Role of calcium desensitization in the treatment of myocardial dysfunction after deep hypothermic circulatory arrest

Deep hypothermic circulatory arrest (DHCA) is widely used during repair of the aortic arch and congenital heart defects as a method of cerebral protection. However, the effect of myocardial ischemia-reperfusion injury combined with hypothermia-rewarming has important clinical implications and remains a significant contributor to perioperative morbidity and mortality [1].

Hypotension and low cardiac output are common after rewarming and up to 50 to 80% of patients have hemodynamic instability [2].

Because of the limited knowledge of the pathophysiology of hypothermia- and rewarming-induced cardiac dysfunction either in the setting of cardiac surgical intervention or in accidental hypothermia, guidelines for pharmacological treatment are missing [3]. Among the mechanisms underlying cardiac dysfunction after hypothermia-rewarming, a pivotal role has been assigned to Ca²⁺ overload and the paradoxical myofilaments Ca²⁺ desensitization. Ca²⁺-dependent interactions among myofilament regulatory proteins primarily control cardiac muscle contraction. Among them, troponin and tropomyosin confer not only Ca²⁺ sensitivity to contraction, but also modulation by phosphorylation of troponin I (cTnI) [4]. The latter represents a well-established mechanism for decreased myofilament Ca²⁺ sensitivity and could be mediated by phosphokinase A activated by β-adrenergic stimulation [5].

Thus, agents that target the regulatory apparatus by increasing the calcium sensitivity without deleterious effects on the intracellular calcium concentration or oxygen consumption are useful in the treatment of cardiac dysfunction. Epinephrine is currently used in the treatment of hypothermia-induced cardiac dysfunction [6]. However, the efficacy of epinephrine in this setting has been questioned [7–9].

Agents that exert a positive-inotropic effect, by directly acting on thin filament regulatory proteins or on cross-bridge cycling, are commonly referred to as calcium sensitizers. Among them, levosimendan, in contrast to epinephrine, improves ventricular function without increasing oxygen demand [10]. We previously demonstrated that levosimendan compared to epinephrine has better inotropic and lusitropic effects when administrated during rewarming from DHCA [11].

The present study was designed to investigate treatment with levosimendan or epinephrine after rewarming from DHCA when acute myocardial dysfunction represents a clinical problem. We aimed to investigate the effects of levosimendan and epinephrine on calcium desensitization mediated by cTnI phosphorylation. Moreover, we purposed to evaluate the acute overall hemodynamic, biochemical and metabolic effects of the two pharmacologic agents after rewarming from DHCA.

The Institutional Animal Care and Use Committee of the University Animal Research Laboratory approved this study. All animals received standardized care in accordance with the National Institute of Health Guidelines. Adult male Wistar rats (400 to 450 g, Harlan, Udine, Italy) were used for all experiments.

Fifty rats were used for this study; 40 completed the experimental protocol and were included. Ten animals were excluded, including five rats due to their inability to wean from CPB and five due to instrumentation or technical failure during animal preparation.

Functional indices derived from pressure-volume analysis at different preloads are less influenced by loading conditions. In particular, Ees and PRSW were calculated as load-independent indexes of left ventricular contractility. Ees was significantly higher after both levosimendan and epinephrine infusion than control, indicating a steeper end-systolic pressure-volume relationship and a better systolic performance (Figure 1). Only levosimendan significantly improved PRSW; the slope was steeper after levosimendan compared to epinephrine treatment, indicating an improved systolic function determined by levosimendan in this setting (Figure 1). Compared with the corresponding control animals, the overall PRSW values did not reach statistical significance in the epinephrine group (Figure 1).

The diastolic function after rewarming was described by the left ventricular relaxation index (Tau) and the stiffness index end-diastolic pressure-volume relationship (EDPVR). Only levosimendan significantly improved EDPVR and Tau (Figure 1).

Lactate levels were significantly higher after treatment with epinephrine as well as in the control group than after levosimendan infusion (8.2 ± 3.8 mmol/L and 9.3 ± 4.4 vs 4.1 ± 3.2 mmol/L respectively; P<0.001) (Figure 2).

In addition, levosimendan better preserved the high-energy phosphate status of the left ventricle compared to epinephrine. The levosimendan group showed a significantly higher ATP content (25.25 ± 1.91 vs 15.8 ± 2.11 nmol ATP/mg protein; P<0.001) (Figure 3) and energy charge (0.80 ± 0.03 vs 0.65 ± 0.03; P<0.001; Figure 4). The high energy phosphate, hypoxanthine and xanthine values are summarized in Table 2. In line with the above findings, the epinephrine group had significantly higher AMP values compared to the levosimendan- and control group.

