Oligophrenin1 protects mice against myocardial ischemia and reperfusion injury by modulating inflammation and myocardial apoptosis

    Oligophrenin1 protects mice against myocardial ischemia and reperfusion injury by modulating inflammation and myocardial apoptosis Christina Niermann, Simone Gorressen, Meike Klier, Nina S. Gowert, Pierre Billuart, Malte Kelm, Marc W. Merx, Margitta Elvers PII: DOI: Reference:

S0898-6568(16)30093-6 doi: 10.1016/j.cellsig.2016.04.008 CLS 8670

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

18 December 2015 11 April 2016 21 April 2016

Please cite this article as: Christina Niermann, Simone Gorressen, Meike Klier, Nina S. Gowert, Pierre Billuart, Malte Kelm, Marc W. Merx, Margitta Elvers, Oligophrenin1 protects mice against myocardial ischemia and reperfusion injury by modulating inflammation and myocardial apoptosis, Cellular Signalling (2016), doi: 10.1016/j.cellsig.2016.04.008

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OPHN1 and Myocardial Infarction

Oligophrenin1 protects mice against myocardial ischemia and reperfusion

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injury by modulating inflammation and myocardial apoptosis

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Christina Niermann1, Simone Gorressen2, Meike Klier1, Nina S. Gowert1, Pierre

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Billuart3, Malte Kelm2, Marc W. Merx2,4, Margitta Elvers1

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Department of Clinical and Experimental Hemostasis, Hemotherapy and Transfusion

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Medicine, Heinrich-Heine-University, Düsseldorf, Germany 2

Department of Cardiology, Pulmonology and Vascular Medicine, Heinrich Heine

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University, Düsseldorf, 3

Institut Cochin, Dpt. DRC, 24 rue du Fg St Jacques, 75014 Paris, France

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Department of Cardiology, Vascular Medicine and Intensive Care Medicine, Robert

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Koch Krankenhaus, Klinikum Region Hannover, Hannover, Germany

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Short title: OPHN1 and Myocardial Infarction Key words: Oligophrenin1, Rho GTPases, Myocardial Infarction, Inflammation, Migration

Word count: 4.842 Abstract word count: 223

Correspondence

to:

Margitta

Elvers,

Ph.D.,

Department

of

Clinical

and

Experimental Hemostasis, Hemotherapy and Transfusion Medicine, Heinrich-HeineUniversity Duesseldorf, Moorenstr. 5, 40225 Duesseldorf, Germany. Phone: +49 (0)211 81-08851 Fax: +49 (0)211 81-17498. Email: [email protected] 1

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Abstract The Rho family of small GTPases has been analyzed in cardiac physiology and pathophysiology including myocardial infarction (MI) in the last years. Contradictory results

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show either a protective or a declined effect of RhoA and the RhoA effector Rho-associated protein kinase (ROCK) in myocardial ischemia and reperfusion injury that is associated with

ischemia

reperfusion

injury

in

diabetic

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cardiomyocyte survival and caspase-3 activation. Cardiac-specific deletion of Rac1 reduced hearts,

whereas

cardiomyocyte

specific

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overexpression of active Rac1 predisposes the heart to increased myocardial injury with enhanced contractile dysfunction. GTPase-activating proteins (GAPs) control the activation

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of Rho proteins through stimulation of GTP hydrolysis. However, the impact of GAPs in myocardial ischemia and reperfusion injury remains elusive. Here we analyzed the role of oligophrenin1 (OPHN1), a RhoGAP with Bin/Amphiphysin/Rvs (BAR) domain known to

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regulate the activity of RhoA, Rac1 and Cdc42 in MI. The expression of Ophn1, RhoA and Rac1 is strongly upregulated 24 h after myocardial ischemia. Loss of OPHN1 induced

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enhanced activity of Rho effector molecules leading to elevated cardiomyocyte apoptosis and increased migration of inflammatory cells into the infarct border zone of OPHN1 deficient mice. Consequently, echocardiography 24 h after myocardial ischemia revealed declined left

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ventricle function in OPHN1 deficient mice. Our results indicate that OPHN1 mediated regulation of RhoA, Rac1 and Cdc42 is crucial for the preservation of cardiac function after myocardial injury.

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1. Introduction Cardiovascular disease is a major cause of morbidity and mortality in Western countries [31].

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Myocardial infarction (MI) results from coronary atherosclerosis, a chronic disease with

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plaques vulnerable to rupture or erosion accounting for more than 70 % of acute events [10,

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28]. This thrombotic process reduces microcirculatory perfusion by decreased coronary artery flow through epicardial stenosis and distal embolisation of thrombi. After ischemia, myocardial inflammation with neutrophils and monocytes/macrophages and repair following

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reperfusion is crucial for the preservation of myocardial function [13, 23]. The development of heart failure after MI is characterized by the size of the necrotric area, the wound healing

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process in the first days (acute) and weeks and the remodeling of the collagen-rich scar and the remote, non-infarcted myocardium after the fatal event [23]. In recent years, extensive

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efforts have been made to identify important mechanisms to improve myocardial repair and

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reduce heart failure.

