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Targeted inhibition of calpain reduces myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes.

Li Y, Ma J, Zhu H, Singh M, Hill D, Greer PA, Arnold JM, Abel ED, Peng T - Diabetes (2011)

Bottom Line: Calpain activity, cardiomyocyte cross-sectional areas, and myocardial collagen deposition were significantly increased in both STZ-induced and OVE26 diabetic hearts, and these were accompanied by elevated expression of hypertrophic and fibrotic collagen genes.These effects were associated with a normalization of the nuclear factor of activated T-cell nuclear factor-κB and matrix metalloproteinase (MMP) activities in diabetic hearts.Thus targeted inhibition of calpain represents a potential novel therapeutic strategy for reversing diabetic cardiomyopathy.

View Article: PubMed Central - PubMed

Affiliation: Critical Illness Research, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada.

ABSTRACT

Objective: Recently we have shown that calpain-1 activation contributes to cardiomyocyte apoptosis induced by hyperglycemia. This study was undertaken to investigate whether targeted disruption of calpain would reduce myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes.

Research design and methods: Diabetes in mice was induced by injection of streptozotocin (STZ), and OVE26 mice were also used as a type 1 diabetic model. The function of calpain was genetically manipulated by cardiomyocyte-specific knockout Capn4 in mice and the use of calpastatin transgenic mice. Myocardial hypertrophy and fibrosis were investigated 2 and 5 months after STZ injection or in OVE26 diabetic mice at the age of 5 months. Cultured isolated adult mouse cardiac fibroblast cells were also investigated under high glucose conditions.

Results: Calpain activity, cardiomyocyte cross-sectional areas, and myocardial collagen deposition were significantly increased in both STZ-induced and OVE26 diabetic hearts, and these were accompanied by elevated expression of hypertrophic and fibrotic collagen genes. Deficiency of Capn4 or overexpression of calpastatin reduced myocardial hypertrophy and fibrosis in both diabetic models, leading to the improvement of myocardial function. These effects were associated with a normalization of the nuclear factor of activated T-cell nuclear factor-κB and matrix metalloproteinase (MMP) activities in diabetic hearts. In cultured cardiac fibroblasts, high glucose-induced proliferation and MMP activities were prevented by calpain inhibition.

Conclusions: Myocardial hypertrophy and fibrosis in diabetic mice are attenuated by reduction of calpain function. Thus targeted inhibition of calpain represents a potential novel therapeutic strategy for reversing diabetic cardiomyopathy.

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Related in: MedlinePlus

Characterization of mice with cardiac-specific Capn4 knockout. A: Representative RT-PCR for Cre and Capn4 mRNA from two of six mice in each group. Cre mRNA was detected in Capn4−/− hearts (lanes 3 and 4). Levels of Capn4 mRNA were significantly reduced in Capn4−/− hearts. In contrast, the Capn4 mRNA was not decreased in Capn4−/− lungs. B: Capn4 mRNA was quantified by real-time RT-PCR in Capn4+/+ and Capn4−/− hearts (B1) and lung tissues (B2). C1 and C2 are the quantification of Capn4 mRNA in isolated cardiomyocytes (C1) and cardiac fibroblasts (C2) from Capn4+/+ and Capn4−/− mice. D: Representative casein zymography for calpain-1 and calpain-2 activities from two of six hearts and lungs, respectively, showing downregulation of calpain-1 and calpain-2 activities in Capn4−/− hearts but not in lungs. Data are mean ± SD; n = 5–9. *P < 0.05 vs. +/+. OD, optical density.
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Figure 2: Characterization of mice with cardiac-specific Capn4 knockout. A: Representative RT-PCR for Cre and Capn4 mRNA from two of six mice in each group. Cre mRNA was detected in Capn4−/− hearts (lanes 3 and 4). Levels of Capn4 mRNA were significantly reduced in Capn4−/− hearts. In contrast, the Capn4 mRNA was not decreased in Capn4−/− lungs. B: Capn4 mRNA was quantified by real-time RT-PCR in Capn4+/+ and Capn4−/− hearts (B1) and lung tissues (B2). C1 and C2 are the quantification of Capn4 mRNA in isolated cardiomyocytes (C1) and cardiac fibroblasts (C2) from Capn4+/+ and Capn4−/− mice. D: Representative casein zymography for calpain-1 and calpain-2 activities from two of six hearts and lungs, respectively, showing downregulation of calpain-1 and calpain-2 activities in Capn4−/− hearts but not in lungs. Data are mean ± SD; n = 5–9. *P < 0.05 vs. +/+. OD, optical density.

