This study demonstrates alterations in myocardial substrate metabolism and impaired myocardial function in a rat model of early DCM. In particular, using
in vivo PET and echocardiography, our data extend previous
in vitro studies that assessed function [
12,
13], metabolism [
22] or both [
9,
10]. In particular, the present data showed increased myocardial FA oxidation with a concomitant decrease in insulin-mediated myocardial glucose utilisation. In addition, changes in myocardial function were associated with systemic insulin sensitivity and reduced myocardial glucose utilisation, whereas no relation was found between function and myocardial FA oxidation.
Emerging evidence supports the concept that alterations in myocardial substrate metabolism contribute to myocardial dysfunction. In this regard it is conceivable that alterations in substrate metabolism and underlying molecular changes precede the development of overt myocardial function. Previously, the earliest reported time point at which there was evidence of heart failure in ZDF rats was at 20 weeks of age [
13], 6 weeks after the age of the rats reported here. In addition to metabolic alterations, echocardiographic diastolic and systolic functional impairment and
in vitro myofilament dysfunction in isolated cardiomyocytes were found. Impaired myocardial carbohydrate utilisation in the absence of systolic dysfunction in 11-weeks-old ZDF rats has been observed using
13C-nuclear magnetic resonance [
10], while at 12 weeks of age, ZDF rats have shown increased FA oxidation and decreased carbohydrate oxidation in isolated hearts with depressed systolic function [
9], suggesting that changes in myocardial substrate metabolism precede myocardial dysfunction. The present
in vivo data demonstrate that myocardial glucose utilisation, but not FA oxidation, is associated with both diastolic and systolic function. Thus, myocardial dysfunction seems closely related to the severity of altered myocardial substrate metabolism in early diabetes.
The information provided by
in vitro studies regarding myocardial substrate metabolism in relation to myocardial function is restricted by experimental conditions. In most studies, the choice of substrate concentrations may not truly reflect what the heart utilises
in vivo. In contrast, PET provides a means for directly measuring true substrate metabolism
in vivo. Here, [
11C]palmitate and
18FDG PET were used to assess myocardial FA and glucose metabolism under fasting and hyperinsulinaemic euglycaemic clamp conditions, respectively. Previously, [
11C]palmitate PET was performed in 12-weeks-old ZDF rats [
22], showing increased FA oxidation. In spite of a reciprocal relationship between FA en glucose utilisation in the heart [
1,
3,
4], increased myocardial glucose utilisation measured with [
11C]glucose was reported. In contrast, the same research group, now using
18FDG PET, found a decrease in myocardial glucose uptake rate and utilisation in 19-weeks-old ZDF rats [
23], possibly due to the difference in age and in glucose tracer. These differences might also be explained by the conditions under which glucose utilisation was measured, i.e. under fasting conditions, resulting in FA being the primary substrate for myocardial energy. In contrast, in the present study glucose utilisation was measured using
18FDG under controlled hyperinsulinaemic euglycaemic conditions, as it is known that these conditions yield the best
18FDG image quality and the highest glucose utilisation in comparison with a glucose load or an insulin bolus [
24,
25]. Taking this approach, increased FA oxidation and decreased glucose utilisation were found in this early stage of DCM in the hearts of ZDF rats. In agreement with physiological data, impaired myocardial insulin sensitivity in ZDF hearts was demonstrated by an impaired ability of insulin to phosphorylate Akt. Similar results were reported in hearts from high-fat diet fed rats [
20], Zucker rats [
26] and ob/ob mice [
27], whereby the latter showed that impaired insulin signaling is associated with alterations in myocardial glucose metabolism measured in isolated working hearts. Further, we showed that systemic insulin sensitivity, measured as whole-body insulin sensitivity (
M-value), was significantly associated with
in vivo systolic and diastolic function. Also, myocardial insulin sensitivity, measured as insulin-mediated myocardial glucose utilisation, correlated with systolic and diastolic function. To the best of our knowledge, this is the first report in rats showing an association between
