Supplementary Material
Luedde et al. “Decreased contractility due to energy… “Supplementary Material
J Mol Med 2009 ______
Supplementary Material and methods
Animals
Transgenic Sprague-Dawley-rats expressing a human cTnT deletion mutation (DEL-TNT) were generated as described [1]. Hearts of these rats stably express the mutated protein at a level of 4-5% in relation to endogenous TNT and exhibit significant systolic and diastolic dysfunction in the working heart model in the absence of cardiac hypertrophy [1].
All rats were synchronized to a 12-hour cycle of alternating light-dark cycles with free access to water and food. The investigation conforms to the Guide for the Care and Use of Laborat- ory Animals by the US National Institutes of Health.
Magnetic resonance studies
Experiments were performed on a Bruker DRX 9.4T wide-bore (89 mm) NMR spectrometer operating at frequencies of 400.13 MHz for 1H and 161.97 MHz for 31P measurements. A
Bruker microimaging unit (Mini 0.5) equipped with an actively shielded 57-mm gradient set
(capable of 200 mT/m maximum gradient strength and 110 µs rise time at 100% gradient switching) was used. Data were taken from a double-tuned 1H/31P 38-mm birdcage resonator suitable for rats up to 80 g. Rats at the age of 4 weeks were anesthetized with 1.5 % isoflurane in a water-saturated gas mixture of 20% oxygen in nitrogen applied at a rate of 200 ml/min via a home-build nose cone. Throughout the experiment, rats were respiring spontaneously at Luedde et al., “Decreased contractility due to energy… “Supplementary Material
J Mol Med 2009 a rate of approximately 80 min-1 and were kept at 37° C. An ECG- and repiratory-triggered fast gradient-echo cine sequence was used to acquire high-resolution 1H images of the rat hearts [2]. Six to eight adjacent ventricular short-axis slices were obtained to map the func- tion of the entire heart. Functional parameters were analyzed as previously described [2].
After acquisition of functional data the spectroscopic slice was positioned in short axis orientation to cover the entire heart (slice thickness usually 8-10 mm). A sine-bell acquisition- weighted 2D 31P chemical shift imaging (CSI) sequences was used to acquire a matrix of
16x16 31P spectra over the rat’s thorax (total of 18432 acquisitions within 75 min). CSI data sets were recorded from a field of view (FOV) of 30x30 mm2 resulting in a spatial resolution of 1.875 mm in the x and y directions. 2D data sets were analyzed by a self-developed soft- ware module allowing quantification of spatially localized 31P MR spectra in direct correlation to the morphological 1H MR image [3]. 31P peak areas were obtained by integration and
PCr/ATP ratios were calculated for each voxel to obtain a direct in vivo measurement of car- diac energetics. Chemical shifts were referenced to the PCr resonance at -2.52 ppm.
Quantification of ATP and PCr concentrations
ATP peak areas were converted to concentrations on basis of the heart extract data (see be- low). Subsequently, PCr concentrations were calculated from the PCr/ATP ratio measured in vivo. Absolute quantification and tissue extracts were essentially performed as previously de- scribed [3]. In brief, rats were anesthetized by intraperitoneous injection of Thiopental (™
Trapanal, 100mg/kg body weight), and immediately after thoracotomy hearts were arrested with ice-cold cardioplegic solution. Hearts were rapidly excised, snap-frozen, and extracted with 1 mol/L perchloric acid (PCA). Extracts were neutralized, lyophilized, and stored at 20
°C. Lyophilized PCA extracts were redissolved in 0.6 ml D2O and transferred into a 5-mm Luedde et al., “Decreased contractility due to energy… “Supplementary Material
J Mol Med 2009 NMR tube. Subsequently, the pool size of various metabolites (in particular total creatine and
ATP) was quantified from fully relaxed high resolution 1H and 31P MR spectra as previously described [3].
