Screening of supernatant proteins using mass spectrometry

A total of 115 proteins were identified in the ISC group of CF cells in comparison to control with an overall log2 FC ranging from -28 to + 30 (Fig. 1A) (Supplementary Table 1). Among these, the FC of 30 proteins (26.09%) ranged from + 20 to + 30 (Fig. 1A and B), 66 proteins (57.39%) were in the range of –3 to + 5 (Fig. 1A and C), and 19 proteins (16.52%) were in the range of –20 to –27 (Fig. 1A and D). Interestingly, all 30 proteins that there upregulated with FC >  + 20 were absent expression in the control group (Fig. 1B). Similarly, 19 proteins that were downregulated with FC < –20 were completely absent in the ischemic group (Fig. 1D). Additionally, 138 proteins were identified in the ISC/R group in comparison to control with an overall log2 FC ranging from –28 to + 28 (Fig. 1E) (Supplementary Table 1). Among these, 53 proteins (38.41%) ranged from + 19 to + 28 (Fig. 1E and F), 71 proteins (51.45%) were in the range of –3 to + 5 (Fig. 1E and G), and 14 proteins (10.14%) ranged from –20 to –28 (Fig. 1E and H). Interestingly, the 53 proteins that were upregulated with FC >  + 19 were completely absent in the control group (Fig. 1E). Also, the 14 proteins that were downregulated with FC < –20 were completely absent in the ISC/R group (Fig. 1E). Moreover, 139 proteins were identified in the ISC/R group in comparison to the ISC group with an overall log2 FC ranging from –30 to + 28 (Fig. 1I) (Supplementary Table 1). Among these, 43 proteins (30.94%) ranged from + 19 to + 28 (Fig. 1I and J), 81 proteins (58.27%) ranged from –3 to + 5 (Fig. 1I and K), and 15 (10.79%) ranged from –20 to –30 (Fig. 1I and L). Interestingly, the 43 proteins that were upregulated with FC >  + 19 were completely absent in the ISC group (Fig. 1I). The major protein mediators identified based on their cardiac function is shown in the Table 2. Among the upregulated proteins CRSP2, HSP27, and IL-8 and among the downregulated proteins Cofilin-1, and HSP90 in the ISC group were considered for further studies. In addition, the cardiac biomarker Troponin-I and antioxidant regulator Nrf2 were included for detailed evaluations owing to their established pathological significance.

Fig. 1

The level of secreted proteins of ischemia challenged CF using mass spectrometry. A Pie diagram showing the proportion of proteins based on the FC expression in the ISC vs Control groups, B Scatter diagram of newly upregulated proteins in ISC group which were absent in the control, C Scatter diagram showing the FC distribution of proteins expressed in both ISC and control groups. D Scatter diagram of newly downregulated proteins in ISC group which were present exclusively in the control. E Pie diagram showing the proportion of proteins based on the FC expression in the ISC/R vs Control groups, F Scatter diagram of newly upregulated proteins in ISC/R group which were absent in the control, G Scatter diagram showing the FC distribution of proteins expressed in both ISC/R and control groups. H Scatter diagram of newly downregulated proteins in ISC/R group which were present exclusively in the control. I Pie diagram showing the proportion of proteins based on the FC expression in the ISC vs ISC/R groups, J Scatter diagram of newly upregulated proteins in ISC group which were absent in the ISC/R, K Scatter diagram showing the FC distribution of proteins expressed in both ISC and ISC/R groups. L Scatter diagram of newly downregulated proteins in ISC group which were present exclusively in the ISC/R. M The histomorphology evaluations using von Kossa staining and Movat’s Pentachrome staining showing (1) calcification, (2) intact ECM, (3) ECM disorganization, (4) collagen deposition, (5) muscle, (6) elastic fibers, and (7) mucin

Table 2 Details of the major secreted protein mediators identified in the ISC group compared to the control (C) and ISC/R groupsLV tissues following MI and CABG sustains ischemic pathology

The von Kossa staining of LV tissues displayed increased calcium deposits in the LV-MI and moderate calcification in the LV-CABG tissues compared to their respective controls (Fig. 1M). The pentachrome staining revealed extreme ECM disorganization, fibrosis, limited muscle fibers, increased mucin deposition, and inflammation in the LV-MI tissues suggesting the severe ischemic injury. Interestingly, the LV-CABG tissues displayed ECM disorganization and collagen deposition along with muscle fibers suggesting inflammatory episodes; however, without mucin deposits (Fig. 1M). On the other hand, the control LV tissues displayed intact ECM without the histological features of inflammation and fibrosis (Fig. 1M).

