Peifeng Li

miR-499 regulates mitochondrial dynamics by targeting calcineurin and dynamin-related protein-1.

Myocardial infarction is a leading cause of mortality worldwide. Here we report that modulation of microRNA-499 (miR-499) levels affects apoptosis and the severity of myocardial infarction and cardiac dysfunction induced by ischemia-reperfusion. We found that both the α- and β-isoforms of the calcineurin catalytic subunit are direct targets of miR-499 and that miR-499 inhibits cardiomyocyte apoptosis through its suppression of calcineurin-mediated dephosphorylation of dynamin-related protein-1 (Drp1), thereby decreasing Drp1 accumulation in mitochondria and Drp1-mediated activation of the mitochondrial fission program. We also found that p53 transcriptionally downregulates miR-499 expression. Our data reveal a role for miR-499 in regulating the mitochondrial fission machinery and we suggest that modulation of miR-499 levels may provide a therapeutic approach for treating myocardial infarction.

miR-30 regulates mitochondrial fission through targeting p53 and the dynamin-related protein-1 pathway.

miRNAs participate in the regulation of apoptosis. However, it remains largely unknown as to how miRNAs are integrated into the apoptotic program. Mitochondrial fission is involved in the initiation of apoptosis. It is not yet clear whether miRNAs are able to regulate mitochondrial fission. Here we report that miR-30 family members are able to regulate apoptosis by targeting the mitochondrial fission machinery. Our data show that miR-30 family members can inhibit mitochondrial fission and the consequent apoptosis. In exploring the underlying molecular mechanism, we identified that miR-30 family members can suppress p53 expression. In response to the apoptotic stimulation, the expression levels of miR-30 family members were reduced, whereas p53 was upregulated. p53 transcriptionally activated the mitochondrial fission protein, dynamin-related protein-1 (Drp1). The latter conveyed the apoptotic signal of p53 by initiating the mitochondrial fission program. miR-30 family members inhibited mitochondrial fission through suppressing the expression of p53 and its downstream target Drp1. Our data reveal a novel model in which a miRNA can regulate apoptosis through targeting the mitochondrial fission machinery.

miR-9 and NFATc3 Regulate Myocardin in Cardiac Hypertrophy

Myocardial hypertrophy is frequently associated with poor clinical outcomes including the development of cardiac systolic and diastolic dysfunction and ultimately heart failure. To prevent cardiac hypertrophy and heart failure, it is necessary to identify and characterize molecules that may regulate the hypertrophic program. Our present study reveals that nuclear factor of activated T cells c3 (NFATc3) and myocardin constitute a hypertrophic pathway that can be targeted by miR-9. Our results show that myocardin expression is elevated in response to hypertrophic stimulation with isoproterenol and aldosterone. In exploring the molecular mechanism by which myocardin expression is elevated, we identified that NFATc3 can bind to the promoter region of myocardin and transcriptionally activate its expression. Knockdown of myocardin can attenuate hypertrophic responses triggered by NFATc3, suggesting that myocardin can be a downstream mediator of NFATc3 in the hypertrophic cascades. MicroRNAs are a class of small noncoding RNAs that mediate post-transcriptional gene silencing. Our data reveal that miR-9 can suppress myocardin expression. However, the hypertrophic stimulation with isoproterenol and aldosterone leads to a decrease in the expression levels of miR-9. Administration of miR-9 could attenuate cardiac hypertrophy and ameliorate cardiac function. Taken together, our data demonstrate that NFATc3 can promote myocardin expression, whereas miR-9 is able to suppress myocardin expression, thereby regulating cardiac hypertrophy.

Foxo3a Inhibits Cardiomyocyte Hypertrophy through Transactivating Catalase

The forkhead transcription factor Foxo3a is able to inhibit cardiomyocyte hypertrophy. However, its underlying molecular mechanism remains to be fully understood. Our present study demonstrates that Foxo3a can regulate cardiomyocyte hypertrophy through transactivating catalase. Insulin was able to induce cardiomyocyte hypertrophy with an elevated level of reactive oxygen species (ROS). The antioxidant agents, including catalase and N-acetyl-l-cysteine, could inhibit cardiomyocyte hypertrophy induced by insulin, suggesting that ROS is necessary for insulin to induce hypertrophy. Strikingly, we observed that the levels of catalase were decreased in response to insulin treatment. The transcriptional activity of Foxo3a depends on its phosphorylation status with the nonphosphorylated but not phosphorylated form to be functional. Insulin treatment led to an increase in the phosphorylated levels of Foxo3a. To understand the relationship between Foxo3a and catalase in the hypertrophic pathway, we characterized that catalase was a transcriptional target of Foxo3a. Foxo3a bound to the promoter region of catalase and stimulated its activity. The inhibitory effect of Foxo3a on cardiomyocyte hypertrophy depended on its transcriptional regulation of catalase. Finally, we identified that myocardin was a downstream mediator of ROS in conveying the hypertrophic signal of insulin or insulin-like growth factor-1. Foxo3a could negatively regulate myocardin expression levels through up-regulating catalase and the consequent reduction of ROS levels. Taken together, our results reveal that Foxo3a can inhibit hypertrophy by transcriptionally targeting catalase.

Control of mitochondrial activity by miRNAs

Mitochondria supply energy for physiological function and they participate in the regulation of other cellular events including apoptosis, calcium homeostasis, and production of reactive oxygen species. Thus, mitochondria play a critical role in the cells. However, dysfunction of mitochondria is related to a variety of pathological processes and diseases. MicroRNAs (miRNAs) are a class of small noncoding RNAs about 22 nucleotides long, and they can bind to the 3′‐untranslated region (3′‐UTR) of mRNAs, thereby inhibiting mRNA translation or promoting mRNA degradation. We summarize the molecular regulation of mitochondrial metabolism, structure, and function by miRNAs. Modulation of miRNAs levels may provide a new therapeutic approach for the treatment of mitochondria‐related diseases. J. Cell. Biochem. 113: 1104–1110, 2012.

