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Executioner Caspase-3 and 7 Deficiency Reduces Myocyte Number in the Developing Mouse Heart.

Cardona M, López JA, Serafín A, Rongvaux A, Inserte J, García-Dorado D, Flavell R, Llovera M, Cañas X, Vázquez J, Sanchis D - PLoS ONE (2015)

Bottom Line: Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult myocardium.Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase- myocardium.The results reveal that executioner caspases can modulate heart's cellularity and maturation during development, contributing novel information about caspase biology and heart development.

View Article: PubMed Central - PubMed

Affiliation: Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida-IRBLLEIDA, Av. Rovira Roure, 80, Lleida, 25198, Spain.

ABSTRACT
Executioner caspase-3 and -7 are proteases promoting cell death but non-apoptotic roles are being discovered. The heart expresses caspases only during development, suggesting they contribute to the organ maturation process. Therefore, we aimed at identifying novel functions of caspases in heart development. We induced simultaneous deletion of executioner caspase-3 and -7 in the mouse myocardium and studied its effects. Caspase knockout hearts are hypoplastic at birth, reaching normal weight progressively through myocyte hypertrophy. To identify the molecular pathways involved in these effects, we used microarray-based transcriptomics and multiplexed quantitative proteomics to compare wild type and executioner caspase-deficient myocardium at different developmental stages. Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult myocardium. Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase- myocardium. Correlation between mRNA expression and protein abundance of relevant genes was confirmed, but transcriptomics and proteomics indentified complementary molecular pathways influenced by caspases in the developing heart. Forced expression of wild type or proteolytically inactive caspases in cultured cardiomyocytes induced expression of genes promoting cell division. The results reveal that executioner caspases can modulate heart's cellularity and maturation during development, contributing novel information about caspase biology and heart development.

No MeSH data available.


Related in: MedlinePlus

Executioner caspase deficiency in the developing myocardium triggers changes in gene expression in accordance to reduced myocyte proliferation.(A) Clustering of differentially expressed genes between wild type and caspase-3 and -7 double knockout mice by biological processes. Ploted is the–log of P values adjusted by the Benjamini and Hochberg method. Highlighted is the process most significantly affected in newborns and young mice. (B) Representation of top up-regulated genes (towards the red region of the bar) or down-regulated genes (towards the blue region of the bar) in newborn and young knockout mice, colored by molecular function. (C) Reverse-transcription/qPCR was performed to validate the microarray results for the expression of a set of genes downregulated (blue) or upregulated (red) in caspase KO hearts. N: newborn, Y: young. *, p<0.05, Student’s-t test KO vs. WT for the same age. n = 5–7 cardiac RNA extracts per age and genotype. (D) Changes of protein abundance for a set of the differentially expressed genes (selected by availability of antibodies with confirmed specificity). Upper panel: representative Western Blots (WB) featuring two WT and two KO independent cardiac protein extracts per age. Graphs: Densitometric analysis of WB. AU = Arbitrary units. *, p<0.05 KO vs. WT (Student’s-test) comparing the same age (N: newborn, Y: young). N = 6 per age and genotype. Mef2a expression is used as a control of unaffected gene and GAPDH is used as loading control. Bars are means ± s.e.m.
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pone.0131411.g002: Executioner caspase deficiency in the developing myocardium triggers changes in gene expression in accordance to reduced myocyte proliferation.(A) Clustering of differentially expressed genes between wild type and caspase-3 and -7 double knockout mice by biological processes. Ploted is the–log of P values adjusted by the Benjamini and Hochberg method. Highlighted is the process most significantly affected in newborns and young mice. (B) Representation of top up-regulated genes (towards the red region of the bar) or down-regulated genes (towards the blue region of the bar) in newborn and young knockout mice, colored by molecular function. (C) Reverse-transcription/qPCR was performed to validate the microarray results for the expression of a set of genes downregulated (blue) or upregulated (red) in caspase KO hearts. N: newborn, Y: young. *, p<0.05, Student’s-t test KO vs. WT for the same age. n = 5–7 cardiac RNA extracts per age and genotype. (D) Changes of protein abundance for a set of the differentially expressed genes (selected by availability of antibodies with confirmed specificity). Upper panel: representative Western Blots (WB) featuring two WT and two KO independent cardiac protein extracts per age. Graphs: Densitometric analysis of WB. AU = Arbitrary units. *, p<0.05 KO vs. WT (Student’s-test) comparing the same age (N: newborn, Y: young). N = 6 per age and genotype. Mef2a expression is used as a control of unaffected gene and GAPDH is used as loading control. Bars are means ± s.e.m.

