Experimental procedures were approved by the St Vincent's Hospital Animal Ethics Committee, Melbourne, Australia in accordance with the National Health and Medical Research Council of Australia's Code of the Care and Use of Animals for scientific purposes: Ethics Approval ID. 063/03 (PC2 certificate number. 1480/2003, Permit number. SPPL120).

Six week old male homozygous transgenic m(Ren-2)27 rats (St. Vincent's Hospital Animal Resource Centre, Melbourne, Victoria, Australia) were randomized to receive either 55 mg/kg of streptozotocin (STZ; Sigma, St Louis, MO, USA) diluted in 0.1 mol/L citrate buffer pH 4.5 (diabetic) to induce experimental type 1 diabetes or citrate buffer alone (non-diabetic control) by tail vein injection following overnight fasting. Diabetic and control rats (n = 10) were further randomized to receive either an orally active synthetic antioxidant, DiOHF (3′,4′-dihydroxyflavonol; Indofine Chemical Co., USA) at 1 mg/kg or vehicle (1% carboxy methyl cellulose solution; CMC) by daily gavage for six weeks post STZ. DiOHF was a gift from NeuProtect Pty Ltd, Melbourne VIC. Animals were housed in a stable environment maintained at 21±1°C (12 hour light/dark cycle commencing at 6 am). Animals had free access to standard rat chow (GR2 Clark-King and Co, Gladesville, NSW, Australia) and drinking water.

Each week, rats were weighed and their blood glucose levels were measured (Accucheck Advantage II Blood Glucose Monitor, Roche Diagnostics, USA). Only STZ-treated animals with blood glucose greater than 15 mmol/L were considered diabetic. Prior to the induction of diabetes and every three weeks post randomization, systolic blood pressure (SBP) was assessed in preheated conscious rats by tail cuff plethysmography using a non-invasive blood pressure controller and Powerlab system (AD Instruments Pty Ltd, NSW, Australia) ^[23], ^[24]. Diabetic animals received 2–4 units of isophane insulin (Humulin NPH; Eli Lilly and Co., NSW, Australia) intraperitoneally 3 times per week to maintain blood glucose levels, promote weight gain and reduce mortality.

At the end of the experimental period, animals were anaesthetized (pentobarbitone sodium 30 mg/kg body weight i.p.; Virbac, Peakhurst, NSW, Australia). The abdomen, neck, and chest were shaved, and echocardiography performed followed by in vivo left ventricular pressure-volume loop acquisition.

Detailed two dimensional and Doppler echocardiography were performed to assess systolic and diastolic function using a Vivid 7 Dimension (GE Vingmed, Horten, Norway) echocardiograph with a 10 MHz phased array probe, as previously published ^[21]. Animals underwent echocardiographic interrogation in the left recumbent position, after being anaesthetized using the protocol above. All data were acquired and analyzed by a single blinded observer using EchoPAC (GE Vingmed) offline processing.

Cardiac catheterization was performed as previously published ^[21]. Post echocardiography, animals were placed on a warming pad (37°C), intubated using a 14 gauge catheter, and ventilated using positive pressure with a tidal volume of 8 to 10% body weight at 70 breaths/min using room air. The right internal carotid artery was identified, ligated, and a 2F miniaturized combined conductance catheter-micromanometer (Model SPR-838 Millar instruments, Houston, TX) was inserted to obtain aortic blood pressure, then advanced into the LV until stable PV loops were obtained. Data were acquired under steady state conditions and during preload reduction. Using the pressure conductance data, a range of functional parameters was then calculated (Millar analysis software PVAN 3.4). These included end diastolic pressure (EDP), end systolic pressure (ESP), the time constant of the isovolumic-pressure decline (Tau, τ Weiss), the slope of end diastolic pressure volume relationship (EDPVR), and the slope of the preload recruitable stroke work relationship (PRSW), defined as the relationship between stroke work (SW) and end diastolic volume (EDV) ^[21]. The active and passive phases of diastole were resolved by the assessment of Tau and EDPVR, respectively.