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The Rho family of small GTPases includes 22 Rho genes that encode for more than 25 proteins [18]. The prominent members of Rho proteins are RhoA, Rac1 and Cdc42 and play an important role in cytoskeletal reorganization because they induce the formation of actin

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stress fibers, filopodia and lamellipodia [32]. Besides, Rho proteins are involved in platelet secretion, phospholipase C2 (PLC2) activation and the regulation of GPIb-mediated signal transduction [18, 34, 35]. Moreover, Rho GTPases modulate different cellular processes such as cell cycle, gene transcription, enzyme activation, polarity and vesicle transport [21]. In recent years the impact of Rho GTPases in cardiac physiology and pathophysiology including MI has been studied extensively. Interestingly, different studies show either a protective or a declined effect of RhoA or the RhoA effector Rho kinase (ROCK) related to cardiomyocyte survival and caspase-3 activation [8, 19, 27, 37, 42]. Long term treatment of mice with the Rho-associated protein kinase (ROCK) inhibitor fasudil showed suppressed left ventricle (LV) remodeling and significantly reduced expression of the proinflammatory cytokines transforming growth factor (TGF)-2, TGF-3 and Macrophage migration inhibitory 3

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factor (MIF) after MI compared to controls [17]. A benefit of ROCK inhibition in the acute phase of MI was shown by Bao and colleagues. Treatment of mice with the ROCK inhibitor Y-27632 led to reduced infarct size, enhanced cardiac function and reduced myocardial

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apoptosis demonstrating a protective effect of ROCK inhibition 24 h after MI [2]. Thus inhibition of ROCK by treatment of mice with either fasudil or Y-27632 confirmed that ROCK

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is involved in the pathogenesis of MI and suggests therapeutic importance of

ROCK

inhibition to improve cardiac function. Xiang and colleagues provided evidence that RhoA

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protects the heart against ischemia reperfusion injury after MI. Transgenic mice with conditional in vivo postnatal expression of modest levels of constitutively activated RhoA in

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cardiomyocates showed increased tolerance to injury with reduced infarct size and improved LV function [20, 46]. In contrast, cardiac-specific knock-out mice are more susceptible to

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myocardial injury. Mechanistically, RhoA activation leads to PLC mediated protein kinase

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D1 (PKD1) phosphorylation to inhibit mitochondrial translocation and proapoptotic function of cofilin2 and Bax and thus protect mitochondria from oxidative stress in the heart [45].

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Cardiomyocyte specific overexpression of active Rac predisposes the heart to increased ischemia reperfusion injury with enhanced contractile dysfunction [43]. In diabetic hearts the cardiac-specific deletion of Rac1 reduced ischemia reperfusion injury via calpain activation

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as shown by decreased infarct size and production of reactive oxygen species (ROS) [41]. Interestingly, so far nothing is known about a direct impact of Cdc42 in ischemia and reperfusion injury although different studies show changes in Cdc42 protein expression upon myocardial infarction [16, 24, 29]. Rho GTPases act as molecular switches in different signaling pathways because they cycle between an active and an inactive state [4]. GTPase-activating proteins (GAPs) are important modulators of Rho GTPases and control the activation of Rho proteins in part by switching off Rho GTPases through stimulation of GTP hydrolysis [4, 38, 39]. Oligophrenin1 (OPHN1) is a RhoGAP with a Bin/Amphiphysin/Rvs (BAR) domain and binds to GTPases of the Rho family [3, 9]. OPHN1 is associated with X-linked mental retardation, actin filament organization and exo- and endocytosis [3, 11, 25, 26]. Recently, we identified OPHN1 in 4

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platelets and found a pivotal role for OPHN in the regulation of Rho GTPase activity, cytoskeletal reorganization and platelet activation upon thrombus formation [9, 12]. However, the impact of OPHN1 in myocardial ischemia and reperfusion injury remains elusive.

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To analyze the role of Rho GTPases and GAP-mediated regulation of Rho activity after myocardial infarction, we investigated OPHN1 deficient mice in an experimental model of

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myocardial ischemia and reperfusion injury.

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2. Material and methods 2.1. Animals

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Mice with constitutively inactivated OPHN1 were analyzed. Animals were backcrossed to

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C57BL/6J genetic background (>10 generations) and genotyped by PCR. [25]. All animal

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experiments were conducted according to the German law for the welfare of animals and approved by local authorities (regional board Düsseldorf).

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2.2. Myocardial I/R by Closed-Chest-Model

A closed-chest model of reperfused MI was utilized in order to exclude that any inflammatory

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reaction following reperfused MI was due to the surgical trauma itself as described elsewhere [30]. Briefly, ten to twelve week old male ophn+/y and ophn-/y mice were anesthetized by a

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weight adapted intraperitoneal injection of Ketanest S (60mg/kg BW) and (10mg/kg body

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weight) and were ventilated with 2 Vol% isoflurane by an endotracheal tubus. Mice were placed in a supine position on a warmed plate. Body temperature was maintained at 37°C,

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and electrocardiography (ECG) (Hugo Sachs Apparatus) was monitored. To ligate the left anterior descending artery (LAD) chest was opened between the third and the fourth rib. A 07 prolene fiber is placed around the LAD and put in a pocket under the skin. The chest was

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closed with four interrupted stitches utilizing 7-0 suture. Anesthesia was turned off while closing the skin. Animal received buprenorphine (0.05 – 0.1mg/kg, s.c.) every 8 hours for first 3 days. At 3 days post instrumentation, the animals were re-anesthetized by mask inhalation of isoflurane 2.0 Vol.% and a mixture of one third oxygen and two thirds room air. The coronary artery was completely ligated proximally by this 7-0 prolene fiber. The occlusion was maintained for 60 min under ECG control, followed by a reperfusion time of 24 h. Reperfusion was confirmed by resolution of ST-elevation. Animal received buprenorphine (0.05 – 0.1mg/kg, s.c.) every 8 hours for one day.

2.3. Myocardial Infarct Size and Area at risk

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After reperfusion heart was again ligated at the same place and coronary arteries were injected with 1% Evans Blue above the ligation, to differentiate non-affected tissue from tissue/area at risk. Infarcted tissue (infarct size) and area at risk were detected by

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triphenyltrazolium (TTC) staining. Areas were photographed and quantified by Diskus

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Software, Hilgers.

2.4. Heart function/Echocardiography

Cardiac function after 24 h reperfusion was acquired using Vevo 2100 (VisualSonics Inc.) as

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descripted previously [30]. Analysis of ejection fraction (%), fractional shortening (%), cardiac

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output (mL/min), endsystolic volume (µL) and enddiastolic volume (µL) were derived from Bmode echocardiography.

