The Underlying Mechanisms Involving Instability Of Cardiac Electricity And Abnormal Calcium Signaling For Severe Burn Trauma Generated Cardiac Dysfunction

Patch clamp and laser confocal scaning microscopy techniques were conjugatedly used in the experiment to investingate the change of cardiac electricity and calcium signaling in severe burn trauma rat ventricular myocyte.Part 1: Severe burn trauma prolongs AP in burn traumatized cardiac myocytesSevere burn trauma causes prolongation of action potential duration (APD) of cardiomyocytes, which results in cardiac dysfunction by inducing disturbance of calcium dyshomeostasis in cardiac myocytes. However, the underlying mechanism for APD prolongation remains unclear. In the present study, we examined the major AP repolarization-related ion channel currents in rat ventricular cardiomyocytes, including transient outward current (I_to), inward rectifier K⁺ current (I_K1) and L-type Ca²⁺ current (I_Ca-L) to investigate the alterations of these channels, which might account for the pathogenesis of APD prolongation induced by burn injury. Twelve hours after producing full-thickness cutaneous burn injury of about 40% total body surface area in rats, ventricular cardiomyocytes were isolated from the hearts with systolic and diastolic dysfunction. AP was found to be markedly prolonged, while APD₅₀ and APD₉₀ in ventricular cardiomyocytes from rats with burn injury were 21.16±1.59 ms and 108.52±10.39 ms(n=22), respectively, significantly longer than 7.58±0.48 ms and 55.78±3.09 ms(n=17,P＜0.000) in ventricular cardiomyocytes isolated from sham rats. Burn injury remarkably suppressed I_to density in ventricular cardiomyocyte. I_to density at +60 mV was decreased from 34.15±3.78 pA/pF (n=20) in sham group to 20.39±1.98 pA/pF (n=25, P＜0.01 ) in burn injury group, and the decrease extended through the whole voltages tested (from -30 to 60 mV). Similarly, current densities of I_K1 at the voltages ranging from -120 to -90 mV in burn injury group were also significantly smaller than that in sham group. In contrast, we failed to detect any alterations in I_Ca-L density and voltage-dependence of activation in burn injury group, compared with that in sham group. Taken together, our data suggest that burn injury results in downregulation of I_to and I_K1, which contributes, at least in part, to the APD prolongation and subsequent cardiac dysfunction.Part 2: Abnormal calcium signaling in severe burn trauma generated cardiac dysfunctionBurn trauma causes precipitate deterioration of cardiac function characterized by dysregulation of intracellular Ca²⁺ signaling and impaired contractility. However, much of the underlying cellular and molecular mechanism remains elusive. In the present study, we demonstrate roles of excessive SR Ca²⁺ leakage and oxidative stress in burn-associated acute heart failure.2.1 Alterations of Action Potenital-Elicited Intracellular Ca²⁺ Transients andCell Shortening Contractility of burns was significantly decreased in peak amplitude VS shams. The average percentage of maximum shortening of cardiomyocytes was decreased from 10.51±0.38 % (n=123) in sham group to 5.75±0.18 % (n=159, P＜0.000) in burn group. Consistent with the suppression of myocyte contractility, action potential induced Ca²⁺ transient was reduced in burns VS shams. The Ca²⁺ transient amplitude (â–³F/F₀) was decreased from 4.01±0.07 (n=123) in shams to 3.04±0.07 (n=159, P＜0.000) in burns.2 Reduction of Gain of E-C coupling in Voltage-Clamped Cells To distinguish whether alterations in Ca²⁺ influx or SR Ca²⁺ release contributes to the decreased systolic Ca²⁺ transient, we measured depolarization-induced I_Ca density and spatially resolved intracellular Ca²⁺ transients in cardiomyocytes simultaneously. The results showed that the amplitude of Ca₂₊ transients was significantly reduced in burn myocytes through the voltage range from -20 mV to +40 mV, whereas the I_Ca density did not change through the whole range of membrane potential tested, indicating alteration in SR Ca²⁺ release, rather than I_Ca contributed to the suppressed systolic Ca²⁺ transient. The ratio of peak Ca²⁺ transients to peak I_Ca density, which represented the gain of E-C coupling, was reduced in burns versus shams, suggesting that the efficiency of E-C coupling was reduced in burn myocytes.2.3 Partially Depleted SR Ca²⁺ Content in Burn SR Ca²⁺ content has been known to be an important factor in determining the amplitude of intracellular Ca²⁺ release. We therefore estimated SR Ca²⁺ content from recordings of Ca²⁺ transients and integrating the resulting INCX on short puffs of caffeine. The average amplitude (â–³F/F₀) of caffeine-induced Ca²⁺ transients were 4.71±0.09 (n=153) in shams and 3.67±0.07 in burns (n=256 P＜0.000). The results indicated that reduced CICR during E-C coupling was dominated by depletion of SR Ca²⁺ content by burn injury.2.4 Hyperactive Ca²⁺ Sparks in Burn The triggered [Ca²⁺]_i transient arises as a stochastic summation of sparks and thus Ca²⁺ sparks are largely responsible for prominent features of global Ca²⁺ release signal. We observed 299 sparks in 132 cells from 6 sham rats and 926 sparks in 168 cells from 6 burn rats. The results showed that burn significantly increased sparks frequency, but decreased sparks amplitude in cardiomyocytes. On average, the Ca²⁺ sparks frequency was 2.49±0.14/100μms in burns, much higher than that of 1.05±0.06/100μm.s in shams (P＜0.000). The sparks amplitude (F/F₀) was reduced from 2.32±0.03 in shams to 2.09±0.02 in burns(P＜0.000).2.5 Ca²⁺ instability in burn As the properties of Ca²⁺ sparks are determined by SR Ca²⁺ content as well as RyRs behavior, we detected RyRs releasing activity under the SR Ca²⁺ overload state by exposing the cells to higher extracellular Ca²⁺ (i.e., 10 mmol/L) which should induce cell-wide propagation of Ca²⁺ waves in cardiomyocytes with normal SR function. The results revealed that the Ca²⁺ waves could be more readily induced in burns (with a frequency of 1.17±0.07/100μm's, n=131) than in shams (with a frequency of 0.40±0.03/100μm's, n=109, P＜0.01) when extracellular Ca²⁺ was elevated to 10 mmol/L, consistent with the change in Ca²⁺ sparks frequency at normal extracellular Ca²⁺. Ca²⁺ sparks frequency was also significantly increased in shams and burns, but more obvious in burns.2.6 Antioxidants Protected Hearts From Burn-Induced Ca²⁺ Dysregulation andCardiac Dysfunction To explore the possible role of oxidative stress in calcium dysregulation and impaired EC coupling modification, we applied antioxidant vitamin C and E treatment in sham (sham+vit) and burn rats (burn+vit). Vitamin C (38mg/kg/day) and Vitamin E (27u/kg/day) were administered 4 days before and 5 to 10 min after burn injury by intragastric administration. Amazingly, this antioxidant regimen prevented the occurrence of high frequent Ca²⁺ sparks in burn. Although the sparks rate in burn+vit group (1.21±0.06/100μm's, n=422 sparks) was still slightly higher than 1.02±0.06/100μm's, (n=309 sparks, P＜0.01) in sham+vit group, it was significantly lower than 2.45±0.13/100μm's in burn group (P＜0.000). The amplitude of Ca²⁺ transient stimulated by 20 mM caffeine in cardiomyocytes from burn+vit rats was 5.40±0.14 (n=47), almost equal to the value of 5.45±0.21(n=34) in cardiomyocytes from sham+vit rats, but significantly higher than that in burn. The result suggested that enhanced Ca²⁺ sparks-mediated Ca²⁺ leak is the major reason for reduced SR Ca²⁺ content. Parallel to the restoring of SR Ca²⁺ content, the amplitude of AP-induced Ca²⁺ transients was also increased by antioxidant vitamins treatment. Althoughâ–³F/F0 of Ca²⁺ transients in burn+vit cells (4.96±0.14, n=68) had a mild decrease in comparison with that in sham+vit cells (5.34±0.17, n=62), it was significantly increased versus burn (P＜0.01). Consistently, the maximum shortening of myocytes in burn+vit group was increased versus burn.In consistent with the improvement of intracellular Ca²⁺ signaling and contractility in single cardiomyocytes, the antioxidant treatment remarkably improved cardiac function in burn rats. Although burn injury still caused a decrease in cardiac function in antioxidant vitamins treated rats, the occurrence of cardiac insufficiency was postponed and the degree of cardiac injury was remarkably lessened versus untreated rats.3,Conclusion:3.1 Consistent with the decrease of contractility in bums, the amplitude of Ca²⁺ transient (â–³F/FO) evoked by membrane depolarization decreased, suggesting that decreased Ca²⁺ transient contributes, at least in part, to deficit of cardiac contractility in burn rats.3.2 Global SR Ca²⁺ release, rather than I_Ca was significantly reduced, which contributed to the decrease of Ca²⁺ transient in burns.3.3 Deficit in global SR Ca²⁺ release was dominantly determined by partial SR Ca²⁺ depletion.3.4 The Ca²⁺ sparks frequency dramatically increased in burns, despite the reduced SR Ca²⁺ content. Enhanced Ca²⁺ spark-mediated SR Ca²⁺ leak might play an important role in reduced SR Ca²⁺ content, as well as cytosolic Ca²⁺ accumulation at the diastolic phase. 3.5 SR leakage due to oxidative stress is a major candidate mechanism underlying burn-associated acute heart failure. Antioxidant therapy in burn trauma provides cardioprotection, at least in part, by protecting RyRs from oxidative stress-induced hypersensitivity.