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Suppression of Induced microRNA-15b Prevents Rapid Loss of Cardiac Function in a Dicer Depleted Model of Cardiac Dysfunction.

Roy S, Banerjee J, Gnyawali SC, Khanna S, He G, Pfeiffer D, Zweier JL, Sen CK - PLoS ONE (2013)

Bottom Line: Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function.Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy.Anti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.

View Article: PubMed Central - PubMed

Affiliation: Davis Heart and Lung Research Institute and Department of Surgery, The Ohio State University Medical Center, Columbus, Ohio, United States of America.

ABSTRACT

Background: Dicer endonuclease, critical for maturation of miRNAs, is depleted in certain forms of cardiomyopathy which results in differential expression of certain microRNAs. We sought to elucidate the mechanisms underlying the rapid loss of cardiac function following cardiac-specific Dicer depletion in adult mice.

Results: Conditional Dicer deletion in the adult murine myocardium demonstrated compromised heart function, mitochondrial dysfunction and oxidant stress. Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function. Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy.

Conclusions: Anti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.

No MeSH data available.


Related in: MedlinePlus

miR-15b over expression in HL-1 cells compromises mitochondrial membrane potential.(A,B) HL-1 cells were transfected (72 hours) with mimic negative control or miRNA-15b mimic. Untransfected cells were used as negative control and cells transfected with carbonyl cyanide m-chlorophenylhydrazone (CCCP) were used as positive control (left panel –8 nM tetramethylrhodamine methyl ester (TMRM); center panel - 0.5 µl/ml plasma membrane potential indicator (PMPI); right panel - merged image). Bar graph shows significant decrease in TMRM with no change in PMPI. Solid bars represent mimic negative control while open bars represent miRNA-15b mimic. (C,D) Loss of mitochondrial membrane potential in HL-1 cells transfected with miRNA-15b mimic, as assessed by JC-1 flow cytometry 72 h post-transfection. Cells were transfected with a (i) mimic control, (ii) miRNA-15b mimic or (iii) treated with CCCP Arrows (K gate) indicate cells containing JC-1 aggregates resulting from intact mitochondria; M gate indicates cells with low or collapsed mitochondrial membrane potential. Ratio of polarized to depolarized cells calculated as a ratio between cells in K gate to M gate. Data indicate decrease in membrane potential upon up-regulation of miRNA-15b. (n = 3).
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pone-0066789-g005: miR-15b over expression in HL-1 cells compromises mitochondrial membrane potential.(A,B) HL-1 cells were transfected (72 hours) with mimic negative control or miRNA-15b mimic. Untransfected cells were used as negative control and cells transfected with carbonyl cyanide m-chlorophenylhydrazone (CCCP) were used as positive control (left panel –8 nM tetramethylrhodamine methyl ester (TMRM); center panel - 0.5 µl/ml plasma membrane potential indicator (PMPI); right panel - merged image). Bar graph shows significant decrease in TMRM with no change in PMPI. Solid bars represent mimic negative control while open bars represent miRNA-15b mimic. (C,D) Loss of mitochondrial membrane potential in HL-1 cells transfected with miRNA-15b mimic, as assessed by JC-1 flow cytometry 72 h post-transfection. Cells were transfected with a (i) mimic control, (ii) miRNA-15b mimic or (iii) treated with CCCP Arrows (K gate) indicate cells containing JC-1 aggregates resulting from intact mitochondria; M gate indicates cells with low or collapsed mitochondrial membrane potential. Ratio of polarized to depolarized cells calculated as a ratio between cells in K gate to M gate. Data indicate decrease in membrane potential upon up-regulation of miRNA-15b. (n = 3).

