This multi-phase prospective study utilized Data-Independent Acquisition proteomics on a nested subset (15 SIC vs. 15 matched sepsis) to map early molecular alterations. Guided by gene-set enrichment evidence of extracellular matrix (ECM) disruption as a pathway-derived biomarker. Admission matrix metalloproteinase-3 was validated in 567 critically ill patients (417 sepsis, 150 non-septic controls) across three hospital campuses.

This prospective, multi-phase observational cohort study was conducted across three intensive care units (ICUs) at Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China). Recruitment occurred during two distinct periods: June 2017 to September 2018 and September 2023 to October 2024. The study was conducted in strict compliance with the Declaration of Helsinki and received ethical approval from the Institutional Review Board of Tongji Hospital. Written informed consent was obtained from all participants or their legally authorized representatives prior to enrollment. Throughout the study period, all enrolled patients received protocolized care in strict accordance with the contemporaneous Surviving Sepsis Campaign (SSC) guidelines, including timely fluid resuscitation, vasopressor administration, and appropriate antimicrobial therapy.

Participants and Eligibility Criteria To ensure adherence to the STROBE guidelines, consecutive adults admitted to the ICUs were prospectively screened. Following predefined criteria, we excluded patients with: 1) paraquat poisoning; 2) age under 18 years at diagnosis; 3) acute cardiovascular and cerebrovascular diseases unrelated to inflammation; 4) history of cardiac surgery; 5) pregnancy or breastfeeding; and 6) intellectual or psychological disorders precluding suitable study participation.

Enrolled patients were categorized into non-sepsis and sepsis groups according to Sepsis-3.0 criteria23. The non-sepsis control group comprised critically ill individuals without evidence of infection and a Sequential Organ Failure Assessment (SOFA) score below 2 points. Within the sepsis cohort, patients were further evaluated for sepsis-induced cardiomyopathy (SIC), defined as meeting at least one diagnostic criterion within the initial 24 hours of ICU admission: echocardiographic evidence of left ventricular dysfunction (ejection fraction < 50%), troponin T elevation exceeding 0.05 ng/mL, or B-type natriuretic peptide (BNP) levels greater than 500 pg/mL. All echocardiographic assessments were performed by accredited sonographers and interpreted by board-certified cardiologists.

Clinical Data Collection and Biomarker Measurement To ensure temporality for risk prediction, primary exposure variables and baseline clinical data-including demographics, SOFA score, lactate, and standard inflammatory markers (CRP, IL-6, PCT)-were measured within the initial 24 hours of ICU admission (Day 1). For the longitudinal tracking subcohort (n=151), plasma samples were serially collected on Day 1, Day 3, and Day 7 to map kinetic trajectories. For the full validation cohort, Day 1 plasma was utilized. Plasma MMP3 concentrations were quantified using specific enzyme-linked immunosorbent assay (ELISA) kits (catalog no. ab100607; Abcam, Cambridge, UK) in strict accordance with the manufacturer's instructions. The intra-assay and inter-assay coefficients of variation (CV) were rigorously monitored to ensure analytical reliability by researchers blinded to the clinical outcomes.

Outcomes and Sample Size Justification The primary clinical outcome was the incidence of SIC during the ICU stay. The secondary outcome was 28-day all-cause mortality, prospectively monitored from the onset of disease. The sample size for the clinical validation phase was determined to satisfy the rigorous requirements of multivariable predictive modeling. Following the rule of thumb requiring at least 10 outcome events per predictor variable, the final enrollment of 417 sepsis patients-including 104 individuals who developed SIC-provided sufficient statistical power to support robust multivariable logistic regression and biomarker evaluation without overfitting.

Inclusion Criteria:

1. Age >= 18 years old.

2. Admitted to the Intensive Care Unit (ICU) with an anticipated length of stay exceeding 24 hours.

3. Patient or legally authorized representative provides written informed consent prior to enrollment.

4. Categorized into one of the following three mutually exclusive cohorts within 24 hours of ICU admission:

   • Cohort 1 (Non-sepsis Controls): Admitted for definitive non-infectious etiologies (e.g., severe trauma, major non-cardiac surgery) with no clinical or microbiological evidence of infection throughout the ICU stay.
   • Cohort 2 (Sepsis without SIC): Diagnosed with sepsis according to the Sepsis-3 criteria (acute change in SOFA score >= 2 points driven by infection), but with normal cardiac troponin levels and preserved cardiac function.
   • Cohort 3 (Sepsis-Induced Cardiomyopathy, SIC): Diagnosed with sepsis according to the Sepsis-3 criteria, accompanied by new-onset myocardial injury (elevated cardiac troponin above the upper limit of normal) and/or echocardiographic evidence of myocardial dysfunction directly attributable to sepsis.

Exclusion Criteria:

1. Pre-existing severe chronic cardiac conditions, including history of cardiac surgery, severe pre-existing heart failure (NYHA Class III or IV), persistent severe arrhythmias, or known primary cardiomyopathy (e.g., hypertrophic or dilated cardiomyopathy).
2. Acute non-infectious cardiovascular or cerebrovascular events prior to or upon ICU admission, such as acute myocardial infarction (Type 1), acute ischemic/hemorrhagic stroke, or cardiac arrest.
3. Severe end-stage comorbidities, including end-stage renal disease (ESRD) requiring chronic maintenance dialysis prior to this illness, Child-Pugh Class C hepatic cirrhosis, or advanced malignant tumors with a life expectancy < 3 months.
4. History of paraquat poisoning or other toxic ingestions known to directly cause profound myocardial or pulmonary toxicity.
5. Pregnant or breastfeeding women.
6. Indeterminate or ambiguous infectious status (e.g., cases treated with empiric antibiotics for suspected infection but where infection could neither be confirmed nor ruled out), excluded to prevent misclassification bias.
7. Intellectual, psychological, or neurological disorders that preclude necessary clinical examinations or compliance with study procedures.

Critical ill patients without sepsis; Patients with sepsis and patients diagnosed with septic cardiomyopathy.