Proarrhythmic changes in human cardiomyocytes during hypothermia by milrinone and isoprenaline, but not levosimendan: an experimental in vitro study.

BACKGROUND: Accidental hypothermia, recognized by core temperature below 35 °C, is a lethal condition with a mortality rate up to 25%. Hypothermia-induced cardiac dysfunction causing increased total peripheral resistance and reduced cardiac output contributes to the high mortality rate in this patient group. Recent studies, in vivo and in vitro, have suggested levosimendan, milrinone and isoprenaline as inotropic treatment strategies in this patient group. However, these drugs may pose increased risk of ventricular arrhythmias during hypothermia. Our aim was therefore to describe the effects of levosimendan, milrinone and isoprenaline on the action potential in human cardiomyocytes during hypothermia. METHODS: Using an experimental in vitro-design, levosimendan, milrinone and isoprenaline were incubated with iCell2 hiPSC-derived cardiomyocytes and cellular action potential waveforms and contraction were recorded from monolayers of cultured cells. Experiments were conducted at temperatures from 37 °C down to 26 °C. One-way repeated measures ANOVA was performed to evaluate differences from baseline recordings and one-way ANOVA was performed to evaluate differences between drugs, untreated control and between drug concentrations at the specific temperatures. RESULTS: Milrinone and isoprenaline both significantly increases action potential triangulation during hypothermia, and thereby the risk of ventricular arrhythmias. Levosimendan, however, does not increase triangulation and the contractile properties also remain preserved during hypothermia down to 26 °C. CONCLUSIONS: Levosimendan remains a promising candidate drug for inotropic treatment of hypothermic patients as it possesses ability to treat hypothermia-induced cardiac dysfunction and no increased risk of ventricular arrhythmias is detected. Milrinone and isoprenaline, on the other hand, appears more dangerous in the hypothermic setting.

Moderate but not severe hypothermia increases intracellular cyclic AMP through preserved production and reduced elimination.

Rewarming from accidental hypothermia could be complicated by acute cardiac dysfunction but providing supportive pharmacotherapy at low core temperatures is challenging. Several pharmacological strategies aim to improve cardiovascular function by increasing cAMP in cardiomyocytes as well as cAMP and cGMP levels in vascular smooth muscle, but it is not clear what effects temperature has on cellular elimination of cAMP and cGMP. We therefore studied the effects of differential temperatures from normothermia to deep hypothermia (37 °C-20 °C) on cAMP levels in embryonic H9c2 cardiac cells and elimination of cAMP and cGMP by PDE-enzymes and ABC-transporter proteins. Our experiments showed significant elevation of intracellular cAMP in H9c2-cells at 30 °C but not 20 °C. Elimination of both cAMP and cGMP through ABC transport-proteins and PDE-enzymes showed a temperature dependent reduction. Accordingly, the increased cardiomyocyte cAMP-levels during moderate hypothermia appears an effect of preserved production and reduced elimination at 30 °C. This correlates with earlier in vivo findings of a positive inotropic effect of moderate hypothermia.

Pharmacodynamic properties for inhibition of cAMP- and cGMP elimination by pentoxifylline remain unaltered in vitro during hypothermia.

