SUMMARY We studied DNA (mtDNA) replication in adult female rat hearts undergoing hypertrophy secondary to constriction of the ascending aorta. MtDNA was measured in isolated mitochondria by a fiuorometric method adapted for that purpose. The conditions for removal of contaminating nuclear DNA were developed, and the purity of the mtDNA was assessed from its molecular conformation (open and closed circles) and by renaturation-kinetic analysis. The mtDNA concentration in mitochondria, expressed as micrograms of DNA per milligram of mitochondrial protein, increased 2, 4, and 7 days postoperatively by 21, 73, and 98%, respectively. Similar results were obtained when mtDNA was expressed per nanomole of cytochrome a. The population of replicative intermediates of mtDNA was analyzed by electron microscopy. In normal hearts, we observed molecular forms characteristic of animal mtDNA, such as circular monomers and dimers, catenated molecules, D-loops, expanded Dloops, and gapped molecules. D-loop frequency, which was near 50% in the mtDNA of control hearts, was markedly reduced to 5-7% in hypertrophying hearts. This result indicates that the increase in replicative flux of mtDNA is associated with the removal of a block in the conversion of D-loops to other intermediates. Ore Res. 46: 1979 IT is now well established that increased tension in the ventricular wall is correlated with increased metabolic activity of the heart. It is also known that the energy requirement of the myocardium is met completely by aerobic metabolism during both rest and strenuous activity (Neely and Morgan, 1974). This dependence of the myocardium on oxidative processes is reflected by the high content of mitochondria, which occupy 35% of the cardiac cell volume (Page et al., 1972).The increased requirement for ATP during acute work overload is met effectively by the respiratory control mechanism: the accumulating ADP stimulates oxidative phosphorylation. If the hemodynamic overload is sustained, however, the capacity of the existing contractile and energy-producing apparatus is exceeded, and a second adaptive process, cardiac growth, is activated. This adaptive growth is reflected in increased incorporation of labeled amino acids into cardiac subcellular fractions, including mitochondria, as measured both in vivo (Zak and Fischman, 1971) and in vitro (Shahab and Wollenberger, 1970). Quantitative electronmicroscopic analysis (Page et al., 1972) and measurements of mitochondrial cytochromes and respiratory enzymes (Albin et al., 1973) indicate that, in early hypertrophy, mitochondria accumulate in preference to other organelles. Thus, the study of processes leading to mitochondrial proliferation may provide clues about the still elusive feedback mechanism that couples hemodynamic load with biosynthetic pathways. In this respect, the report of Meerson and Pomoinitsky (1972) that the amount of mtDNA, expressed per unit of mitochondrial protein, is increased strikingly after aortic constriction in the rat is of great interest. The mtDNA concentra...