Response of the secreted mediators in the tissues and cells from ischemic myocardiumIschemia decreased the expression of Cofilin 1 in the ischemic LV tissues and CF

The level of Cofilin-1 was decreased in both LV-CABG (P = 0.0943) and LV-MI (P = 0.2913) groups compared to the control; however, was statistically not significant. Also, the variation between LV-CABG and LV-MI groups were statistically not significant (Fig. 2A and B). The mRNA transcripts of Cofilin-1 were significantly decreased in the ISC/R CF (P < 0.0001) compared to the control whereas the decrease in the ISC group (P = 0.8768) was statistically not significant. The extent of decrease was significantly more in the ISC/R group compared to the ISC group (P = 0.0002) (Fig. 2C). Similarly, the level of Cofilin-1 was significantly decreased in the ISC group (P = 0.0552) of cultured CF and decreased non-significantly in the ISC/R group (P = 0.1127) compared to the control. Cofilin-1 was significantly decreased in the ISC group than ISC/R group (P = 0.0040) as evident from immunostaining (Fig. 2D and E). Western Blot analysis displayed upregulation of Cofilin-1 in ISC (P = 0.3360) CF with concomitant downregulation in ISC/R (P = 0.2935) CF compared to the control; however, were statistically not significant. Notably, ISC/R displayed significant downregulation of Cofilin-1 compared to the ISC (P = 0.0308) CF (Fig. 2F and G).

Fig. 2

Representative images for the immunofluorescence analysis for the expression of Cofilin 1 in the LV (A) (N = 3) and the quantification of protein expression of Cofilin 1 in LV tissues calculated from MFI (B). C Quantification of Cofilin 1 gene expression in CF (N = 8) following the treatment with ISC and ISC/R using qRT-PCR relative to the housekeeping gene GAPDH normalized to the control and presented as Log2 FC. Representative images for the immunofluorescence and quantification for the expression of Cofilin 1 in the CF (N = 3) following the treatment with ISC and ISC/R (D and E). Representative image of Western blot and quantification showing the expression status of Cofilin 1 in the ISC and ISC/R groups compared to the control in CF (N = 4) (F and G). Single cell genomics of Cofilin 1 + CF showing the (H) (split and combined views of Control, ISC and ISC/R groups of t-SNE plot) showing the distribution of cells within all clusters based on the global expression of 4601 genes. Violin plot (I) (FC 5 and 5.5) indicating the alteration of genes in the 3 clusters. J Scatter plot FC expression indicating the alteration of Cluster 1 genes reflecting the contrasting phenotype compared to Clusters 2 and 3. K Scatter plot FC expression indicating the alteration of Cluster 2 genes reflecting the contrasting phenotype compared to Clusters 1 and 3. L Scatter plot FC expression indicating the alteration of Cluster 3 genes reflecting the contrasting phenotype compared to Clusters 1 and 2. (**** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 and unlabeled groups represent NS)