BACKGROUND: Rewarming from hypothermia is associated with severe complications, one of which is hypothermia-induced cardiac dysfunction. This condition is characterized by decreased cardiac output accompanied by increased total peripheral resistance. This contributes to mortality rate approaching 40%. Despite this, no pharmacological interventions are recommended for these patients below 30 °C. Raising the intracellular levels of cAMP and/or cGMP, through PDE3- and PDE5-inhibitors respectively, have showed the ability to alleviate hypothermia-induced cardiac dysfunction in vivo. Drugs that raise levels of both cAMP and cGMP could therefore prove beneficial in patients suffering from hypothermia-induced cardiac dysfunction. METHODS: The unselective PDE-inhibitor pentoxifylline was investigated to determine its ability to reach the intracellular space, inhibit PDE3 and PDE5 and inhibit cellular efflux of cAMP and cGMP at temperatures 37, 34, 30, 28, 24 and 20 °C. Recombinant human PDE-enzymes and human erythrocytes were used in the experiments. IC50-values were calculated at all temperatures to determine temperature-dependent changes. RESULTS: At 20 °C, the IC50-value for PDE5-mediated enzymatic breakdown of cGMP was significantly increased compared to normothermia (IC50: 39.4 µM ± 10.9 µM vs. 7.70 µM ± 0.265 µM, p-value = 0.011). No other significant changes in IC50-values were observed during hypothermia. CONCLUSIONS: This study shows that pentoxifylline has minimal temperature-dependent pharmacodynamic changes, and that it can inhibit elimination of both cAMP and cGMP at low temperatures. This can potentially be effective treatment of hypothermia-induced cardiac dysfunction. TRIAL REGISTRATION: Not applicable.

Treatment of Cardiovascular Dysfunction with PDE3-Inhibitors in Moderate and Severe Hypothermia-Effects on Cellular Elimination of Cyclic Adenosine Monophosphate and Cyclic Guanosine Monophosphate.

Introduction: Rewarming from accidental hypothermia is often complicated by hypothermia-induced cardiovascular dysfunction, which could lead to shock. Current guidelines do not recommend any pharmacological treatment at core temperatures below 30°C, due to lack of knowledge. However, previous in vivo studies have shown promising results when using phosphodiesterase 3 (PDE3) inhibitors, which possess the combined effects of supporting cardiac function and alleviating the peripheral vascular resistance through changes in cyclic nucleotide levels. This study therefore aims to investigate whether PDE3 inhibitors milrinone, amrinone, and levosimendan are able to modulate cyclic nucleotide regulation in hypothermic settings. Materials and methods: The effect of PDE3 inhibitors were studied by using recombinant phosphodiesterase enzymes and inverted erythrocyte membranes at six different temperatures-37°C, 34°C, 32°C, 28°C, 24°C, and 20°C- in order to evaluate the degree of enzymatic degradation, as well as measuring cellular efflux of both cAMP and cGMP. The resulting dose-response curves at every temperature were used to calculate IC50 and Ki values. Results: Milrinone IC50 and Ki values for cGMP efflux were significantly lower at 24°C (IC50: 8.62 ± 2.69 µM) and 20°C (IC50: 7.35 ± 3.51 µM), compared to 37°C (IC50: 22.84 ± 1.52 µM). There were no significant changes in IC50 and Ki values for enzymatic breakdown of cAMP and cGMP. Conclusion: Milrinone, amrinone and levosimendan, were all able to suppress enzymatic degradation and inhibit extrusion of cGMP and cAMP below 30°C. Our results show that these drugs have preserved effect on their target molecules during hypothermia, indicating that they could provide an important treatment option for hypothermia-induced cardiac dysfunction.

Resistance to ventricular fibrillation predicted by the QRS/QTc - Ratio in an intact rat model of hypothermia/rewarming.

Accidental hypothermia is associated with increased risk for arrhythmias. QRS/QTc is proposed as an ECG-marker, where decreasing values predict hypothermia-induced ventricular arrhythmias. If reliable it should also predict nonappearance of arrhythmias, observed in species like rat that regularly tolerate prolonged hypothermia. A rat model designed for studying cardiovascular function during cooling, hypothermia and subsequent rewarming was chosen due to species-dependent resistance to ventricular arrhythmias. ECG was recorded throughout the protocol. No ventricular arrhythmias occurred during experiments. QRS/QTc increased throughout the cooling period and remained above normothermic baseline until rewarmed. Different from the high incidence of hypothermia-induced ventricular arrhythmias in accidental hypothermia patients, where QRS/QTc ratio is decreased in moderate hypothermia; hypothermia and rewarming of rats is not associated with increased risk for ventricular fibrillation. This resistance to lethal hypothermia-induced arrhythmias was predicted by QRS/QTc.