Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 424 (1), 99-107

Importance of the Bioenergetic Reserve Capacity in Response to Cardiomyocyte Stress Induced by 4-hydroxynonenal

Affiliations

Importance of the Bioenergetic Reserve Capacity in Response to Cardiomyocyte Stress Induced by 4-hydroxynonenal

Bradford G Hill et al. Biochem J.

Abstract

Mitochondria play a critical role in mediating the cellular response to oxidants formed during acute and chronic cardiac dysfunction. It is widely assumed that, as cells are subjected to stress, mitochondria are capable of drawing upon a 'reserve capacity' which is available to serve the increased energy demands for maintenance of organ function, cellular repair or detoxification of reactive species. This hypothesis further implies that impairment or depletion of this putative reserve capacity ultimately leads to excessive protein damage and cell death. However, it has been difficult to fully evaluate this hypothesis since much of our information about the response of the mitochondrion to oxidative stress derives from studies on mitochondria isolated from their cellular context. Therefore the goal of the present study was to determine whether 'bioenergetic reserve capacity' does indeed exist in the intact myocyte and whether it is utilized in response to stress induced by the pathologically relevant reactive lipid species HNE (4-hydroxynonenal). We found that intact rat neonatal ventricular myocytes exhibit a substantial bioenergetic reserve capacity under basal conditions; however, on exposure to pathologically relevant concentrations of HNE, oxygen consumption was increased until this reserve capacity was depleted. Exhaustion of the reserve capacity by HNE treatment resulted in inhibition of respiration concomitant with protein modification and cell death. These data suggest that oxidized lipids could contribute to myocyte injury by decreasing the bioenergetic reserve capacity. Furthermore, these studies demonstrate the utility of measuring the bioenergetic reserve capacity for assessing or predicting the response of cells to stress.

