Metabolic reprogramming via mitochondrial delivery for enhanced maturation of chemically induced cardiomyocyte-like cells

Abstract

Heart degenerative diseases pose a significant challenge due to the limited ability of native heart to restore lost cardiomyocytes. Direct cellular reprogramming technology, particularly the use of small molecules, has emerged as a promising solution to prepare functional cardiomyocyte through faster and safer processes without genetic modification. However, current methods of direct reprogramming often exhibit low conversion efficiencies and immature characteristics of the generated cardiomyocytes, limiting their use in regenerative medicine. This study proposes the use of mitochondrial delivery to metabolically reprogram chemically induced cardiomyocyte-like cells (CiCMs), fostering enhanced maturity and functionality. Our findings show that mitochondria sourced from high-energy-demand organs (liver, brain, and heart) can enhance structural maturation and metabolic functions. Notably, heart-derived mitochondria resulted in CiCMs with a higher oxygen consumption rate capacity, enhanced electrical functionality, and higher sensitivity to hypoxic condition. These results are related to metabolic changes caused by increased number and size of mitochondria and activated mitochondrial fusion after mitochondrial treatment. In conclusion, our study suggests that mitochondrial delivery into CiCMs can be an effective strategy to promote cellular maturation, potentially contributing to the advancement of regenerative medicine and disease modeling.

Keywords: cardiomyocytes; cell reprogramming; metabolic regulation; mitochondrial transfer.

FIGURE 1

Mitochondrial characteristics and functional analysis across different tissues. (A) Flow cytometry analysis using MitoTracker Red to verify mitochondrial purity. (B) Transmission electron microscopy (TEM) images of B‐Mito, L‐Mito, and H‐Mito (scale bars = 500 nm). (C) Size (n = 6) and (D) zeta potential (n = 10) of isolated mitochondria. (E) ATP levels of the freshly isolated mitochondria (n = 8). (F) Oxidative phosphorylation (OXPHOS) activities of isolated mitochondria determined by extracellular oxygen consumption rate (OCR) assay (n = 4). (G) Cytochrome c oxidase activity reflecting an initial burst of cytochrome c oxidation (n = 3). (H) Western blot analysis of mitochondrial OXPHOS complex protein subunits and TOM20 as a loading control. Complex I, NDUFB8 (20 kDa); Complex II, SDHB (30 kDa); Complex III, UQCRC2 (48 kDa); Complex IV, MTCO1 (40 kDa); Complex V, ATP5A (55 kDa). (I) Relative protein expression of mitochondria isolated from different tissues quantified by densitometry and normalized to that of TOM20 (n = 3). All data are expressed as the means ± SD. Statistical difference between the groups was determined by two‐tailed t‐test (*p < 0.05 and **p < 0.01).

FIGURE 2

Uptake of tissue‐derived mitochondria by chemically induced cardiomyocyte‐like cells (CiCMs). (A) Schematic timeline of generating induced CiCMs by chemical induction and mitochondrial delivery isolated from adult tissues. (B) Fluorescent images illustrating CiCMs during chemical cardiac reprogramming, 24 h post‐transfection with MitoTracker Green‐labeled mitochondria isolated from various adult tissues (scale bars = 10 µm). (C) Analysis of MitoTracker Green fluorescence intensity per cell, representing internalized mitochondria (n = 4). (D) Images showing co‐localization of exogenous mitochondria (Exog.), labeled with MitoTracker Green, and the cell's endogenous mitochondria (Endog.), marked with MitoTracker Red. A higher magnification image (right) illustrates the coexistence of transfected and native mitochondria (scale bars = 10 µm for the main image, and 1 µm for the magnified image). (E) Transmission electron microscopy (TEM) images of CiCMs after 3 days of transfection with H‐Mito (scale bars = 1 µm). Elongated mitochondria are marked with red arrows. Quantitative analysis of (F) mitochondrial count per µm² (n = 15), and (G) average mitochondrial size in terms of area (n = 24). (H) Representative TEM images of CiCMs at 3 days post‐transfection with H‐Mito, depicting mitochondrial fusion/fission events (indicated by yellow arrows, scale bars = 500 nm), and (I) quantitative analysis of these mitochondrial dynamics (n = 10 at each time point). All data are expressed as the means ± SD. Statistical difference between the groups was determined by (C, F, G) two‐tailed t‐test or (I) Two‐way ANOVA followed by Bonferroni correction (*p < 0.05 and **p < 0.01 versus NT group).

FIGURE 3

Enhanced maturation of chemically induced cardiomyocyte‐like cells (CiCMs) induced by mitochondrial treatment. (A) Immunostaining for alpha‐actinin (α‐ACT) and t‐tubules (detected by FITC‐conjugated WGA) in PMEFs and CiCMs, both untreated (No Treat; NT) and those treated with mitochondria derived from brain, liver, and heart tissues (B‐Mito, L‐Mito, H‐Mito) for 7 days (scale bars = 10 µm). (B) Sarcomere length (SL) analysis derived from the region of interest on α‐ACT immunostained images (indicated by a white line in the second‐row images). Quantifications of (C) α‐ACT+ area (n = 4), (D) cells with sarcomere structures (n = 7), (E) average SL (n = 10), and (F) coefficient of variation of SL (n = 5) based on α‐ACT immunostained images. (G) Immunostaining for cardiac troponin T (cTnT) and WGA in PMEF, B‐Mito, L‐Mito, and H‐Mito groups after 7 days of mitochondrial treatment (corresponding to 15 days of CiCM culture) (scale bars = 10 and 2 µm for magnified images). (H) Quantification of cTnT‐positive area (n = 4). (I) Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) analysis of cardiac‐specific genes in various CiCM groups (n = 3). All data are expressed as the means ± SD. Statistical difference between the groups was determined by two‐tailed t‐test (*p < 0.05 and **p < 0.01 versus NT, #p < 0.05 and ##p < 0.01 versus B‐Mito, and $$p < 0.01 versus L‐Mito group).

