; 36(3): 466-74, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26769049

ABSTRACT

OBJECTIVE: Hypercholesterolemia and hypertension are associated with aortic valve stenosis (AVS) in humans. We have examined aortic valve function, structure, and gene expression in hypercholesterolemic/hypertensive mice. APPROACH AND RESULTS: Control, hypertensive, hypercholesterolemic (Apoe(-/-)), and hypercholesterolemic/hypertensive mice were studied. Severe aortic stenosis (echocardiography) occurred only in hypercholesterolemic/hypertensive mice. There was minimal calcification of the aortic valve. Several structural changes were identified at the base of the valve. The intercusp raphe (or seam between leaflets) was longer in hypercholesterolemic/hypertensive mice than in other mice, and collagen fibers at the base of the leaflets were reoriented to form a mesh. In hypercholesterolemic/hypertensive mice, the cusps were asymmetrical, which may contribute to changes that produce AVS. RNA sequencing was used to identify molecular targets during the developmental phase of stenosis. Genes related to the structure of the valve were identified, which differentially expressed before fibrotic AVS developed. Both RNA and protein of a profibrotic molecule, plasminogen activator inhibitor 1, were increased greatly in hypercholesterolemic/hypertensive mice. CONCLUSIONS: Hypercholesterolemic/hypertensive mice are the first model of fibrotic AVS. Hypercholesterolemic/hypertensive mice develop severe AVS in the absence of significant calcification, a feature that resembles AVS in children and some adults. Structural changes at the base of the valve leaflets include lengthening of the raphe, remodeling of collagen, and asymmetry of the leaflets. Genes were identified that may contribute to the development of fibrotic AVS.


Subject(s)
Aortic Valve Stenosis/etiology , Aortic Valve/pathology , Hypercholesterolemia/complications , Hypertension/complications , Angiotensinogen/genetics , Angiotensinogen/metabolism , Animals , Aortic Valve/metabolism , Aortic Valve/physiopathology , Aortic Valve Stenosis/metabolism , Aortic Valve Stenosis/pathology , Aortic Valve Stenosis/physiopathology , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Disease Models, Animal , Female , Fibrosis , Gene Expression Regulation , Hypercholesterolemia/genetics , Hypercholesterolemia/metabolism , Hypertension/genetics , Hypertension/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Plasminogen Activator Inhibitor 1/genetics , Plasminogen Activator Inhibitor 1/metabolism , Renin/genetics , Renin/metabolism , Severity of Illness Index
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