During normothermia epinephrine was associated with a three-fold increase in cTnI phosphorylation, whereas levosimendan maintained cTnI phosphroylation at a physiological ratio (Figure 5). After DHCA, cTnI phosphroylation increased five-fold compared with normothermia. Epinephrine was unable to reverse this increase. On the contrary, this change was reversed by levosimendan, which re-established the physiological cTnI phosphorylation ratio observed during normothermia (Figure 5).

Data presented here clearly show that levosimendan administered after rewarming from DHCA prevents Ca²⁺ desensitization mediated by cTnI phosphorylation. Levosimendan, compared to epinephrine, led to a significantly better preservation of myocardial ATP content as well as energy charge and to a reduction in plasma lactate concentrations. In comparison, Levosimendan was superior in improving myocardial systolic and diastolic functions as demonstrated by PV-analysis.

Clinically deep hypothermia, either accidental or induced as a neuroprotective measure during aortic arch interventions, leads to an overall myocardial depression that aggravates ischemia-reperfusion injury [2]. Although systolic and diastolic dysfunctions are often transient, more permanent injury in the form of necrosis and apoptosis might occur. Pulmonary dysfunction, which is frequently associated with hypothermia-induced myocardial dysfunction, worsens the low cardiac output state [1].

Experimental models of deep hypothermia and rewarming confirm and explain these clinical findings [15, 16]. In an experimental animal model of DHCA, left ventricle contractility, relaxation and afterload were markedly, but transiently, depressed early after reperfusion and mildly depressed late after reperfusion [17]. During cooling, end systolic volume decreases, but during the rewarming phase, isovolumetric pressure is depressed and ventricular wall shortening reduced, whereas diastolic function is reported to be affected to a lesser extent. Hence, the observed hypothermia-induced cardiac dysfunction is mainly due to compromised systolic function. Based on recent reports, it is possible to advocate that one of the main underlying mechanisms of hypothermia- rewarming-induced contractile dysfunction could be Ca²⁺ overload, which is defined as mechanical or electrical contractile failure induced by elevated intracellular Ca²⁺ content in myocytes [3, 16, 18].

Ca²⁺ overload may induce cardiac contractile dysfunction through different pathways. In particular after hypothermia and rewarming, increase in reactive oxygen species, decreased ATP synthesis and damaged mitochondrial ultrastructure have been reported [3].

YS Han and colleagues have observed that papillary muscle twitch force was reduced by 35% after rewarming despite a 15% increase in the intracellular Ca²⁺ content, indicating that reduced contractile force after rewarming is related to a decrease in myofibrillar Ca²⁺ sensitivity rather than impairment in intracellular Ca²⁺ regulation [4]. Significant increases in the temporal parameters of contraction and relaxation in twitch force after rewarming likely reflect a change in myosin/actin cross-bridge cycling kinetics. By plotting phase-plane loops (the relationship between twitch force and intracellular Ca²⁺ concentration), which represent coarse dynamic indices of myofilaments Ca²⁺ sensitivity during the relaxation phase of the twitch force, a rightward shift was observed in the relaxation phase during stable hypothermia, as well as after rewarming, in intact papillary muscle [4]. This indicates a decrease in myofilaments Ca²⁺ sensitivity due to hypothermia and rewarming.

These data propose that hypothermia decreases cardiac myofilaments Ca²⁺ sensitivity, which is not reversed by rewarming, thus contributing to post-hypothermic cardiac contractile dysfunction.

Numerous studies have demonstrated that increased phospho-mediated phosphorylation of the sarcomeric protein cTnI reduces myofilament Ca²⁺ sensitivity and shifts the force-Ca2+ relationship rightward under normothermic circumstances [5, 19, 20].

One way to increase the contractile force of the sarcomere is to increase cytosolic Ca²⁺; another is to modulate the response of sarcomeres to Ca²⁺. The unique position of cTnI to regulate and modulate cardiac function under the influence of adrenergic signaling is well documented [21]. In more detail, cTnI has a NH2-terminal extension of approximately 30 amino acids that houses Ser23/24, which if phosphorylated by phosphokinase A depresses sarcomere sensitivity to Ca²⁺and increases cross-bridge kinetics.

In the present study, we found that after rewarming from DHCA, there was a significant increase in cTnI phosphorylation at phosphokinase A sites, as shown by Western blot analysis. Phosphorylation of cTnI is mediated by several kinases (phosphokinase A, C, G), but particularly phosphokinase A-mediated phosphorylation via-adrenergic stimulation plays a major physiological role in meeting the circulatory demand in the physiological state [21].

In general, changes in the cTnI phosphorylation status, as reported in the failing human heart are considered a change in the balance between kinase and phosphatase activities, and the optimal balance is realized during normal cardiac function. Thus, hypothermia and rewarming, depending on severity and depth, appear to tip the balance between kinase and phosphatase activities, leading to alterations in myofilament Ca²⁺ sensitivity and myocardial contractility.

