Pol γ is responsible for replication of mtDNA (2
). Mutations in POLG
, the gene for the catalytic subunit cause CPEO, Alper’s syndrome, ataxia-neuropathy, Parkinsonism (2
), and other heritable conditions (24
Targeted cardiac transgenic mice were used to define features of cardiac dysfunction and CM (26
), and were applied to explore features of CM in AIDS (44
) where nucleoside reverse transcriptase inhibitors (NRTIs) cause mitochondrial dysfunction in the heart (45
). Transgenic targeting of human Pol γ harboring the Y955C pathogenic mutation here resulted in depleted mtDNA, oxidative stress, pathological cardiomegaly, cardiac mitochondrial ultrastructural damage, CM with LV dysfunction, bradycardia, and premature death. These findings forge a pathogenetic link between defective mtDNA replication and cardiac dysfunction with overwhelming depletion of mtDNA and mutant human Pol γ expressed in the murine heart.
TG experiments here causally linked transgenically targeted cardiac expression of Y955C Pol γ enzyme (derived from a known pathogenic mutation in POLG) to mtDNA depletion, oxidative stress, and dysfunction (in the form of CM). In the targeted TG, it is reasonable that Y955C Pol γ affected mtDNA replication at the level of enzyme:template interface by reducing relative availability of native Pol γ compared to the mutant. The Y955C mutant polypeptide was sufficiently abundant in this model to disrupt mtDNA homeostasis by overwhelming native Pol γ and becoming the dominant enzyme in the system but with substantially diminished enzyme activity. The subcellular outcome was mitochondrial oxidative stress documented by increased abundance of 8-OHdG.
Homeostasis of the mtDNA replicative machinery is regulated (18
). In the present model, genetic disruption with expression of this mutant Pol γ resulted in organ dysfunction and premature death (). Based on mass action of the mutant Y955C Pol γ, a “dominant negative” phenotype was the first step in organellar dysfunction and ultimately cardiac dysfunction. It also may be inferred that, at least at the level of mtDNA replication machinery in the mouse, human Y955C mutant Pol γ may substitute effectively for native murine Pol γ or interfere with the murine mtDNA replicon. Transgenic substitution here allows mutant human Pol γ to participate directly in mtDNA replication with resultant depletion. Data here do not directly explain the organ specific nature of mtDNA defects in CPEO. However, they can apply to and reinforce the “OXPHOS paradigm” of Wallace in which organs that require significant energy from oxidative phosphorylation may be principal targets for genetic mitochondria diseases in which oxidative phosphorylation is limited (46
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 (more ...)
The present work extends previous studies that focused on disruption of mtDNA biogenesis at the level of the mitochondrial nucleotide pools. In those studies, TGs were treated with NRTIs that compete with native nucleotides for intramitochondrial phosphorylation and transport (31
). Data indicated transport of nucleotides into mitochondria may be disrupted by NRTIs.
Genetic “mtDNA depletion syndromes” (49
) offer support for the reasoning used in generating these experimental models and for the data obtained from them. In those illnesses, mitochondrial and cytoplasmic nucleotide pools are disturbed by mutations of kinases, transporters, and enzymes involved in nucleotide pool homeostasis. Such mutations yield mtDNA depletion. “Acquired mtDNA depletion” from administration of NRTIs for AIDS (31
) offers a pharmacological model to deplete mtDNA in vivo
. NRTI chemical structure, dose, and duration of therapy each impacted the extent of mtDNA depletion and tissue target (31
This TG model strengthens the pathogenetic link between defects in mtDNA replication and cardiac dysfunction, particularly CM (51
). Although not proven here (owing to the severity of the TG phenotype), it is conceivable that subtle genetic mutations in Pol γ (already described or yet to be uncovered) may offer further mechanistic insights if examined transgenically. Presently there are over 70 documented disease mutations and a handful of single nucleotide polymorphisms in the POLG
). Such Pol γ mutations may exhibit only mild enzyme dysfunction without a phenotype in the native, undisturbed condition. However, a mitochondrial dysfunction phenotype could occur in selected tissues if a challenge with NRTIs that deplete mtDNA (32
) and cause oxidative stress (36
) were added. If these events (mutation of polymerase and disturbed nucleotide pools from NRTIs) were to coincide, significant side effects could result. The combination of genetic predisposition and environmental effects serves as a cornerstone of the threshold effect seen in mitochondrial genetic diseases and related systems, as articulated by Wallace and colleagues (46
In summary, this study utilized TGs that expressed human Y955C Pol γ in the murine heart to define defective mtDNA replication in vivo at the level of the enzyme machinery that replicates mtDNA. Targeted cardiac transgenic expression of Y955C Pol γ was sufficient to yield a molecular phenotype of mtDNA depletion, a biochemical phenotype with increased abundance of the mutant enzyme in the target, increased 8-OHdG, histopathological and mitochondrial ultrastructural changes in cardiac cells of TGs, and organ dysfunction with cardiomegaly, increased ventricular volume, increased cardiac mass on ECHO and MRI. Together, these interrelated findings underscore mtDNA replication as the nexus for dysfunction. It is reasonable to suggest that when a threshold of genetic mtDNA replication defects occurs, particularly from ineffective Pol γ function, cardiac dysfunction and pathological features of CM is an outcome. The role of defective mtDNA replication in various CMs merits deeper investigation in the future.