Abstract
Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) (iPSC-CMs) are a promising cell source for myocardial regeneration, disease modeling and drug assessment. However, iPSC-CMs exhibit immature fetal CM-like characteristics that are different from adult CMs in several aspects, including cellular structure and metabolism. As an example, glycolysis is a major energy source for immature CMs. As CMs mature, the mitochondrial oxidative capacity increases, with fatty acid β-oxidation becoming a key energy source to meet the heart’s high energy demand. The immaturity of iPSC-CMs thereby limits their applications. The aim of this study was to investigate whether the energy substrate fatty acid-treated iPSC-CMs exhibit adult CM-like metabolic properties. After 20 days of differentiation from human iPSCs, iPSC-CMs were sequentially cultured with CM purification medium (lactate+/glucose-) for 7 days and maturation medium (fatty acids+/glucose-) for 3–7 days by mimicking the adult CM’s preference of utilizing fatty acids as a major metabolic substrate. The purity and maturity of iPSC-CMs were characterized via the analysis of: (1) Expression of CM-specific markers (e.g., troponin T, and sodium and potassium channels) using RT-qPCR, Western blot or immunofluorescence staining and electron microscopy imaging; and (2) cell energy metabolic profiles using the XF96 Extracellular Flux Analyzer. iPSCs-CMs (98% purity) cultured in maturation medium exhibited enhanced elongation, increased mitochondrial numbers with more aligned Z-lines, and increased expression of matured CM-related genes, suggesting that fatty acid-contained medium promotes iPSC-CMs to undergo maturation. In addition, the oxygen consumption rate (OCR) linked to basal respiration, ATP production, and maximal respiration and spare respiratory capacity (representing mitochondrial function) was increased in matured iPSC-CMs. Mature iPSC-CMs also displayed a larger change in basal and maximum respirations due to the utilization of exogenous fatty acids (palmitate) compared with non-matured control iPSC-CMs. Etomoxir (a carnitine palmitoyltransferase 1 inhibitor) but not 2-deoxyglucose (an inhibitor of glycolysis) abolished the palmitate pretreatment-mediated OCR increases in mature iPSC-CMs. Collectively, our data demonstrate for the first time that fatty acid treatment promotes metabolic maturation of iPSC-CMs (as evidenced by enhanced mitochondrial oxidative function and strong capacity of utilizing fatty acids as energy source). These matured iPSC-CMs might be a promising human CM source for broad biomedical application.
Highlights
Despite advances in medical technology and treatment, heart disease remains the leading cause of mortality worldwide partially due to the lack of appropriate human cardiomyocyte (CM) sources for repairing injured hearts, studying disease mechanisms, and screening drugs for efficacy/toxicity.For example, myocardial infarction is characterized by CM death following restricted blood flow [1].As adult CMs have extremely limited proliferation capacity, injured heart function is progressively worsened, leading to severe long-term consequences such as arrhythmia, congestive heart failure, or even death
We observed that the maturation medium significantly increased the oxygen consumption rate (OCR) that were linked to basal respiration, ATP production, maximal respiration and spare respiratory capacity (Figure 4B,C)
The resultscould showed that fatty maturation of induced pluripotent stem cell (iPSC)-CMs in cell structure, morphology, expression, and energy metabolism
Summary
As adult CMs have extremely limited proliferation capacity, injured heart function is progressively worsened, leading to severe long-term consequences such as arrhythmia, congestive heart failure, or even death. There has been a long list of high-profile drugs that have been withdrawn from the market due to their off-target and clinical cardiac toxicities even after years of laboratory and preclinical testing. These drug withdrawals are partly attributable to inadequate screening using less than ideal animal models and non-CM cell lines (e.g., human embryonic kidney or Chinese hamster ovary cell lines), resulting in a tremendous burden on the healthcare system [7,8]. Human pluripotent stem cell (iPSC)-derived CMs (iPSC-CMs) are an ideal solution to these challenges by providing unlimited human CMs for myocardial regeneration, drug screening, and disease modeling [9,10,11]
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