Cofilin-1 + Cluster 1 CF represent a pro-survival phenotype

Single cell genomics revealed that 622 CF cells were positive for Cofilin-1 (FC > 5), which constituted 2.07% of the parent population where 120 cells were in the control group (19.13% of Cofilin-1 + population), 352 cells in the ISC group (56.59% of Cofilin-1 + population), and 150 cells in the ISC/R group (23.95% of Cofilin-1 + population). Overall, the Cofilin-1 + cells favored the ISC group and were mapped in 3 cluster based on the expression level of 4601 genes (Fig. 2H and I). Cluster-1 displayed 352 cells where 100% cells were displayed in the ISC CF, and the violin plot revealed the distribution of 4601 genes in cluster 1 CF based on expression status in ISC group (Fig. 2H and I) (Supplementary File 1). ADAMTS5 (ADAM metallopeptidase with thrombospondin type 1 motif 5) (FC + 5.07, P < 0.0001), IGFBP5 (insulin like growth factor binding protein 5) (FC + 4.66, P < 0.0001) and IGF1 (insulin like growth factor 1) (FC + 4.51, P < 0.0001) were the major genes highly altered in the ISC group of Cluster 1 (Fig. 2J) (Supplementary File 1). On the other hand, CCNB1 (Cyclin B1) (FC –6.08, P < 0.0001), ECHDC1 (ethyl-malonyl-CoA decarboxylase 1) (FC –6.24, P < 0.0001), and CCNB2 (Cyclin B2) (FC –6.37, P < 0.0001) were the major genes highly altered in the cluster 1 compared to the other clusters (Fig. 2J) (Supplementary File 1). Interestingly, most of the upregulated genes in the cluster 1 cells were significantly downregulated in the Clusters 2 and 3 (and vice versa) reflecting the contrasting phenotypes (Fig. 2J). The pathway analysis of Cofilin-1 + Cluster 1 cells displayed multiple pathways associated with (1) calcium homeostasis with a concomitant operation of cell proliferation, metabolism, and exocytosis (Supplementary Fig. 1A), (2) ischemia tolerance via HIF signaling accelerating the matrix function, oxygen delivery, angiogenesis, and anabolic pathways with a concomitant reduction in oxygen consuming pathways (Supplementary Fig. 1B), and (3) p53 mediated cell survival (Supplementary Fig. 1C). Overall, Cofilin-1 + Cluster 1 cells represents a pro-survival/regenerative phenotype.

Cofilin-1 + Cluster 2 CF signify a pro-inflammatory phenotype

Cluster 2 CF displayed 150 cells where 99.33% cells were mapped in the ISC/R group (Fig. 2H and I) (Supplementary File 1). HPS5 (Hermansky-Pudlak syndrome-5) (FC + 5.82, P < 0.0001), MGP (matrix gla protein) (FC + 3.89, P < 0.0001), and MX2 (MX dynamin like GTPase 2) (FC + 3.81, P < 0.0001) were significantly upregulated in the ISC/R group of Cofilin-1 + Cluster 2 CF compared to the Clusters 1 and 3 (Fig. 2K) (Supplementary File 1). The major downregulated genes were CCNB2 (FC –4.66, P < 0.0001), ECHDC1 (FC –6.24, P < 0.0001), and IGFBP5 (insulin like growth factor binding protein 5) (FC –6.38, P < 0.0001) in the cluster 2 compared to the other two clusters (Fig. 2K) (Supplementary File 1). Interestingly, most of the upregulated genes in the cluster 2 cells were significantly downregulated and vice versa in the Clusters 1 and 3 suggesting the contrasting phenotypes (Fig. 2K). The pathway analysis of Cofilin-1 + Cluster 2 cells displayed the inflammatory signaling associated with (1) chemokine signaling such as chemotaxis, cell migration, and ROS production (Supplementary Fig. 1D), (2) TNF signaling accelerating the apoptosis and leukocyte activation (Supplementary Fig. 1E), and (3) NOD-like signaling pathways aggravating the pro-inflammatory responses (Supplementary Fig. 1F). Overall, Cofilin-1 + Cluster 2 cells represents a pro-inflammatory phenotype.

