**1. Introduction**

Deaths arising from ventricular arrhythmias (VA) after acute cardiac events, in Germany, have increased by 100% over the last twenty years [1]. Therefore, new therapeutic methods for the prevention of VA following myocardial infarction (MI) need to be developed. There are reports that have indicated that transplanting of various stem cell populations post MI can influence the heart rhythm. Some have been defined as pro-arrhythmogenic cells, i.e., skeletal myoblasts, while others have shown antiarrhythmic effects, such as embryonic cardiomyocytes and mesenchymal stem cells (MSC) [2–5]. Our research group has focused on the role of human bone marrow (BM) derived hematopoiesis such as MSC in regenerative medicine [6–9]. In previous studies we have shown that it is safe to use CD117<sup>+</sup> stem cells for myocardial regeneration, as their electrophysiological properties make them very unlikely to produce hazardous action potentials or contribute to arrhythmias [10,11]. However, the availability of a realistic small animal model reflecting the situation in patients is of utmost importance. For testing the rhythmological behavior of human cells, Rag2−/<sup>−</sup> γc−/<sup>−</sup> has been selected. These mice (strain C, 129S4-*Rag2tm1.1FlvIl2rgtm1.1Flv*/J) have been derived from a V17 embryonic stem cell line (BALB/c × 129 heterozygote) targeted for the Rag2 and Il2rg genes and lack B, T, and natural killer (NK) cells [12]. They have a deletion of the common cytokine receptor γ chain (γc) gene, and thus reduced numbers of peripheral T and B lymphocytes, and absent natural killer cell (NK) activity. A genetic cross with a recombinase activating gene 2 (RAG2) deficient strain produced mice doubly homozygous for the γc and RAG2 null alleles (Rag2−/<sup>−</sup> γc−/−) with a stable phenotype characterized by the absence of all T lymphocyte, B lymphocyte, and NK cell function [13]. This phenotype makes the completely a lymphoid strain useful for studies on human tissue xenotransplantation [14].

As previously shown, stem cell antiarrhythmic effects appear after completion of an approximately one-week engraftment period [4]. The major objective of the present study was to develop a mouse model that enables us to reproduce VA several days after an initial MI. In vivo electrophysiological mouse models are well established: Previously, Berul et al. established an open chest epicardial study of conduction properties with the ability to induce second and third degree heart blocks, but no induction of tachyarrhythmias after programmed stimulation [15]. Hagendorff et al. used a rapid transesophageal atrial stimulation with the ability to induce some types of heart block and atrial tachyarrhythmias with induction of VA [16]. Roell et al. used a method relying on burst and extra stimulus pacing mediated induction of VA, leading to induction of ventricular tachycardia (VT) in 38.9% of the non-infarcted control group and 96.4% of the infarcted group [4].

This mouse model can also be considered representative for patients that suffer from severe ischemic heart disease and related arrhythmias. In this respect the application of the second MI at various points in time will be of interest.

#### **2. Materials and Methods**

#### *2.1. Bone Marrow Aspiration*

Informed donors gave written consent to the aspiration of their BM according to the Declaration of Helsinki. The ethical committee of the University of Rostock approved the presented study (registered as no. A201023) as of 29 April 2010. The BM samples were obtained by sternal aspiration from patients undergoing coronary artery bypass graft surgery at Rostock University, Germany. The donors had no BM or hematological diseases and were not receiving any immune suppressing medication. The amount of the aspirated BM ranged from 60–100 mL. The BM samples were received from four donors. Two donors got more than 200,000 isolated CD271<sup>+</sup> MSC in their BM samples and each one has been used for 2 mice (100,000 cells/animal). Anticoagulation was achieved by heparinization with 250 i.E./mL sodium heparine (B. Braun Melsungen AG, Melsungen, Germany).

#### *2.2. Cell Isolation*

The mononuclear cells (MNC) were isolated by density gradient centrifugation using Lymphocyte Separation Medium (LSM; 1.077g/L, PAA Laboratories GmbH, Pasching, Austria). The MNC were indirectly labeled with CD271 APC and anti-APC micro beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD271<sup>+</sup> cells were enriched by positive magnetic selection using the magnet activated cell sorting (MACS) system (Miltenyi Biotec).

