<p dir="ltr"><b>Background</b></p><p dir="ltr">Heart failure (HF) is a chronic clinical syndrome characterized by symptoms including breathlessness, fatigue, swelling of the ankles, and signs such as edema pulmonary crackles etc. During HF, pathogenic mechanisms including hemodynamic overload, ventricular remodeling, aberrant calcium handling, excessive neurohormonal stimulation contribute to the worsening and progression of the condition. Ventricular arrhythmias are the common cause of sudden cardiac death (SCD) in HF patients.</p><p dir="ltr">Hyperactivation of the sympathetic nervous system (SNS), a characteristic of HF, causes an increase in circulating catecholamines which becomes detrimental to-adrenergic receptors (-AR) leading to signaling dysfunction, and decrease in contractility and the ionotropic reserve. Expression of calcium/calmodulin-dependent protein kinase II (CaMKII), a downstream effector of-AR and a key regulator of calcium homeostasis, has been shown to be enhanced in HF. CaMKII-mediated mechanisms have been demonstrated to contribute to cardiac remodeling, arrhythmias by pathological regulation of ion channels, and contractile dysfunction.</p><p dir="ltr">The human ether-a-go-go related gene (hERG) encodes the pore-forming subunit of the voltage-gated potassium channel that conduct the rapid component of the delayed rectifier potassium current, <i>I</i>_Kr. The gating kinetics of <i>I</i>_Krmakes it a crucial determinant of the duration of the plateau phase of atrial and ventricular action potential (AP). Reduced <i>I</i>_Kr density due to loss-of-function mutations or pharmacological blockage of hERG channels precipitate arrhythmias. Downregulation of <i>I</i>_Kr density and protein have been reported in HF. Recent studies suggest that microRNAs (miRNAs) are involved in pathological downregulation of hERG.</p><p dir="ltr">miRNA are small non-coding RNAs of approximately 22 nucleotides in length that function as gene expression regulatory elements by repression translation. Aberrant miRNA expression has associated with cancer, cardiovascular, autoimmune, and inflammatory disorders.</p><p dir="ltr"><b>Objective</b></p><p dir="ltr">The overarching objective of this study is to investigate the mechanisms of CaMKII-mediated regulation of hERG function, including assessment of an interplay with miR-362-3p during sustained β-AR stimulation. In Specific Aim 1, the effect of CaMKII activation through sustained β-AR stimulation on hERG function and miR-362-3p expression will be assessed. The mechanism of miR-362-3p upregulation will be evaluated in Specific Aim 2, and in Specific Aim 3, the interactome of miR-362-3p and binding sites will be characterized and predicted, respectively.</p><p dir="ltr"><b>Methods</b></p><p dir="ltr">Whole-cell, voltage clamp electrophysiology experiments were performed in HEK 293 cells stably expressing hERG (hERG-HEK) and both hERG and wild-type CaMKIIδ<br>(hERG/CaMKII-HEK) following treatment with isoproterenol for 48 hours, and after transfection with miR-362-3p. The effect of CaMKII activation on miR-362-3p was assessed using real-time quantitative polymerase chain reaction (RT-qPCR). Total RNA was isolated 48 hours after isoproterenol treatment and the TaqMan assay was used to reverse transcribe and analyze miR-362-3p expression. Cells were transfected with cJun siRNA and precursor miR-362-3p to assess the role of cJun miR-362-3p upregulation during sustained β-AR stimulation with isoproterenol. The interactome of miR-362-3p was assessed in both cell lines using enhanced crosslinking immunoprecipitation (eCLIP) assay. miR-362-3p binding sites were predicted using RNAStructure Duplexfold after identification of miR-362-3p chimeric molecules from eCLIP experiment. Interaction analysis was performed using GeneMania in Cytoscape to identify genes that were potentially downregulated by miR-362-3p and been reported to interact with hERG.