Expression of FFAR2/3 in the human heart
In the hca dataset consisting of 486,134 cardiac tissue cells, there were 1,030 (0.21%) FFAR2+ cells and 525 (0.11%) FFAR3+ cells. Most of these FFAR2/3+ cells were myeloid cells and pericytes, distributed across the whole heart (Fig. 1a-e). In particular, FFAR3+ endothelial was not found in the left ventricle, while FFAR3+ pericytes were not found in the right atrium. The FFAR2+ cardiomyocytes (n = 70, 0.014%) were located in all of the heart with equal distribution in atrium (12 in 23,248 cells, 0.051%) and ventricle 58 in 64510 cells, 0.046%). Very low number of FFAR3+ cardiomyocytes (n = 1, 0.000206%) were found in the interventricular septum.
Figure 1f presents the subcluster distribution of FFAR2/3+ in various cell types. The FFAR2/3+ myeloid cells were mainly CD16+ Mo (CD16+ monocytes), and FFAR2/3+ ventricular cardiomyocytes were mainly vCM1, which marker genes were PCDH7, FHL2, and MYH7.24 The FFAR2/3+ also distributed on SMC1_basic (basic Smooth muscle cells), PC1_vent (ventricle-enriched pericytes), PC3_str (stromal pericytes), and CD14+ Mo (CD14+ monocytes). The significant proportion of FFAR2/3+ PC3_str, which may be a transitional state between pericytes and endothelial cells (ECs),24 indicates the potential effect of SCFA in endothelial differentiation. The SCFA might also play roles in Smooth muscle cell (SMC) development, due to the expression of FFAR2/3 on SMC1_basic which represents an immature subtype of SMC. FFAR3-positive fibroblasts were the ventricular-specific FB1 and the FB3 that enriched with high levels of cytokine receptors24. Notably, there were no FFAR2+ mesothelial cells, FFR3+ adipocytes, and FFAR3+ atrial cardiomyocytes, and FFAR2/3+ neuronal cells observed.
We also presented the expression level of FFAR2 and FFAR3 in each subcluster of cell types via the dot plot (Fig. 1g). The DOCK4+ MΦ1 (DOCK4+ macrophage sets 1), FB3, FB4, LYVE1+ MΦ2 (LYVE1+ macrophages sets 2), and NΦ (neutrophils) had a higher level of FFAR2 than other cell subclusters. On the other hand, the Mo_pi (pro-inflammatory monocytes), CD4+ T_cytox (CD4+ cytotoxic T cells), FB3, and Mast cells had higher levels of FFAR3 expression.
We explored specifically upregulated genes (marker genes) in FFAR2/3+ cells. These marker genes were mainly from the myeloid cells (Supplemental Fig. 1a-d). Top marker genes of FFAR2+ cells, FFAR3+ cells, non-FFAR2/3 cells, and cells positive with both FFAR2 and FFAR3 for myeloid, lymphoid, ventricular cardiomyocytes and atrial cardiomyocytes were illustrated in Supplemental Fig. 1 3-h. Multi types of heat shock proteins were observed in the FFAR2/3+ myeloid and cardiomyocytes (Supplemental Fig. 1f-g).
There were five gene expression modules found in both FFAR2+ cells (ffar2_M1 to ffar2_M5) and FFAR3+ cells (ffar3_M1 to ffar3_M5) (Supplemental Figs. 2 & 3). When screened at the cell subcluster level, the FFAR2+ CD16+ Mo was mainly enriched with ffar2_M1 module that was annotated with the biological process (BP) of SRP-dependent cotranslational protein targeting to membrane, the cellular component (CC) of cytosolic large ribosomal subunit, and the molecular function (MF) of ubiquitin ligase inhibitor activity (Supplemental Fig. 2c-g). The FFAR3+ CD16+ Mo was enriched with the ffar3_M5 module that was annotated with neutrophil degranulation (BP), MHC class II protein complex (CC), and RAGE receptor binding (MF) (Supplemental Fig. 3c-g).
