Background: The sensitivity to drug-induced QT prolongation is highly variable in heart failure (HF) patients. QT interval prolongation can lead to a life-threatening ventricular arrhythmia known as torsade de Pointes (TdP), which can result in sudden cardiac death. Although QT prolongation is a surrogate marker for sudden cardiac death, the extent of drug-induced QT prolongation, and thus TdP, is largely unpredictable. Therefore, developing a biomarker to predict patients’ sensitivity to drug-induced QTc prolongation could have a profound clinical impact. MicroRNA (miR) are recognized as important regulators of cardiovascular function as they shape the transcriptome by targeting mRNAs for repression of translation. Our multidisciplinary research group has demonstrated that miR-362-3p regulates a potassium channel (i.e., hERG) that is the most widely implicated in drug-induced QTc prolongation. The primary objects of this analysis focus on characterizing serum miR-362-3p expression in the circulation as a potential biomarker to predict subject’s susceptibility to ibutilide exposure induced QT-interval prolongation.<div><br></div><div>Methods: The dataset utilized to develop the PK-PD models were collected from a previous clinical study carried out by Tisdale et al. (Tisdale,et al. 2020).A total of 22 adult subjects who met the inclusion and exclusion criteria were enrolled and divided into three groups: a group of patients with heart failure with preserved ejection fraction (HFpEF, n=10), a group of patients with heart failure with reduced ejection fraction (HFrEF, n=2), and ten healthy subjects in the control group who were matched to subjects in the HFpEF group for age and sex. Following a baseline day of triplicate 12-lead ECGs, all subjects received ibutilide 0.003mg/kg intravenously infused over 10 minutes. Serial collection of blood samples to determine serum Ibutilide concentrations (HPLC/MS), serum miR-362-3 expression (qPCR), with triplicate ECG readings were obtained pre-and-post ibutilide administration. To describe ibutilide serum concentration exposure and the9relationship with Fridericia-corrected QT (QTF) intervals, a non-linear mixed effect modeling approach was used along with clinical and demographic data, and serum miR-362-3p expression was evaluated as potential covariates on the PK/PD model.<div><br></div><div>Results: A three-compartment model best described the time course of ibutilide concentrations profile with a proportional residual error. The individual ibutilide concentrations time profile was then used in an indirect response model where ibutilide concentrations are indirectly driving the QT interval prolongation through inhibition of the output (Kout) parameters linked to an indirect response model with zero‐order input parameter best described the ibutilide concentrations QT interval lengthening relationship. The Individual PK/PD parameters using the base model for the Imax and IC50 were 11.4% (9.9%RES) and 0.36(8.4% RES)ng/mL, respectively. Following stepwise forwarding inclusion steps, the final covariate analyses identified circulating miR-362-3p expression associated with a history of myocardial infarction covariate influencing both the Imax and IC50( p<0.05). <div><br></div><div>Conclusions: An indirect response model has been developed to describe the effects of ibutilide concentrations on QT-intervals. Although the semi-mechanistic model could not be developed; serummiR-362-3p expression was identified as a significant predictor for ibutilide-induced QT-interval prolongation. Moreover, the upregulation of serum miR-362-3p expression enhanced IC50 seen after ibutilide administration. The potential use of miR-362-3p as a biomarker warrants further investigation to identify patients at the greatest risk of TdP </div></div></div>

<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>