236 3 Journal of S W Park et al. CRABP1 protects the heart 236:3 151–165 Endocrinology RESEARCH CRABP1 protects the heart from isoproterenol-induced acute and chronic remodeling Sung Wook Park1, Shawna D Persaud1, Stanislas Ogokeh1, Tatyana A Meyers2, DeWayne Townsend2 and Li-Na Wei1 1Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota, USA 2Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, Minnesota, USA Correspondence should be addressed to L-N Wei: [email protected] Abstract Excessive and/or persistent activation of calcium-calmodulin protein kinase II (CaMKII) is Key Words detrimental in acute and chronic cardiac injury. However, intrinsic regulators of CaMKII f CaMKII activity are poorly understood. We find that cellular retinoic acid-binding protein 1 f CRABP1 (CRABP1) directly interacts with CaMKII and uncover a functional role for CRABP1 in f heart failure regulating CaMKII activation. We generated Crabp1-null mice (CKO) in C57BL/6J background f cardiac remodeling for pathophysiological studies. CKO mice develop hypertrophy as adults, exhibiting significant left ventricular dilation with reduced ejection fraction at the baseline cardiac function. Interestingly, CKO mice have elevated basal CaMKII phosphorylation at T287, and phosphorylation on its substrate phospholamban (PLN) at T17. Acute isoproterenol (ISO) challenge (80 mg/kg two doses in 1 day) causes more severe apoptosis and necrosis in CKO hearts, and treatment with a CaMKII inhibitor KN-93 protects CKO mice from this injury. Chronic (30 mg/kg/day) ISO challenge also significantly increases hypertrophy and fibrosis in CKO mice as compared to WT. In wild-type mice, CRABP1 expression is increased in early stages of ISO challenge and eventually reduces to the basal level. Mechanistically, CRABP1 directly inhibits CaMKII by competing with calmodulin (CaM) for CaMKII interaction. This study demonstrates increased susceptibility of CKO mice to ISO-induced acute and chronic cardiac injury due to, at least in part, elevated CaMKII activity. Deleting Crabp1 results in reduced baseline cardiac function and aggravated damage challenged with acute and persistent β-adrenergic stimulation. This is the first report of a physiological role of CRABP1 Journal of Endocrinology as an endogenous regulator of CaMKII, which protects the heart from ISO-induced damage. (2018) 236, 151–165 Introduction Neuroendocrine stimulation of β-adrenergic receptors ventricular remodeling marked by cell death, inflammation, (β-AR) in the heart augments muscle contractility and heart fibrosis, hypertrophy and cardiac dilation, leading to rate, thereby increasing cardiac output during fight-or-flight arrhythmias and heart failure (Nichtova et al. 2012, response (Mann & Bristow 2005). Excess and/or persistent Garg & Khanna 2014). β-Adrenergic stimulation activates stimulation by catecholamines, e.g. isoproterenol (ISO) – a cAMP-protein kinase A (PKA) signaling and CaMKII synthetic agonist of β-ARs, causes decompensation and left (Grimm & Brown 2010) through multiple mechanisms. http://joe.endocrinology-journals.org © 2018 Society for Endocrinology https://doi.org/10.1530/JOE-17-0613 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/07/2021 08:04:00AM via free access 10.1530/JOE-17-0613 Journal of S W Park et al. CRABP1 protects the heart 236:3 152 Endocrinology Initial activation of CaMKII by Ca2+/calmodulin physiological assessment. Interestingly, the basal cardiac (CaM) induces autophosphorylation at T287 that function of CKO mice is significantly reduced and their provides molecular memory to sustain its activity. hearts display evidence of adverse remodeling. We CaMKII substrates include ryanodine receptor 2a conclude that CKO mice are more susceptible to both (RyR2a) at S2814 and phospholamban (PLN) at T17 on acute and chronic ISO challenges due to significantly the sarcoplasmic reticulum (SR) membrane to augment elevated CaMKII activity in cardiomyocyte, and that a excitation–contraction coupling (Mattiazzi et al. 2015). physiological function of CRABP1 is to modulate CaMKII Ca2+ surge caused by excessive and/or persistent CaMKII activity through direct interaction with CaMKII and activity leads to diastolic SR Ca2+ leak and arrhythmias competing with CaM for CaMKII activation. This study (Ferrero et al. 2007, Chelu et al. 2009). Indeed, CaMKII reports a physiological role for apo-CRABP1 in modulating inhibition by specific inhibitors and genetic ‘loss of heart health. function’ in animal models have demonstrated that excessive CaMKII activation underlines β-AR stimulated cardiac damage (Zhang et al. 2005, Kreusser et al. Materials and methods 2016), ischemia/reperfusion injury (Said et al. 