Smooth muscle protein 22α-Cre recombination in resting cardiac fibroblasts and hematopoietic precursors

The Cre-loxP system has been widely used for cell- or organ-specific gene manipulation, but it is important to precisely understand what kind of cells the recombination takes place in. Smooth muscle 22α (SM22α)-Cre mice have been utilized to alter genes in vascular smooth muscle cells (VSMCs), activated fibroblasts or cardiomyocytes (CMs). Moreover, previous reports indicated that SM22α-Cre is expressed in adipocytes, platelets or myeloid cells. However, there have been no report of whether SM22α-Cre recombination takes place in nonCMs in hearts. Thus, we used the double-fluorescent Cre reporter mouse in which GFP is expressed when recombination occurs. Immunofluorescence analysis demonstrated that recombination occurred in resting cardiac fibroblasts (CFs) or macrophages, as well as VSMCs and CMs. Flow cytometry showed that some CFs, resident macrophages, neutrophils, T cells, and B cells were positive for GFP. These results prompted us to analyze bone marrow cells, and we observed GFP-positive hematopoietic precursor cells (HPCs). Taken together, these results indicated that SM22α-Cre-mediated recombination occurs in resting CFs and hematopoietic cell lineages, including HPCs, which is a cautionary point when using SM22α-Cre mice.

Furthermore, we analyzed whether differentiation facilitates SM22α-Cre recombination. Flow cytometric analysis showed that differentiation had no effect on recombination under TGF-β stimulation (Fig. 4f,g).

