The animal study was conducted according 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 1996) and the protocol was approved by the Animal Research Committee of Wuhan University (Wuhan, China).

A total of 24 18-week-old male SH rats weighing 330–360 g were randomly divided into two groups (12 rats in each): Losartan-treated [los-SH group, 10 mg/kg⁻¹/d⁻¹, intragastric administration (ig)] (8) and SH group. A total of 24 18-week-old male WKY rats weighing 330–360 g were randomly divided into two groups (12 rats in each): Losartan-treated (los-WKY; 10 mg/kg⁻¹/d⁻¹, ig) and WKY. Two losartan-treated groups were administered losartan dissolved in tap water for eight weeks, and SH and WKY groups received only tap water during the same time period. All rats were supplied by the experimental animal center of Wuhan University Medical College (Wuhan, China) and bred in our laboratory. All rats were housed in individual cages and fed a standard diet and tap water ad libitum. They were maintained in a quiet room at constant temperature (20–22°C) and humidity (50–60%) with 12-h light/dark cycle.

LV myocytes were isolated according to methods previously described (5). Briefly, rats were sacrificed by cervical dislocation. The heart was rapidly removed and mounted on a Langendorff apparatus by aortic cannulation. The heart was first perfused for 5 min at 35°C with calcium-free Tyrode’s solution containing NaCl 140 mmol/l⁻¹, KCl 5.4 mmol/l⁻¹, NaH₂PO₄ 0.33 mmol/l⁻¹, MgCl₂ 0.5 mmol/l⁻¹, HEPES 5 mmol/l⁻¹ and glucose 5.5 mmol/l⁻¹ (pH 7.4 with NaOH), and then the heart was perfused for 12–15 min with the same solution containing 80 μmol/l⁻¹ Ca²⁺ and collagenase I 0.33 g/l⁻¹, bovine serum albumin (BSA) 0.25 g/l⁻¹ and protease E 0.25 g/l⁻¹. Next, the heart was washed out with calcium-free Tyrode’s solution. The heart was removed from the Langendorff apparatus and the LV tissue was dissected into small sections and gently agitated in 2 mmol/l⁻¹ CaCl₂-containing Tyrode’s solution. The cells were suspended in modified Kreb’s solution (K-glutamate 100 mmol/l⁻¹, K-aspartate 10 mmol/l⁻¹, KCl 25 mmol/l⁻¹, glucose 20 mmol/l⁻¹, KH₂PO₄ 10 mmol/l⁻¹, HEPES 5 mmol/l⁻¹, MgSO₄ 2 mmol/l⁻¹, taurine 20 mmol/l⁻¹, creatine 5 mmol/l⁻¹, EGTA 0.5 mmol/l⁻¹ and 0.1% BSA, pH 7.4 with KOH) and stored at room temperature for 2 h prior to use and used within 10 h of isolation. The reagents are products of Sigma-Aldrich (St. Louis, MO, USA).

The ruptured patch whole-cell configuration was used as described previously (9). The isolated cells were transferred to an open perfusion chamber mounted on the stage of an inverted microscope (model IMT2; Olympus Corporation, Tokyo, Japan). Following being set to the bottom of the chamber, cells were perfused with Tyrode’s solution that contained NaCl 140 mmol/l⁻¹, KCl 5.4 mmol/l⁻¹, Na₂HPO₄ 1 mmol/l⁻¹, HEPES 5 mmol/l⁻¹, glucose 10 mmol/l⁻¹, MgCl₂ 1 mmol/l⁻¹ and CaCl₂ 1 mmol/l⁻¹ (pH 7.35 with NaOH) to wash out the dead cells. Only quiescent rod-shaped cells showing clear cross striations were used. The external solutions were gassed with 100% O₂. Whole-cell membrane currents were recorded with a patch-clamp technique using Pulse+Pulsefit software (version 8.31; HEKA Elektronik, Lambrecht, Germany) and a patch amplifier (EPC-9; HEKA Elektronik). Glass pipette electrodes were forged by a micropipette puller (PB-7; Narishige, Tokyo, Japan) and the resistance was 2–4 MΩ. The current signal was filtered with a low-pass filter and digitized by an A/D converter (Labmaster 1600; Labmaster Oy Ltd., Aura, Finland) under the control of a computer (IBM PC/AT; IBM, Armonk, NY, USA) and stored on a hard disk for later analysis. The liquid junction potential was corrected following immersion of the pipette in the solution. Following establishment of a tight pipette-membrane seal (seal resistance >1 GW), fast capacitance was compensated, the membrane was ruptured with gentle suction to obtain the whole-cell voltage-clamp configuration and slow capacitance and series resistance were compensated. Data were analyzed using the software program pCLAMP (Axon Instruments, Inc., Sunnyvale, CA, USA). Temperature was maintained at 20°C.

