Tim Parr

The calpain system and cancer

The calpains are a conserved family of cysteine proteinases that catalyse the controlled proteolysis of many specific substrates. Calpain activity is implicated in several fundamental physiological processes, including cytoskeletal remodelling, cellular signalling, apoptosis and cell survival. Calpain expression is altered during tumorigenesis, and the proteolysis of numerous substrates, such as inhibitors of nuclear factor-κB (IκB), focal adhesion proteins (including, focal adhesion kinase and talin) and proto-oncogenes (for example, MYC), has been implicated in tumour pathogenesis. Recent evidence indicates that the increased expression of certain family members might influence the response to cancer therapies, providing justification for the development of novel calpain inhibitors.

Tenderness - an enzymatic view.

One of the most common causes of unacceptability in meat quality is toughness. Toughness is attributed to a range of factors including the amount of intramuscular connective tissue, intramuscular fat, and the length of the sarcomere. However, it is apparent that the extent of proteolysis of key proteins within muscle fibres is significant determinant of ultimate tenderness. The objective of this manuscript is to describe the main endogenous proteolytic enzyme systems that have the potential to be involved in muscle post-mortem proteolysis and whether the experimental evidence available supports this involvement.

The relationship between slow and fast myosin heavy chain content, calpastatin and meat tenderness in different ovine skeletal muscles.

The present study investigated the relationship between fibre type distribution and slow (MHC-s) and fast (MHC-f) myosin heavy chain content on calpastatin and meat tenderness in longissimus dorsi (LD), tensor fasciae latae (TFL), semitendinosus (ST), trapezius (TZ) and supraspinatus (SS) muscles from six Mule×Charolais rams. Samples taken at slaughter were frozen either in liquid N(2) for analysis of MHC-s and MHC-f by immunoblotting, or in cooled isopentane for histochemical fibre typing. Calpastatin activity and an immunoreactive 135 kDa calpastatin band were analysed in samples taken 24 h postmortem. Shear force was determined on muscle chops taken at 24 h postmortem and conditioned until day 14. The intensity of MHC-s and MHC-f immunopositive bands correlated with %Type I and %Type II fibres identified histochemically (r(2)=0.612 and 0.366, respectively, p<0.001). Muscle specific differences were observed in MHC-s and MHC-f immunoreactivity, fibre type distribution, calpastatin activity, calpastatin 135 kDa immunoreactivity and shear force. MHC-s correlated positively with calpastatin activity (r(2)=0.725, p<0.001) and 135 kDa calpastatin (r(2)=0.228, p<0.01) across all muscle types. The data show that detection of MHC-s can be used to identify fibre type differences between ovine muscles and that this correlates with differences in calpastatin content and inhibitory activity, but not tenderness.

Calpain system protein expression in basal-like and triple-negative invasive breast cancer

BACKGROUND Basal-like and triple-negative breast tumours encompass an important clinical subgroup and biomarkers that can prognostically stratify these patients are required. MATERIALS AND METHODS We investigated two breast cancer tissue microarrays for the expression of calpain-1, calpain-2 and calpastatin using immunohistochemistry. The first microarray was comprised of invasive tumours from 1371 unselected patients, and the verification microarray was comprised of invasive tumours from 387 oestrogen receptor (ER)-negative patients. RESULTS The calpain system contains a number of proteases and an endogenous inhibitor, calpastatin. Calpain activity is implicated in important cellular processes including cytoskeletal remodelling, apoptosis and survival. Our results show that the expression of calpastatin and calpain-1 are significantly associated with various clinicopathological criteria including tumour grade and ER expression. High expression of calpain-2 in basal-like or triple-negative disease was associated with adverse breast cancer-specific survival (P=0.003 and <0.001, respectively) and was verified in an independent cohort of patients. Interestingly, those patients with basal-like or triple-negative disease with a low level of calpain-2 expression had similar breast cancer-specific survival to non-basal- or receptor- (oestrogen, progesterone or human epidermal growth factor receptor 2 (HER2)) positive disease. CONCLUSIONS Expression of the large catalytic subunit of m-calpain (calpain-2) is significantly associated with clinical outcome of patients with triple-negative and basal-like disease.Background Basal-like and triple-negative breast tumours encompass an important clinical subgroup and biomarkers that can prognostically stratify these patients are required. Materials and methods We investigated two breast cancer tissue microarrays for the expression of calpain-1, calpain-2 and calpastatin using immunohistochemistry. The first microarray was comprised of invasive tumours from 1371 unselected patients, and the verification microarray was comprised of invasive tumours from 387 oestrogen receptor (ER)-negative patients. Results The calpain system contains a number of proteases and an endogenous inhibitor, calpastatin. Calpain activity is implicated in important cellular processes including cytoskeletal remodelling, apoptosis and survival. Our results show that the expression of calpastatin and calpain-1 are significantly associated with various clinicopathological criteria including tumour grade and ER expression. High expression of calpain-2 in basal-like or triple-negative disease was associated with adverse breast cancer-specific survival (P = 0.003 and <0.001, respectively) and was verified in an independent cohort of patients. Interestingly, those patients with basal-like or triple-negative disease with a low level of calpain-2 expression had similar breast cancer-specific survival to non-basal- or receptor- (oestrogen, progesterone or human epidermal growth factor receptor 2 (HER2)) positive disease. Conclusions Expression of the large catalytic subunit of m-calpain (calpain-2) is significantly associated with clinical outcome of patients with triple-negative and basal-like disease.

