Evaluating the Reparative Potential of Secretome from Patient-Derived Induced Pluripotent Stem Cells during Ischemia-Reperfusion Injury in Human Cardiomyocytes

Abstract

During a heart attack, ischemia causes losses of billions of cells; this is especially concerning given the minimal regenerative capability of cardiomyocytes (CMs). Heart remuscularization utilizing stem cells has improved cardiac outcomes despite little cell engraftment, thereby shifting focus to cell-free therapies. Consequently, we chose induced pluripotent stem cells (iPSCs) given their pluripotent nature, efficacy in previous studies, and easy obtainability from minimally invasive techniques. Nonetheless, using iPSC secretome-based therapies for treating injured CMs in a clinical setting is ill-understood. We hypothesized that the iPSC secretome, regardless of donor health, would improve cardiovascular outcomes in the CM model of ischemia-reperfusion (IR) injury. Episomal-generated iPSCs from healthy and dilated cardiomyopathy (DCM) donors, passaged 6-10 times, underwent 24 h incubation in serum-free media. Protein content of the secretome was analyzed by mass spectroscopy and used to treat AC16 immortalized CMs during 5 h reperfusion following 24 h of hypoxia. IPSC-derived secretome content, independent of donor health status, had elevated expression of proteins involved in cell survival pathways. In IR conditions, iPSC-derived secretome increased cell survival as measured by metabolic activity (p < 0.05), cell viability (p < 0.001), and maladaptive cellular remodelling (p = 0.052). Healthy donor-derived secretome contained increased expression of proteins related to calcium contractility compared to DCM donors. Congruently, only healthy donor-derived secretomes improved CM intracellular calcium concentrations (p < 0.01). Heretofore, secretome studies mainly investigated differences relating to cell type rather than donor health. Our work suggests that healthy donors provide more efficacious iPSC-derived secretome compared to DCM donors in the context of IR injury in human CMs. These findings illustrate that the regenerative potential of the iPSC secretome varies due to donor-specific differences.

Keywords: cardiomyocytes; cell-free therapeutics; dilated cardiomyopathy; induced pluripotent stem cells; ischemia–reperfusion; proteomics; secretome.

Figure 1

Proteomic profiling of iPSC-derived secretome from healthy donors and dilated cardiomyopathy patients. (A) Venn diagram comparing identified proteins in secretome. (B) Volcano plot identifying differentially expressed proteins. (C) GO pathway analysis of iPSC secretome from all iPSC lines. (D) Heatmap of differentially expressed proteins involved in cell survival, (E) mitochondrial Ca²⁺ homeostasis, and (F) intracellular Ca²⁺ and contractility. Data are presented in number of peptides. DCM sec = dilated cardiomyopathy patient-derived secretome; HE sec = healthy donor-derived secretome.

Figure 2

Effect of secretome on viability as measured by metabolic activity after hypoxia-reperfusion injury. Percentage of resazurin reduction at 2, 3, and 4 h post-ischemia–reperfusion injury comparing control to secretome (A) grouped (n = 1 with six repeats and 6 with six repeats each, respectively), and (B) separated based on donor health status (n = 1 with six repeats, with six repeats, and 3 with six repeats). Relative transcript abundance of Hypoxia inducible factor subunit 1 alpha with secretome (C) grouped (n = 7, 1, and 6), and (D) ungrouped (n = 7, 1, 3, and 3); of superoxide dismutase 1 with secretome (E) grouped (n = 6, 1, and 5), and (F) ungrouped (n = 6, 1, 2, and 3); and of superoxide dismutase 2 with secretome (G) grouped (n = 6, 1, and 5), and (H) ungrouped (n = 6, 1, 2, and 3). * p < 0.05, ** p < 0.01. HE Sec = healthy donor secretome; DCM Sec = dilated cardiomyopathy donor secretome; HIF1A = hypoxia inducible factor subunit 1 alpha; SOD = superoxide dismutase; IR = ischemia–reperfusion. Relative abundance was calculated by normalization of raw quantitative data against the mean of the normoxia-treated group.