</p><p dir="ltr"><b>Results</b></p><p dir="ltr">In Specific Aim 1, the effect of sustained β-AR stimulation on hERG currents and endogenous miR-362-3p was assessed in hERG-HEK and hERG/CaMKII-HEK cells. Using whole-cell voltage clamp electrophysiology, we demonstrated that 48 hours treatment with 100 nM isoproterenol reduced hERG currents in hERG/CaMKII-HEK cells (p = 0.032) but had no effect on the voltage dependence of activation (p = 0.61) relative to control vehicle. Isoproterenol treatment for 48 hours, however, had no effect on hERG currents (p = 0.58) and the voltage dependence of activation (p = 0.99) in hERG-HEK cells. The effect of sustained isoproterenol treatment on miR-362-3p was also assessed using RT-qPCR. In hERG/CaMKII cells, 48 hours isoproterenol treatment increased miR-362-3p expression (2.3 folds; p = 0.038) relative to control vehicle. hERG/CaMKII-HEK cells were also treated with 500 nM KN-93 or its inactive analogue, KN-92, in an attempt to reverse CaMKII effect on miR-362-3p expression. Treatment with KN-93 decreased miR-362-3p expression (0.5-fold; p = 0.002) relative KN-92 treatment. Isoproterenol treatment had no effect on miR-362-3p expression in hERG-HEK cells (p = 0.38).</p><p dir="ltr">The regulatory mechanism of miR-362-3p expression was evaluated in Specific Aim 2. The role of an activator protein-1 (AP-1)-like sequence located at 98 base pairs upstream of miR-362-3p transcription start site was probed using siRNA inhibition of cJun, a central protein of the AP-1 complex, and deletion of the site sequence. The effect of exogenous miR-362-3p on hERG currents were first assessed. Precursor miR-362-3p decreased hERG currents (p = 0.003) compared to control plasmid. The effect of CaMKII overexpression was also assessed on exogenous miR-363-3p expression. Isoproterenol treatment in hERG/CaMKII-HEK cells transfected with precursor miR-362-3p increased mature miR-362-3p expression (0.029) compared to control vehicle treatment. Inhibition of cJun inhibition with cJun-specific siRNA decreased mature miR-362-3p expression (0.5-fold; p = 0.027) compared to scramble siRNA in hERG-HEK cells. In hERG-HEK cells transfected with mutated precursor miR-362-3p (AP-1-like site deleted), cJun inhibition with siRNA had no effect on miR-362-3p expression (p = 0.40).</p><p dir="ltr">The focus of Specific Aim 3 was to characterize the interactome of miR-362-3p as well as predict the miRNA response element (MRE) of its target mRNAs using enhanced crosslinking immunoprecipitation. A network analysis was also performed to identify miR-362-3p targets that have been reported to interact with hERG. Approximately 23% of miR-362-3p mRNA targets from the eCLIP assay have also been catalogued in miRNA database, TargetScanHuman, as miR-362-3p targets. miR-362-3p chimeric molecules with 853 unique targets, of which 75 were identified to interact with hERG through the network analysis. Four unique chimeric molecules between miR-362-3p and hERG mRNA were identified, but the interactions were non-canonical (located in the coding sequence of hERG and outside the seed region of miR-362-3p). Thirty five of the 75 miR-362-3p targets that were identified to interact had a chimeric read ≥ 3, a cutoff number indicating non-random chimeric formation. Using RNAStructure DuplexFold, miR-362-3p was predicted to form canonical binding with 12 of 35 mRNA targets. HSPA4, a heat shock protein involved in the maturation and trafficking of hERG, was identified in a canonical interaction (8-mer) with miR-362-3p.</p><p dir="ltr"><b>Conclusion</b>:</p><p dir="ltr">Sustained β-AR stimulation increases miR-362-3p expression and decreases hERG currents in CaMKII overexpressing cells. cJun mediates miR-362-3p upregulation by interacting with an AP-1-like sequence upstream of miR-362-3p transcription start site. Pathological regulation of <i>I</i>_Kr by CaMKII mediated by miR-362-3p during sustained-AR may contribute to increased risk of arrhythmias in states of increase catecholaminergic activity, such as HF.</p>

<p dir="ltr"><b>Background</b></p><p dir="ltr">Recent developments in cancer therapeutics have allowed increased use of Oral Anticancer Medications (OAMs), including in the treatment of breast cancer. Breast cancer is the most common cancer among women in the United States. Patients with breast cancer may face key barriers in managing their OAMs at home. These challenges can lead to sub-optimal adherence and lower the overall quality of life. Designing interventions that enhance the patient experience with use of OAMs requires a deeper understanding of barriers faced by patients as they navigate their cancer care journey. The objective of this study was to identify the unmet medication management needs of patients with breast cancer who are receiving OAMs and co-design an early prototype intervention with patients to support medication management needs of patients with breast cancer.