Doxorubicin Increases Oxidative Metabolism in HL-1 Cardiomyocytes as Shown by 13C Metabolic Flux Analysis

Abstract

Doxorubicin (DXR), an anticancer drug, is limited in its use due to severe cardiotoxic effects. These effects are partly caused by disturbed myocardial energy metabolism. We analyzed the effects of therapeutically relevant but nontoxic DXR concentrations for their effects on metabolic fluxes, cell respiration, and intracellular ATP. (13)C isotope labeling studies using [U-(13)C(6)]glucose, [1,2-(13)C(2)]glucose, and [U-(13)C(5)]glutamine were carried out on HL-1 cardiomyocytes exposed to 0.01 and 0.02 μM DXR and compared with the untreated control. Metabolic fluxes were calculated by integrating production and uptake rates of extracellular metabolites (glucose, lactate, pyruvate, and amino acids) as well as (13)C-labeling in secreted lactate derived from the respective (13)C-labeled substrates into a metabolic network model. The investigated DXR concentrations (0.01 and 0.02 μM) had no effect on cell viability and beating of the HL-1 cardiomyocytes. Glycolytic fluxes were significantly reduced in treated cells at tested DXR concentrations. Oxidative metabolism was significantly increased (higher glucose oxidation, oxidative decarboxylation, TCA cycle rates, and respiration) suggesting a more efficient use of glucose carbon. These changes were accompanied by decrease of intracellular ATP. We conclude that DXR in nanomolar range significantly changes central carbon metabolism in HL-1 cardiomyocytes, which results in a higher coupling of glycolysis and TCA cycle. The myocytes probably try to compensate for decreased intracellular ATP, which in turn may be the result of a loss of NADH electrons via either formation of reactive oxygen species or electron shunting.

Doxorubicin-induced carbonylation and degradation of cardiac myosin binding protein C promote cardiotoxicity

“…We used HL-1 cells for in vitro study of MyBPC protein carbonylation. HL-1 cells have been used to study normal cardiomyocyte functions (34) and to study Dox-induced cytotoxicity and metabolic changes (35). Consistent with previous observations (35), less than 200 nM Dox was not toxic to cells within 24 h and significant MyBPC protein carbonylation and degradation were not observed under this concentration.…”

“…HL-1 cells have been used to study normal cardiomyocyte functions (34) and to study Dox-induced cytotoxicity and metabolic changes (35). Consistent with previous observations (35), less than 200 nM Dox was not toxic to cells within 24 h and significant MyBPC protein carbonylation and degradation were not observed under this concentration. We observed that 500 nM is the optimal concentration of Dox required to induce MyBPC carbonylation, degradation, and cytotoxicity in HL-1 cells at 24 h. Carbonylation and degradation of MyBPC were also observed in SHRs under cardiotoxic conditions (23).…”

Loss of Krüppel-like factor 6 cripples podocyte mitochondrial function

“…In human podocytes, knockdown of SCO2 expression increased cleaved caspase-9 levels and release of cytochrome c, adding some support to the proposal by Mallipattu and colleagues that SCO2 may be a key KLF6 target for mediating protection against mitochondrial injury (22). The expression of a number of other mitochondrial genes was also reduced in podocyte-specific Klf6-knockout mice, including nuclear oxidation, and this may impose oxidative stress (25). This suggests that KLF6 contributes to progression of glomerular injury, at least in this model.…”

Non-stationary 13C metabolic flux analysis of Chinese hamster ovary cells in batch culture using extracellular labeling highlights metabolic reversibility and compartmentation

“…Metabolic flux analysis has been applied for mammalian cells for a long time already but mostly only using metabolite balancing [8]. The labelling of extracellular excreted lactate and CO 2 has already been used in the past but only at metabolic steady state [8-11]. The use of other labelled metabolites, e.g.…”