The
type 2 diabetes mouse model was established using db/db
leptin receptor-deficient mice and high-fat diet/
streptozotocin-induced mice. Cardiac-specific knockout of USP28 in the db/db background mice was generated by crossbreeding db/m and Myh6-Cre+/USP28fl/fl mice. Recombinant adeno-associated virus serotype 9 carrying USP28 under cardiac
troponin T promoter was injected into db/db mice. High
glucose plus
palmitic acid-incubated neonatal rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes were used to imitate
diabetic cardiomyopathy in vitro. The molecular mechanism was explored through
RNA sequencing, immunoprecipitation and mass spectrometry analysis,
protein pull-down,
chromatin immunoprecipitation sequencing, and
chromatin immunoprecipitation assay.
RESULTS: Microarray profiling of the UPS (
ubiquitin-
proteasome system) on the basis of db/db mouse hearts and diabetic patients' hearts demonstrated that the diabetic ventricle presented a significant reduction in USP28 expression. Diabetic Myh6-Cre+/USP28fl/fl mice exhibited more severe progressive cardiac dysfunction,
lipid accumulation, and mitochondrial disarrangement, compared with their controls. On the other hand, USP28 overexpression improved systolic and diastolic dysfunction and ameliorated
cardiac hypertrophy and
fibrosis in the diabetic heart. Adeno-associated virus serotype 9-USP28 diabetic mice also exhibited less
lipid storage, reduced
reactive oxygen species formation, and mitochondrial impairment in heart tissues than adeno-associated virus serotype 9-null diabetic mice. As a result, USP28 overexpression attenuated cardiac remodeling and dysfunction,
lipid accumulation, and mitochondrial impairment in high-fat diet/
streptozotocin-induced
type 2 diabetes mice. These results were also confirmed in neonatal rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes.
RNA sequencing, immunoprecipitation and mass spectrometry analysis,
chromatin immunoprecipitation assays,
chromatin immunoprecipitation sequencing, and
protein pull-down assay mechanistically revealed that USP28 directly interacted with PPARα (
peroxisome proliferator-activated receptor α), deubiquitinating and stabilizing PPARα (Lys152) to promote Mfn2 (mitofusin 2) transcription, thereby impeding mitochondrial morphofunctional defects. However, such cardioprotective benefits of USP28 were largely abrogated in db/db mice with PPARα deletion and conditional loss-of-function of Mfn2.
CONCLUSIONS: Our findings provide a USP28-modulated mitochondria homeostasis mechanism that involves the PPARα-Mfn2 axis in diabetic hearts, suggesting that USP28 activation or adeno-associated virus
therapy targeting USP28 represents a potential therapeutic strategy for
diabetic cardiomyopathy.