Clinical Utility of Bioelectrical Impedance Analysis Parameters for Evaluating Patients with Lower Limb Lymphedema after Lymphovenous Anastomosis.

BACKGROUND: In lymphedema, lymphatic fluid accumulates in the interstitial space, and localized swelling appears. Lymphovenous anastomosis (LVA) is the most widely used surgery to rebuild a damaged lymphatic system; however, assessing outcome of LVA involves performing volume measurements, which provides limited information on body composition changes. Therefore, we analyzed the bioelectrical impedance analysis (BIA) parameters that can reflect the status of lymphedema patients who underwent LVA. METHODS: We retrospectively reviewed records of 42 patients with unilateral lower extremity lymphedema who had LVA. We measured the perioperative BIA parameters such as extracellular water (ECW) ratio and volume as defined by the percentage of excess volume (PEV). We evaluated the relationship between the amount of change in PEV and in BIA parameters before and after surgery. We confirmed the correlation between ΔPEV and BIA parameters using Spearman's correlation. RESULTS: Most patients included had secondary lymphedema due to cancer. Average age was 51.76 years and average body mass index was 23.27. PEV and all BIA parameters after surgery showed a significant difference (p < 0.01) compared with preoperative measurements. The ECW ratio aff/unaff showed the strongest correlation with PEV with a correlation coefficient of 0.473 (p < 0.01). CONCLUSION: Our findings suggest that BIA parameters, especially ECW ratio aff/unaff could reflect the status of patients with lower limb lymphedema after LVA. Appropriate use of BIA parameters may be useful in the postoperative surveillance of patients.

An analysis of fall incidence rate and risk factors in an inpatient rehabilitation unit: A retrospective study.

BACKGROUND: Accurate prediction of fall likelihood is advantageous for instituting fall prevention program in rehabilitation facilities. OBJECTIVE: This study was designed to determine the clinical measures, which can predict the risk of fall events in a rehabilitation hospital. METHODS: Medical records of 166 patients (114 males and 52 females) who were hospitalized in an adult inpatient unit of a rehabilitation hospital were retrospectively analyzed for this study. As predictor variables for assessing fall risk, demographic data and the following measurements were selectively collected from patient's medical records: Tinetti Performance-Oriented Mobility Assessment-Ambulation (POMA-G), Timed Up and Go test (TUG), 10 m walk test, 2 min walk test, Korean version Mini-Mental State Examination (K-MMSE), Korean version of the Modified Barthel Index (KMBI), Berg Balance Scale (BBS), Global Deterioration Scale (GDS), and Morse Fall Scale (Morse FS). RESULTS: The Morse FS, TUG, and age were found to be risk factors for the classification of faller and non-faller groups. CONCLUSION: This study suggests Morse FS, TUG, and age in the routine initial assessment upon admission in a rehabilitation setting, as key variables for screening the risk of fall. Additionally, the cutoff scores of Morse FS and TUG were observed to be more rigid than other clinical settings.

The test-retest reliability and minimal detectable change of the short-form Barthel Index (5 items) and its associations with chronic stroke-specific impairments.

[Purpose] To establish the test-retest reliabilities, minimal detectable change of the Short form Barthel Index and associations with stroke-specific impairments. [Subjects and Methods] The Short form-Barthel Index assessment was tested on 24 chronic stroke patients twice, 7 days apart. A relative reliability index (ICC2,1), Weighted Kappa Coefficients was used to examine the level of agreement of test-retest reliability for SF-BI, Absolute reliability indices, including the standard error of measurement and the minimal detectable change. The validity was demonstrated by spearman correlation of SF BI-total score with Postural Assessment Scale for Storke, Fugl Meyer Assessment. [Results] There was excellent agreement between test-retest for individual items of BI and total score ICC2,1=0.91 and it all showed acceptable SEM and MDC were 2.83 score, 7.84 score respectively. The item-to-total correlations were all significant, ranging from r=0.83-0.92. SF-BI showed good internal consistency. Individual items also possessed high internal consistency 0.82-0.86. The SF-BI and total score were demonstrated high concurrent validity with the PASS, FMA. [Conclusion] This study has demonstrated that the SF-BI is a useful instrument with high test-retest reliability, Absolute reliability indices, internal consistency and validity.