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2.5. Survival of bone marrow cells

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Femoral bones of ophn+/y and ophn-/y mice were extracted and cut on both ends to wash cells

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out under aseptic conditions. Cells were given through a 100 µm filter and centrifuged for 10 sec. at 500g. Erythrocytes were lysed 5’’ by 155 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA, followed by 10 sec. 500g centrifugation. Pellet was resuspended in PBS.

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Respectively 200,000 bone marrow cells of ophn+/y and ophn-/y mice were incubated under aseptic conditions at 37°C in culture medium (Glutamax, Gibco with vitamin, Pen/Strep, NEAA and FCS) for 12 h and then incubated with new culture medium for another 6 h.

2.6. Flow cytometry Apoptosis of bone marrow cells was analyzed using the Apoptosis Detection Kit I (Annexin V, FITC, BD Bioscienes, Cat.# 556547) as descripted in the manufacturer’s manual and analyzed with a FACSCalibur flow cytometer (BD Biosciences, CA, USA). Cells were measured directly after preparation and after 18 h incubation in culture medium.

2.7. Real-time PCR

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One day after myocardial ischemia mice were killed after tissue was perfused with PBS to wash out blood cells. mRNA was purified from left ventricular myocardial tissue of ophn+/y and ophn-/y mice and from healthy controls with RNeasy Mini Kit (Qiagen, Cat.# 74104).

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Reverse Transciption was performed with ImProm-II Reverse Transcription System (Promega, Cat.# A3800). To determine relative quantitave levels of endogenously expressed

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oligophrenin1, RhoA, Rac1, TNF-, IL-6, Bax, Bcl-2 and Bcl-XL real-time PCR was performed using 7500 Fast Real-Time PCR System and Fast SybrGreen Master Mix (Life

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Technologies, Cat.# 4385610). The Assessment of expression levels was normalized to mRNA levels of glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) or actin as indicated

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using the following oligonucleotide primers:

forward 5’-CCAAGGCTTTCGCGGCTCTT-3’, reverse 5’- TTGGTGCCCCCACCTTTCCC-3’ (oligophrenin1);

forward

5’-

reverse

GCGGCCTCTCTCTTATCCAG-3’

5’-

3’;

reverse

5’-

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CATTTTGGCCAACTCCCGTC-3’ (RhoA); forward 5’- ATGCAGGCCATCAAGTGTGTGGTGTTACAACAGCAGGCATTTTCTCTTCC-3’

forward

reverse

forward

5’-GGGGCTGGCTCTGTGAGGAA-3’

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GCCCCCACTCTGACCCCTTT-3’,

(Rac1);

5’-ACTCGGCAAACCTAGTGCGTTATG-3’,

reverse

5’-

(TNF); 5’-

ACATTCCAAGAAACCATCTGGCTAG-3’ (IL-6); forward 5’-ATGTGTGTGGAGAGCGTCAA5’-CATGCTGGGGCCATATAGTT-3’

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3’, reverse

(Bcl-2);

forward

5’-

GACAAGGAGATGCAGGTATTGG-3’, reverse 5’-TCCCGTAGAGATCCACAAAAGT-3’ (BclxL); forward 5’-TGAAGACAGGGGCCTTTTTG-3’, reverse 5’-AATTCGCCGGAGACACTCG3’

(Bax);

forward

5’-

GGTGAAGGTCGGTGTGAACG-3’,

reverse

5’-

CTCGCTCCTGGAAGATGGTG-3’ (GAPDH); forward 5’- CTAAGGCCAACCGTGAAAAG-3’, reverse 5’-ACCAGAGGCATACAGGGACA-3’ (actin).

2.8. Hematoxylin and Eosin (HE) staining, immunostaining, esterase-staining Histology has been performed as described previously [40]. Briefly, paraffin-embedded cardiac sections were taken one day after ischemia. Staining with hematoxylin and eosin was performed according to a standard protocol. Immunostaining was performed with labelled 8

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streptavidin

OPHN1 and Myocardial Infarction

biotin/horseradish

peroxidase

(LSAB2

System

HRP,

Dako)

and

Diaminobenzidine (DAB) - Chromogen (Dako). As primary antibodies cleaved Caspase-3 (Asp175) antibody (Cell Signaling), mouse anti-Rac1 Mab antibody (BD Biosciences), RHOA

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Poly antibody (Proteintech), Phospho-PAK1 antibody (cell signaling), and Phospho-ERM antibody (Cell Signaling). The assessment of granulocytes and monocytes was performed

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with specific esterase kits (Naphthol AS-D Chloroacetate Esterase and α-Naphthyl Acetate Esterase, Sigma-Aldrich). To evaluate caspase-3 staining and the number of granulocytes,

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the numbers of cells that were positively labeled for the respective protein were counted in six different fields pro-section and expressed as cells/mm2. To detect monocytes, dark blue

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positive cells were counted because these cells stain dark blue due to an enzymatic reaction.

2.9. IL-6 ELISA

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The quantification of IL-6 in the plasma of ophn+/y and ophn-/y mice after MI was measured following the manufacturer´s protocol (DuoSet ELISA development system, mouse IL-6, R&D

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systems, UK).

2.10. G-LISA; Rac1 and RhoA activation assay

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To determine the level of activated Rac1 (G-LISA™ Rac1 Activation Assay Biochem Kit, Cytoskeleton) and RhoA (antibody, Cell Signaling) hearts of ophn+/y and ophn-/y mice were lysed 24 h after MI. A G-LISA assay (Cytoskeleton) and a standard pull-down assay (Cell signaling) was carried out following the manufacturer´s protocol as described previously [12].

