Cardiac-targeted transgenic mutant mitochondrial enzymes: mtDNA defects, antiretroviral toxicity and cardiomyopathy

Abstract

Mitochondrial (mt) DNA biogenesis is critical to cardiac contractility. DNA polymerase gamma (Pol gamma) replicates mtDNA, whereas thymidine kinase 2 (TK2) monophosphorylates pyrimidines intramitochondrially. Point mutations in POLG and TK2 result in clinical diseases associated with mtDNA depletion and organ dysfunction. Pyrimidine analogs (NRTIs) inhibit Pol gamma and mtDNA replication. Cardiac "dominant negative" murine transgenes (TGs; Pol gamma Y955C, and TK2 H121N or I212N) defined the role of each in the heart. mtDNA abundance, histopathological features, histochemistry, mitochondrial protein abundance, morphometry, and echocardiography were determined for TGs in "2 x 2" studies with or without pyrimidine analogs. Cardiac mtDNA abundance decreased in Y955C TGs ( approximately 50%) but increased in H121N and I212N TGs (20-70%). Succinate dehydrogenase (SDH) increased in hearts of all mutants. Ultrastructural changes occurred in Y955C and H121N TGs. Histopathology demonstrated hypertrophy in H121N, LV dilation in I212N, and both hypertrophy and dilation in Y955C TGs. Antiretrovirals increased LV mass ( approximately 50%) for all three TGs which combined with dilation indicates cardiomyopathy. Taken together, these studies demonstrate three manifestations of cardiac dysfunction that depend on the nature of the specific mutation and antiretroviral treatment. Mutations in genes for mtDNA biogenesis increase risk for defective mtDNA replication, leading to LV hypertrophy.

Fig. 1

Transgenic mutant TK2 lines. Southern blot analysis confirmed TK2 mutants (+), WT (-), and control (C). Gels document transgenesis in each of two mutant TK2 lines (H121N and I212N)

Fig. 2

Real-time PCR mtDNA/nDNA ratios with NRTI treatment: TGs and WT cohorts were treated with combination antiretroviral (AZT, 3TC, IDV; HAART) or vehicle control for 35d. Tissue samples were analyzed using real-time PCR. (a) I212N TGs with vehicle- or AZT-HAART treatment had increased mtDNA/nDNA ratios compared to WT littermates. (b) Vehicle-treated H121N TGs also had increased mtDNA/nDNA ratio while AZT-HAART TGs have little change compared to WTs. (c) Vehicle- or AZT-HAART treated Y955C TGs had decreased mtDNA abundance compared to WTs

Fig. 3

SDH histochemical staining in cardiac tissues from both TGs and WTs: Frozen cardiac tissues from representative I212N, H121N, and Y955C TGs and WTs, with or without antiretroviral treatment (HAART). Representative stained tissues for vehicle-treated (upper panels) and AZT-HAART (lower panels) are shown. Increase in dark blue staining is indicative of increase in SDH enzyme activity. Darker blue staining occurs in all TG cohorts including WT following AZT-HAART treatment, with the exception of H121N TG. The relative intensity of the immunohistochemistry staining for SDH using light microscopy (20S magnification) is indicated from a relative score of 0 (vehicle-treated WT) to +4

Fig. 4

Electron photomicrographs of mitochondria from cardiac myocytes of H121N and Y955C TG compared to WT: Shown are representative images for each cohort selected from minimum of 12 captured EM images/ mouse from 2-4 randomly selected mice from each cohort. AZT-HAART treated WTs have slightly enlarged mitochondria containing fewer more prominent and discernable cristae compared to vehicle-treated WTs (upper right and left panels, respectively). Spherical or oval mitochondria in the vehicle-treated and AZT-HAART treated H121N TGs appear to be accompanied by an occasional irregular shaped mitochondrion (middle panels). Vehicle-treated and AZT-HAART treated Y955C TGs have profound increase in mitochondrial number, smaller size, and irregular shape and structure with substantially reduced numbers of prominent and often truncated cristae (lower panels). (original magnification 26,000×. Marker indicates 1μ)

Fig. 5

Comparison of mitochondrial volumes of H121N and Y955C TG and WT cardiac sections: Relative mitochondrial/total field section volumes were determined on EM sections. H121N transgenesis alone resulted in increased volume over WT while antiretroviral treatment in H121N TGs was lower than treated WTs. Treated Y955C TGs had a higher mitochondrial volume than vehicle-treated WTs, but were no different from treated WTs

Fig. 6

Steady state abundance of 20-kd mitochondrial subunit of complex I polypeptide by Western blot in H121N and Y955C TGs with AZT-HAART. Infrared scans were performed using antibody probes to the 20-kd (mitochondrial-encoded) subunit of complex I using the Odyssey infrared imaging system. Band signal intensities were normalized to porin control on the same blot. (a) H121N and Y955C TGs (males) with vehicle or AZT-HAART had slight increase (non-statistical) in Complex I/ porin ratio compared to WTs. (b) Vehicle- and AZT-HAART treated (5 weeks) Y955C TGs (females) had a decrease in steady state abundance of 20-kd mitochondrial subunit compared to H121N TGs and WT littermates

Fig. 7

Histologic analysis of sectioned hearts: H121N TGs demonstrated evidence of LV hypertrophy with decreased cavity % (7.7%), whereas the I212N TGs exhibited LV dilation with increased cavity % (18.2%) compared to WT (15.1%). Y955C TGs had the largest change in cavity % (20.9%) suggesting both hypertrophy and dilation. (original magnification 2×; Masson Trichrome)

Fig. 8

Quantitative analyses of ECHO images: LV mass was calculated in a blinded fashion, code was broken, and data tabulated. (a) I212N TGs only had an increase in LV mass following combination antiretroviral treatment. (b) H121N TGs without anti-retrovira1 treatment (HAART) had significant increase (∼25-50%) in LV mass from WT. (c) Y955C TGs also demonstrated an increase in LV mass (both vehicle and AZT-HAART treated) with an apparent additive impact in hearts from treated Y955C TGs. Data were normalized to body weight (mg/g) and plotted as mean ± SEM