Proteomic investigation of acute and chronic hypoxia/reoxygenation responsive proteins and pathways in H9C2 cardiomyoblasts.

Abstract

Background/aim: Ischemic heart diseases continue to be a significant global cardiovascular problem in today's world. Myocardial reperfusion (R) is provided with an effective and rapid treatment; however, it can lead to fatal results, as well as ischemia (I). This study aims to use proteomic analysis to assess proteins and pathways in H9C2 cardiomyoblast cells exposed to hypoxic conditions, followed by reoxygenation, representing I/R injury for both short and long terms, reflecting acute and chronic hypoxia, respectively. Utilizing advanced techniques, our goal is to identify and characterize key proteins undergoing alterations during these critical phases.

Materials and methods: H9C2 cardiomyoblasts, a commonly used cell line for simulating in vivo I/R damage, were exposed to normoxia and hypoxia (0.4% O₂) in six experimental groups: normoxia (3h), acute hypoxia (3h), acute hypoxia (3h) + reoxygenation (3h), normoxia (21h), chronic hypoxia (21h), and chronic hypoxia (21h) + reoxygenation (3h). Analyses were conducted using Nano LC/MSMS from tryptic digest of the whole cell lysates. Proteins were quantified using the label-free quantification (LFQ) algorithm in Proteome Discoverer 2.4.

Results: Proteomic analysis resulted in identification of 2383 protein groups. Proteins that differentially expressed in the various groups were identified (p < 0.05 among mean values for groups). Short-term hypoxia induces mitochondrial damage, energy demand, and cytoskeletal modifications. Chronic hypoxia triggers metabolic shifts, stress-response proteins, and extracellular matrix alterations. Data are available via ProteomeXchange with identifier PXD047994.

Conclusion: Our research provides in-depth insights into how H9C2 cardiomyoblasts respond to both short-term and prolonged oxygen deprivation. Understanding hypoxia-related pathophysiology provides avenues for therapeutic intervention in hypoxia-related disorders.