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1.
Int J Cardiol ; 187: 325-33, 2015.
Article in English | MEDLINE | ID: mdl-25841124

ABSTRACT

BACKGROUND: Mitochondria constitute 30% of cell volume and are engaged in two dynamic processes called fission and fusion, regulated by Drp-1 (dynamin related protein) and mitofusin 2 (Mfn2). Previously, we showed that Drp-1 inhibition attenuates cardiovascular dysfunction following pressure overload in aortic banding model and myocardial infarction. As dynamic organelles, mitochondria are capable of changing their morphology in response to stress. However, whether such changes can alter their function and in turn cellular function is unknown. Further, a direct role of fission and fusion in cardiomyocyte contractility has not yet been studied. In this study, we hypothesize that disrupted fission and fusion balance by increased Drp-1 and decreased Mfn2 expression in cardiomyocytes affects their contractility through alterations in the calcium and potassium concentrations. METHODS: To verify this, we used freshly isolated ventricular myocytes from wild type mouse and transfected them with either siRNA to Drp-1 or Mfn2. Myocyte contractility studies were performed by IonOptix using a myopacer. Intracellular calcium and potassium measurements were done using flow cytometry. Immunocytochemistry (ICC) was done to evaluate live cell mitochondria and its membrane potential. Protein expression was done by western blot and immunocytochemistry. RESULTS: We found that silencing mitochondrial fission increased the myocyte contractility, while fusion inhibition decreased contractility with simultaneous changes in calcium and potassium. Also, we observed that increase in fission prompted decrease in Serca-2a and increase in cytochrome c leakage leading to mitophagy. CONCLUSION: Our results suggested that regulating mitochondrial fission and fusion have direct effects on overall cardiomyocyte contractility and thus function.


Subject(s)
Mitochondrial Dynamics/physiology , Myocardial Contraction/physiology , Myocytes, Cardiac/physiology , Animals , Cells, Cultured , Mice
2.
Neuroscience ; 252: 302-19, 2013 Nov 12.
Article in English | MEDLINE | ID: mdl-23912038

ABSTRACT

High levels of homocysteine (Hcy), known as hyperhomocysteinemia are associated with neurovascular diseases. H2S, a metabolite of Hcy, has potent anti-oxidant and anti-inflammatory activities; however, the effect of H2S has not been explored in Hcy (IC)-induced neurodegeneration and neurovascular dysfunction in mice. Therefore, the present study was designed to explore the neuroprotective role of H2S on Hcy-induced neurodegeneration and neurovascular dysfunction. To test this hypothesis we employed wild-type (WT) males ages 8-10 weeks, WT+artificial cerebrospinal fluid (aCSF), WT+Hcy (0.5 µmol/µl) intracerebral injection (IC, one time only prior to NaHS treatment), WT+Hcy+NaHS (sodium hydrogen sulfide, precursor of H2S, 30 µmol/kg, body weight). NaHS was injected i.p. once daily for the period of 7 days after the Hcy (IC) injection. Hcy treatment significantly increased malondialdehyde, nitrite level, acetylcholinestrase activity, tumor necrosis factor-alpha, interleukin-1 beta, glial fibrillary acidic protein, inducible nitric oxide synthase, endothelial nitric oxide synthase and decreased glutathione level indicating oxidative-nitrosative stress and neuroinflammation as compared to control and aCSF-treated groups. Further, increased expression of neuron-specific enolase, S100B and decreased expression of (post-synaptic density-95, synaptosome-associated protein-97) synaptic protein indicated neurodegeneration. Brain sections of Hcy-treated mice showed damage in the cortical area and periventricular cells. Terminal deoxynucleotidyl transferase-mediated, dUTP nick-end labeling-positive cells and Fluro Jade-C staining indicated apoptosis and neurodegeneration. The increased expression of matrix metalloproteinase (MMP) MMP9, MMP2 and decreased expression of tissue inhibitor of metalloproteinase (TIMP) TIMP-1, TIMP-2, tight junction proteins (zonula occulden 1) in Hcy-treated group indicate neurovascular remodeling. Interestingly, NaHS treatment significantly attenuated Hcy-induced oxidative stress, memory deficit, neurodegeneration, neuroinflammation and cerebrovascular remodeling. The results indicate that H2S is effective in providing protection against neurodegeneration and neurovascular dysfunction.


