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

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.

Fig 1. Histopathology of myocardial tissue after CLP.

Myocytolysis and contraction bands become evident in the SSI group (upper right panel) compared to the SSI+ALLN group (bottom right panel) 24 h after surgery. The SHAM (upper left panel) and SHAM+ALLN (bottom left panel) groups revealed no morphological alterations. Scale bars indicate 50 mm.

Fig 2. 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.

Fig 3. Immunofluorescence and western blot analysis of cardiac dystrophin after CLP.

(A) Immunofluorescence signal for dystrophin is significantly reduced in the SSI heart (bottom left panel) compared with the immunofluorescent signal in the SHAM heart (upper left panel), and the SHAM+ALLN (upper right panel) and SSI+ALLN (bottom right panel) myocardium. (B) Protein levels of dystrophin in the SHAM, SSI, SHAM+ALLN and SSI+ALLN hearts were measured 24 h after the CLP procedure and were expressed in arbitrary units (AUs). α-Tubulin was used to determine equivalent loading conditions. The results (n = 6 per group) are representative of three different experiments. Scale bars indicate 50 μm.

Fig 4. Western blot analysis of cardiac β-dystroglycan, sarcomeric actin and myosin after CLP.

Protein levels of β-dystroglycan (A), sarcomeric actin (B) and myosin (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. The results (n = 6 per group) are representative of three different experiments.

Fig 5

(A) Western blot analysis of cardiac mTOR protein levels after CLP. Cardiac mTOR protein levels in the SHAM, SSI, SHAM+ALLN and SSI+ALLN groups were measured 24 h after surgery and were expressed in arbitrary units (AUs). GAPDH was used to determine equivalent loading conditions. (B) Analysis of cardiac dystrophin mRNA expression. Dystrophin 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.