Activation Of β3-adrenergic Receptor Mediated Negative Inotropic Action Through Decreasing Calcium Sensitivity Of Rat Ventricular Myocytes

Objectives: 1.By anterior descending coronary artery ligation in rats, resulting in ischemic heart failure, making heart failure rat models; 2.After blockade of β1 and β2 receptors, to observe the function of the hearts by giving the β3 receptor agonists BRL37344 and antagonists SR59230 A in normal and heart failure in rats; 3.Acutely isolated rat cardiomyocytes from failure hearts, measuring single cardiomyocyte contractility and calcium transients by giving β3 receptor agonists BRL37344 and antagonists SR59230 A,then analyze calcium sensitivity of myocardial cells, and further explore the mechanism of β3 receptors in normal and heart failure rats.Methods: 1.100 SD rats(weighting 180-220 g) were randomly divided into sham group(n = 30) and HF group(n = 70). Before surgery by injection of 10% chloral hydrate(0.3 ml / 100g) to rats’ intraperitoneal. Then open their chests, coronary artery ligation. Normal ECG tracings before surgery, continuous electrocardiographic monitoring during the surgery. consecutive three days by intraperitoneal injection of 200 thousands of units of penicillin to prevent wound infection. Open the chest of sham group in the same way, but no coronary artery ligation, the rest step of the operation is the same with HF group. After four weeks, both the sham group and HF group were randomly divided into BRL37344 group and SR59230 A group. 2.Hung the isolated heart on the Lengendorff device with 1.8m M of calcium of Tyrode’s Solution and fully 95% O2 and 5% O2 saturated, perfusion pressure is about 60mm Hg, 39 ℃, p H7.38. During the experiment giving BRL37344 and SR59230 A. Detection indexs including left ventricular systolic pressure(LVSP), left ventricular diastolic pressure(LVEDP), maximum increase rate of left ventricular isovolumic systolic period(+ dp / dtmax) and maximum decrease rate of left ventricular isovolumic diastolic period(- dp / dtmax), analysis with Lab Chart software. 3.Clip the surrounding areas of ligation portion from the left ventricle anterior wall of the sham and HF group.Put the tissue into the Formalin solution, paraffin embedded, sliced, then dewaxing, antigen retrieval,add to anti-β3 receptor, the second antibody, closed by SABC, DAB reagent, the expression of β3 adrenergic receptor is observed. 4.Freshly isolated single cardiomyocytes, protect it from light with Fura-2. Tyrode’s solution with calcium(1.5 mmol / L) perfusion.Then with the Ion Optix ion imaging system to simultaneous decet a single cell contractility and calcium transients. bl%peak h: relative contraction amplitude of the cell; dep v: the maximum systolic rate; ret v: the maximum diastolic rate; △ Ca: amplitude of calcium transient; +d[Ca]/dtmax: The maximum rise rate of calcium in systolic myocytes;-d[Ca]/dtmax : The maximum decrease rate of calcium in diastolic myocytes; Ca S: Calcium sensitivity.Results: 1.Compared with the sham group, after four weeks、eight weeks and twelve weeks the index of ventricular HW / BW and LVW / BW were significantly decreased(P<0.05). Left ventricular end-diastolic diameter(LVEDD) and left ventricular end-systolic diameter(LVESD) were significantly increased(P<0.05), left ventricular ejection fraction(LVEF) and fractional shortening(LVFS) were significantly decreased(P<0.05). 2.The cells in left ventricular myocardial of the sham group were almost neatly arranged,there hardly were myocardial injury. In the HF group, there were myocardial necrosis in the left ventricular anterior wall as well as gap expansion between the cells, and with a mild to moderate hypertrophy. The collagen fibers grew and thicker, and there were angiogenesis. 3.With immunohistochemical detected, we can see that there is a relatively large amount expression of β3 receptors near vicinity of the cells under the microscope, there were also expression in the cytoplasm of the cells.4.After blockade of β1 and β2 receptors, the heart rate(HR), left ventricular systolic pressure(LVSP), left ventricular systolic isovolumic ventricular pressure maximal rate of rise(+dp/dtmax) and left ventricular isovolumic relaxation pressure and maximal rate of decrease(-dp/dtmax) were significantly lowered(P<0.05), while left ventricular diastolic pressure(LVEDP) was increased(P<0.05). After blockade of β3 receptor, hemodynamic parameters were reversed significantly(P<0.05). In the isolated single cardiomyocyte, the amplitude of sarcomere contraction( bl%peak h) and calcium transient(F340/380) decreased significantly in presence of BRL37344 in HF rats. The calcium sensitivity decreased after β3 adrenoceptor activation. 5.In the isolated single cardiomyocyte,the amplitude of sarcomere contraction( bl%peak h) and calcium transient(F340/380) decreased significantly after giving BRL37344. 6.Giving L-NAME, the blocker of NOS, amplitude of sarcomere contraction( bl%peak h) and calcium transient(F340/380) were no longer decline, calcium sensitive enhanced(P <0.05), described NOS signal transduction pathways was associated and β3 receptors.Conclusion: While had very low expression in normal rat heart,β3-adrenoceptor had much higher expression in HF hearts of rat. After blocking of β1 and β2-adrenoceptors, the cardiac function of normal and HF rats were inhibited with administration of BRL37344. In the failure heart, activation of β3-adrenoceptor led to decreased bl%peak/h and calcium transients. The results showed that β3-adrenoceptor played more significant negative inotropic effect in the HF heart of rats. Activation of β3-adrenoceptor led to decrease of calcium sensitivity. L-NAME and β3 receptor signaling pathway has partial relevance, and plays a protective role in its signaling pathway in the heart.