An alternative renin isoform is cardioprotective by modulating mitochondrial metabolism

Abstract

The renin-angiotensin system promotes oxidative stress, apoptosis, necrosis, fibrosis, and thus heart failure. Secretory renin plays a central role in these processes, initiating the generation of angiotensins. Nevertheless, alternative renin transcripts exist, which code for a cytosolically localized renin isoform (cyto-renin) that is cardioprotective. We tested the hypothesis that the protective effects are associated with a beneficial switch of metabolic and mitochondrial functions. To assess H9c2 cell mitochondrial parameters, we used the Seahorse XF analyser. Cardiac H9c2 cells overexpressing cyto-renin exhibited enhanced nonmitochondrial oxygen consumption, lactate accumulation, and LDH activity, reflecting a switch to more aerobic glycolysis known as Warburg effect. Additionally, mitochondrial spare capacity and cell respiratory control ratio were enhanced, indicating an increased potential to tolerate stress conditions. Renin knockdown induced opposite effects on mitochondrial functions without influencing metabolic parameters. Thus, the protective effects of cyto-renin are associated with an altered bioenergetic profile and an enhanced stress tolerance, which are favourable under ischaemic conditions. Therefore, cyto-renin is a promising new target for the prevention of ischaemia-induced myocardial damage.

Keywords: H9c2 cells; Warburg effect; aerobic glycolysis; cytosolic renin; exon(1A-9)renin; exon-b renin; heart; mitochondrial spare capacity.

Figure 1

Basal oxygen consumption rate increases with cell number. Oxygen consumption rate (OCR) was analysed using the Seahorse technology. Real‐time measurements were obtained from H9c2 cells seeded at different numbers per well (n = 7). A, Distinct modules of cellular oxygen consuming processes. B, Real time OCRs obtained from H9c2 cells seeded with different cell numbers/well (n=6). Data represent mean ± SEM values

Figure 2

Mitochondria of cyto‐renin overexpressing cells exhibit a beneficial bioenergetic profile. Oxygen consumption rate (OCR) was analysed after injection of inhibitors of the respiratory chain at indicated time‐points. A, Real‐time OCRs obtained from H9c2 cells (n = 11), pIRES control cells (n = 12) and ren(2‐9) cells (n = 20). B, OCRs in cells after injection of rotenone (R) and antimycin A (AA) (cellular respiration) according to mitochondrial (blue) and nonmitochondrial components (yellow). C, OCRs in mitochondria after injection of oligomycin (Oligo) (mitochondrial respiration) according to ATP‐linked respiration (orange) and proton leak (grey). D, Maximal OCRs obtained after injection of the uncoupler FCCP (maximal respiration) (green) and spare capacity (green shaded). Spare capacity was calculated by subtracting mitochondrial respiration from maximal respiration. E, Coupling efficiency (CE) of oxidative phosphorylation was determined as the oligomycin‐sensitive portion of mitochondrial respiration. F, Maximal cell respiratory control (CRC_Max) was derived from the ratio of maximal respiration to oligomycin‐insensitive proton leak OCR. Data represent mean ± SEM values with ***P < 0.001 or **P < 0.01 control cell lines vs ren(2‐9) cells; #P < 0.05 total cellular respiration control cell lines vs the ren(2‐9) cells

Figure 3

Bioenergetic profile of H9c2 cells is affected disadvantageously by renin knockdown. OCR was analysed after injection of inhibitors of the respiratory chain at indicated time‐points. A, Real‐time OCRs obtained from scramble controls (n = 9) and cells after knockdown of renin using 80 nmol siRNA (n = 9). B, Cellular respiration after injection of rotenone (R) and antimycin A (AA) representing mitochondrial (blue) and nonmitochondrial components (yellow). C, OCRs in mitochondria after injection of oligomycin (Oligo) (mitochondrial respiration) according to ATP‐linked respiration (orange) and proton leak (grey). D, Maximal OCRs (green) measured after injection of the uncoupler FCCP and spare capacity (green shaded). Spare capacity was calculated by subtracting mitochondrial respiration from maximal respiration. E, Coupling efficiency (CE) of oxidative phosphorylation was determined as the oligomycin‐sensitive portion of mitochondrial respiration. F, Maximal cell respiratory control (CRC_Max) was derived from the ratio of maximal respiration to oligomycin‐insensitive proton leak OCR. Data represent mean ± SEM values with **P < 0.01 or *P < 0.05 scramble control vs siRenin‐treated H9c2 cells. #P < 0.05 total cellular respiration scramble control vs siRenin‐treated cells

Figure 4

Influence of renin on the mitochondrial membrane potential. Mitochondrial membrane potential was determined as mean fluorescence intensity after addition of MitoTracker red CMXRos (A, B) or JC1 (C, D). Both mitochondrial probes were added either to H9c2, pIRES and ren(2‐9) cells (each n = 10) or scramble controls and renin knockdown cells (n = 10‐14). Fluorescence intensity of the cells was measured by flow cytometry. Data represent mean ± SEM values with ***P < 0.001 or *P < 0.05 ren(2‐9) cells or siRenin‐treated cells to the corresponding controls

Figure 5

Visualization of the mitochondrial network in H9c2 cell lines. Mitochondrial distribution in pIRES and ren(2‐9) cells was evaluated by the labelling with MitoTracker red CMXRos probe (red) combined with the membrane dye wheat germ agglutinin (WGA, green) and the nuclear dye 4′,6′‐diamidino‐2‐phenylindole (DAPI, blue) (A). Magnification amounted to 100x. Location of mitochondria in relation to the nucleus and the cell membrane was determined by the Line Profile software of the BZ II 9000 microscop (B)

Figure 6

Glycolysis‐related parameters are increased in ren(2‐9) cells. A, Extracellular lactate concentration. B, Glucose uptake. C, Real‐Time measurement of extracellular acidification rate (ECAR, mpH/min) determined by the Seahorse analyser. D, Lactate dehydrogenase (LDH) activity in lysed cells. Data represent mean ± SEM of n = 12 measurements with ***P < 0.01 and **P < 0.01 control H9c2 and pIRES cell lines vs ren(2‐9) cells

Figure 7

Glycolysis‐related parameters are unchanged in H9c2 cells pretreated by siRNA to renin. A, Extracellular lactate concentration. B, Glucose uptake. C, Real‐Time measurement of extracellular acidification rate (ECAR, mpH/min) determined by the Seahorse analyser. D, Lactate dehydrogenase (LDH) activity in lysed cells. Data represent mean ± SEM of n = 9 values