Age Related Bioenergetics Profiles in Isolated Rat Cardiomyocytes Using Extracellular Flux Analyses

Abstract

Mitochondrial dysfunction is increasingly recognized and studied as a mediator of heart disease. Extracellular flux analysis (XF) has emerged as a powerful tool to investigate cellular bioenergetics in the context of cardiac health and disease, however its use and interpretation requires improved understanding of the normal metabolic differences in cardiomyocytes (CM) at various stages of maturation. This study standardized XF analyses methods (mitochondrial stress test, glycolytic stress test and palmitate oxidation test) and established age related differences in bioenergetics profiles of healthy CMs at newborn (NB1), weaning (3WK), adult (10WK) and aged (12–18MO) time points. Findings show that immature CMs demonstrate a more robust and sustained glycolytic capacity and a relative inability to oxidize fatty acids when compared to older CMs. The study also highlights the need to recognize the contribution of CO2 from the Krebs cycle as well as lactate from anaerobic glycolysis to the proton production rate before interpreting glycolytic capacity in CMs. Overall, this study demonstrates that caution should be taken to assure that translatable developmental time points are used to investigate mitochondrial dysfunction as a cause of cardiac disease. Specifically, XF analysis of newborn CMs should be reserved to study fetal/neonatal disease and older CMs (≥10 weeks) should be used to investigate adult disease pathogenesis. Knowledge gained will aid in improved investigation of developmentally programmed heart disease and stress the importance of discerning maturational differences in bioenergetics when developing mitochondrial targeted preventative and therapeutic strategies for cardiac disease.

DJ-1 is indispensable for the S-nitrosylation of Parkin, which maintains function of mitochondria

“…2j). Furthermore, we evaluated mitochondrial respiration and glycolytic function by measuring the cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells, as described in the Materials and Methods section 21 . There were significant differences in the "basal OCR", "proton leak", "spare respiratory capacity", "non-mito respiration" and "ATP production" aspects of mitochondrial respiration ( Fig.…”

“…For analyses of mitochondrial respiration and glycolytic function, the cellular OCR and ECAR were measured using an XFp extracellular flux analyzer and an XFp extracellular flux cartridge (Seahorse Bioscience, North Billerica, MA) in combination with an XFp Cell Mito Stress Test Kit and XFp Glycolysis Stress Test Kit, respectively 21 . Cells were plated in an XFp miniplate (Seahorse Bioscience) at a concentration of 3.0-4.0 × 10 4 cells/well (three wells/sample) and incubated at 37 °C for 24 h in a 5% CO 2 atmosphere.…”

Maternal High Fat Diet and Diabetes Disrupts Transcriptomic Pathways that Regulate Cardiac Metabolism and Cell Fate in Newborn Rat Hearts

“…A limitation of this study is that we did not replicate the same severity of glycolytic impairment, but found impaired aerobic glycolysis, higher GSK3β, and more cardiac glycogen deposition, consistent with a chronic shift from glycolysis to gluconeogenesis. This switch carries signi cant bioenergetic consequences as the newborn heart relies primarily on aerobic glycolysis for ATP production [57,58]. Our ndings demonstrate that diabetic pregnancy, especially combined with hyperlipidemia precipitates cardiac "insulin resistance" in the developing fetal heart.…”

Evaluation of mitochondrial respiratory function in rat cardiomyocytes under different glucose conditions, using an extracellular flux analysis method

“…MRF was evaluated via a mitochondrial stress test that was performed by observing the responses to injections of oligomycin (3 μmol/L), FCCP (2.5 μmol/L) and rotenone (1 μmol/L) plus antimycin A (1 μmol/L). The respiratory parameters described below were analysed according to the methods described in past reports …”