Keijo J. Peuhkurinen

Epidemiology of A3243G, the Mutation for Mitochondrial Encephalomyopathy, Lactic Acidosis, and Strokelike Episodes: Prevalence of the Mutation in an Adult Population

Mitochondrial diseases are characterized by considerable clinical variability and are most often caused by mutations in mtDNA. Because of the phenotypic variability, epidemiological studies of the frequency of these disorders have been difficult to perform. We studied the prevalence of the mtDNA mutation at nucleotide 3243 in an adult population of 245,201 individuals. This mutation is the most common molecular etiology of MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes), one of the clinical entities among the mitochondrial disorders. Patients with diabetes mellitus, sensorineural hearing impairment, epilepsy, occipital brain infarct, ophthalmoplegia, cerebral white-matter disease, basal-ganglia calcifications, hypertrophic cardiomyopathy, or ataxia were ascertained on the basis of defined clinical criteria and family-history data. A total of 615 patients were identified, and 480 samples were examined for the mutation. The mutation was found in 11 pedigrees, and its frequency was calculated to be >=16. 3/100,000 in the adult population (95% confidence interval 11.3-21. 4/100,000). The mutation had arisen in the population at least nine times, as determined by mtDNA haplotyping. Clinical evaluation of the probands revealed a syndrome that most frequently consisted of hearing impairment, cognitive decline, and short stature. The high prevalence of the common MELAS mutation in the adult population suggests that mitochondrial disorders constitute one of the largest diagnostic categories of neurogenetic diseases.

Mechanisms of Ischemic Preconditioning in Rat Myocardium

BACKGROUNDnAdenosine has been proposed as one mediator for the preconditioning effect in the myocardium of some animals, but recent investigations have shown that this may not be the mechanism in the rat heart, although the effect itself is clearly demonstrable. The cellular energy state has been shown to be better in preconditioned hearts, and the role of ATP consumption has been discussed. The role of inhibition of mitochondrial F1F0-ATPase as a mechanism for the preservation of ATP in preconditioned hearts remains controversial.nnnMETHODS AND RESULTSnThree-minute global ischemia followed by 9 minutes of reperfusion was used to precondition Langendorff-perfused rat hearts, and control hearts were perfused under normoxic conditions for the same time. The duration of sustained ischemia in both groups of hearts was 21 minutes, after which the hearts were reperfused for 15 minutes to evaluate their mechanical and metabolic recovery. Separate experiments were performed for tissue metabolite determinations, mitochondrial ATPase activity measurements, and 31P nuclear magnetic resonance studies. The recovery of the rate-pressure product was better in the preconditioned group. Three-minute preconditioning ischemia caused inhibition of the mitochondrial ATPase that persisted throughout the 9-minute intervening reperfusion so that at the early stages of sustained ischemia the enzyme activity was still more inhibited in preconditioned hearts. ATP was better preserved in preconditioned hearts than in control hearts during sustained ischemia. The accumulation of adenosine and its degradation products during sustained ischemia was greater in the control group. More lactate and H+ ions accumulated in this group, indicating higher anaerobic glycolysis. Also, inhibition of mitochondrial ATPase by oligomycin slowed ATP depletion during ischemia.nnnCONCLUSIONSnThe results indicate that preconditioning causes inhibition of rat heart mitochondrial ATPase that persists during reperfusion so that the enzyme is inhibited from the very beginning of the sustained ischemia. This inhibition leads to sparing of high-energy phosphates and improves the time-averaged energy state during ischemia. Although a causal relationship is difficult to prove, this reversible inhibition may contribute to postischemic recovery of the heart.

Collagen scar formation after acute myocardial infarction: relationships to infarct size, left ventricular function, and coronary artery patency.