2.11. Western Blot analysis We determined the expression level of RhoA (antibody, Proteintech), Rac1 (antibody, BD), Bax (antibody, cell signaling), Bcl-2 (Bcl-2 (50E3) rabbit mAB, Cell Signaling), Bcl-xl (bcl-xL antibody, Cell Signaling), TNF-α (antibody, Cell Signaling) and α-tubulin (antibody, Cell Signaling) used as control, in lysed hearts of ophn+/y and ophn-/y mice 24 h after MI via immuno(western)blotting. The lysates were prepared with reducing sample buffer (Laemmli 9

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buffer) and denatured at 95 °C for 5 min, separated on SDS-polyacrylamide gel and transferred onto nitrocellulose blotting membrane (GE Healthcare Life Sciences). Subsequently, the membrane was blocked and probed with appropriate blocking medium

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and antibody following the equal manufacturer´s protocol. Followed membranes were incubated with peroxidase-conjugated goat anti-rabbit IgGs (1:2500). Protein bands were

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visualized by the use of Immobilon™ Western Chemiluminescent HRP Substrate solution

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(BioRad).

2.12. Statistical analysis

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Data are given as arithmetic means ± s.e.m (standard error of mean) from at least three individual experiments (n represents the number of experiments). All data were tested for significance using the student’s paired t-test, where applicable. P-values < 0.05 were

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considered to be statistically significant.

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3. Results 3.1. OPHN1 expression is enhanced after MI in mice. After ligation of the LAD for 60 min, hearts from C57Bl6/J mice were isolated and mRNA

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from the LV was prepared. Real-time PCR was performed to quantify the relative mRNA expression of Ophn1 in the LV after ischemia and reperfusion injury and compared to healthy

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control mice. As shown in figure 1A, Ophn1 is strongly upregulated 24 h after MI suggesting an important role for OPHN1 in cardiovascular inflammatory disease (0.97 ±0.68 versus

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18.55 ±9.54, p=0.0522, fig. 1A). For many years, OPHN1 was thought to be restricted to the central nervous system (CNS), therefore we performed real-time PCR and confirmed

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expression of Ophn1 in platelets, macrophages and thymocytes known to play a pivotal role in processes of myocardial ischemia and reperfusion (fig. 1B). OPHN1 is known to regulate the activity of RhoA, Rac1 and Cdc42 [3, 9]. Therefore we measured the expression level of

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OPHN1 target proteins RhoA and Rac1 in the LV of mice after myocardial infarction. As shown in figure 1C-D, RhoA and Rac1 expression in the LV is upregulated 24 h after

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myocardial ischemia and reperfusion in mice. However, no siginificant alterations were observed between OPHN1 wildtype (ophn1+/y) and deficient mice (RhoA expression: 42.79 ±2.89 before MI versus 108.76 ±11.68, 24 h after MI, ophn1+/+ mice, p=0.0134; and 57.47

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±8.49 before MI versus 91.37 ±5.13 24 h after MI, ophn1-/y mice, p<0.05); Rac1 expression: 3.35 ±0.68 before MI versus 7.62 ±0.24, 24 h after MI, ophn1+/+ mice, p=0.002) and 3.22 ±0.13 before MI versus 7.01 ±1.14 24 h after MI, ophn1-/y mice, p=0.0151); fig. 1C-D).

3.2. Increased activation of the RhoA effector ERM and the Rac1/Cdc42 effector PAK1 in cells of the infarct zone of ophn1-/y mice 24 h after ischemia. As an important modulator of Rho GTPase activity, OPHN1 is able to switch off the activity of RhoA, Rac1 and Cdc42 [3, 9]. Therefore we wanted to know if the deficiency of OPHN1 leads to enhanced activity of Rho proteins after myocardial ischemia. According to the data obtained by real-time PCR (fig. 1C-D) we found unaltered protein expression of the OPHN1 target proteins RhoA and Rac1 in cardiac tissue 24 h after ischemia (RhoA: 100% ±13% 11

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versus 108% ±28%; Rac1: 100% ±13% versus 104% ±20%, not significant, fig. 2A-D). In contrast, the activation of RhoA before and 24h after MI was increased in the left ventricle of OPHN1 deficient mice compared to control mice as observed by determination of active

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RhoA (RhoA-GTP, fig. 2E) using pulldown assay. However, the level of active Rac1 (Rac1GTP) as determined by G-LISA (pulldown assay, Cytoskeleton) was significantly enhanced

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in OPHN1 deficient mice only before MI compared to control mice. 24 h after MI, the amount of active Rac1 did not increase in OPHN1 deficient mice (100% ±2.45% versus 133%

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±2.35% before MI, p=0.0154; 125.67% ±8.59% versus 123.21% ±11.29% 24 h after MI, not significant, fig. 2F). Furthermore, immunhistostaining of cardiac sections showed increased

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activation of the RhoA effector ROCK as detected by ERM (Ezrin/Radixin/Moesin) phosphorylation 24 h after MI (100% ±47% versus 347% ±94%, p=0.0003). In contrast to the results of active Rac1 by G-LISA, the activation of the Rac1/Cdc42 effector PAK1 was

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increased as well (100% ±25% versus 365% ±155%, p=0.0054; fig. 2G-J) demonstrating that loss of OPHN1 leads to increased activation of Rho, Rac and Cdc42 target proteins in the

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infarct zone.