Inhibition of VEGF-dependent angiogenesis and tumor angiogenesis by an optimized antibody targeting CLEC14a.

The C-type lectin-like domain of CLEC14a (CLEC14a-C-type lectin-like domain [CTLD]) is a key domain that mediates endothelial cell-cell contacts in angiogenesis. However, the role of CLEC14a-CTLD in pathological angiogenesis has not yet been clearly elucidated. In this study, through complementarity-determining region grafting, consecutive deglycosylation, and functional isolation, we generated a novel anti-angiogenic human monoclonal antibody that specifically targets CLEC14a-CTLD and that shows improved stability and homogeneity relative to the parental antibody. We found that this antibody directly inhibits CLEC14a-CTLD-mediated endothelial cell-cell contact and simultaneously downregulates expression of CLEC14a on the surface of endothelial cells. Using various in vitro and in vivo functional assays, we demonstrated that this antibody effectively suppresses vascular endothelial growth factor (VEGF)-dependent angiogenesis and tumor angiogenesis of SNU182 human hepatocellular carcinoma, CFPAC-1 human pancreatic cancer, and U87 human glioma cells. Furthermore, we also found that this antibody significantly inhibits tumor angiogenesis of HCT116 and bevacizumab-adapted HCT116 human colorectal cancer cells. These findings suggest that antibody targeting of CLEC14a-CTLD has the potential to suppress VEGF-dependent angiogenesis and tumor angiogenesis and that CLEC14a-CTLD may be a novel anti-angiogenic target for VEGF-dependent angiogenesis and tumor angiogenesis.

Arteriovenous malformation of the external ear: a clinical assessment with a scoping review of the literature / Malformação arteriovenosa da orelha externa: avaliação clínica com revisão abrangente da literatura

Abstract Introduction: Auricular Arteriovenous Malformation of the external ear is a rarely encountered disease; in particular, arteriovenous malformation arising from the auricle, with spontaneous bleeding, has seldom been reported. Objective: In the current study, we report an unusual case of late-onset auricular arteriovenous malformation originating from the posterior auricular artery that was confirmed by computed tomographic angiography. The case was successfully managed by pre-surgical intravascular embolization followed by total lesion excision. Prompted by this case, we also present a scoping review of the literature. Methods: A case of a 60 year-old man with right auricular arteriovenous malformation treated in our tertiary care center, and 52 patients with auricular arteriovenous malformation described in 10 case reports and a retrospective review are presented. Auricular arteriovenous malformation can manifest as swelling of the ear, pulsatile tinnitus, pain, and/or bleeding. On physical examination, a pulsatile swelling and/or a tender mass is evident. When arteriovenous malformation is suspected, the lesions should be visualized using imaging modalities that optimally detect vascular lesions, and managed via embolization, mass excision, or auricular resection. Effectiveness of the various diagnostic methods used and the treatment outcomes were analyzed. Results: Various imaging modalities including Doppler sonography, computed tomographic angiography, magnetic resonance angiography, and/or transfemoral cerebral angiography were used to diagnose 38 cases reported in the literature. In another 15 cases, no imaging was performed; treatment was determined solely by physical examination and auscultation. Of the total of 53 cases, 12 were not treated (their symptoms were merely observed) whereas 20 underwent therapeutic embolization. In total, 32 patients, including 1 patient who was not treated and 10 with persistent or aggravated arteriovenous malformation after previous embolization, underwent mass excision or auricular resection depending on the extent of the lesion. No major postoperative complication was recorded. The postoperative follow-up duration varied from 1 month to 19 years, and only one case of unresectable, residual cervicofacial arteriovenous malformation was recorded. Conclusion: Auricular arteriovenous malformation is a rarely encountered disease, but should be suspected if a patient presents with a swollen ear and pulsatile tinnitus. Appropriate imaging is essential for diagnosis and evaluation of the extent of disease. As embolization affords only relatively poor control, total surgical removal of the vascular mass is recommended.