BACKGROUNDnLeft ventricular function after acute myocardial infarction (AMI) is determined by the expansion of the infarct zone and remodeling of the noninfarcted myocardium. An occluded infarct-related artery (IRA) is an independent risk factor for remodeling.nnnMETHODS AND RESULTSnChanges in myocardial collagen metabolism were evaluated in 36 patients with suspected AMI. The plasma creatine kinase MB fraction and myoglobin release curves were analyzed for assessment of early reperfusion and infarct size. Collagen scar formation was evaluated by measurement of serum concentrations of the aminoterminal propeptide of type III procollagen (PIIINP), the aminoterminal propeptide of type I procollagen (intact PINP), and the carboxyterminal propeptide of type I procollagen (PICP). Plasma renin activity and urine excretion of cortisol and aldosterone were also measured. Coronary angiography and left ventricular cineangiography were performed during early hospitalization. The serum concentration of PIIINP increased from 3.50+/-0.20 to a maximum of 5.08+/-0.36 microg/L (n=32) in the patients with AMI, whereas the concentrations of intact PINP and PICP tended to decrease. The area under the curve (AUC) of PIIINP during the first 10 postinfarction days was larger in patients with severe heart failure or ejection fractions < or = 40% than in those with no heart failure or with an ejection fraction > 40% (P<.05 and P<.01, respectively), and it was also larger in the patients with TIMI grade 0 to 2 flows than in those with TIMI 3 flows (P<.05), despite similar enzymatically determined infarct sizes. No significant correlations between PIIINP and neurohumoral parameters were observed. The AUC of PIIINP and the change in PIIINP during the first 4 days were significantly correlated with indices of cardiac function.nnnCONCLUSIONSnCollagen scar formation after AMI can be quantified by measurement of serum PIIINP concentrations. Scar formation is more prominent in large infarctions causing left ventricular dysfunction and in patients with occluded IRAs.

Thrombolytic therapy with streptokinase stimulates collagen breakdown.

BackgroundPlasmin is capable of degrading extracellular matrix components such as collagen in vitro. To evaluate the significance of this for in vivo conditions, we set out to study the effect of streptokinase, which acts by converting plasminogen to plasmin, on the serum concentrations of the amino-terminal propeptide of type III procollagen (PIIINP) and the carboxy-terminal propeptide of type I procollagen (PICP). Methods and ResultsTwenty-three patients with suspected acute myocardial infarction were included in the study; 17 of them received thrombolytic therapy, and six were treated conservatively. PIIINP and PICP were assayed with radioimmunoassays. Kinetics of creatine kinase-MB release were determined to differentiate reperfusers from nonreperfusers. Composite curves of creatine kinase-MB release were constructed for different patient subgroups. During streptokinase infusion the serum concentrations of PIIINP increased rapidly, with a maximum mean increase of 50% (from 2.2 ± 0.2 to 3.3 ± 0.3 μg/l) in 45 minutes. A similar increase was also observed in two patients who received thrombolytic therapy but did not subsequently develop any myocardial infarction determined on the basis of enzyme release. The relative increase in PIIINP during streptokinase treatment was higher in those acute myocardial infarction patients with probable reperfusion than those with nonprobable reperfusion. Corresponding changes in PIIINP were not seen in the control group. Two days later there was a second increase in serum PIIINP for both patient groups. This change coincidedwith a similar increase in PICP. ConclusionsWe conclude that streptokinase, probably by activation of plasminogen to plasmin, stimulates the breakdown of type III collagen during thrombolytic therapy. This phenomenon may decrease the risk of rethrombosis of the affected artery if the exposed collagen is responsible for thrombosis formation, but it could also be involved in the development of hemorrhagic complications during thrombolytic therapy. The second increase in PIIINP levels probably indicates type III collagen synthesis of the infarcted area. This investigation represents a pilot study, and more studies on the effects of various thrombolytic agents on interstitial collagen metabolism are obviously needed. (Circulation 1991;83:1969—1975)

Regulation of the tricarboxylic acid cycle pool size in heart muscle

The anaplerotic mechanisms are mainly responsible for the intricate control of the size of the total tricarboxylic acid cycle pool, since cataplerotic fluxes leading out of the cycle can even increase during net synthesis of the cycle intermediates. Pyruvate carboxylation is probably the most important anaplerotic mechanism regulating the pool size of the citric acid cycle. However, not much is known of the regulation of pyruvate carboxylation or other ana- or cataplerotic mechanisms, and further research in this area is needed. The evidence accumulated so far indicates that regulation of the tricarboxylic acid cycle pool size serves to optimize energy transduction in heart muscle. This is seen at the level of the tricarboxylic acid cycle, where the tricarboxylic acid cycle intermediates can be taken to act in a catalytic manner to adjust the activities of the individual enzymes of the cycle at an appropriate level for a certain metabolic situation. On the other hand, increase in the mitochondrial citrate content serves as the mediator between the mitochondrial and cytosolic spaces and leads to diminished glucose utilization in glycolysis due to feed-back inhibition. However, net anaplerosis does not constitute a major energy-releasing system in the ischemic myocardium.

