Myocardial Frequency Dependence Of Action Potential Duration And Force And Its Influences On TRPCs Expression

Functional activity of the heart is the integrity of action potential and myocardial contractility, which is affected by heart rate. Myocardial excitation-contraction process is accompanied by changes of intracellular ion concentration, and the calcium ion is the most important inorganic ion involved in cardiac activity. TRPC (transient receptor potential canonical) is one subfamily of TRP, including7isoforms (TRPC1-7). TRPC is widely distributed in the cardiovascular system, which is a non-selective cation channel with permeability for calcium. Abnormal function and expression of TRPC can be seen in various kinds of cardiovascular diseases, such as cardiac hypertrophy, arrhythmia, myocardial ischemia. Heart rate changes can affect the transmembrane movement of intracellular ions, as well as the myocardial action potential duration (APD) and contractile force. Whether TRPC participate in this process, it is worth being investigated further.Objective:Controlling heart rate through the electric stimulation, we observe the relationship between myocardial APD or contractile force and stimulation frequency in different kinds of animal, and investigate frequency dependence of APD associated with the expression of TRPC. We further analysis the roles of TRPC in this process, and whether TRPC is involved in the drug effects of APD prolonging.Methods:1. The relationship between myocardial APD and stimulation frequency in different species of animalTo observe the relationship between APD and stimulation frequency in different species of animal, monophasic action potentials (MAP) were recorded using contact electrode technique in different programmed stimulus frequencies on rats, guinea pigs, or rabbits ventricular epicardium in vivo. And transmembrane action potentials were recorded using conventional microelectrode techniques on ventricular papillary muscle of rats.2. Frequency dependence of action potential duration and its influences on TRPC expression of rabbit myocardiumPacing at the stimulus frequency of4.5Hz、6Hz、7.5Hz, we recorded MAP using contact electrode technique on rabbit ventricular epicardium in vivo. Ten minutes later, hearts were taken out and the expressions of TRPCs were detected using RT-PCR or Western blotting.3. Effects of neferine on TRPCs expression and frequency dependence of action potential duration of rabbit myocardium(1) Given intravenous injection of neferine (2.5mg/kg), amiodarone (5mg/kg) in rabbits, the changes of MAP were recorded and compared at different stimulus frequencies (4.5Hz～7Hz) before and after administration.(2) Given intravenous injection of neferine (2.5mg/kg), amiodarone (2.5mg/kg) in rabbits, a continuous electrical stimulation of10min was applied on rabbit myocardium after administration. Then the hearts were taken out rapidly and the expressions of TRPC were detected.4. Force-frequency relationship of ventricular papillary muscle in different species of animal(1) Ventricular papillary muscles were isolated from heart of rats or rabbits and immediately perfused with organ bath. Different frequency electrical stimulations were applied on the preparation in sequence and the contraction force was recorded. (2) In order to investigate the influences of post-rest twitch, different rest intervals between1and240s were prescribed from a basal stimulation rate of1Hz for3min, and the first contraction amplitude after resume was recorded.Results:1. The relationship between myocardial APD and stimulation frequency in different species of animal(1) At the frequency of4.5Hz,5Hz,5.5Hz,6Hz,6.5Hz,7Hz, myocardial APD gradually shortened with the increase of stimulation frequency in the MAP recording of guinea pigs. The APD90from the control level96.84±5.43ms (4.5Hz), respectively reduced to91.77±3.88ms (5Hz),87.07±3.78ms (5.5Hz),81.79±3.92ms (6Hz),76.84±2.91ms (6.5Hz),76.52±1.40ms (7Hz). Compared with the control (4.5Hz), the shorten percentages correspondingly were5.2±0.9%,10.1±1.1%,15.6±1.5%,20.5±1.3%,20.4±3.0%.