Resumo Introdução: Malformação Arteriovenosa Auricular da orelha externa é uma doença raramente observada, em particular, na região da aurícula, com hemorragia espontânea, tem sido infrequentemente relatada. Objetivo: No presente estudo, relatamos um caso incomum de malformação arteriovenosa auricular de início tardio originária da artéria auricular posterior confirmada por angiotomografia computadorizada. O caso foi controlado com sucesso por embolização endovascular pré-cirúrgica seguida por excisão completa da lesão. Além disso, nós também apresentamos uma revisão abrangente da literatura. Método: Um homem de 60 anos de idade com malformação arteriovenosa auricular direita tratado em nosso centro de atendimento terciário e 52 pacientes com malformação arteriovenosa auricular descritos em 10 relatos de casos e um estudo de revisão são apresentados. A malformação arteriovenosa auricular pode manifestar-se como inchaço da orelha, zumbido pulsátil, dor e/ou sangramento. Ao exame físico, um edema pulsátil e/ou uma massa sensível é evidente. Quando há suspeita de malformação arteriovenosa, as lesões devem ser visualizadas usando modalidades de imagem que detectam de maneira ideal as lesões vasculares, e tratadas por meio de embolização, excisão total da lesão, ou ressecção auricular. A eficácia dos vários métodos de diagnóstico utilizados e os desfechos do tratamento foram analisados. Resultados: Várias modalidades de imagem, incluindo ultrassonografia Doppler, angiotomografia computadorizada, angiografia por ressonância magnética e/ou angiografia cerebral transfemoral foram usadas para diagnosticar 38 casos relatados na literatura. Em outros 15 casos, nenhuma imagem foi realizada; o tratamento foi determinado unicamente pelo exame físico e ausculta. Do total de 53 casos, 12 não foram tratados (os seus sintomas foram apenas observados), enquanto que 20 foram submetidos a embolização terapêutica. No total, 32 doentes, incluindo um paciente que não foi tratado e 10 com malformação arteriovenosa persistente ou agravada após a embolização anterior, foram submetidos a excisão completa da lesão ou ressecção auricular, dependendo da extensão da lesão. Nenhuma complicação pós-operatória importante foi registrada. O tempo de seguimento pós-operatório variou de 1 mês a 19 anos, e apenas um caso de malformação arteriovenosa cervicofacial irressecável, residual foi registrado. Conclusão: A malformação arteriovenosa auricular é uma doença raramente encontrada, mas deve ser suspeitada se um paciente apresentar orelha inchada e zumbido pulsátil. A imagem apropriada é essencial para o diagnóstico e avaliação da extensão da doença. Como a embolização proporciona apenas um controle relativamente precário, a remoção cirúrgica total da lesão vascular é recomendada.

Arteriovenous malformation of the external ear: a clinical assessment with a scoping review of the literature.