Figures

Figure 1
Figure 1. Measurement of bioenergetic parameters in myocytes using extracellular flux technology
Oxygen consumption rate (OCR) from isolated neonatal rat ventricular myocytes (NRVM): (A) NRVM were seeded at 25,000–75,000 cells/well and the oxygen consumption rate (OCR) was measured. y=0.0027x – 25.244, R2 = 0.99. (B) In situ mitochondrial function assay: After three baseline OCR measurements, oligomycin (1 μg/ml), FCCP (1 μM), and antimycin A (10 μM) are injected sequentially with OCR measurements recorded after each injection. ATP-linked oxygen consumption (ATP) and the OCR due to proton leak (leak) can be calculated using the basal and the oligomycin-sensitive rate. Injection of the uncoupling agent, FCCP, is then used to determine the maximal respiratory capacity. Lastly, injection of antimycin A allows for the measurement of non-mitochondrial oxygen consumption. The reserve capacity is calculated by subtracting the maximal rate of oxygen consumption by the pre-oligomycin rate.
Figure 2
Figure 2. HNE promotes protein damage and myocyte cell death
NRVM were treated with the indicated concentrations of HNE or nonanal for 90 min for measurement of protein-HNE adducts or for 8 and 16 h for cell death assay. (A) Western blots of HNE-modified proteins: After treatment with the indicated concentrations of HNE, myocytes were lysed, and protein-HNE adducts were detected by immunoblotting. Actin was used as a normalization control for Western blotting experiments, and nonanal was used as a non-electrophilic analog control for HNE. (B) Relative quantification of protein-HNE modifications: Protein-HNE antibody immunoreactivity was quantified by densitometry, and the fold change of immunoreactivity over non-treated cells was plotted as a function of HNE concentration; y=1.4127x – 0.1261, R2=0.99. (C) Cell death assay of HNE-treated myocytes: NRVM were exposed to 0–30 μM HNE for 8h (■) and 16h (□), followed by measurement of cell death by MTT assay. The inset shows the micrograph of the control and HNE treated cells (magnification 10 x). D) Total GSH content was measured in NRVM cell lysates exposed to 0–20μM HNE for 90 min. Data are expressed as the nmol GSH/mg protein. All data shown are means ± sem, n≥3; *p<0.01 vs. non-HNE treated myocytes from each time point.
Figure 3
Figure 3. HNE increases oxygen consumption in isolated myocytes
Oxygen consumption rate (OCR) plots from myocytes exposed to 0–30 μM HNE: (A) The basal OCR was measured followed by addition of 0 (filled squares, solid line), 5 (open squares, solid line), 10 (filled squares, dashed line), 20 (open squares, dashed line, and 30 μM HNE (closed squares, dotted line), as indicated by the arrow. The rate of oxygen consumption was then measured for the indicated time. The OCR values are shown as the percent of baseline for each group. For visual clarity, statistical indicators were omitted from the graph. (B) The slope of the initial increase in OCR due to HNE treatment was then measured and plotted as the rate of OCR increase; y=0.1065x + 0.1283, R2=0.99. (C) Area under the curve analyses were used to determine the overall amount of oxygen consumed with each treatment. In panels A–C, data shown are means ± SEM, n≥3. *p<0.05 vs. cells not treated with HNE; #p<0.05 vs. cells treated with 5–10 μM HNE.
Figure 4
Figure 4. HNE increases glycolytic rate in isolated myocytes
Measurements of proton production from myocytes exposed to 0–30 μM HNE: (A) The basal extracellular acidification rate (ECAR) was measured followed by addition of 0 (filled squares, solid line), 5 (open squares, solid line), 10 (filled squares, dashed line), and 20 μM HNE (open squares, dashed line), as indicated by the arrow. The rates of extracellular acidification, indicative of changes in glycolytic flux, were then measured for the indicated time. For visual clarity, the 30 μM HNE group and statistical indicators were omitted from the graph. (B) The slope of the initial increase in ECAR due to HNE treatment was then measured and plotted as the rate of ECAR increase; y=0.0534x + 0.2751, R2=0.97. (C) Area under the curve analyses of the proton production rate were used to determine the overall amount of protons produced with each treatment. In panels A–C, data shown are means ± SEM, n≥3. *p<0.05 vs. cells not treated with HNE; #p<0.05 vs. cells treated with 7.5 and 10 μM HNE.
Figure 5
Figure 5. Changes in the mitochondrial and glycolytic profiles due to HNE treatment
(A) Metabolic profile of the stimulatory effect of HNE on aerobic and anaerobic respiration: The OCR and ECAR were plotted against one another at the time where OCR was increased to the greatest extent in cells treated with 20 μM HNE (from experiment in panel B – dotted line at ~80 min timepoint). (B) After measurement of the basal OCR, HNE was injected to 0 (filled squares, solid line), 5 (open squares, solid line), 10 (filled squares, dashed line), and 20 μM (open squares, dashed line) final concentrations as indicated. Mitochondrial function assay was then performed by sequential injections of oligomycin, FCCP, and antimycin A to determine the level of proton leak and ATP-linked oxygen consumption, the maximal OCR, and the non-mitochondrial OCR, respectively. Data in panels A and B are means ± SEM, n≥3. *p<0.05 vs. cells not treated with HNE; #p<0.05 vs. cells treated with 5 μM HNE; @p<0.05 vs. cells treated with 5–10 μM HNE.
Figure 6
Figure 6. Specific defects in mitochondrial function caused by HNE
The HNE-induced changes in the following parameters derived from the data in Figure 5 are shown: (A) The HNE–dependent change in OCR relative to the initial basal OCR assessed immediately before the addition of oligomycin (B) the HNE-dependent change in the Oligomycin-insensitive OCR (C) The OCR ascribed to proton leak (D) The OCR ascribed to ATP-synthesis (E) bioenergetic reserve capacity, and (F) the non-mitochondrial OCR. Data represent means ± SEM. N ≥ 3/group. *p<0.05 vs. myocytes not treated with HNE.

Similar articles

See all similar articles

Cited by 137 PubMed Central articles

See all "Cited by" articles

Publication types

Feedback