Detailed studies correlating changes in mitochondrial optical density, packed volume, and ultrastructure associated with osmotically-induced swelling were performed . Various swelling states were established by incubating mitochondria (isolated in 0 .25 M sucrose) at 0°C for 5 min in series of KCI and sucrose solutions ranging in tonicity from 250 to 3 milliosmols . Reversibility of swelling was determined by examining mitochondria exposed to 250 milliosmols media after they had been induced to swell . Swelling induced by lowering the ambient tonicity to approximately 130 (liver mitochondria) and 90 (heart mitochondria) milliosmols involves primarily swelling of the inner compartment within the intact outer membrane . Decreasing the ambient tonicity beyond this level results in rupture of the outer membrane and expansion of the inner compartment through the break . The maximum extent of swelling, corresponding with complete unfolding of the cristae and an increase in over-all mitochondrial volume of approximately 6-fold (liver mitochondria) and 11-fold (heart mitochondria), is reached at approximately 15 (liver mitochondria) and 3 (heart mitochondria) milliosmols . Exposure of liver mitochondria to media of lower tonicity results in irreversibility of inner compartment swelling and escape of matrix material . These changes appear to result from increased inner membrane permeability, possibly due to stretching .

The inner membranes of isolated bovine heart mitochondria undergo pronounced contraction upon being exposed to exogenous adenosine diphosphate (ADP), adenosine triphosphate (ATP), and certain other hlgh-energy phosphate compounds. Contraction results in decrease of inner membrane expanse which in turn results in decrease of intracristal space and increase of mitochondrial optical density (OD). The magnitude of the OD change appears to be proportional to the degree of contraction Half-maximal contraction can be achieved with ADP or ATP at concentrations as low as about 0 3 /zM. Atractyloside at concentrations as low as about 1.2 nmol/mg mitochondrial protein completely inhibits the contraction. It is concluded from these and other observations that inner membrane contraction occurs as a result of adenine nucleotide binding to the carrier involved in the exchange of adenine nucleotides across the inner mitochondrial membrane.

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In bovine heart mitochondria bongkrekic acid at concentrations as tow as about 4 nmol/mg protein (a) completely inhibits phosphorylation of exogenous adenosine diphosphate (ADP) and dephosphorylation of exogenous adenosine triphosphate (ATP), (b) completely reverses atractyloslde inhibition of inner membrane contraction induced by exogenous adenine nucleotides, and (c) decreases the amount of adenine nucleotide required to elicit maximal exogenous adenine nucleotide-induced inner membrane contraction to a level which appears to correspond closely with the concentration of contractile, exogenous adenine nucleotide binding sites Bongkrekic acid at concentrations greater than 4 nmol/mg protein induces inner membrane contraction which seems to depend on the presence of endogenous ADP and/or ATP. The findings appear to be consistent with the interpretations (a) that the inner mitochondrial membrane contains two types of contractile, adenine nucleotide binding sites, (b) that the two sites differ markedly with regard to adenine nucleotide affinity, kc) that the high affinity site is identical with the adenine nucleotide exchange carrier, (d) that the low affinity site is accessible exclusively to endogenous adenine nucleotides and is largely unoccupied in the absence of bongkrekic acid, and (e) that bongkrekic acid increases the affinity of both sites in proportion to the amount of the antibiotic bound to the inner membrane.

Oxidative and phosphorylative activities were measured polarographically in mitochondria isolated from the right and left ventricles of normal and chronically stressed dog hearts. Chronic myocardial stress was produced experimentally by surgical procedures (combined tricuspid insufficiency and pulmonary stenosis, pulmonary insufficiency, aortic stenosis, aortic insufficiency, Potts's anastomosis) and by inducing thyrotoxicosis. Experimental stress periods ranged from 332 to 608 days. Some of the dogs had overt symptoms of congestive heart failure at the time they were killed.Mitochondria isolated from the stressed hearts had abnormally high values for oxidative activity and respiratory control ratios when incubated in the presence of malate-pyruvate. In the presence of succinate, they had either normal or slightly elevated values for oxidative activity and respiratory control ratios. No differences were found between mitochondria from normal and stressed hearts with regard to the efficiency (ADP/O) of oxidative phosphorylation. Estimates of mitochondrial protein per gram of myocardial tissue indicated that the stressed hearts contained normal amounts of mitochondria. The results of this study suggest that the mitochondrial oxidative and phosphorylative capabilities of the chronically stressed myocardium are not impaired.ADDITIONAL KEY WORDS mitochondrial function mitochondrial respiration mitochondrial respiratory control experimental chronic congestive heart failure mitochondrial metabolism• Chronic stress on the heart often results in a gradual deterioration of myocardial performance as evidenced clinically by congestive heart failure. Although this deterioration has long been considered to be a reflection of a biochemical abnormality in the myocardium, positive identification of this abnormality has not been achieved. The present report describes a search for biochemical abnormalities in mitochondria isolated from chronically stressed dog hearts, some of which appeared to be in chronic failure.From the Heart Biochemistry Research Laboratory of the Cardiovascular Service, Division of Thoracic Surgery, and Departments of Surgery and Physiological Chemistry, Ohio State University College of Medicine, Columbus, Ohio 43210. These studies were supported by U. S. Public Health Service Research Grant HE 05273-06 from the National Heart Institute.Accepted for publication May 20, 1968. The results of previous studies on the condition of mitochondria in the chronically stressed or failing heart are conflicting. Mitochondrial function was reported to be normal in some of these studies (1-4), whereas in others a reduction in oxidative activity (5-7), respiratory control ratios (6) and efficiency of oxidative phosphorylation (5-7) was observed. The factors responsible for these divergent results have not been clear. To test the possibility that some of the conflict in these reports is due to differences in the means by which myocardial stress was produced, we have measured oxidative activity, respiratory control...