Changes in interstitial collagen metabolism during acute myocardial infarction treated with streptokinase or tissue plasminogen activator

Collagen plays a specific role in the maintenance of vascular integrity and in the thrombosis and scar formation processes. Therefore we found it interesting to study the changes in interstitial collagen metabolism during acute myocardial infarction treated with thrombolytic agents. Changes in collagen synthesis were evaluated by obtaining assays of the serum concentrations of the carboxyterminal propeptide of type I procollagen. Except fibrin plasmin is capable of degrading extracellular matrix components including collagen, and this capability was evaluated by monitoring the serum concentrations of the aminoterminal propeptide of type III procollagen. Twenty-four patients with suspected acute myocardial infarction and indications for thrombolytic therapy were randomized to receive either streptokinase (n = 11) or tissue plasminogen activator (n = 13). The patient groups were identical in their clinical characteristics. Serum levels of the aminoterminal propeptide of type III collagen increased rapidly on infusion of the thrombolytic agents, with the maximal mean increases of 44% and 16% in the streptokinase and TPA-treated groups, respectively. Levels of the carboxyterminal propeptide of type I collagen did not change during the thrombolytic therapy. A transient decrease occurred in the type I propeptide concentration at postinfarction day 2, and this decrease was followed by a secondary increase at days 4 to 6 in both patient groups studied. We conclude that thrombolytic agents stimulate the breakdown of interstitial collagen and that the collagen-degrading activity of TPA is lower than that of streptokinase. This factor may contribute to the relatively higher rethrombosis rate seen after TPA, because exposed collagen in the affected vascular wall stimulates thrombosis formation. On the other hand, increased collagen degradation followed by inhibition of collagen synthesis in the infarcted myocardium might increase the risk for cardiac rupture, especially after streptokinase treatment.

Regulation of pyruvate dehydrogenase during infusion of fatty acids of varying chain lengths in the perfused rat heart.

The effects of a homologous series of fatty acids with a chain length of two to eight on the rate of pyruvate oxidation and covalent interconversions of the pyruvate dehydrogenase complex (PDH) were studied in isolated perfused rat hearts. In the Langendorff-perfused heart beating at 5 Hz against an aortic pressure of 59 mmHg (7.85 kPa), a positive linear correlation was found between the fraction of PDH existing in the active non-phosphorylated form of pyruvate dehydrogenase complex (PDHa) and the pyruvate oxidation rate until the PDHa fraction increased to 48%. This value resulted in a saturation of the citric acid cycle and further activation did not increase the metabolic flux. The PDHa content of the tissue was higher during infusion of odd carbon number fatty acids than during infusion of even carbon number fatty acids. Propionate caused an almost maximal (93%) activation of PDH. A negative correlation was found between the mitochondrial NADH/NAD+ ratio and the PDHa content. A negative correlation was also found between the acetyl-CoA/CoA ratio and the tissue PDHa content. The rate of labelled CO2 production, the specific radioactivity of tissue alanine and the metabolic balance sheet demonstrated that the alanine aminotransferase reaction in the total tissue does not reach equilibrium with the mitochondrial pyruvate pool during propionate oxidation, but the equilibrium is reached during the oxidation of even-number carbon fatty acids. This suggests that pyruvate is formed from propionate-derived metabolites also in the cytosol, although the primary metabolism of propionate occurs in the mitochondria. The results indicate that the rate of pyruvate oxidation in the myocardium is mainly regulated by covalent interconversion of PDH. During propionate oxidation the PDHa content in the tissue can increase beyond the point of saturation of the citric acid cycle and this indicates that feedback inhibition of the enzyme is rate-determining under these conditions.

