| Heart failure (HF) is the main cardiovascular disease in the world. The genesis of HF syndromes is complex and multifactorial, while ventricular arrhythmias and degression of the myocardial contractility are the main causes of human heart failure, but the altered cellular Ca2+ regulation may be a final common pathway in both contractile dysfunction and arrhythmogenesis [1].In a heart that has suffered from myocardial damage, regardless of the initial cause of the damage (hypertension, myocardial ischemia, cardiomyopathy, etc), HF eventually occurs if such damage persists for a prolonged period. In the initial stages, compensation for the depressed myocardial contractility due to myocardial damage can occur via activation of both the sympathetic nervous system and the renin-angiotensin system, resulting in left ventricle dilatation and/or hypertrophy. However, if the depressed cardiac function persists, with a parallel activation of neurohumoral factors, the myocardial damage becomes progressive and irreversible, and the heart can no longer meet the metabolic demand of the body, resulting in HF [2].Over the past 50 years, various mechanisms, including: (1) defects in energy production and utilization, (2) increased preload and afterload, (3) altered neurohormonal profile and signal transduction, and (4) occurrence of intracellular Ca2+-overload and Ca2+-handling abnormalities, have been indicated to explain cardiac dysfunction in HF. Since HF is invariably associated with changes in the shape and size of the heart, it has been suggested that the myocardial damage may be lead to cardiac remodelling. HF induced by pressure overload, or volume overload is a consequence of cardiac remodelling [3]. During cardiac hypertrophy and HF, a rise in intracellular Na+ concentration ([Na+]i) could potentially contribute to increasing or maintaining contractility, as it would increase Ca2+ influx via the Na+/Ca2+ exchanger. During the pathophysiological states of HF, expression and function of several of the Na transporters are affected, which can lead to an increase in [Na+]i. Several studies have reported a decrease in activity of the Na+/K+-pump and the up-regulation of the Na+/H+ exchanger would result in an increase in [Na+]i [4].The cardiac Na+/K+-pump regulates intracellular Ca2+ concentration ([Ca2+]i) in the heart by regulating [Na+]i [5]. The Na+/Ca2+ exchanger is closely associated with the Na+/K+-pump, which suggests that inhibition of the Na+/K+-pump in the heart, through effects on the Na+/Ca2+ exchanger, raises the [Ca2+]i and strengthens cardiac contraction [6-9]. Hence, the alterations of Na+/K+-pump activity can have profound effects on cardiac myocytes contractility [10,11].The active Na+/K+-pump contains a catalyticαsubunit (~110 kDa) and a smallβsubunit (~55 kDa). Theαsubunit mediates the catalytic activity of the enzyme and contains the specific binding sites for Na+, K+, ATP and cardiac glycosides [12-14]. Four isoforms of theαsubunit (α1,α2,α3,α4) have been identified and posssess tissue specific distribution and species differences [15].α1 andα2 isoforms are expressed in rat, guinea pig and mouse heart, while three isoforms (α1,α2, andα3) are present in human heart [16,17].In human with heart failure, the Na+/K+-pump subunitsα1,α3, andβ1 were significantly reduced in ventricular myocardium and the Na+/K+-pump activity decreased [18]. The Na+/K+-pump function is unchanged in rabbit with heart failure[19]. Immunoblots showed a ~19% lower expression ofα1 isoform and a ~74% lower expression ofα2 isoform in HF rat [20]. Svein reported the expressions of theα1 andβ1 subunits (mRNA and protein) of the Na+/K+-pump were not significantly different in CHF, and the mRNA and protein levels of theα2 isoform were lower by 25 and 55%, respectively [21].Because the reports about the activity and the protein expression of the Na+/K+-pump in HF models are not coincident, we established rat model HF induced by Adriamycin (ADR), measured the Na+/K+-pump current (Ip) of the single myocardial cell by the whole-cell patch-clamp technique, and detected the protein expression of Na+/K+-pumpα1 andα2 isoform by Western Blot analysis. The aim of this study was to investigate the changes of Na+/K+-pump activity and the protein expressions in HF rats. We also analyze the role of the Na+/K+-pump in the development of HF.Objective: The purpose of this study was to investigate the changes of the activity of Na+/K+-pump and the expression of Na+/K+-pumpαisoforms in HF rats.Methods: (1) The establishment of the HF rats: The SD rats were randomly divided into two groups: control group (NOR) and HF group (HF). The model of HF was established by the ip of ADR once every two days, at a dose of 1 mg/kg for the first two times, 2 mg/kg for the third and forth time, 3 mg/kg for the last four times. The NOR received an equivalent volume of the saline solution. During about twenty days after ADR administration, the indexes of HF, behavior,body weight (BW), ratio of left ventricular weigh and body weight (LVW/BW), ratio of lung weight and body weight (LW/BW) were evaluated. The parameters of heart function such as heart rate (HR), left ventricular systolic pressure (LVSP),left ventricular end-diastolic pressure (LVEDP),±dP/dtmax were measured.