Cofilin-1 + Cluster 3 CF reflect a proliferative phenotype

More than 99% of the Cluster 3 cells were mapped in the control group (Fig. 2H and I) (Supplementary File 1). CCNB2 (FC + 8.26, P < 0.0001), ECHDC1 (FC + 8.13, P < 0.0001), and SHCBP1 (SHC binding, and spindle associated 1) (FC + 7.12, P < 0.0001) were significantly upregulated in the ISC/R group of Cofilin-1 + Cluster 3 CF compared to Clusters 2 and 3 (Fig. 2L) (Supplementary File 1). The major downregulated genes were GHSR (growth hormone secretagogue receptor) (FC –6.57, P < 0.0001), ADAMTS5 (FC –7.23, P < 0.0001), and AKR1C2 (aldo–keto reductase family 1 member C2) (FC –8.26, P < 0.0001) in the cluster 3 compared to the other two clusters (Fig. 2L) (Supplementary File 1). Interestingly, most of the upregulated genes in the cluster 3 cells were significantly downregulated and vice versa in the Clusters 1 and 3 suggesting the contrasting phenotypes (Fig. 2L). The pathway analysis of Cofilin-1 + Cluster 3 cells displayed the survival responses including (1) PI3K-AKT signaling mediated through the increased protein synthesis, glucose metabolism, cell proliferation and repair (Supplementary Fig. 1G), and (2) cell cycle progression (Supplementary Fig. 1H). Overall, Cofilin-1 + Cluster 3 cells represents a proliferative phenotype whereas the Clusters 2 and 3 represent non-proliferative and secretory phenotypes.

Ischemia increased the expression of CRSP2 in the ischemic LV tissues and CF

CRSP2 was significantly increased in LV-CABG (P < 0.0001) and LV MI tissues (P < 0.0001) compared to control. Also, the variation between LV-CABG and LV-MI groups were statistically significant (P < 0.0001) (Fig. 3A and B). The mRNA expression of CRSP2 was significantly decreased in both ISC (P < 0.0001) and ISC/R (P < 0.0001) CF compared to the control and ISC/R CF exhibited a significant reduction in CRSP2 transcripts than ISC CF (P < 0.0001) (Fig. 3C). Similar trend was observed in the CF cells in ISC and ISC/R group as evident from immunostaining (Fig. 3D and E). The level of CRSP2 protein was increased in the ISC CF (P = 0.6524) and decreased in the ISC/R group (P = 0.8335) compared to the control; however, was statistically not significant. Also, the alteration between ISC and ISC/R groups (P = 0.3566) were statistically not significant (Fig. 3D and E).

Fig. 3

Representative images for the immunofluorescence analysis for the expression of CRSP2 in the LV (A) (N = 3) and the quantification of protein expression of CRSP2 in LV tissues calculated from MFI (B). C Quantification of CRSP2 gene expression in CF (N = 4) following the treatment with ISC and ISC/R using qRT-PCR relative to the housekeeping gene GAPDH normalized to the control. Representative images for the immunofluorescence and quantification for the expression of CRSP2 in the CF (N = 3) following the treatment with ISC and ISC/R (D and E). Single cell genomics of CRSP2 + CF showing the (F) (split and combined views of Control, ISC and ISC/R groups of t-SNE plot) showing the distribution of cells within all clusters based on the global expression of 6821 genes. Violin plot (G) (FC > 2) indicating the alteration of genes in the 6 clusters. J Scatter plot FC expression indicating the alteration of Cluster 1 genes reflecting the expression of the key signature genes. (**** P < 0.0001, and unlabeled groups represent NS)

CRSP2 + Cluster 1 CF suggest a non-proliferative phenotype

Single cell genomics revealed that 63 CF were positive for CRSP2 (FC > 2), which constituted 0.2094% of the parent population where 45 cells in the ISC group (71.43% of CRSP2 + population) and the CRSP2 + cells favored the ISC group and were mapped in six clusters based on the expression status of 6821 genes (Fig. 3F and G). IGFBP5 (FC + 4.02, P < 0.0001), IGF1 (FC + 3.60, P < 0.0001), and ADAMTS5 (FC = 3.50, P < 0.0001) were the most significantly upregulated genes in the ISC group of CF (Fig. 3H) (Supplementary File 1). CENPF (centromere protein F) (FC –6.85, P < 0.0001), HMMR (hyaluronan mediated motility receptor) (FC –6.74, P = 0.0001), and DLGAP5 (DLG associated protein 5) (FC –6.28, P = 0.0001) were the most significantly downregulated genes (Fig. 3H) (Supplementary File 1). The pathway analysis of CRSP2 + Cluster 1 cells displayed the downregulation multiple key mediators of cell cycle pathways suggesting a non-proliferative phenotype (Supplementary Fig. 2A).

