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<p>Hypoxia, caused by various factors, significantly threatens human health by damaging the heart and cardiomyocytes. Revascularization strategies can sometimes precipitate adverse clinical outcomes due to myocardial ischemia/reperfusion (I/R) injury. The primary objective of this investigation was to explore the mechanism underlying myocardial I/R injury to identify novel alleviating strategies. CRISPR/Cas9 technology was used for high-throughput screening of hypoxia/reoxygenation (H/R)-tolerant genes; afterward, the genes were intersected with differentially expressed genes in the GSE61592 dataset. Subsequent validation identified SPRY domain-containing SOCS box protein 3 (SPSB3) as a critical gene that confers resistance to H/R injury. Increased apoptosis and worse heart function were observed in mouse and cardiomyocyte models of myocardial I/R injury. These pathological alterations coincided with a marked elevation in SPSB3 expression. Furthermore, SPSB3 inhibition substantially attenuated the myocardial I/R injury-induced increase in cardiomyocyte apoptosis, decrease in cardiac function, and mitochondrial dysfunction. To identify SPSB3 substrates, we combined the results from mass spectrometry, ubiquitin-modified proteomics, and the MitoCarta3.0 database (cardiac mitochondrial proteins), yielding six candidate molecules. Co-immunoprecipitation and western blot analyses indicated that SPSB3 may bind to Tu translation elongation factor, mitochondrial (TUFM). In SPSB3-knockdown cardiomyocytes, additional TUFM knockdown partially reversed the protective effect of SPSB3 knockdown alone. Additionally, MG132 and Cycloheximide treatment effectively inhibited TUFM degradation. Further amino acid site mutagenesis and other analyses revealed that SPSB3 inhibition prevents K48- and K63-linked ubiquitination at the K259 residue of TUFM and TUFM degradation, providing a promising therapeutic avenue for mitigating myocardial I/R injury.</p> Graphical Abstract <p>1. Myocardial ischemia/reperfusion (I/R) injury upregulates SPSB3 expression, increases cell damage and apoptosis, and reduces cardiac function.</p> <p>2. Knockdown of SPSB3 alleviates myocardial I/R injury-induced cell damage and apoptosis, restoring cardiac function.</p> <p>3. In myocardial I/R injury, SPSB3 interacts with TUFM, promoting K48 and K63-linked ubiquitination at TUFM K259, which enhances apoptosis.</p> <p></p>

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SPSB3-mediated K48- and K63- linked ubiquitination and degradation of TUFM promote apoptosis induced by myocardial ischemia/reperfusion injury

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月4日

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  • Yuyao Yin,
  • Yaofeng Xie,
  • Xiao Wang,
  • Yuan Tan,
  • Xiaodong Li,
  • Yilong Pan

摘要

Hypoxia, caused by various factors, significantly threatens human health by damaging the heart and cardiomyocytes. Revascularization strategies can sometimes precipitate adverse clinical outcomes due to myocardial ischemia/reperfusion (I/R) injury. The primary objective of this investigation was to explore the mechanism underlying myocardial I/R injury to identify novel alleviating strategies. CRISPR/Cas9 technology was used for high-throughput screening of hypoxia/reoxygenation (H/R)-tolerant genes; afterward, the genes were intersected with differentially expressed genes in the GSE61592 dataset. Subsequent validation identified SPRY domain-containing SOCS box protein 3 (SPSB3) as a critical gene that confers resistance to H/R injury. Increased apoptosis and worse heart function were observed in mouse and cardiomyocyte models of myocardial I/R injury. These pathological alterations coincided with a marked elevation in SPSB3 expression. Furthermore, SPSB3 inhibition substantially attenuated the myocardial I/R injury-induced increase in cardiomyocyte apoptosis, decrease in cardiac function, and mitochondrial dysfunction. To identify SPSB3 substrates, we combined the results from mass spectrometry, ubiquitin-modified proteomics, and the MitoCarta3.0 database (cardiac mitochondrial proteins), yielding six candidate molecules. Co-immunoprecipitation and western blot analyses indicated that SPSB3 may bind to Tu translation elongation factor, mitochondrial (TUFM). In SPSB3-knockdown cardiomyocytes, additional TUFM knockdown partially reversed the protective effect of SPSB3 knockdown alone. Additionally, MG132 and Cycloheximide treatment effectively inhibited TUFM degradation. Further amino acid site mutagenesis and other analyses revealed that SPSB3 inhibition prevents K48- and K63-linked ubiquitination at the K259 residue of TUFM and TUFM degradation, providing a promising therapeutic avenue for mitigating myocardial I/R injury.

Graphical Abstract

1. Myocardial ischemia/reperfusion (I/R) injury upregulates SPSB3 expression, increases cell damage and apoptosis, and reduces cardiac function.

2. Knockdown of SPSB3 alleviates myocardial I/R injury-induced cell damage and apoptosis, restoring cardiac function.

3. In myocardial I/R injury, SPSB3 interacts with TUFM, promoting K48 and K63-linked ubiquitination at TUFM K259, which enhances apoptosis.