ARC is a critical cardiomyocyte survival switch in doxorubicin cardiotoxicity

Despite its complexity of action, doxorubicin (Dox)-induced cardiomyopathy eventually results in loss of cardiac myocytes which further contributes to the development of overt heart failure. In the present study, we examined the relevance of the apoptosis repressor with caspase recruitment domain (ARC) on cardiac myocyte survival and its underlying mechanisms in a model of Dox-induced cardiotoxicity. Exposure of neonatal rat ventricular cardiomyocytes with Dox resulted in a downregulation of ARC mRNA and protein levels that occurred in a pre-translational and post-translational manner and led to a significant induction of apoptosis. Proteasomal inhibitors partially rescued both Dox-induced downregulation of ARC protein and induction of apoptosis. Knockdown of endogenous ARC sensitised cardiomyocytes to undergo apoptosis upon treatment with Dox. In contrast, enforced expression of ARC by adenoviral-mediated gene transfer dramatically increased the resistance of cardiomyocytes to undergo apoptotic cell death following Dox administration. In response to Dox, Bax translocated from cytosol to mitochondria where it resulted in dissipation of the mitochondrial membrane potential, cytochrome c release and activation of caspases -3 and -9. ARC prevented Bax translocation to the mitochondrium and thereby blocked the activation of the mitochondrial apoptotic death pathway in a t-Bid and caspase-8-independent manner. In this study, we provide evidence for the protective role of anti-apoptotic ARC in Dox-induced cardiotoxicity, which makes this molecule an interesting target for future therapies.

Novel cardiac apoptotic pathway: the dephosphorylation of apoptosis repressor with caspase recruitment domain by calcineurin.

Background— Apoptosis repressor with caspase recruitment domain (ARC) is abundantly expressed in cardiomyocytes. Protein kinase CK2 can phosphorylate ARC at threonine-149, thereby enabling ARC to antagonize apoptosis. ARC phosphorylation occurs in a constitutive manner. Nevertheless, cardiomyocytes still undergo apoptosis that is related to cardiac diseases such as myocardial infarction and heart failure. Whether the occurrence of apoptosis is related to the loss of protection by ARC under pathological conditions remains unknown. Methods and Results— ARC phosphorylation levels are decreased in cardiomyocytes treated with isoproterenol or aldosterone. We explored the molecular mechanism by which ARC phosphorylation levels are decreased. Our results reveal that either direct incubation or coexpression with calcineurin leads to a decrease in ARC phosphorylation levels. Inhibition of calcineurin can attenuate the reduction in ARC phosphorylation levels on treatment with isoproterenol or aldosterone. These data indicate that the reduction in ARC phosphorylation levels is related to its dephosphorylation by calcineurin. Our results further reveal that ARC can prevent isoproterenol- and aldosterone-induced apoptosis, but this function depends on its phosphorylation status. Isoproterenol and aldosterone upregulate Fas ligand expression, and Fas ligand and caspase-8 are required for isoproterenol and aldosterone to induce apoptosis. However, phosphorylated but not dephosphorylated ARC is able to inhibit caspase-8–mediated apoptosis. Phosphorylated ARC exerts its effects against caspase-8 by directly associating with procaspase-8 and inhibiting its interaction with Fas-associated protein with death domain. Conclusions— Our study identifies a novel cardiac apoptotic pathway in which ARC is dephosphorylated by calcineurin. This pathway could be a component in the cardiac apoptotic machinery.

Mitochondrial fission controls DNA fragmentation by regulating endonuclease G.

Mitochondria constantly undergo fusion and fission that are necessary for the maintenance of organelle fidelity. However, growing evidence has shown that abnormal mitochondrial fusion and fission participate in the regulation of apoptosis. Mitochondrial fusion is able to inhibit apoptosis, whereas mitochondrial fission is involved in the initiation of apoptosis. It remains elusive as to whether mitochondrial fission can regulate DNA fragmentation during apoptosis. Mitochondrial fission is triggered by dynamin-related protein-1 (Drp1), whereas mitofusin 1 (Mfn 1) is able to induce mitochondrial fusion. Here, we report that Drp1 is required for the release of endonuclease G from mitochondria. Knockdown of Drp1 can attenuate DNA fragmentation. Our data further show that Mfn 1 prevents endonuclease G release from mitochondria and the consequent DNA fragmentation. Intriguingly, Mfn 1 could inhibit the activation of caspase-3 and caspase-9, which are necessary for endonuclease G translocation to the nucleus. Our results provide novel evidence that DNA fragmentation is regulated by the mitochondrial fission machinery.

MADD Knock-Down Enhances Doxorubicin and TRAIL Induced Apoptosis in Breast Cancer Cells

The Map kinase Activating Death Domain containing protein (MADD) isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.

MiR-23a binds to p53 and enhances its association with miR-128 promoter

Apoptosis plays an important role in cardiac pathology, but the molecular mechanism by which apoptosis regulated remains largely elusive. Here, we report that miR-23a promotes the apoptotic effect of p53 in cardiomyocytes. Our results showed that miR-23a promotes apoptosis induced by oxidative stress. In exploring the molecular mechanism by which miR-23a promotes apoptosis, we found that it sensitized the effect of p53 on miR-128 regulation. It promoted the association of p53 to the promoter region of miR-128, and enhanced the transcriptional activation of p53 on miR-128 expression. miR-128 can downregulate prohibitin expression, and subsequently promote apoptosis. Our data provides novel evidence revealing that miR-23a can stimulate transcriptional activity of p53.