The level of HSP27 was higher in the ischemic LV tissues and CF

HSP27 was significantly increased in LV-CABG (P = 0.0570) and significantly decreased in LV-MI (P = 0.0566) groups compared to the respective controls and LV-CABG displayed a significant increase in HSP27 compared to LV-MI (P = 0.0025) (Fig. 4A and B). The transcript levels of HSP27 in CF were significantly increased in ISC (P = 0.0039) and ISC/R (P = 0.0307) group compared to the control whereas the increase in ISC CF was statistically not significant compared to the ISC/R (P = 0.2040) CF (Fig. 4C). Immunostaining revealed the increased level of HSP27 in both ISC (P = 0.0809) and ISC/R (P = 0.0501) CF compared to the control; however, was statistically not significant in the ISC group. Also, the alteration between ISC and ISC/R groups (P = 0.9230) was statistically not significant (Fig. 4D and E). Western blot analysis revealed that HSP27 was significantly downregulated in both ISC (P = 0.0271), and ISC/R (P = 0.0038) CF compared to controls and the alteration between ISC, and ISC/R groups (P = 0.9853) was statistically not significant (Fig. 4F and G).

Fig. 4

Representative images for the immunofluorescence analysis for the expression of HSP27 in the LV (A) (N = 3) and the quantification of protein expression of HSP27 in LV tissues calculated from MFI (B). C Quantification of HSP27 gene expression in CF (N = 3) following the treatment with ISC and ISC/R using qRT-PCR relative to the housekeeping gene GAPDH normalized to the control. Representative images for the immunofluorescence and quantification for the expression of HSP27 in the CF (N = 3) following the treatment with ISC and ISC/R (D and E). Representative image of Western blot and quantification showing the expression status of HSP27 in the ISC and ISC/R groups compared to the control in CF (N = 4) (F and G). Single cell genomics of HSP27 + CF showing the (H) (split and combined views of Control, ISC and ISC/R groups of t-SNE plot) showing the distribution of cells within all clusters based on the global expression of 6821 genes. Violin plot (I) (FC > 5) indicating the alteration of genes in the 2 clusters. J Scatter plot FC expression indicating the alteration of Cluster 1 and 2 genes reflecting the contrasting level of expression of the key signature genes. (* P < 0.05, ** P < 0.01, and unlabeled groups represent NS)

HSP27 + CF represent contrasting phenotypes

Single cell genomics revealed that 334 CF were positive for HSP27 (FC > 5), which constituted 1.1100% of the parent population where 10 cells were in the control group (2.99% of HSP27 + population), 196 cells in the ISC group (58.68% of HSP27 + population), and 124 cells in the ISC/R group (37.13% of HSP27 + population). Overall, the HSP27 + CF cells favored the ISC group and were mapped in two clusters (Fig. 4H and I). Cluster 1 CF displayed 196 cells where 100% cells were mapped in the ISC group, and the violin plot revealed the distribution of 4144 genes in HSP27 + CF based on expression status in ISC group (Fig. 4H and I) (Supplementary File 1). IGFBP5 (FC + 5.26, < 0.0001), ARL4D (ADP ribosylation factor like GTPase 4D) (FC + 4.43, P < 0.0001), and IGF1 (FC + 4.14, P < 0.0001) were the key genes significantly upregulated in the ISC group (Fig. 4J) (Supplementary File 1). ACTA1 (Actin, alpha skeletal muscle) (FC –8.75, P < 0.0001), HPS5 (FC –5.55, P < 0.0001), and MX2 (FC –5.16, P < 0.0001) were the most significantly downregulated genes (Fig. 4J) (Supplementary File 1). The pathway analysis of HSP27 + Cluster 1 cells displayed the (1) chemokine-receptor signaling (Supplementary Fig. 2B), (2) chemokine activation for cell migration, apoptosis, and ROS production (Supplementary Fig. 2C), and (3) PI3K-AKT signaling for cell survival suggesting a pro-survival phenotype (Supplementary Fig. 2D). Surprisingly, the HSP27 + Cluster 2 cells displayed exactly contrasting Log2 FC values for the same set of genes displayed by HSP27 + Cluster 1 cells and favored ISC/R group suggesting an anti-survival phenotype (Fig. 4J) (Supplementary File 1).