Animals were euthanized with a further dose of anaesthetic (pentobarbitone sodium 60 mg/kg of body weight i.p.; Virbac, Peakhurst, NSW, Australia) and their hearts and lungs excised. The whole heart and lung were blotted dry and weighed. The heart was then dissected into atria, right ventricle and left ventricle, inclusive of the interventricular septum, and weighed separately. The apex was removed, bisected and snap frozen in −80°C liquid nitrogen for chemiluminescence. The top portion of the LV was fixed in 10% neutral buffered formalin overnight for subsequent processing and the mid portion was embedded in Tissue-Tek® OCT compound (Sakura Finetek USA, Inc., Torrance, CA, USA) and stored in a −80°C freezer. The formalin-fixed LV sections were paraffin-embedded and sectioned to 4 µm thick using a rotary microtome (Leica Microsystems GmbH Wetzlar, Germany).

Changes in cardiac structure were assessed in a masked protocol in at least 10 randomly selected tissue sections from each group studied. Sections were stained with picrosirius red to demonstrate collagenous matrix and fibrillar collagen types I and III were assessed using specific antibodies (anti-type I collagen 1∶100: Southern Biotechnology Associates Inc., Birmingham, AL, USA; anti-type III collagen antibody: Biogenex, San Ramon, CA, USA). Indication of oxidative damage within the myocardium was assessed using anti-nitrotyrosine 1∶40 (Upstate, NY, USA). In brief, 4 µm LV sections were dewaxed in histolene, rehydrated in graded ethanol, and immersed in distilled water. Each LV section was incubated in 100 µL of picrosirius red (Merck Pty Ltd, Kilsyth, Victoria, Australia) solution for 1 hour at room temperature to aid visualization of collagenous matrix. Sections were then briefly rinsed in 2 changes of acidified water (1% acetic acid), dehydrated in 2× 100% ethanol for 3 minutes each and 2× histolene for 2 minutes each, and coverslipped with DPX mounting media. Immunohistochemistry was performed on 4 µm sections, as previously described ^[21].

The extent of cardiac myocyte hypertrophy, as measured by cross sectional area (CSA), was determined on haemotoxylin-eosin stained sections as previously published ^[21]. In brief, only myofibers with intact cellular membranes from field with circular capillary profiles and myofiber shapes were assessed and the circumferences of 20–30 cells per LV were traced and digitized to calculate mean CSA.

The accumulation of matrix (picrosirius red) and the extent of immunostaining of collagen I, collagen III, and 3-nitrotyrosine were quantified using computer-assisted image analysis software, as previously described ^[21]. Sections were scanned and digitized using Aperio ScanScope Console v.8.0.0.1058 (Aperio Technologies, Inc). Ten random non-overlapping fields from each section around the subendocardial region were captured using Aperio ImageScope v.8.0.39.1059 (Aperio Technologies, Inc) or Axioimager.A1 microscope attached to an AxioCam MRe5 digital camera (Carl Zeiss AxioVision, Germany) at ×230 magnification. Images were then exported and loaded onto a Pentium D Dell computer. An area of red on picrosirius red-stained (for matrix) or brown on immunostained sections (for collagen I and collagen III) were highlighted using a selective colour tool. Calculation of proportional area stained red or brown was determined using image analysis software AIS (Analytic Imaging Station version 6.0, Imaging Research Inc., Ontario, Canada) or AxioVs40LE (AxioVision version 4.5.0.0).

NADPH oxidase-dependent superoxide production in LV tissues was measured using lucigenin-enhanced chemiluminescence (LEC) as previously described ^[25]. Equal size of LV tissues were homogenized using glass homogenizer in the buffer containing 250 mM sucrose and 10 mM HEPES with protease inhibitors. Lysate was collected by centrifugation at 12,000 rpm for 10 mins at 4°C. Protein was quantitated and equal amount of lysate was transferred into a 96-well OptiPlate (Packard BioScience, Australia) with 200 µl Krebs-HEPES buffer in each well. 5 µM lucigenin and 100 µM NADPH were added into each well and the chemiluminescence was detected using a POLARstar OPTIMA plate reader (BMG Labtech, Offenburg, Germany). All the chemiluminescence data were normalised to protein concentration.

