The key findings of this study are that in the early stages of type 2 diabetes, myocardial expression of SERCA2a is markedly elevated, whereas the expression of PLB mRNA is reduced. These changes go along with a significant increase in sarcoplasmatic Ca2+ uptake. Interestingly, the SERCA/PLB ratio in diabetic animals was further increased by insulin treatment. In vitro, we were able to demonstrate that insulin treatment of isolated cardiac myocytes led to a concentration-dependent increase in SERCA2a expression. This effect was also seen in a more complex model of engineered heart tissue and correlated positively with cardiac relaxation in vitro. . In addition, Akt was significantly stimulated by insulin in cardiomyocyte cell culture. This approach allows us to directly determine contractile alterations caused by insulin treatment without considering insulin-related systemic changes which might also affect the cardiovascular system. However we must consider that in vitro data can not totally be compared to in vivo data. Nevertheless they give essential additional information. Together, this suggests that insulin, which is particularly elevated in premature type 2 diabetes mellitus, may be involved in the induction of SERCA2a mRNA expression and may be an early step in the pathogenesis of diabetic cardiomyopathy.
Alterations of SERCA, PLB and ca uptake in diabetes mellitus
Diastolic relaxation of the heart is mediated to a large extent by the uptake of Ca2+ into the sarcoplasmic reticulum. Several studies have shown that diabetic cardiomyopathy is associated with decreased contractility and impaired relaxation [
19]. These changes have been attributed to a reduced ability to sequester calcium into the SR, which primarily determines the speed of cardiac relaxation. Consecutive experiments have shown that SERCA2a mRNA expression, protein level and activity is down-regulated in streptozotocin-induced (STZ) type 1 diabetes mellitus [
10,
11]. In another model Wold and co-workers demonstrated in cardiomyocytes of rats with sucrose-induced insulin resistance that impaired SERCA activity with normal protein content contributes to cardiomyocyte dysfunction, whereas NCX function and expression are normal [
20]. The authors concluded that subtle changes in Ca2+ regulation which occur prior to overt ventricular dysfunction and/or failure, may be common to early stages of a number of disorders involving insulin resistance. Furthermore, it has been demonstrated that reduced Ca2+ signaling in vascular smooth muscle cells from diabetic animals is related to a decline and/or redistribution in the IP3R Ca2+ channels and SERCA proteins. These changes could be repeated in cell culture experiments with high glucose levels [
21]. The mechanism of alteration of SR proteins in STZ- induced diabetes, however, is unclear at present. The same is true for the
Otsuka-Long-Evans Tokushima Fatty rats (OLETF), a type 2 diabetes model. Here in late stages of diabetes, after additional sucrose feeding, the ventricular relaxation rate was significantly slower and was associated with reduced SERCA2a level [
22]. These experiments are therefore in apparent contrast to our results. This discrepancy is most likely due to the different models used and the rather advanced duration of the diabetes and older age (60 weeks in the latter experiments as compared to 19-week-old diabetic rats in our study). OLETF rats present a milder form of diabetes mellitus with later onset and milder hyperglycemia at the beginning [
23]. We examined ZDF rats at earlier diabetic stages, and our previous experiments have shown that C-peptide levels are high at this age [
7].
Insulin directly up-regulates SERCA and preserves cardiac function
Most of the previous studies used a model with type 1 diabetes which differs fundamentally in pathophysiology. Whereas type 1 diabetes mellitus results from selective destruction of the insulin- producing beta cells of the pancreas, type 2 diabetes is primarily characterized by insulin resistance followed by progressive beta-cell dysfunction, resulting in low insulin levels in the long term. As recently shown, obese ZDF rats are insulin-resistant and have basal hyperinsulinemia that is due mainly to hypersecretion of insulin, as indicated by their elevated basal C-peptide levels [
7,
24]. Obese pre- diabetic and diabetic rats also show a reduction in insulin clearance, as indicated by their lower C- peptide/insulin ratio. Interestingly, the decrease in SERCA2a activity in STZ-treated rats can be reversed by insulin treatment [
9,
11], suggesting a direct stimulatory effect of insulin on SERCA2a. This hypothesis is further supported by other experiments demonstrating an up-regulation of SERCA1 in skeletal muscle after stimulation with insulin [
1]. These observations demonstrate a possible link between insulin and expression of SR calcium ATPase, which is further confirmed by the present in vitro studies showing a direct effect of insulin on SERCA2a transcription in isolated cardiac myocytes. The important role of insulin for heart function is further supported by Kim et al., who showed that insulin preserves heart function in streptozotocin-induced diabetic heart failure with and without transplantation of smooth muscle cells [
25].
