PURPOSE: This study was designed to investigate the characteristics of Korean patients with calpainopathy. MATERIALS AND METHODS: Thirteen patients from ten unrelated families were diagnosed with calpainopathy via direct or targeted sequencing of the CAPN3 gene. Clinical, mutational, and pathological spectra were then analyzed. RESULTS: Nine different mutations, including four novel mutations (NM_000070: c.1524+1G>T, c.1789_1790inA, c.2184+1G>T, and c.2384C>T) were identified. The median age at symptom onset was 22 (interquartile range: 15-28). Common clinical findings were joint contracture in nine patients, winged scapula in four, and lordosis in one. However, we also found highly variable clinical features including early onset joint contractures, asymptomatic hyperCKemia, and heterogeneous clinical severity in three members of the same family. Four of nine muscle specimens revealed lobulated fibers, but three showed normal skeletal muscle histology. CONCLUSION: We identified four novel CAPN3 mutations and demonstrated clinical and pathological heterogeneity in Korean patients with calpainopathy.
Adolescent ; Adult ; Amino Acid Sequence ; Asian Continental Ancestry Group/*genetics ; Calpain/*genetics ; Female ; Genetic Testing ; Humans ; Male ; Molecular Sequence Data ; Muscle Proteins/*genetics ; Muscle, Skeletal/pathology ; Muscular Dystrophies, Limb-Girdle/ethnology/*genetics/*pathology ; *Mutation ; Republic of Korea

The purpose of this study was to investigate the effect of inhibition of calpain on retinal ganglion cell-5 (RGC-5) necroptosis following oxygen glucose deprivation (OGD). RGC-5 cells were cultured in Dulbecco's-modified essential medium and necroptosis was induced by 8-h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. The calpain expression was detected by Western blotting and immunofluorescence staining. The calpain activity was tested by activity detection kit. Flow cytometry was used to detect the effect of calpain on RGC-5 necroptosis following OGD with or without N-acetyl-leucyl-leucyl-norleucinal (ALLN) pre-treatment. Western blot was used to detect the protein level of truncated apoptosis inducing factor (tAIF) in RGC-5 cells following OGD. The results showed that there was an up-regulation of the calpain expression and activity following OGD. Upon adding ALLN, the calpain activity was inhibited and tAIF was reduced following OGD along with the decreased number of RGC-5 necroptosis. In conclusion, calpain was involved in OGD-induced RGC-5 necroptosis with the increased expression of its downstream molecule tAIF.
Animals ; Apoptosis Inducing Factor ; biosynthesis ; genetics ; Calpain ; biosynthesis ; genetics ; Gene Expression Regulation ; drug effects ; Glucose ; metabolism ; Humans ; Leupeptins ; administration & dosage ; Mice ; Oxygen ; metabolism ; Retinal Ganglion Cells ; metabolism ; pathology ; Retinal Necrosis Syndrome, Acute ; genetics ; pathology

OBJECTIVE: To explore the role of calpain in pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension and the underlying mechanisms.
 METHODS: Sprague-Dawley rats were randomly divided into the hypoxia group and the normoxia control group. Right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were monitored by a method with right external jugular vein cannula. Right ventricular hypertrophy index was presented as the ratio of right ventricular weight to left ventricular weight (left ventricle plus septum weight). Levels of calpain-1, -2 and -4 mRNA in pulmonary artery were determined by real-time PCR. Levels of calpain-1, -2 and -4 protein were determined by Western blot. Primary rat pulmonary arterial smooth muscle cells (PASMCs) were divided into 4 groups: a normoxia control group, a normoxia+MDL28170 group, a hypoxia group and a hypoxia+MDL28170 group. Cell proliferation was detected by MTS and flow cytometry. Levels of Ki-67 and proliferating cell nuclear antigen (PCNA) mRNA were determined by real-time PCR.
 RESULTS: RVSP, mPAP and right ventricular remodeling index were significantly elevated in the hypoxia group compared to those in the normoxia group. In the hypoxia group, pulmonary vascular remodeling was significantly developed, accompanied by up-regulation of calpain-1, -2 and -4. MDL28170 significantly inhibited hypoxia-induced proliferation of PASMCs concomitant with the suppression of Ki-67 and PCNA mRNA expression.
 CONCLUSION Calpain mediates vascular remodeling via promoting proliferation of PASMCs in hypoxia-induced pulmonary hypertension.
Animals ; Calpain ; genetics ; physiology ; Cell Proliferation ; Dipeptides ; physiology ; Hypertension, Pulmonary ; chemically induced ; genetics ; physiopathology ; Hypertrophy, Right Ventricular ; Hypoxia ; Ki-67 Antigen ; drug effects ; Myocytes, Smooth Muscle ; physiology ; Proliferating Cell Nuclear Antigen ; drug effects ; Pulmonary Artery ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Up-Regulation ; Vascular Remodeling ; genetics ; physiology

