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Background: Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3 , regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure. Methods: We generated the knock-in mice ( Mylk3 +/fs and Mylk 3 fs/fs ) with a familial dilated cardiomyopathy–associated MYLK3 frameshift mutation ( MYLK3 +/fs ) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell–derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154). Results: Both mice ( Mylk3 +/fs and Mylk 3 fs/fs ) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose–dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_ MYLK3 vector. MYLK3 +/fs induced pluripotent stem cell–derived cardiomyocytes reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_ MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the V max for ventricular myosin regulatory light chain phosphorylation without affecting the K m . LEUO-1154 treatment of human MYLK3 +/fs induced pluripotent stem cell–derived cardiomyocytes restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3 / PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes. Conclusions: cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.

Dilated cardiomyopathy (DCM) is a major cause of heart failure, characterized by ventricular dilatation and systolic dysfunction. Familial DCM is reportedly caused by mutations in more than 50 genes, requiring precise disease stratification based on genetic information. However, the underlying genetic causes of 60 to 80% of familial DCM cases remain unknown. Here, we identified that homozygous truncating mutations in the gene encoding Bcl-2–associated athanogene (BAG) co-chaperone 5 ( BAG5 ) caused inherited DCM in five patients among four unrelated families with complete penetrance. BAG5 acts as a nucleotide exchange factor for heat shock cognate 71 kDa protein (HSC70), promoting adenosine diphosphate release and activating HSC70-mediated protein folding. Bag5 mutant knock-in mice exhibited ventricular dilatation, arrhythmogenicity, and poor prognosis under catecholamine stimulation, recapitulating the human DCM phenotype, and administration of an adeno-associated virus 9 vector carrying the wild-type BAG5 gene could fully ameliorate these DCM phenotypes. Immunocytochemical analysis revealed that BAG5 localized to junctional membrane complexes (JMCs), critical microdomains for calcium handling. Bag5 -mutant mouse cardiomyocytes exhibited decreased abundance of functional JMC proteins under catecholamine stimulation, disrupted JMC structure, and calcium handling abnormalities. We also identified heterozygous truncating mutations in three patients with tachycardia-induced cardiomyopathy, a reversible DCM subtype associated with abnormal calcium homeostasis. Our study suggests that loss-of-function mutations in BAG5 can cause DCM, that BAG5 may be a target for genetic testing in cases of DCM, and that gene therapy may potentially be a treatment for this disease.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that shows progressive muscle weakness. A few treatments exist including symptomatic therapies, which can prolong survival or reduce a symptom; however, no fundamental therapies have been found. As a therapeutic strategy, enhancing muscle force is important for patients’ quality of life. In this study, we focused on skeletal muscle-specific myosin regulatory light chain kinase (skMLCK), which potentially enhances muscle contraction, as overexpression of skMLCK was thought to improve muscle function. The adeno-associated virus serotype 6 encoding skMLCK (AAV6/skMLCK) and eGFP (control) was produced and injected intramuscularly into the lower limbs of SOD1G37R mice, which are a familial ALS model. AAV6/skMLCK showed the successful expression of skMLCK in the muscle tissues. Although the control did not affect the muscle force in both of the WT and SOD1G37R mice, AAV6/skMLCK enhanced the twitch force of SOD1G37R mice and the tetanic force of WT and SOD1G37R mice. These results indicate that overexpression of skMLCK can enhance the tetanic force of healthy muscle as well as rescue weakened muscle function. In conclusion, the gene transfer of skMLCK has the potential to be a new therapy for ALS as well as for other neuromuscular diseases.

