Objective:

The mitochondrial isoform of CK (MtCK) catalyses the transfer of a phosphoryl group from ATP onto creatine to form phosphocreatine (PCr) and ADP. The supply of cellular energy in the form of ATP is closely matched to demand the creatine kinase (CK) phosphagen system acts to buffer ATP levels and signals for increased energy production. Previous studied investigated the potential for MtCK to protect against cellular stress: Increase in MtCK activity improves post-ischaemic recovery of contractile function in the intact heart and protects against myocardial injury in vivo. However, although activity of MtCK as well as evidence on transcriptomic level were given, still proof on protein level was low. Here, we were aiming to fill this gap by performing a mass spectrometry approach to measure MtCK in a ischemia/reperfusion setting in mice.

Methods and Results:

Each n=5 mice (C57BL/6J) underwent either myocardial ischemia without reflow (NR), ischemia for 45 min followed by reperfusion for 23h (I/R), sham-surgery or no surgery at all (baseline). In each group, plasma 24h after intervention was collected, followed by protein extraction and mass spectrometry measurements. Here, common ischemia markers like lactate were elevated significantly in both I/R and NR compared to sham control (p=.0032 resp. .0079, Figure 1A). Although in both I/R and NR release of cytochrome c as marker of mitochondrial cell death increased in comparison to sham (Figure 1B, p = .016 resp. .0079), only reperfusion was strongly associated with elevated release of the mitochondrial isoform of CK (MtCK/Ckmt2; p = .036, Figure 1C), while NR was not (p = .13).

Conclusions:

We could show for the first time using a mass-spectrometry approach that release of mitochondrial isoform of CK (Ckmt2) is associated to reperfusion injury in mice. This adds evidence to the hypothesis that the CK system could be used as a specific marker for reperfusion injury. Future work will focus on the identification of small molecules that increase creatine kinase activity for the translation of these findings.