Mentions: We screened for miRNAs targeting mitochondrial function among the significantly differentially expressed miRNAs, using publicly available prediction softwares like TargetScan™ v 5.1 and Pictar™. In our search, elevated miRNA-15b emerged as a key candidate as it was computationally predicted to silence some key proteins like Pim-1 kinase, ESRRG, Mfn-2 which are required for preserving proper mitochondrial function and integrity. We used laser capture microdissection [22], [42], [43], [44] to confirm that miR-15b was indeed elevated in cardiomyocytes. We recognize that the other differentially expressed miRs may also play a significant role in development of the pathology in Dicer deleted hearts and needs to be investigated as a future work. Our study was directed to Pim-1 kinase, silencing of which is known to directly cause cardiac dysfunction. Pim-1 is a crucial requirement for downstream cardio protective effects of Akt signaling [45]. Increase in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload does not protect the myocardium in Pim-1-deficient mice [45]. Loss of Pim-1 also prevents the long term structural and functional benefits obtained from cardiac progenitor cell based therapies indicating its importance in myocardial repair and regeneration [46], [47]. Transgenic over-expression of Pim-1 in the myocardium protects mice against infarction [45]. Specifically, Pim-1 kinase is required for preserving mitochondrial integrity in cardiomyocytes [48], and also blunts cardiac hypertrophy [49]. Pim-1 kinase over-expression also reduces calcium mediated mitochondrial swelling, t-bid-induced cytochrome-C release and peroxide-induced membrane depolarization [50]. Pim-1 inhibits apoptosis-related mitochondrial dysfunction [51] and Pim-1 inhibition results in apoptosis and is closely related to the decrease in Akt and Bad phosphorylation and increase in cleaved caspase-3/−9 activities [52]. Delivery of miR-15b to HL-1 cells using a mimic compromised mitochondrial membrane potential (TMRM fluorescence) similar to what was observed upon Dicer knockdown (Fig. 5A,B). Mitochondrial membrane potential changes were also assessed using the JC-1 assay which demonstrated that over expression of miRNA-15b in HL-1 cells compromised ΔΨ (Fig. 5C,D). Pim-1 kinase protein level was observed to be low in heart tissue isolated from Dicer−/− mice by western blot (Fig. 6A, p<0.05, n = 6) and immunohistochemistry (Fig. 6B). HL-1 cells transfected with si-Dicer showed lower Pim-1 protein expression. This effect was ameliorated when cells were co-transfected with a miRNA-15b inhibitor pointing towards a direct role of miRNA-15b in silencing Pim-1 (Fig. 6C, p<0.05, n = 3). In order to validate whether miRNA-15b silences Pim-1 kinase, HL-1 cells were transfected with miRNA-15b mimic and lowered Pim-1 kinase protein levels was observed (Fig. 6D; p<0.05, n = 6). To validate the direct binding between miRNA-15b and the Pim-1 3′ UTR region, miRNA target reporter luciferase assay was performed using the pLuc-Pim1–3′UTR plasmid (Fig. 6E) in HL-1 cells. 40% reduction of luciferase activity from the pLuc-Pim1–3′UTR plasmid was observed in miRNA-15b mimic transfected HL-1 cells compared with control (Fig. 6F). No reduction in luciferase activity was observed using a mutated pLuc-Pim1-mutated 3′UTR (Fig. 6G). Thus, elevated levels of miRNA-15b silence Pim-1 kinase which may be causatively linked to the observed mitochondrial dysfunction in Dicer deleted heart of adult mice.


Suppression of Induced microRNA-15b Prevents Rapid Loss of Cardiac Function in a Dicer Depleted Model of Cardiac Dysfunction.

Roy S, Banerjee J, Gnyawali SC, Khanna S, He G, Pfeiffer D, Zweier JL, Sen CK - PLoS ONE (2013)

miR-15b over expression in HL-1 cells compromises mitochondrial membrane potential.(A,B) HL-1 cells were transfected (72 hours) with mimic negative control or miRNA-15b mimic. Untransfected cells were used as negative control and cells transfected with carbonyl cyanide m-chlorophenylhydrazone (CCCP) were used as positive control (left panel –8 nM tetramethylrhodamine methyl ester (TMRM); center panel - 0.5 µl/ml plasma membrane potential indicator (PMPI); right panel - merged image). Bar graph shows significant decrease in TMRM with no change in PMPI. Solid bars represent mimic negative control while open bars represent miRNA-15b mimic. (C,D) Loss of mitochondrial membrane potential in HL-1 cells transfected with miRNA-15b mimic, as assessed by JC-1 flow cytometry 72 h post-transfection. Cells were transfected with a (i) mimic control, (ii) miRNA-15b mimic or (iii) treated with CCCP Arrows (K gate) indicate cells containing JC-1 aggregates resulting from intact mitochondria; M gate indicates cells with low or collapsed mitochondrial membrane potential. Ratio of polarized to depolarized cells calculated as a ratio between cells in K gate to M gate. Data indicate decrease in membrane potential upon up-regulation of miRNA-15b. (n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3686742&req=5