Figure 3

Effect of secretome on cell survival and relative transcript abundance of genes related to apoptosis. (A) Representative pictures of AC16 cardiomyocytes at 4× magnification dyed with crystal violet prior to solubilization with methanol after being cultured in normoxia conditions or after ischemia–reperfusion injury with control media treatment or secretome treatment wherein purple represents viable cells still attached to the plate. Percentage of viability following ischemia–reperfusion injury comparing control to secretome (B) grouped (n = 1 with six repeats and 6 with six repeats each, respectively) and (C) ungrouped (n = 1 with six repeats, 3 with six repeats each, and 3 with six repeats each). Relative transcript abundance of B-cell lymphoma 2-associated X/B-cell lymphoma 2 ratio with secretome (D) grouped (n = 7, 1, and 5), and (E) ungrouped (n = 7, 1, 3, and 2); and of caspase 3 with secretome (F) grouped (n = 7, 1, and 6), and (G) ungrouped (n = 7, 1, 3, and 3). *** p < 0.001, **** p < 0.0001. HE Sec = healthy donor secretome; DCM Sec = dilated cardiomyopathy donor secretome; BCL2 = B-cell lymphoma 2; BAX = B-cell lymphoma 2-associated X; CASP3 = caspase 3; IR = ischemia–reperfusion. Relative abundance was calculated by normalization of raw quantitative data against the mean of the normoxia-treated group.

Figure 4

Effect of secretome on the hypertrophic response after ischemia–reperfusion injury. (A) Representative pictures of AC16 CMs with the nucleus and actin-dyed. Relative cell area following ischemia–reperfusion injury comparing control to secretome (B) grouped (n = 7 with 3–4 repeats, n = 1 with 36 repeats, and n = 6 with 47–77 repeats), and (C) ungrouped (n = 7 with 3–4 repeats, n = 1 with 36 repeats, n = 3 with 47–77 repeats, and n = 3 with 55–77 repeats). Cell area following ischemia–reperfusion injury comparing control to secretome (D) grouped (n = 7 with 3–4 repeats, n = 1 with 36 repeats, and n = 6 with 47–77 repeats), and (E) ungrouped (n = 7 with 3–4 repeats, n = 1 with 36 repeats, n = 3 with 47–77 repeats, and n = 3 with 55–77 repeats). HE Sec = healthy donor secretome; DCM Sec = dilated cardiomyopathy donor secretome; IR = ischemia–reperfusion.

Figure 5

Effect of secretome on calcium handling after hypoxia–reperfusion injury. (A) Representative fluorescence imaging at 20× magnification of cardiomyocytes where calcium was stained with Fluo-4, AM, after being cultured in normoxia conditions or after ischemia–reperfusion with control media treatment or secretome treatment. Relative intracellular calcium fluorescence intensity in cardiomyocytes treated with secretome (B) grouped (n = 7 with 28–101 repeats, n = 1 with 54 repeats, and n = 6 with 27–83 repeats), and (C) ungrouped (n = 7 with 28–101 repeats, n = 1 with 54 repeats, n = 3 with 27–43 repeats, and n = 3 with 33–83 repeats). Relative change in intracellular calcium fluorescence intensity in cardiomyocytes treated with secretome (D) grouped (n = 7 with 28–101 repeats, n = 1 with 58 repeats, and n = 6 with 28–29 repeats), and (E) ungrouped (n = 7 with 28–101 repeats, n = 1 with 58 repeats, n = 3 with 32–33 repeats, and n = 3 with 24–25 repeats). ** p < 0.01, **** p < 0.0001. HE Sec = healthy donor secretome; DCM Sec = dilated cardiomyopathy donor secretome; IR = ischemia–reperfusion.

Figure 6

Relative abundance transcript abundance of genes involved in calcium contractility. Transcripts identified have various roles relating to contractility and calcium homeostasis and hypoxia response. Relative transcript abundance of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a with secretome (A) grouped (n = 6, 1, and 5), and (B) ungrouped (n = 6, 1, 3, and 2); of calcium voltage-gated channel subunit alpha 1 C, with secretome (C) grouped (n = 7, 1, and 6), and (D) ungrouped (n = 7, 1, 3, and 3); of Cardiac troponin T 2 with secretome (E) grouped (n = 7, 1, and 6), and (F) ungrouped (n = 7, 1, 3, and 3); and of connexin 43 with secretome (G) grouped (n = 6, 1, and 5), and (H) ungrouped (n = 6, 1, 2, and 3). * p < 0.05, ** p < 0.01, *** p < 0.001. HE Sec = healthy donor secretome; DCM Sec = dilated cardiomyopathy donor secretome; SERCA2A = sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a; CACNA1C = calcium voltage-gated channel subunit alpha 1 C; TNNT2 = cardiac troponin T 2; CNX43 = connexin 43; IR = ischemia–reperfusion. Relative abundance was calculated by normalization of raw quantitative data against the mean of the normoxia-treated group.