After invasion of red blood cells, malaria matures within the cell by degrading hemoglobin avidly. For enormous protein breakdown in trophozoite stage, many efficient and ordered proteolysis networks have been postulated and exploited. In this study, a potential interaction of a 60-kDa Plasmodium falciparum (Pf)-heat shock protein (Hsp60) and Pf-calpain, a cysteine protease, was explored. Pf-infected RBC was isolated and the endogenous Pf-Hsp60 and Pf-calpain were determined by western blot analysis and similar antigenicity of GroEL and Pf-Hsp60 was determined with anti-Pf-Hsp60. Potential interaction of Pf-calpain and Pf-Hsp60 was determined by immunoprecipitation and immunofluorescence assay. Mizoribine, a well-known inhibitor of Hsp60, attenuated both Pf-calpain enzyme activity as well as P. falciparum growth. The presented data suggest that the Pf-Hsp60 may function on Pf-calpain in a part of networks during malaria growth.
Amino Acid Sequence ; Calpain/genetics/*metabolism ; Chaperonin 60/chemistry/genetics/*metabolism ; Erythrocytes/parasitology ; Humans ; Malaria, Falciparum/parasitology ; Molecular Sequence Data ; Plasmodium falciparum/chemistry/enzymology/genetics/*metabolism ; Protein Binding ; Protozoan Proteins/chemistry/genetics/*metabolism ; Sequence Alignment

The purpose of this study was to study the role of neurofilament (NF) mRNA and calpain in NF reduction of acrylamide (ACR) neuropathy. Male Wistar adult rats were injected i.p. every other day with ACR (20 mg/kg·bW or 40 mg/kg·bW) for 8 weeks. NF mRNA expression was detected using RT-PCR and the calpain concentration was determined using western blot analysis. The NF mRNA expression significantly decreased while the level of m-calpain and μ-calpain significantly increased in two ACR-treated rats groups regardless of the ACR dose. The light NF (NF-L) protein expression was significantly correlated with NF-L mRNA expression. Combined with previous data, the concentrations of three NF subunits were negatively correlated with the calpain levels. These findings suggest that NF-L mRNA and calpain mediated the reduction in NF of ACR neuropathy.
Acrylamide ; toxicity ; Animals ; Calpain ; metabolism ; Gene Expression Regulation ; drug effects ; Intermediate Filaments ; genetics ; Male ; Peripheral Nervous System Diseases ; chemically induced ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.
Caco-2 Cells ; Calcium-Binding Proteins ; Calpain/genetics/metabolism ; Caspase 3/genetics/metabolism ; Caspases ; *Cell Death ; Colon/cytology ; Entamoeba histolytica/*physiology ; Epithelial Cells/cytology/parasitology ; Humans ; I-kappa B Proteins/metabolism ; Intestinal Mucosa/cytology ; NF-kappa B/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering ; STAT3 Transcription Factor/genetics/*metabolism ; STAT5 Transcription Factor/genetics/*metabolism ; Signal Transduction

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and micro-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and micro-calpain may be involved in colon epithelial cell death induced by E. histolytica.
Calpain/antagonists & inhibitors/genetics/*metabolism ; *Cell Death ; Cell Line ; Cell Survival/drug effects ; Dipeptides/metabolism ; Entamoeba histolytica/*pathogenicity ; Epithelial Cells/*parasitology ; Gene Knockdown Techniques ; Humans

OBJECTIVETo investigate the splice variants of the calpain 3 gene existing in human skeletal muscle tissue and white blood cells, and to explore the feasibility of gene diagnosis using CAPN3 mRNA extracted from peripheral leukocytes.

METHODSTotal RNA was extracted from peripheral blood and skeletal muscle tissue in healthy individuals. CAPN3 cDNAs were determined by reverse transcriptase polymerase chain reaction and DNA sequencing. CAPN3 cDNAs from peripheral leukocytes were compared with sequences obtained from skeletal muscle tissue.

RESULTSRT-PCR and DNA sequencing showed that the CAPN3 cDNAs comprised 24 exons in human skeletal muscle tissue, while the number of exons was 23 in white blood cells. Exon 15 was spliced out in human white blood cells.