pone-0066789-g005: miR-15b over expression in HL-1 cells compromises mitochondrial membrane potential.(A,B) HL-1 cells were transfected (72 hours) with mimic negative control or miRNA-15b mimic. Untransfected cells were used as negative control and cells transfected with carbonyl cyanide m-chlorophenylhydrazone (CCCP) were used as positive control (left panel –8 nM tetramethylrhodamine methyl ester (TMRM); center panel - 0.5 µl/ml plasma membrane potential indicator (PMPI); right panel - merged image). Bar graph shows significant decrease in TMRM with no change in PMPI. Solid bars represent mimic negative control while open bars represent miRNA-15b mimic. (C,D) Loss of mitochondrial membrane potential in HL-1 cells transfected with miRNA-15b mimic, as assessed by JC-1 flow cytometry 72 h post-transfection. Cells were transfected with a (i) mimic control, (ii) miRNA-15b mimic or (iii) treated with CCCP Arrows (K gate) indicate cells containing JC-1 aggregates resulting from intact mitochondria; M gate indicates cells with low or collapsed mitochondrial membrane potential. Ratio of polarized to depolarized cells calculated as a ratio between cells in K gate to M gate. Data indicate decrease in membrane potential upon up-regulation of miRNA-15b. (n = 3).
Mentions: We screened for miRNAs targeting mitochondrial function among the significantly differentially expressed miRNAs, using publicly available prediction softwares like TargetScan™ v 5.1 and Pictar™. In our search, elevated miRNA-15b emerged as a key candidate as it was computationally predicted to silence some key proteins like Pim-1 kinase, ESRRG, Mfn-2 which are required for preserving proper mitochondrial function and integrity. We used laser capture microdissection [22], [42], [43], [44] to confirm that miR-15b was indeed elevated in cardiomyocytes. We recognize that the other differentially expressed miRs may also play a significant role in development of the pathology in Dicer deleted hearts and needs to be investigated as a future work. Our study was directed to Pim-1 kinase, silencing of which is known to directly cause cardiac dysfunction. Pim-1 is a crucial requirement for downstream cardio protective effects of Akt signaling [45]. Increase in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload does not protect the myocardium in Pim-1-deficient mice [45]. Loss of Pim-1 also prevents the long term structural and functional benefits obtained from cardiac progenitor cell based therapies indicating its importance in myocardial repair and regeneration [46], [47]. Transgenic over-expression of Pim-1 in the myocardium protects mice against infarction [45]. Specifically, Pim-1 kinase is required for preserving mitochondrial integrity in cardiomyocytes [48], and also blunts cardiac hypertrophy [49]. Pim-1 kinase over-expression also reduces calcium mediated mitochondrial swelling, t-bid-induced cytochrome-C release and peroxide-induced membrane depolarization [50]. Pim-1 inhibits apoptosis-related mitochondrial dysfunction [51] and Pim-1 inhibition results in apoptosis and is closely related to the decrease in Akt and Bad phosphorylation and increase in cleaved caspase-3/−9 activities [52]. Delivery of miR-15b to HL-1 cells using a mimic compromised mitochondrial membrane potential (TMRM fluorescence) similar to what was observed upon Dicer knockdown (Fig. 5A,B). Mitochondrial membrane potential changes were also assessed using the JC-1 assay which demonstrated that over expression of miRNA-15b in HL-1 cells compromised ΔΨ (Fig. 5C,D). Pim-1 kinase protein level was observed to be low in heart tissue isolated from Dicer−/− mice by western blot (Fig. 6A, p<0.05, n = 6) and immunohistochemistry (Fig. 6B). HL-1 cells transfected with si-Dicer showed lower Pim-1 protein expression. This effect was ameliorated when cells were co-transfected with a miRNA-15b inhibitor pointing towards a direct role of miRNA-15b in silencing Pim-1 (Fig. 6C, p<0.05, n = 3). In order to validate whether miRNA-15b silences Pim-1 kinase, HL-1 cells were transfected with miRNA-15b mimic and lowered Pim-1 kinase protein levels was observed (Fig. 6D; p<0.05, n = 6). To validate the direct binding between miRNA-15b and the Pim-1 3′ UTR region, miRNA target reporter luciferase assay was performed using the pLuc-Pim1–3′UTR plasmid (Fig. 6E) in HL-1 cells. 40% reduction of luciferase activity from the pLuc-Pim1–3′UTR plasmid was observed in miRNA-15b mimic transfected HL-1 cells compared with control (Fig. 6F). No reduction in luciferase activity was observed using a mutated pLuc-Pim1-mutated 3′UTR (Fig. 6G). Thus, elevated levels of miRNA-15b silence Pim-1 kinase which may be causatively linked to the observed mitochondrial dysfunction in Dicer deleted heart of adult mice.

Bottom Line: Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function.Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy.Anti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.

View Article: PubMed Central - PubMed

Affiliation: Davis Heart and Lung Research Institute and Department of Surgery, The Ohio State University Medical Center, Columbus, Ohio, United States of America.

ABSTRACT

Background: Dicer endonuclease, critical for maturation of miRNAs, is depleted in certain forms of cardiomyopathy which results in differential expression of certain microRNAs. We sought to elucidate the mechanisms underlying the rapid loss of cardiac function following cardiac-specific Dicer depletion in adult mice.

Results: Conditional Dicer deletion in the adult murine myocardium demonstrated compromised heart function, mitochondrial dysfunction and oxidant stress. Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function. Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy.

Conclusions: Anti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.

No MeSH data available.


Related in: MedlinePlus