Contractile function and substrate metabolism were characterized in perfused hearts from genetically diabetic C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and their non-diabetic lean littermates. Contractility was assessed in working hearts by measuring left ventricular pressures and cardiac power. Rates of glycolysis, glucose oxidation, and fatty acid oxidation were measured using radiolabeled substrates ([5-(3)H]glucose, [U-(14)C]glucose, and [9,10-(3)H]palmitate) in the perfusate. Contractile dysfunction in db/db hearts was evident, with increased left ventricular end diastolic pressure and decreased left ventricular developed pressure, cardiac output, and cardiac power. The rate of glycolysis from exogenous glucose in diabetic hearts was 48% of control, whereas glucose oxidation was depressed to only 16% of control. In contrast, palmitate oxidation was increased twofold in db/db hearts. The hypothesis that altered metabolism plays a causative role in diabetes-induced contractile dysfunction was tested using perfused hearts from transgenic db/db mice that overexpress GLUT-4 glucose transporters. Both glucose metabolism and palmitate metabolism were normalized in hearts from db/db-human insulin-regulatable glucose transporter (hGLUT-4) hearts, as was contractile function. These findings strongly support a causative role of impaired metabolism in the cardiomyopathy observed in db/db diabetic hearts.

Glucose and palmitate metabolism and contractile function were measured with ex vivo perfused working hearts from control (db/؉) and diabetic (db/db) female mice at 6, 10 -12, and 16 -18 weeks of age. Palmitate oxidation was increased by 2.2-fold in 6-week-old db/db hearts and remained elevated in 10-to 12-and 16-to 18-week-old hearts. Carbohydrate oxidation was normal at 6 weeks but was reduced to 27 and 23% of control at 10 -12 and 16 -18 weeks, respectively. At 6 weeks, db/db hearts exhibited a slight reduction in mechanical function, whereas marked signs of dysfunction were evident at 10 -12 and 16 -18 weeks. Mechanical function after ischemia-reperfusion was examined in hearts from male mice; at 6 weeks, db/db hearts showed normal recovery, whereas at 12 weeks it was markedly reduced. Fatty acid oxidation was the predominant substrate used after reperfusion. Thus, diabetic db/db hearts exhibit signs of a progressive cardiomyopathy; increased fatty acid oxidation preceded reductions in carbohydrate oxidation. Postischemic recovery of function was reduced in db/db hearts, in parallel with age-dependent changes in normoxic contractile performance. Finally, peroxisome proliferator-activated receptor-␣ treatment (3 weeks) did not affect sensitivity to ischemiareperfusion, even though carbohydrate oxidation was increased and palmitate oxidation was decreased. Diabetes 52:434 -441, 2003 N on-insulin-dependent (type 2) diabetes is a prevalent disease that results in a marked increase in cardiovascular complications (1) that are in part due to a specific cardiomyopathy, characterized by ventricular dysfunction in the absence of atherosclerotic coronary heart disease or hypertension (2,3).Diabetic db/db mice provide an animal model of type 2 diabetes, with obesity and insulin resistance (4,5). Recently, we have reported that isolated perfused hearts from db/db mice at 10 -14 weeks of age exhibited characteristics of a diabetic cardiomyopathy, with decreased contractile performance and altered cardiac metabolism (6,7).The natural history of db/db mice follows a distinct pattern (8,9). Initially, peripheral insulin resistance is overcome by increased insulin secretion, so hyperinsulinemia produces normoglycemia. Hyperglycemia develops when enhanced insulin secretion can no longer compensate for insulin resistance. The maximal extent of hyperinsulinemia occurs at 2-3 months of age. Subsequently, insulin levels fall rapidly as ␤-cells exhibit a severe secretory defect, resulting in a progressive increase in hyperglycemia. Thus, the metabolic features of db/db mice are similar to the pathogenesis of type 2 diabetes in humans (10).The first objective of this investigation was to study age-dependent changes of cardiac function and metabolism in db/db mice assessed with ex vivo perfused hearts (7,11,12). Of particular interest was to establish whether the onset of metabolic alterations coincided with contractile dysfunction. Previously, evidence for contractile dysfunction in perfused db/db hearts has been obtained from norm...

Rationale: In the present study we explored the mechanisms behind excitation-contraction (EC) coupling defects in cardiomyocytes from mice with type-2 diabetes (db/db 1 This has severe implications, because cardiovascular mortality is Ϸ2-to 4-fold higher in diabetic compared to nondiabetic patients 2 and accounts for Ϸ80% of the mortality in type 2 diabetes, 3 of which Ϸ50% die of sudden cardiac death. 4 Furthermore, diabetics are 2.5 times more likely to develop congestive heart failure compared to nondiabetics. 5 The db/db diabetic mouse model develops cardiomyopathy in a similar manner as type 2 diabetes in humans, 6 and presents with reduced whole-heart 7 and isolated cardiomyocyte 8 11,12 In contrast, exercise training in healthy mice increases the level of phosphorylated cytosolic CaMKII␦ and in so doing increases CaMKII␦ activity. Under these circumstances, increased phosphorylation of CaMKII␦ was associated with an increased cardiac performance. 13 Alongside increased SR Ca 2ϩ leak, reduced transverse (T)-tubule structure leading to less synchronous SR Ca 2ϩ release contributes further to the depressed EC coupling in models of cardiac dysfunction. 14 The mechanism for increased SR Ca 2ϩ leak, and whether T-tubule structure in diabetic cardiomyopathy is conserved, has currently not been studied.In the present study, we explored the mechanisms behind the impaired cardiomyocyte function and increased SR Ca 2ϩ leak in db/db cardiomyocytes, and then reexamined the same parameters in the db/db mice after an aerobic interval exercise training program. Because the activities of both CaMKII␦ and PKA are associated with both pathological and physiological remodeling, we also investigated the contributions of CaMKII␦ and PKA for the observed exercise training-induced changes. MethodsFor a detailed description, see the data supplement (available online at http://circres.ahajournals.org). Mouse Model of Diabetes and Exercise TrainingThe db/db mice model has been proven to be a suitable model to study the consequences of diabetes on the heart. Here we studied the male diabetic (BKS.Cg-m ϩ/ϩ Lepdb/Bom Tac; 20 exercised and 20 sedentary mice) and sedentary (nϭ23) and exercise trained (nϭ6) nondiabetic healthy heterozygote (BKS.Cg-m ϩ/ϩ Lepdb/ϩ lean); all age-matched (7 weeks at study start). To determine maximal oxygen uptake (VO 2max ), mice ran until exhaustion on a customized treadmill in a metabolic chamber, and high-intensity aerobic interval training was performed as uphill running, alternating between 4 minutes at 85% to 90% of VO 2max and 2 minutes at 50% of VO 2max for 80 minutes/day, 5 days/wk, for 13 weeks. 15 Cardiomyocyte Isolation and Ca 2؉ MeasurementsLeft ventricular myocytes were isolated as previously described. 15 Fura-2/AM-loaded cardiomyocytes were stimulated by bipolar electric pulses for Ca 2ϩ handling measurements including SR Ca 2ϩ leak. CaMKII inhibitor and PKA inhibitor were used to determine the influence of the 2 kinases. Contractility was recorded by video-based sarcomere spacing. ...