#### *2.3. Flow Cytometric Analysis*

Purity and viability of all cell isolations were verified by flow cytometry. Antibodies were all mouse anti-human and appropriate mouse isotype antibodies were applied for validation of our gating strategy. Anti-CD271 allophycocyanine was obtained from Miltenyi Biotec. Anti-CD45 allophycocyanin-H7, anti-CD45 Horizon V500, anti-CD29 allophycocyanin, anti-CD44 peridinin-chlorophyll-protein Cyanine 5.5 tandem dye, anti-CD73 phycoerythrin, as well as 7-aminoactinomycin were from BD Biosciences

(Heidelberg, Germany) and anti-CD105 Alexa Fluor 488 was purchased from AbD Serotec GmbH (Puchheim, Germany). Near-IR live dead stain and 4',6-diamidino-2-phenylindole (DAPI) were obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA).

Cells were suspended in MACS buffer containing PBS, 2mM EDTA, and 0.5% bovine serum albumin. To reduce unspecific binding, FcR blocking reagent (Miltenyi Biotec) was added to all samples. Cells were incubated with the antibodies for 10 min in the dark at 4 ◦C. RBC lysis buffer (red blood cell, eBioscience GmbH, Frankfurt/Main, Germany) was added for 10 min on ice. Samples were analyzed by BD LSRII flow cytometer and data were analyzed using FACSDiva software, version 6.1.2 (both Becton Dickinson, Franklin Lakes, New Jersey, USA). For optimal multicolor setting and correction of the spectral overlap, single stained controls were utilized, and gating strategy was performed with matched isotype/fluorescence minus one control. The CD271<sup>+</sup> cells with a low granularity (SSClow) were included for further flow cytometric analysis and positivity for mesenchymal markers such as CD29, CD44, CD73, CD105, and CD271 was evaluated based on viable CD45− cells in an established 6-fold staining, as previously described [17]. After performing antibody staining, as described above, 15 μM DAPI was added; cells were incubated for 2 min and then immediately acquired.

#### *2.4. Animals*

All animal procedures were performed according to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. The federal animal care committee of LALLF Mecklenburg-Vorpommern (Germany) approved the study protocol (approval number LALLF M-V/TSD/7221.3-1-020/14). The Rag2−/−γc−/<sup>−</sup> mice (strain C, 129S4-*Rag2tm1.1FlvIl2rgtm1.1Flv*/J) were purchased from the Jackson Laboratory (USA).

#### *2.5. Ambulatory ECG Monitoring and Ligation of the Left Anterior Descending Artery*

For ambulatory electrocardiogram (ECG) monitoring, twelve-to-fourteen-week-old female Rag2−/−γc−/<sup>−</sup> mice (*n* = 22) were implanted with a telemetric device (Data Sciences International, DSI, New Brighton, Minnesota, USA) 7 days before performing the myocardial intervention. The mice were anesthetized with pentobarbital (50 mg/kg, intraperitoneal). An incision was made on the left thorax along the ribs to insert a telemetric transmitter (TA11ETA-F10 Implant; DSI) into a subcutaneous pocket with paired wire electrodes placed over the thorax with leads tunneled to the right upper and left lower thorax. The heart rate, PR interval and QRS complex as the entities of an ECG were recorded using Ponemah Physiology Platform (DSI) until 48 h post infarction. Telemetric ECG signal was qualitatively analyzed, and VA were detected and counted with ECG auto 1.5.11.26 software (EMKA Technologies, Paris, France).

Mice were randomly assigned into three groups: (untreated reinfarction, URI; stem cell treated reinfarction, SRI; and MI control, MIC). First, animals of all three groups underwent thoracotomy, as well as the ligation of the left anterior descending (LAD) artery. After 45 min, each mouse received an intramyocardial application of 20 μL BD MatrigelTM Matrix (BD Biosciences, San Jose, CA, USA) and MACS Buffer in a ratio of 1/2. For stem cell treatment (SRI group) a total of 100,000 CD271<sup>+</sup> MSC were injected at 4 sites along the border of the blanched myocardium (25,000 cells per injection point). Subsequently, the node was reopened (here, ischemia-reperfusion) leaving the ligature in the heart looped around the LAD with their ends kept inserted in a soft flexible tube placed subcutaneously (Figure 1A).

After an observation period of 7 days, every mouse underwent a second thoracotomy and, subsequently, animals of URI, as well as the SRI group, underwent permanent LAD ligation at the same site and using the suture from the first ligation (here, reinfarction). The rubber tube has facilitated finding of the suture material which was easily exempted from the surrounding tissue. MIC operated mice underwent an identical second surgical procedure without LAD ligation.

**Figure 1.** Induction of ventricular arrhythmias (VA). Schematic drawing of the loose left anterior descending (LAD) ligature positioning (**A**). Mouse ECG changes in relation to the time of the firs LAD ligation during ischemia-reperfusion infarction (**B**). Mouse ECG changes in relation to the time of the permanent second LAD-ligation (reinfarction and URI group **C**). Mouse ECG strips showing different types of observed VA (**D**).

