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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

Dicer deletion in the adult heart leads to oxidative stress.(A) Representative EPR imaging of nitroxide radical decay in (A) Dicer+/+ and (B) Dicer−/− hearts. (B) Time course of average % signal change in the region of interest (ROI). Logarithmic values of signal change (normalized to the initial signal at time = 0) in the ROIs are plotted with respect to time. Decay rate constants were obtained from the slope of linear decay after peak. Line with black circles represent Dicer+/+ and line with grey circles represent Dicer−/−. (C) Bar-graph showing the measured rate constants of nitroxide reduction in the tissues. (D) Thiobarbituric acid–reactive substances (TBARS), an indicator of lipid peroxidation was measured from Dicer+/+ and Dicer−/− hearts and was significantly higher in the later. (E) Total GSSG to GSH ratio in Dicer+/+ mice heart compared to Dicer−/− heart. (F) Lactate levels measured in Dicer+/+ and Dicer−/− mice hearts.
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pone-0066789-g002: Dicer deletion in the adult heart leads to oxidative stress.(A) Representative EPR imaging of nitroxide radical decay in (A) Dicer+/+ and (B) Dicer−/− hearts. (B) Time course of average % signal change in the region of interest (ROI). Logarithmic values of signal change (normalized to the initial signal at time = 0) in the ROIs are plotted with respect to time. Decay rate constants were obtained from the slope of linear decay after peak. Line with black circles represent Dicer+/+ and line with grey circles represent Dicer−/−. (C) Bar-graph showing the measured rate constants of nitroxide reduction in the tissues. (D) Thiobarbituric acid–reactive substances (TBARS), an indicator of lipid peroxidation was measured from Dicer+/+ and Dicer−/− hearts and was significantly higher in the later. (E) Total GSSG to GSH ratio in Dicer+/+ mice heart compared to Dicer−/− heart. (F) Lactate levels measured in Dicer+/+ and Dicer−/− mice hearts.

Mentions: Hypertrophy and cardiac failure has often been associated with mitochondrial dysfunction and oxidative stress [26], [27], [28]. Mitochondrial energy deficiency is known to underlie dilated cardiomyopathy characterized by early mechanical dysfunction followed by a decline in left ventricular systolic function [29]. We therefore wanted to determine the redox and mitochondrial health of the Dicer depleted hearts. Redox state of the Dicer deleted adult heart was determined by EPR spectroscopy. After intramuscular injection of 2, 2, 5, 5,-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid (PCA) to the heart, decay of nitroxyl radicals was studied. A faster decay in Dicer deleted hearts was seen that has been considered indicative of a higher abundance of reactive oxygen species [9] (Fig. 2A–C, p<0.05, n = 3). This observation was consistent with elevated lipid peroxidation (Fig. 2D; p<0.05, n = 3) and glutathione oxidation (Fig. 2E; p<0.05, n = 4), indices of oxidative stress, in the hearts of Dicer−/− mice. Consistent with this observation demonstrating impairment in oxidative metabolism it was noted that lactate levels in the heart of Dicer−/− mice were significantly higher (Fig. 2F; p<0.05, n = 4). Overt oxidative stress in Dicer-deficient hearts evident in this study pointed towards underlying mitochondrial dysfunction [28], [30]. In order to further elucidate the cause and effect relationship, we used beating HL-1 cells which have been demonstrated be physiologically and functionally similar to adult cardiomyocytes [15], [31]. Knocking down Dicer in HL-1 cells using a siRNA compromised mitochondrial membrane potential (determined by TMRM fluorescence) (Fig. 3A). To further confirm the in-vitro findings, we performed in-vivo TMRM staining on Dicer+/+ and Dicer−/− heart sections and observed similar results (Fig. 3B). We analyzed mitochondrial structure by transmission electron microscope and observed a loss of mitochondrial structural integrity in cardiac tissue from Dicer−/− mice (Fig. 3C). Indeed, Dicer deficient cardiac tissue showed evidence of matrix swelling represented by greater inter-cristae space and sometimes unfolded cristae localized at one pole of the organelle as compared to intact cristae uniformly distributed across the organelle in Dicer+/+ tissue. This contention was supported by analysis of respiration of isolated heart mitochondria which led to the observation that the respiratory control ratio (RCR) was significantly decreased (p<0.05, n = 5) in cardiac mitochondria from Dicer−/− mice compared with Dicer+/+ animals (Fig. 3D–E). Taken together, these findings demonstrate that Dicer deletion induced loss of cardiac function of the adult heart is associated with mitochondrial dysfunction and oxidative stress.