(2)Enzymatic isolation of rats ventricular myocytes: The rats were anaesthetized by intraperitoneal injection of sodium pentobarbitone (50 mg/kg) and heparin (2500 U·kg-1) solutions. Hearts were excised, perfused in a retrograde fashion through Langendorff apparatus and ventricular myocytes were isolated by enzymatic dispersion (0.6 mg/mL Type-2 collagenase) .(3)The record of the Na+/K+-pump current: Whole-cell patch-clamp technique was performed to record the Na+/K+-pump current (Ip), the Na+/K+-pump exchanges three intracellular Na+ for two external K+ aross the cell membrane during each active transport process and generates a net outward current (Ip). With selected external and pipette solutions, membrane current through K+ channel, Ca2+ channel, Na+/Ca2+ exchanger were minimized. Under the above experimental conditions, Ip was measured as the ouabain-blocked current. 1 mmol·L-1 ouabain (OUA) can completely block the activity of Na+/K+-pump, so the current generated by 1 mmol·L-1 OUA is defined as Ip. The Ip density is the ratio of Ip and capacitance. TheΔIp–OUA relation curve was plotted by normalized Na+/K+-pump current vs log OUA concentration and fitted by a two-site binding model formula.(4)Western Blot analysis was used to detect the protein expression of Na+/K+-pumpα1 andα2 isoforms: Membrane proteins were extrcted from the left ventricles of NOR and HF rats and the concentration of the protein was measured by BCA protein assay. Membrane proteins were electrophoresed into 10% SDS-PAGE gels and blotted onto PVDF membrane. Membrane were blocked for 1 h in 5% nonfat dry milk in TBST, then incubated overnight with Na+/K+-pumpα1 andα2 isoforms antibody at 4℃, washed three times with TBST, and incubated with secondary antibody at 37℃for 1 h, washed with TBS for three times. The resulting bands were finally visualized using DAB.Results: (1)The evaluation of HF indexes: The HF rats appeared poisoned symptom on 10th day after ADR injection, showed inanimate behavior, and BW, HR, LVSP, +dp/dtmax, and -dp/dtmax were decreased (238±15 g vs 207±13 g, 483±23次/min vs 388±34次/min, 129±4 mmHg vs 80±4 mmHg, 3357±101 mmHg/s vs 1727±55 mmHg/s, 3314±127 mmHg/s vs 1550±45 mmHg/s, p<0.05), but the LVW/BW, LW/BW and LVEDP were increased significantly (2.14±0.11 mg·g-1 vs 2.61±0.04 mg·g-1, 4.11±0.27 mg·g-1 vs 5.51±0.31 mg·g-1, 5.9±0.9 mmHg vs 11.3±1.2 mmHg, p<0.05).(2)The change of Ip of venticular myocytes from HF rats: Compared with those from NOR rats, the Na+/K+-pump current density of venticular myocytes from HF rats was decreased (0.5070+0.06297 pA/pF vs 0.2106+0.04890 pA/pF, p<0.05). TheΔIp–OUA relation curve was plotted by normalized Na+/K+-pump current vs log OUA concentration and fitted by a two-site binding model formula. The results indicates that, there are two kinds ofαisoforms in both NOR rats and HF rats, which are with high affinity (α2 isoform) and low affinity (α1 isoform) for OUA binding. But in NOR rats, the K ofα1 isoform are 2.85×10-5 M andα2 isoform are 6.74×10-8 M; and in HF rats the K ofα1 isoform are 3.55×10-5 M andα2 isoform are 2.97×10-7 M, indicating that HF can mainly reduce the high affinity Na+/K+-pump of hearts.(3)The change of protein expression ofα1 andα2 isoform of Na+/K+-pump in the HF rats left ventricles: In the NOR and HF rats left ventricles, we used Western blot to detect theα1 andα2 isoforms (112 KD). Compared with NOR rats, the protein expression ofα1 isoform of HF rats had no change, but the protein expression ofα2 isoform showed a significant decrease, about 50% (p<0.05). The results imply the mechanism of HF is closely related to the reduction of Na+/K+-pumpα2 isoform, which is in accordance with the results of electrophysiology.Conclusion: In HF rats, the density of Na+/K+-pump current was significant decreased, the high affinity Na+/K+-pump was changed, and theα2 isoforms showed a significant decrease. These results imply the pathogenesis of HF is closely related to the reduction expression of Na+/K+-pumpα2 isoforms.
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