Isolation and culture of adult rat ventricular cardiomyocytes
Adult rat cardiomyocytes (ARCM) were isolated from Sprague-Dawley rats (~300g) using the collagenase digestion method [4] and plated on laminin-coated dishes. ARCM were cultured in a HEPES-modified medium 199 (M199, Sigma S7528, supplemented with 5mM taurine, 5mM carnitine, 5mM creatine, 5mM N-mercaptoproprionyl glycine, 0.1µM insulin,
10,000 U/ml penicillin and 10 mg/ml streptomycin, pH 7.25).
Measurement of contractile parameters
Contractile parameters in ARCM were obtained 24 hours after plating by video edge detection, as previously described [5]. Contraction amplitude and rates of contraction and re-
+ laxation were assayed in the HEPES-modified medium 199 described above at 1.8mM [Ca² ]e and at 0.5 Hz field stimulation. Analysis of steady-state twitches was performed by LabView
4.01 (National Instruments).
Transmission Electron Microscopy
The detailed procedures to prepare samples for electron microscopy scanning have been performed as described elsewhere [6,7].
Hearts from DEL-TNT rats and WT controls were fixated with 10% neutral formalin immediately after explantation. After fixation was completed, several small blocks ( 5 mm diameter) were cut from the middle layer of the transmural myocardium for scanning electron microscopy. The cuboidal blocks were subsequently washed several times in 0.1 mol/L phos- phate buffer solution at pH 7.2 and then immersed in 8N hydrochloric acid in a test tube, Luedde et al., “Decreased contractility due to energy… “Supplementary Material
J Mol Med 2009 shaken in a water bath at 60°C for 40 to 60 minutes, and then placed in buffer overnight. After dehydration through a graded series of alcohol up to 100% ethanol, the specimens were pro- cessed for critical-point drying in liquid carbon dioxide.
Specimens from each animal were analyzed using a Hitachi S-800 scanning electron microscope at an accelerating voltage of 20 kV. Photographic final magnification was x2100.
Following random sampling of specimens (via blocks and microscopic fields), the fraction of cell volume occupied by mitochondria was determined using a lattice of points arranged as
2cmx2cm squares on the scanning microscopic photographs. Fractions of cell volume occu- pied by mitochondria were summed over all images from each animal. The examiner was blinded to the genotype of the respective animals (transgenic vs. WT).
RNA isolation
Total RNA was isolated from rat heart tissue using the TRIzolTM reagent (Invitrogen) according to the manufacturer’s protocol. RNA was resuspended in diethyl pyrocarbonate– treated H2O. RNA integrity was electrophoretically verified by ethidium bromide staining and confirmed by OD260/OD280 absorption. RNA was pooled from several specimens to re- duce biologic variability.
Quantitative real-time PCR analysis of gene expression
5 µg total RNA of each condition were transcribed to first strand cDNAs with 200 U
TM reverse transcriptase Superscript III (Invitrogen) using 1µl of 50 µM (dT)(20) oligonuc- leotides and 1 µl of 10 µM of deoxynucleotide triphophates. Reaction conditions were set as recommended by the supplier. Realtime PCR primers (MWG Biotech) were designed as shown in supplemental table 1. The target genes were normalized using oligonucleotide Luedde et al., “Decreased contractility due to energy… “Supplementary Material
J Mol Med 2009 primers for β-actin as an internal standard. The ABI Prism 7700 Sequence Detection System
(Perkin Elmer Applied Biosystems) and the Platinum SYBR Green qPCR SuperMix-UDG
(Invitrogen) were used for performing the real-time PCR from reverse transcribed cDNA samples. Specificity of the reactions was confirmed by performing a dissociation protocol after each cycle and comparison of the results with the expected melting-point temperature of the amplicon as well as by verification of the expected size of the product on a 2% agarose gel. Each PCR amplification was carried out in triplicate wells, using the following conditions unless stated differently: 2 minutes at 95°C, followed by a total of 40 three temperature cycles
(15 seconds at 95°C, 15 seconds at 57°C and 1 minute at 72°C).
