The cytochrome P450 3As (CYP3As) are abundantly expressed in the liver and metabolize many commonly prescribed medications. Their expression is highly variable between individuals with little known genetic cause. Despite extensive investigation, cis-acting genetic elements that control the expression of the CYP3As remain uncharacterized. Using chromatin conformation capture (4C assays), we detected reciprocal interaction between a distal regulatory region (DRR) and the CYP3A4 promoter. The DRR colocalizes with a variety of enhancer marks and was found to promote transcription in reporter assays. CRISPR-mediated deletion of the DRR decreased expression of CYP3A4, CYP3A5, and CYP3A7, supporting its role as a shared enhancer regulating the expression of three CYP3A genes. Using reporter gene assays, we identified two single-nucleotide polymorphisms (rs115025140 and rs776744/rs776742) that increased DRR-driven luciferase reporter expression. In a liver cohort (n = 246), rs115025140 was associated with increased expression of CYP3A4 mRNA (1.8-fold) and protein (1.6-fold) and rs776744/rs776742 was associated with 1.39-fold increased expression of CYP3A5 mRNA. The rs115025140 is unique to the African population and in a clinical cohort of African Americans taking statins for lipid control rs115025140 carriers showed a trend toward reduced statin-mediated lipid reduction. In addition, using a published cohort of Chinese patients who underwent renal transplantation taking tacrolimus, rs776744/rs776742 carriers were associated with reduced tacrolimus concentration after adjusting for CYP3A5*3. Our results elucidate a complex regulatory network controlling expression of three CYP3A genes and identify two novel regulatory variants with potential clinical relevance for predicting CYP3A4 and CYP3A5 expression. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?There is limited knowledge concerning distal cis-acting regulatory elements and genetic variants controlling expression of the CYP3As.

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Tyrosine Kinase Inhibitors (TKIs) are used by tens of thousands of patients and are efficacious for treating renal cell carcinoma . However, TKIs have been reported to have numerous off-target effects, including arrhythmias and heart failure. Many patients who receive TKIs are over 60 years of age and have existing cardiac diseases, thus increasing their risk of cardiotoxicity. However, the exact mechanism to how these TKIs cause cardiotoxicity remains unclear. Our research goal is to understand how TKIs lead to cardiotoxicity by learning what proteins are being targeted and what are potential upstream regulators so we can introduce possible treatments that can combat these effects. Recent literature has stated that the TKI Sorafenib led to increased reactive oxygen species (ROS) production and Calcium (Ca 2+ )/Calmodulin dependent kinase II (CaMKII) activity in rat ventricular cardiomyocytes. ROS can activate CaMKII, and increased CaMKII activity can lead to hyperphosphorylation of the voltage-gated sodium channel, leading to pathophysiological enhancement of late sodium current (I Na,L ). Increased activity of CaMKII and I Na,L can be seen in arrhythmias and heart failure. We hypothesize that TKIs lead to an increase enhancement of I Na,L due to an increase in ROS production and increased CaMKII activity. To test this hypothesis, we treated H9c2 myoblasts with Pazopanib (n=3) or DMSO (n=3) and wild-type mice with Pazopanib (n=5) or DMSO (n=5). After treatment, we assessed cytosolic and mitochondrial ROS production in vitro and functional affects, CaMKII activity, and I Na,L current in vivo . Results showed that Pazopanib led to a significant increase in ROS production in H9c2 cells and more arrhythmogenic events in WT mice. While CaMKII activity or I Na,L expression was not significantly changed between the two groups, with electrophysiology studies Pazopanib did lead to alterations in amplitude, peak current, ultra-slow inactivation, and recovery. In conclusion, Pazopanib leads to cardiovascular dysfunction and this could be due to an increase in ROS production and altered sodium channels. Our study indicates a potential mechanism as to how TKI treatment leads to cardiotoxicity in patients.

Background: The cardiac dyad and associated calcium (Ca 2+ ) release units (CRUs) are integral for cardiac excitation-contraction (E-C) coupling. The cardiac dyad is a multicomponent complex composed of the transverse-tubules, junctional sarcoplasmic reticulum (jSR), and CRU-associated proteins. Proper E-C coupling requires Ca 2+ influx via long-lasting-L-type Ca 2+ channels (Cav1.2), which triggers Ca 2+ release from adjacent ryanodine receptor 2 (RyR2) located in jSR. βII-spectrin loss alters RyR2 localization, but its contribution to cardiac dyad organization and E-C coupling remains unclear. Hypothesis: We hypothesized that βII-spectrin is required for cardiac dyad organization and proper cardiomyocyte calcium signaling. Methods: Cardiomyocyte-specific βII-spectrin knockout (βII-cKO) and control mice (βII-flox) were generated and harvested for immunoblotting, qRT-PCR, and imaging by transmission electron microscopy (TEM). Mice hearts were also interrogated by echocardiography following transaortic constriction. Results: Cardiac dyad structure and CRU components are significantly altered in βII-cKO hearts. Ultrastructural analysis of βII-cKOhearts by TEM showed fragmented cardiac dyads, dilated jSR and reduced jSR contact with T-tubules. Quantitative RT-PCR showed increased expression of calsequestrin 1 and 2, decreased phospholambam, Ncx and Cacna1c (major Cav1.2 subunit), while Ryr2 , Jph2 , Serca2a are unchanged. Protein expression of NCX was decreased, while expression of CAV1.2, RYR2, CASQ2, and SERCA2A remained unchanged. Furthermore, pressure-induced overload of βII-cKO hearts promoted accelerated heart failure (HF). Conclusions: This is the first demonstration that the βII-spectrin cytoskeletal protein is critically important for maintaining the structural integrity of the cardiac dyad. βII-spectrin loss and the resulting alterations in CRU structure, genes, and protein composition promoting accelerated HF.