Mentions: To investigate the role of calpain, we generated mice with cardiomyocyte-specific disruption of calpain by crossing floxed Capn4PZ mice with transgenic mice overexpressing Cre under the control of the α-MHC promoter (Tg-Cre). All of the mice used in this study, including controls, were littermates of the same generation. PCR analysis of tail DNA confirmed untargeted (Capn4+/+), either homozygous (Capn4PZ/PZ) or heterozygous (Capn4+/PZ) for the floxed Capn4 gene, and the Cre transgene (Supplementary Fig. 2A). Mice homozygous or heterozygous for the floxed Capn4 gene and positive for the α-MHC-Cre transgene were identified as potential homozygous (Capn4−/−) or heterozygous knockouts (Capn4+/−), respectively. To confirm decreased Capn4 expression in the heart, Capn4 mRNA was analyzed in Capn4+/+ and Capn4−/− mice using primers specifically amplifying the deleted region of Capn4 mRNA. Myocardial Capn4 mRNA was reduced by 78% in Capn4−/− mice, as compared with Capn4+/+ mice (Fig. 2A and B). In contrast, Capn4 mRNA levels in lung tissues were similar between Capn4−/− and Capn4+/+ mice (Fig. 2A and B), supporting cardiac-specific Capn4 knockout. To further confirm that the deletion of Capn4 was restricted in cardiomyocytes, the mRNA levels of Capn4 were significantly decreased in isolated cardiomyocytes of Capn4−/− mice, whereas their levels were comparable between Capn4−/− and Capn4+/+ fibroblasts (Fig. 2C). Because the Capn4-encoded regulatory subunit is required for the stability and proteolytic activities of calpain-1 and calpain-2 catalytic subunits (10), Capn4 deletion results in the disruption of calpain-1 and calpain-2 activities. Indeed, casein zymography analysis of myocardial tissues showed a robust reduction of both calpain-1 and calpain-2 activities in Capn4−/− compared with Capn4+/+ mice (Fig. 2D), confirming the functional knockout of Capn4. Similar to Capn4 mRNA expression, both calpain-1 and calpain-2 activities were not decreased in Capn4−/− lung tissues (Fig. 2D).


Targeted inhibition of calpain reduces myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes.

Li Y, Ma J, Zhu H, Singh M, Hill D, Greer PA, Arnold JM, Abel ED, Peng T - Diabetes (2011)

Characterization of mice with cardiac-specific Capn4 knockout. A: Representative RT-PCR for Cre and Capn4 mRNA from two of six mice in each group. Cre mRNA was detected in Capn4−/− hearts (lanes 3 and 4). Levels of Capn4 mRNA were significantly reduced in Capn4−/− hearts. In contrast, the Capn4 mRNA was not decreased in Capn4−/− lungs. B: Capn4 mRNA was quantified by real-time RT-PCR in Capn4+/+ and Capn4−/− hearts (B1) and lung tissues (B2). C1 and C2 are the quantification of Capn4 mRNA in isolated cardiomyocytes (C1) and cardiac fibroblasts (C2) from Capn4+/+ and Capn4−/− mice. D: Representative casein zymography for calpain-1 and calpain-2 activities from two of six hearts and lungs, respectively, showing downregulation of calpain-1 and calpain-2 activities in Capn4−/− hearts but not in lungs. Data are mean ± SD; n = 5–9. *P < 0.05 vs. +/+. OD, optical density.
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Related In: Results  -  Collection