2011, Ling et al. 2013), workload and pressure overload injury Plasmid constructs and cell culture (Grimm et al. 2015), hypertrophy (Sucharov et al. 2006) and cell apoptosis and necrosis (Vila-Petroff et al. 2007). Mouse CaMKII alpha Venus was a gift from Steven Consistently, CaMKII expression and its autonomous Vogel (Addgene plasmid # 29427) (Thaler et al. 2009). activity are significantly elevated in hypertrophy and Mouse cDNA for Camk2D (MMM1013-202805730, GE heart failure (Zhang et al. 2003, Mollova et al. 2015). Dharmacon, Lafayette, CO, USA) was amplified with Therefore, a consensus has emerged that CaMKII PCR and subcloned into a modified pCMX-PL1 vector activation underlines various heart diseases and can containing Flag tag. Mouse Crabp1 cDNA was PCR be a therapeutic target for heart failure. To this end, amplified with primers for cDNA and cloned into a several small-molecule and peptide inhibitors have modified pCMX-PL1 vector containing Flag tag and into been tested experimentally, but not for clinical use due pET-15b vector containing His tag for Flag-CRABP1 and to concerns over the inhibition of other kinases and His-CRABP1, respectively. Calmodulin was cloned through channels (Pellicena & Schulman 2014). Understanding PCR of mouse cDNA into pGEX-2T containing GST tag. physiological regulation of CaMKII is important for HEK293T cells were maintained in DMEM supplanted with managing the cardiovascular system. This study attempts 10% fetal bovine serum and 1% penicillin–streptomycin. to address a newly discovered negative physiological Expression vectors for GFP-CaMKIIα and Flag-CRABP1 control of CaMKII activity in the heart. were transfected by calcium phosphate method. Cellular retinoic acid (RA)-binding protein 1 (CRABP1) is a highly conserved cytosolic protein that can bind RA Experimental mice, ISO injection and with a high affinity Fiorella( 1993). Its biological et al. tissue preparation activity is classically measured by specific RA binding in vitro and implicated in modulating the biological availability of Experimental procedures were conducted according RA (Donovan et al. 1995, Venepally et al. 1996). However, to NIH guidelines and approved by the University of its physiological function has remained elusive due to Minnesota Institutional Animal Care and Use Committee. the lack of apparent phenotype of Crabp1-null mice We used a Crabp1-targeted DE3 (ES) clone 22 to generate (de Bruijn et al. 1994, Gorry et al. 1994). Nevertheless, CKO mice. CKO Crabp1 allele contains 5 bp insertion transgenic mice ectopically expressing CRABP1 exhibited generating a Not1 restriction site (Gorry et al. 1994). Male abnormal cell growth and differentiation in the eye and WT and CKO mice (16–20 weeks of age) were divided into the liver (Wei & Chen 1991, Perez-Castro et al. 1993), two groups each: control and ISO groups. We applied two indicating certain physiological function of CRABP1 that ISO treatment strategies: acute and chronic injection. For remains to be uncovered. acute treatment, ISO groups of WT and CKO mice were In a proteomic screening, we found CRABP1 directly intraperitoneally injected with 80 mg/kg of ISO (I6504, interacts with CaMKII and confirmed its interaction with Sigma-Aldrich) twice with 8-h interval and were killed endogenous CaMKII in cardiomyocytes. We thus generated at 24 h after first injection. For chronic treatment, ISO Crabp1-null mice (CKO) in C57BL/6J background for groups of WT and CKO mice were treated with daily http://joe.endocrinology-journals.org © 2018 Society for Endocrinology https://doi.org/10.1530/JOE-17-0613 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 10/07/2021 08:04:00AM via free access Research Journal of S W Park et al. CRABP1 protects the heart 236:3 153 Endocrinology intraperitoneal injections of ISO (30 mg/kg/day) for (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.2% NP40, 15 days or 5 weeks. Control groups were treated with daily 10% glycerol, and 1 mM EDTA) at cold room overnight. injections of 0.9% saline. KN-92 and KN-93 (5 µmol/kg, Or proteins from HEK293T ectopically expressing 2 doses, MedChem Express, Monmouth Junction, NJ, GFP-CaMKIIα with or without Flag-CRABP1 were USA) (Zhang et al. 2005) were injected 30 min before extracted in RIPA buffer (25 mM Tris–HCl, pH 7.4, ISO injection (80 mg/kg, 2 doses) into CKO mice. At the 150 mM NaCl, 0.1% SDS, 1% NP-40 and 0.5% sodium end of ISO injection period, hearts were quickly excised, deoxycholate). GFP-CaMKIIα was precipitated with anti- cross-cut in the middle of ventricles and ventricles were GFP antibody and protein G beads. After washing with weighed after blood, aorta and atria were removed. Base the binding