in vivo function and
in vivo myocardial metabolism. The myocardium is a metabolic omnivore that under healthy conditions will rely on FA oxidation for the largest part of its ATP production. However, as glucose is the more energetically efficient substrate, the myocardium should be readily able to switch to glucose under conditions of stress (e.g. ischemia, myocardial functional impairment such as in heart failure). As insulin resistance impacts on myocardial substrate supply (e.g. by increasing triglyceride-rich lipoprotein- and glucose output from the liver and adipose tissue-derived free fatty acids through unsuppressed lipolysis), it is clear that both systemic and organ-specific impairment of insulin action will influence substrate utilisation and reduce myocardial "metabolic flexibility" [
28‐
31]. Thus, in the stressed heart insulin resistance may finally hamper energy metabolism and as such myocardial function [
28,
29,
31]. Conversely, improvement of insulin sensitivity may ameliorate myocardial function. In humans, Iozzo
et al. found an inverse association of myocardial insulin-mediated glucose utilisation and systolic function [
31]. Our group showed that the insulin sensitizer pioglitazone improved whole-body insulin sensitivity and insulin-mediated myocardial glucose utilisation together with an improvement in myocardial diastolic function in patients with well-controlled T2DM [
29]. These findings support the role of insulin sensitivity in myocardial metabolism and function in DCM.
A restriction to glucose utilisation in the diabetic heart is the slow rate of glucose transport across the sarcolemmal membrane into the myocardium [
32]. In line with previous reports [
6,
33], decreased GLUT4 expression was found. PDK4 has an inhibitory effect on glucose oxidation via the pyruvate dehydrogenase complex (PDH) [
34], however, no differences were found in PDK4 protein expression. Similar PDK4 mRNA levels were found in ZDF rats [
6] and Zucker rats [
5]. However, Chatham
et al. [
10] showed increased PDH activity in ZDF hearts. AMPK is known as a major regulator of metabolic myocardial energy substrate acting as a metabolic sensor following an energetic imbalance. Decreased AMPKα1/2 phosphorylation was found, which is consistent with previous results [
35,
36]. In addition, a significant increase in sarcolemmal localisation of the FA transporter FAT/CD36 in ZDF hearts was seen, which is compatible with the observed enhancement of myocardial FA oxidation. These data are in line with those reported in high-fat diet fed [
14] and Zucker [
37,
38] rats, in both of which relocation of FAT/CD36 to the sarcolemmal membrane was associated with increased myocardial FA uptake. Collectively, these molecular findings are compatible with the measured shift in myocardial substrate metabolism towards increased myocardial FA metabolism and decreased myocardial glucose metabolism.
In isolated cardiomyocytes of ZDF rats, significant reductions were observed in both maximal force and maximal rate of force redevelopment. A previous study by Ren
et al. [
39] showed reduction in peak shortening, prolonged duration of re-lengthening and unaltered resting intracellular calcium levels in intact cardiomyocytes from 14-weeks-old Zucker rats. The reduction in maximal force generating capacity of myofilaments observed in the present study may well explain reduced cardiomyocyte shortening. Moreover, the reduction in maximal rate of force redevelopment may, at least in part, underlie prolonged duration of cellular re-lengthening (i.e. cardiomyocyte relaxation). No differences were found in calcium sensitivity as well as SERCA2a expression. However, a trend towards decreased phosphorylation of phospholamban was found. Hence, cellular dysfunction in early DCM could be the result of myofilament dysfunction. Nevertheless, the exact mechanisms leading to depressed myocardial function in early diabetes remain elusive. Several potential options to be responsible for the pathogenesis of myocardial dysfunction in early diabetes have been postulated, including (1) lipotoxicity via ceramide dependent pathways, (2) increased accumulation of advanced glycation end products, and (3) generation of reactive oxygen species via increased flux through mitochondrial pathways. Further studies are necessary to reveal the exact transition from compensatory myocardial function to overt myocardial dysfunction during alterations in myocardial substrate metabolism.