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Local IGF-1 isoform protects cardiomyocytes from hypertrophic and oxidative stresses via SirT1 activity.

Vinciguerra M, Santini MP, Claycomb WC, Ladurner AG, Rosenthal N - Aging (Albany NY) (2009)

Bottom Line: Here we show that locally acting mIGF-1 increases SirT1 expression/activity, whereas circulating IGF-1 isoform does not affect it, in cultured HL-1 and neonatal cardiomyocytes. mIGF-1-induced SirT1 activity exerts protection against angiotensin II (Ang II)-triggered hypertrophy and against paraquat (PQ) and Ang II-induced oxidative stress.Conversely, circulating IGF-1 triggered itself oxidative stress and cardiomyocyte hypertrophy.Interestingly, potent cardio-protective genes (adiponectin, UCP-1 and MT-2) were increased specifically in mIGF-1-overexpressing cardiomyocytes, in a SirT1-dependent fashion.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory-Mouse Biology Unit, Campus A. Buzzati-Traverso, Monterotondo-Scalo, Rome 00016, Italy. Manlio.Vinciguerra@embl.it

ABSTRACT
Oxidative and hypertrophic stresses contribute to the pathogenesis of heart failure. Insulin-like growth factor-1 (IGF-1) is a peptide hormone with a complex post-transcriptional regulation, generating distinct isoforms. Locally acting IGF-1 isoform (mIGF-1) helps the heart to recover from toxic injury and from infarct. In the murine heart, moderate overexpression of the NAD(+)-dependent deacetylase SirT1 was reported to mitigate oxidative stress. SirT1 is known to promote lifespan extension and to protect from metabolic challenges. Circulating IGF-1 and SirT1 play antagonizing biological roles and share molecular targets in the heart, in turn affecting cardiomyocyte physiology. However, how different IGF-1 isoforms may impact SirT1 and affect cardiomyocyte function is unknown. Here we show that locally acting mIGF-1 increases SirT1 expression/activity, whereas circulating IGF-1 isoform does not affect it, in cultured HL-1 and neonatal cardiomyocytes. mIGF-1-induced SirT1 activity exerts protection against angiotensin II (Ang II)-triggered hypertrophy and against paraquat (PQ) and Ang II-induced oxidative stress. Conversely, circulating IGF-1 triggered itself oxidative stress and cardiomyocyte hypertrophy. Interestingly, potent cardio-protective genes (adiponectin, UCP-1 and MT-2) were increased specifically in mIGF-1-overexpressing cardiomyocytes, in a SirT1-dependent fashion. Thus, mIGF-1 protects cardiomyocytes from oxidative and hypertrophic stresses via SirT1 activity, and may represent a promising cardiac therapeutic.

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mIGF-1 prevents Ang II-, PQ- and IGF-1-induced increase in reactive oxygen species (ROS) generation in HL-1 cardiomyocytes and in mouse neonatal primary cardiomyocytes.(A,                                                    B) HL-1 cardiomyocytes were transfected or treated as in Legend of                                            Figure 2A, except that Ang II (1 μM)                                            or PQ (100 μM) were added                                            for only 60 min. Untransfected cells were used as control (CTL). (C, D) Neonatal                                            primary cardiomyocytes from wild type or mIGF-1 Tg mice were treated as in                                            Legend of Figure 2A, except that Ang II (1 μM) or PQ (100 μM)                                            were added for only 60 min. (A-D) ROS production was monitored with                                            the fluorescent probe dichlorofluorescein diacetate (CM-DCFDA) and                                            fluorescence values were normalized to protein content. Results are means ± SE of 3 independent experiments                                            (*,**,***p versus unstimulated                                            control cells or untreated WT cardiomyocytes).
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Figure 6: mIGF-1 prevents Ang II-, PQ- and IGF-1-induced increase in reactive oxygen species (ROS) generation in HL-1 cardiomyocytes and in mouse neonatal primary cardiomyocytes.(A, B) HL-1 cardiomyocytes were transfected or treated as in Legend of Figure 2A, except that Ang II (1 μM) or PQ (100 μM) were added for only 60 min. Untransfected cells were used as control (CTL). (C, D) Neonatal primary cardiomyocytes from wild type or mIGF-1 Tg mice were treated as in Legend of Figure 2A, except that Ang II (1 μM) or PQ (100 μM) were added for only 60 min. (A-D) ROS production was monitored with the fluorescent probe dichlorofluorescein diacetate (CM-DCFDA) and fluorescence values were normalized to protein content. Results are means ± SE of 3 independent experiments (*,**,***p versus unstimulated control cells or untreated WT cardiomyocytes).