Real-time quantitative PCR was used to assess transcript levels of thioredoxin-interacting protein (TXNIP, NM_006472), Cu/Zn superoxide dismutase (SOD1, NM_017050), and glutathione peroxidase (Gpx1, NM_030826). A 20–25 µL of real-time PCR reaction included Brilliant SYBR Green QRT-PCR Master Mix as per manufacturer's instructions (Stratagene, USA). Real-time quantitations were performed using ABI Prism 7000 Sequence Detection System (Applied Biosystems, CA, USA) according to manufacturer's instructions. The fluorescence threshold value was calculated and the calculation of relative change in mRNA was performed using the delta-delta C_T method, with normalization for the housekeeping gene 18s. Nucleotide sequences of primers were as follows:

TXNIP (forward) 5′-AGGATTCTGTGAAGGTGATG-3′

TXNIP (reverse) 5′-TCTGACTGAGGACAGCTTCT-3′

SOD1 (forward) 5′-CGGTGCAGGGCGTCATTCACTT-3′

SOD1 (reverse) 5′-CCGCTGGACCGCCATGTTTCTT-3′

Gpx1 (forward) 5′-GGTGCTGGGCTCTGACTGCG-3′

Gpx1 (reverse) 5′-GCGCGCGGAGAAGGCATACA-3′

18S (forward) 5′-TCGAGGCCCTGTAATTGGAA-3′

18S (reverse) 5′-CCCTCCAATGGATCCTCGTT-3′

Synthesis of riboprobes and in situ hybridization were performed as previously described ^[25], ^[26]. Briefly, ³³P-labelled antisense RNA probe for Txnip was generated by in vitro transcription (Promega, WI, USA) from linearized templates. Primers were designed using Oligo 6 primer design software (Molecular Biology Insight, CO, USA). Two rat Txnip probes were generated by RT-PCR, using rat kidney cDNA as template, to span the regions 1481–2004 (540 bp) and 2008–2384 (377 bp) of the rat Txnip sequence (NM_001008767). Purified riboprobe length was adjusted to approximately 150 bases by alkaline hydrolysis. In situ hybridization was performed on 4 µm thick sections of formaldehyde-fixed, paraffin-embedded LV tissue. Briefly, tissue sections were dewaxed in histolene, rehydrated in graded ethanol, and microwaved in 10 mM citrate buffer pH 6.0 on medium-high (600 to 700 W) for 5 min. Sections were washed in 0.1 M sodium phosphate buffer pH 7.2, fixed in 4% paraformaldehyde for 10 min, and washed again in phosphate buffer and milliQ water. After equilibration in P buffer (50 mM Tris-HCl pH 7.2 and 5 mM EDTA pH 8.0), slides were incubated with 125 µg/ml Pronase E (Sigma) in P buffer pH 7.2, refixed in 4% paraformaldehyde for 10 min, rinsed in milliQ water, dehydrated in 70% ethanol, and air dried. Hybridization of the riboprobe to the pretreated tissue was performed overnight at 60°C in 50% formamide-humidified chambers. Sense probes were used on an additional set of tissue sections as controls for nonspecific binding. After hybridization, slides were washed, incubated with RNase A, dehydrated in graded ethanol, air dried, and exposed to Kodak Biomax MR autoradiographic film (Kodak, Rochester, NY) for 3 days.

Densitometry of autoradiographic images obtained by in situ hybridization was performed by computer-assisted image analysis using Micro Computing Imaging Device (MCID; Imaging Research, Ontario, Canada) as previously described ^[25]. In brief, in situ autoradiographic images were placed on a uniformly illuminating fluorescent light box (Northern Light Precision Luminator model C60) and captured using a video camera (Dage MTI CCD72) connected to an IBM AT computer with a 512×512 pixel array imaging board with 256 grey levels. After calibration by construction of a curve of optical density versus radioactivity density using Amersham ¹⁴C microscale autoradiography standard, which were co-exposed with the hybridized sections, quantification of digitalized autoradiographic images was performed with the MCID software and expressed as nCi/g.