Subject(s)
Brain/drug effects , Gasotransmitters/metabolism , Hydrogen Sulfide/metabolism , Nerve Degeneration/metabolism , Oxidative Stress/physiology , Animals , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/pathology , Blotting, Western , Brain/metabolism , Brain/pathology , Gasotransmitters/administration & dosage , Homocysteine/administration & dosage , Homocysteine/toxicity , Hydrogen Sulfide/administration & dosage , Inflammation/metabolism , Inflammation/pathology , Injections, Intraventricular , Male , Mice , Nerve Degeneration/pathology , Oxidative Stress/drug effects , Reverse Transcriptase Polymerase Chain Reaction
3.
Pharmacology ; 91(5-6): 287-96, 2013.
Article in English | MEDLINE | ID: mdl-23736684

ABSTRACT

BACKGROUND: Vasomotor responses conducted from terminal arterioles to proximal vessels may contribute to match tissue demands and blood supply during skeletal muscle contraction. Conduction of vasodilatation (CVD) from distal resistance arterioles to the proximal arterioles and feeding arteries during metabolic demand is mediated by intercellular gap junctions in the vascular endothelium. The role of hyperhomocysteinemia (HHcy) in the musculoskeletal system during CVD is unclear. We hypothesize that during HHcy, there is impaired CVD due to decreased expression of endothelial-associated connexins and thus decreased tissue perfusion to the contracting skeletal muscles. METHODS: CVD studies were performed in a gluteus maximus muscle preparation of wild-type (C57BL6/J) and CBS-/+ (HHcy) mice using intravital microscopy. Expression of connexins and myostatin protein (an antiskeletal muscle statin) was studied by Western blot and immunohistochemistry methods. Tissue perfusion to acetylcholine was assessed by the laser Doppler technique. RESULTS: There was decreased CVD and tissue perfusion in response to acetylcholine in CBS-/+ mice compared to wild-type controls. There was decreased expression of connexins 37, 40 and 43 and increased expression of myostatin in CBS-/+ mice compared to wild-type controls. CONCLUSION: Our findings suggest that CVD in skeletal muscle is decreased during HHcy due to decreased expression of gap junction connexins.


Subject(s)
Arterioles/physiopathology , Hyperhomocysteinemia/physiopathology , Muscle, Skeletal/blood supply , Animals , Collagen/metabolism , Connexins/metabolism , Hyperhomocysteinemia/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscle, Skeletal/metabolism , Myostatin/metabolism , Vasodilation
4.
Arch Physiol Biochem ; 119(2): 65-74, 2013 May.
Article in English | MEDLINE | ID: mdl-23398532

ABSTRACT

Pressure overload induces cardiac extracellular matrix (ECM) remodelling and results in heart failure. ECM remodelling by matrix metalloproteinases (MMPs) is primarily regulated by their target inhibitors, tissue inhibitor of matrix metalloproteinases (TIMPs). It is known that TIMP-2 is highly expressed in myocardium and is required for cell surface activation of pro-MMP-2. We and others have reported that imbalance between angiogenic growth factors and anti-angiogenic factors results in transition from compensatory cardiac hypertrophy to heart failure. We previously reported the pro-angiogenic role of MMP-2 in cardiac compensation, however, the specific role of TIMP-2 during pressure overload is yet unclear. We hypothesize that genetic ablation of TIMP-2 exacerbates the adverse cardiac matrix remodelling due to lack of pro-angiogenic MMP-2 and increase in anti-angiogenic factors during pressure overload stress and results in severe heart failure. To verify this, ascending aortic banding (AB) was created to mimic pressure overload, in wild type C57BL6/J and TIMP-2-/- (model of MMP-2 deficiency) mice. Left ventricular (LV) function assessed by echocardiography and pressure-volume loop studies showed severe LV dysfunction in TIMP-2-/- AB mice compared to controls. Expression of MMP-2, vascular endothelial growth factor (VEGF) was decreased and expression of MMP-9, anti-angiogenic factors endostatin and angiostatin was increased in TIMP-2-/- AB mice compared with wild type AB mice. Connexins (Cx) are the gap junction proteins that are widely present in the myocardium and play an important role in endothelial-myocyte coupling. Our results showed that expression of Cx 37 and 43 was decreased in TIMP-2-/- AB mice compared with corresponding wild type controls. These results suggest that genetic ablation of TIMP-2 decrease the expression of pro-angiogenic MMP-2, VEGF and increases anti-angiogenic factors that results in exacerbated abnormal ventricular remodelling leading to severe heart failure.