Ischemic insults resulted in the downregulation of IL8 in the LV tissues and CF

The level of IL8 was decreased in LV-CABG (P = 0.3214) and increased in LV-MI (P = 0.9408) compared to control; however, the alterations were statistically not significant. Also, the variation between LV-CABG and LV-MI (P = 0.2134) groups were statistically not significant (Fig. 5A and B). The gene expression of IL8 was significantly decreased in both ISC (P = 0.0005), and ISC/R (P < 0.0001) CF compared to the control and ISC/R CF exhibited a significant reduction in IL8 transcripts than ISC CF (P < 0.0001) (Fig. 5C). The level of IL8 was decreased in both ISC (P = 0.8160), and ISC/R (P = 0.0514) CF compared to the control as evident from immunostaining where the level of IL8 was statistically significant in the ISC/R group. However, the alteration between ISC and ISC/R groups (P = 0.1119) was statistically not significant (Fig. 5D and E). Also, IL8 was downregulated in both ISC (P = 0.8138), and ISC/R (P = 0.7191) CF as determined by the Western blotting compared to the control; however, was not significant. Also, the variation between ISC and ISC/R groups (P = 0.9836) was statistically not significant (Fig. 5F and G).

Fig. 5

Representative images for the immunofluorescence analysis for the expression of IL8 in the LV (A) (N = 3) and the quantification of protein expression of IL8 in LV tissues calculated from MFI (B). C Quantification of IL8 gene expression in CF (N = 4) following the treatment with ISC and ISC/R using qRT-PCR relative to the housekeeping gene GAPDH normalized to the control. Representative images for the immunofluorescence and quantification for the expression of HSP27 in the CF (N = 3) following the treatment with ISC and ISC/R (D and E). Representative image of Western blot and quantification showing the expression status of IL8 in the ISC and ISC/R groups compared to the control in CF (N = 3) (F and G). Single cell genomics of IL8 + CF showing the (H) (split and combined views of Control, ISC and ISC/R groups of t-SNE plot) showing the distribution of cells within all clusters based on the local expression of 2654 genes. Violin plot (I) (FC > 2) indicating the alteration of genes in the 2 clusters. J Scatter plot FC expression indicating the alteration of Cluster 1 genes reflecting the expression of key signature genes. (* P < 0.05, *** P < 0.001, **** P < 0.0001, and unlabeled groups represent NS)

IL8 + Cluster 1 CF reflect a proliferative pro-survival phenotype

Single cell genomics revealed that 99 CF cells were positive for IL8 (FC > 2), which constituted 0.3290% of the parent population where 11 cells were in the control group (12.22% of IL8 + population), 6 cells in the ISC group (6.67% of IL8 + population), and 80 cells in the ISC/R group (88.89% of IL8 + population). Overall, the IL8 + cells favored the ISC/R group and were mapped in two clusters (Fig. 5H and I). Cluster 1 displayed 86 CF cells where 6.97% cells were mapped in the ISC group, and 93.02% cells with the distribution of 2654 genes in the cluster 1 CF based on expression status in ISC/R group (Fig. 5H and I) (Supplementary File 1). MYBPH (Myosin Binding Protein H) (FC + 3.84, P = 0609), SFRP1 (Secreted frizzled-related protein 1) (FC + 3.31, P = 0609), and GLIPR1 (FC + 3.02, P = 0.0321) were the key genes significantly upregulated in the ISC/R group; however, the expression status of MYBPH and SFRP1 were statistically not significant (Fig. 5J) (Supplementary File 1). SLP1 (secretory leukocyte peptidase inhibitor) (FC –9.15, P = 0.0168), ECSCR (endothelial cell surface expressed chemotaxis and apoptosis regulator) (FC –8.03, P = 0.0026), and EMCN (endomucin) (FC –6.80, P = 0.0210) were the most significantly downregulated genes (Fig. 4J) (Supplementary File 1). The pathway analysis of IL8 + Cluster 1 cells displayed the (1) MAPK signaling activating cell proliferation and differentiation (Supplementary Fig. 2E), (2) cytokine-cytokine receptor interaction (Supplementary Fig. 2F), and (3) PI3K-AKT signaling for cell survival suggesting a proliferative pro-survival phenotype (Supplementary Fig. 2G).