Transcriptome analysis of mRNA and miRNA in skeletal muscle indicates an important network for differential Residual Feed Intake in pigs

Feed efficiency (FE) can be measured by feed conversion ratio (FCR) or residual feed intake (RFI). In this study, we measured the FE related phenotypes of 236 castrated purebred Yorkshire boars, and selected 10 extreme individuals with high and low RFI for transcriptome analysis. We used RNA-seq analyses to determine the differential expression of genes and miRNAs in skeletal muscle. There were 99 differentially expressed genes identified (q ≤ 0.05). The down-regulated genes were mainly involved in mitochondrial energy metabolism, including FABP3, RCAN, PPARGC1 (PGC-1A), HK2 and PRKAG2. The up-regulated genes were mainly involved in skeletal muscle differentiation and proliferation, including IGF2, PDE7A, CEBPD, PIK3R1 and MYH6. Moreover, 15 differentially expressed miRNAs (|log2FC| ≥ 1, total reads count ≥ 20, p ≤ 0.05) were identified. Among them, miR-136, miR-30e-5p, miR-1, miR-208b, miR-199a, miR-101 and miR-29c were up-regulated, while miR-215, miR-365-5p, miR-486, miR-1271, miR-145, miR-99b, miR-191 and miR-10b were down-regulated in low RFI pigs. We conclude that decreasing mitochondrial energy metabolism, possibly through AMPK - PGC-1A pathways, and increasing muscle growth, through IGF-1/2 and TGF-β signaling pathways, are potential strategies for the improvement of FE in pigs (and possibly other livestock). This study provides new insights into the molecular mechanisms that determine RFI and FE in pigs.

Dose-dependent effects of vitamin D on transdifferentiation of skeletal muscle cells to adipose cells

Fat infiltration within muscle is one of a number of features of vitamin D deficiency, which leads to a decline in muscle functionality. The origin of this fat is unclear, but one possibility is that it forms from myogenic precursor cells present in the muscle, which transdifferentiate into mature adipocytes. The current study examined the effect of the active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), on the capacity of the C2C12 muscle cell line to differentiate towards the myogenic and adipogenic lineages. Cells were cultured in myogenic or adipogenic differentiation media containing increasing concentrations (0, 10−13, 10−11, 10−9, 10−7 or 10−5 M) of 1,25(OH)2D3 for up to 6 days and markers of muscle and fat development were measured. Mature myofibres were formed in both adipogenic and myogenic media, but fat droplets were only observed in adipogenic media. Relative to controls, low physiological concentrations (10−13 and 10−11 M) of 1,25(OH)2D3 increased fat droplet accumulation, whereas high physiological (10−9 M) and supraphysiological concentrations (≥10−7 M) inhibited fat accumulation. This increased accumulation of fat with low physiological concentrations (10−13 and 10−11 M) was associated with a sequential up-regulation of Pparγ2 (Pparg) and Fabp4 mRNA, indicating formation of adipocytes, whereas higher concentrations (≥10−9 M) reduced all these effects, and the highest concentration (10−5 M) appeared to have toxic effects. This is the first study to demonstrate dose-dependent effects of 1,25(OH)2D3 on the transdifferentiation of muscle cells into adipose cells. Low physiological concentrations (possibly mimicking a deficient state) induced adipogenesis, whereas higher (physiological and supraphysiological) concentrations attenuated this effect.