Both the FFAR2+ SMC1_basic and FFAR2+ PC1_vent cells were enriched with the ffar2_M5 module, which was annotated with oxygen transport (BP), collagen-containing extracellular matrix (CC), and platelet-derived growth factor binding (MF) (Supplemental Fig. 2c-g). The FFAR3+ SMC1_basic and PC1_vent cells were enriched with the ffar3_M3 module, which GO annotation results was the cellular response to low-density lipoprotein particle stimulus (BP), collagen-containing extracellular matrix (CC), and platelet-derived growth factor binding (MF) (Supplemental Fig. 3c-g). The FFAR3+ PC3_str was detected to express the ffar3_M1, which GO terms were blood vessel endothelial cell migration (BP), G protein-coupled receptor dimeric complex (CC), and calcitonin family receptor activity (MF) (Supplemental Fig. 3c-g).
The FFAR2+ ECs gene module was the ffar2_M4 that enriched mainly in the EC10_CM-like subcluster (ECs with cardiomyocyte features) and annotated as caveola assembly (BP), G protein-coupled receptor dimeric complex (CC), and calcitonin family receptor activity (MF) (Supplemental Fig. 2c-g). The ffar3_M4 was recognized as the gene module of FFAR3+ ECs, which were mainly enriched in the subcluster of EC8_ln (lymphatic ECs) (Supplemental Fig. 3c). GO terms of the ffar3_M4 were the same as the ffar2_M1 module that was expressed in the FFAR2+ CD16+ Mo (Supplemental Fig. 3d-g).
Expression of FFAR2/3 in the human heart with MI
In the human heart MI sma dataset consisting of 191,795 cells (Fig. 2a), there were 85 (0.04%) FFAR2+ cells and no FFAR3+ cells were found (Fig. 2b). Among the FFAR2+ cells, the proportion of myeloid was 38.82% (n = 33), followed by fibroblasts (n = 18, 21.18%) and cardiomyocytes (n = 13, 15.29%) (Fig. 2c). All the sma dataset samples come from cardiac ventricles.25 So, the proportion of FFAR2+ cells in ventricular cardiomyocytes was 0.020% (13 in 64,510 cells). Most of the FFAR2+ cells came from control samples (CTRL) and the remote zone (RZ) but were not detected in the fibrotic zone (FZ) (Fig. 2d). The ischemic zone FFAR2+ cells were myeloid, cycling cells, fibroblasts, and endothelial cells, while the border zone FFAR2+ cells were myeloid, fibroblasts, cardiomyocytes, and lymphoid (Fig. 2d). The myeloid, occupied the most proportion of the FFAR2+ cells, followed by the fibroblast, and cardiomyocyte (Fig. 2d). Similar to the gene expression pattern in normal heart in the hca dataset, the FFAR2+ cells DEGs were mainly on myeloid cells (Supplemental Fig. 4a-b).
CD55 and CYBB were the top enriched genes in FFAR2+ myeloid, SORCS1 and RTN3 were enriched in FFAR2+ fibroblast, while FFAR2+ cardiomyocytes’ top enriched genes were LOC100506869 and PNPLA8 (Supplemental Fig. 4c, e, & g). Differentially expressed genes were also observed among the border zone, ischemic zone, remote zone (the unaffected left ventricular myocardium), and control cardiac tissue (control samples from non-transplanted donor hearts) in FFAR2+ cells (Supplemental Fig. 4d, f, & h). We also checked the expression of immune cell type marker genes in different cardiac regions and found the enrichment of NCAM1 (neural cell adhesion molecule 1, also known as CD56) on both lymphoid and myeloid cells in the border zone (Fig. 2e).
There were seven gene expression modules (ffar2_M1 to ffar2_M7) found in FFAR2+ cells (Supplemental Fig. 5a-c). The ffar2_M2 was enriched in cardiomyocytes, while the ffar2_M5 and ffar2_M6 were fibroblast gene modules that related to cell respiration and mitochondria activity (Supplemental Fig. 5a, d-f). GO terms of the ffar2_M2 were negative regulation of cardiac muscle cell proliferation (BP), PRC1 complex (CC), and oxidoreductase activity (MF) (Supplemental Fig. 5d-f). The biological process of FFAR2+ cycling cells was chromatin maintenance, while FFAR2+ lymphocytes presented with the biological process of sodium ion transmembrane transporter activity regulation, positive regulation of ERAD pathway, and the microtubule anchoring at the microtubule organizing center (Supplemental Fig. 5a, d-f).
