Browsing by Author "Romano, Migachelle"
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Item THE EFFECT OF CARDIAC-SPECIFIC HSP90 KNOCKOUT ON THE GENE AND PROTEIN EXPRESSIONS OF CALCIUM-HANDLING PROTEINS(University of Delaware, 2023-05) Romano, MigachelleFor the heart to function and pump blood to the body, it must contract following a series of events called excitation-contraction (E-C) coupling to link the electrical excitation of the cardiomyocytes to actin-myosin cross-bridge formation. Regulation of cytosolic calcium levels is crucial to E-C coupling. Particularly, sarcoplasmic reticulum calcium reuptake, governed by the calcium ATPase (SERCA2A), has known to be the main dictator for the strength of contraction and relaxation. Indeed, β-Adrenergic (βA) stimulation during fight-or-flight response increases contractility by phosphorylation of phospholamban (P-PLN), a SERCA2A inhibitor during basal conditions. Increasing P PLN will enhance SERCA2A activity, thus cardiac contractility. Previously, our lab has shown that Heat Shock protein 90 (HSP90) can form a complex with PLN/SERCA2A via interaction with a hematopoietic associated protein X-1 (HAX-1). However, the functional consequences of this interaction remain to be elucidated. Thus, my goal is to study the role of Heat shock protein 90 (HSP90) on each individual calcium handling proteins such as PLN, SERCA2A and HAX-1 and its effect on heart contraction. In addition, HSP90 is involved in regulating G-protein coupled receptor kinase-2 (GRK2), a protein that can deactivate the β-adrenergic receptor by phosphorylation. So, understanding the expression of GRK2 in KO mice could give me more insight into the role of HSP90 in the βARs regulation on calcium kinetics as well. Because of the importance of calcium recycling in heart contraction, I hypothesize that HSP90 KO may increase calcium recycling by causing the downregulation of PLN, a SERCA2A inhibitor, without affecting the protein expression of SERCA2A, HAX-1 and GRK2. To test the hypothesis, I used cardiac-specific HSP90 knock-out (KO) mice to study the expression of the calcium recycling proteins of interest: GRK2, SERCA2A, PLN, and HAX-1. After extracting the heart of HSP90 KO, I performed a western blot and quantified the data. In addition, I performed a reverse transcription-quantitative polymerase chain reaction (RT-PCR) to analyze the effect of the KO mice on the mRNA expression of the same calcium handling proteins. The data showed no significant change in protein and gene expressions for both GRK2 and SERCA2A. The hax-1 mRNA expression decreased significantly but its protein expression remained the same across the groups. In addition, PLN protein expression decreased significantly in the KO group, but the P-PLN did not change. After comparing the ratio of P-PLN to PLN to gain an insight on the activity of the βA signaling pathway, the data revealed no change in phosphorylation before or after HSP90 KO. In this regard, I proved that when HSP90 is KO, only PLN protein is downregulated. The downregulation of PLN may result in increased contraction by relieving SERCA2A function. Therefore, HSP90 appears to have a direct effect on calcium cycling in the heart, which could subsequently impact contractility