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3158 Sunitinib-Induced Cardiotoxicity in an Engineered Cardiac Microtissue Model
- Carissa Livingston, Abhinay Ramachandran, Elise Corbin, Alexia Vite, Alexander Bennett, Kenneth Margulies
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- Journal:
- Journal of Clinical and Translational Science / Volume 3 / Issue s1 / March 2019
- Published online by Cambridge University Press:
- 26 March 2019, pp. 114-115
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OBJECTIVES/SPECIFIC AIMS: The aims of this study are threefold. Firstly, we are examining the effects of increased in vitro afterload (a proxy for hypertension) on human induced pluripotent stem cell cardiomyocyte (hiPSC-CM) response to sunitinib in a durable and dynamic cardiac microtissue culture system. Secondly, we are exploring effects of repeat exposure and recovery of both sunitinib and afterload throughout the lifetime of the hiPSC-CM microtissue. Finally, we are assessing methods to prevent and treat sunitinib induced cardiotoxicity. Primary outcomes for this study are commonly utilized metrics of cardiotoxicity: degree of caspase activation, electrophysiology benchmarks for minimum voltage threshold and maximum capture rate, and microtissue force generation. METHODS/STUDY POPULATION: HiPSC-CMs are cultured and matured as 3D cardiac microtissues (CMTs) on a microtissue array. After maturation, cells are exposed to sunitinib doses of 0µM, 0.5µM, 1µM or 5µM for 12 hours. Concurrently with sunitinib dosing, increases in microtissue array stiffness are created with application of an external magnetic field. Afterload spring constants are fixed at pre-determined physiologic values ranging from 0.5µN/µm, to 5µN/µm. For Aim 1: Half of the CMTs are harvested at 8 hours after sunitinib dosing to conduct the caspase 3/7 assay, and the remainder are examined for 3 days following drug exposure to track temporal changes in electrophysiology and force generation. For Aim 2: After CMT maturation, 12-hour exposures to sunitinib are repeated three times at a fixed dose, with doses separated by one week. Concurrently with sunitinib dosing, increases or decreases in microtissue stiffness are created by changing the strength of an applied external magnetic field to create “ramp up” or “ramp down” stiffness conditions. Caspase assay and contractility metrics are measured at each timepoint. For Aim 3: Experimental conditions are conducted as described in Aim 1. Prior to the introduction of sunitinib, either carvedilol or an AMP-kinase activator is added to the CMT culture media at physiologic concentrations. Primary outcomes are examined as in Aim 1. RESULTS/ANTICIPATED RESULTS: Aim 1: We hypothesize that increases in microtissue afterload, synchronized with sunitinib exposure will augment sunitinib toxicity in cardiomyocytes resulting in elevations of caspase 3/7 activity and minimum voltage capture as well as decreases in maximum capture rate and maximum force generation. Aim 2: We hypothesize that repeat exposures to both sunitinib and to increases in afterload will augment sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. Additionally, we hypothesize that decreases in afterload will decrease effective sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. Aim 3: We hypothesize that exposure to an AMP-kinase activator but not carvedilol will decrease the effects of sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. DISCUSSION/SIGNIFICANCE OF IMPACT: The use of small molecule, targeted chemotherapeutic agents is increasingly common. Many of these agents cause cardiotoxic side effects, the mechanisms of which are incompletely understood. Our lab has developed a novel 3D tissue engineering platform capable of supporting durable in vitro cardiac microtissues that experience dynamic alterations in their biomechanical load. By using this platform to examine the cardiotoxic effects of sunitinib, insight into treatment and prevention of this common problem will be developed.
Influence of dopamine on cyclic nucleotide enzymes in bovine retinal membrane fractions
- Ari Sitaramayya, Lorraine Lombardi, Alexander Margulis
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- Journal:
- Visual Neuroscience / Volume 10 / Issue 6 / November 1993
- Published online by Cambridge University Press:
- 02 June 2009, pp. 991-996
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Dopamine is a major neurotransmitter and neuromodulator in vertebrate retina. Although its pharmacological and physiological actions are well understood, the biochemical mechanisms of its signal transduction are less clear. Acting via D1 receptors, dopamine was shown to increase cyclic AMP levels in intact retina and to activate adenylate cyclase in retinal homogenates. The action via activation of D2 receptors is controversial: it was reported to decrease cyclic AMP levels in intact retina but inhibition of cyclase could not be demonstrated in retinal homogenates; also it was reported to activate rod outer segment cyclic GMP phosphodiesterase in vitro but did not decrease cyclic GMP levels in aspartate-treated retinas. We made an attempt to fractionate bovine retinal membranes and to investigate the effects of dopamine, via Dl and D2 receptors, on the synthesis and hydrolysis of cyclic AMP and cyclic GMP. Activation of cyclic AMP synthesis was noted in all fractions, but no effects were evident on cyclic nucleotide hydrolysis or cyclic GMP synthesis in any fraction. Also, D2 agonist did not inhibit cyclic AMP synthesis. These observations suggest that D2 receptors may not be directly coupled to cyclic nucleotide metabolizing enzymes in bovine retina.
