Male Zucker Diabetic Fatty rats (body weight (BW) range: 106–158 g, n = 13) and male Zucker lean rats (BW 85–118 g, n = 7) were obtained at the age of five weeks from Genetic Models (Indianapolis, USA). Animals were maintained on RMH-B rat chow from Hope Farms (Woerden, Netherlands) with water ad libitum. All animals were individually housed in a 12 h dark/light cycle controlled room. The protocol had been approved by the local committee on animal research and conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85–23; revised 1985). At the age of 13 weeks, one week after developing hyperglycemia, the animals were divided into three groups: (1) Zucker lean rats (control group, n = 7), (2) Zucker Diabetic Fatty rats without insulin treatment (ZDF; n = 7), and (3) Zucker Diabetic Fatty rats treated with insulin (ZDF + Ins; n = 6). Insulin treatment (Actrapid HM U500, Novo Nordisk, Mainz, Germany) was initiated at a dose of 25.0.
U/kg/day with subcutaneously implanted Alzet osmotic minipumps (Model 2ML2 and 2ML4, Charles River Wiga, Sulzfeld, Germany). Pumps were changed after 2 weeks and the insulin dose was adapted to normalize blood glucose levels. Body weight was determined every week, and blood glucose levels every 2–3 weeks (Accu-Chek Plus Roche, Mannheim, Germany). At the age of 18 weeks, systolic blood pressure and heart rate were measured by indirect tail-cuff method as described  using an automated cuff inflator-pulse detection system (W + W electronic AG, BP recorder No. 8005, Basel, Switzerland). After 6 weeks of insulin treatment, at the age of 19 weeks, the animals were killed.
Hearts were rapidly excised, rinsed with saline and blotted dry. The whole heart weight was determined. The heart was dissected free from the atria, cut into right and left ventricular tissue, frozen in liquid nitrogen within 3 minutes and stored at −80 C until analyzed, as described earlier .
Neonatal rat cardiac myocytes
Rat cardiac myocytes were isolated from 1- to 3-day-old neonatal Wistar rats (University of Regensburg breed, from Charles River, Sulzfeld, Germany) as described earlier . Briefly, hearts from 50–70 pups were minced and subjected to serial trypsin digestion to release single cells. After the final digestion, cells were washed and pre-plated for 1–2 h in complete culture medium (MEM supplemented with 10% fetal calf serum and 1% penicillin/streptomycin). Unattached cells were pelleted and suspended in culture medium containing 0.1 mmol/l 5´-bromo-2´-desoxyuridine (BrdU) to suppress overgrowth of non- myoctes. Cells were then plated on culture dishes at a density of 150,000 cells/cm2 and incubated for 5 days at 37°C before being stimulated with insulin (0.1-3.0 μmol/l), wortmannin (300 and 1000 nM) or triiodothyronine (3.0 nmol/l) as a positive control for up-regulation of SERCA2a mRNA expression .
Engineered heart tissues
Engineered heart tissues (EHT) were prepared as described previously . Briefly, circular EHT were prepared by mixing freshly isolated cardiac myocytes from neonatal rats with collagen type 1 prepared from rat tails, a basement membrane mixture (Matrigel, tebu, Offenbach, Heidelberg, Germany), and concentrated serum containing culture medium (2xDMEM, 20% horse serum, 4% chicken embryo extract, 200 U/ml penicillin and 200 μg/ml streptomycin); pH was neutralized by titration with NaOH (0,1 N). The reconstitution mix was pipetted into circular casting molds and incubated for 30 to 45 min at 37oC and 5% CO2 to allow hardening of the reconstitution mix. Thereafter, 5 ml serum-containing culture medium (DMEM, 10% horse serum, 2% chicken embryo extract, 100 U/ml penicillin and 100 μg/ml streptomycin) was added to each dish. Culture was performed as described earlier . After 7 days in culture, EHTs were transferred to a stretch device and subjected to phasic stretch (to 110% of their original length) for 5 days. Culture medium was changed 12 hours after EHT casting and then every other day. After transfer to the stretch device, the culture medium was changed every day and supplemented with insulin at a high physiological concentration (0.1 μg/ml; Sigma-Aldrich, Taufkirchen, Germany).
After 12 days (7 days in casting molds and 5 days of stretching), the EHTs were transferred to thermostated organ baths containing gassed Tyrode´s solution and subjected to isometric force measurement as described elsewhere . Briefly, electrically stimulated EHTs (2 Hz) were stretched to the length at which force of contraction was maximal and inotropic and lusitropic responses to cumulative concentrations of isoprenaline (0.1-1000 nM) in the presence of 0.2 mM calcium were recorded. Contractile activity was evaluated with a PC-assisted system (BMON2, Ingenieurbüro Jäckel, Hanau, Germany).
