Susceptibility of murine induced pluripotent stem cell-derived cardiomyocytes to hypoxia and nutrient deprivation

Abstract

IntroductionInduced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) may be suitable for myocardial repair. While their functional and structural properties have been extensively investigated, their response to ischemia-like conditions has not yet been clearly defined.MethodsiPS-CMs were differentiated and enriched from murine induced pluripotent stem cells expressing enhanced green fluorescent protein (eGFP) and puromycin resistance genes under the control of an α-myosin heavy chain (α-MHC) promoter. iPS-CMs maturity and function were characterized by microscopy, real-time PCR, calcium transient recordings, electrophysiology, and mitochondrial function assays, and compared to those from neonatal murine cardiomyocytes. iPS-CMs as well as neonatal murine cardiomyocytes were exposed for 3 hours to hypoxia (1% O2) and glucose/serum deprivation, and viability, apoptosis markers, reactive oxygen species, mitochondrial membrane potential and intracellular stress signaling cascades were investigated. Then, the iPS-CMs response to mesenchymal stromal cell-conditioned medium was determined.ResultsiPS-CMs displayed key morphological and functional properties that were comparable to those of neonatal cardiomyocytes, but several parameters indicated an earlier iPS-CMs maturation stage. During hypoxia and glucose/serum deprivation, iPS-CMs exhibited a significantly higher proportion of poly-caspase-active, 7-aminoactinomycin D-positive and TUNEL-positive cells than neonatal cardiomyocytes. The average mitochondrial membrane potential was reduced in “ischemic” iPS-CMs but remained unchanged in neonatal cardiomyocytes; reactive oxygen species production was only increased in “ischemic” iPS-CMs, and oxidoreductase activity in iPS-CMs dropped more rapidly than in neonatal cardiomyocytes. In iPS-CMs, hypoxia and glucose/serum deprivation led to upregulation of Hsp70 transcripts and decreased STAT3 phosphorylation and total PKCε protein expression. Treatment with mesenchymal stromal cell-conditioned medium preserved oxidoreductase activity and restored pSTAT3 and PKCε levels.ConclusioniPS-CMs appear to be particularly sensitive to hypoxia and nutrient deprivation. Counteracting the ischemic susceptibility of iPS-CMs with mesenchymal stromal cell-conditioned medium may help enhance their survival and efficacy in cell-based approaches for myocardial repair.

Guidelines for experimental models of myocardial ischemia and infarction

“…Thus, assays of cell viability are often performed to assess the role of a particular genetic or pharmacological intervention in exacerbating or protecting the cell from H/R-induced cell death. Viability may be measured with a variety of assays, including lactate dehydrogenase (LDH) release or propidium iodide exclusion as an indicator of membrane integrity ( 24 , 31 , 58 ). Apoptosis is assessed with TUNEL or annexin V staining ( 24 , 58 , 166 ).…”

“…Viability may be measured with a variety of assays, including lactate dehydrogenase (LDH) release or propidium iodide exclusion as an indicator of membrane integrity ( 24 , 31 , 58 ). Apoptosis is assessed with TUNEL or annexin V staining ( 24 , 58 , 166 ). Mitochondrial damage, including disruption of mitochondrial membrane potential, is also a key component of cellular injury after H/R and may be assessed using fluorescent dyes, such as tetramethylrhodamine methyl ester (TMRM).…”

“…Mitochondrial damage, including disruption of mitochondrial membrane potential, is also a key component of cellular injury after H/R and may be assessed using fluorescent dyes, such as tetramethylrhodamine methyl ester (TMRM). The loss of mitochondrial membrane potential causes TMRM to leak from the mitochondria, decreasing fluorescence ( 24 , 31 ). In addition, reactive oxygen species (ROS) have been implicated in H/R injury ( 275 ); thus, ROS production is another common assessment ( 24 , 58 , 124 , 125 , 227 ).…”

MicroRNA-210 suppresses glucocorticoid receptor expression in response to hypoxia in fetal rat cardiomyocytes

“…For treatment with 2-methoxyestradiol (2ME; Sigma-Aldrich, St. Louis, MO), an inhibitor of HIF-1α accumulation [ 29 , 30 ], cells were cultured in fully supplemented M199 containing 3 μM 2ME under normoxic (21% O 2 ) or hypoxic (1%) conditions for 24 hours. The 1% O 2 (~7 mmHg) is commonly used to induce hypoxic treatment on tissue or cells [ 8 , 54 , 57 , 58 ]. Our previous studies showed that 1% O 2 induced cellular and molecular responses in cardiomyocytes such as the nuclear accumulation of HIF-1α [ 8 , 54 ].…”

Mesenchymal Stem Cell-Based Therapy for Cardiovascular Disease: Progress and Challenges

“…Combining cell and gene therapy offers the advantage that cells can be genetically engineered ex vivo prior to transplantation, offering a safer alternative and more precise control of gene expression compared to gene therapy alone. 27,38,[45][46][47] Another such approach involves the ex vivo engineering of cell grafts with mixtures of physiologically relevant cell types, such as cardiomyocytes; cardiac precursors; and vascular, neuronal, immune, and mesenchymal cell types, 35,41,[48][49][50][51][52][53][54] which may offer a more comprehensive regenerative strategy compared to each cell type alone, given that both cardiomyogenic and non-cardiomyogenic cells are necessary for heart function and regeneration.…”