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)

Dicer deletion in the adult heart leads to oxidative stress.(A) Representative EPR imaging of nitroxide radical decay in (A) Dicer+/+ and (B) Dicer−/− hearts. (B) Time course of average % signal change in the region of interest (ROI). Logarithmic values of signal change (normalized to the initial signal at time = 0) in the ROIs are plotted with respect to time. Decay rate constants were obtained from the slope of linear decay after peak. Line with black circles represent Dicer+/+ and line with grey circles represent Dicer−/−. (C) Bar-graph showing the measured rate constants of nitroxide reduction in the tissues. (D) Thiobarbituric acid–reactive substances (TBARS), an indicator of lipid peroxidation was measured from Dicer+/+ and Dicer−/− hearts and was significantly higher in the later. (E) Total GSSG to GSH ratio in Dicer+/+ mice heart compared to Dicer−/− heart. (F) Lactate levels measured in Dicer+/+ and Dicer−/− mice hearts.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3686742&req=5

pone-0066789-g002: Dicer deletion in the adult heart leads to oxidative stress.(A) Representative EPR imaging of nitroxide radical decay in (A) Dicer+/+ and (B) Dicer−/− hearts. (B) Time course of average % signal change in the region of interest (ROI). Logarithmic values of signal change (normalized to the initial signal at time = 0) in the ROIs are plotted with respect to time. Decay rate constants were obtained from the slope of linear decay after peak. Line with black circles represent Dicer+/+ and line with grey circles represent Dicer−/−. (C) Bar-graph showing the measured rate constants of nitroxide reduction in the tissues. (D) Thiobarbituric acid–reactive substances (TBARS), an indicator of lipid peroxidation was measured from Dicer+/+ and Dicer−/− hearts and was significantly higher in the later. (E) Total GSSG to GSH ratio in Dicer+/+ mice heart compared to Dicer−/− heart. (F) Lactate levels measured in Dicer+/+ and Dicer−/− mice hearts.
Mentions: Hypertrophy and cardiac failure has often been associated with mitochondrial dysfunction and oxidative stress [26], [27], [28]. Mitochondrial energy deficiency is known to underlie dilated cardiomyopathy characterized by early mechanical dysfunction followed by a decline in left ventricular systolic function [29]. We therefore wanted to determine the redox and mitochondrial health of the Dicer depleted hearts. Redox state of the Dicer deleted adult heart was determined by EPR spectroscopy. After intramuscular injection of 2, 2, 5, 5,-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid (PCA) to the heart, decay of nitroxyl radicals was studied. A faster decay in Dicer deleted hearts was seen that has been considered indicative of a higher abundance of reactive oxygen species [9] (Fig. 2A–C, p<0.05, n = 3). This observation was consistent with elevated lipid peroxidation (Fig. 2D; p<0.05, n = 3) and glutathione oxidation (Fig. 2E; p<0.05, n = 4), indices of oxidative stress, in the hearts of Dicer−/− mice. Consistent with this observation demonstrating impairment in oxidative metabolism it was noted that lactate levels in the heart of Dicer−/− mice were significantly higher (Fig. 2F; p<0.05, n = 4). Overt oxidative stress in Dicer-deficient hearts evident in this study pointed towards underlying mitochondrial dysfunction [28], [30]. In order to further elucidate the cause and effect relationship, we used beating HL-1 cells which have been demonstrated be physiologically and functionally similar to adult cardiomyocytes [15], [31]. Knocking down Dicer in HL-1 cells using a siRNA compromised mitochondrial membrane potential (determined by TMRM fluorescence) (Fig. 3A). To further confirm the in-vitro findings, we performed in-vivo TMRM staining on Dicer+/+ and Dicer−/− heart sections and observed similar results (Fig. 3B). We analyzed mitochondrial structure by transmission electron microscope and observed a loss of mitochondrial structural integrity in cardiac tissue from Dicer−/− mice (Fig. 3C). Indeed, Dicer deficient cardiac tissue showed evidence of matrix swelling represented by greater inter-cristae space and sometimes unfolded cristae localized at one pole of the organelle as compared to intact cristae uniformly distributed across the organelle in Dicer+/+ tissue. This contention was supported by analysis of respiration of isolated heart mitochondria which led to the observation that the respiratory control ratio (RCR) was significantly decreased (p<0.05, n = 5) in cardiac mitochondria from Dicer−/− mice compared with Dicer+/+ animals (Fig. 3D–E). Taken together, these findings demonstrate that Dicer deletion induced loss of cardiac function of the adult heart is associated with mitochondrial dysfunction and oxidative stress.

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