Serum proteome profiles in patients treated with targeted temperature management after out-of-hospital cardiac arrest

Abstract

Background: Definition of temporal serum proteome profiles after out-of-hospital cardiac arrest may identify biological processes associated with severe hypoxia-ischaemia and reperfusion. It may further explore intervention effects for new mechanistic insights, identify candidate prognostic protein biomarkers and potential therapeutic targets. This pilot study aimed to investigate serum proteome profiles from unconscious patients admitted to hospital after out-of-hospital cardiac arrest according to temperature treatment and neurological outcome.

Methods: Serum samples at 24, 48, and 72 h after cardiac arrest at three centres included in the Target Temperature Management after out-of-hospital cardiac arrest trial underwent data-independent acquisition mass spectrometry analysis (DIA-MS) to find changes in serum protein concentrations associated with neurological outcome at 6-month follow-up and targeted temperature management (TTM) at 33 °C as compared to 36 °C. Neurological outcome was defined according to Cerebral Performance Category (CPC) scale as "good" (CPC 1-2, good cerebral performance or moderate disability) or "poor" (CPC 3-5, severe disability, unresponsive wakefulness syndrome, or death).

Results: Of 78 included patients [mean age 66 ± 12 years, 62 (80.0%) male], 37 (47.4%) were randomised to TTM at 36 °C. Six-month outcome was poor in 47 (60.3%) patients. The DIA-MS analysis identified and quantified 403 unique human proteins. Differential protein abundance testing comparing poor to good outcome showed 19 elevated proteins in patients with poor outcome (log₂-fold change (FC) range 0.28-1.17) and 16 reduced proteins (log₂(FC) between - 0.22 and - 0.68), involved in inflammatory/immune responses and apoptotic signalling pathways for poor outcome and proteolysis for good outcome. Analysis according to level of TTM showed a significant protein abundance difference for six proteins [five elevated proteins in TTM 36 °C (log₂(FC) between 0.33 and 0.88), one reduced protein (log₂(FC) - 0.6)] mainly involved in inflammatory/immune responses only at 48 h after cardiac arrest.

Conclusions: Serum proteome profiling revealed an increase in inflammatory/immune responses and apoptosis in patients with poor outcome. In patients with good outcome, an increase in proteolysis was observed, whereas TTM-level only had a modest effect on the proteome profiles. Further validation of the differentially abundant proteins in response to neurological outcome is necessary to validate novel biomarker candidates that may predict prognosis after cardiac arrest.

Keywords: Heart arrest; Hypothermia; Out-of-hospital cardiac arrest; Prognostication; Proteomics; Targeted temperature management; Temperature control.

Fig. 1

Flowchart of the patients and serum samples included in the study. *One patient was mislabeled between the mass spectrometry- and clinical data sheet; included 3 samples. A second patient was incorrectly identified between the mass spectrometry- and clinical data sheet; included 3 samples. TTM Target Temperature Management after out-of-hospital cardiac arrest trial