Subject(s)
Heart Failure/etiology , Hypertension/complications , Matrix Metalloproteinase 2/deficiency , Tissue Inhibitor of Metalloproteinase-2/deficiency , Ventricular Dysfunction, Left/etiology , Angiogenesis Inhibitors , Animals , Aorta , Cardiomegaly , Connexin 43/genetics , Connexins/genetics , Constriction , Extracellular Matrix/physiology , Gene Expression , Heart Failure/physiopathology , Hypertension/etiology , Hypertrophy, Left Ventricular , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardium/ultrastructure , Neovascularization, Physiologic/physiology , Tissue Inhibitor of Metalloproteinase-2/physiology , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Ventricular Remodeling/physiology , Gap Junction alpha-4 Protein
5.
Am J Physiol Gastrointest Liver Physiol ; 303(3): G281-90, 2012 Aug 01.
Article in English | MEDLINE | ID: mdl-22595990

ABSTRACT

Elevated levels of plasma homocysteine (Hcy) called hyperhomocysteinemia (HHcy) have been implicated in inflammation and remodeling in intestinal vasculature, and HHcy is also known to aggravate the pathogenesis of inflammatory bowel disease (IBD). Interestingly, colon is the pivotal site that regulates Hcy levels in the plasma. We hypothesize that HHcy decreases intestinal motility through matrix metalloproteinase-9 (MMP-9)-induced intestinal remodeling leading to constipation. To verify this hypothesis, we used C57BL/6J or wild-type (WT), cystathionine ß-synthase (CBS(+/-)), MMP-9(-/-), and MMP-9(-/-) + Hcy mice. Intestinal motility was assessed by barium meal studies and daily feces output. Plasma Hcy levels were measured by HPLC. Expression of ICAM-1, inducible nitric oxide synthase, MMP-9, and tissue inhibitors of MMPs was studied by Western blot and immunohistochemistry. Reactive oxygen species (ROS) including super oxide were measured by the Invitrogen molecular probe method. Tissue nitric oxide levels were assessed by a commercially available kit. Plasma Hcy levels in the treated MMP-9 group mice were comparable to CBS(+/-) mice. Barium meal studies suggest that intestinal motility is significantly decreased in CBS(+/-) mice compared with other groups. Fecal output-to-body weight ratio was significantly reduced in CBS(+/-) mice compared with other groups. There was significant upregulation of MMP-9, iNOS, and ICAM-1 expression in the colon from CBS(+/-) mice compared with WT mice. Levels of ROS, superoxide, and inducible nitric oxide were elevated in the CBS(+/-) mice compared with other groups. Results suggest that HHcy decreases intestinal motility due to MMP-9-induced intestinal remodeling leading to constipation.


Subject(s)
Colon/physiology , Constipation/etiology , Gastrointestinal Motility/drug effects , Hyperhomocysteinemia/physiopathology , Matrix Metalloproteinase 9/metabolism , Animals , Cystathionine beta-Synthase/genetics , Feces , Hyperhomocysteinemia/complications , Intercellular Adhesion Molecule-1/biosynthesis , Male , Matrix Metalloproteinase 9/biosynthesis , Mice , Mice, Inbred C57BL , Nitric Oxide Synthase Type II/biosynthesis , Tissue Inhibitor of Metalloproteinase-1/biosynthesis
6.
Arch Physiol Biochem ; 117(1): 1-7, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20839901

ABSTRACT

Although protease activated receptor-1 (PAR-1) and matrix metalloproteinase-9 (MMP-9) play significant role in vascular remodelling in hyperhomocysteinemia (HHcy due to cystathionine beta synthase deficiency, CBS-/+) and diabetes, mechanism remains nebulous. We hypothesized that differential vascular density and remodelling in different vascular beds in HHcy and diabetes were responsible for an adaptive metabolic homeostasis during the pathogenesis. To test this hypothesis, vascular density in mice lacking PAR-1, MMP-9, CBS and Insulin-2 gene mutant (Ins2-/+, Akita) was measured and compared with wild type (WT, C57BL/6J) mice. The vascular density was detected by x-ray angiography using KODAK 4000 MM image station, using barium sulphate as contrasting agent. The % vascular density in the hearts of WT, CBS-/+ (HHcy), MMP-9-/-, PAR-1-/+ and Ins2-/+ (type-1 diabetes) was 100 ± 2.8, 85 ± 3.3, 90 ± 3.3, 95 ± 3.8 and 73 ± 1.7, respectively. The vascular density in CBS-/+ and Akita hearts decreased while it was increased in lungs of CBS-/+ and MMP-9-/-.There was decreased vascular density in liver and kidney of Akita mice. Vascular density in brain, kidney and mesentery was decreased in CBS-/+ mice. These findings support the notation that metabolic derangement in diabetes and HHcy causes the chronic decline and/or rarefaction in vascular density.


