Positive inotropic effect of Ivabradine in rats and its underlying mechanism / 医学研究生学报

ObjectiveIvabradine reduces heart rate by inhibiting If current of cardiomyocyte and is used clinically to treat stable angina pectoris and myocardial ischemia. However, the mechanism of positive inotropic effect by Ivabradine is still not well understood. This study aims to investigate the Ivabradine's positive inotropic effect both in vivo and in vitro and the underlying mechanism involved.Methods①A Millar catheter with double-pressure was inserted into the right carotid artery of general anesthesia rats. The pressure-volume of left ventricle, HR (heart rate) and aortic pressure were recorded as a blank group (n=7). The effect of Ivabradine (1 mg/kg) administrated via left external jugular vein was recorded as a drug treated group (n=7). The cardiac output, left ventricular and aortic pressure of the rats in the blank group A and the administration group A were compared, and the results were used to analyze the Ivabradine's inotropic effectin vivo.②Langendorff setup was used to analyze the left ventricular pressure of the isolated heart. The normal perfusion solution was used as the blank group (n=6), while the Ivabradine (10 μmol/L) perfusion was used as the treated group (n=6). In addition, the treatment of H89 (200 nmol/L) (a PKA inhibitor) was recorded as the blank group (n=6) and the combined use of H89 (200 nmol/L) and Ivabradine (10 μmol/L) was recorded as drug treated group (n=6). Following the above protocol, KN-93 (500 nmol/L) (a CaMKII inhibitor) or CA (10 nmol/L) (a protein phosphatase 1 and 2A inhibitor) was used to analyze the inhibitory effect on inotropic effect of Ivabradine (n=6 for each group). ③The field stimulation induced Ca2+ transient from cardiomyocyte was used to investigate the mechanism underlying the positive inotropic effect of Ivabradine (10 μmol/L).The perfusion orders and concentrations of Ivabradine or/and H89, KN-93 and CA were the same as that in isolated rat heart experiment (n= 6 for each group).Results① Ivabradine (1 mg/kg) significantly increased the left ventricular develop pressure (from 102.43±11.06 in blank group to 109.86±11.65 mmHg in ivabradine treated group, P<0.01, n=7) and cardiac output (from 33.72±1.96 in blank group to 36.27±2.22 mL/min in ivabradine treated group, P<0.01, n=7). It reduced the heart rate (from 348.56±10.02 in blank group to 324.17±11.33 beats/min in ivabradine treated group, P<0.01, n=7) and increased the systolic blood pressure (from 99.74±8.67 in blank group to 108.57±9.24 mmHg in Ivabradine treated group, P<0.01, n=7) without significant change in diastolic blood pressure. ② Ivabradine (1, 10 μmol/L) significantly increased the left ventricular developed pressure (LVDP) (P<0.05, n=6). The positive inotropic effect of Ivabradine was blocked by CaMKⅡ inhibitor of KN-93. ③ Ivabradine (10 μmol/L) significantly increased the amplitude of SR Ca2+ transient (P<0.01,n=6). The enhanced amplitude of Ca2+ transient was blocked by CaMKⅡ inhibitor of KN-93.ConclusionIvabradine shows a positive inotropic effect in rat hearts both in vivo and in vitro and its underlying mechanism involved the action which was mediated by CaMKⅡ.

A Computational Model of Cytosolic and Mitochondrial Ca2+ in Paced Rat Ventricular Myocytes

We carried out a series of experiment demonstrating the role of mitochondria in the cytosolic and mitochondrial Ca2+ transients and compared the results with those from computer simulation. In rat ventricular myocytes, increasing the rate of stimulation (1~3 Hz) made both the diastolic and systolic [Ca2+] bigger in mitochondria as well as in cytosol. As L-type Ca2+ channel has key influence on the amplitude of Ca2+-induced Ca2+ release, the relation between stimulus frequency and the amplitude of Ca2+ transients was examined under the low density (1/10 of control) of L-type Ca2+ channel in model simulation, where the relation was reversed. In experiment, block of Ca2+ uniporter on mitochondrial inner membrane significantly reduced the amplitude of mitochondrial Ca2+ transients, while it failed to affect the cytosolic Ca2+ transients. In computer simulation, the amplitude of cytosolic Ca2+ transients was not affected by removal of Ca2+ uniporter. The application of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) known as a protonophore on mitochondrial membrane to rat ventricular myocytes gradually increased the diastolic [Ca2+] in cytosol and eventually abolished the Ca2+ transients, which was similarly reproduced in computer simulation. The model study suggests that the relative contribution of L-type Ca2+ channel to total transsarcolemmal Ca2+ flux could determine whether the cytosolic Ca2+ transients become bigger or smaller with higher stimulus frequency. The present study also suggests that cytosolic Ca2+ affects mitochondrial Ca2+ in a beat-to-beat manner, however, removal of Ca2+ influx mechanism into mitochondria does not affect the amplitude of cytosolic Ca2+ transients.

Effects of electric stimulation applied during absolute refractory period on contraction and relaxation of ventricular myocytes in normal and failure guinea-pigs / 西安交通大学学报(医学版)

Objective To investigate the effect of electric stimulation applied during absolute refractory period on contraction and relaxation and Ca 2+ transient of ventricular myocytes from normal guinea-pigs. Methods The ventricular myocytes of guinea-pigs were obtained by enzyme digesting, and the extent of the contraction and relaxation and Ca 2+ transient were recorded through the motion edge detection system. The Ca 2+ transient was reflected by fluorescence ratio (360/380) ( F 360/F 380). Results The ventricular myocytes from the normal guinea-pigs: ①The contraction extent of guinea-pig ventricular myocytes increased by (16.55?5.49)%. The peak velocity of shorting (contraction) and the peak velocity of relengthening (relaxation) increased by (17.43?7.08)% and (19.74?9.08)%, respectively(n=10); ②The extent of F 360/F 380 increased (25.79?6.88)%. The peak velocity of F 360/F 380 increased by (29.47?9.25)% and (22.52?7.81)% during shortening and relengthening, respectively(n=10). The ventricular myocytes from failure guinea-pigs: ①The contraction extent of guinea-pig ventricular myocytes increased by (15.53?5.31)%. The peak velocity of shortening (contraction) and the peak velocity of relengthening (relaxation) increased by (10.60?3.02)% and (23.32?8.26)%, respectively(n=6); ②The extent of F 360/F 380 increased (16.82?7.03)%. The peak velocity of F 360/F 380 increased by (16.27?5.91)% and (10.32?2.46)% during shorenting and relengthening, respectively(n=6). Conclusion Appropriate electric stimulation applied during absolute refractory period might strengthen the contracting and relaxing function of normal and failing ventricular myocytes in guinea-pigs.