Responses of hypertrophied myocytes to reactive species: implications for glycolysis and electrophile metabolism

Abstract

Synopsis During cardiac remodeling, the heart generates higher levels of reactive species; yet an intermediate “compensatory” stage of hypertrophy is associated with a greater ability to withstand oxidative stress. The mechanisms underlying this protected myocardial phenotype are poorly understood. We examined how a cellular model of hypertrophy deals with electrophilic insults, such as would occur upon ischemia or in the failing heart. For this, we measured energetics in control and phenylephrine (PE)-treated rat neonatal cardiac myocytes (NRCMs) under basal conditions and when stressed with 4-hydroxynonenal (HNE). PE treatment caused hypertrophy as indicated by augmented atrial natriuretic peptide and increased cellular protein content. Hypertrophied myocytes demonstrated a 2.5-fold increase in ATP-linked oxygen consumption and a robust augmentation of oligomycin-stimulated glycolytic flux and lactate production. Hypertrophied myocytes displayed a protected phenotype that was resistant to HNE-induced cell death and a unique bioenergetic response characterized by a delayed and abrogated rate of oxygen consumption and a twofold increase in glycolysis upon HNE exposure. This augmentation of glycolytic flux was not due to increased glucose uptake, suggesting that electrophile stress results in utilization of intracellular glycogen stores to support the increased energy demand. Hypertrophied myocytes also had an increased propensity to oxidize HNE to 4-hydroxynonenoic acid and sustained less protein damage due to acute HNE insults. Inhibition of aldehyde dehydrogenase resulted in bioenergetic collapse when myocytes were challenged with HNE. The integration of electrophile metabolism with glycolytic and mitochondrial energy production appears to be important for maintaining myocyte homeostasis under conditions of increased oxidative stress.

Standardized bioenergetic profiling of adult mouse cardiomyocytes

“…In contrast to spontaneously beating isolated neonatal rat cardiomyocytes (NRCMs), isolated ACMs appear to be largely oligomycin-insensitive. This indicates that proton leak is a major oxygen-and substrate-consuming process in isolated, nonbeating ACMs and they, once isolated, remain in a state 4-like state (21).…”

“…The bioenergetic response of ACMs was measured with the Seahorse Bioscience XF24 Flux Analyzer (5,10,20,21). For seeding density experiments, the plating media were changed to 675 l unbuffered DMEM supplemented with 4 mmol/l glutamine and 1 mmol/l pyruvate 1 h before assay.…”

“…Therefore, studying bioenergetics in intact cells may provide a more physiological view of mitochondrial function. Recently, bioenergetic profiling of intact cells has been described in multiple cell lines (1,9,14,16) and primary cells (6,7,10,11,20,21,28) using a microplate-based system that measures extracellular flux (XF). This automated system measures the two major cellular energyproducing pathways, glycolysis and mitochondrial respiration.…”

Cardiomyocyte Ogt is essential for postnatal viability

“…The Seahorse Flux Analyzer (North Billerica, MA) was used to measure the O 2 consumption of isolated mitochondria, similar to previously described reports of intact cells (31,33). During sensor calibration, isolated mitochondria were seeded in a 50-l volume of isolation buffer containing 5 g protein/ well in XF24 V7 cell culture microplates.…”

High glucose induces mitochondrial dysfunction independently of protein O-GlcNAcylation

“…The bioenergetics of intact adherent NRCMs that were seeded at 75000 cells per well was measured using a Seahorse Bioscience XF24 Flux Analyzer [42,47,50,52,53]. For these experiments, the treatment medium was replaced with 675 µl of assay medium: unbuffered DMEM supplemented with glucose (5 or 33 mM; or appropriate concentration of mannitol osmotic control), 4 mM glutamine and 1 mM pyruvate, and plates were placed in a non-CO 2 supplemented incubator 1 h before assay.…”