Expression of FFAR2/3 across human organs
We also explored FFAR2/3+ cells in whole human body via the tsc dataset. There were 12,346 (2.56%) FFAR2 positive cells and 1,799 (0.37%) FFAR3 positive cells in the tsc human dataset consisting of 483,152 cells, and most of the FFAR2/3+ cells were immune cells distributed in spleen and blood (Fig. 3a-c, Supplemental Table 1). Most of the FFAR2+ cells were detected from the spleen, followed by blood, liver, bone marrow, and trachea. The expression of FFAR2 was mainly enriched in the spleen and trachea (Fig. 3d), and the FFAR3 was mainly enriched in the liver, which had the highest expression level (Fig. 3d). When presented with cell types, both FFAR2 and FFAR3 were mainly enriched in immune cells (Fig. 3e). In the heart, there were only 6 FFAR2+ cells and 2 FFAR3+ cells detected.
Expression of FFAR2/3 in the mice heart
We combined five public single-cell RNA sequence datasets and built a dataset of normal mice heart tissue that consisted of 79,880 cells, among which 54.14% (43,249 cells) were newborn mice and the other 45.86% (36,631 cells) were from adult mice (Supplemental Fig. 6, Fig. 4). No FFAR3+ cells were observed. There were 339 (0.42%) FFAR2+ cells detected, among which 27.14% (92 cells) were in the newborn group, which are mainly muscle cells (pericyte and smooth muscle cells), fibroblasts, and lymphoid, and 72.86% (247 cells) were in the adult group (Fig. 4c-f). Myeloid cells (n = 146) occupied 43.07% of the FFAR2+ cells and mainly came from adult samples (Fig. 4d). There was only 1 FFAR2+ Cardiomyocyte detected in the newborn samples (Fig. 4d). When divided by postnatal days and months, most of the FFAR2+ cells came from age 3 months individuals and were mainly myeloid cells (Fig. 4g).
The differentially expressed genes (DEGs) compared to non-FFAR2 cells distributed in myeloid, lymphoid, and fibroblast (Supplemental Fig. 7a-b). There were four subclusters detected in the myeloid (Mye0-3) (Supplemental Fig. 7d). The B cell marker gene, Ifitm1, was the top enriched DEG in the FFAR2+ myeloid and also the marker gene of the Mye1 subcluster (Supplemental Fig. 7c-e). The top enriched DEGs in FFAR2+ lymphoid cells were Tmem176b, Tmem176a, and Scart1 (Supplemental Fig. 7f), and in FFAR2+ fibroblasts were Camp, Ngp, and BC100530 (Supplemental Fig. 7j). Distinct subclusters that represent the adult and newborn states were observed both for FFAR2+ lymphoid and fibroblasts (Supplemental Fig. 7f-m). The Scart1 was both FFAR2+ lymphoid enriched DEG and also the marker gene of newborn state lymphoid subcluster, lym0 (Supplemental Fig. 7f-i). Similarly, the Ngp was both FFAR2+ fibroblast enriched DEG and the marker gene of newborn state fibroblast subcluster (Supplemental Fig. 7j-m). We also detected co-expression of the lymphoid cell markers, Cd3d and Il7r, The T cell lineage differentiation required transcription factor (TF), Gata3, and other genes specific in T cell differentiation (Icos, Cd25 (Il2ra), Cxcr6, Cd44, Cd45ra (Ptprc), and Cst3) (Fig. 4h).
There were 11 gene co-expression modules observed in the mice FFAR2+ cardiac tissue cells (ffar2_M1 to ffar2_M11) (Supplemental Fig. 8a-d). The module of myeloid ffar2_M2 was annoated as the SRP-dependent cotranslational protein targeting to membrane (BP), cytosolic large ribosomal subunit (CC), and ubiquitin ligase inhibitor activity (MF) (Supplemental Fig. 8e-g). The mice FFAR2+ fibroblast, the second most FFAR2+ cells, was related to endopeptidase activity involved in the apoptotic signaling pathway, elastic fiber assembly, TORC1 signaling, and the dermatan sulfate proteoglycan metabolic process (Supplemental Fig. 8e). The ffar2_M3 was a gene module of pericyte and was annotated with extracellular structure organization (BP), collagen-containing extracellular matrix (CC), and platelet-derived growth factor binding (MF) (Supplemental Fig. 8b-g).