Decreased mtDNA, oxidative stress, cardiomyopathy, and death from transgenic cardiac targeted human mutant polymerase gamma

Abstract

POLG is the human gene that encodes the catalytic subunit of DNA polymerase gamma (Pol gamma), the replicase for human mitochondrial DNA (mtDNA). A POLG Y955C point mutation causes human chronic progressive external ophthalmoplegia (CPEO), a mitochondrial disease with eye muscle weakness and mtDNA defects. Y955C POLG was targeted transgenically (TG) to the murine heart. Survival was determined in four TG (+/-) lines and wild-type (WT) littermates (-/-). Left ventricle (LV) performance (echocardiography and MRI), heart rate (electrocardiography), mtDNA abundance (real time PCR), oxidation of mtDNA (8-OHdG), histopathology and electron microscopy defined the phenotype. Cardiac targeted Y955C POLG yielded a molecular signature of CPEO in the heart with cardiomyopathy (CM), mitochondrial oxidative stress, and premature death. Increased LV cavity size and LV mass, bradycardia, decreased mtDNA, increased 8-OHdG, and cardiac histopathological and mitochondrial EM defects supported and defined the phenotype. This study underscores the pathogenetic role of human mutant POLG and its gene product in mtDNA depletion, mitochondrial oxidative stress, and CM as it relates to the genetic defect in CPEO. The transgenic model pathophysiologically links human mutant Pol gamma, mtDNA depletion, and mitochondrial oxidative stress to the mtDNA replication apparatus and to CM.

Figure 1

Y955C TG Pol γ protein expression and gene copy dosage: Protein from purified mitochondrial lysates of TG and control hearts were separated on a 4–12% poly-acrylamide gel and either transferred to Immobilon-P for Western blot analysis or Coomassie Blue staining. A) Western blot analysis of the heart mitochondrial extracts. Lane 1, Purified recombinant human Pol γ as control, Lanes 2, 4 and 6, mitochondrial lysates from Y955C TG hearts of 33, 25 and 10 week old mice, respectively. Lanes 3, 5 and 7, mitochondrial lysates from control TG negative hearts of 33, 25, and 10 week old mice, respectively. Arrow indicates pol γ. B) Coomassie Blue stained gel to demonstrate that equivalent portions of heart mitochondrial proteins were analyzed by Western blot. Lanes 1–7 are identical to that in panel A except that 5 times the amount of protein was loaded and separated in the gel for detection by Coomassie Blue staining. C. Relative gene copy number of mutant Y955C Pol γ was determined from cardiac tissues for each of the 4 lines generated. Arrow indicates pol γ.C) Relative gene copy number of human mutant Y955C Pol γ was determined from cardiac tissues for each of the 4 lines generated. Levels of POLG were analyzed semi-quantitatively from murine tail DNA extracts using real-time PCR and Light Cycler TaqMan Master kit and normalized to GAPDH from WT.

Figure 2

Kaplan-Meier survival curve and histopathologic assessment of Y955C TG: A) Differential survival in the different TG lines was found. Median survival was 90d in line D, 210d in line C, and >420d in line E, >600d in line B which most resembled the WT. B) Histopathological (Masson’s Trichrome stain) correlate with death in TG line D: Autopsy performed on 21 day old TG from line D. Hemi-section of thorax reveals massive atrial enlargement (A) suggesting heart failure (arrows) and compressing the lungs (L).

Figure 3

Real time PCR mtDNA/nDNA ratios and steady state abundance of 2dG and 8-OHdG in mitochondrial extracts from WT and Y955C TG mice: A) WT and TG cohorts’ cardiac samples (N ≥ 8) were analyzed using real time PCR. Decrease in mtDNA/nDNA ratio was found with Y955C Pol γ TG (C line) (*p<0.01). B) Mitochondria were isolated according to standard methods and mtDNA extracted. Nucleotide abundance was expressed as a mean molar ratio and standard error of the mean (SEM) of 8-OHdG to 2dG x10⁻⁵. Steady state abundance of 8-OHdG in Y955C hearts was 3-fold that found in WT (p< 0.05), (N=6).

Figure 4

Quantitative analysis of ECHO images in Y955C Pol γ TG: LV mass was calculated in a blinded fashion, code was broken, and data tabulated from the Y955C Pol γ TG lines at 120 days. Data were normalized to body weight (mg/g) and plotted as mean ± SEM. At 120 days, line C and D had survivors. They exhibited cardiac mass that was >100% increased above that of WT (*p<0.01).

Figure 5

MRI of TG and WT hearts: MRI images of the murine heart were performed using methods as described previously. Four chamber enlargement in TG Y955C (left) and WT littermate (right) at 9 months of age. TG demonstrates 4 chamber enlargement of the heart with both right ventricle (open arrow) and left ventricle (filled arrow) enlargement.

Figure 6

Histopathological (A, B) and EM (C) images of hearts and of mitochondria from Y955C Pol γ TGs: Gender matched littermates expressing Y955C TG or WT controls were used. TG over-expression in the heart caused cardiomyopathy with bi-ventricular thickening and cardiomegaly. Arrow points to lytic change with increased granularity. (Y955C: A, original magnification 1X, and B, original magnification 600 X). Histopathological results (hematoxylin and eosin staining) were supported by mitochondrial damage and enlargement as seen by EM (C, original magnification on EM: 26,000. Marker is 1μ).

Figure 7

Schematic Summary of Effects of Y955C Pol γ in murine model. Cardiac targeted over-expression of Y955C mutant of Pol γ results in altered mtDNA biogenesis leading to cardiac dysfunction and ultimately premature death. Demonstrated are presence of both native Pol γ and over-expressed Y955C mutants.