| The transient receptor potential canonical channels (TRPCs) are a subfamily of transient receptor potential (TRP) channel, including TRPC1-7. TRPCs are widely distributed in the cardiovascular system, and they are a class of cation channels which are permeable for cations such as Na+and Ca2+. The disfunction and abnormal expression of TRPCs could induce cardiovascular diseases, such as myocardial hypertrophy, myocardial ischemia, cardiac arrhythmias, hypertension and so on. TRPCs are considered to be participants of the stretch-activated ion channels (SACs). Compared with normal, the expression of TRPCs were varied in the hypertrophied myocardium. But the significance of TRPCs for cardiac contractile function remains unclear.Objective:The objective of this research was to establish the model of myocardial hypertrophy rat, and observe the changes of TRPCs expression. At the cellular level, we researched the increase of the intracellular calcium ion concentration ([Ca2+]i) in the myocardium, and the action potential of myocardium. We researched the ventricular muscle contraction force at the tissue level, and studied the function of cardiac pump at the organ level. We investigated the expression change of TRPCs and the role of TRPCs in the contractile function of hypertrophic myocardium. We help our research could provide theoretical basis for development of new drugs against heart failure. Methods:1. The isoproterenol-induced rat model of cardiac hypertrophyMale Sprague-Dawley rats were randomly divided into the cardiac hypertrophy (CH) group and the control group. The CH group rats were administered isoproterenol, while the control group rats received saline solution (0.9%NaCl). After5weeks, the rats were weighed and anesthetized. The hearts were excised for research and weigh, and left ventricle samples were prepared for the paraffin sections. After stained with hematoxylin and eosin, the mean myocyte diameter was calculated from each section by the light microscopic evaluation.2. Western blot determine the expression of TRPCs and NCX1proteinsWe determined the expression of TRPC1/3/6and NCX1proteins in the myocardium by western blot analysis.3. Measure the change of [Ca2+]i with Fura-2/AMThe ventricular myocytes were freshly isolated. After incubated with Fura-2/AM, the cells were illuminated using a calcium imaging system. The SACs activated by the hyposmotic swollen, which induced increase of [Ca2+]i. Compared the ascendant amplitude of [Ca2+]i increase between the hypertrophic myocardium and normal myocardium. Observed the effects of the TRPC blocker, gadolinium chloride (Gd3+10μmol/L), the NCX inhibitor, nickel chloride (Ni2+5.0mmol/L) and the L-type Ca2+channel inhibitor, verapamil (Ver0.1μmol/L) on the increase of [Ca2+]i.4. Record the action potential of myocardiumThe papillary muscles were excised from the right ventricles, and perfused with Tyrode’s solution. The muscles were attached to a micrometer for length adjustments, and the action potential (AP) was recorded by standard microelectrode recording technique. The muscles were first set to the original length without stretch (Lo). and then stretched to an increase of10%of the length (1.1L0). Recorded AP at L0and1.1L0. Compared AP between CH group and control group, and observe the effects of Gd3+and Ver on the AP.5. Record the length-dependent contractile force of myocardiumThe papillary muscles were excised from the left ventricles, and perfused with Tyrode’s solution. The contractile force of myocardium was observed at the lengths of Lo and1.1L0. Compared the developed contractile force (Fdev) between CH group and control group, and observed the effects of Gd3+and Ver on the cardiac contractility of the preparations before and after being stretched.6. Research the Frank-Starling effect of heart by Langendorff techniqueThe isolated working heart preparations were perfused by Langendorff technique, and the cardiac output was recorded at different ventricular preloads. Frank-Starling curves of the relationship between the ventricular preload and the stroke volume were drawn. The changes of curves were observed when the SACs were blocked by Gd3+.Results:1. Heart weights and diameter of cardiomyocyte in CH and control groupCompared with control group, the atria and ventricles were much heavier in the CH group of rats. The mean myocyte diameter of the CH group was more than that of the control group, and cardiac hypertrophy was observed by morphological examination. 2. Expression of TRPC1, TRPC3, TRPC6and NCX1in hypertrophic myocardiumThe Western blot analysis revealed that the expression of TRPC1was upregulated in the CH group, while the levels of TRPC3, TRPC6and NCX1remained stationary.3. Increase of [Ca2+]i induced by stretching cytomembraneAfter the cytomembrane was stretched by hyposmotic cell swelling, the SACs were activated and [Ca2+]i was increased. The ascendant amplitude of [Ca2+]i increase in the hypertrophic myocardium was30.75±4.16%, and obviously higher than that in the control group (11.65±1.43%). The administration of Gd3+and Ni2+suppressed the increase of [Ca2+]i in both groups, and Ver had no effect.4. The effects of stretch on the action potential of myocardium(1) The results revealed that the hypertrophic myocardium had longer action potential duration (APD) than normal. The APD50were prolongated by stretching in both CH and control groups and shortened by Gd3+. The increase of APD50induced by stretching (AAPD50) in the CH group (8.86±1.16ms) was more significant than that in the control group (3.48±0.41ms). The administration of Gd3+decreased the APD and restrained the prolongation induced by stretching.(2) The resting membrane potential (RMP) was depolarized by stretched in both CH and control groups, and the amplitude of depolarization in the CH group (from-60.12±0.70mV to±1.12mV)was more significant than that in the control group(from-61.77±1.05mV to-58.04±1.02mV). The administration of Gd3+hyperpolarized the RMP and restrained the depolarization induced by stretching in both groups. In our observation, neither the APD nor the AP configuration was obviously affected by Ver. 5. Role of SACs in length-dependent contractile forceWhen the muscles were stretched from Lo to1.1Lo, the developed contractile force (Fdev) of the control and the CH groups was undifferentiated when perfused with Tyrode’s solution. After the SACs were blocked by Gd3+, the increases of Fdev were different, and obviously decreased in the hypertrophic myocardium (CH group vs. control group:37.25±1.62%vs.25.07±1.86%). The administration of0.1μmol/L Ver decreased the basal contraction force but did not affect the increase of Fdev in both groups. But Ver enhanced the effect of Gd3+in suppressing the increase of Fdev, especially in the hypertrophic myocardium.6. Role of SACs in the Frank-Starling effectThe Frank-Starling curves of control and CH hearts were similar when perfused with Tyrode’s solution, but in the CH group the ascending limb of curves was decreased after SACs were blocked by Gd3+. More serious decline was observed in the CH group induced by Gd3+, compared with the control group.Conclusions:1. Hypertrophic myocardium expresses more TRPCl to enhance the capacity of the SACs in the cytomembrane.2. The non-selective cation channel SACs were activated by stretched. Na+was the main carrier of the stretch-activated currents, and the increased [Na+]i caused Ca2+influx through NCX1, Na+/Ca2+exchange caused the increase of [Ca2+];.3. In the hypertrophic myocardium, enhanced SACs facilitated Ca2+influx to maintain the contractile force when the preload increases, which was a type of compensatory mechanism to avoid heart failure occurring. |
PDF Full Text Request