Myosin heavy chain mRNA isoforms are expressed in two distinct cohorts during C2C12 myogenesis

The regulation of muscle fibre transitions has mainly been studied in vivo using conventional histological or immunohistochemical techniques. In order to investigate the molecular regulation of myosin heavy chain (MyHC) isoform expression in cell culture studies, we first characterised the normal transitions in endogenous expression of the MyHC isoforms and the myogenic regulatory factors during differentiation of C2C12 muscle cells. Interestingly, across the time course of differentiation, MyHC mRNA isoforms were expressed in a distinct temporal pattern as two distinct cohorts, one including MyHC I, embryonic and neonatal, the other including MyHC IIa, IIx and IIb. The pattern of expression suggests a transition in MyHC isoforms, from one cohort to another, occurs during muscle cell differentiation and that these transitions occur independent of nerve innervation. To our knowledge, this is the most comprehensive analysis of in vitro MyHC mRNA isoform transitions and provides important information for studying the regulation of transitions in MyHC isoforms in cell culture systems.

The effect of altered growth rates on the calpain proteolytic system and meat tenderness in cattle

The present study was conducted to determine the effects of growth pattern on the calpain system and meat tenderization. Twenty-four Friesian calves were randomly allocated to three treatment groups: FAST (fast growth rate), SLOW (severely restricted growth rate) and ALTER (restricted growth for 30 days followed by fast growth rate). Four animals from each group were slaughtered on day 32 or 45 after altering the growth rates. Samples of M. longissimus dorsi were rapidly frozen at slaughter for protein analysis by Western blotting. Restricted growth reduced the immunoreactivity of a calpastatin band (135 kDa) measured at 24 h postmortem. Immunoreactivity associated with the large subunit of μ- or m-calpain appeared to be unaffected by growth patterns. Shear force measurements taken after 14 days of conditioning were positively related to 135 kDa calpastatin at 24 h postmortem. In this study there was no clear relationship between shear force and growth pattern.

The effect of recombinant caspase 3 on myofibrillar proteins in porcine skeletal muscle.

The objective of this study was to investigate the potential role of the caspase protease family in meat tenderisation by examining if caspase 3 was capable of causing myofibril protein degradation. Full-length human recombinant caspase 3 (rC3) was expressed in Escherichia coli and purified. The rC3 was active in the presence of myofibrils isolated from porcine longissimus dorsi muscle (LD) and retained activity in a buffer system closely mimicking post mortem conditions. The effect of increasing concentrations of rC3, incubation temperature, as well as incubation time on the degradation of isolated myofibril proteins were all investigated in this study. Myofibril protein degradation was determined by SDS-PAGE and Western blotting. There was a visible increase in myofibril degradation with a decrease in proteins identified as desmin and troponin I and the detection of protein degradation products at approximately 32, 28 and 18 kDa with increasing concentrations of rC3. These degradation products were analysed using MALDI-TOF mass spectrometry and identified to occur from the proteolysis of actin, troponin T and myosin light chain, respectively. The production of these degradation products was not inhibited by 5 mM EDTA or semi-purified calpastatin but was inhibited by the caspase-specific inhibitor Ac-DEVD-CHO. The temperature at which isolated myofibrils were incubated with rC3 was also found to affect degradation, with increasing incubation temperatures causing increased desmin degradation and cleavage of pro-caspase 3 into its active isoform. Incubation of isolated myofibrils at 4°C for 5 days with rC3 resulted in the visible degradation of a number of myofibril proteins including desmin and troponin I. This study has shown that rC3 is capable of causing myofibril degradation, hydrolysing myofibril proteins under conditions that are similar to those found in muscle in the post mortem conditioning period.

Comparison of the relative expression of caspase isoforms in different porcine skeletal muscles.

The present study investigated the relationship between muscle type and components of the caspase protease system in porcine trapezius (TZ), psoas (PS), longissimus dorsi (LD) and semitendinosus (ST) muscles. Muscles were classified according to slow and fast myosin heavy chain (MHC) content determined by western blotting. MHC slow, but not MHC fast protein expression was significantly different between muscles (p<0.001). Protein levels of caspases 3, 8 and 12 and the caspase inhibitor apoptosis repressor with caspase recruitment domain (ARC) were determined. In addition the level of caspase 3 mRNA and activity levels of caspase 3/7 were determined. There was a significant difference in protein levels and activity between muscles (p<0.01), although no difference was observed in mRNA abundance. The data show that multiple components of the caspase system are expressed in porcine skeletal muscle and that their levels are variable, but there is not a distinct association of expression with a particular muscle.