3.3. Decreased cardiac function in OPHN1 deficient mice 24 h after MI.

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To assess the effects of OPHN1 deficiency on mycocardial function in vivo, we used a mouse model of myocardial ischemia and reperfusion injury. In this model, myocardial damage was induced by ligation of the LAD for 60 min and reperfusion was allowed for 24 h (fig. 3A). Infarct size was investigated by 2,3,5-triphenyltetrazolium chloride (TTC) staining to discriminate between metabolically active and inactive tissue. Infarct size of OPHN1 deficient mice normalized to the area of risk was enhanced by trend without reaching significance (25.17 ±4.61 versus 39.19 ±8.16, not significant, fig. 3B-D). Echocardiography 24 h after MI showed significantly enhanced declined cardiac LV function of OPHN1 deficient mice as measured by ejection fraction (53.18 ±4.34 before MI versus 43.26 ±8.78, 24 h after MI, ophn1+/+ mice, p=0.0226; and 56.05 ±3.34 before MI versus 40.35 ±5.13 24 h after MI, ophn1-/y mice, p=0.00042), fractional shortening (12.53 ±2.21 before MI versus 8.26 ±2.2 24 12

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h after MI, ophn1+/+ mice; and 12.12 ±3.19 before MI versus 5.64 ±2.47 24 h after MI, ophn1/y

mice, p=0.00047) and cardiac output (18.65 ±1.86 before MI versus 16.24 ±3.02 24 h after

MI, ophn1+/+ mice; and 17.87 ±1.55 before MI versus 15.22 ±1.69 24 h after MI, ophn1-/y

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mice, p=0.00556) compared to control mice (fig. 3E-H). Additional hemodynamic parameters

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such as endsystolic (39.6 ±7.03 before MI versus 44.05 ±13.84 24 h after MI, ophn1+/+ mice;

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and 35.79 ±7.38 before MI versus 49.52 ±7.89 24 h after MI, ophn1-/y mice, p=0.00293, fig. 3I) and enddiastolic volume indicate that impairment of hemodynamic function in OPHN1

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deficient mice is increased compared to control mice (fig. 3J). These data clearly show that the absence of OPHN1 augments impaired hemodynamic function in response to cardiac

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injury.

3.4. Loss of OPHN1 led to enhanced migration of inflammatory cells into the infarct border

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zone 24 h after ischemia.

OPHN1 regulates the activity of Rho GTPases known to be important for cytoskeletal

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reorganization and cell migration [9, 12, 14, 36]. Thus we investigated the migration of inflammatory cells into the infarct border zone 24 h after MI. Quantitative analysis of cell migration revealed a significantly enhanced number of cells in the infarct border zone of

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OPHN1 deficient mice (5.908 ±361 versus 7.085 ±215, p=0.0128, fig. 4A-B). To determine the nature of these migrating cells, we stained myocardial tissue with a-Naphtylacetatesterase and found increased neutrophil recruitment in OPHN1 deficient mice (395 ±24 versus 494 ±37, p=0.0434, fig. 4C-D). Similarly, migration of monocytes/macrophages into the infarct border zone of OPHN1 deficient mice was significantly enhanced as well (515 ±86 versus 619 ±77, p=0.0428, fig. 4E-F).

3.5.

OPHN1

deficiency

suppresses

ischemia

reperfusion

induced

production

of

proinflammatory cytokines. Acute phase cytokines such as TNF- and IL-6 are important mediators of inflammation and implicated in the acute phase after myocardial ischemia. Thus we analyzed cytokine 13

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expression 24 h after MI in OPHN1 deficient and wildtype mice. Real-time PCR revealed an increase of TNF-and IL-6 expression in the LV after MI in both, OPHN1 wildtype and OPHN1 deficient mice (TNF-expression: 0.25 ±0.01 (ophn1+/+) versus 0.37 ±0.03 (ophn1-/)y)

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before MI, p=0.019; 2.28 ±0.31 (ophn1+/+) versus 1.76 ±0.29 (ophn1-/)y) 24 h after MI, not significant; IL-6 expression: 0.03 ±0.01 (ophn1+/+) versus 0.01 ±0.01 (ophn1-/)y) before MI, not

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significant; 1.34 ±0.26 (ophn1+/+) versus 0.73 ±0.05 (ophn1-/)y) 24 h after MI, p=0.0533, fig. 5A-B). However, the increase in IL-6 expression level was significantly lower in OPHN1

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deficient compared to wildtype mice (fig. 5B). Subsequent analysis of cytokine levels by Western blot analysis and ELISA revealed no alterations in the TNF- level 24 h after MI (fig.

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5C) while the amount of IL-6 was significantly reduced 6 h after MI (51.37 ±15.03 (ophn1+/+) versus 19.23 ±4.46 (ophn1-/)y) 6 h after MI, p=0.0339; and 2.34 ±0.74 (ophn1+/+) versus 5.43

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±2.34 (ophn1-/)y) 24 h after MI, not significant, fig. 5D).

3.6. Increased cell apoptosis in the infarct zone of ophn1-/y mice 24 h after ischemia.

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Cell apoptosis is characterized by caspase-3 expression in target cells [44]. Quantitative analysis of caspase-3 positive cells revealed a significant increase in the number of apoptotic cells in the infarct zone of OPHN1 deficient mice compared to wildtype mice (515 ±94 versus

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790 ±159, p=0.0025, fig. 6A-B). In a further set of experiments we measured the expression of different anti- and pro-apoptotic markers 24 h after ischemia and reperfusion using realtime PCR. The BCL-2 (B-cell lymphoma 2) protein family determines the commitment of cells to apoptosis [7]. Bcl-2 is specifically considered an important anti-apoptotic protein and is thus classified as an oncogene [6]. 24 h after MI, we found increased expression of the antiapoptotic marker Bcl-2 in the LV of wildtype mice. In OPHN1 deficient mice, we only detected a slightly increase in the expression of Bcl-2 in the LV after myocardial ischemia that was significantly lower compared to wildtype mice (0.52 ±0.2 before MI versus 1.56 ±0.58 24 h after MI, ophn1+/+ mice, p=0.0533; and 0.11 ±0.04 before MI versus 0.45 ±0.2 24 h after MI, ophn1-/y mice, p=0.0497; ophn1+/+ versus ophn1-/y p=0.0529, fig. 6C). Subsequent analysis of Bcl-2 protein expression by Western blot analysis confirmed increased Bcl-2 expression in 14

