Ann-Dorit Andersen:
Hypoxia and Acidosis – A study of myocardial ischemia/reperfusion and cardioprotection in the murine atrial cardiomyocyte cell culture line, HL-1

Date: 20-07-2011    Supervisor: Stine Falsig Pedersen

Acute myocardial infarction (AMI) is among the leading causes of death in the western world. AMI is most commonly the result of coronary artery occlusion. Ischemic postconditioning (IPoC) i.e. brief repeated cycles of ischemia/reperfusion (I/R) within the first minutes of reperfusion following an ischemic insult is a widely studied way to limit cardiac damage after AMI. As such, elucidating mechanisms behind injuries induced by I/R and events underlying cardioprotective procedures such as IPoC is of immense interest. To study these cellular events we employed one of the few established cell culture lines with a cardiac phenotype, HL-1. HL-1 is, in second generation, derived from a mouse atrial SV40 large T antigen induced tumor. The protocol of simulated I/R (SI/R) was conducted with authentic hypoxia in a closed incubator/workchamber system with tightly controlled gas supply.

As a specific aim we wanted to examine the role of pH during SI/R. In my earliest published manuscript, “HL-1 mouse cardiomyocyte injury and death after simulated ischemia and reperfusion: roles of pH, Ca2+-independent phospholipase A2, and Na+/H+ exchange”, we focused on the effect of extracellular pH (pHo) during simulated ischemia (SI) in regulation of the pH sensitive proteins Ca2+-independent phospholipase A2 group VI (PLA2-VIA) and Na+/H+ exchanger 1 (NHE1); in integrity of cytoskeletal components and mitochondria; and in cell death after SI/R. We found that the mode of cell death and the roles and regulation of PLA2-VIA and NHE1 are at least in part determined by pHo during cardiac ischemia, with acidic pHo during SI being cardioprotective compared to pH-neutral SI.

Subsequently we addressed the potential role of pHo during reperfusion. One of the protective mechanisms behind IPoC is suggested to be delayed pH-recovery upon reperfusion. In a collaborative work we, besides HL-1 cells, implemented experiments on isolated perfused rat hearts. This work is reported in my latest published manuscript, “The cardioprotective effect of acidic reperfusion after ischemia in perfused rat hearts is not mimicked by inhibition of the Na+/H+ exchanger NHE1”. Here we demonstrate strong protective effects of acidic reperfusion in perfused hearts, but not in HL-1 cells. This in conjunction with previous reports on HL-1 cell metabolism lead us to question the application of HL-1, at least in protocols of simulated acidic reperfusion.

To examine the diverging effects between HL-1 and whole hearts experiments I next addressed signaling in HL-1 in response to acidic reperfusion, presented in a manuscript currently in preparation. We have so far shown that SI/R induced activation of cardioprotective kinases in HL-1 do not differ significantly from that reported in intact hearts or primary cardiomyocytes, but in contrast, kinase activation in HL-1 does not respond to acidic reperfusion. We speculate that HL-1 cells are in a constitutively protected state, compared to normal cardiomyocytes. This was supported by our demonstration of STAT3 being constitutively activated in HL-1 which is a typical cancer cell characteristic. Previous reports point to several cancerous characteristics of HL-1 cells and we suggest that the apparent loss of susceptibility to I/R injuries might be related to these properties, probably related to the transformed origin of these cells. Interestingly if pH regulation was compromised in HL-1 (buffering devoid of bicarbonate) I/R induced apoptotic cell death increased and acidic reperfusion, as well as inhibition of NHE1 and mitochondrial permeability transition pore opening was protective.

These considerations, in conjunction with the relevance of hypoxia and acidosis not only in ischemic heart disease but equally so in cancer, drew my attention to the field of cancer research and I recently joined in a collaboration regarding pancreatic cancer. Preliminary results were published in a meeting abstract in Acta Physiologica primo 2010 and are part of an ongoing study, with a paper currently in preparation. My contribution was to construct the experimental setup design and execute the experiments in examination of expression and regulation of relevant pH-regulating ion transporters by hypoxia and acidosis in pancreatic cancer cell lines.

In a broader perspective, these findings both call for caution and may suggest some new potential applications of HL-1 cells. My findings strongly indicate that HL-1 cells are poor models for the normal cardiomyocyte response to I/R. On the other hand, however, the low susceptibility of HL-1 cells to irreversible I/R injuries may be relevant to explore further in order to gain novel insight into mechanisms that might be mimicked pharmacologically in order to increase normal cardiomyocyte survival during I/R. Finally, a novel application possibility for HL-1 cells may be as a tool in basal cancer research, with particular focus on the mechanisms and consequences of changes in metabolism during cancer cell development.