In this context it has been shown that transgenic (TG) mice with cardiac-specific overexpression of active Akt not only exhibit hypertrophy and enhanced left ventricular function but also show a 6.6-fold increase in SERCA2a protein levels, which could be recapitulated in vitro by adenovirus-mediated overexpression of Akt in cultured adult ventricular myocytes [
26]. We demonstrated on isolated cardiac myocytes a strong and rapid phosphorylation of Akt after stimulation with insulin. Conversely, inhibiting SERCA2a with either ryanodine or thapsigargin affected myocyte contraction and relaxation and Ca2+ channel kinetics more in TG than in WT. Thus, myocytes from mice with overexpressed Akt demonstrated enhanced contractility and relaxation, Fura-2 Ca2+ transients, and Ca2+ channel currents [
26]. Furthermore, increased protein expression of SERCA2a plays an important role in mediating enhanced left ventricular function by Akt. Interestingly, insulin stimulation led to a significant increase in SERCA2a, co-immunoprecipitated with insulin receptor substrate proteins (IRS-1 and IRS-1) in isolated cardiac muscle demonstrating a link between insulin, insulin receptor and SERCA2a [
27] in cardiac tissue.
Recent experiments showed that insulin-like growth factor 1 (IGF-1) activates multiple signaling pathways, which involve the activation of the phosphatidylinositol (PI)3-kinase and Akt [
28]. It is well known that the PI3-kinase-Akt cascade modulates diverse cellular functions. Furthermore, it has been shown that that IGF-I caused increases in myocyte contraction and relaxation function, increases in intracellular Ca2+ transients, and an upregulation of SERCA2a [
29]. The same group demonstrated that transgenic mice with cardiac-specific overexpression of Akt showed an enhanced left ventricular function, associated with an increased expression of SERCA2a [
30]. These data are in accord with the studies of von Lewinski and co-workers [
31](4), who suggested that Akt contribute to the acute inotropic effect of IGF-I in myocytes from human failing hearts.
In the present study, the insulin induced increase in phosphorylated Akt in isolated cardiomyocytes was abolished by the PI3-kinase inhibitor wortmannin, which provided evidence for a role of PI3-kinase. This finding suggests that the underlying cellular mechanism for up regulation of SERCA2a is mediated by the PI3-kinase-Akt-SERCA2a signaling cascade.
From a pathophysiological point of view, insulin-induced up-regulation of myocardial SERCA2a may be seen as a feedback mechanism in handling the volume overload caused by high glucose levels in the early phase of type 2 diabetes, when insulin levels are high. With progression of the disease and decreasing levels of insulin the expression of SERCA2a in the heart becomes impaired. The reduction of SERCA2a, as typically seen in the late phase of type 2 diabetes, is a major cause of reduced diastolic and systolic function of the heart. This hypothesis is reinforced by the findings of Sakata et al. who demonstrated that cardiac SERCA2a gene transfer restores systolic and diastolic function to normal in diabetic rats [
8]. Furthermore, in-vitro experiments provide evidence that high glucose levels also impair cytosolic Ca2+ removal involving slowed SR Ca2+ uptake. It has been speculated that slowed SR Ca2+ uptake results from depressed protein kinase A (PKA) down-regulating SERCA2a, rather than through depressed SERCA expression. Both expression and function of the Na-Ca-exchanger (NCX) appear to be normal in these experiments [
16]. In summary, the up-regulation of SERCA2a in the early phase of type 2 diabetes is an important physiological adaptation of the heart allowing it to handle volume overload caused by high glucose levels.