Mentions: ROS generation and oxidative stress contribute to the progression of pathological cardiac hypertrophy and heart failure. Indeed, oxidative stress and hypertrophy are intimately linked in cardiac muscle[3]. It is increasingly appreciated that the Ang II hypertrophic effects on cardiomyocytes are strictly dependent on the generation of ROS [32]. IGF-1 also triggers ROS production, although it is controversial if this growth factor antagonizes or favors oxidative stress in cardiomyocytes[12,13]. Since SirT1 overexpression has been reported to protect the murine heart from PQ-induced oxidative stress [16], we measured ROS content by dichlorofluorescein diacetate (CM-DCFDA) method in mouse cardiomyocytes to shed light on the impact of IGF-1/SirT1 signaling on oxidative stress generated by Ang II and by PQ. To this end, HL-1 or neonatal cardiomyocytes were pretreated with superoxide anion scavenger Tiron before exposure to Ang II or PQ for 1 hour (Figure 6A-D). In both cardiomyocytes models, Ang II and PQ triggered a significant augmentation in intracellular ROS compared to untreated control cells, which was fully blocked by Tiron (Figure 6A-B, and 6C-D, for HL-1 and neonatal cardiomyocytes, respectively). mIGF-1 did not induce ROS production and efficiently prevented ROS generation by Ang II and PQ in both cardiomyocytes models (Figure 6A-D). Similarly, also SirT1 overexpression reversed ROS production in HL-1 cardiomyocytes (Figure 6A and B). In addition, blocking SirT1 enzymatic activity, by overexpression of SirT1 H363Y in HL-1 cells or incubation with SirT1 inhibitors in neonatal cardiomyocytes, abrogated the protective effects of mIGF-1 against Ang II- and PQ-induced intracellular ROS generation (Figure 6A-D). In contrast to locally acting mIGF-1 isoform, incubation of cardiomyocytes with the circulating IGF-1 isoform triggered a significant rise in ROS content, however less sustained than that generated by Ang II or by PQ (Figure 6A and C). Taken together, these data clearly indicate that mIGF-1, but not IGF-1, shields mouse cardiomyocytes from a rise of intracellular ROS generated by oxidative stressors. To ascertain if mIGF-1 exerts a cardio-protective role against oxidative stress as well in vivo, we injected peritoneally wild type and mIGF-1 Tg mice with PQ, and we assessed lipid and protein peroxidation levels, normally increasing upon ROS generation in cardiomyocytes [33]. Immunoblot analyses of lipid peroxidation 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) protein adducts in the heart showed that the levels of both protein adducts were significantly increased in the heart of PQ-injected wild type mice, whereas hearts of mIGF-1 Tg mice were to some extent protected from forming these compounds upon PQ injection (Figure 7). These data indicate that mIGF-1 protects the murine heart from oxidative stress as well in vivo.


Local IGF-1 isoform protects cardiomyocytes from hypertrophic and oxidative stresses via SirT1 activity.

Vinciguerra M, Santini MP, Claycomb WC, Ladurner AG, Rosenthal N - Aging (Albany NY) (2009)