Mentions: To determine the molecular changes underlying reduced cardiomyocyte number in the absence of executioner caspase expression, we performed a microarray-based gene expression analysis comparing wild type and caspase-3 and -7 double knockout hearts in newborns, an age in which wild type hearts still express caspases and in thirty-day-old mice, an age in which wild type hearts have downregulated caspase signaling and myocytes do not further divide). Gene functional categories most significantly affected in newborns by the lack of executioner caspases were those regulating DNA replication and cell cycle, whereas in young mice the most affected genes were those involved in tissue development (Fig 2A). Detailed inspection of transcription changes showed that newborn caspase-deficient myocardium transcribed abnormally low levels of genes coding for key proteins involved in DNA replication, recombination and repair (e.g. pold1 coding for DNA polymerase subunit delta, topbp1 coding for DNA topoisomerase-2 binding protein-1; BRCA1, PCNA, etc.), centromere and kinetochore formation (e.g. CENP genes, INCEMP, auroraB, etc.) and cell cycle regulation (e.g. cell division cycle-6, ccne1 coding for cyclin-E), as well as some genes involved in metabolism (e.g. slc2a4 coding for GLUT4, and hif3a, among others) (Fig 2B). In young hearts, lack of executioner caspases during development resulted in abnormally high expression of genes involved in cell cycle inhibition (such as fam107a, which was also increased in newborn knockouts), genes coding for non-cardiac isoforms of sarcomeric proteins (myosin heavy chain-11 and tropomyosin-2), as well as genes involved in embryonic development (hand2, gata5) and also resulted in slightly reduced expression of genes coding for components of the contraction machinery (e.g. titin, dystrophin, obscurin) (Fig 2B, Figure C in S3 File). Gene expression changes detected by the microarray study were confirmed using specific quantitative PCR (qPCR) assays for a set of key genes involved in the above mentioned functions (Fig 2C). Of note, the mRNA of natriuretic peptide A (nppa), which is a marker of myocyte hypertrophy [29], was highly expressed in caspase-3 and -7 double mutant hearts and the opposite effect was observed for ndrg4, which is involved in cardiomyocyte proliferation [30]. Western blotting analysis revealed that changes in expression of selected proteins were coherent with the alterations observed in mRNA abundance related to age and genotype (Fig 2D). The expression changes observed in double mutant hearts were present yet modest in caspase-3 deficient hearts, while lack of caspase-7 did not affect the expression of any gene checked (Figure D in S3 File). Gene expression was also unaffected by Cre expression (Figure D in S3 File), discarding that changes observed were due to expression of the recombinase used to interrupt caspase-3. Taken together, the results obtained by the microarray-based gene expression study show that lack of caspase-3 and -7 in the developing myocardium deeply modifies the expression of genes involved in DNA replication, recombination and repair, chromatin organization and cell cycle progression in accordance with reduced cell proliferation. The results also show increased expression of non-muscular and smooth muscle structural gene isoforms in hearts of young caspase knockouts, in accordance with the gene expression changes occurring during hypertrophy previously described elsewhere [31].


Executioner Caspase-3 and 7 Deficiency Reduces Myocyte Number in the Developing Mouse Heart.

