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Cardioprotective Signature of Short-Term Caloric Restriction.

Noyan H, El-Mounayri O, Isserlin R, Arab S, Momen A, Cheng HS, Wu J, Afroze T, Li RK, Fish JE, Bader GD, Husain M - PLoS ONE (2015)

Bottom Line: This short-term model of CR was associated with cardio-protection, as evidenced by decreased infarct size (18.5±2.4% vs. 26.6±1.7%, N=10/group; P=0.01). mRNA and miR profiles pre-MI (N=5/group) identified genes modulated by short-term CR to be associated with circadian clock, oxidative stress, immune function, apoptosis, metabolism, angiogenesis, cytoskeleton and extracellular matrix (ECM).Western blots pre-MI revealed CR-associated increases in phosphorylated Akt and GSK3ß, reduced levels of phosphorylated AMPK and mitochondrial related proteins PGC-1α, cytochrome C and cyclooxygenase (COX) IV, with no differences in the levels of phosphorylated eNOS or MAPK (ERK1/2; p38).CR regimen was also associated with reduced protein abundance of cleaved caspase 3 in the infarcted heart and improved cardiac function.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Therapeutics, Toronto General Research Institute, Toronto, Ontario, Canada.

ABSTRACT

Objective: To understand the molecular pathways underlying the cardiac preconditioning effect of short-term caloric restriction (CR).

Background: Lifelong CR has been suggested to reduce the incidence of cardiovascular disease through a variety of mechanisms. However, prolonged adherence to a CR life-style is difficult. Here we reveal the pathways that are modulated by short-term CR, which are associated with protection of the mouse heart from ischemia.

Methods: Male 10-12 wk old C57bl/6 mice were randomly assigned to an ad libitum (AL) diet with free access to regular chow, or CR, receiving 30% less food for 7 days (d), prior to myocardial infarction (MI) via permanent coronary ligation. At d8, the left ventricles (LV) of AL and CR mice were collected for Western blot, mRNA and microRNA (miR) analyses to identify cardioprotective gene expression signatures. In separate groups, infarct size, cardiac hemodynamics and protein abundance of caspase 3 was measured at d2 post-MI.

Results: This short-term model of CR was associated with cardio-protection, as evidenced by decreased infarct size (18.5±2.4% vs. 26.6±1.7%, N=10/group; P=0.01). mRNA and miR profiles pre-MI (N=5/group) identified genes modulated by short-term CR to be associated with circadian clock, oxidative stress, immune function, apoptosis, metabolism, angiogenesis, cytoskeleton and extracellular matrix (ECM). Western blots pre-MI revealed CR-associated increases in phosphorylated Akt and GSK3ß, reduced levels of phosphorylated AMPK and mitochondrial related proteins PGC-1α, cytochrome C and cyclooxygenase (COX) IV, with no differences in the levels of phosphorylated eNOS or MAPK (ERK1/2; p38). CR regimen was also associated with reduced protein abundance of cleaved caspase 3 in the infarcted heart and improved cardiac function.

No MeSH data available.


Related in: MedlinePlus

Short-term CR is associated with a complex miR expression profile.(A) qPCR validation of selected miRs from the microRNA array showing significant down-regulation of miR-21 and miR-92a and significant up-regulation of miR-27, miR-29, miR-208 and miR-214 in CR compared to Ad lib. miR-24 and miR-486 were used as negative controls. miR levels were normalized to U6. (B & C) Enrichment Map of CR vs Ad lib. Nodes represent enriched pathways and edges the similarity between those pathways calculated based on an overlap statistic > 0.5. Enrichments were filtered by p-value < 0.01, FDR < 0.05. The enrichment map was further annotated using a post analysis using mir-29 and mir-27 gene sets to highlight enriched pathways (blue or red nodes) that contain a statistically significant amount of B) miR-27 or C) mir-29 targets (as calculated using the hypergeometric distribution, p-value < 0.05). Post analysis edges are colored in pink, and enrichment map edges are colored in green.
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pone.0130658.g006: Short-term CR is associated with a complex miR expression profile.(A) qPCR validation of selected miRs from the microRNA array showing significant down-regulation of miR-21 and miR-92a and significant up-regulation of miR-27, miR-29, miR-208 and miR-214 in CR compared to Ad lib. miR-24 and miR-486 were used as negative controls. miR levels were normalized to U6. (B & C) Enrichment Map of CR vs Ad lib. Nodes represent enriched pathways and edges the similarity between those pathways calculated based on an overlap statistic > 0.5. Enrichments were filtered by p-value < 0.01, FDR < 0.05. The enrichment map was further annotated using a post analysis using mir-29 and mir-27 gene sets to highlight enriched pathways (blue or red nodes) that contain a statistically significant amount of B) miR-27 or C) mir-29 targets (as calculated using the hypergeometric distribution, p-value < 0.05). Post analysis edges are colored in pink, and enrichment map edges are colored in green.

