Study On Cardiac Myocytes Intracellular Calcium Signaling: The Effects Of Extracellular Calcium,β Receptor Agonist And Antagonist

BackgroundAs the most extensive and important second messenger, intracellular Ca²⁺ plays an important role in cardiac excitation-contraction coupling, substance metabolism, cell cycle regulation, cell communication and other activities. Cardiac myocyte calcium signalling is an in important research field for pathophysiological mechanism of heart diseases. Because of the biological importance of intracellular calcium ([Ca²⁺]i) signaling, lots of scientists tried to establish many different techniques to investigate the cellular or subcellular calcium signalling, such as single-photon and two-photon confocal microscopy, total internal reflection fluorescence microscopy, wide vision microscope and low noise CCD camera and etc. Over the last decade, with the development of the laser scanning confocal microscopy and calcium fluorescent indicators,cardiac calcium imaging and and its three dimension reconstruction were improved greatly with good accuracy and high resolution. Calcium imaging makes cellular function study more conveniently and directly, and enhances our understanding of intracellular signaling greatly.In the present study, we used the laser scanning confocal microscope, by fast-scanning mode and line-scanning mode, to measure the effects of high extracellular Ca²⁺, low extracellular Ca²⁺, isoproterenol (ISO, aβreceptor agonist), and propranolol (aβreceptor antagonist) on the cardiac myocytes'resting calcium and evoked calcium transients by electrical field stimulation. The mechanisms of the effects (relationship between calcium signaling and cardiac myocytes'function) were discussed. These may be theoretically helpful for the treatment and prevention of cardiovascular diseases.Objectives1. To establish a research model and method for cardiac myocytes'intracellular calcium signaling, using the confocal laser scanning microscopy system.2. To explore the effects of extracellular low Ca²⁺ and high Ca²⁺ on cardiac myocytes'intracellular calcium signals. 3. To investigate the effects ofβreceptor agonists and antagonist on cardiac myocytes'intracellular calcium signals.Materials and MethodsAdult SD rats, weighing 200-300 g, were employed to get single cardiac myocytes acutely, using an improved Langendorff device,reverse-aortic perfusion of collagenase plus protease solution. After a three-step gradient of calcium for calcium recovery, single cardiac myocytes were kept for 1 to 2 h. Then the cells were incubated with Fluo4-AM and placed on the stage of Zeiss510- META inverted laser scanning confocal microscope. A NEC-7203 electronic stimulator was used to provide electrical field stimulation onto cardiac myocytes, working with the confocal system synchronously by a trigger signal. Intracellular calcium transients were activated and recoded by the confocal system simultaneously. Chemical reagents were delivered to the cells by a self-made multi-channel system. Effects of extracellular calcium in different concentration (including 0.0, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mmol \ L Ca²⁺ respectively),βreceptor agonist (including 0.1, 0.2, 0.5, 1.0μmol \ L ISO respectively),βreceptor agonist and antagonist(1.0μmol\L ISO+4.0μmol\L propranolol) on cardiac myocytes'intracellular calcium signals (including resting [Ca²⁺]i and evoked calcium transients) were observed. Experimental data were collected before, during and after the delivery of chemical reagents from the same single cells. Computer programs for Calcium signal image analysis was coded with IDL6.2 (Research Systems, Inc, Boulder, CO) software. Resting intracellular calcium level was measured by the background fluorescence intensity (F0). And the evoked calcium transients were measured by normalized fluorescence (F/F0), Where F0 refers to the present fluorescence signal intensity, and F0 the background fluorescence signal intensity.Results1. By using an improved Langendorff device, about 85% -95% of the single isolated heart cells were long-rod-shaped, among which 40% to 60% were calcium homeostasis cardiac cells. After incubation with fluo4-AM, a calcium fluorescent dye,single cardiac myocytes were triggered by electrical field stimulation and typical calcium transient images were successfully recorded by the confocal microscope system.2. Effects of extracellular low calcium on Myocardial intracellular calcium signalling: In this study, the calcium concentration in normal perfusion of extracellular is 1mM. When extracellular calcium concentration lowered to 0.5 mM, about 3/7 induced calcium transient of the cells did not change significantly, 2/7 induced calcium transient significantly enhanced, and 2/7 induced calcium transient has weakened; majority (6/7) induced calcium transient time-range (up from the peak of the half fell to half of the premises sustained peak time) has extended,the forefront of calcium transient phenomenon didn'tvary obviously.When extracellular calcium concentration has gradually lowered to 0.2 mM, induced calcium transient have increased about 40% (2/5), and the rest did not change significantly; calcium transients time-range extension ( P<0.05) * (by normal perfusion fluid 222.5 + / -36.9 ms 0.2 mM into the 247.4 + / -37.9 ms), the calcium transient forefront are becoming vary (because some sites at the beginning of calcium flow is too weak to trigger the release of intracellular calcium; later, near the site of calcium signals enhanced through calcium-induced calcium release from the