Cardona M, López JA, Serafín A, Rongvaux A, Inserte J, García-Dorado D, Flavell R, Llovera M, Cañas X, Vázquez J, Sanchis D - PLoS ONE (2015)

Executioner caspase deficiency in the developing myocardium triggers changes in gene expression in accordance to reduced myocyte proliferation.(A) Clustering of differentially expressed genes between wild type and caspase-3 and -7 double knockout mice by biological processes. Ploted is the–log of P values adjusted by the Benjamini and Hochberg method. Highlighted is the process most significantly affected in newborns and young mice. (B) Representation of top up-regulated genes (towards the red region of the bar) or down-regulated genes (towards the blue region of the bar) in newborn and young knockout mice, colored by molecular function. (C) Reverse-transcription/qPCR was performed to validate the microarray results for the expression of a set of genes downregulated (blue) or upregulated (red) in caspase KO hearts. N: newborn, Y: young. *, p<0.05, Student’s-t test KO vs. WT for the same age. n = 5–7 cardiac RNA extracts per age and genotype. (D) Changes of protein abundance for a set of the differentially expressed genes (selected by availability of antibodies with confirmed specificity). Upper panel: representative Western Blots (WB) featuring two WT and two KO independent cardiac protein extracts per age. Graphs: Densitometric analysis of WB. AU = Arbitrary units. *, p<0.05 KO vs. WT (Student’s-test) comparing the same age (N: newborn, Y: young). N = 6 per age and genotype. Mef2a expression is used as a control of unaffected gene and GAPDH is used as loading control. Bars are means ± s.e.m.
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pone.0131411.g002: Executioner caspase deficiency in the developing myocardium triggers changes in gene expression in accordance to reduced myocyte proliferation.(A) Clustering of differentially expressed genes between wild type and caspase-3 and -7 double knockout mice by biological processes. Ploted is the–log of P values adjusted by the Benjamini and Hochberg method. Highlighted is the process most significantly affected in newborns and young mice. (B) Representation of top up-regulated genes (towards the red region of the bar) or down-regulated genes (towards the blue region of the bar) in newborn and young knockout mice, colored by molecular function. (C) Reverse-transcription/qPCR was performed to validate the microarray results for the expression of a set of genes downregulated (blue) or upregulated (red) in caspase KO hearts. N: newborn, Y: young. *, p<0.05, Student’s-t test KO vs. WT for the same age. n = 5–7 cardiac RNA extracts per age and genotype. (D) Changes of protein abundance for a set of the differentially expressed genes (selected by availability of antibodies with confirmed specificity). Upper panel: representative Western Blots (WB) featuring two WT and two KO independent cardiac protein extracts per age. Graphs: Densitometric analysis of WB. AU = Arbitrary units. *, p<0.05 KO vs. WT (Student’s-test) comparing the same age (N: newborn, Y: young). N = 6 per age and genotype. Mef2a expression is used as a control of unaffected gene and GAPDH is used as loading control. Bars are means ± s.e.m.
Mentions: To determine the molecular changes underlying reduced cardiomyocyte number in the absence of executioner caspase expression, we performed a microarray-based gene expression analysis comparing wild type and caspase-3 and -7 double knockout hearts in newborns, an age in which wild type hearts still express caspases and in thirty-day-old mice, an age in which wild type hearts have downregulated caspase signaling and myocytes do not further divide). Gene functional categories most significantly affected in newborns by the lack of executioner caspases were those regulating DNA replication and cell cycle, whereas in young mice the most affected genes were those involved in tissue development (Fig 2A). Detailed inspection of transcription changes showed that newborn caspase-deficient myocardium transcribed abnormally low levels of genes coding for key proteins involved in DNA replication, recombination and repair (e.g. pold1 coding for DNA polymerase subunit delta, topbp1 coding for DNA topoisomerase-2 binding protein-1; BRCA1, PCNA, etc.), centromere and kinetochore formation (e.g. CENP genes, INCEMP, auroraB, etc.) and cell cycle regulation (e.g. cell division cycle-6, ccne1 coding for cyclin-E), as well as some genes involved in metabolism (e.g. slc2a4 coding for GLUT4, and hif3a, among others) (Fig 2B). In young hearts, lack of executioner caspases during development resulted in abnormally high expression of genes involved in cell cycle inhibition (such as fam107a, which was also increased in newborn knockouts), genes coding for non-cardiac isoforms of sarcomeric proteins (myosin heavy chain-11 and tropomyosin-2), as well as genes involved in embryonic development (hand2, gata5) and also resulted in slightly reduced expression of genes coding for components of the contraction machinery (e.g. titin, dystrophin, obscurin) (Fig 2B, Figure C in S3 File). Gene expression changes detected by the microarray study were confirmed using specific quantitative PCR (qPCR) assays for a set of key genes involved in the above mentioned functions (Fig 2C). Of note, the mRNA of natriuretic peptide A (nppa), which is a marker of myocyte hypertrophy [29], was highly expressed in caspase-3 and -7 double mutant hearts and the opposite effect was observed for ndrg4, which is involved in cardiomyocyte proliferation [30]. Western blotting analysis revealed that changes in expression of selected proteins were coherent with the alterations observed in mRNA abundance related to age and genotype (Fig 2D). The expression changes observed in double mutant hearts were present yet modest in caspase-3 deficient hearts, while lack of caspase-7 did not affect the expression of any gene checked (Figure D in S3 File). Gene expression was also unaffected by Cre expression (Figure D in S3 File), discarding that changes observed were due to expression of the recombinase used to interrupt caspase-3. Taken together, the results obtained by the microarray-based gene expression study show that lack of caspase-3 and -7 in the developing myocardium deeply modifies the expression of genes involved in DNA replication, recombination and repair, chromatin organization and cell cycle progression in accordance with reduced cell proliferation. The results also show increased expression of non-muscular and smooth muscle structural gene isoforms in hearts of young caspase knockouts, in accordance with the gene expression changes occurring during hypertrophy previously described elsewhere [31].