Western blots were performed as previously described ^[27]. Protein concentration of heart tissue homogenates were determined using Bio-Rad Bradford Protein Assay (Bio-Rad, CA, USA). Samples with equal concentration of protein were subjected to SDS-PAGE and western blot analysis with rabbit polyclonal Txnip antibody overnight at 4°C (1∶1000: Invitrogen, CA, USA). Following incubation with anti-rabbit secondary antibody (1∶2500: Dako, Golstrup, Denmark) for 1 hour at room temperature, proteins were detected by ECL detection system. The membranes were reprobed with pan-actin (1∶500: Neomarkers, CA, USA), which served as loading control. The bands corresponding to Txnip (50 kDa) and pan-actin (42 kDa) were quantitated by densitometry using Quantity One Software (Bio-Rad, CA, USA) and expressed as the ratio of the loading control. At least six samples were analysed from each group in four separate gels.

Data were expressed as mean ± standard error of mean (SE) unless otherwise stated. Differences between groups were determined by one-way analysis of variance (ANOVA) with Fisher PLSD post hoc comparison using Statview II + Graphics package (Abacus Concepts, Berkeley, CA). A value of P<0.05 was considered as statistically significant.

All rats were hypertensive with elevated systolic blood pressure. Blood glucose was elevated to a similar extent in diabetic animals. Body weight and heart weight, indexed to body weight (HW∶BW) were reduced in diabetic rats. DiOHF treatment did not significantly alter BGL, SBP, BW or HW∶BW in either control or diabetic animals (Table 1).

Chamber compliance, a measure of diastolic function was significantly reduced in diabetic rats (P<0.05) when compared to control rats (Table 2b, Fig. 1). Chamber compliance is inversely proportional to the slope of end-diastolic pressure volume relationship (EDPVR). Treatment of diabetic rats with DiOHF improved chamber compliance to levels comparable to control rats (P<0.05; Table 2b, Fig. 1). Active relaxation, as measured by Tau τ, was significantly prolonged in diabetic compared to control rats. DiOHF treatment significantly attenuated the increase in Tau τ in diabetic animals (P<0.05; Table 2b).

Diastolic function, assessed using Doppler echocardiographic examination, showed a significant reduction in E to A ratio in diabetic animals when compared to control rats (P<0.05) with a trend to improvement following DiOHF treatment (Table 3, Fig. 2). Diabetic rats demonstrated impaired relaxation with prolonged deceleration time (DT) of early (E) diastolic filling compared to control rats (P<0.05), which was significantly improved with DiOHF treatment (P<0.05; Table 3). There were no significant differences in any cardiac parameters measured in control rats when compared to control rats treated with DiOHF.

Heart rate was significantly reduced in diabetic rats when compared to control rats (P<0.05). DiOHF treatment did not significantly alter heart rate in either diabetic or control animals (Table 2a). End-diastolic pressure (EDP) was significantly elevated in diabetic compared to control rats (P<0.05) and was attenuated by DiOHF in diabetic rats.

Diabetes was associated with a significant increase in myocardial fibrosis as evidenced by greater proportional area of interstitial collagen I and III immunostaining (Fig. 3 and 4) and extracellular matrix deposition (data not shown). Diabetic rats also displayed cellular hypertrophy with increased cardiomyocyte cross-sectional area when compared with control rats (P<0.05; Fig. 5). DiOHF treatment significantly attenuated myocardial fibrosis and cellular hypertrophy (P<0.05 versus diabetic).

The presence of diabetes resulted in significant induction of intracellular oxidative stress as measured by increased tissue localisation of 3-nitrotyrosine and NADPH oxidase-dependent superoxide production (P<0.05 versus control), which were significantly attenuated by DiOHF in diabetic rats (P<0.05; Fig. 6). Similarly, Txnip mRNA and protein expression levels were significantly increased in diabetic compared to control rats (P<0.05 versus control) and were attenuated by DiOHF treatment (Fig. 8 and 9). No significant differences were observed in the control rats when compared to control-treated rats.

Diabetic animals demonstrated a marked increase in the gene expression of intracellular antioxidant enzymes such as superoxide dismutase (SOD1) and glutathione peroxidase (Gpx1) when compared to control rats (Fig. 7). SOD1 and Gpx1 mRNA levels were not changed by DIOHF treatment in diabetic rats.