Subject(s)
Blood Vessels , Diabetes Mellitus, Type 2 , Hyperhomocysteinemia , Matrix Metalloproteinase 9 , Receptor, PAR-1 , Angiography , Animals , Barium Sulfate/analysis , Blood Vessels/pathology , Brain/blood supply , Cystathionine beta-Synthase/deficiency , Cystathionine beta-Synthase/genetics , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/physiopathology , Disease Models, Animal , Hyperhomocysteinemia/genetics , Hyperhomocysteinemia/metabolism , Hyperhomocysteinemia/physiopathology , Matrix Metalloproteinase 9/deficiency , Matrix Metalloproteinase 9/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Organ Size/genetics , Receptor, PAR-1/deficiency , Receptor, PAR-1/genetics , Renal Circulation , Splanchnic Circulation , X-Rays
7.
Mol Cell Biochem ; 348(1-2): 99-108, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21076854

ABSTRACT

Remodeling by its very nature implies synthesis and degradation of extracellular matrix components (such as elastin, collagen, and connexins). Most of the vascular matrix metalloproteinase (MMP) are latent because of the presence of constitutive nitric oxide (NO). However, during oxidative stress peroxinitrite (ONOO-) activates the latent MMPs and instigates vascular remodeling. Interestingly, in mesenteric artery, homocysteine (Hcy) decreases the NO bio-availability, and folic acid (FA, an Hcy-lowering agent) mitigates the Hcy-mediated mesentery artery dysfunction. Dimethylarginine dimethylaminohydrolase-2 (DDAH-2) and endothelial nitric oxide synthase (eNOS) increases NO production. The hypothesis was that the Hcy decreased NO bio-availability, in part, activating MMP, decreasing elastin, DDAH-2, eNOS and increased vasomotor response by increasing connexin. To test this hypothesis,the authors used 12-week-old C57BJ/L6 wild type (WT) and hyperhomocysteinemic (HHcy)-cystathione beta synthase heterozygote knockout (CBS+/-) mice. Blood pressure measurements were made by radio-telemetry. WT and MMP-9 knockout mice were administered with Hcy (0.67 mg/ml in drinking water). Superior mesenteric artery and mesenteric arcade were analyzed with light and confocal microscopy. The protein expressions were measured by western blot analysis. The mRNA levels for MMP-9 were measured by RT-PCR. The data showed decreased DDAH-2 and eNOS expressions in mesentery in CBS-/+ mice compared with WT mice. Immuno-fluorescence and western blot results suggest increased MMP-9 and connexin-40 expression in mesenteric arcades of CBS-/+ mice compared with WT mice. The wall thickness of third-order mesenteric artery was increased in CBS-/+ mice compared to WT mice. Hcy treatment increased blood pressure in WT mice. Interestingly, in MMP-9 KO, Hcy did not increase blood pressure. These results may suggest that HHcy causes mesenteric artery remodeling and narrowing by activating MMP-9 and decreasing DDAH-2 and eNOS expressions, compromising the blood flow, instigating hypertension, and acute abdomen pain.


Subject(s)
Extracellular Matrix Proteins/metabolism , Hyperhomocysteinemia/metabolism , Hypertension/metabolism , Mesenteric Artery, Superior/metabolism , Abdominal Pain/etiology , Amidohydrolases/metabolism , Animals , Blood Pressure , Blood Pressure Monitoring, Ambulatory , Blotting, Western , Connexins/metabolism , Cystathionine beta-Synthase/genetics , Cystathionine beta-Synthase/metabolism , Disease Models, Animal , Elasticity , Elastin/metabolism , Fluorescent Antibody Technique , Homocysteine , Hyperhomocysteinemia/chemically induced , Hyperhomocysteinemia/genetics , Hyperhomocysteinemia/pathology , Hyperhomocysteinemia/physiopathology , Hypertension/genetics , Hypertension/pathology , Hypertension/physiopathology , Male , Matrix Metalloproteinase 9/deficiency , Matrix Metalloproteinase 9/genetics , Mesenteric Artery, Superior/pathology , Mesenteric Artery, Superior/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Microscopy, Video , Nitric Oxide Synthase Type III/metabolism , Nitrites/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Splanchnic Circulation , Telemetry , Vascular Resistance , Gap Junction alpha-5 Protein
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