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Online supplement methods.
1. siRNA transfection.
Cardiomyocytes were grown on 48-well cell culture plate. After 24 hours in culture,
the cardiomyocytes were ready for transfection for control siRNA (Santa Cruz) or
siRNA specific for mouse PKC (Santa Cruz) with Lipofectamine 2000 (Invitrogen)
according to manufecturer’s instructions. Briefly, the myocytes were washed with
transfection medium (serum and antibiotic free) followed by adding the siRNAlipofectamine 2000 mixture (final concentration for the siRNA was 80 nM) to the
myocytes and cultured with transfection medium for 24 hours. Subsequently, the
medium was challenged to M199 containing 100% fetal calf serum. Myocytes were
harvested 48hrs after the transfection for evaluation of knock down efficiency with
Western blot.
2. Aldose reductase (AR) activity.
Aldose reductase activity was measured as previously studies1,2. Briefly,
cardiomyocytes were harvested after washed with ice-cold PBS. Subsequently, the
cells were incubated with 20mM sodium phosphate buffer (pH 7.0) containing 2 mM
dithiothreitol, 5 M leupeptin, 2 M pepstatin, and 20M phenylmethylsulfonyl
fluoride. The cell lysates were centrifugated at 4oC, 2000g for 10 min and the
supernatant was used for enzyme analysis. The AR activity was determined in
reaction mixtures containing 0.1M sodium phosphate buffer (pH=6.2), 150M
NADPH, 10mM DL-glyceraldehyde, and the enzyme solution in a total of 100L
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volume. The activity of AR was measured with a spectrophotometer by estimating
NADPH oxidation from the decrease in absorbance at 340nm. Assays were conducted
at room temperature with an appropriate blank and a series of NADPH concentrations
as standard. The concentration of protein was measured and one unit of AR activity
was defined as the amount of enzyme catalyzing the oxidation of 1 nmol of
NADPH/min under the present conditions.
3. Intracellular ATP assay.
Cardiomyocyte ATP levels were measured with a commercially available
fluorometric assay system (Biovision, cat# K354-100) according to manufacturer’s
instruction. Briefly, the cardiomyocytes (1 x 106) were lysed with 100 µl ice cold
lysis buffer followed by centrifugation (15,000 g for 5 min at 4oC). Subsequently, 50
µl of the supernatants were added to 50 µl of ATP reaction mixture which containing
ATP fluorescence probe, ATP converter, Developer mixture and assay buffer. The
mixed reaction solution was incubated at room temperature for 30 min in light free
environment. Fluorescence intensity was determined with Victor 3 multi-label
counter (Perkin Elmer) at excitation and emission wavelengths of 502 and 523 nm,
respectively. ATP concentration was calculated based on standard curve and
normalized to the protein content and expressed as nmole per mg protein.
4. Determine intracellular lactate/pyruvate ratio.
To determine lactate/pyruvate ratio in cardiomyocytes, the cells were washed with ice
cold PBS followed by lysed with a 0.8 M perchloric acid. The cell lysates were
harvested and centrifuged at 15,000 g for 5 min at 4 oC. The supernatants were
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collected for determination of the lactate and pyruvate levels with a Lactate Assay kit
(Biovision, cat# K607-100) or a Pyruvate Assay kit (Biovision, cat# K609-100)
according the manufacturer’s instructions, respectively. Finally, the lactate/pyruvate
ratio is calculated.
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Online Figure legend.
Figure 1. Transfection of cardiomyocytes with siRNA specific for the PKCβII decrease
the PKCβII expression. Cardiomyocytes were harvested at 48 hrs after the tranfection for
protein expression assay with Western blot. Upper panel: representative blots of 2
separate experiments. Lower panel: densitometry analysis data.
Figure 2. Aldose reductase activity (A) and lactate/pyruvate ratio (B) were determined in
cardiomyocyte with HG or HG with A/R. n=3, p<0.05 vs mannital/N/R, +p<0.05 vs
HG/N/R, #p<0.05 vs HG/N/R, $p<0.05 vs HG/A/R. Intracellular ATP levels were
measured in HG treated cardiomyocytes after the A/R (C). n=3, *p<0.05 vs HG/N/R,
#p<0.05 vs HG/A/R.
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References
1. Hwang YC, Sato S, Tsai JY, Yan S, Bakr S, Zhang H et al. Aldose reductase
activation is a key component of myocardial response to ischemia. FASEB J, 2002;
16:243-5.
2. Kang ES, Iwata K, Ikami K, Ham SA, Kim HJ, Chang KC et al. Aldose reductase
in keratinocytes attenuates cellular apoptosis and senescence induced by UV
radiation. Free Radic Biol Med. 2011;50:680-8.
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