ORIGINAL RESEARCH CaMK4 Gene Deletion Induces Hypertension Gaetano Santulli, MD; Ersilia Cipolletta, MD; Daniela Sorriento, PhD; Carmine Del Giudice, MS; Antonio Anastasio, MS; Sara Monaco, PhD; Angela Serena Maione, BS; Gianluigi Condorelli, MD, PhD; Annibale Puca, MD, PhD; Bruno Trimarco, MD; Maddalena Illario, MD, PhD; Guido Iaccarino, MD, PhD Background-—The expression of calcium/calmodulin-dependent kinase IV (CaMKIV) was hitherto thought to be confined to the nervous system. However, a recent genome-wide analysis indicated an association between hypertension and a single-nucleotide polymorphism (rs10491334) of the human CaMKIV gene (CaMK4), which suggests a role for this kinase in the regulation of vascular tone. Methods and Results-—To directly assess the role of CaMKIV in hypertension, we characterized the cardiovascular phenotype of CaMK4À/À mice. They displayed a typical hypertensive phenotype, including high blood pressure levels, cardiac hypertrophy, vascular and kidney damage, and reduced tolerance to chronic ischemia and myocardial infarction compared with wild-type littermates. Interestingly, in vitro experiments showed the ability of this kinase to activate endothelial nitric oxide synthase. Eventually, in a population study, we found that the rs10491334 variant associates with a reduction in the expression levels of CaMKIV in lymphocytes from hypertensive patients. Conclusions-—Taken together, our results provide evidence that CaMKIV plays a pivotal role in blood pressure regulation through the control of endothelial nitric oxide synthase activity. ( J Am Heart Assoc. 2012;1:e001081 doi: 10.1161/JAHA.112.001081) Key Words: angiogenesis • arrhythmia • endothelium • hypertension • hypertrophy growing body of evidence bears out the rising interest in single-nucleotide polymorphism (SNP) of the human CaMKIV A the function of calcium/calmodulin-dependent kinases gene (CaMK4). Such a finding suggests that this kinase, the (CaMKs) in cardiovascular pathophysiology. In particular, expression of which once was thought to be confined to the although it is now established that CaMKII is an important nervous tissue,7–10 has a yet unidentified role in the control of player in the regulation of cardiac responses, both in terms of vascular tone. electrophysiology and of cardiac myocyte hypertrophy,1–4 less We therefore hypothesized that CaMKIV could affect is known about the role of other members of the CaMK family, endothelial functions, such as the control of vascular such as CaMKIV, in the cardiovascular system.5 resistance, and that changes in its level of expression or The recent genome-wide analysis of the Framingham Heart activity in endothelial cells (ECs) might alter the fine regulation Study 100K Project6 showed an association between elevated of vascular responses, causing hypertension. To ascertain diastolic blood pressure (BP) and the rs10491334 T/C whether CaMKIV signaling is involved in endothelial dysfunction, a hallmark of the hypertensive state,11–14 we used a murine model of genetic deletion of CaMK4. Finally, a From the Department of Clinical Medicine, Cardiovascular and Immunologic population study was carried out in normotensive and Sciences (G.S., E.C., D.S., C.D.G., A.A., B.T.), Department of Cellular and Molecular Biology and Pathology (S.M., A.S.M., M.I.), “Federico II” University of hypertensive patients to investigate the effects of the CaMK4 Naples, Naples, Italy; Istituto clinico Humanitas IRCCS and Istituto Ricerca rs10491334 SNP in humans. Genetica Biomedica, National Research Council, Rozzano, Italy (G.C.); Multi- medica Research Hospital, Milan, Italy (G.C., A.P., G.I.); Department of Medicine and Surgery, University of Salerno, Salerno, Italy (A.P., G.I.). Correspondence to: Guido Iaccarino, MD, PhD, FESC, Department of Methods Medicine and Surgery, University of Salerno, Via Salvador Allende, 84081 Baronissi (Salerno), Italy. E-mail [email protected] In Vivo Studies Received April 17, 2012; accepted June 21, 2012. ª 2012 The Authors. Published on behalf of the American Heart Association, Animals Inc., by Wiley-Blackwell. This is an Open Access article under the terms of the All animal procedures were performed in accordance with the Creative Commons Attribution Noncommercial License, which permits use, “ distribution and reproduction in any medium, provided the original work is policies and guidelines of the Position of the American Heart 15 properly cited and is not used for commercial purposes. Association on Research Animal Use” and were approved by DOI: 10.1161/JAHA.112.001081 Journal of the American Heart Association 1 CaMK4 and Hypertension Santulli et al ORIGINAL RESEARCH the Ethics Committee of the “Federico II” University. The view.19,20 Intraventricular septal and LV