title: Increased myocardial SERCA expression in early type 2 diabetes mellitus is insulin dependent: In vivo and in vitro data.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Klinik und Poliklinik für Innere Medizin II, Universität Regensburg, Regensburg, Germany. sabine.fredersdorf@klinik.uni-r.de
authors.forename: Sabine
authors.initials: S
authors.lastname: Fredersdorf
authors.forename: Christian
authors.initials: C
authors.lastname: Thumann
authors.forename: Wolfram H
authors.initials: WH
authors.lastname: Zimmermann
authors.forename: Roland
authors.initials: R
authors.lastname: Vetter
authors.forename: Tobias
authors.initials: T
authors.lastname: Graf
authors.forename: Andreas
authors.initials: A
authors.lastname: Luchner
authors.forename: Günter Aj
authors.initials: GA
authors.lastname: Riegger
authors.forename: Heribert
authors.initials: H
authors.lastname: Schunkert
authors.forename: Thomas
authors.initials: T
authors.lastname: Eschenhagen
authors.forename: Joachim
authors.initials: J
authors.lastname: Weil
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015152:Blotting, Northern; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000097:blood / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000503:physiopathology; D004195:Disease Models, Animal; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000201:enzymology*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011924:Rats, Zucker; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D015854:Up-Regulation; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D007004:Hypoglycemic Agents; D007328:Insulin; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban; C506948:Atp2a2 protein, rat