Tyrode’s solution for I_to (normal solution plus 0.5 mmol/l⁻¹ CdCl₂, used to block calcium current). The intracellular solution contained KCl 20 mmol/l⁻¹, K-aspartate 110 mmol/l⁻¹, MgCl₂ 1.0 mmol/l⁻¹, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid 10 mmol/l⁻¹, ethylene glycol tetraacetic acid 5 mmol/l⁻¹ and bisodium adenosine triphosphate 5 mmol/l⁻¹ (pH 7.3 with KOH). The I_to was evoked by steps in the range between −40 and 70 mV from a holding potential (HP) of −70 mV (sampling rate, 5 kHz); a prestep to −40 mV was used to inactivate the sodium current. Series resistance and membrane capacitance were compensated by 80% to minimize the capacitive transient. I_to was measured as the peak outward current at the beginning of the depolarizing step and normalized with respect to the membrane capacitance value. Recovery from inactivation was evaluated by applying double pulses to 60 mV, separated by intervals of 5–300 msec. APs were elicited by a brief (2 msec) suprathreshold pulse applied at a frequency of 2 Hz. Cell membrane capacitance was measured by applying a ±10 mV pulse starting from a HP of −70 mV.

The gene-specific sequences of oligonucleotide primers (Table I) were used to check the expression of respective genes with a ABI-Prism 7700 Sequence Detection system (Applied Biosystems, Inc., Foster City, CA, USA) and a 1× final concentration of SYBR^®-Green PCR Master mix containing SYBR-Green I dye, AmpliTaq Gold DNA Polymerase, dNTPs and optimized buffer components, and 0.25 U/ml MultiScribe Reverse Transcriptase, 0.4 U/ml RNase inhibitor and 10 ng tissue RNA in a 50 μl PCR mixture, according to the manufacturer’s instructions (Applied Biosystems, Inc.). PCR amplification was performed on a PTC-200 Peltier Thermal cycler (MJ Research, Edison, NJ, USA). The temperature profile included an initial 30 min cycle at 48°C (for cDNA synthesis) and denaturation at 95°C for 10 min to deactivate the reverse transcription and activate the ThermoScript Taq polymerase. This was immediately followed by 40 cycles of denaturation at 95°C for 15 sec, 60 sec at 60°C annealing and 60 sec at 72°C elongation using the optical function for fluorescence monitoring. The relative quantification of gene expression by qPCR in a sample was determined by comparing the target-amplified product against GAPDH (internal standard) within the same sample.

Membrane proteins were extracted from isolated LV cardiomyocytes. Following isolation, cells were centrifuged at 1,000 × g for 5 min. The supernatant was discarded and the cells were resuspended in lysis buffer (Tris-HCl 5 mmol/l⁻¹, EDTA 2 mmol/l⁻¹ and benzamidine 10 μg/ml, leupeptin 5 μg/ml and soybean trypsin inhibitor 5 μg/ml). The suspension was homogenized and centrifuged at 1,000 × g to pellet cellular debris and the remaining supernatant was centrifuged at 45,000 × g at 4°C for 20 min. The pellet was resuspended in resuspension buffer (Tris-HCl 75 mmol/l⁻¹, EDTA 2 mmol/l⁻¹, MgCl₂ 12.5 mmol/l⁻¹ and soybean trypsin inhibitor 5 μg/ml). Total protein concentration was determined using the Bradford assay.

Western blot analysis was performed to measure Kv4.2, Kv4.3, KChIP2 and GAPDH proteins. Following separation by 10% SDS-PAGE, proteins were transferred to polyvinylidene sulfonyl fluoride membranes (Bio-Rad, Hercules, CA, USA) in Tris/glycine transfer buffer containing 5% methanol and 0.05% sodium dodecyl sulfate. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline (PBS) supplemented with 0.05% Tween-20 (PBS-T), for 1 h at room temperature and incubated with primary antibody (1:200) [rabbit polyclonal antibodies against Kv4.2 or Kv4.3 were purchased from Chemicon (Temecula, CA, USA), KChIP2 from Affinity BioReagents (Golden, CO, USA) and a mouse monoclonal antibody against GAPDH was purchased from Research Diagnostics, Inc. (Flanders, NJ, USA)] at 4°C overnight. The membrane was washed six times for 5 min with PBS-T and incubated with primary and secondary antibodies (anti-rabbit or anti-mouse antibodies conjugated with horseradish peroxidase were purchased from Amersham Pharmacia Biotech, Amersham, UK) diluted at 1:500 in PBS-T. Following six further washes in PBS-T, blots were visualized with the Chemi-Imager 5500 (Alpha Innotech, San Leandro, CA, USA). Finally, the amount of target protein, normalized to an endogenous reference GAPDH, was calculated.