</p><p dir="ltr"><b>Methods</b></p><p dir="ltr">Two phases comprise this study. Phase 1 involved patient-journey mapping to characterize the longitudinal experience of OAMs use among patients diagnosed with breast cancer. In phase 2, we conducted participatory design (PD) workshops to develop a prototype tool to address OAM needs identified in phase 1. All participants were recruited from an outpatient breast cancer clinic in Indianapolis. Eligible participants were: 18 years of age or older, diagnosed with breast cancer, and currently receiving an OAM. All participants completed a brief sociodemographic and health information questionnaire. In phase 1, enrolled persons participated in a journey mapping exercise through semi-structured interviews. Interviews were conducted either in-person or remotely via Zoom, based on participant preference. For each interview, two researchers and the participant collaborated to create individual patient journey maps to generate a concise visual storyboard focused on medication use experiences related to OAMs. The journey maps helped capture treatment timelines, key markers of medication use, and specific barriers faced by patients. Individual journey maps were consolidated to generate personas representing groups of patients with related characteristics, treatment types, goals, and unmet needs. In phase 2, three rounds of PD workshops were conducted using the focus group format to develop an early prototype intervention. In round one (inspiration stage), participants defined the problem space and prioritized a list of challenges amenable to solutions; in round two (ideation stage), participants generated multiple possible solutions and design ideas; and in round three (convergence stage), two design concepts were selected and evaluated by participants.</p><p dir="ltr"><b>Results</b></p><p dir="ltr">In phase 1, 12 interviews (11 females and 1 male) were completed. The median age of participants was 65.5 years (range, 37-75). Participants were divided into two groups based on their prescribed medication types: (1) specialty medication (palbociclib or ribociclib; n=4 patients) and (2) traditional medication (tamoxifen, anastrozole, or exemestane; n=8 patients). We defined ‘Specialty’ medications as those that require specialty pharmacies and ‘traditional’ medications as those obtainable in local community pharmacies. To represent participants across these two broad categories of medications, two personas were created. Participants who had been prescribed specialty medication reported difficulty navigating the insurance process during medication fills, while participants who prescribed traditional medication did not. Notably, the word “prior authorization” was not used by participants to explain the issues they experienced. While all participants reported having side effects from their medications, sub-optimal adherence (n=2) was reported among the traditional medication group only. Other participants taking traditional medications either found their own ways to manage side effects or simply reported: “dealing with side effects as I don’t want cancer.” Participants expressed coping with side effects by enduring them. Participants had few strategies to manage their side effects, often stating that “they didn’t think of reaching out to the doctor,” when asked. Additionally, participants mentioned needing more financial and emotional support during their treatment journey. In phase 2, each PD session was conducted with 4-5 participants and 2 researchers (the design panel). Participants identified key challenges including difficulties navigating resources and information as well as managing medication side effects. The design panel prioritized two design concepts, which were subsequently developed into two prototypes: 1) a physical breast cancer handbook; and 2) an interactive treatment navigation app for use on tablet and smartphone devices. Our team plans to consolidate, further develop, and evaluate these prototypes in subsequent work as a follow up to this pilot study.</p><p dir="ltr"><b>Conclusion</b></p><p dir="ltr">This study provides insight into the patient experience with OAMs. The personas created can be applied in designing interventions tailored to breast cancer patients’ needs and goals, while the consolidated journey maps identify potential areas for improvement. Adequate patient education and enhanced tools and processes are necessary to manage medication side effects effectively, ultimately leading to improved medication outcomes and assisting patients in navigating their treatment. The two design concepts require further revision prior to implementation and pilot testing.</p>