Hyperglycemia-Driven Inhibition of AMP-Activated Protein Kinase α2 Induces Diabetic Cardiomyopathy by Promoting Mitochondria-Associated Endoplasmic Reticulum Membranes In Vivo

Abstract

FUN14 domain containing 1 (Fundc1), an outer mitochondrial membrane protein, is important for mitophagy and mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs). The roles of Fundc1 and MAMs in diabetic hearts remain unknown. The aims of this study therefore, were to determine if the diabetes-induced Fundc1 expression could increase MAM formation, and whether disruption of MAM formation improves diabetic cardiac function. Levels of FUNDC1 were examined in the hearts from diabetic patients and non-diabetic donors. Levels of Fundc1-induced MAMs, and mitochondrial and heart function were examined in mouse neonatal cardiomyocytes exposed to high glucose (HG, 30 mmol/L D-glucose for 48 h), as well as in streptozotocin (STZ)-treated cardiac-specific Fundc1 knockout (KO) mice and cardiac-specific Fundc1 KO diabetic Akita mice. FUNDC1 levels were significantly elevated in cardiac tissues from diabetic patients compared to those in non-diabetic donors. In cultured mouse neonatal cardiomyocytes, HG conditions increased levels of Fundc1, the inositol 1,4,5-trisphosphate type 2 receptor (Ip 3 r2), and MAMs. Genetic downregulation of either Fundc1 or Ip 3 r2 inhibited MAM formation, reduced ER-mitochondrial Ca 2+ flux, and improved mitochondrial function in HG-treated cardiomyocytes. Consistently, adenoviral overexpression of Fundc1 promoted MAM formation, mitochondrial Ca 2+ increase, and mitochondrial dysfunction in cardiomyocytes exposed to normal glucose (5.5 mmol/L D-glucose). Compared with non-diabetic controls, levels of Fundc1, Ip 3 r2, and MAMs were significantly increased in hearts from STZ-treated mice and Akita mice. Further, compared with control hearts, diabetes markedly increased co-immunoprecipitation of Fundc1 and Ip 3 r2. The binding of Fundc1 to Ip 3 r2 inhibits Ip 3 r2 ubiquitination and proteasome-mediated degradation. Cardiomyocyte-specific Fundc1 deletion ablated diabetes-induced MAM formation, prevented mitochondrial Ca 2+ increase, mitochondrial fragmentation, and apoptosis with improved mitochondrial functional capacity and cardiac function. In mouse neonatal cardiomyocytes, HG suppressed AMP-activated protein kinase (Ampk) activity. Furthermore, in cardiomyocytes of Prkaa2 KO mice, expression of Fundc1, MAM formation, and mitochondrial Ca 2+ levels were significantly increased. Finally, adenoviral overexpression of a constitutively active mutant Ampk ablated HG-induced MAM formation and mitochondrial dysfunction. We conclude that diabetes suppresses Ampk, initiating Fundc1-mediated MAM formation, mitochondrial dysfunction, and cardiomyopathy, suggesting that Ampk-induced Fundc1 suppression is a valid target to treat diabetic cardiomyopathy.