Ischemic insults resulted in the upregulation of HSP90 in the LV tissues and CF

HSP90 was decreased in LV-CABG (P = 0.1032) and increased in LV-MI (P = 0.2516) compared to the control; however, was statistically not significant. Interestingly, LV-CABG displayed significantly higher level of HSP90 compared to LV-MI (P = 0.0123) (Fig. 6A and B). The transcript level of HSP90 (P = 0.2038) was increased in the ISC; however, was statistically not significant and was significantly decreased ISC/R (P < 0.0001) CF compared to the control. Also, the ISC/R displayed statistically significant decrease in HSP90 mRNA level compared to ISC (P < 0.0001) CF (Fig. 6C). Immunostaining revealed significant increase of HSP90 in both the ISC (P = 0.0016) and ISC/R (P < 0.0001) groups compared to the control CF, and the level of HSP90 was significantly decreased in the ISC/R (P < 0.0001) groups compared to the ISC CF (P = 0.0016) (Fig. 6D and E). Interestingly, HSP90 was downregulated in both ISC (P = 0.2196), and ISC/R (P = 0.0033) CF compared to the control; however, was not statistically significant in the ISC cells as evident from Western blotting. Also, the level of HSP90 was significantly decreased in the ISC/R groups compared to the ISC CF (P = 0.0511) (Fig. 6F and G).

Fig. 6

Representative images for the immunofluorescence analysis for the expression of HSP90 in the LV (N = 3) (A) and the quantification of protein expression of HSP90 in LV tissues calculated from MFI (B). C Quantification of HSP90 gene expression in CF (N = 4) following the treatment with ISC and ISC/R using qRT-PCR. Representative images for the immunofluorescence and quantification for the expression of HSP90 in the CF (N = 3) (D and E) (N = 3) following the treatment with ISC and ISC/R. F Representative image of Western blot and (G) quantification showing the expression status of HSP90 in the ISC and ISC/R groups compared to the control in CF (N = 4). Single cell genomics of HSP90 + CF showing the (H) (split and combined views of Control, ISC and ISC/R groups of t-SNE plot) showing the distribution of cells within all clusters based on the local expression of 4272 genes. Violin plot (I) (FC > 5) indicating the alteration of genes in the 2 clusters. J Scatter plot FC expression indicating the alteration of Cluster 1 genes reflecting the expression of key signature genes. (* P < 0.05, ** P < 0.01, **** P < 0.0001, and unlabeled groups represent NS)

HSP90 + Cluster 1 CF reflect pro-survival and Cluster 2 CF reflect pro-inflammatory phenotypes

Single cell genomics revealed that 102 cells were positive for HSP90 (FC > 5), which constituted 0.32% of the parent population where 55 cells were mapped in the ISC group (57.29% of HSP90 + population), and 41 cells in the ISC/R group (40.63% of HSP90 + population). Overall, the HSP90 + cells favored the ISC group and were mapped in 3 clusters based on the expression status of 4272 genes (Fig. 6H and I). Cluster 1 displayed 55 cells where 100% cells were mapped in the ISC group. IGFBP5 (FC + 7.63, < 0.0001), ADAMTS5 (FC + 5.40, P = 0.0009), AKR1C2 (FC + 5.10, P = 0.0060), and IGF1 (FC + 4.90, P < 0.0001) were the key genes significantly upregulated in the ISC group (Fig. 6J) (Supplementary File 1). SST (somatostatin) (FC –6.06, P = 1.0000), HPS5 (FC –5.35, P < 0.0001), CCL5 (FC –5.18, P = 0.9050), and MX2 (FC –5.00, P = 0.0001) were the most downregulated genes; however, was statistically not significant for SST and CCL5 (Fig. 6J) (Supplementary File 1). The pathway analysis of HSP90 + Cluster 1 cells displayed the downregulation of key inflammatory mediators in the pathways associated with (1) cytokine—cytokine-receptor signaling (Supplementary Fig. 3A), (2) NLR signaling (Supplementary Fig. 3B), and (3) TNF signaling suggesting a pro-survival phenotype (Supplementary Fig. 3C). Cluster 2 displayed 41 cells where 95.12% cells were mapped in the ISC/R group. HPS5 (FC + 5.61, < 0.0001), CCL5 (FC + 5.44, P = 0.7275), MX2 (FC + 5.26, P < 0.0001), and IGF1 (FC + 4.90, P < 0.0001) were the key genes upregulated in the ISC group; however, the upregulation in CCL5 was not significant (Fig. 6K) (Supplementary File 1). IGFBP5 (FC –7.96, P < 0.0001), ADAMTS5 (FC –5.14, P = 0.0049), SST (FC –5.09, P = 1.0000), and ARLD (FC –4.99, P < 0.0001) were the most downregulated genes; however, was statistically not significant for SST (Fig. 6K) (Supplementary File 1). The pathway analysis of HSP90 + Cluster 2 cells displayed the (1) cytokine—cytokine-receptor signaling (Supplementary Fig. 3A), (2) NLR signaling (Supplementary Fig. 3B), and (3) TNF signaling suggesting a pro-inflammatory phenotype (Supplementary Fig. 3C) as in Cluster 1.

