Cardiac fibroblasts play pivotal roles in the pathogenesis of heart failure. Mitochondrial dysfunction contributes to calcium imbalance, increased oxidative stress, apoptosis and cardiac fibrosis. Accordingly, modulating mitochondrial defects in fibroblasts is a potential strategy in treating heart failure. Histone deacetylase (HDAC) causes cardiovascular disease through modulation of oxidative stress, inflammation and metabolic derangement. Studies have shown that inhibition of HDAC can reduce myocardial fibrosis and improve metabolic disorders of heart failure and diabetic myocardium. Our preliminary data showed that inhibition of class I and IIb HDACs can improve mitochondrial respiration of pathological cardiomyocytes, revealing the important roles of HDAC in mitochondrial activity. However, the profibrotic role of mitochondria activity in fibroblasts has not yet been clarified. MicroRNA (miRNA) is a critical regulator of fibroblasts activity that can regulate mitochondrial activity and calcium homeostasis. However, whether HDAC can affect mitochondrial activity and calcium homeostasis through regulating miRNA in fibroblasts remains to be clarified. Therefore, the purposes of this study are to investigate the role of HDAC in calcium homeostasis and metabolic derangements of mitochondrial function, and study the therapeutic potential of HDAC inhibition on mitochondria in cardiac fibroblasts. The first year experiment will investigate whether HDAC directly regulates the mitochondrial activity of fibroblasts and analyzes the associated miRNA to identify the regulating signaling pathway. The second year experiment will investigate whether inhibition of HDAC activity alters the mitochondrial activity and calcium retention capacity of cardiac fibroblasts in heart failure rats. The third year experiment will investigate whether HDAC regulates the cytokine secretion from fibroblasts that further affect the calcium homeostasis, energy metabolism and cellular hypertrophy of ventricular myocytes.Methods: The first year study, cardiac fibroblasts will be treated with different HDAC inhibitors, including MS-275 (class I inhibitor), MC-1568 (class II inhibitor) and Tubacin (HDAC6 inhibitor) each 1 uM for 48 hours. The Seahorse XF24 analyzer and transmission electron microscopy will be used to assess the different energy metabolism substrates on the respiration rate and morphology of mitochondria. The miRNA expression will be analyzed by small RNA sequencing and to identify the potential regulatory mechanism. In the second year study, heart failure rats will be received intraperitoneally injection of MS-275 (10 mg/kg/day), MC-1568 (20 mg/kg/day), or Tubacin (20 mg/kg/day) for 14 consecutive days. Cardiac structure and function will be evaluated by electrocardiogram and cardiac echography. The left ventricular fibroblasts will be isolated to investigate the mitochondrial respiratory efficiency and calcium retention capacity. In the third year study, calcium imaging will be used to assess whether the conditioned medium from fibroblasts treated with different classes HDAC inhibitor could regulate the calcium homeostasis of cardiac myocytes. Seahorse XF24 analyzer will be used to investigate the effects of the different energy metabolism substrates on mitochondria respiration rate. Immunoblot blot will be used to analyze protein expressions which are related to calcium homeostasis, myocardial energy metabolism and cardiac hypertrophy.Preliminary Results: Class I HDAC inhibition in cardiac fibroblasts significantly reduced ATP production but increased mitochondrial reactive oxygen species as compared to control fibroblasts.
|Effective start/end date||8/1/18 → 7/1/19|
- Cardiac fibroblast
- Heart failure
- Histone deacetylase
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