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Figure 2: Characterization of mice with cardiac-specific Capn4 knockout. A: Representative RT-PCR for Cre and Capn4 mRNA from two of six mice in each group. Cre mRNA was detected in Capn4−/− hearts (lanes 3 and 4). Levels of Capn4 mRNA were significantly reduced in Capn4−/− hearts. In contrast, the Capn4 mRNA was not decreased in Capn4−/− lungs. B: Capn4 mRNA was quantified by real-time RT-PCR in Capn4+/+ and Capn4−/− hearts (B1) and lung tissues (B2). C1 and C2 are the quantification of Capn4 mRNA in isolated cardiomyocytes (C1) and cardiac fibroblasts (C2) from Capn4+/+ and Capn4−/− mice. D: Representative casein zymography for calpain-1 and calpain-2 activities from two of six hearts and lungs, respectively, showing downregulation of calpain-1 and calpain-2 activities in Capn4−/− hearts but not in lungs. Data are mean ± SD; n = 5–9. *P < 0.05 vs. +/+. OD, optical density.
Mentions: To investigate the role of calpain, we generated mice with cardiomyocyte-specific disruption of calpain by crossing floxed Capn4PZ mice with transgenic mice overexpressing Cre under the control of the α-MHC promoter (Tg-Cre). All of the mice used in this study, including controls, were littermates of the same generation. PCR analysis of tail DNA confirmed untargeted (Capn4+/+), either homozygous (Capn4PZ/PZ) or heterozygous (Capn4+/PZ) for the floxed Capn4 gene, and the Cre transgene (Supplementary Fig. 2A). Mice homozygous or heterozygous for the floxed Capn4 gene and positive for the α-MHC-Cre transgene were identified as potential homozygous (Capn4−/−) or heterozygous knockouts (Capn4+/−), respectively. To confirm decreased Capn4 expression in the heart, Capn4 mRNA was analyzed in Capn4+/+ and Capn4−/− mice using primers specifically amplifying the deleted region of Capn4 mRNA. Myocardial Capn4 mRNA was reduced by 78% in Capn4−/− mice, as compared with Capn4+/+ mice (Fig. 2A and B). In contrast, Capn4 mRNA levels in lung tissues were similar between Capn4−/− and Capn4+/+ mice (Fig. 2A and B), supporting cardiac-specific Capn4 knockout. To further confirm that the deletion of Capn4 was restricted in cardiomyocytes, the mRNA levels of Capn4 were significantly decreased in isolated cardiomyocytes of Capn4−/− mice, whereas their levels were comparable between Capn4−/− and Capn4+/+ fibroblasts (Fig. 2C). Because the Capn4-encoded regulatory subunit is required for the stability and proteolytic activities of calpain-1 and calpain-2 catalytic subunits (10), Capn4 deletion results in the disruption of calpain-1 and calpain-2 activities. Indeed, casein zymography analysis of myocardial tissues showed a robust reduction of both calpain-1 and calpain-2 activities in Capn4−/− compared with Capn4+/+ mice (Fig. 2D), confirming the functional knockout of Capn4. Similar to Capn4 mRNA expression, both calpain-1 and calpain-2 activities were not decreased in Capn4−/− lung tissues (Fig. 2D).

Bottom Line: Calpain activity, cardiomyocyte cross-sectional areas, and myocardial collagen deposition were significantly increased in both STZ-induced and OVE26 diabetic hearts, and these were accompanied by elevated expression of hypertrophic and fibrotic collagen genes.These effects were associated with a normalization of the nuclear factor of activated T-cell nuclear factor-κB and matrix metalloproteinase (MMP) activities in diabetic hearts.Thus targeted inhibition of calpain represents a potential novel therapeutic strategy for reversing diabetic cardiomyopathy.

View Article: PubMed Central - PubMed

Affiliation: Critical Illness Research, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada.

ABSTRACT

Objective: Recently we have shown that calpain-1 activation contributes to cardiomyocyte apoptosis induced by hyperglycemia. This study was undertaken to investigate whether targeted disruption of calpain would reduce myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes.

Research design and methods: Diabetes in mice was induced by injection of streptozotocin (STZ), and OVE26 mice were also used as a type 1 diabetic model. The function of calpain was genetically manipulated by cardiomyocyte-specific knockout Capn4 in mice and the use of calpastatin transgenic mice. Myocardial hypertrophy and fibrosis were investigated 2 and 5 months after STZ injection or in OVE26 diabetic mice at the age of 5 months. Cultured isolated adult mouse cardiac fibroblast cells were also investigated under high glucose conditions.

Results: Calpain activity, cardiomyocyte cross-sectional areas, and myocardial collagen deposition were significantly increased in both STZ-induced and OVE26 diabetic hearts, and these were accompanied by elevated expression of hypertrophic and fibrotic collagen genes. Deficiency of Capn4 or overexpression of calpastatin reduced myocardial hypertrophy and fibrosis in both diabetic models, leading to the improvement of myocardial function. These effects were associated with a normalization of the nuclear factor of activated T-cell nuclear factor-κB and matrix metalloproteinase (MMP) activities in diabetic hearts. In cultured cardiac fibroblasts, high glucose-induced proliferation and MMP activities were prevented by calpain inhibition.

Conclusions: Myocardial hypertrophy and fibrosis in diabetic mice are attenuated by reduction of calpain function. Thus targeted inhibition of calpain represents a potential novel therapeutic strategy for reversing diabetic cardiomyopathy.

Show MeSH
Related in: MedlinePlus