(2) At the frequency of4.5Hz,5Hz,5.5Hz,6Hz,6.5Hz,7Hz,7.5Hz, a clear frequency dependence of APD was showed in the MAP recording of rabbit myocardium in vivo. Myocardial APD90from the control level (4.5Hz)141.53±3.85ms, respectively, reduced to138.44±3.45ms (5Hz),131.65±3.23ms (5.5Hz),125.94±3.11ms(6Hz),119.77±3.31ms (6.5Hz),112.64±1.83ms (7Hz),104.69±1.58ms (7.5Hz). Compared with the control (4.5Hz), the shorten percentages were2.1±1.5%,6.9±1.3%,10.9±1.7%,15.3±1.8%,20.2±2.1%,25.8±2.0%.(3) There was no obvious frequency dependence of action potential duration in rat MAP in vivo within the range of5-8.5Hz or isolated ventricular papillary muscle TAP (transmembrane action potentials) at the range of1～8Hz.2. Frequency dependence of action potential duration and its influences on TRPC expression of rabbit myocardiumIn the groups of4.5,6and7.5Hz, TRPC3mRNA and protein expression of rabbit myocardial was gradually raised with the increase of stimulus frequency. In contrast with control level (4.5Hz), the mRNA expression of TRPC3remarkably grew in the6Hz and7.5Hz group (P<0.05), while that of myocardial TRPC1-2, TRPC4-7showed no significant change. Compared with4.5Hz group, the protein expression of TRPC3obviously increased in the7.5Hz group (P<0.05). The expression of TRPC1and TRPC6had no significant difference in the three groups.3. Effects of neferine on frequency dependence of action potential duration and TRPCs expression of rabbit myocardium(1) At different stimulus frequencies, neferine and amiodarone could prolong APD in rabbit myocardial, and showed characters of rate independence. Compared with that before administration at the frequency of4.5Hz,5Hz,5.5Hz,6Hz,6.5Hz, the prolonging percentage were respectively11.4±4.1%,10.6±3.4%,6.5±1.6%,7.7±1.8%,8.8±3.0%after intravenous injection of amiodarone (5mg/kg) in rabbits. At the frequency of4.5Hz,5Hz,5.5Hz,6Hz,6.5Hz,7Hz, the prolonging percentage of neferine (2.5mg/kg) injection correspondingly were5.8±1.6%,2.0±1.7%,2.4±0.4%,3.1±0.5%,2.5±0.3%,6.2±1.2%compared with that before administration.(2) Neferine (2.5mg/kg) could significantly increase the expression of TRPC3mRNA and protein in the stimulation of4.5Hz, while amiodarone (5mg/kg) had no such effects. Under the same stimulus conditions, the medication administration groups had no significant difference on the mRNA expression of TRPC1-2and TRPC4-7, and on the protein expression of TRPC1and TRPC6compared with4.5Hz control group.4. Force-frequency relationship of ventricular papillary muscle in different species of animal(1)With the increase of frequency (0.2-3Hz), contraction force gradually weakened in papillary muscles of rats, while enhanced in that of rabbits within0.2-1.5Hz range. At the frequency of0.4Hz,0.6Hz,0.8Hz,1.0Hz,1.5Hz,2Hz and3Hz, contraction force percentage of rat papillary muscles from the control level (0.2Hz)100%, respectively reduced to78.2±3.4%,66.3±4.9%,60.4±5.6%,56.0±6.0%,51.5±6.8%,49.3±6.9%and 43.7±5.8%. Under the same stimulus conditions, that of rabbit papillary muscles from the control level (0.2Hz)100%, correspondingly raised to109.6±3.5%,120.2±5.4%,130.6±7.5%,141.4±10.4%,142.7±12.4%,137.3±13.9%and125.5±14.4%.(2) There was clear rest decay of twitch amplitude in rabbit papillary muscles, while rat ventricular muscles exhibited rest potentiation of twitch force after different rest intervals between1and240s. After rest15s,30s,60s,120s,180s and240s, contraction force percentage of rat papillary muscles from the control level (rest Is)100%, respectively raised to299.8±7.3%,386.7±14.9%,451.3±32.3%,455.7±35.8%,451.1±44.0%and445.9±46.6%. Under the same rest conditions, that of rabbit papillary muscles from the control level (rest Is)100%, correspondingly reduced to88.1±2.3%,82.7±1.9%,78.7±2.0%,77.8±2.6%,76.7±3.1%and76.5±3.5%.Conclusions:1. There are species differences in the relationship between myocardial APD and stimulation frequency. An obviously APD shorten with the increase of frequency is showed in guinea pigs and rabbits, while this phenomenon is not observed in rat at the frequency ranges in our experiments. There are also distinctions between rat and rabbit papillary muscles in the force-frequency relationship.2. Frequency dependent shorten of APD is associated with the increase of TRPC3expression in rabbit myocardium.3. Neferine could significantly increase the expression of TRPC3rabbit myocardial, and the prolongation of APD showed characters of rate independence.