Connexin 36 in bovine retina: Lack of phosphorylation but evidence for association with phosphorylated proteins
- ARI SITARAMAYYA, JOHN W. CRABB, DIANE F. MATESIC, ALEXANDER MARGULIS, VINITA SINGH, SADHONA PULUKURI, LOAN DANG
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- Journal:
- Visual Neuroscience / Volume 20 / Issue 4 / July 2003
- Published online by Cambridge University Press:
- 18 November 2003, pp. 385-395
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In vertebrate retina interneuronal communication through gap junctions is involved in light adaptation and in the transfer of visual information from the rod pathway to the cone pathway. Reports over the last two decades have indicated that these gap junctions are regulated by cyclic nucleotide-dependent protein kinases suggesting that the gap junction proteins, connexins, are phosphorylated. Though all the connexins involved in light adaptation and information transfer from rod to cone pathway are not yet known, connexin 36 has been shown to be definitively involved in the latter process. We have therefore attempted to investigate the cyclic nucleotide-dependent phosphorylation of this connexin in bovine retina. We found several soluble and membrane proteins in bovine retina whose phosphorylation was regulated by cyclic nucleotides. However, no protein of about 36 kDa with cyclic nucleotide-regulated phosphorylation was found in gap junction-enriched membrane preparations. A 36-kDa phosphorylated protein was found in gap junction-enriched membranes phosphorylated in the presence of calcium. However, this protein was not immunoprecipitated by anti-connexin 36 antibodies indicating that it was not connexin 36 in spite of its similarity in molecular weight. Immunoprecipitation did reveal phosphorylated proteins coimmunoprecipitated with connexin 36. Two of these proteins were identified as beta and alpha tubulin subunits. Though cyclic GMP and calcium did not greatly influence the association of these proteins with connexin 36, the results suggest the possibility of connexin 36 associating with other proteins. Together, these observations indicate that interneuronal communication at gap junctions made by connexin 36 may not be regulated by direct phosphorylation of connexin 36, but possibly by phosphorylation of associated proteins.
Soluble guanylate cyclase and nitric oxide synthase in synaptosomal fractions of bovine retina
- ALEXANDER MARGULIS, NIKOLAY POZDNYAKOV, LOAN DANG, ARI SITARAMAYYA
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- Journal:
- Visual Neuroscience / Volume 15 / Issue 5 / May 1998
- Published online by Cambridge University Press:
- 01 May 1998, pp. 867-873
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Cyclic GMP has been shown in recent years to directly activate ion channels in bipolar and ganglion cells, and to indirectly regulate coupling between horizontal cells, and between bipolar and amacrine cells. In all of these cases, the effects of cyclic GMP are mimicked by nitric oxide. An increase in calcium concentration stimulates the production of nitric oxide by neuronal and endothelial forms of nitric oxide synthase, which in turn activates soluble guanylate cyclases, enhancing the synthesis of cyclic GMP. Though some effects of nitric oxide do not involve cyclic GMP, the nitric oxide-cyclic GMP cascade is well recognized as a signaling mechanism in brain and other tissues. The widespread occurrence of nitric oxide/cyclic GMP-regulated ion channel activity in retinal neurons raises the possibility that nitric-oxide-sensitive soluble guanylate cyclases play an important role in cell–cell communication, and possibly, synaptic transmission. Immunohistochemical studies have indicated the presence of soluble guanylate cyclase in retinal synaptic layers, but such studies are not suitable for determination of the density or quantitative subcellular distribution of the enzyme. Microanalytical methods involving microdissection of frozen retina also showed the presence of cyclase activity in retinal plexiform layers but these methods did not permit distinction between nitric oxide-sensitive and insensitive cyclases. In this study, we fractionated retinal homogenate into the cytosolic and synaptosomal fractions and investigated the specific activity and distribution of soluble guanylate cyclase and nitric oxide synthase. The results show that both enzymes are present in the synaptosomal fractions derived from inner and outer plexiform layers. The synaptosomal fraction derived from inner retina was highly enriched in cyclase activity. Nitric oxide synthase activity was also higher in the inner than outer retinal synaptosomal fraction. The results suggest that the nitric oxide-cyclic GMP system is operational in both synaptic layers of retina and that it may play a more significant role in the inner retina.