mIGF-1 prevents Ang II-, PQ- and IGF-1-induced increase in reactive oxygen species (ROS) generation in HL-1 cardiomyocytes and in mouse neonatal primary cardiomyocytes.(A,                                                    B) HL-1 cardiomyocytes were transfected or treated as in Legend of                                            Figure 2A, except that Ang II (1 μM)                                            or PQ (100 μM) were added                                            for only 60 min. Untransfected cells were used as control (CTL). (C, D) Neonatal                                            primary cardiomyocytes from wild type or mIGF-1 Tg mice were treated as in                                            Legend of Figure 2A, except that Ang II (1 μM) or PQ (100 μM)                                            were added for only 60 min. (A-D) ROS production was monitored with                                            the fluorescent probe dichlorofluorescein diacetate (CM-DCFDA) and                                            fluorescence values were normalized to protein content. Results are means ± SE of 3 independent experiments                                            (*,**,***p versus unstimulated                                            control cells or untreated WT cardiomyocytes).
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Figure 6: mIGF-1 prevents Ang II-, PQ- and IGF-1-induced increase in reactive oxygen species (ROS) generation in HL-1 cardiomyocytes and in mouse neonatal primary cardiomyocytes.(A, B) HL-1 cardiomyocytes were transfected or treated as in Legend of Figure 2A, except that Ang II (1 μM) or PQ (100 μM) were added for only 60 min. Untransfected cells were used as control (CTL). (C, D) Neonatal primary cardiomyocytes from wild type or mIGF-1 Tg mice were treated as in Legend of Figure 2A, except that Ang II (1 μM) or PQ (100 μM) were added for only 60 min. (A-D) ROS production was monitored with the fluorescent probe dichlorofluorescein diacetate (CM-DCFDA) and fluorescence values were normalized to protein content. Results are means ± SE of 3 independent experiments (*,**,***p versus unstimulated control cells or untreated WT cardiomyocytes).
Mentions: ROS generation and oxidative stress contribute to the progression of pathological cardiac hypertrophy and heart failure. Indeed, oxidative stress and hypertrophy are intimately linked in cardiac muscle[3]. It is increasingly appreciated that the Ang II hypertrophic effects on cardiomyocytes are strictly dependent on the generation of ROS [32]. IGF-1 also triggers ROS production, although it is controversial if this growth factor antagonizes or favors oxidative stress in cardiomyocytes[12,13]. Since SirT1 overexpression has been reported to protect the murine heart from PQ-induced oxidative stress [16], we measured ROS content by dichlorofluorescein diacetate (CM-DCFDA) method in mouse cardiomyocytes to shed light on the impact of IGF-1/SirT1 signaling on oxidative stress generated by Ang II and by PQ. To this end, HL-1 or neonatal cardiomyocytes were pretreated with superoxide anion scavenger Tiron before exposure to Ang II or PQ for 1 hour (Figure 6A-D). In both cardiomyocytes models, Ang II and PQ triggered a significant augmentation in intracellular ROS compared to untreated control cells, which was fully blocked by Tiron (Figure 6A-B, and 6C-D, for HL-1 and neonatal cardiomyocytes, respectively). mIGF-1 did not induce ROS production and efficiently prevented ROS generation by Ang II and PQ in both cardiomyocytes models (Figure 6A-D). Similarly, also SirT1 overexpression reversed ROS production in HL-1 cardiomyocytes (Figure 6A and B). In addition, blocking SirT1 enzymatic activity, by overexpression of SirT1 H363Y in HL-1 cells or incubation with SirT1 inhibitors in neonatal cardiomyocytes, abrogated the protective effects of mIGF-1 against Ang II- and PQ-induced intracellular ROS generation (Figure 6A-D). In contrast to locally acting mIGF-1 isoform, incubation of cardiomyocytes with the circulating IGF-1 isoform triggered a significant rise in ROS content, however less sustained than that generated by Ang II or by PQ (Figure 6A and C). Taken together, these data clearly indicate that mIGF-1, but not IGF-1, shields mouse cardiomyocytes from a rise of intracellular ROS generated by oxidative stressors. To ascertain if mIGF-1 exerts a cardio-protective role against oxidative stress as well in vivo, we injected peritoneally wild type and mIGF-1 Tg mice with PQ, and we assessed lipid and protein peroxidation levels, normally increasing upon ROS generation in cardiomyocytes [33]. Immunoblot analyses of lipid peroxidation 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA) protein adducts in the heart showed that the levels of both protein adducts were significantly increased in the heart of PQ-injected wild type mice, whereas hearts of mIGF-1 Tg mice were to some extent protected from forming these compounds upon PQ injection (Figure 7). These data indicate that mIGF-1 protects the murine heart from oxidative stress as well in vivo.

Bottom Line: Here we show that locally acting mIGF-1 increases SirT1 expression/activity, whereas circulating IGF-1 isoform does not affect it, in cultured HL-1 and neonatal cardiomyocytes. mIGF-1-induced SirT1 activity exerts protection against angiotensin II (Ang II)-triggered hypertrophy and against paraquat (PQ) and Ang II-induced oxidative stress.Conversely, circulating IGF-1 triggered itself oxidative stress and cardiomyocyte hypertrophy.Interestingly, potent cardio-protective genes (adiponectin, UCP-1 and MT-2) were increased specifically in mIGF-1-overexpressing cardiomyocytes, in a SirT1-dependent fashion.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory-Mouse Biology Unit, Campus A. Buzzati-Traverso, Monterotondo-Scalo, Rome 00016, Italy. Manlio.Vinciguerra@embl.it

ABSTRACT
Oxidative and hypertrophic stresses contribute to the pathogenesis of heart failure. Insulin-like growth factor-1 (IGF-1) is a peptide hormone with a complex post-transcriptional regulation, generating distinct isoforms. Locally acting IGF-1 isoform (mIGF-1) helps the heart to recover from toxic injury and from infarct. In the murine heart, moderate overexpression of the NAD(+)-dependent deacetylase SirT1 was reported to mitigate oxidative stress. SirT1 is known to promote lifespan extension and to protect from metabolic challenges. Circulating IGF-1 and SirT1 play antagonizing biological roles and share molecular targets in the heart, in turn affecting cardiomyocyte physiology. However, how different IGF-1 isoforms may impact SirT1 and affect cardiomyocyte function is unknown. Here we show that locally acting mIGF-1 increases SirT1 expression/activity, whereas circulating IGF-1 isoform does not affect it, in cultured HL-1 and neonatal cardiomyocytes. mIGF-1-induced SirT1 activity exerts protection against angiotensin II (Ang II)-triggered hypertrophy and against paraquat (PQ) and Ang II-induced oxidative stress. Conversely, circulating IGF-1 triggered itself oxidative stress and cardiomyocyte hypertrophy. Interestingly, potent cardio-protective genes (adiponectin, UCP-1 and MT-2) were increased specifically in mIGF-1-overexpressing cardiomyocytes, in a SirT1-dependent fashion. Thus, mIGF-1 protects cardiomyocytes from oxidative and hypertrophic stresses via SirT1 activity, and may represent a promising cardiac therapeutic.

Show MeSH
Related in: MedlinePlus