BACKGROUND: Calcium (Ca2+) handling proteins are known to play a pivotal role in 
the pathophysiology of cardiomyopathy. However little is known about early 
changes in the diabetic heart and the impact of insulin treatment (Ins).
METHODS: Zucker Diabetic Fatty rats treated with or without insulin (ZDF ± Ins, 
n = 13) and lean littermates (controls, n = 7) were sacrificed at the age of 
19 weeks. ZDF + Ins (n = 6) were treated with insulin for the last 6 weeks of 
life. Gene expression of Ca2+ ATPase in the cardiac sarcoplasmatic reticulum 
(SERCA2a, further abbreviated as SERCA) and phospholamban (PLB) were determined 
by northern blotting. Ca2+ transport of the sarcoplasmatic reticulum (SR) was 
assessed by oxalate-facilitated 45Ca-uptake in left ventricular homogenates. In 
addition, isolated neonatal cardiomyocytes were stimulated in cell culture with 
insulin, glucose or triiodthyronine (T3, positive control). mRNA expression of 
SERCA and PLB were measured by Taqman PCR. Furthermore, effects of insulin 
treatment on force of contraction and relaxation were evaluated by 
cardiomyocytes grown in a three-dimensional collagen matrix (engineered heart 
tissue, EHT) stimulated for 5 days by insulin. By western blot phosphorylations 
status of Akt was determed and the influence of wortmannin.
RESULTS: SERCA levels increased in both ZDF and ZDF + Ins compared to control 
(control 100 ± 6.2 vs. ZDF 152 ± 26.6* vs. ZDF + Ins 212 ± 18.5*# % of control, 
*p < 0.05 vs. control, #p < 0.05 vs. ZDF) whereas PLB was significantly 
decreased in ZDF and ZDF + Ins (control 100 ± 2.8 vs. ZDF 76.3 ± 13.5* vs. 
ZDF + Ins 79.4 ± 12.9* % of control, *p < 0.05 vs control). The increase in the 
SERCA/PLB ratio in ZDF and ZDF ± Ins was accompanied by enhanced Ca2+ uptake to 
the SR (control 1.58 ± 0.1 vs. ZDF 1.85 ± 0.06* vs. ZDF + Ins 2.03 ± 0.1* 
μg/mg/min, *p < 0.05 vs. control). Interestingly, there was a significant 
correlation between Ca2+ uptake and SERCA2a expression. As shown by in-vitro 
experiments, the effect of insulin on SERCA2a mRNA expression seemed to have a 
direct effect on cardiomyocytes. Furthermore, long-term treatment of engineered 
heart tissue with insulin increased the SERCA/PLB ratio and accelerated 
relaxation time. Akt was significantly phosphorylated by insulin. This effect 
could be abolished by wortmannin.
CONCLUSION: The current data demonstrate that early type 2 diabetes is 
associated with an increase in the SERCA/PLB ratio and that insulin directly 
stimulates SERCA expression and relaxation velocity. These results underline the 
important role of insulin and calcium handling proteins in the cardiac 
adaptation process of type 2 diabetes mellitus contributing to cardiac 
remodeling and show the important role of PI3-kinase-Akt-SERCA2a signaling 
cascade.