INTRODUCTION: Auricular Arteriovenous Malformation of the external ear is a rarely encountered disease; in particular, arteriovenous malformation arising from the auricle, with spontaneous bleeding, has seldom been reported. OBJECTIVE: In the current study, we report an unusual case of late-onset auricular arteriovenous malformation originating from the posterior auricular artery that was confirmed by computed tomographic angiography. The case was successfully managed by pre-surgical intravascular embolization followed by total lesion excision. Prompted by this case, we also present a scoping review of the literature. METHODS: A case of a 60 year-old man with right auricular arteriovenous malformation treated in our tertiary care center, and 52 patients with auricular arteriovenous malformation described in 10 case reports and a retrospective review are presented. Auricular arteriovenous malformation can manifest as swelling of the ear, pulsatile tinnitus, pain, and/or bleeding. On physical examination, a pulsatile swelling and/or a tender mass is evident. When arteriovenous malformation is suspected, the lesions should be visualized using imaging modalities that optimally detect vascular lesions, and managed via embolization, mass excision, or auricular resection. Effectiveness of the various diagnostic methods used and the treatment outcomes were analyzed. RESULTS: Various imaging modalities including Doppler sonography, computed tomographic angiography, magnetic resonance angiography, and/or transfemoral cerebral angiography were used to diagnose 38 cases reported in the literature. In another 15 cases, no imaging was performed; treatment was determined solely by physical examination and auscultation. Of the total of 53 cases, 12 were not treated (their symptoms were merely observed) whereas 20 underwent therapeutic embolization. In total, 32 patients, including 1 patient who was not treated and 10 with persistent or aggravated arteriovenous malformation after previous embolization, underwent mass excision or auricular resection depending on the extent of the lesion. No major postoperative complication was recorded. The postoperative follow-up duration varied from 1 month to 19 years, and only one case of unresectable, residual cervicofacial arteriovenous malformation was recorded. CONCLUSION: Auricular arteriovenous malformation is a rarely encountered disease, but should be suspected if a patient presents with a swollen ear and pulsatile tinnitus. Appropriate imaging is essential for diagnosis and evaluation of the extent of disease. As embolization affords only relatively poor control, total surgical removal of the vascular mass is recommended.

A Human Antibody That Binds to the Sixth Ig-Like Domain of VCAM-1 Blocks Lung Cancer Cell Migration In Vitro.

Vascular cell adhesion molecule-1 (VCAM-1) is closely associated with tumor progression and metastasis. However, the relevance and role of VCAM-1 in lung cancer have not been clearly elucidated. In this study, we found that VCAM-1 was highly overexpressed in lung cancer tissue compared with that of normal lung tissue, and high VCAM-1 expression correlated with poor survival in lung cancer patients. VCAM-1 knockdown reduced migration of A549 human lung cancer cells into Matrigel, and competitive blocking experiments targeting the Ig-like domain 6 of VCAM-1 (VCAM-1-D6) demonstrated that the VCAM-1-D6 domain was critical for VCAM-1 mediated A549 cell migration into Matrigel. Next, we developed a human monoclonal antibody specific to human and mouse VCAM-1-D6 (VCAM-1-D6 huMab), which was isolated from a human synthetic antibody library using phage display technology. Finally, we showed that VCAM-1-D6 huMab had a nanomolar affinity for VCAM-1-D6 and that it potently suppressed the migration of A549 and NCI-H1299 lung cancer cell lines into Matrigel. Taken together, these results suggest that VCAM-1-D6 is a key domain for regulating VCAM-1-mediated lung cancer invasion and that our newly developed VCAM-1-D6 huMab will be a useful tool for inhibiting VCAM-1-expressing lung cancer cell invasion.

Ig-like domain 6 of VCAM-1 is a potential therapeutic target in TNFα-induced angiogenesis.

Tumor necrosis factor alpha (TNFα)-induced angiogenesis plays important roles in the progression of various diseases, including cancer, wet age-related macular degeneration, and rheumatoid arthritis. However, the relevance and role of vascular cell adhesion molecule-1 (VCAM-1) in angiogenesis have not yet been clearly elucidated. In this study, VCAM-1 knockdown shows VCAM-1 involvement in TNFα-induced angiogenesis. Through competitive blocking experiments with VCAM-1 Ig-like domain 6 (VCAM-1-D6) protein, we identified VCAM-1-D6 as a key domain regulating TNFα-induced vascular tube formation. We demonstrated that a monoclonal antibody specific to VCAM-1-D6 suppressed TNFα-induced endothelial cell migration and tube formation and TNFα-induced vessel sprouting in rat aortas. We also found that the antibody insignificantly affected endothelial cell viability, morphology and activation. Finally, the antibody specifically blocked VCAM-1-mediated cell-cell contacts by directly inhibiting VCAM-1-D6-mediated interaction between VCAM-1 molecules. These findings suggest that VCAM-1-D6 may be a potential novel therapeutic target in TNFα-induced angiogenesis and that antibody-based modulation of VCAM-1-D6 may be an effective strategy to suppress TNFα-induced angiogenesis.

