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Activation of Both the Calpain and Ubiquitin-Proteasome Systems Contributes to Septic Cardiomyopathy through Dystrophin Loss/Disruption and mTOR Inhibition

View Article: PubMed Central - PubMed

ABSTRACT

Cardiac dysfunction caused by the impairment of myocardial contractility has been recognized as an important factor contributing to the high mortality in sepsis. Calpain activation in the heart takes place in response to increased intracellular calcium influx resulting in proteolysis of structural and contractile proteins with subsequent myocardial dysfunction. The purpose of the present study was to test the hypothesis that increased levels of calpain in the septic heart leads to disruption of structural and contractile proteins and that administration of calpain inhibitor-1 (N-acetyl-leucinyl-leucinyl-norleucinal (ALLN)) after sepsis induced by cecal ligation and puncture prevents cardiac protein degradation. We also tested the hypothesis that calpain plays a role in the modulation of protein synthesis/degradation through the activation of proteasome-dependent proteolysis and inhibition of the mTOR pathway. Severe sepsis significantly increased heart calpain-1 levels and promoted ubiquitin and Pa28β over-expression with a reduction in the mTOR levels. In addition, sepsis reduced the expression of structural proteins dystrophin and β-dystroglycan as well as the contractile proteins actin and myosin. ALLN administration prevented sepsis-induced increases in calpain and ubiquitin levels in the heart, which resulted in decreased of structural and contractile proteins degradation and basal mTOR expression levels were re-established. Our results support the concept that increased calpain concentrations may be part of an important mechanism of sepsis-induced cardiac muscle proteolysis.

No MeSH data available.


Related in: MedlinePlus

Western blot analysis of cardiac calpain-1, ubiquitin and Pa28β after CLP.Protein levels of calpain-1 (A), ubiquitin (B) and Pa28β (C) in the SHAM, SSI, SHAM+ALLN and SSI+ALLN groups were measured 24 h after the CLP procedure and expressed in arbitrary units (AUs). GAPDH was used to determine equivalent loading conditions. (D) Calpain-1 mRNA expression was measured 24 h after the CLP procedure and expressed in arbitrary units (AUs). GAPDH gene expression was used as the internal control for gene expression normalization. The results (n = 6 per group) are representative of three different experiments.
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pone.0166839.g002: Western blot analysis of cardiac calpain-1, ubiquitin and Pa28β after CLP.Protein levels of calpain-1 (A), ubiquitin (B) and Pa28β (C) in the SHAM, SSI, SHAM+ALLN and SSI+ALLN groups were measured 24 h after the CLP procedure and expressed in arbitrary units (AUs). GAPDH was used to determine equivalent loading conditions. (D) Calpain-1 mRNA expression was measured 24 h after the CLP procedure and expressed in arbitrary units (AUs). GAPDH gene expression was used as the internal control for gene expression normalization. The results (n = 6 per group) are representative of three different experiments.

Mentions: We found that protein levels of calpain-1 in the myocardium of animals receiving CLP (SSI) were increased by approximately 25% compared with SHAM mice (1.23±0.26 vs. 0.91±0.11) 24 hours after the CLP procedure. The SSI+ALLN group demonstrated a marked reduction in calpain-1 levels of approximately 31% compared to the SSI group (0.82±0.06 vs. 1.23±0.26), which exhibited values similar to those of the control groups (Fig 2A). Sepsis increased the ubiquitin protein levels by greater than 901% in the cardiac tissue obtained from the SSI group compared with that of the SHAM group (2.01±0.32 vs. 1.05±0.16) (Fig 2B), whereas in the sepsis group treated with ALLN (SSI+ALLN), the levels of ubiquitin were similar to those of the controls (1.45±0.09 vs. 1.05±0.16). Similarly, the protein levels of Pa28β is increased by approximately 15% in the group (SSI) when compared to the sham group (SHAM) (0.71±0.06 vs. 0.62±0.05), where Pa28β levels remained similar in septic group treated with ALLN (SSI+ALLN) compared to the SHAM values (Fig 2C), suggesting a correlation between increased calpain-1 and ubiquitin-proteasome protein levels during sepsis.


