Moderate hypothermia initiated during oxygen–glucose deprivation preserves HL-1 cardiomyocytes☆
Introduction
Ischemic heart disease, estimated to cause 7.1 million deaths per year, is the leading cause of death worldwide [23]. Despite advancements in accelerated reperfusion time and optimized management of complications such as arrhythmias, which have improved survival rates after acute ischemic events, the rate of long-term heart failure is increasing [33], [37]. Therapeutic hypothermia (TH), routinely applied to attenuate neurological impairment following traumatic brain injury (TBI) [17], anoxic brain injury [16], [27], or cardiac arrest [34], is a promising intervention strategy to decrease mortality and morbidity after myocardial ischemia. In fact, TH played a prominent role in the vision of Dr. Peter Safar, considered by most as the “Father of Resuscitation”, for optimized resuscitation and neurointensive care [17], [35].
The pathomechanisms of ischemia/reperfusion injury (I/R) are similar in both the brain and heart, including mitochondrial dysfunction, intracellular acidosis, and disturbance in ion homeostasis [4], [24]. If ischemia is followed by reperfusion, the re-introduction of oxygen and nutrients leads to excessive formation of reactive oxygen species (ROS) followed by inflammatory responses and subsequent cell death [26]. Moderate therapeutic hypothermia (32–34 °C) may effectively influence many stages of this deleterious I/R-induced injury cascade [32].
The data from clinical and animal studies investigating the possible cardioprotective effects of moderate hypothermia are inconclusive. Randomized controlled trials applying TH to patients suffering from acute myocardial infarction (AMI) found no significant decrease in infarct size [5], [10], [13], [31]. However, a re-analysis of the subpopulations of clinical trials using a more rigorous cooling treatment (rapid endovascular catheter core cooling combined with cold saline) reported patients diagnosed with anterior myocardial infarction that were cooled to temperatures below 35 °C before reperfusion showed a significant reduction in infarct size and/or release of adverse cardiac biochemical markers [9], [10], [12], [13], [31]. Attaining targeted temperatures below 35 °C was also shown to be feasible and well tolerated by all patients in some of these studies [10], [12]. Likewise, a re-analysis of the data from the Hypothermia after Cardiac Arrest Study (HACA) [38], one of the landmark studies promoting TH for cardiac arrest survivors, reported a favorable effect on myocardial damage when target temperature between 32 and 34 °C was reached within 8 h after cardiac arrest [18]. In various animal studies of acute myocardial infarction and cardiac arrest, TH effectively reduced infarct sizes and/or improved cardiac functions when initiated during the ischemic event but failed when applied after the induction of reperfusion [3], [11], [14], [25], [41].
In this study we developed an in vitro model for investigating hypothermia-induced cardioprotection using HL-1 cardiomyocytes, a cell line that has been proven to be an excellent model for investigating cardiac functions [20], [28]. In vitro ischemia/reperfusion injury was simulated by exposing the cardiomyocytes to oxygen glucose deprivation (OGD) followed by re-oxygenation and re-supply of nutrients (OGD/R). Moderate TH (33.5 °C) was initiated post and intra-OGD and maintained throughout the re-oxygenation phase. We sought to contribute new insights regarding the optimal timing of targeted temperature management and identify the effects of hypothermia-induced cellular protection.
Section snippets
HL-1 cell culture
HL-1 mouse cardiomyocytes derived from AT-1 atrial cardiomyocyte tumor cells were generously donated by William C. Claycomb, Ph.D (LSU Health Sciences Center, New Orleans, La., USA). HL-1 cells contract spontaneously and show biochemical, electrophysiological, and morphological characteristics of adult cardiomyocytes while dividing in culture [2]. The cells were cultured at 37 °C and 5% CO2 in Claycomb Medium supplemented with 100 μM/ml penicillin/streptomycin, 10% fetal bovine serum (FBS), 0.1 mM
Cell morphology
In our established model for cardiac OGD/R-induced injury, exposure of the HL-1 mouse cardiomyocytes to 6 h of 0.2% O2 and glucose/serum deprivation followed by 24 h of replenished oxygen and nutrients at 37 °C resulted in observable cellular damage as compared to normoxic controls. Post-OGD therapeutic hypothermia, i.e. cooling to 33.5 °C for 24 h initiated after OGD and continued till the end of the re-oxygenation phase, did not attenuate OGD/R-induced damage in the cardiomyocytes, as observed in
Discussion
Rapid initiation of therapeutic hypothermia is believed to confer protection against oxygen–glucose deprivation/re-oxygenation-induced injuries. However, the optimal application timing and efficacy of TH remains to be validated, as the beneficial effects observed in experimental studies have not consistently been translated to the clinical setting. We, hereby, present a reproducible model of cultured HL-1 cardiomyocytes, which allows for further investigation of the effects of hypothermia in
Conclusion
In summary, we introduced a reproducible model consisting of HL-1 cardiomyocytes with sufficient responses to OGD and OGD/R-induced injuries, which were effectively attenuated by moderate intra-OGD therapeutic hypothermia but not by post-OGD therapeutic hypothermia. Moderate hypothermia initiated during OGD and maintained after re-oxygenation efficaciously preserved energy sources, improved mitochondrial activity, and mitigated cell death by attenuating OGD/R-stimulated apoptosis. Our findings
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Statement of funding: This work was supported by the Fördergemeinschaft Deutsche Kinderherzzentren e.V. (Amtsgericht Bonn: VR 5868), Elsa-Brändström-Str. 21, 53225 Bonn, Germany.
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Both authors contributed equally to this work.