Characterization of smooth muscle, enteric nerve, interstitial cells of Cajal, and fibroblast-like cells in the gastric musculature of patients with diabetes mellitus.

AIM: To investigate histologic abnormalities in the gastric smooth muscle of patients with diabetes mellitus (DM). METHODS: Full-thickness gastric specimens were obtained from patients undergoing surgery for gastric cancer. H&E stain and Masson's Trichrome stain were performed to assess the degree of fibrosis. Immunohistochemical staining using various antibodies was also performed [antibodies against protein gene product 9.5 (PGP9.5), neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP), neurokinin-1 (NK1) receptor, c-Kit, and platelet-derived growth factor receptor-alpha, (PDGFRα)]. Immunofluorescent staining and evaluation with confocal microscopy were also conducted. RESULTS: Twenty-six controls and 35 diabetic patients (21 short-duration patients and 14 long-duration patients) were included. There were no significant differences in basic demographics between the two groups except in mean body mass index (BMI) (higher in the DM group). Proportions of moderate-to-severe intercellular fibrosis in the muscle layer were significantly higher in the DM group than in the control group (P < 0.01). On immunohistochemical staining, c-Kit- and PDGFRα-positive immunoreactivity were significantly decreased in the DM group compared with the control group (P < 0.05). There were no statistically significant differences in PGP9.5, nNOS, VIP, and neurokinin 1 expression. On immunofluorescent staining, cellularity of interstitial cells of Cajal (ICC) was observed to decrease with increasing duration of DM. CONCLUSION: Our study suggests that increased intercellular fibrosis, loss of ICC, and loss of fibroblast-like cells are found in the smooth muscle of DM patients. These abnormalities may contribute to changes in gastric motor activity in patients with DM.

Reliability and validity of the modified functional ambulation category scale in patients with hemiparalysis.

[Purpose] This study aimed to examine the inter- and intra-rater reliability and validity of the modified functional ambulation category (mFAC) scale. [Subjects and Methods] The participants were 66 stroke patients with hemiparalysis. The inter- and intra-rater validity of the mFAC was calculated using the Spearman correlation coefficient. A score comparison of the stable or maximum gait speed with regard to mFAC and modified Rivermead Mobility Index (mRMI) performances was performed as a univariate linear regression analysis to determine how the Kruskal-Wallis test affects the mRMI and stable/maximum gait speed with regard to mFAC. [Results] The inter-rater reliability of the mFAC (intraclass coefficient [ICC]) was 0.982 (0.971-0.989), with a kappa coefficient of 0.923 and a consistency ratio of 94%. In contrast, the intra-rater reliability of the mFAC (ICC) was 0.991 (0.986-0.995), with a kappa coefficient of 0.961 and a consistency ratio of 96%, showing higher reliability. Moreover, there was a significant difference in stable/maximum gait speed between the mFAC and the mRMI. [Conclusion] Since the mFAC has sufficient inter- and intra-reliability and high validity, it can be used as an assessment tool that reflects the gait performance and mobility of stroke patients.

Characterization of Ca(2+)-Dependent Protein-Protein Interactions within the Ca(2+) Release Units of Cardiac Sarcoplasmic Reticulum.

