Cardiac effects of OPA1 protein promotion in a transgenic animal model

Abstract

Mitochondria form a dynamic network in cells, regulated by the balance between mitochondrial fusion and fission. The inhibition of mitochondrial fission can have positive effects in acute ischemic/reperfusion injury models by preventing the fall in mitochondrial membrane potential associated with fission processes. However, inhibition of fission in chronic models is disadvantageous because it obstructs the elimination of damaged mitochondrial fragments. OPA1, in view of previous results, is a possible therapeutic target as a fusion promoter and structure stabilizer protein. We used transgenic mice in which the OMA1 cleavage sites of OPA1 were deleted. This resulted in a higher representation of L-OPA1 compared to S-OPA1. After genotyping and model validation, all animals were examined by echocardiograph on two occasions, at weeks 11 and 36. Histological samples were taken from hearts to examine mitochondrial morphology and structure remodeling. The signaling pathways related to mitochondrial dynamic processes were evaluated. Cardiomyocytes were isolated from neonatal mice to determine the efficiency of mitochondrial respiration using the SeaHorse assay method. OPA1 protein promotion has a negative effect on systolic function during aging. We confirmed that volume overload and ventricular remodeling did not manifest. The reason behind the loss of pump function might be, at least partly, due to the energy deficit caused by mitochondrial respiratory failure and damage in mitochondrial quality control pathways.

Fig 1. Schematic picture of mitochondrial quality control processes.

Mitochondrial quality control consists of biogenesis, fusion, fission and mitophagy. To maintain optimal mitochondrial function, these processes are in dynamic equilibrium. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha and beta (PGC1 α and β) regulate mitochondrial biogenesis. Mitochondrial fusion promoted by OPA1 and Mfn 1,2. Cleavage of mitochondrial fragments is facilitated by DRP1 and Fis1 proteins. Mitophagy is mediated Parkin and PINK1 proteins. Cytochrome C is released from the damaged mitochondria in case of insufficient mitophagy, which induces apoptosis.

Fig 2. Effect of OPA1 transgenic phenotype on the extent of interstitial fibrosis at the age of 36-weeks.

Representative histological sections stained with Masson’s trichrome (A) (n = 6). Scale bar: 50 μm, magnification: 10-fold. Densitometric evaluation of the section is shown (B). Data are expressed as mean ± SEM. WT vs Positive Control ¥¥ p < 0.01, TG vs Positive Control ## p < 0.01.

Fig 3. Effect of OPA1 transgenic phenotype on cardiomyocyte cell diameter at the age of 36 weeks.

Representative histological sections stained with Picrosirius red (A) (n = 6). Scale bar: 20 μm, magnification: 40-fold. The average cellular diameter in the two groups is shown (B). WT: wild-type mice, TG: OPA1 transgenic mice.

Fig 4. Effect of OPA1 transgenic phenotype on the oxidative stress.

Representative immunohistochemical staining and densitometric evaluation of the section for nitrotyrosine (A) and 8-Oxoguanine (B) in the heart (n = 6). Scale bar: 50 μm, magnification 20-fold. WT: wild-type mice, TG: OPA1 transgenic mice.

Fig 5. Effect of OPA1 transgenic phenotype on interfibrillar mitochondria of the myocardium.

(A-B) Representative electron micrograph of interfibrillar mitochondria of wild-type (A) and OPA1 transgenic (B) mice (magnification 12 k, scale bar: 0.5 μm). (C-D) Ultrastructure of interfibrillar mitochondria in the myocardium of wild-type (C) and transgenic (D) animals (magnification: 25 k, scale bar: 0.5 μm). (E) Means of area values in given groups (~500 mitochondria/group). (F) Relative frequencies of measured mitochondrial areas in each arbitrary interval. WT: wild-type mice, TG: OPA1 transgenic mice. Data are expressed as mean ± SEM. * p < 0.05 vs. WT, n = 6.

Fig 6. Fluorescent staining of the mitochondrial network.

The neonatal mice cardiomyocyte cells were stained with 100 nM MitoTracker Red CMXRos, scale bar: 25 μm, magnification: 60-fold. Groups: WT: wild-type cardiomyocytes, TG: NMCM cells from OPA1 transgenic mice.

Fig 7. Quantification analysis of the mitochondrial branch length.

The neonatal mice cardiomyocyte cells were stained with 100 nM MitoTracker Red CMXRos, Mitochondria Network Analysis tool (MiNA) on the ImageJ interface. scale bar: 25 μm, magnification: 60-fold. Groups: WT: wild-type cardiomyocytes, TG: NMCM cells from OPA1 transgenic mice.

Fig 8. Effect of OPA1 transgenic phenotype on mitochondrial membrane potential in NMCM cells.

Cells were stained with 5 μg/mL of JC-1, which is a membrane potential sensitive dye. The dye was loaded and after 90 minutes incubation, fluorescent microscopic images were taken using both the red and green channels. A: representative images are presented. Scale bar: 50 μm, magnification: 40-fold, WT: wild-type cardiomyocytes, TG: NMCM cells from OPA1 transgenic mice. WT+FCCP: wild-type NMCM cells treated with 10 μM FCCP. B: quantitative analysis of mitochondrial polarization. Data are presented as the mean ± SEM of four independent measurements, n = 6. WT vs TG **p<0.01, WT vs WT+FCCP ##p<0.01, TG vs WT+FCCP ¥¥ p<0.01.

Fig 9. Changes in the oxygen consumption rate of OPA1 transgenic NMCM cells.

A: oxygen consumption rate (OCR) in NMCM cells, measured by Seahorse XFp Analyser, B: basal respiration, C: maximal respiration, D: ATP production and E: spare respiratory capacity, WT: wild-type cardiomyocytes, TG: NMCM cells from OPA1 transgenic mice. *p<0.05 vs. WT. Values are means ± SEM, n = 6.

Fig 10. Effect of OPA1 transgenic phenotype on mtDNA copy number.

A: Average number of copies, B: Average number of copies fold change, Using GenElute^™ Mammalian DNA Miniprep kit For quantification of mtDNA copy number, real-time PCR analysis was performed with the NovaQUANT^™ Mouse Mitochondrial to Nuclear DNA Ratio Kit. WT: wild-type, TG: OPA1 transgenic mice. Values are means ± SEM, n = 8.

Fig 11. Changes of OPA1, OMA1 and YME1L1 level Representative Western blot analysis of OPA1, DYKDDDDK Tag (FLAG), VDAC, BNIP3, OMA1, YME1L1 and densitometric evaluation are shown.

GAPDH was used as a loading control. WT: wild-type mice(n = 8), TG: OPA1 transgenic mice (n = 8). Values are mean ± SEM. ** p < 0.01 vs. WT.

Fig 12. Changes in protein levels of mitochondrial dynamics.

Representative Western blot analysis of Mfn1, Mfn2, DRP1, Fis1, PINK1, Parkin and densitometric evaluation are shown. GAPDH was used as a loading control. WT: wild-type mice (n = 8), TG: OPA1 transgenic mice (n = 8). Values are mean ± SEM. * p < 0.01 vs. WT.