posterior wall thick- European Commission Directive 2010/63/EU was followed. ness were estimated at end diastole. LV fractional shortening We studied male mice with global homozygous deletion of the was calculated as follows: LVFS = [(LVEDD À LVESD)/ CaMK4 gene (CaMK4À/À), backcrossed >12 generations onto LVEDD] 9 100, where LVFS indicates LV fractional shorten- a C57Bl/6J background. The mice were kindly provided by ing; LVEDD, LV end-diastolic diameter; and LVESD, LV Anthony Means (Duke University, Durham, NC).9 Age-matched end-systolic diameter. LV ejection fraction was calculated wild-type littermates (CaMK4+/+) were used as controls. The automatically by the echocardiography system. All measure- animals were housed in a 22°C room with a 12-hour light/ ments were averaged on 10 consecutive cardiac cycles dark cycle and were allowed food and tap water ad libitum. per experiment and were analyzed by one experienced Two groups of mice (CAMK4+/+ and CAMK4À/À) were investigator. subjected to a long-term furosemide treatment (0.35 mg/kg per day) from the age of 3 months until 6 or 18 months. The Electrocardiography drug was added directly to the drinking water. The develop- Electrocardiography (ECG) was performed under isoflurane ment of the typical target-organ damage (vascular, cardiac, (1.5%) anesthesia. Mice were placed on a thermocontrolled 16–18 and kidney damage) was evaluated in 18-month-old plate (37±1°C) and were given 10 minutes to acclimate +/+ mice. Longitudinal survival observation on CAMK4 and before ECG recording. Signal-averaged ECG tracings were À/À CAMK4 mice was performed over a period of 24 months. obtained by subcutaneous placement of 27-gauge steel Genotypes were determined by polymerase chain reaction needle electrodes in each limb, secured with tape. ECG was fi ampli cation of tail DNA. The individual performing all recorded for 60 minutes with the PowerLab Chart 7.1 system experiments was blinded to the mouse genotype until all (ADInstruments, Sydney, Australia) and then was analyzed data were fully analyzed. offline. Invasive Arterial BP Measurement Angiogenic Response After Peripheral Chronic Mice were anesthetized by isoflurane (4%) inhalation and Ischemia fl maintained by mask ventilation (iso urane 1.8%). Direct BP Peripheral chronic ischemia was induced in 6-month-old mice and heart rate measurements were performed with the use of by means of surgical ligation and excision of the right a 1.0F Mikro-Tip catheter (SPR1000, Millar Instruments, common femoral artery. We have published a detailed Houston, TX), which was advanced through the right external description of the procedure.21–23 The angiogenic response carotid artery and placed in the descending aorta. After was assessed on postoperative days 3, 7, 14, and 21 by laser implantation, the catheter was connected to a transducer Doppler (Perimed Instruments, Järfälla, Sweden).22 Further- (Gould Instruments Systems, Cleveland, OH) to record BP and more, 3 weeks after surgery, we performed (1) ultrasound heart rate for 15 minutes. The pressure catheter then was Doppler analysis of the posterior tibial artery with a VeVo770 advanced through the aortic valve into the left ventricle (LV). imaging system equipped with a 20- to 60-MHz scanhead fl fi Subsequent of ine evaluation provided the rst derivative of (VisualSonics, Inc, Toronto, Canada), (2) dyed-microbead the LV pressure curve (maximum and minimum dP/dt). All assay on the gastrocnemius muscle, and (3) histological data were analyzed with dedicated software (PowerLab-Chart analysis of the anterior tibial muscle. 7.1, ADInstruments, Sydney, Australia). Myocardial Infarction Echocardiography Reproducible infarcts of the anterior LV wall were imposed on Transthoracic echocardiography was performed with a small- 6-month-old mice by cryogeny with a 6-mm2 cryoprobe. animal high-resolution imaging system (VeVo770, VisualSon- Briefly, after isofluorane (2%) anesthesia, a thoracotomy was ics, Inc, Toronto, Canada) equipped with a 30-MHz transducer performed through the fourth left intercostal space, the (Real-Time Micro Visualization, RMV-707B). The mice, anes- pericardium was opened, and the heart was exposed. thetized as described previously for BP measurement, were Cryoinfarction was produced by applying the cryoprobe to placed in a shallow left lateral decubitus position, with strict the anterior LV free wall, followed by freezing for 10 seconds. thermoregulation (37±1°C) to optimize physiological condi- The exact position of the probe was set carefully by using the tions and reduce hemodynamic variability. Fur was removed left atrium and pulmonary artery as anatomic landmarks. from the chest by application of a cosmetic cream (Veet, Rinsing with room-temperature saline was performed to allow