In the heart, excitation-contraction (E-C) coupling is mediated by Ca(2+) release from sarcoplasmic reticulum (SR) through the interactions of proteins forming the Ca(2+) release unit (CRU). Among them, calsequestrin (CSQ) and histidine-rich Ca(2+) binding protein (HRC) are known to bind the charged luminal region of triadin (TRN) and thus directly or indirectly regulate ryanodine receptor 2 (RyR2) activity. However, the mechanisms of CSQ and HRC mediated regulation of RyR2 activity through TRN have remained unclear. We first examined the minimal KEKE motif of TRN involved in the interactions with CSQ2, HRC and RyR2 using TRN deletion mutants and in vitro binding assays. The results showed that CSQ2, HRC and RyR2 share the same KEKE motif region on the distal part of TRN (aa 202-231). Second, in vitro binding assays were conducted to examine the Ca(2+) dependence of protein-protein interactions (PPI). The results showed that TRN-HRC interaction had a bell-shaped Ca(2+) dependence, which peaked at pCa4, whereas TRN-CSQ2 or TRN-RyR2 interaction did not show such Ca(2+) dependence pattern. Third, competitive binding was conducted to examine whether CSQ2, HRC, or RyR2 affects the TRN-HRC or TRN-CSQ2 binding at pCa4. Among them, only CSQ2 or RyR2 competitively inhibited TRN-HRC binding, suggesting that HRC can confer functional refractoriness to CRU, which could be beneficial for reloading of Ca(2+) into SR at intermediate Ca(2+) concentrations.

Generation of a human antibody that inhibits TSPAN8-mediated invasion of metastatic colorectal cancer cells.

Tetraspanin 8 (TSPAN8) is a tumor-associated antigen implicated in tumor progression and metastasis. However, the validation of TSPAN8 as a potential therapeutic target in metastatic colorectal cancer (mCRC) has not yet been studied. In this study, through several in vitro methodologies, we identified a large extracellular loop of TSPAN8 (TSPAN8-LEL) as a key domain for regulating mCRC invasion. Using phage display technology, we developed a novel anti-TSPAN8-LEL human antibody with subnanomolar affinity that specifically recognizes amino acids 140-205 of TSPAN8-LEL in a conformation-dependent manner. Finally, we demonstrated that the antibody specifically reduces invasion in the HCT116 and LoVo mCRC cell lines more potently than in the HCT-8 and SW480 non-mCRC cell lines. Our data suggest that TSPAN8-LEL may play an important role in mCRC cell invasion, and that the antibody we have developed could be a useful tool for inhibiting the invasion of TSPAN8-expressing mCRCs.

Stromal interaction molecule 1 (STIM1) regulates sarcoplasmic/endoplasmic reticulum Ca²âº-ATPase 1a (SERCA1a) in skeletal muscle.

Stromal interaction molecule 1 (STIM1) mediates Ca2+ movements from the extracellular space to the cytosol through a store-operated Ca2+ entry (SOCE) mechanism in various cells including skeletal muscle cells. In the present study, to reveal the unidentified functional role of the STIM1 C terminus from 449 to 671 amino acids in skeletal muscle, binding assays and quadrupole time-of-flight mass spectrometry were used to identify proteins binding in this region along with proteins that mediate skeletal muscle contraction and relaxation. STIM1 binds to sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) via this region (called STIM1-SBR). The binding was confirmed in endogenous full-length STIM1 in rabbit skeletal muscle and mouse primary skeletal myotubes via co-immunoprecipitation assay and immunocytochemistry. STIM1 knockdown in mouse primary skeletal myotubes decreased Ca2+ uptake from the cytosol to the sarcoplasmic reticulum (SR) through SERCA1a only at micromolar cytosolic Ca2+ concentrations, suggesting that STIM1 could be required for the full activity of SERCA1a possibly during the relaxation of skeletal muscle. Various Ca2+ imaging experiments using myotubes expressing STIM1-SBR suggest that STIM1 is involved in intracellular Ca2+ distributions between the SR and the cytosol via regulating SERCA1a activity without affecting SOCE. Therefore, in skeletal muscle, STIM1 could play an important role in regulating Ca2+ movements between the SR and the cytosol.

The effects of additional action observational training for functional electrical stimulation treatment on weight bearing, stability and gait velocity of hemiplegic patients.