Fig. 2

Proteomic analysis results for protein abundance according to neurological outcome. a Heatmap of the significantly abundant proteins at 24, 48 and 72 h after return of spontaneous circulation for poor vs. good neurological outcome. Positive log₂-fold change (FC) indicates elevated proteins (red colour) in patients with poor outcome when compared with good outcome patients. Negative log₂(FC) indicates reduced proteins (blue colour) in poor outcome compared with good outcome patients. b-d Volcano plots for the differential abundance of proteins for neurological outcome at 24 h (b), 48 h (c) and 72 h (d), respectively. Positive log₂(FC) indicates good outcome, negative log₂(FC) indicates poor outcome. Statistically significant proteins (adjusted p-value ≤ 0.05) and regulated proteins (absolute log₂(FC) > 1) are labelled in red. Proteins with a significant adjusted p-value with a log₂(FC) between − 1 and 1 are labelled in yellow, and statistically non-significant proteins with an absolute log₂(FC) < − 1 or > 1 are labelled in green. Individual protein descriptions with their biological functions are presented in Additional file 4: Table S1. AFM afamin; AGT angiotensinogen; ASL argininosuccinate lyase; B2M beta-2-microglobulin; C3 complement C3; C7 complement component C7; CCL14 C–C motif chemokine 14; CFD complement factor D; CHGA chromogranin-A; CHI3L1 chitinase-3-like protein 1; CST3 cystatin-C; EFEMP1-EGF containing fibulin-like extracellular matrix protein 1; FETUB fetuin-B; FGA fibrinogen alpha chain; FGB fibrinogen beta chain; FGG fibrinogen gamma chain; GPLD1 phosphatidylinositol-glycan-specific phospholipase D; GSN gelsolin; IGFBP2 insulin-like growth factor-binding protein 2; IGFBP4 insulin-like growth factor-binding protein 4; IGHV3-13 immunoglobulin heavy variable 3-13; IGHV3-23 immunoglobulin heavy variable 3-23; ITIH1 inter-alpha-trypsin inhibitor heavy chain H1; ITIH2 inter-alpha-trypsin inhibitor heavy chain H2; ITIH3 inter-alpha-trypsin inhibitor heavy chain H3; KLKB1 plasma kallikrein; LCN2 neutrophil gelatinase-associated lipocalin; LPA apolipoprotein (a); NIBAN protein Niban 3; PEPD Xaa-Pro dipeptidase; PLA2G7 platelet-activating factor acetylhydrolase; PLG plasminogen; PROS vitamin K-dependent protein S; RNASE1 ribonuclease pancreatic; SBSN suprabasin; SERPINA3 alpha-1-antichymotrypsin; SERPINA4 kallistatin; SFTPB pulmonary surfactant-associated protein B; TNXB tenascin-X; UBB/UBC polyubiquitin-B/polyubiquitin-C

Fig. 3

Biological processes for the differentially abundant proteins associated with neurological outcome. Proteins enhanced for neurological outcome were annotated by selected gene ontology terms for biological process. The average log₂-fold change for all proteins included in each term is plotted to show the direction of average change for poor outcome (in red) as compared to good outcome (in blue)

Fig. 4

Proteomic analysis results for protein abundance according to temperature treatment of 36 °C to 33 °C. a Heatmap of the significantly differentially abundant proteins for comparison of temperature treatment of 36 °C to 33 °C at 24, 48 and 72 h after return of spontaneous circulation. Positive log₂-fold change (FC) indicates elevated proteins (red colour) in patients treated at target temperature 36 °C compared to 33 °C. Negative log₂(FC) indicates proteins reduced (blue colour) in patients treated at target temperature 36 °C versus 33 °C. b Volcano plots for the differential abundance of proteins between temperature treatment at 24, 48, and 72 h. Positive log₂(FC) indicates treatment with 36 °C, negative log₂(FC) indicates treatment with 33 °C. Proteins with a significant adjusted p-value, and with a log₂(FC) between − 1 and 1 are labelled in yellow, and statistically non-significant proteins with an absolute log₂(FC) <  − 1 or > 1 are labelled in green. Individual protein descriptions with their biological functions are presented in Additional file 4: Table S1. ANG angiogenin; COL6A collagen alpha-1(VI) chain; CPB1 carboxypeptidase B; FCN2 Ficolin-2; ICAM1 intercellular adhesion molecule 1; IGLV7-43 immunoglobulin lambda variable 7–43; ITIH4 inter-alpha-trypsin inhibitor heavy chain family member 4; MASP1 isoform 2 of mannan-binding lectin serine protease 1; PCSK9 proprotein convertase subtilisin/kexin type 9

Fig. 5

Interaction plot for Extracellular superoxide dismutase (EC-SOD) levels according to neurological outcome and temperature treatment. Results are displayed for 24, 48, and 72 h after return of spontaneous circulation. EC-SOD levels were significantly increased in poor outcome patients treated with a targeted temperature of 36 °C at 48 h compared to patients with good outcome, indicating an antioxidative response. No statistically significant differences could be seen between good and poor outcome in patients treated with targeted temperature of 33 °C. CPC Cerebral Performance Category