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wildtype mice and only a slight increase in protein expression in OPHN1 deficient mice 24 h after MI (fig. 6F). Furthermore, the expression of the anti-apoptotic marker Bcl-xL [22] showed an increase in OPHN1 wildtype mice after myocardial ischemia compared to healthy

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mice (3.71 ±0.21 before MI versus 9.1 ±0.44 24 h after MI, ophn1+/+ mice, p=0.0001; and

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7.84 ±0.69 before MI versus 7.35 ±0.46 24 h after MI, ophn1-/y mice, not significant, ophn1+/+

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versus ophn1-/y p=0.0004 (before MI) and p=0.0253 (24 h after MI), fig. 6D). In contrast, in OPHN1 deficient mice high basal expression of Bcl-xL in the LV was observed in healthy

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mice. MI did not lead to a further increase of Bcl-xL expression in these mice. In addition, its relative expression 24 h after MI was significantly lower in OPHN1 deficient mice compared

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to wildtype mice (fig. 6D). Results obtained by realtime PCR were confirmed by Western blot analysis showing again higher basal levels of Bcl-xL expression in OPHN1 deficient mice compared to controls (fig. 6F). We next compared the expression level of the apoptosis

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regulator Bcl-2-associated X protein (Bax), another member of the Bcl-2 gene family [33] known to exhibit pro-apoptotic function. An increase of Bax expression was measured 24 h

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after MI in both, OPHN1 wildtype and OPHN1 deficient mice. No significant differences were observed in the level of Bax expression in both groups (5.49 ±0.51 before MI versus 15.69 ±2.05 24 h after MI, ophn1+/+ mice, p=0.0042; and 3.88 ±0.23 before MI versus 10.51 ±1.61

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24 h after MI, ophn1-/y mice, p=0.0074, fig. 6E). Western blot analysis confirmed comparable protein expression of Bax before MI in both groups, while we found a slightly enhanced level of Bax in wildtype mice compared to OPHN1 deficient mice 24 h after MI (fig. 6F). To investigate whether hematopoetic stem cells are affected by increased apoptosis in OPHN1 deficient mice after MI, we isolated bone marrow cells and performed an apoptosis assay. As shown in figure 6G-H, OPHN1 deficiency does not induce alterations in the survival of progenitor cells as measured by Annexin-V binding used as an early apopotic marker (47.81 ±22.46 versus 45.97 ±11.34, basal; 484 ±79.91 versus 527.76 ±63.32, apoptotic conditions, fig. 6G) and propidium iodide staining to investigate late apoptosis (14.72 ±0.46 versus 17.31 ±1.97, basal; 74.37 ±9.8 versus 56.69 ±4.2, apoptotic conditions, fig. 6H) of cells under basal as well as under apoptotic conditions in minimal medium. 15

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4. Discussion The present study demonstrates for the first time that well-regulated activity of Rho GTPases is critical for the preservation of myocardial function. Loss of OPHN1 led to increased activity

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of RhoA and Rac1 effector molecules resulting in enhanced cardiomyocyte apoptosis, enhanced migration of inflammatory cells into the infarct zone and declined LV function 24 h

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after myocardial ischemia and reperfusion.

OPHN1 was thought to be neuron-specific for decades and first identified in patients

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suffering from X-linked mental retardation [3]. Because OPHN1 is abundantly expressed in the brain in neurons of the hippocampus, cortex and present in axons, dendrites and spines

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[15], mutations in the OPHN1 gene impact neuronal function [1]. When OPHN1 was overexpressed, the observed phenotypes were mostly mediated by impaired RhoA signaling; accordingly, a rescue of the phenotype was achieved by the inhibition of RhoA effector

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ROCK [9, 15]. Consequently, increased levels of activated RhoA were measured in platelets

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of OPHN1 deficient mice and ophn1-/y fibroblasts [12, 25, 26]. In platelets an OPHN1

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mediated cytoskeletal phenotype with a strong relation to Rac1 and RhoA was determined, because spreading experiments revealed enhanced numbers of lamellipodia and increased adhesion of ophn1-/y platelets on a fibrinogen matrix [12]. In the present study, enhanced

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activation of the RhoA effector ERM and the Rac1/Cdc42 effector PAK were observed suggesting increased activity of RhoA, Rac1 and Cdc42 also in the myocardium after myocardial ischemia and reperfusion. Increased levels of active RhoA (RhoA-GTP) were confirmed by pulldown assay showing increased activity of RhoA in OPHN1 deficient mice before and 24 h after MI. Determination of active Rac1 (Rac1-GTP) revealed only enhanced levels of Rac1-GTP before MI in OPHN1 deficient mice while no alterations between OPHN1 deficient and control mice were observed 24 h after MI. In contrast the levels of activated ERM and PAK in OPHN1 deficient mice 24 h after MI suggest enhanced activity of both, RhoA and Rac1, in the absence of OPHN1. This might be due to experimental conditions because we determined active ERM and PAK in the infarct zone while the whole left ventricle was lysed to perform pulldown assays that might weaken the signal of active Rac1 in the 16

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whole left ventricle. Increased activity of Rho proteins resulted in declined cardiac function with increased cardiomyocyte apoptosis and enhanced infiltration with inflammatory cells providing evidence that dysregulated activation of Rho proteins worsens myocardial ischemia

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and reperfusion injury. In platelets enhanced Rho activity upon OPHN1 deficiency led to enhanced platelet activation and thrombus formation on collagen under low shear conditions

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ex vivo and promoted arterial thrombus formation in ophn1-/y mice that might be also relevant upon cardiac injury, where platelets play a pivotal role.