#### *2.6. Basic ECG Parameters*

To evaluate the antiarrhythmic mechanism of the CD271<sup>+</sup> MSC the basic ECG parameters of the mouse groups were measured at different time points. This was performed automatically with the use of ecgAUTO v3.3.0.28 software after manual definition of the start of P wave, the start of Q wave, R wave, end of S wave, and end of T wave. Then, the analysis was done for many subsequent beats at

a specific time point. We measured the RR-interval, QRS duration, and QT-interval in milliseconds. The corrected QT-intervals (QTc) were measured using the formula QTc = QT/(RR/100)1/2 , as previously described [18]. The basic ECG parameters were measured at the time points (1) 48 h after the first LAD ligation, (2) immediately before, and (3) 48 h after the second intervention.

#### *2.7. Definitions of Various Ventricular Arrhythmias*

The first temporary occlusion of the LAD resulted in a rapid onset of hyper acute T waves along with ST-segment elevation, as observed in the ECG record. Through reopening of the LAD 45 min post ligation, and subsequent myocardial reperfusion, ST-segment returned back to the isoelectric line and a development of Q waves could be observed. Figure 1B shows the recorded ECG before LAD occlusion, at the time of LAD occlusion, immediately after reopening of the LAD and reperfusion, as well as 12 h after the induction of the MI with clear Q waves and recognizable T waves.

Figure 1C shows the onset of the second infarction after permanently ligating the suture already placed at the time of the first ischemia one week earlier. The ECG immediately prior to performing reinfarction through the second LAD ligation shows deep S waves with deeply depressed ST-segment 7 days after the first operation (ischemia-reperfusion). The induction of the second MI with permanent ligation of the LAD led into an immediate ST-segment elevation in the ECG with subsequent development of deep Q waves. Two hours after reinfarction the ECG shows, a still elevated ST-segment reflecting the permanent LAD ligation. At 12 h post the second MI, Q waves developed with still elevated ST-segment with reappearance of T waves.

Both, the first and second infarctions resulted in the development of different types of VA, namely ventricular premature beats (VPB), ventricular bigeminies and trigeminies (BG/TG), ventricular salvos, as well as ventricular tachycardia (VT) (Figure 1D).

We have analyzed, named, and defined the observed VA in this mouse model in accordance with a standard that fits the previously established guidelines for labeling and describing each type of VA [19] as follows:

#### 2.7.1. Ventricular premature beats

We have defined VPB, as a ventricular electrical complex which varies in voltage (i.e., height) or duration (i.e., width) from the preceding non-VPB ventricular complex and occurs prematurely in relation to it. This means that VPB are not preceded by a P wave if they appear during the early cardiac cycle or they have shorter PR-intervals than those of non-VPB if they appear later in the cardiac cycle.

#### 2.7.2. Bigeminies and trigeminies

A minimum sequence of VPB, normal sinus beat, and VPB repeated at least three times has been defined as bigeminy. The number of repetitions has been defined as three in our model. Additionally, we have defined a sequence of VPB, two normal sinus beats, and a consecutive VPB with a repetition of three times as trigeminy.

#### 2.7.3. Salvos

The observation of two or three consecutive VPB has been defined as salvo [19].

#### 2.7.4. Ventricular tachycardia

VT is defined as a sequence of four or more consecutive VPB [19,20]. This varies between three or more consecutive VPB to ten or more consecutive VPB [21,22]. A detailed description of the morphological and durational subtypes and classifications of VT has not been performed in these mouse models.

#### *2.8. Organ Harvesting*

Every mouse underwent euthanization, 48 h after the second intervention by cervical dislocation. Each heart was removed, embedded in O.C.T.TM Compound (Sakura Finetek, Alphen aan den Rijn, Netherlands) and snap frozen in liquid nitrogen. For further histological examination of the infarction area the heart tissue was divided into four horizontal levels from the apex to the base and cut into 5 μm thick slices.

#### *2.9. Human Cells Detection*

For detection of human cells on mouse heart cryosections, monoclonal anti-human nuclei primary antibody (Chemicon, Temecula, CA, USA) was used in combination with M.O.M.TM Mouse Ig Blocking Reagent and M.O.M.TM Protein Concentrate following the instructions of Vector® M.O.M.TM Immunodetection Kit (Linaris, Dossenheim, Germany). Donkey anti-mouse Alexa Fluor® 594 served as conjugated secondary antibody followed by counterstaining with DAPI (both from Thermo Fisher Scientific).