Furthermore, we have previously reported a lack of effect of pharmacological agents to support cardiac contractile function mediated via the receptor - cAMP - phosphokinase A pathway during, as well as after, rewarming [11]. Likewise, if hypothermia and rewarming per se activate the receptor - cAMP - phosphokinase A pathway, one may conclude that an additional pharmacological stimulus of the receptor under these circumstances would achieve a markedly reduced cardiac inotropic effect.

Moreover, cardiovascular effects mediated by β-adrenoceptors are significantly diminished after hypothermia, suggesting that the use of the β-adrenoceptor specific agonist should be reconsidered [7–9]. Additionally, pharmacologic therapy with catecholamines has substantial limitations because these are associated with Ca²⁺ overload, elevated myocardial oxygen consumption, arrhythmogenesis and regional hypoperfusion leading to organ damage [8–10].

Levosimendan is a pyridazinone dinitrite derivative belonging to the new inotropic drugs class of Ca²⁺ sensitizers. It exerts its positive inotropic effect by sensitizing cardiac troponin C to calcium during systole, thus increasing cardiac performance without increasing myocardial oxygen consumption. Levosimendan also has a weak arrhythmogenic effect [22]. Our group demonstrated for the first time the efficacy of levosimendan in improving myocardial dysfunction after deep hypothermia-rewarming [11].

This study was conducted in a previously described and validated rat model of CPB [12]. DHCA and CPB-assisted rewarming reproduces the clinical setting of both cardiac surgery interventions and extracorporeal resuscitation after accidental hypothermia. The results of this study demonstrated that levosimendan treatment after rewarming produced a significant improvement of clinically used parameters of cardiac functions (HR, MAP, SV and CO). In the same manner, more precise parameters derived from pressure-volume analysis being insensitive to preload effects confirmed significantly improved systolic function after levosimendan treatment when compared with epinephrine.

Epinephrine produced an increased MAP without an improvement in SV. This increase in afterload during the rewarming phase may explain the significantly higher lactate plasma levels with epinephrine compared to levosimendan after rewarming from hypothermia. Moreover, levosimendan was superior in improving diastolic function, which is clearly evident from the left ventricular relaxation indexes. In line with these findings, levosimendan also significantly better preserved high-energy phosphates shown by energy charge and ATP myocardial content than epinephrine. The differences concerning phosphocreatine, hypoxanthine and xanthine between groups are due to the high standard deviation values (control- and epinephrine group; Table 2) not significant. However, the mean values are lowest for phosphocreatine and highest for xanthine in the epinephrine group. In addition, there was a significant rise of AMP contents of the hearts in the epinephrine group compared to the control - as well as levosimendan group. These findings are in line with the observed significantly increased arterial lactate levels after treatment with epinephrine.

Energy charge uses the total measured concentrations of AMP, ADP and ATP determined after careful digestion (liquid nitrogen, ball mill and neutralization of then sample) by HPLC and, therefore, do not directly reflect the bioenergetic situation of the free metabolites in the cytosol. The assessment of free fluctuating concentrations of these metabolites would require 31P NMR and a number of near equilibrium expressions using the creating kinase and adenylatekinase equilibrium [23]. However, energy charge as calculated value from the absolute concentrations of AMP, ADP and ATP has been proven to be a valuable parameter for energetic situations of cells and tissue [24–26].

In the present study and differently from the previously reported [11], there were no major arrhythmic events in the epinephrine-treated group or in levosimendan-treated one. No other downsides related to treatment were documented.

Further investigations are needed to validate these results in the clinical setting and to evaluate the cost/benefit ratio in this setting of high-risk patients. Nonetheless, on the basis of these findings, use of Ca²⁺ sensitizer drugs, such as levosimendan, seems rational in induced or accidental deep hypothermia.

Finally, some important limitations remain. The animal model does not faithfully reproduce the clinical setting. Therefore, the outcome of this study in a rat model of CPB remains to be demonstrated in large-animal and clinical studies. However, because of the small animal size, this model allows rapid and precise control over body temperature, which is difficult to achieve in clinical practice at present, and it may be particularly appropriate for defining optimal hypothermia parameters. Another limitation of the present study is that we only investigated a single dose of epinephrine and levosimendan. One, therefore, cannot rule out the possibility that different dosages or treatment schemes might have generated different functional outcomes from the one herein. Nevertheless, the particular infusion rate for levosimendan and for epinephrine was deliberately chosen because it is the highest dose regimen suggested in the clinical setting.

Levosimendan due to prevention of calcium desensitization by cTnI phosphorylation is more effective than epinephrine for treatment of myocardial dysfunction after rewarming from DHCA.