Activation of Both the Calpain and Ubiquitin-Proteasome Systems Contributes to Septic Cardiomyopathy through Dystrophin Loss/Disruption and mTOR Inhibition
Western blot analysis of cardiac calpain-1, ubiquitin and Pa28β after CLP.Protein levels of calpain-1 (A), ubiquitin (B) and Pa28β (C) in the SHAM, SSI, SHAM+ALLN and SSI+ALLN groups were measured 24 h after the CLP procedure and expressed in arbitrary units (AUs). GAPDH was used to determine equivalent loading conditions. (D) Calpain-1 mRNA expression was measured 24 h after the CLP procedure and expressed in arbitrary units (AUs). GAPDH gene expression was used as the internal control for gene expression normalization. The results (n = 6 per group) are representative of three different experiments.
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Related In: Results  -  Collection

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pone.0166839.g002: Western blot analysis of cardiac calpain-1, ubiquitin and Pa28β after CLP.Protein levels of calpain-1 (A), ubiquitin (B) and Pa28β (C) in the SHAM, SSI, SHAM+ALLN and SSI+ALLN groups were measured 24 h after the CLP procedure and expressed in arbitrary units (AUs). GAPDH was used to determine equivalent loading conditions. (D) Calpain-1 mRNA expression was measured 24 h after the CLP procedure and expressed in arbitrary units (AUs). GAPDH gene expression was used as the internal control for gene expression normalization. The results (n = 6 per group) are representative of three different experiments.
Mentions: We found that protein levels of calpain-1 in the myocardium of animals receiving CLP (SSI) were increased by approximately 25% compared with SHAM mice (1.23±0.26 vs. 0.91±0.11) 24 hours after the CLP procedure. The SSI+ALLN group demonstrated a marked reduction in calpain-1 levels of approximately 31% compared to the SSI group (0.82±0.06 vs. 1.23±0.26), which exhibited values similar to those of the control groups (Fig 2A). Sepsis increased the ubiquitin protein levels by greater than 901% in the cardiac tissue obtained from the SSI group compared with that of the SHAM group (2.01±0.32 vs. 1.05±0.16) (Fig 2B), whereas in the sepsis group treated with ALLN (SSI+ALLN), the levels of ubiquitin were similar to those of the controls (1.45±0.09 vs. 1.05±0.16). Similarly, the protein levels of Pa28β is increased by approximately 15% in the group (SSI) when compared to the sham group (SHAM) (0.71±0.06 vs. 0.62±0.05), where Pa28β levels remained similar in septic group treated with ALLN (SSI+ALLN) compared to the SHAM values (Fig 2C), suggesting a correlation between increased calpain-1 and ubiquitin-proteasome protein levels during sepsis.

View Article: PubMed Central - PubMed

ABSTRACT

Cardiac dysfunction caused by the impairment of myocardial contractility has been recognized as an important factor contributing to the high mortality in sepsis. Calpain activation in the heart takes place in response to increased intracellular calcium influx resulting in proteolysis of structural and contractile proteins with subsequent myocardial dysfunction. The purpose of the present study was to test the hypothesis that increased levels of calpain in the septic heart leads to disruption of structural and contractile proteins and that administration of calpain inhibitor-1 (N-acetyl-leucinyl-leucinyl-norleucinal (ALLN)) after sepsis induced by cecal ligation and puncture prevents cardiac protein degradation. We also tested the hypothesis that calpain plays a role in the modulation of protein synthesis/degradation through the activation of proteasome-dependent proteolysis and inhibition of the mTOR pathway. Severe sepsis significantly increased heart calpain-1 levels and promoted ubiquitin and Pa28β over-expression with a reduction in the mTOR levels. In addition, sepsis reduced the expression of structural proteins dystrophin and β-dystroglycan as well as the contractile proteins actin and myosin. ALLN administration prevented sepsis-induced increases in calpain and ubiquitin levels in the heart, which resulted in decreased of structural and contractile proteins degradation and basal mTOR expression levels were re-established. Our results support the concept that increased calpain concentrations may be part of an important mechanism of sepsis-induced cardiac muscle proteolysis.

No MeSH data available.


Related in: MedlinePlus