FIGURE 4

Mitochondrial reprogramming enhances ultrastructure and bioenergetics in chemically induced cardiomyocyte‐like cells (CiCMs). (A) Transmission electron microscopy (TEM) images of CiCMs after 10 days of treatment with H‐Mito (Annotations: mito = mitochondria, Z = Z‐line, M = M‐line, MF = myofibril, scale bars = 2 µm). Quantitative analysis of (B) mitochondrial count per 10 µm² (n = 15), and (C) average mitochondrial size in terms of area (n = 13). (D) Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) analysis of metabolism‐related genes in various CiCM groups (n = 3). (E) Oxygen consumption rate (OCR) measurements in untreated CiCMs (NT), and those treated with mitochondria from brain, liver, and heart tissues (n = 5). Basal respiration was observed for 72 min. Oligomycin, which inhibits ATP production via oxidative phosphorylation, was introduced at 16 min. Subsequently, carbonyl cyanide p‐trifluoro methoxyphenylhydrazone (FCCP) was injected at 36 min, followed by complex I and III inhibitors, rotenone and antimycin A, respectively. The quantitative analysis includes (F) basal respiration (n = 5), (G) ATP production (n = 5), (H) maximal respiration (n = 5), and (I) spare respiratory capacity (n = 5). Statistical significance between groups was determined using a two‐tailed t‐test (*p < 0.05 and **p < 0.01 versus NT, #p < 0.05 and ##p < 0.01 versus B‐Mito, and $P < 0.05 versus L‐Mito group).

FIGURE 5

Calcium influx characterization of metabolically reprogrammed chemically induced cardiomyocyte‐like cells (CiCMs). (A) Representative time‐lapse calcium imaging in CiCMs loaded with the intracellular Ca²⁺ indicator Fluo‐4 AM (scale bars = 20 µm). The traces depicting changes in fluorescence intensity are shown in the area marked by a white dotted circle. (B) Average number of cells per square millimeter exhibiting spontaneous Ca²⁺ transients (n = 7). (C) Mean frequency of Ca²⁺ transients occurring per minute (n = 11). (D) Coefficient of variation for the duration of Ca²⁺ transients (n = 6). (E) Average amplitude of Ca²⁺ transients (n = 14). (F) Representative traces of Ca²⁺ fluorescence pre‐ and post‐treatment with 10 nM carbamylcholine (CCh). (G) Change in beating per minute (BMP) normalized to pre‐treatment of CCh (n = 4). (H) Quantification of CCh‐responsive cells, demonstrated by a reduction in beats per minute (BPM). Statistical significance between groups was determined using (B‐E) a two‐tailed t‐test or (G, H) two‐way ANOVA followed by Tukey (*p < 0.05 and **p < 0.01 versus NT, and #p < 0.05 and ##p < 0.01 versus B‐Mito group).

FIGURE 6

Metabolically matured chemically induced cardiomyocyte‐like cells (CiCMs) via mitochondrial delivery are more sensitive to hypoxia. (A) Live/dead assay by EthD‐1/calcein AM staining of CiCMs in NT, B‐Mito, L‐Mito, and H‐Mito groups exposed to 1% O₂ for 6, 12, and 24 h (n = 4). (B) Quantitative analysis of the number of dead cells in terms of fluorescence area in different groups (n = 4). Immunostaining for alpha‐actinin (α‐ACT) in different CiCM groups exposed to 1% hypoxia for (C) 12 h, and (D) 24 h (scale bars = 10 and 2 µm for magnified images). (E) Real‐time oxygen consumption rate (OCR) of CiCMs in different groups exposed to 1% hypoxia for 6 and 12 h (n = 9). Cells were treated with oligomycin, carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP), and antimycin A and rotenone to measure mitochondrial respiration. The quantitative analysis includes (F) basal respiration (n = 9), (G) ATP production (n = 9), and (H) maximal respiration (n = 9). Statistical significance between groups was determined using (B), (F), (G), and (H) two‐way ANOVA followed by Tukey **p < 0.01 versus NT, and #p < 0.05 and ##p < 0.01 versus B‐Mito group, and &&p < 0.01 versus L‐Mito group).

FIGURE 7

Proposed mechanistic model of mitochondrial delivery inducing metabolic reprograming and maturation of chemically induced cardiomyocyte‐like cells (CiCMs). During the process of direct reprogramming, mitochondrial delivery modulates bioenergetics of immature CMs inducing maturation of the cells. After the treatment of functional mitochondria with robust oxidative phosphorylation (OXPHOS) activity, recipient mitochondria are replaced to mature status, and in turn induces CiCMs metabolically active. As a result, the mitochondria‐treated CiCMs can exhibit enhanced energy production, respiratory activity, and electrophysiological functionality with organized intracellular structures and cardiac‐specific characteristics, resulting in mature CMs.