*3.4. Antiarrhythmic E*ff*ects of CD271*<sup>+</sup> *MSC Engraftment*

The intramyocardial implantation of human CD271<sup>+</sup> MSC did not lead to a significant difference in the number of VA early after the first MI (Figure 3A), but showed antiarrhythmic effects by significantly reducing the quantitatively measured VPB which occurred after a reinfarction during the time frame between LAD ligation until 12 h, thereafter (URI 1105.0 ± 1146.72 vs. SRI 178.16 ± 370.12, Figure 3B). Such significant antiarrhythmic behavior was also reflected in a reduction of the number of VT 45 min after the second LAD ligation until 12 h post reinfarction (URI 32.6 ± 52.51 vs. SRI 0 ± 0, Figure 3C). Moreover, there was no significant difference in BG/TG and salvos occurrence between the two groups.

In order to further assess the mechanism of antiarrhythmic effect, ECG monitoring was used. Accordingly, we measured the mean QRS duration and the corrected QT-interval (Figure 3D). Immediately prior to the second LAD ligation (seven days after the first intervention), the stem cell treated animals had a significantly shorter QRS duration (URI 20.77 ± 1.98 vs. SRI 14.66 ± 0.61 and MIC 19.83 ± 0.54 milliseconds, Figure 3E). The significant shorter QTc-interval in SRI (URI 64.25 ± 3 vs. SRI 53.54 ± 3.52 and MIC 67.25 ± 4.14 milliseconds, Figure 3F) could be secondary to the shorter QRS duration as the QRS duration is an integral part of QT duration. There was no statistically significant difference in the mean QRS durations and QTc between URI and MIC immediately prior to the second intervention, as well as between any of the groups 48 h after the first LAD ligation.

After nine days, the engrafted human cells were successfully detected on SRI mouse heart cryosections predominantly in the peri-infarct area by immunofluorescent staining (Figure 3G).

**Figure 3.** Comparison of developed VA. Until 12 h post LAD ligation (**A**,**B**). At the time period 45 min to 12 h post the second LAD ligation (**C**). ECG monitoring immediately prior to the second LAD ligation (**D**). QRS duration and QTc-interval 48 h post the first infarction and immediately prior to the second intervention (**E**,**F**). Mean <sup>±</sup> SD, \* *p* <sup>≤</sup> 0.05 as compared with MIC, # *p* <sup>≤</sup> 0.05 as compared with SRI (Mann–Whitney *U* Test). Representative images illustrate the engrafted human stem cells 9 days post transplantation performed for the SRI experimental group which the remaining human MSC were found predominantly in the peri-infarct area as with the Fast Green and Sirius Red staining method (the left picture) confirmed (**G**).

#### *3.5. Alterations of the Infarct Scar*

The first ligation of the LAD and opening of the node after 45 min (ischemia-reperfusion) consistently resulted in a transmural MI with its typical histologic changes including the thinning of the left ventricular free wall (Fast Green) and extensive collagen deposition (Sirius Red) nine days post intervention (Figure 4A). After stem cell injection we observed no significant reduction in infarct scar

formation (SRI, 14.78% ± 5.85%) in contrast to untreated infarction (URI 17.28% ± 5.10% URI), as well as to control group (MIC, 19.42% ± 3.66%, Figure 4C).

The first ligation of the LAD followed by a permanent second LAD ligation resulted in leukocyte infiltration within the infarction area as depicted in the images of Hematoxylin and Eosin stained slices from hearts 48 h post infarction (Figure 4B). This cellular intervention consequently led to a significant enlargement of the myocardial scar area in URI (38.54% ± 14.28%), as well as SRI (29.36% ± 8.63%) in comparison to MIC (20.1% ± 4.04%, Figure 4C).

**Figure 4.** Alterations in MI size. Representative images show the infarction area (enclosed within the yellow border) for URI, SRI, and MIC 9 days after the first LAD ligation (Fast Green and Sirius Red staining) (**A**), as well as 48 h after the second LAD ligation (Hematoxylin and Eosin staining) (**B**). Significant increase of the infarction size after the second LAD ligation, mean ± SD, \* *p* ≤ 0.009 as compared with the first LAD ligation, # *<sup>p</sup>* <sup>≤</sup> 0.041 in contrast to MIC (Mann–Whitney *<sup>U</sup>* test) (**C**).

#### **4. Discussion**

The purpose of this study was to develop a mouse model to reproduce VA that typically occurs in patients several days after an initial MI. To study engraftment and development of human cells in a murine model it was necessary to use an immunodeficient mouse strain. During development of an in vivo model of immunodeficient mice for the study of human stem cell transplantation, we found that VA are mostly seen only on the first day after induced MI. Our results supported the hypothesis, that a therapeutic stem cell treatment could have no such antiarrhythmic effect early after their transplantation, as previously shown for other cells [4]. For testing potential antiarrhythmic effects of intramyocardially transplanted cells, Roell et al. induced VA in vivo through transjugular venous catheter mediated burst stimulation of the myocardium after inducing MI [4]. However, this conventional approach could not be utilized in immunodeficient mice, because of the sensitivity of these animals as we observed. For this reason, there was an urgent need for the development of a new mouse model that used immunodeficient mice for in vivo study of antiarrhythmic effects exerted by various human-derived stem cells. On the basis of this, we aimed to develop a new mouse model with re-induced VA by performing a permanent second LAD ligation seven days after re-perfused of the first MI. Indeed, performing the second ligation, one week after the first infarction in Rag2−/−γc−/<sup>−</sup> mice, reproduced cardiac arrhythmias. Other previously described small animal models in this field

have utilized immune competent mice or rats subjected to higher stress after performing recurrent transient ischemia following an operatively instrumented mice with the capability of subsequent recurrent transient closure of the LAD [25,26].