Bottom Line: Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult myocardium.Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase- myocardium.The results reveal that executioner caspases can modulate heart's cellularity and maturation during development, contributing novel information about caspase biology and heart development.

View Article: PubMed Central - PubMed

Affiliation: Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida-IRBLLEIDA, Av. Rovira Roure, 80, Lleida, 25198, Spain.

ABSTRACT
Executioner caspase-3 and -7 are proteases promoting cell death but non-apoptotic roles are being discovered. The heart expresses caspases only during development, suggesting they contribute to the organ maturation process. Therefore, we aimed at identifying novel functions of caspases in heart development. We induced simultaneous deletion of executioner caspase-3 and -7 in the mouse myocardium and studied its effects. Caspase knockout hearts are hypoplastic at birth, reaching normal weight progressively through myocyte hypertrophy. To identify the molecular pathways involved in these effects, we used microarray-based transcriptomics and multiplexed quantitative proteomics to compare wild type and executioner caspase-deficient myocardium at different developmental stages. Transcriptomics showed reduced expression of genes promoting DNA replication and cell cycle progression in the neonatal caspase-deficient heart suggesting reduced myocyte proliferation, and expression of non-cardiac isoforms of structural proteins in the adult myocardium. Proteomics showed reduced abundance of proteins involved in oxidative phosphorylation accompanied by increased abundance of glycolytic enzymes underscoring retarded metabolic maturation of the caspase- myocardium. Correlation between mRNA expression and protein abundance of relevant genes was confirmed, but transcriptomics and proteomics indentified complementary molecular pathways influenced by caspases in the developing heart. Forced expression of wild type or proteolytically inactive caspases in cultured cardiomyocytes induced expression of genes promoting cell division. The results reveal that executioner caspases can modulate heart's cellularity and maturation during development, contributing novel information about caspase biology and heart development.

No MeSH data available.


Related in: MedlinePlus