Total RNA from left ventricles or cultured cardiac myocytes was isolated with Trizol® (Canadian Life Technologies Inc., Burlington, Ontario, Canada) according to the manufacturer’s instructions. The concentration was determined photometrically at 260 nm. Total RNA was stored at 80°C. For Northern blot analysis 20 μg of total RNA were denatured, size-fractionated by electrophoresis on 1% agarose gels under denaturing conditions, transferred to nylon membranes (Gene Screen Plus, NEN, Dreieich, Germany) and immobilized by ultraviolet irradiation. Blots were prehybridized and hybridized using standard protocols as described previously . Hybridized filters were washed and exposed at −80 C° to x-ray films (XAR-5, Eastman Kodak, N.Y., USA) by using intensifying screens. Different exposures of all autoradiograms were obtained to ensure that laser scanning (Personal Densitometer No. 50301, Molecular Dynamics) was performed within the linear range of densitometry. For hybridization cDNA probes for rat SERCA, PLB (kindly gifted by K.R. Boeheler) and GAPDH were radiolabelled with α32-P dCTP (specific activity 3000 Ci/mmol, Amersham, Dreieich, Germany) for Northern blot analysis. Values were normalized to these house-keeping gene GAPDH. The rat cDNA of GAPDH was cloned by reverse transcriptase PCR using the following primers: forward 5´-CTTCACCACCATGGAGAAGG-3´; and reverse 5´-ATTGAGAGCAATGCCAGCC-3´.
For quantitative RT-PCR (qRT-PCR), total RNA was transcribed with SuperScriptII RT (Invitrogen, CA, USA). Individual samples of 20 ng cDNA were amplified with AmpliTaqGold Polymerase (Applied Biosystems, CA, USA) utilizing gene specific primers and fluorogenic probes (5’ FAM and 3’ TAMRA; see below for complete primer/probe sequence information) in an ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems). Probes were designed to cross exon/intron boundaries with primer annealing sites being located in the adjacent exons to eliminate the possibility of genomic DNA amplification. . Standard curves were performed in duplicate with serially diluted cDNA from neonatal rat heart tissue (1.5 - 50 ng) to determine PCR efficiency, which was similar in all groups SERCA and Phospholamban expression were evaluated as SERCA/PLB ratio and correlated to EHT twitch tension and relaxation time (T2). Quantification was performed by the standard curve and 2-ΔΔCt methods .
SERCA2a: forward primer 5´- AGT GGC TGA TGG TGC TGA AA-3´.
reverse primer 5´- GCA CCC GAA CAC CCT TAC AT-3.
probe 5´ FAM- TTA CTC CAG TAT TGC AGG CTC CAG GTA -TAMRA 3´.
PLB: forward primer 5´- GCA GCT GAG CTC CCA GAC TT-3.
reverse primer 5´- TTT CCA TGA TGC CAG GAA GAC-3´.
probe 5´ FAM- CAC AGA AGC CAA GGC CTC CTA AAA GGA G -TAMRA 3´.
We checked 18 S, GAPDH, and CSQ2 (data not shown). However, corrections are not necessary because we have determined PLB and SERCA from the same cDNA samples.
Western blot analysis
20 μl of cell suspension of the cardiomyocyte cell culture were separated on 10% SDS-polyacrylamide gels. Gels were run andseparated proteins were transferred to nitrocellulose membranes in 50 mM sodium phosphate buffer, pH 7.4, for 20 h at 300 mA, and 4°C. Nitrocellulose sheets were incubated with a rabbit polyclonal anti-human antisera (Sigma) at a 1:2000 dilution. Phospho-Akt and Akt antibodies (1:1000, from New England Biolabs) were visualized colorimetrically by using horseradish peroxidase- (HRP) conjugated goat anti-rabbit immunoglobulin G at a 1:1000 dilution. After phospho-Akt blotting, the blot was stripped for 30 min at 50oC and then blotted for Akt, serving also as loading control. Apparent molecular weights were determined by using a prestained standard (kaleidoscope prestained standard, Biorad, USA).
Oxalate-supported Ca2+ uptake
Oxalate-supported SR Ca2+ uptake was measured in left ventricular homogenates as described previously . Briefly, the Ca2+ uptake medium of 0.2 ml contained 40 mmol/l imidazole (pH 7.0), 100 mmol/l KCl, 5 mmol/l MgCl2, 5 mmol/l TrisATP, 6 mmol/l phosphocreatine, 10 mmol/l K + -oxalate, 10 mmol/l NaN3, 10 μM synthetic protein kinase A-inhibitor peptide [PKI(6–22)amide; GIBCO-BRL, Eggenstein, Germany], 0.2 mmol/l EGTA, and 0.08 or 0.250 mmol/l 45CaCl2 corresponding to 0.34 or 3.68 μmol/l free Ca2+, respectively After 2 min of preincubation at 37°C, the measurement was started by addition of homogenate (30 μg protein) and 2 min later a 0.15-ml sample was filtered through 0.45- μm Millipore filters using a vacuum pump. The filter was immediately washed twice with 3 ml ice-cold solution containing 100 mM KCl, 2 mM EGTA, and 40 mM imidazole (pH 7.0). Radioactivity bound to dry filters was determined by liquid scintillation counting. All measurements were done in duplicate Ca uptake was measured within the linear range of the reaction. Calculated Ca2+ uptake values were expressed as nmoles of Ca2+ per mg of protein per min or μmoles of Ca2+ per g wet LV wt min.
Statistical analysis was performed using GraphPad PRIZM 5.0. Results are expressed as mean ± SEM. Comparisons between multiple groups were assessed by one-way analysis ANOVA-test and post-hoc analysis by Bonferroni. The strength of the relationship between two variables was assessed by calculating the product–moment correlation coefficient r. Statistical significance was accepted at p < 0.05.