The previously described development of VPB in the control group after anesthesia and surgery without performing LAD ligation [27] has also been seen in our mouse model in the time period between 15 and 45 min after re-thoracotomy (without the second LAD ligation) in MIC group. These developed VPB were statistically not significant as compared with the SRI and URI groups.

Our stem cell therapy resulted in shorter QRS duration, shorter QTc-intervals, and decreased occurrence of VT in the early period after the second MI. The observed shortening of QRS indicates the ability of MSC to improve cardiac electric conduction, which is in line with findings from Boink et al. who showed shortened QRS duration in a canine model of MI after MSC transplantation. [28]. However, antiarrhythmic behaviors of human MSC have also been shown in a clinical trial after their intravenous injection following a re-perfused MI [29]. The positive effect may be due to the possible coupling of the MSC with cardiomyocytes through connexin 43 (Cx43) bridges, as it has been found in an in vitro study by our research group [17]. This may be especially relevant as Cx43 plays a major role in post ischemia and post infarction cardiac arrhythmias in the time period between 45 min and 12 h after MI in different animal models [24].

As there is a close relationship between the infarct size and the incidence of VA [30], the significant reduction of the developed VA in the SRI group could not be attributed to the infarct size because there was no significant difference in the infarction area between the SRI and the URI group, early after the second infarction. The Hematoxylin and Eosin staining of the heart sections 48 h after the second intervention (second LAD ligation in SRI and URI groups, re-thoracotomy in MIC group) shows no infarct area expansion and new inflammatory cell infiltration in the MIC group. This indicates that the stained inflammatory cells in SRI and URI groups cannot be remaining cells of the initial MI and their infiltration is the early consequence of the second infarction. Additionally, the lack of intensive leukocyte infiltration in the MIC group after re-thoracotomy (no second LAD ligation) seen with Hematoxylin and Eosin staining of the heart sections nine days after the first operation (LAD ligation) in our mouse strain supports the finding of Yang et al. [31]. The described initial neutrophil infiltration in the infarct border one to two days after LAD ligation in immunocompetent C57BL/6J mice used by Yang and associates has been clearly seen in our SRI and URI groups 48 h after the second infarction. However, the described later lymphocytic infiltration, seven to 14 days after myocardial injury could not be noticeable in our complete alymphoid mouse strain, Rag2−/−γc−/<sup>−</sup> nine days after induced MI.

In this study, we introduce our novel mouse model for testing arrhythmias in the Rag2−/−γc−/<sup>−</sup> mouse. Importantly, CD271<sup>+</sup> MSC transplanted into the infarcted area were retained and did not bear any proarrhythmic properties but rather antiarrhythmic effects. We have successfully utilized the model to test potential antiarrhythmic effects of human BM derived CD271<sup>+</sup> stem cells in vivo and showed their safety and efficacy. Therefore, we conclude, that human BM derived MSC, i.e., CD271<sup>+</sup> are suitable cell types to prevent arrhythmias after MI. This finding supports the previous observations on the safety of intramyocardial transplantation of BM derived stem cells and can be further evaluated for future clinical implication of cell transplantation in the field of electrophysiology [32]. Overall, our novel mouse model offers a new option for testing potential antiarrhythmic effects of cell transplantation therapies with high clinical relevance.

**Author Contributions:** H.S., R.G., G.S., and R.D. designed and supervised the research study; H.S., R.G., B.A., A.S., C.A.L., S.S., and P.V. conducted the experiments and acquired the data; H.S. and R.G. contributed to the analysis and interpretation of the results; H.S., R.G., G.S., and R.D. were involved in writing and correction of the manuscript, which all authors reviewed and approved.

**Funding:** This work was supported by the Federal Ministry of Education and Research Germany (FKZ 0312138A and FKZ 316159), the State Mecklenburg-Western Pomerania with EU Structural Funds (ESF/IVWM-B34-0030/10 and ESF/IVBM-B35-0010/12) and the DFG (DA1296/6-1) and the German Heart Foundation (F/01/12). In addition, R.D. is supported by the EU Structural Fund (ESF/14-BM-A55-0024/18), the FORUN Program of Rostock University

Medical Centre (889001 and 889003), the Josef and Käthe Klinz Foundation (T319/29737/2017), the DAMP Foundation, and the BMBF (VIP+ 00240).

**Conflicts of Interest:** The authors declare that they have no conflict of interest.