CONCLUSIONSplice variants exist in human skeletal muscle tissue and white blood cells. Gene diagnosis may omit the mutations of exon 15 using mRNA extracted from peripheral leukocytes. These findings suggest that mutation analysis of the CAPN3 cDNA should use skeletal muscle tissue as materials instead of peripheral blood.

Calpain ; genetics ; DNA Mutational Analysis ; DNA, Complementary ; genetics ; Exons ; genetics ; Humans ; Leukocytes ; metabolism ; Muscle Proteins ; genetics ; Muscle, Skeletal ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Calpain ; genetics ; Diabetes, Gestational ; genetics ; Female ; Genotype ; Haploidy ; Humans ; Polymorphism, Single Nucleotide ; Pregnancy

OBJECTIVEThe mechanisms of hypoxic-ischemic brain damage (HIBD) are still largely unknown. Elevation of intracellular calcium concentration and subsequent calcium-dependent proteases activation such as calpains seem to play an important role in the process of neuronal death. Calpain inhibitors showed neuroprotective effects in adult rat cerebral ischemia models. This study aimed to investigate the protective effect and associated mechanisms of calpain inhibitor-3 (MDL28170) on HIBD of neonatal rats.

METHODSSeven-day old Sprague-Dawley rats were randomly divided into three groups: the control group (n = 18), HIBD group (n = 48) and calpain inhibitor-3 treated group (MDL group, n = 48). The mice in the latter two groups were subjected to hypoxia-ischemia (HI) insult. The puppies in MDL group were intraperitoneally injected with MDL28170 (25 mg/kg) at 0, 2 and 4 h after HI, while those in the other two groups were intraperitoneally injected with normal saline instead. All the pupies were sacrificed at 6 h, 24 h and 72 h after HI. Quantitative real-time fluorescent polymerase chain reaction was employed to detect micro-calpain gene expression, immunoblotting technique was used to measure mu-calpain and caspase-3 protein activation, apoptosis of ipsilateral cortex was detected by terminal deoxynucleotidyl transferase mediated d-UTP nick end labeling staining (TUNEL). CA1 neuronal loss was counted 24 h after HI by light microscopy.

RESULTSAfter HI mu-calpain mRNA began to increase at 6 h and reached peak at 24 h compared to the control (1.805 and 4.83 vs. 1, P < 0.05); mu-calpain was activated through autolysis, the ratio of its activated fragment (76 000) vs. whole fragment (80 000) was significantly higher at 6 h (0.547 +/- 0.095) compared to the control (0.095 +/- 0.016, P < 0.05), it reached peak at 24 h (0.921 +/- 0.058, P < 0.01) and was still at a high level at 72 h (0.708 +/- 0.025, P < 0.05). Expression of activated caspase-3 protein reached peak at 24 h (3.78 +/- 0.30, P < 0.01), decreased to the same level as the control (1.56 +/- 0.07) at 72 h (1.82 +/- 0.11, P > 0.05). Apoptotic cells in the cortex ipsilateral to HI insult increased after HIBD, reached peak at 24 h (135.46 +/- 17.52/visual field) and was still markedly higher at 72 h (79.32 +/- 17.79/visual field) compared with the control (5.33 +/- 1.53/visual field, P < 0.01). At 24 h after HI CA1 neuronal loss (30.0 +/- 6.2/oil immersion lens field) in the HIBD group was significantly higher than that of the control (2.4 +/- 0.3/oil immersion lens field, P < 0.01). However, in the MDL group the expressions of mu-calpain and caspase-3 proteins were diminished, TUNEL positive cells at 6 h and 24 h were decreased and CA1 neuronal loss (18.2 +/- 2.4/oil immersion lens field, P < 0.05) was alleviated. The amount of micro-calpain mRNA was decreased in the MDL group, but there was no significant difference compared with the HIBD group.

CONCLUSIONmu-calpain gene and protein expressions increased after HI, which may contribute to the pathogenensis of HIBD. Calpain inhibitor-3 may intervene neural necrosis and apoptosis by diminishing expressions of mu-calpain and caspase-3 to play a protective role after HI insult of neonatal brain.

Animals ; Animals, Newborn ; Brain ; metabolism ; Calpain ; genetics ; metabolism ; therapeutic use ; Caspase Inhibitors ; Enzyme Inhibitors ; therapeutic use ; Female ; Hypoxia, Brain ; genetics ; metabolism ; prevention & control ; Hypoxia-Ischemia, Brain ; drug therapy ; genetics ; metabolism ; prevention & control ; In Situ Nick-End Labeling ; Injections, Intraperitoneal ; Isoenzymes ; therapeutic use ; Male ; Muscle Proteins ; therapeutic use ; Neurons ; drug effects ; enzymology ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley