Figure 1.
Crif1Δ/Δ and CAP treated Crif1+/+ MEFs show mitochondrial abnormalities.
(A) Western blot analysis of Crif1 expression in Crif1+/+ and Crif1Δ/Δ MEFs. (B) Schematic presentation of experimental procedures. At day 0 (D0), MEFs were infected with retrovirus that expresses Cre recombinase. After puromycin selection, mitochondrial characteristics were analyzed after the treatment of either ethanol (EtOH) vehicle or chloramphenicol (CAP). (C) At day 8, cells were immunostained with Cox I antibody (in green) and DAPI (in blue) (upper panel), or stained with Mitotracker Red (bottom panel). Scale bars, 10 µm. (D, E) To measure mitochondrial membrane potential (MMP), MEFs were stained with tetramethylrhodamine ethyl ester (TMRE), a cationic fluorescent dye sensitive to MMP, and analyzed by flow cytometry on the FL2 channel at day 8 (D) and day 11 (E). Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a proton ionophore that disrupts proton gradient, was treated to gain the control peak of MMP decrease.
Figure 2.
Inducible excision of Crif1 in adult cardiac myocytes causes cardiac hypertrophy.
(A) Scheme of tamoxifen injections. 6 week old Crif f/f (Crif1WT) and Myh6-cre/Esr1;Criff/f (Crif1iCKO) mice were administrated with tamoxifen intraperitoneally at a dose of 20 mg/kg/day, for 8 days. Black arrows, days with injections. White arrows, days without injections. Day 0, start of days counted. (B) Western blot analysis of Crif1 expression in Crif1WT and Crif1iCKO hearts at 1∼5 months post tamoxifen injections (C) The survival graph of Crif1WT and Crif1iCKO mice. Survivality were checked every week after tamoxifen (txm) injections. n = 12 for both groups. (D) H&E staining of the heart sections from Crif1WT and Crif1iCKO mice at 5 months post tamoxifen injections. Scale bar, 1 mm. (E) heart weight per body weight ratio, (F) body weight and (G) heart weight per tibia length ratio of Crif1WT and Crif1iCKO mice at 3 ∼ 5 months. The tibia length of Crif1iCKO mice was not different from that of Crif1WT mice. n = 5 for each genotype at 3 months, n = 7 for Crif1WT at 4 months, n = 9 for Crif1iCKO at 4 months, n = 10 for Crif1WT at 5 months, n = 12 for Crif1WT at 5 months. Error bars show SD. *P<0.05, ***P<0.005.
Figure 3.
Crif1 loss impairs in vivo heart function.
(A) Echocardiographic tracings of Crif1WT and Crif1iCKO mice at 4 months post tamoxifen injections. Scale bar = 0.1 sec. (B) Values of ejection fraction (EF) and fractional shortening (FS) obtained from echocardiography. n = 8 for Crif1WT and n = 11 for Crif1iCKO mice. Error bars show SD. ***P<0.0001.
Figure 4.
Crif1 is required for mitochondrial respiratory activities in cardiac muscle.
(A∼C) Oxygen consumption rates and (D∼F) ATP synthesis rates using the saponin-permeablized cardiac fibers from Crif1WT and Crif1iCKO mice at 4.5 months post tamoxifen injections. (A, D) Pyruvate-malate respiration. n = 6 for each genotype. (B, E) PC-malate respiration. n = 6 for each genotype. (C, F) Glutamate-malate respiration. n = 5 for Crif1WT and n = 6 for Crif1iCKO mice. Error bars show SE. *P<0.05, **P<0.01, ***P<0.005. No significant changes were observed in ATP/O with all substrates (P>0.05). (G) COX, (H) SDH and (I) H&E stain on the cardiac sections of Crif1WT and Crif1iCKO mice at indicated time points. Scale bar, 100 µm. Quantification of (J) COX and (K) SDH staining intensities. Microscope images were analyzed using ImageJ software. The total intensity of the Crif1iCKO heart (black) is shown as the relative ratio to the Crif1WT stain (white). Microscope images were taken from two different sections for each heart sample. Three heart samples were collected for each genotype. Error bars show SE. *P<0.05, ***P<0.005.
Figure 5.
Crif1 loss increases the mass of abnormal mitochondria.
(A) Low and (B) high magnification images of transmission electron microscopy. Experiments were performed using the cardiac apex from Crif1WT and Crif1iCKO mice at 5 months post tamoxifen injections. Scale bar, 2 µm (A) and 0.2 µm (B).
Figure 6.
Crif1 deletion in the cardiac muscle using the Ckmm-cre transgene leads to cardiac hypertrophy.
(A) Western blot analysis of Crif1 expression in isolated hearts and soleus muscles from Crif1f/f and Ckmm-cre;Crif1f/f mice. Protein lysates were prepared from P0 or P10 mouse tissues. (B) H&E staining of heart sections from Crif1f/f (W1 and W2) and Ckmm-cre;Crif1f/f mice (K1 and K2) at P10. Scale bar, 1 mm. (C) Heart weight per body weight ratios of P10 mice. n = 7 for each genotype. Error bars show SD. ***P = 0.00001. (D) COX and SDH stain on snap-frozen P10 heart sections. Scale bars, 100 µm. (E) Quantification of COX and SDH staining intensities using ImageJ. The total intensity of the Ckmm-cre;Crif1f/f heart (black) is shown as the relative ratio to the Crif1f/f stain (white). Microscope images were taken from two different sections for each heart sample. Three heart samples were collected for each genotype. Error bars show SE. *P<0.05, ***P<0.005.