Discussion
SM22α-Cre recombination has not been fully assessed in murine hearts, although SM22α-Cre has been widely used to alter VSMCs, activated fibroblasts and CMs in cardiovascular research [2][3][4] . This study offers further insights into the target of SM22α-Cre and how to lead recombination in some cells. First, SM22α-Cre recombination occurs in 65% of resting CFs. Second, SM22α-Cre recombination occurs in cardiac T and B cells, in addition to myeloid cells. Third, recombination also takes place in putative HSCs. Finally, CFs have the capacity to express SM22α-Cre. However, TGF-β, which is well known to induce Tagln 15-17 , cannot induce SM22α-Cre recombination in BMDMs.
One important finding in the present study is that there are some GFP-positive populations among resting CFs from embryos. SM22α is a well-known marker of activated fibroblasts 18 . In vivo, SM22α-Cre cannot be used to target only activated CFs, but could be used to manage quiescent CFs. As stated in previous papers 5,6,19 , we recommend other Cre recombination systems, such as Postn-Cre when focusing on activated CFs.
Next, SM22α-Cre is expressed in some immune cells, including phenotypically defined putative HSCs. It was unlike the results of a previous paper 5 , but Zovein et al. observed SM22α-Cre recombination at E11.5 in the aortic-gonado-mesonephros (AGM) region, the origin of HSCs 20 . This is likely to support the results on the recombination in HSCs, but further in vivo repopulating experiments would confirm this conclusion.
Considering the results, we recommend that when examining inflammation models such as myocardial infarction and heart failure with SM22α-Cre, bone marrow transplantation should be performed first.
Finally, in vitro, SM22α-Cre is a good tool to modify genes of murine fibroblasts. However, in BMDMs and other immune cells, we cannot induce SM22α-Cre recombination during adulthood because the RNA level of Tagln is very low, similar to the open RNA-seq data 21 . This may be due to the expression of Transgelin-2, which is one of the homologues of SM22α 22 , in these cells. Transgelin-2, the only transgelin isoform expressed in immune cells, acts as a molecular staple to stabilize the actin cytoskeleton; this function may replace the function of SM22α 23 .
The reason for recombination in immune cells is unclear. In addition to the SM22α-Cre recombination in the AGM region as mentioned above, recent single-cell data about preHSCs may reveal this answer 24 . The data indicated that recombination in immune cells may occur via the endothelial to hematopoietic transition (Supplementary Fig. 3a-c). This effect may be because the downregulation of Erg1, a key transcription factor at the endothelial to hematopoietic transition, upregulates SM22α 25 .   CFs were isolated as described previously 26 . Media and buffers were prepared according to a previous paper 26 .
The descending aortas and inferior venae cavae were cut. The hearts were perfused with EDTA buffer from the right ventricle. The ascending aortas were clamped. The clamped hearts were removed, transferred to a dish containing EDTA buffer, and perfused with EDTA buffer from the left ventricle (LV). The hearts were transferred to a dish of perfusion buffer, and perfused with perfusion buffer from the LV. The hearts were transferred to a dish of collagenase buffer and perfused with collagenase buffer from the LV. The ventricles were transferred to the other dish of collagenase buffer, gently teased apart into pieces, and triturated. Stop solution was added to the cell-tissue cell suspension. The supernatants obtained via gravity settling three times for 10 min in perfusion buffer were collected as nonCMs. The nonCMs were centrifuged at 300×g for 5 min, resuspended in DMEM containing 10% fetal bovine serum (FBS) and penicillin-streptomycin (Wako, #168-23191), plated on cell culture dishes and cultured for 6-7 days. Almost all cells were fibroblasts after the culture. Bone marrow was extracted from the femurs and tibias of euthanized mice and differentiated in bone marrow macrophage differentiation media (RPMI 1640 containing 10% fetal bovine serum (FBS), penicillin-streptomycin, 20 μg/mL recombinant mouse M-CSF (Biolegend, #576404), and 0.1 mM/L 2-mercaptoethanol (Wako, #133-14571)). Seven days after being harvested, BMDMs were stimulated with recombinant mouse TGF-β1 (Biolegend, # 763102) for 1 or 7 days, for RNA or for Flow cytometric analysis, respectively. For RNA analysis, cells were cultured under serum-free media (RPMI +1% Bovine serum albumin) (Merck, #A9418) during stimulation. To investigate whether Cre-recombination occurs during the differentiation process from HSCs to BMDMs, bone marrow cells were harvested and cultured in bone marrow macrophage differentiation media containing with TGF-β.
Immunostaining. Hearts in 6-week-old mice were perfused with cold phosphate-buffered saline (PBS) and 4% paraformaldehyde, removed, and fixed with 4% paraformaldehyde (PFA) for 3 h. The hearts of Embryos at 17.5 dpc were removed, washed with cold PBS and fixed with 4% PFA for 3 h. The hearts were incubated in 10%, 20% and 30% sucrose diluted in PBS. The samples were then embedded in OCT compound (Sakura Finetek Japan), frozen and stored at − 80°. Cryosections (8 µm thick) were obtained using a Leica cryostat.
Immunofluorescence images were acquired on an Axio Observer (Carl Zeiss) (5 × objective) or SP8 Falcon (Leica) (63 × objective) and analyzed with Zen software (Carl Zeiss) or LAS X (Leica), respectively. Flow cytometric analysis. Single cardiac cell suspensions were generated as described previously 27 .
Peripheral blood samples were collected from the inferior vena cava of anesthetized mice using a heparincontained syringe. Red blood cell lysis was performed with ACK lysis buffer. The samples were washed with FACS buffer and resuspended. Peripheral blood samples were blocked with TruStain FcX Plus for 5 min at 4°.
Bone marrow cells were obtained by flushing femurs and tibias with RPMI supplemented with 25 mM HEPES and 10% FBS. The suspensions were passed through a 40 μm cell strainer. After the red blood cells were lysed, the samples were washed with FACS buffer and resuspended.
BMDMs were collected after harvesting as described before and were blocked with TruStain FcX Plus for 5 min at 4°.
Cells were stained with monoclonal antibodies at 4 °C for 20 min in the dark. The samples were washed twice, and the final resuspension was made in 500 μL of FACS buffer. 7-AAD was used to exclude dead cells. Flow cytometric analysis was performed on BD FACS ARIAII platforms. Complete lists of antibodies and flow cytometry gating strategies are provided in Supplementary Tables 1 and 2, respectively. RNA extraction and qRT-PCR. Total RNA was isolated and purified using TRIzol reagent (Ther-moFisher), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOY-OBO, #FSQ-301) in accordance with the manufacturer's instructions. For quantitative real-time PCR (qRT-