Abnormal Intracellular Ca~2+ Handling In Chronic Rat Heart Failure And Effect Of Oxymatrine

Objective:Altered intracellular Ca²⁺ handling by the sarcoplasmic reticulum (SR) plays a crucial role in the pathogenesis of heart failure (HF). Despite extensive effort, the underlying causes of abnormal SR Ca²⁺ handling in HF are not clarified. To determine whether the diastolic SR Ca²⁺ leak along with reduced Ca²⁺ reuptake are required for decreased contractility, we investigated the cytosolic Ca²⁺ transients,SR Ca²⁺ content and assessed the expression of Ca²⁺ handling genes and proteins, using SD-rat model of chronic heart failure.Methods:28 male SD rats were randomly divided into two groups: sham-operated rats (n=10), heart failure rats (n=18). Rats were deeply anesthetized, following intubation and placement on a respirator, a left lateral thoracotomy was performed and the left anterior descending (LAD) coronary artery was permanently ligated. Sham-operated animal sunder went the same procedure without ligation of the LAD. Twenty-eight days after myocardial infarction (MI), survival rate, hemodynamic and heart weight of rats were assessed. Single cardiomyocytes of the rat heart were isolated by an enzymatic dissociation method. Patch-clamp and laser scanning confocal microscope synchronous recording system software was used to record transmembrane Ca²⁺ currents (I_Ca·L) and intracellular Ca²⁺ transients simultaneously. SR Ca²⁺ content can be measured by caffeine-induced Ca²⁺ transients (CCT) indirectly and can be measured by permeabilized myocytes with SR-entrapped Fluo-5N/AM directly. The total RNA was extracted from the homogenate and the mRNA levels of Ca²⁺ handling proteins were measured using real-time quantitative RT-PCR. The levels of Ca²⁺ handling proteins were determined by immunoblot analysis. SPSS11.5 software was used for the statistical analyses. All data are presented as mean±SD and t-test or Fisher's Exact test was used for the statistical analyses. P-values of <0.05 were considered significant. Results:(1) According to the experiment, the survival is 83.3% in HF group and 100% in sham group(P>0.05). There was a significant increase in the left ventricular end-diastolic pressure (LVEDP) in HF rats (8.3±0.42 mmHg, n=15) compared with sham rats (4.7±0.65 mmHg, n=10, P<0.01). There was a significant decrease in the maximal change of systolic pressure over time (dp/dt_max) in HF rats (2,140.41±118.38 mmHg/s, n=15) compared with control rats (4,355.75±259.71 mmHg/s, n=10, P<0.01) and there was a significant decrease in the maximum change in the rate of relaxation over time (dp/dt_min) in HF rats (-1,798.33±111.41 mmHg/s, n=15) compared with sham rats (-2,531.22±414.66 mmHg/s, n=10, P<0.01). Furthermore, there was a significant increase in heart weight (HW) divided by body weight (BW) in HF rats (6.76±0.36, n=15) compared with sham rats (3.33±0.41, n=10, P<0.01). (2) The amplitude of I_Ca·L was reduced in HF myocytes (1.01±0.09, n=20) compared with sham (2.18±0.1, n=18, P<0.01). Moreover the amplitude of Ca²⁺ transients was also reduced in HF group. There was a significant decrease in spatial averages (ΔF/F₀) of I_Ca·L-induced Ca²⁺ transients in HF (12.2±0.56, n=12) compared with sham (17.8±0.53, n=10, P<0.01). SR Ca²⁺ content can be measured by caffeine-induced Ca²⁺ transients (CCT) during diastolic phase. The amplitude of Ca²⁺ transients was reduced in HF group. Caffeine-induced Ca²⁺ transients (ΔF/F₀) was significantly decreased in HF (12.4±0.4, n=12) compared with sham (33.2±1.9, n=10, P<0.01). Isolated cardiomyocytes were loaded with Fluo-5N/AM and SR Ca²⁺ content can be measured directly. The result showed that the SR Ca²⁺ content was dramatically reduced in HF myocytes. There was a significant decrease in spatial averages (ΔF/F₀) in HF (26.6±1.87, n=12) compared with sham (49.6±2.02, n=10, P<0.01). (3) We found that the mRNA expression of RyR2 (0.063±0.002 VS 0.064±0.003,n=8, P>0.05) and PLB (0.063±0.003 VS 0.058±0.002,n=8, P>0.05) failed