Mentions: To determine whether the 7d of CR used in our study is accompanied by differential regulation of miR, qPCR arrays were used to determine the expression of 709 distinct miRs in the LV of CR and AL mice. miR array analysis revealed 18 miR that were up-regulated and 24 miR that were down-regulated by CR (Table 1). To validate findings from the miR array, 6 miRs (miR-21, -92a, -27, -29, -208 and -214) were tested by qPCR using SYBR Green or Taqman systems. As shown in Fig 6A, the levels of miR-21 and miR-92a were down-regulated in CR as compared to AL mice (-1.085±0.2465, P<0.01; and -1.798±0.6679, P<0.02; fold change respectively, N = 3). In addition, miR-27, miR-29, miR-208 and miR-214 were significantly up-regulated in CR as compared to AL groups (+2.969±0.5318, P<0.05; +7.483±1.084, P<0.002; +2.483±0.9468, P<0.009; and +2.003±0.5865, P<0.02; fold change respectively, N = 3) (Fig 6A). Our qPCR results demonstrated no change in the level of miR-24 and miR-486 in CR vs. AL groups (N = 3), again confirming results of the miR-array (Fig 6A). Together, these data indicate a strong correlation between miR-array and qPCR. We then annotated the enrichment map with the predicted targets of two miR with the highest fold change in CR (miR-27 and -29) to highlight gene sets that are significantly modulated (hyper-geometric P<0.05) as a percentage of miR targets (Fig 6B and 6C). Both miR-27 and miR-29 showed overlapping targets in pathways associated with ECM organization, angiogenesis, cell migration, adhesion and cytoskeletal organization. We also validated by qPCR the expression levels of known targets of three miR (miR-27, -29 and -214), which were up-regulated in the CR group. Of note, miR-214, which has previously been shown to reduce Ca2+ overload-induced cardiomyocyte death in an ischemia-reperfusion injury model [33], was up-regulated in CR mice. Up- and down-regulation of miR in the present study correlated well with mRNA levels of their known and predicted gene targets as demonstrated by microarray analysis and qRT-PCR results (Fig. B in S2 File). For example, down-regulation of several distinct miR (-34a, -199a, -181a/b and -204), which have been shown to target Sirt1 [34, 35] was associated with up-regulation of the mRNA level of Sirt1 in CR (Fig. A in S2 File). Similarly, while miR-133a and miR-181 were down-regulated, the mRNA level of their target, pro-survival gene Mcl1 [36, 37] was up-regulated in the CR heart (Fig. A in S2 File). On the other hand, up-regulation of miR-214 in the CR group was associated with down-regulation of mRNA levels of its known target genes, Ncx1 and Camk2d (P< 0.01 and P<0.02 respectively) in the hearts of CR as compared to AL mice (Panel B in Fig. B in S2 File). In addition, mRNA levels of Col1a1, Mmp2, and Itg6, known targets of miR-29 (Up-regulated in the CR mice) [38], were also down-regulated (P<0.001, P<0.003 and P<0.0001 respectively) in CR hearts (Panel B in Fig. B in S2 File). Also, mRNA levels of the tumor suppressor gene Fbxw7, a known target of miR-27 [39], was down-regulated (P<0.01) in the hearts of CR vs. AL mice (Panel B in Fig. B in S2 File). These results show, for the first time, that short-term CR for only 7d mediates a cardioprotective gene profile that includes specific miR and their downstream targets. For further details, a large listing of genes regulated by CR is provided in Table C in S1 File.