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In the last years, the impact of Rho GTPases in cardiac pathophysiology was investigated by different groups. Interestingly, contradictory results were obtained in mice where the RhoA

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effector ROCK was inhibited. While Hattori and colleagues found suppressed LV remodelling when mice were treated with fasudil, another study showed a beneficial effect of ROCK inhibition by treatment of mice with Y-27632 [2, 17]. These mice showed reduced infarct size

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and enhanced cardiac function after treatment with Y-27632. Moreover, reduced apoptosis and attenuated downregulation of Bcl-2 in hearts were observed. Consistently, we here show

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the effects of enhanced Rho activity with enhanced apoptosis and reduced Bcl-2 expression. Enhanced phosphorylation of ERM as indicator for increased ROCK activity clearly indicates that abnormal Rho activation is associated with cardiomyocyte apoptosis. In contrast, a

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recent study provided evidence for RhoA to be protective upon cardiac injury. Mice with low constitutively active RhoA levels in the heart displayed increased tolerance to myocardial ischemia and reperfusion injury with reduced infarct size and improved LV function [46]. The authors observed that RhoA promotes survival of cardiomyocytes while cardiac knock-out of RhoA led to increased susceptibility to cardiac injury in mice [46]. The impact of Rac1 in myocardial injury was shown by Talukder and colleagues who found increased ischemia and reperfusion injury with enhanced contractile dysfunction in mice that overexpress active Rac1 in the heart [43]. Concomitantly, mice with a cardiac specific Rac1 knock-out displayed reduced ischemia and reperfusion injury with decreased infarct size [41]. However, OPHN1 regulates all three Rho GTPases, namely RhoA, Rac1 and Cdc42, that makes the correlation of phenotypes to the individual Rho GTPase difficult. Moreover, 17

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nothing so far is known about the impact of Cdc42 in myocardial injury although it is known from different studies in the past that Cdc42 activity is upregulated upon myocardial injury [16, 24, 29].

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In line with the study from Bao and colleagues a RhoA induced regulation of cardiomyocyte apoptosis was observed by Del Re and colleagues. They found an involvement of Bax and

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p53 in RhoA-mediated apoptosis as examined by treatment with a Bax-inhibitory peptide that significantly attenuates DNA fragmentation and caspase-3 activation. Accordingly, dominant

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negative p53 prevented RhoA induced apoptosis [8]. Already in 1999, Sah and colleagues provided evidence for an involvement of RhoA in contractile dysfunction [37]. Overexpression

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of RhoA resulted in bradycardia and induced ventricular failure in mice that constitutively express active cardiac specific RhoA. The observed effects of RhoA might be –at least in part- due to alterations in the activity of the RhoA effector ROCK. Activation of the RhoA

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effector ROCK-1 promoted apoptotic signals in myocardial hypertrophy and failure because ROCK-1 deficient mice showed a marked reduction in myocyte apoptosis while constitutively

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active ROCK-1 expression in cardiomyocytes led to caspase-3 activation [5].

In conclusion we provide strong evidence that OPHN1 mediated regulation of RhoA, Rac1

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and Cdc42 is crucial to improve myocardial repair and reduce heart failure after myocardial ischemia and reperfusion. Loss of OPHN1 induced an increase in the activity of RhoA, Rac1 and Cdc42 leading to enhanced cardiomyocyte apoptosis and increased LV dysfunction. Thus our results imply an important role for OPHN1 in the regulation of cardiomyocyte apoptosis, cytokine release and LV function in vivo.

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5. Conclusion 5.1 Ophn1 expression is upregulated upon myocardial ischemia and reperfusion injury. 5.2 OPHN1 deficiency results in enhanced activation of RhoA, Rac1 and Cdc42 effector

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molecules in the heart after MI.

5.3 Increased inflammatory cell migration and cardiomyocyte apoptosis is responsible for

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declined cardiac LV function 24 h after myocardial ischemia in OPHN1 deficient mice. 5.4 OPHN1 is an important regulator of inflammation, cardiomyocyte survival and LV function

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after cardiac injury.

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Conflict-of-interest

The authors declare no competing financial interests.

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Author contributions

Contributions: C.N., S.G., N.S.G. and M.K. performed experiments. P.B., M.W.M. and M.E.

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analyzed data. M.E. designed research and wrote the manuscript.

Acknowledgements

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We thank Martine Spelleken for excellent technical assistance. This study was supported by grant from the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich Düsseldorf.

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Figure Legends Fig. 1. Ophn1 expression is enhanced after MI in mice. (A) Quantification of mRNA

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expression of Ophn1 in the myocardium 24 h after MI was performed using real-time PCR.

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Myocardial tissue from healthy mice served as controls. Actin was used as reference gene.

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Agarose gel resolves PCR products (left panel), quantification of relative expression of Ophn1 in the myocardium (right panel). (B) Real-time PCR showed Ophn1 expression in brain (positive control), platelets, macrophages and thymocytes. Agarose gels resolved PCR

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products. (C-D) Quantification of mRNA expression of the OPHN1 target proteins RhoA (C) and Rac1 (D) in the myocardium 24 h after MI using real-time PCR. Myocardial tissue from

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healthy mice served as controls. Glyceraldehyde-3-phosphate dehydrogenase was used as reference gene. Bar graphs depict mean values ± S.E.M. (n = 3 per group). MI = myocardial

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infarction, n.s. = not significant.

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Fig. 2. Unaltered expression of the OPHN1 target proteins RhoA and Rac1 but increased

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activation of the Rho effector proteins ERM and PAK in the infarct zone of ophn1-/y mice 24 h after ischemia. (A-D) Immunhistostaining of RhoA (A-B) and Rac1 (C-D) positive cells shows no differences between both groups. Scale bar 200 µm (left panel) and 50 µm (right panel).