to alter in HF compared with sham group. The downregulation of FKBP12.6 (0.018±0.002 VS 0.042±0.002,n=8, P<0.01), SERCA2a (0.14±0.019 VS 0.28±0.016,n=8, P<0.01), NCX (0.07±0.016 VS 0.12±0.019,n=8, P<0.01) and Cav1.2 (0.01±0.0012 VS 0.026±0.0019,n=8, P<0.01) was established by RT-PCR to be significant relative to the sham, respectively. Compared with the sham rat, the expression of RyR2 (0.36±0.028 VS 0.39±0.03,n=8, P>0.05) and PLB (0.5±0.02 VS 0.57±0.03,n=8, P>0.05) failed to alter in HF rat, whereas FKBP12.6 (0.13±0.007 VS 0.9±0.05, n=8, P<0.01) content was significantly reduced in HF rat. Furthermore, the expression of SERCA2a (0.74±0.02 VS 1.36±0.009,n=8, P<0.01), NCX (0.18±0.03 VS 0.78±0.05,n=8, P<0.01) and DHPR (0.11±0.01 VS 1.2±0.05,n=8, P<0.01) were also reduced in HF rat.Conclusion:Diastolic SR Ca²⁺ leak (due to dissociation of FKBP12.6 from RyR2) along with reduced SR Ca²⁺ uptake (due to down-regulation of SERCA2a) and defective E-C coupling(due to down-regulation of DHPR)could contribute to the decreased contractility observed in failing hearts associated with reduced amplitude and slowed decay of the intracellular Ca²⁺ transients.Partâ…¡Protective Effect of Oxymatrine on Chronic Rat Heart FailureObjective:Oxymatrine is one of the alkaloids extracted from Chinese herb Sophora japonica (Sophora flavescens Ait.) with activities of anti-inflammation, antivirus and protecting hepatocytes. However, the effect of oxymatrine on heart failure has not been known yet. In this study, the effect of oxymatrine on heart failure was investigated using SD-rat model of chronic heart failure.Methods:60 male SD rats were randomly divided into five groups: sham-operated rats (n=12), heart failure rats (n=12), high dose group (rats with MI and 100 mg/kg OMT treatment, qd), middle dose group (rats with MI and 50 mg/kg OMT treatment, qd), low dose group (rats with MI and 25 mg/kg OMT treatment, qd). Rats were deeply anesthetized, following intubation and placement on a respirator, a left lateral thoracotomy was performed and the left anterior descending (LAD) coronary artery was permanently ligated. Sham-operated animal sunder went the same procedure without ligation of the LAD. Twenty-eight days after MI, survival rate, hemodynamic and heart weight of rats were assessed. Single cardiomyocytes of the rat heart were isolated by an enzymatic dissociation method. Patch-clamp and laser scanning confocal microscope synchronous recording system software was used to record transmembrane Ca²⁺ currents (I_Ca·L) and intracellular Ca²⁺ transients simultaneously. SR Ca²⁺ content can be measured by caffeine-induced Ca²⁺ transients (CCT) indirectly. The ultrastructure of myocardial cells in left ventricle of rats was studied under transmission electron microscopy. The total RNA was extracted from the homogenate and the mRNA levels of Ca²⁺ handling proteins were measured using real-time quantitative RT-PCR. The levels of Ca²⁺ handling proteins were determined by immunoblot analysis. SPSS11.5 software was used for the statistical analyses. Data were expressed as mean±SD for all the experiments. Statistical analysis were made with ANOVA and followed by LSD test for individual comparisons of means. P-values of <0.05 were considered significant.Results:(1) According to the experiment, the survival is 100% in every group. There was a significant increase in the left ventricular end-diastolic pressure (LVEDP) in HF rats (8.1±0.24 mmHg) compared with sham rats (3.7±0.49 mmHg, n=12, P<0.01). There was a significant decrease in the maximal change of systolic pressure over time (dp/dt_max) in HF rats (2,458.85±124.05 mmHg/s) compared with sham rats (4,164.63±130.69 mmHg/s, n=12, P<0.01) and there was a significant decrease in the maximum change in the rate of relaxation over time (dp/dt_min) in HF rats (-1,594.85±214.22 mmHg/s) compared with sham rats (-2,765.82±152.99 mmHg/s, n=12, P<0.01). Furthermore, there was a significant increase in heart weight (HW) divided by body weight (BW) in