Reversible ischemic inhibition of F1F0-ATPase in rat and human myocardium

The physiological role of F(1)F(0)-ATPase inhibition in ischemia may be to retard ATP depletion although views of the significance of IF(1) are at variance. We corroborate here a method for measuring the ex vivo activity of F(1)F(0)-ATPase in perfused rat heart and show that observation of ischemic F(1)F(0)-ATPase inhibition in rat heart is critically dependent on the sample preparation and assay conditions, and that the methods can be applied to assay the ischemic and reperfused human heart during coronary by-pass surgery. A 5-min period of ischemia inhibited F(1)F(0)-ATPase by 20% in both rat and human myocardium. After a 15-min reperfusion a subsequent 5-min period of ischemia doubled the inhibition in the rat heart but this potentiation was lost after 120 min of reperfusion. Experiments with isolated rat heart mitochondria showed that ATP hydrolysis is required for effective inhibition by uncoupling. The concentration of oligomycin for 50% inhibition (I(50)) for oxygen consumption was five times higher than its I(50) for F(1)F(0)-ATPase. Because of the different control strengths of F(1)F(0)-ATPase in oxidative phosphorylation and ATP hydrolysis an inhibition of the F(1)F(0)-ATPase activity in ischemia with the resultant ATP-sparing has an advantage even in an ischemia/reperfusion situation.

Mitochondrial DNA deletions in dilated cardiomyopathy: A clinical study employing endomyocardial sampling☆

OBJECTIVESnThe aim of this study was to assess the occurrence of the two most commonly encountered mitochondrial DNA (mtDNA) deletions in the hearts of patients with idiopathic dilated cardiomyopathy.nnnBACKGROUNDnThe mutation frequency of mtDNA is high, and sporadic cases of cardiomyopathies associated with mtDNA deletions have been described. Reports of increases in mtDNA deletions with advancing age also exist.nnnMETHODSnWe studied 15 consecutive patients with typical signs of idiopathic dilated cardiomyopathy, without a family history, together with 16 control hearts obtained at autopsy from patients who died of noncardiac causes. The patients underwent both right and left heart catheterization, during which endomyocardial biopsy samples were taken. The mtDNA in these samples and in the control hearts was analyzed by the polymerase chain reaction technique for the occurrence and proportion of 5- and 7.4-kilobase (kb) deletions (Cambridge sequence map positions from nucleotides 8469 to 13447 and 8637 to 16084, respectively).nnnRESULTSnThe 5-kb mtDNA deletion was observed in the hearts of all of the patients with idiopathic dilated cardiomyopathy, accounting for 0.32 +/- 0.05% (mean +/- SEM) of the total mtDNA. The 7.4-kb deletion was found in 7 of the 15 patients with idiopathic dilated cardiomyopathy and comprised 0.28 +/- 0.08% of the total. The 5- and 7.4-kb deletions were detected in 12 and 9 control hearts, respectively, quantitatively similar to the patients with idiopathic dilated cardiomyopathy. A sigmoidal age dependency of the mtDNA deletions was found both in the patients with cardiomyopathy and in the control hearts, but after elimination of the confounding age variable, there was no difference between these groups.nnnCONCLUSIONSnBecause of the similarity of the age-dependent increase in the frequency of mtDNA deletions in cardiomyopathic and control hearts, the deletions have no causal relation with idiopathic dilated cardiomyopathy. The present results confirm the notion of an increase in mtDNA deletions with advancing age and show that endomyocardial tissue sampling is a feasible method for detecting mtDNA defects in affected hearts.

Role of cellular energy state and adenosine in the regulation of coronary flow during variation in contraction frequency in an isolated perfused heart

Regulation of coronary flow as a function of myocardial energy expenditure was investigated in isolated perfused rat hearts electrically paced at the desired frequencies. The sinoatrial node was excised to lower the endogenous heart rate. The main covariants measured were phosphagen, inorganic phosphate, adenosine, inosine and hypoxanthine concentrations in the tissue, washout of nucleosides and hypoxanthine into the perfusate, oxygen consumption and coronary flow. Oxygen consumption was linearly correlated with heart rate and coronary flow, while the correlation between coronary flow and perfusate adenosine was nonlinear. The adenosine concentrations in the tissue and perfusate showed a mirror image curvilinearity reminiscent of a threshold pattern for adenosine washout. The tissue adenosine content had a negative linear correlation with the adenylate phosphorylation potential (long(ATP/ADP X Pi)). Adenosine output was linearly correlated with free AMP concentration in the tissue, the latter being calculated from the equilibrium of the adenylate kinase reaction. The results confirm the correlation between cellular energy state and coronary flow and support the notion that the mediators between the former and the vascular smooth muscle involve the concentration of free AMP in the tissue, suggesting that the formation of adenosine may be limited by the availability of AMP. The results are in agreement with the hypothesis that adenosine is the diffusible extracellular mediator in the energy-linked regulation of coronary flow.