Mitochondrial Creatine Kinase 2 (Ckmt2) as a Plasma-Based Biomarker for Evaluating Reperfusion Injury in Acute Myocardial Infarction

Abstract

Background/objectives: Acute myocardial infarction (AMI), characterized by irreversible heart muscle damage and impaired cardiac function caused by myocardial ischemia, is a leading cause of global mortality. The damage associated with reperfusion, particularly mitochondrial dysfunction and reactive oxygen species (ROS) formation, has emerged as a crucial factor in the pathogenesis of cardiac diseases, leading to the recognition of mitochondrial proteins as potential markers for myocardial damage. This study aimed to identify differentially expressed proteins based on the type of cardiac injury, in particular those with and without reperfusion.

Methods: Male C57Bl/6J mice were either left untreated, sham-operated, received non-reperfused AMI, or reperfused AMI. Twenty-four hours after the procedures, left ventricular (LV) function and morphological changes including infarct size were determined using echocardiography and triphenyl tetrazolium chloride (TTC) staining, respectively. In addition, plasma was isolated and subjected to untargeted mass spectrometry and, further on, the ELISA-based validation of candidate proteins.

Results: We identified mitochondrial creatine kinase 2 (Ckmt2) as a differentially regulated protein in plasma of mice with reperfused but not non-reperfused AMI. Elevated levels of Ckmt2 were significantly associated with infarct size and impaired LV function following reperfused AMI, suggesting a specific involvement in reperfusion damage.

Conclusions: Our study highlights the potential of plasma Ckmt2 as a biomarker for assessing reperfusion injury and its impact on cardiac function and morphology in the acute phase of MI.

Keywords: biomarker; mitochondrial damage; myocardial infarction; reperfusion injury.

Figure 1

Protein abundance in the plasma of mice subjected to experimental AMI with and without reperfusion. A volcano plot illustrating the relative abundance (log2 fold changes) versus probability (−log10 p-values) in the plasma of mice that received either non-reperfused or reperfused AMI. Multiple t-test analyses with False Discovery Rate (FDR) correction were used to determine significance (indicated by red dots). Significant proteins are defined as those with a fold change > | ±1.5| and a p-value < 0.05. Two proteins are significantly upregulated in the non-reperfusion group (Mup3, Mup18), while six proteins are significantly upregulated in the reperfusion-operated mice (Ckmt2, Grn, Got1, Igfbp4, Got1, Actn3, Fermt3). n = 5 samples per group.

Figure 2

The mass spectrometric quantification of eight proteins significantly altered in plasma between non-reperfusion- and reperfusion-operated mice and their abundance in the respective controls. Plasma was isolated 24 h after the induction of sham-operated, non-reperfused AMI, and reperfused AMI and in untreated animals and was analyzed by untargeted mass spectrometry. Each bar plot represents the mean value with standard deviation (SD), with individual data points overlaid to show the distribution of measurements. Statistical significance between groups was determined using paired one-way ANOVA for normally distributed data or Kruskal–Wallis test for non-normally distributed data, with a p-value < 0.05 considered significant. Figure panels correspond to specific proteins: (A) Ckmt2, (B) Actn3, (C) Got1, (D) Igfbp4, (E) Grn, (F) Fermt3, (G) Mup3 and (H) Mup18. (A–F) show proteins that are upregulated in reperfusion-operated mice compared to non-reperfusion-operated mice, while (G,H) show proteins that are downregulated. Protein levels that were not detected are valued as 0. n = 5 per group.

Figure 3

Ckmt2 correlates to infarct size and left ventricular dysfunction 24 h following reperfused myocardial infarction. (A) Ckmt2 plasma levels detected by ELISA show a significant positive correlation with infarct size (INF) relative to the area at risk (AAR), a negative relationship with (B) ejection fraction (EF) and a positive relationship with (C) end-systolic volume (ESV). (D) No significant correlation is observed between Ckmt2 levels and end-diastolic volume (EDV), (E) stroke volume (SV) or (F) heart rate (HR). Correlations are performed using Pearson’s correlation. Each dot represents an individual measurement from one mouse. Significance is defined as p < 0.05, and correlations are displayed with R² values. n = 15.