HF rats (4.39±0.36) compared with sham rats (2.41±0.06, n=12, P<0.01). There was a significant decrease in LVEDP in middle (5.1±0.24 mmHg) and high dose group (4.6±0.34 mmHg) rats compared with HF rats (8.1±0.24 mmHg, n=12, P<0.01). There was a significant increase in dp/dt_max in middle (3,827.77±212.73 mmHg/s) and high dose group (3,910.72±101.36 mmHg/s) rats compared with HF rats (2,458.85±124.05 mmHg/s, n=12, P<0.01) and there was a significant increase in dp/dt_min in middle (-2,537.75±213.09 mmHg/s) and high dose group (-2,548.28±205.65 mmHg/s) rats compared with HF rats (-1,594.85±214.22 mmHg/s, n=12, P<0.01). Furthermore, there was a significant decrease in HW/BW in middle (3.11±0.08) and high dose group (3.09±0.08) rats compared with HF rats (4.39±0.36, n=12, P<0.01). (2) The amplitude of I_Ca·L was reduced in HF myocytes (1.89±0.19) compared with sham (6.48±0.33, n=20, P<0.01). The amplitude of I_Ca·L was enhanced in middle (4.35±0.32) and high dose group (4.21±0.24) myocytes compared with HF myocytes (1.89±0.19, n=20, P<0.01). Moreover the amplitude of Ca²⁺ transients was also reduced in HF group. There was a significant decrease in spatial averages (ΔF/F₀) of I_Ca·L-induced Ca²⁺ transients in HF (12.52±1.38) compared with sham (41.37±2.44, n=12, P<0.01). The amplitude of Ca²⁺ transients was also enhanced in middle and high dose group. There was a significant increase in spatial averages (ΔF/F₀) of I_Ca·L-induced Ca²⁺ transients in middle (26.82±1.02) and high dose group (30.07±0.91) compared with HF group (12.52±1.38,n=12, P<0.01). (3) SR Ca²⁺ content can be measured by CCT during diastolic phase. The amplitude of Ca²⁺ transients was reduced in HF group. Caffeine-induced Ca²⁺ transients (ΔF/F₀) was significantly decreased in HF (17.05±0.61) compared with sham (35.36±0.89, n=12, P<0.01). The amplitude of Ca²⁺ transients was enhanced in middle and high dose group compared with HF group. Caffeine-induced Ca²⁺ transients (ΔF/F₀) was significantly increased in middle (32.3±0.74) and high dose group (32.19±0.51) compared with HF group (17.05±0.61,n=12, P<0.01). (4) The ultrastructure of myocardial cells in left ventricle of rats was studied under TEM. In sham group, the myocardial cell appeared normal with intact sarcomeres, mitochondria contain tightly packed cristae. Numerous glycogen granules were present. In HF group, some parts of the myocardial cell appeared intermyofibrillar lysis and vesicles of varying size. Mitochondria were swollen, and mitochondrial cristae were separated. In group with treated by low dose of OMT, intermyofibrillar lysis slightly restored, but a few mitochondria were still swollen, and mitochondrial cristae were separated. In group with treated by middle dose of OMT, intermyofibrillar lysis disappeared, myofilaments were orderly, closely, and evenly arranged, and mitochondria contain tightly packed cristae. The ultrastructure of myocardial cells, treated by high dose of OMT, was similar with that of treated by middle dose of OMT, and electron density of some mitochondria increased. (5) We found that the mRNA express of SERCA2a was significantly increased in middle (0.2±0.017) and high (0.2±0.019) dose group compared with the HF group (0.12±0.014, n=8, P<0.01). Furthermore, the mRNA content of Cav1.2 was significantly increased in middle (0.02±0.002) and high dose group (0.02±0.002) compared with the HF rat (0.014±0.002, n=8, P<0.01). (6) The expression of SERCA2a was increased in middle (0.94±0.053) and high dose group (1.36±0.059) compared with HF group (0.25±0.032, n=8, P<0.01). The expression of DHPR was also increased in middle (0.3±0.027) and high dose group (0.28±0.019) compared with HF group (0.18±0.015, n=8, P<0.01).Conclusion:The present results suggest that oxymatrine improves heart failure by ameliorating the cardiac function, and that this amelioration is associated with upregulation of SERCA2a and DHPR. We suggest that OMT maybe a novel, effective therapeutic drug for the treatment of heart failure.