The cardiomyocytes of SH and WKY rats were harvested in accordance with the aforementioned procedure. The cell suspension obtained (1–2 drops) was layered onto sterile laminin-coated cover slips and incubated for 30 min (37°C in 5% CO₂/95% O₂) to allow cell attachment. Plating media (Dulbecco’s modified Eagle medium containing 10% fetal bovine serum, 5 U/ml each of penicillin and streptomycin) was gently added. Losartan (10⁻⁷ mol/l) (10) was added to the plating media of cells from six SH and six WKY rats. Experiments were performed between 18–20 h. qPCR, membrane protein extraction and western blot analysis were performed in accordance with the aforementioned methods.

All values are expressed as means ± standard error. Paired and unpaired Student’s t-tests were used as appropriate to evaluate the statistical significance of differences between two group means and analysis of variance was used for multiple groups. The Pearson correlation coefficients between the mRNA, protein expression level of Kv4.2, Kv4.3, KChIP2 and the change from baseline in BP were calculated. All data were normalized prior to statistical analysis. All statistical tests were two-tailed. P<0.05 was considered to indicate a statistically significant difference between values. Statistical analyses were performed using SPSS 11.5 statistical package (SPSS, Inc., Chicago, IL, USA).

There were no differences in body weight among the aforementioned four groups prior to and following the experiment (P>0.05; Table II). Systolic BP values at the age of 18 weeks were significantly higher in the SH and los-SH groups compared with the WKY and los-WKY groups (P<0.05). At the age of 26 weeks, the systolic BP value was higher in SH compared with the los-SH, los-WKY and WKY groups (P<0.05). The heart weight, cardiac index (the heart to body weight ratio) and the mean cell membrane capacitance were significantly greater in los-SH and SH groups compared with the WKY and los-WKY groups following the experiment (P<0.01).

Action potential durations (APD) measured at 50% repolarization (APD₅₀) and APD measured at 90% repolarization (APD₉₀) were significantly shorter in WKY, los-WKY and los-SH groups compared with the SH group (P<0.01; Table III and Fig. 1). APD₅₀ and APD₉₀ were longer in SH compared with the WKY group (P<0.01). APD₅₀ and APD₉₀ were shorter in los-SH and los-WKY comared with the SH and WKY groups (P<0.05). There was no differences in resting potential (RP) and amplitude of action potential (APA) among the aforementioned four groups (P>0.05).

The I_to amplitude was lower in the SH compared with the WKY, los-WKY and los-SH groups (Fig. 2), the amplitude of I_to at 70 mV was lower in SH compared with WKY, los-WKY and los-SH groups (P<0.01) (Fig 3). The I_to amplitude was higher in los-WKY compared with WKY and los-SH groups (P<0.05) (Fig. 3). When normalized to cell membrane capacitance, the I_to current density in SH was smaller compared with that in the WKY, los-WKY and los-SH groups (40–70 mV; P<0.01) (Fig. 4).

The kinetic properties of I_to were analyzed. Inactivation, steady-state inactivation and recovery from inactivation of I_to was recorded from myocytes of rats (Figs. 5–7 and Table IV).

The inactivation time constant was larger in myocytes isolated from SH compared with WKY, los-WKY and los-SH groups (P<0.01) (Fig. 5). There were no differences in average steady-state inactivation curves of I_to (Fig. 6) and recovery time constants among the four groups (P>0.05) (Fig. 7).

The expression levels of Kv4.2 and Kv4.3 were significantly lower in myocytes from SH compared with WKY, los-WKY and los-SH groups (P<0.01; Figs. 8–12), while they were significantly higher in los-WKY compared with WKY and los-SH groups (P<0.05). The expression levels of KChIP2 were significantly higher in SH compared with WKY, los-WKY and los-SH groups (P<0.01), and were significantly higher in WKY and los-SH compared with the los-WKY group (P<0.05). No significant difference was observed in mRNA and protein expression levels of Kv4.2, Kv4.3 and KChIP2 between WKY and los-SH group (P>0.05). In los-SH and los-WKY groups, expression of Kv4.2 and Kv4.3 steadily increased with increasing I_to density and expression of KChIP2 steadily decreased with increasing I_to density.

In order to eliminate the effect of depressurization, isolated myocytes were directly treated with losartan in the in vitro experiments. The expression levels of Kv4.2 and Kv4.3 were significantly lower in SH compared with WKY, los-WKY and los-SH groups (P<0.01; Table V). The expression levels of Kv4.2 and Kv4.3 were significantly lower in WKY and los-SH compared with the los-WKY group (P<0.05). The expression levels of KChIP2 were significantly higher in SH compared with WKY, los-WKY and los-SH groups (P<0.01). The expression levels of KChIP2 were significantly higher in WKY and los-SH compared with los-WKY group (P<0.05). No significant difference in mRNA and protein expression levels of Kv4.2, Kv4.3 and KChIP2 was observed between WKY and los-SH groups (P>0.05).

In the losartan treatment group the Pearson correlation coefficients between expression levels of Kv4.2, Kv4.3 and KChIP2 and change in BP are shown in Table VI; those were not correlated with the change in BP.