[Purpose] The purpose of this study was to evaluate the functional effects of additional action observational training for functional electrical stimulation treatment on weight bearing, stability and gait velocity of hemiplegic patients. [Subjects and Methods] Twenty subjects were randomized into two groups. Subjects more than six months post-stroke participated. Balance and gait velocity were measured at the baseline, and after six weeks of treatment. Both groups received functional electrical stimulation treatment. The experimental group additionally received action observational training. The paired t-test was used to analyze differences in the outcome measures between before and after the intervention. The difference between the groups was compared using the independent t-test. [Results] The experimental group showed significant increases in weight bearing (anterior·posterior, right·left) on the affected side, stability index and gait velocity. The control group showed only a significant increase in anterior·posterior weight bearing on the affected side. Moreover, according to the comparison of training effects between in the two groups, the variables of anterior·posterior weight bearing, stability index and gait velocity revealed a statistically significant difference. [Conclusion] Additional action observational training for functional electrical stimulation treatment should be considered as a therapeutic method in physical therapy for the improvement of weight bearing, stability index and gait velocity of hemiplegic patients.

A massive fibrolipoma in the temple.

We treated a 54-year-old man who presented with a massive mass on his temple. The mass was excised completely and sent to a pathologist. Histopathologic analysis indicated that the mass was a fibrolipoma.Fibrolipoma is a rare subtype of lipoma, and no report of a massive fibrolipoma of the temple has been reported previously. In this study, we provide detailed information and discuss the differential diagnosis of a very large facial mass.

Targeted ablation of the histidine-rich Ca(2+)-binding protein (HRC) gene is associated with abnormal SR Ca(2+)-cycling and severe pathology under pressure-overload stress.

The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild-type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca(2+) transients, and maximal SR Ca(2+) uptake rates in the heart. However, after-contractions were developed in 50 % of HRC-KO cardiomyocytes, compared to 11 % in WT mice under stress conditions of high-frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca(2+) spontaneous SR Ca(2+) release and delayed afterdepolarizations with ISO in HRC-KO, compared to WT cells. The frequency of Ca(2+) sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca(2+) load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca(2+)-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC-null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca(2+)-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.

Mitsugumin 53 attenuates the activity of sarcoplasmic reticulum Ca(2+)-ATPase 1a (SERCA1a) in skeletal muscle.

Mitsugumin 53 (MG53) is a member of the membrane repair system in skeletal muscle. However, the roles of MG53 in the unique functions of skeletal muscle have not been addressed, although it is known that MG53 is expressed only in skeletal and cardiac muscle. In the present study, MG53-binding proteins were examined along with proteins that mediate skeletal muscle contraction and relaxation using the binding assays of various MG53 domains and quadrupole time-of-flight mass spectrometry. MG53 binds to sarcoplasmic reticulum Ca(2+)-ATPase 1a (SERCA1a) via its tripartite motif (TRIM) and PRY domains. The binding was confirmed in rabbit skeletal muscle and mouse primary skeletal myotubes by co-immunoprecipitation and immunocytochemistry. MG53 knockdown in mouse primary skeletal myotubes increased Ca(2+)-uptake through SERCA1a (more than 35%) at micromolar Ca(2+) but not at nanomolar Ca(2+), suggesting that MG53 attenuates SERCA1a activity possibly during skeletal muscle contraction or relaxation but not during the resting state of skeletal muscle. Therefore MG53 could be a new candidate for the diagnosis and treatment of patients with Brody syndrome, which is not related to the mutations in the gene coding for SERCA1a, but still accompanies exercise-induced muscle stiffness and delayed muscle relaxation due to a reduction in SERCA1a activity.

AAV-mediated knock-down of HRC exacerbates transverse aorta constriction-induced heart failure.