Protein analysis and Western Blot
Protein analysis and Western Blot of whole cell extracts were performed as previously de- scribed [1]. The antibodies used were: anti-CD36 (Santa Cruz Biotechnology) and anti- α-tu- bulin (sigma) as loading control. Nuclear extracts were prepared using the Active Motif™ nuclear extract kit according to the manufacturer`s protocol. Western Blot of nuclear extracts was perormed using an anti-PGC-1α antibody (Santa Cruz Biotechnology). An anti-Histon 2B antibody (IMGENEX) served as loading control.
Radiotransmitter implantation and recording of cardiovascular parameters
Radiotransmitters for continuous monitoring of heart rate and blood pressure
(TA11PA-C40, Data Sciences International, St.Paul, Minn., USA) were implanted as de- scribed elsewhere [8].
Data were collected using a receiver panel, (BPR86) that was placed underneath the animal`s cage, and further processed by means of the LabPro programme (Data Sciences, St.- Luedde et al., “Decreased contractility due to energy… “Supplementary Material
J Mol Med 2009 Paul, USA) and the HRV Module, AD Instruments Chart Module Series (Power Lab System).
This system allowed the recording of heart rate and the distinction between sinus rhythm and premature ventricular complexes. Three weeks post insertion of the transmitters, experiments were started: After recording of heart rate and rhythm during a 5-min interval under resting conditions, DEL-TNT rats and WT control animals were stimulated by intraperitoneal injec- tion of either isotonic salt solution or isoproterenol solution in ascending concentrations between 1 and 64 µg/kg body weight. Immediately following the injection, cardiovascular parameters were recorded for another 15 min.
______Luedde et al., “Decreased contractility due to energy… “Supplementary Material
J Mol Med 2009 ______
Supplementary Figure 1:
DEL-TNT cardiomyocytes show decreased fractional shortening after isoproterenol stimulation and glucose depletion
Stimulation of WT and DEL-TNT cardiomyocytes with 10 nm Isoproterenol [ISO] led to a significant increase of fractional shortening in WT and DEL-TNT cardiomyocytes. After one hour glucose depletion, DEL-TNT cells that were stimulated with Isoproterenol revealed a significant decrease in fractional shortening (-47% ± 6.2%) that was comparable to the de- crease of FS in DEL- TNT- CMs that were not stimulated with ISO (-46% ± 9.8%). WT CMs revelealed no significant decrease of FS after one hour glucose depletion with or without ISO stimulation. Data are mean values ±SEM. *P<0.05, †P<0.01, ‡P<0.001 Luedde et al., “Decreased contractility due to energy… “Supplementary Material
J Mol Med 2009 Supplementary Table
Sequences of oligonucleotide primers used in semi-quantitative real time PCR experiments
(species: rattus norvegicus).
Target gene Oligonucletide sequence β-actin Forward: 5’-TCCTGACCCTGAAGTACCCC -3’
Reverse: 5’-CGTCAGGCAGCTCATAGCTC -3’ PGC1 Forward: 5’-GTGCTGCCCTGGTTGGTGAG -3’
Reverse: 5’-CCTCGTTGTCAGTGGTCACG -3’ CPT2 Forward: 5’-GACAGCCTGCCCAGGCTGCC -3’
Reverse: 5’- GGGCCTGAGATGTAGCTGG-3’ CPT1 Forward: 5’-CCAGTCTCAGCCTCTACGGC -3’
Reverse: 5’-CACAGTGTCCTGTCTCCGTG -3’ CD36 Forward: 5’-ACGGGCACCACTGTGTACAG -3’
Reverse: 5’-GGTGCAGCTGCCACAGCCAG -3’ ATP Synthase Fo subunit Forward: 5’-ATGCAGACCACGAAGGCACTG -3’
Reverse: 5’-TCACATGGCGAAGAGGATGAG -3’ Malonyl-CoA decarboxylase Forward: 5’-CATGTGGCTCTGACCGGTGAC -3’
Reverse: 5’-CTGCAGCTCCTTGACCACTC -3’ Malate dehydrogenase Forward: 5’- ACCCAGCCAATACAAACTGC-3’
Reverse: 5’- CTGTCGTCTTTGAGGGC-3’
Luedde et al., “Decreased contractility due to energy… “Supplementary Material
J Mol Med 2009
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