Cardioprotective Signature of Short-Term Caloric Restriction.

Noyan H, El-Mounayri O, Isserlin R, Arab S, Momen A, Cheng HS, Wu J, Afroze T, Li RK, Fish JE, Bader GD, Husain M - PLoS ONE (2015)

Short-term CR is associated with a complex miR expression profile.(A) qPCR validation of selected miRs from the microRNA array showing significant down-regulation of miR-21 and miR-92a and significant up-regulation of miR-27, miR-29, miR-208 and miR-214 in CR compared to Ad lib. miR-24 and miR-486 were used as negative controls. miR levels were normalized to U6. (B & C) Enrichment Map of CR vs Ad lib. Nodes represent enriched pathways and edges the similarity between those pathways calculated based on an overlap statistic > 0.5. Enrichments were filtered by p-value < 0.01, FDR < 0.05. The enrichment map was further annotated using a post analysis using mir-29 and mir-27 gene sets to highlight enriched pathways (blue or red nodes) that contain a statistically significant amount of B) miR-27 or C) mir-29 targets (as calculated using the hypergeometric distribution, p-value < 0.05). Post analysis edges are colored in pink, and enrichment map edges are colored in green.
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pone.0130658.g006: Short-term CR is associated with a complex miR expression profile.(A) qPCR validation of selected miRs from the microRNA array showing significant down-regulation of miR-21 and miR-92a and significant up-regulation of miR-27, miR-29, miR-208 and miR-214 in CR compared to Ad lib. miR-24 and miR-486 were used as negative controls. miR levels were normalized to U6. (B & C) Enrichment Map of CR vs Ad lib. Nodes represent enriched pathways and edges the similarity between those pathways calculated based on an overlap statistic > 0.5. Enrichments were filtered by p-value < 0.01, FDR < 0.05. The enrichment map was further annotated using a post analysis using mir-29 and mir-27 gene sets to highlight enriched pathways (blue or red nodes) that contain a statistically significant amount of B) miR-27 or C) mir-29 targets (as calculated using the hypergeometric distribution, p-value < 0.05). Post analysis edges are colored in pink, and enrichment map edges are colored in green.
Mentions: To determine whether the 7d of CR used in our study is accompanied by differential regulation of miR, qPCR arrays were used to determine the expression of 709 distinct miRs in the LV of CR and AL mice. miR array analysis revealed 18 miR that were up-regulated and 24 miR that were down-regulated by CR (Table 1). To validate findings from the miR array, 6 miRs (miR-21, -92a, -27, -29, -208 and -214) were tested by qPCR using SYBR Green or Taqman systems. As shown in Fig 6A, the levels of miR-21 and miR-92a were down-regulated in CR as compared to AL mice (-1.085±0.2465, P<0.01; and -1.798±0.6679, P<0.02; fold change respectively, N = 3). In addition, miR-27, miR-29, miR-208 and miR-214 were significantly up-regulated in CR as compared to AL groups (+2.969±0.5318, P<0.05; +7.483±1.084, P<0.002; +2.483±0.9468, P<0.009; and +2.003±0.5865, P<0.02; fold change respectively, N = 3) (Fig 6A). Our qPCR results demonstrated no change in the level of miR-24 and miR-486 in CR vs. AL groups (N = 3), again confirming results of the miR-array (Fig 6A). Together, these data indicate a strong correlation between miR-array and qPCR. We then annotated the enrichment map with the predicted targets of two miR with the highest fold change in CR (miR-27 and -29) to highlight gene sets that are significantly modulated (hyper-geometric P<0.05) as a percentage of miR targets (Fig 6B and 6C). Both miR-27 and miR-29 showed overlapping targets in pathways associated with ECM organization, angiogenesis, cell migration, adhesion and cytoskeletal organization. We also validated by qPCR the expression levels of known targets of three miR (miR-27, -29 and -214), which were up-regulated in the CR group. Of note, miR-214, which has previously been shown to reduce Ca2+ overload-induced cardiomyocyte death in an ischemia-reperfusion injury model [33], was up-regulated in CR mice. Up- and down-regulation of miR in the present study correlated well with mRNA levels of their known and predicted gene targets as demonstrated by microarray analysis and qRT-PCR results (Fig. B in S2 File). For example, down-regulation of several distinct miR (-34a, -199a, -181a/b and -204), which have been shown to target Sirt1 [34, 35] was associated with up-regulation of the mRNA level of Sirt1 in CR (Fig. A in S2 File). Similarly, while miR-133a and miR-181 were down-regulated, the mRNA level of their target, pro-survival gene Mcl1 [36, 37] was up-regulated in the CR heart (Fig. A in S2 File). On the other hand, up-regulation of miR-214 in the CR group was associated with down-regulation of mRNA levels of its known target genes, Ncx1 and Camk2d (P< 0.01 and P<0.02 respectively) in the hearts of CR as compared to AL mice (Panel B in Fig. B in S2 File). In addition, mRNA levels of Col1a1, Mmp2, and Itg6, known targets of miR-29 (Up-regulated in the CR mice) [38], were also down-regulated (P<0.001, P<0.003 and P<0.0001 respectively) in CR hearts (Panel B in Fig. B in S2 File). Also, mRNA levels of the tumor suppressor gene Fbxw7, a known target of miR-27 [39], was down-regulated (P<0.01) in the hearts of CR vs. AL mice (Panel B in Fig. B in S2 File). These results show, for the first time, that short-term CR for only 7d mediates a cardioprotective gene profile that includes specific miR and their downstream targets. For further details, a large listing of genes regulated by CR is provided in Table C in S1 File.