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(E-F) Analysis of active GTP-bound RhoA and Rac1 in ophn1+/+ and ophn1-/- lysates of the left ventricle before and 24 h after MI using specific pulldown assays. Western Blots respresent total levels of Rho GTPase expression and specific expression of active RhoA (E). Bar graphs depict mean values ± S.E.M. as percentage of control (ophn1+/+ resting = 100%, F). (G-H) Activation of the RhoA effector Rho-associated protein kinase (ROCK) was detected

by

ERM

(Ezrin/Radixin/Moesin)

immunhistostaining

showing

increased

phosphorylation in the infarct zone of ophn1-/y mice. (I-J) Phospho-PAK positive cells in the infarct zone of wildtype (ophn1+/y) and ophn1-/y mice. Scale bar 200 µm (left panel) and 50 µm (right panel). Bar graphs depict mean values ± S.E.M. (n = 6-7). * P<0.05, *** P<0.001, n.s. = not significant.

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Fig. 3. Decreased cardiac function in OPHN1 deficient mice 24 h after MI. (A) Myocardial ischemia and reperfusion injury was induced by ligation of the LAD for 60 min followed by reperfusion for 24 h. (B) Representative transverse cardiac sections from wildtype (ophn1+/y)

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and ophn1-/y mice are shown. (C) Quantitative analysis of the infarct size in percentage of area at risk. (D) Determination of the area at risk as the percentage of the left ventricle shows

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no differences between both groups. (E) Echocardiographic analysis (baseline versus 24 h after ischemia) of ejection fraction (EF, E-F), fractional shortening (FS, G), cardiac output

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(H), endsystolic (I) and enddiastolic volume (J) of wildtype (ophn1+/y) and ophn1-/y mice to determine LV function after myocardial ischemia and reperfusion (I/R). Bar graphs depict

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mean values ± S.E.M. ΔEjection fraction (EF before I/R – EF 24 h after I/R), ΔEndsystolic/enddiastolic volume (before I/R - 24 h after I/R) in percentage of echocardiographic analysis before and after 24 h of I/R. (basal versus MI; ophn1+/y versus

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ophn1-/y mice). (n = 6-7 per group). * P<0.05, ** P<0.01, *** P<0.001, n.s. = not significant.

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Fig. 4. Loss of OPHN1 led to enhanced migration of inflammatory cells into the infarct border zone 24 h after ischemia. (A-B) Quantitative analysis of cell migration 24 h after ischemia shows significantly more migrated cells in ophn-/y compared to wildtype mice (ophn+/y). (A)

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24h after ischemia, heart tissue of wildtype (ophn+/y) and ophn-/y mice has been stained with hematoxylin and eosin (HE) for quantitave analysis. Pictures were taken with 100x and 400x magnification. Scale bar 200 µm (upper panel) and 50 µm (lower panel). (B) Number of cells in the infarct border zone. (C-D) Quantitative analysis of migrated granulocytes in ophn-/y and wildtype mice (ophn+/y) 24 h after ischemia. Scale bar 200 µm (upper panel) and 50 µm (lower panel). (E-F) Cardiac sections were stained with α-Naphthylacetat-Esterase to analyze the migration of monocytes into the infarct border zone. Representative images (E) and quantitative analysis (F) of wildtype mice (ophn+/y) and ophn-/y mice. Monocytes stain dark blue because of an enzymatic reaction. Scale bar 200 µm (upper panel) and 50 µm (lower panel). (n = 6-7 per group). *p≤0.05.

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Fig. 5. OPHN1 deficiency suppresses the production of the acute phase cytokine IL-6. (A) mRNA expression of TNF- (A) and IL-6 (B) in the left ventricle of ophn+/y and ophn-/y mice 24 h after I/R injury was determined by real-time PCR. Glyceraldehyde-3-phosphate

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dehydrogenase served as control. (C) TNF- protein expression was determined in lysates

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of the left ventricle before and 24 h after MI by Western blot analysis. -tubulin served as loading control. (D) IL-6 plasma levels 6 h and 24 h after MI were determined by commercial

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ELISA. MI = myocardial infarction. (n = 3 per group). *p≤0.05, n.s. = not significant.

Fig. 6. Increased myocardial apoptosis 24 h after myocardial ischemia and reperfusion

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injury. (A) Apoptosis in the infarct zone was determined by caspase-3 staining. (A) Representative cardiac sections and (B) quantitative analysis of caspase-3 positive cells in

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the infarct zone of ophn+/y and ophn-/y mice 24 h after I/R injury. (n = 7). Pictures were taken

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with 100x and 400x magnification. mRNA expression of Bcl-2 (C), Bcl-xL (D) and Bax (E) was determined by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase served as

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control. (n = 3). (F) Protein expression of Bcl-2, Bcl-xL and Bax was measured by Western blot analysis, -tubulin served as loading control. (G-H) Cell survival of bone marrow cells was detected by flow cytometry. BMCs were incubated for 6 h under apoptosis inducing

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culture conditions. Cell survival after 18 h between wildtype (ophn+/y) and ophn-/y mice showed no significant differences. Cell apoptosis was determined by AnnexinV staining (FITC AnnexinV Apoptosis Detection Kit, F) as early apoptosis marker and propidium iodide staining (G) as marker for late apoptosis using flow cytometry. BMCs = bone marrow cells. (n = 3 per group). ** P<0.01, *** P<0.001 (basal versus MI), # P<0.05, ## P<0.01, ### P<0.001 (ophn1+/y versus ophn1-/y mice). n.s. = not significant.

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Highlights ► Ophn1 expression is upregulated upon myocardial ischemia and reperfusion injury (I/R).

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► Loss of OPHN1 results in enhanced activation of Rho effectors in the heart after I/R. ►

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Increased inflammatory cell migration and cardiomyocyte apoptosis 24 h after I/R. ►

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Declined cardiac LV function 24 h after myocardial ischemia in OPHN1 deficient mice. ►These results point to a new role for OPHN1 as an important regulator of LV function after

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