BACKGROUND: Histidine-rich calcium binding protein (HRC) is located in the lumen of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA affecting Ca(2+) cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca(2+) homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca(2+) cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV)-mediated HRC knock-down (KD) systems, respectively. METHODOLOGY/PRINCIPAL FINDINGS: AAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in failing heart (FH). Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH), since predesigned siRNA-mediated HRC-KD enhanced Ca(2+) cycling and increased activities of RyR2 and SERCA2 without change in SR Ca(2+) load in neonatal rat ventricular cells (NRVCs) and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay. CONCLUSIONS/SIGNIFICANCE: Increased Ca(2+) leak and cytosolic Ca(2+) concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca(2+) cycling.

The chemical chaperone 4-phenylbutyric acid attenuates pressure-overload cardiac hypertrophy by alleviating endoplasmic reticulum stress.

Evidence has shown that endoplasmic reticulum stress (ERS) is associated with the pathogenesis of cardiac hypertrophy. The aim of this study was to investigate whether direct alleviation of ER stress by 4-phenylbutyric acid (PBA), a known chemical chaperone drug, could attenuate pressure-overload cardiac hypertrophy in mice. The effects of orally administered PBA (100mg/kg body weight daily for a week) were examined using mice undergoing transverse aortic constriction (TAC-mice), an animal model to produce pressure overload. TAC application for 1 week led to a 1.8-fold increase in the ratio of the heart weight over body weight (HW/BW) and up-regulation of the hypertrophy markers ANF and BNF accompanied by up-regulation of ERS markers (GRP78, p-PERK, and p-elF2α). The oral administration of PBA to the TAC-mice reduced hypertrophy (19%) and severely downregulated the fibrosis-related genes (transforming growth factor-ß1, phospho-smad2, and pro-collagen isoforms). We conclude that ERS is induced as a consequence of remodeling during pathological hypertrophy and that PBA may help to relieve ERS and play a protective role against cardiac hypertrophy and possibly heart failure. We suggest PBA as a novel therapeutic agent for cardiac hypertrophy and fibrosis.

PICOT increases cardiac contractility by inhibiting PKCζ activity.

Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT) has distinct anti-hypertrophic and inotropic functions. We have previously shown that PICOT exerts its anti-hypertrophic effect by inhibiting calcineurin-NFAT signaling through its C-terminal glutaredoxin domain. However, the mechanism underlying the inotropic effect of PICOT is unknown. The results of protein pull-down experiments showed that PICOT directly binds to the catalytic domain of PKCζ through its N-terminal thioredoxin-like domain. Purified PICOT protein inhibited the kinase activity of PKCζ in vitro, which indicated that PICOT is an endogenous inhibitor of PKCζ. The inhibition of PKCζ activity with a PKCζ-specific pseudosubstrate peptide inhibitor was sufficient to increase the cardiac contractility in vitro and ex vivo. Overexpression of PICOT or inhibition of PKCζ activity down-regulated PKCα activity, which led to the elevation of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a activity, concomitant with the increased phosphorylation of phospholamban (PLB). Overexpression of PICOT or inhibition of PKCζ activity also down-regulated protein phosphatase (PP) 2A activity, which subsequently resulted in the increased phosphorylation of troponin (Tn) I and T, key myofilament proteins associated with the regulation of contractility. PICOT appeared to inhibit PP2A activity through the disruption of the functional PKCζ/PP2A complex. In contrast to the overexpression of PICOT or inhibition of PKCζ, reduced PICOT expression resulted in up-regulation of PKCα and PP2A activities, followed by decreased phosphorylation of PLB, and TnI and T, respectively, supporting the physiological relevance of these events. Transgene- or adeno-associated virus (AAV)-mediated overexpression of PICOT restored the impaired contractility and prevented further morphological and functional deterioration of the failing hearts. Taken together, the results of the present study suggest that PICOT exerts its inotropic effect by negatively regulating PKCα and PP2A activities through the inhibition of PKCζ activity. This finding provides a novel insight into the regulation of cardiac contractility.