Bottom Line: This short-term model of CR was associated with cardio-protection, as evidenced by decreased infarct size (18.5±2.4% vs. 26.6±1.7%, N=10/group; P=0.01). mRNA and miR profiles pre-MI (N=5/group) identified genes modulated by short-term CR to be associated with circadian clock, oxidative stress, immune function, apoptosis, metabolism, angiogenesis, cytoskeleton and extracellular matrix (ECM).Western blots pre-MI revealed CR-associated increases in phosphorylated Akt and GSK3ß, reduced levels of phosphorylated AMPK and mitochondrial related proteins PGC-1α, cytochrome C and cyclooxygenase (COX) IV, with no differences in the levels of phosphorylated eNOS or MAPK (ERK1/2; p38).CR regimen was also associated with reduced protein abundance of cleaved caspase 3 in the infarcted heart and improved cardiac function.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Therapeutics, Toronto General Research Institute, Toronto, Ontario, Canada.

ABSTRACT

Objective: To understand the molecular pathways underlying the cardiac preconditioning effect of short-term caloric restriction (CR).

Background: Lifelong CR has been suggested to reduce the incidence of cardiovascular disease through a variety of mechanisms. However, prolonged adherence to a CR life-style is difficult. Here we reveal the pathways that are modulated by short-term CR, which are associated with protection of the mouse heart from ischemia.

Methods: Male 10-12 wk old C57bl/6 mice were randomly assigned to an ad libitum (AL) diet with free access to regular chow, or CR, receiving 30% less food for 7 days (d), prior to myocardial infarction (MI) via permanent coronary ligation. At d8, the left ventricles (LV) of AL and CR mice were collected for Western blot, mRNA and microRNA (miR) analyses to identify cardioprotective gene expression signatures. In separate groups, infarct size, cardiac hemodynamics and protein abundance of caspase 3 was measured at d2 post-MI.

Results: This short-term model of CR was associated with cardio-protection, as evidenced by decreased infarct size (18.5±2.4% vs. 26.6±1.7%, N=10/group; P=0.01). mRNA and miR profiles pre-MI (N=5/group) identified genes modulated by short-term CR to be associated with circadian clock, oxidative stress, immune function, apoptosis, metabolism, angiogenesis, cytoskeleton and extracellular matrix (ECM). Western blots pre-MI revealed CR-associated increases in phosphorylated Akt and GSK3ß, reduced levels of phosphorylated AMPK and mitochondrial related proteins PGC-1α, cytochrome C and cyclooxygenase (COX) IV, with no differences in the levels of phosphorylated eNOS or MAPK (ERK1/2; p38). CR regimen was also associated with reduced protein abundance of cleaved caspase 3 in the infarcted heart and improved cardiac function.

No MeSH data available.


Related in: MedlinePlus