Ischemic insults resulted in the decreased level of Nrf2 in the LV tissues and CF

Nrf2 was decreased in LV-CABG (P = 0.0159) and LV-MI (P = 0.1477) compared to the control; however, the decrease in LV-MI was statistically not significant. Also, the variation between LV-CABG and LV-MI (P = 0.2368) groups were statistically not significant (Fig. 7A and B). PCR data revealed that both the ISC and ISC/R CF displayed significantly decreased expression of Nrf2 compared to the control (P = 0.0034 and P < 0.0001 respectively) and the level of Nrf2 transcript was significantly lower in ISC/R compared to the ISC (P = 0.0014) CF (Fig. 7C). Nrf2 expression was significantly lower in the ISC CF than the control (P = 0.0552) and ISC/R (P = 0.0047) groups as revealed by immunostaining and was increased in the ISC/R (P = 0.1432) than the control group; however, was statistically not significant (Fig. 7D and E). Western blot showed that the level of Nrf2 was significantly upregulated in the ISC (P = 0.0007) CF and was downregulated the in ISC/R (P = 0.8611) CF compared to control. Also, the level of Nrf2 was significantly decreased in the ISC/R groups compared to the ISC CF (P = 0.0004) (Fig. 7F and G).

Fig. 7

Representative images for the immunofluorescence analysis for the expression of Nrf2 in the LV (N = 3) (A) tissues and (B) the quantification of protein expression of Nrf2 in LV tissues calculated from MFI. C Quantification of Nrf2 gene expression in CF (N = 4) following the treatment with ISC and ISC/R using qRT-PCR. Representative images for the immunofluorescence and quantification for the expression of Nrf2 in the CF (N = 3) (D and E) following the treatment with ISC and ISC/R. F Representative image of Western blot and (G) quantification showing the expression status of Nrf2 in the ISC and ISC/R groups compared to the control in CF (N = 4). Single cell genomics of Nrf2 + CF showing the (H) (split and combined views of Control, ISC and ISC/R groups of t-SNE plot) showing the distribution of cells within all clusters based on the local expression of 5817 genes. Violin plot (I) (FC > 4) indicating the alteration of genes in the 2 clusters. J Scatter plot FC expression indicating the alteration of Cluster 1 genes reflecting the expression of key signature genes. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and unlabeled groups represent NS)

Nrf2 + Cluster 1 CF represent proinflammatory phenotype

Single cell genomics revealed that 85 CF cells were positive for Nrf2 (FC > 4), which constituted 0.28% of the parent population where 64 cells were mapped in the ISC group (75.29% of Nrf2 + population), and 21 cells in the ISC/R group (24.71% of Nrf2 + population). Overall, the Nrf2 + cells favored the ISC group and were mapped in 3 clusters (Fig. 7H and I). Nrf2 + Cluster 1 displayed 64 cells where 100% cells were mapped in the ISC group based on the distribution of 5817 genes in Nrf2 + Cluster 1 CF based on the expression status in ISC group (Fig. 7J) (Supplementary File