principle of the whole-cell calcium release).When extracellular calcium concentration has gradually changed to 0.1 mM, about half (3/6)of the cells induced calcium transient significantly weakened, but in the end did not completely disappear; the other half (3/6) in the beginning of reperfusion the calcium transient was soon enhanced and time-extended.When extracellular calcium concentration has gradually changed to 0.05 mM, at the beginning of that induced calcium transient has been enhanced, then gradually weaken, but in the end still visible intracellular calcium signaling weak presence (10/10).When extracellularcalcium concentration has gradually changed 0 mM (without calcium Tyrode's solution), the myocardial cells induced calcium transient gradually weakened, and eventually disappeared (5/5).In short, with the extracellular calcium concentration from the normal circumstances of 1 mM to 0 mM final in the process , myocardial cells were gaven over threshold stimulate, the general process of change was: at the beginning (about 1 minute perfusion) calcium transient and cell contraction cound be induced, and induced calcium transient strength showed increasing trend, the forefront of induced calcium transient became varied; along with the decline of extracellular calcium, induced calcium transient weakened without continuous(Some are intracellular calcium release, some not), then on non-induced calcium transient gradually obviously, until only a few extracellular Ca²⁺without intracellular calcium release was triggered when the extracellular calcium concentration became to 0 mM, induced calcium transient disappeared, myocardial contractility lost.3. Effect of extracellular high calcium on myocardial intracellular calcium signalling:When extracellular calcium concentration has gradually ranged from 1 mM to 2 mM,Resting intracellular calcium levels (fluorescence intensity) has no significant change; calcium transient peak(F/F0)has no significant change also.When extracellular calcium concentration has gradually ranged from 1 mM to 5 mM,Resting intracellular calcium levels change has obvious differences ( P<0.05); calcium transient peak decline obviously ( P<0.05). When extracellular calcium concentration has gradually changed from 1 mM to 10 mM,Resting intracellular calcium levelschange has obvious differences( P<0.05), and above the resting calcium levels of extracellular calcium concentration of 5 mM ( P<0.05); calcium transient peak change has obvious decline( P<0.05), and below the transient peak of extracellular calcium concentration of 5 mM( P<0.05).4. Effect ofβreceptor agonists and antagonist on myocardial intracellular calcium signalling: When the ISO at 0.1μM,Calcium background fluorescence intensity was decreased ( P<0.05), Before and after perfuse 0.1μM ISO, calcium transient peak ratio was 1.24±0.07, peak value significantly increased( P<0.01). When the ISO at 0.2μM,Calcium background fluorescence intensity values were significantly decreased ( P<0.01), after perfuse 0.1μM ISO calcium transient peak significantly increased ( P<0.01). When the ISO at 0.5μM,Calcium background fluorescence intensity values were decreased ( P<0.05), after perfuse 0.5μM ISO calcium transient peak significantly increased also( P<0.01).When the ISO at 1.0μM,Calcium background fluorescence intensity values were decreased ( P<0.05); After perfuse 1.0μM ISO calcium transient peak also increased ( P<0.05).In another experiment, when the ISO at 1.0μM resting intracellular calcium level reduce from the normal 12.18±1.04 (absolute value of the fluorescence intensity) to 10.53±0.90, after added 4.0μM Propranolol (β-receptor antagonist) resting calcium level increased to 11.62±1.11, then, wash away the extracellular ISO and Propranolol resting level of calcium turn to 12.94±1.47 (n=7); This shows that the effects of ISO on the resting levels of intracellular calcium can be block by its antagonist (Propranolol) (P <0.05). After 1.0μM ISO reperfusion, induced calcium transient peak (F/F0) of myocardial cells enhanced from the normal 8.40±0.69 to 10.82±0.77; then adding 4.0μM Propranolol, induced calcium transient peak reduce to 7.70±0.43; and next wash away the extracellular ISO and Propranolol, induced calcium transient peak turn to 7.17±0.79, This shows that the role of ISO induced calcium transient peak enhance may also be blocked by its antagonist Propranolol ( P<0.05).Conclusions1. A research model and method for confocal imaging of cardiac intracellular calcium signalling was established successfully. A series of intracellular calcium signal images were collected, which will be an important part for the digital atlas of intracellular calcium signals.2. Under the condition of lower extracellular calcium, peak of evoked calcium transients decreased, but the duration of calcium transients prolonged, and some excitation-contraction coupling failure could occur in cardiac myocytes.3. Under the condition of higher extracellular calcium, resting intracellular calcium increased, which implied the declined stability of calcium and calcium overload; with the increase in the concentration of extracellular calcium, peak of evoked calcium transients and efficiency of excitation-contraction coupling decreased. It is obviously that the former is the main cause of the latter.4. Low concentration(0.1μM)ofβreceptor agonist (ISO) could make the resting intracellular calcium signal level decrease significantly, and the evoked calcium transient peak increase significantly; this effect of ISO could be completely blocked by its antagonist propranolol.