Arrhythmogenic cardiomyopathy (ACM) caused by TMEM43 p.S358L is a fully penetrant heart disease that results in impaired cardiac function or fatal arrhythmia. However, the molecular mechanism of ACM caused by the TMEM43 variant has not yet been fully elucidated. In this study, we generated knock-in (KI) rats harboring a Tmem43 p.S358L mutation and established induced pluripotent stem cells (iPSCs) from patients based on the identification of TMEM43 p.S358L variant from a family with ACM. The Tmem43-S358L KI rats exhibited ventricular arrhythmia and fibrotic myocardial replacement in the subepicardium, which recapitulated the human ACM phenotype. The four-transmembrane protein TMEM43 with the p.S358L variant (TMEM43S358L ) was found to be modified by N-linked glycosylation in both KI rat cardiomyocytes and patient-specific iPSC-derived cardiomyocytes. TMEM43S358L glycosylation increased under the conditions of enhanced endoplasmic reticulum (ER) stress caused by pharmacological stimulation or age-dependent decline of the ER function. Intriguingly, the specific glycosylation of TMEM43S358L resulted from the altered membrane topology of TMEM43. Moreover, unlike TMEM43WT , which is mainly localized to the ER, TMEM43S358L accumulated at the nuclear envelope of cardiomyocytes with the increase in glycosylation. Finally, our comprehensive transcriptomic analysis demonstrated that the regional differences in gene expression patterns between the inner and outer layers observed in the wild type myocardium were partially diminished in the KI myocardium prior to exhibiting histological changes indicative of ACM. Altogether, these findings suggest that the aberrant accumulation of TMEM43S358L underlies the pathogenesis of ACM caused by TMEM43 p.S358L variant by affecting the transmural gene expression within the myocardium.

The purpose of this study is to revalidate the relationship between total fertility rate (TFR) and labor female participation rate using time series data in Japan. The discussion about the positive correlation between female labor participation and TFR continues, and there is a possibility that this positive relationship is spurious. Considering endogeneity of the two variables, it could be pointed out that a potential variable such as female wage might be the true cause affecting both these variables. The female wage plays an important role in this framework of TFR and labor market of women. Thus, wage is the third variable that has an influence on both the TFR and labor participation rate. From the result of a structural VAR model analysis, female wage has a negative impact on both TFR and labor participation rate simultaneously. In Japan, from the early 1990s, the government implemented various policies to improve fertility rate. To confirm the effectiveness of government policies in improving the TFR, we added policy variable to the structural VAR model and found that government policies have a strong positive impact on the TFR. It could be argued that these policies improve the rather incompatible situation of rearing a child while simultaneously providing labor.

The respiratory chain (RC) transports electrons to form a proton motive force that is required for ATP synthesis in the mitochondria. RC disorders cause mitochondrial diseases that have few effective treatments; therefore, novel therapeutic strategies are critically needed. We previously identified Higd1a as a positive regulator of cytochrome c oxidase (CcO) in the RC. Here, we test that Higd1a has a beneficial effect by increasing CcO activity in the models of mitochondrial dysfunction. We first demonstrated the tissue-protective effects of Higd1a via in situ measurement of mitochondrial ATP concentrations ([ATP]mito) in a zebrafish hypoxia model. Heart-specific Higd1a overexpression mitigated the decline in [ATP]mito under hypoxia and preserved cardiac function in zebrafish. Based on the in vivo results, we examined the effects of exogenous HIGD1A on three cellular models of mitochondrial disease; notably, HIGD1A improved respiratory function that was coupled with increased ATP synthesis and demonstrated cellular protection in all three models. Finally, enzyme kinetic analysis revealed that Higd1a significantly increased the maximal velocity of the reaction between CcO and cytochrome c without changing the affinity between them, indicating that Higd1a is a positive modulator of CcO. These results corroborate that Higd1a, or its mimic, provides therapeutic options for the treatment of mitochondrial diseases.

Oxidative phosphorylation generates most of the ATP in respiring cells. ATP is an essential energy source, especially in cardiomyocytes because of their continuous contraction and relaxation. Previously, we reported that G0/G1 switch gene 2 (G0S2) positively regulates mitochondrial ATP production by interacting with FOF1-ATP synthase. G0S2 overexpression mitigates ATP decline in cardiomyocytes and strongly increases their hypoxic tolerance during ischemia. Here, we show that G0S2 protein undergoes proteasomal degradation via a cytosolic molecular triage system and that inhibiting this process increases mitochondrial ATP production in hypoxia. First, we performed screening with a library of siRNAs targeting ubiquitin-related genes and identified RING finger protein 126 (RNF126) as an E3 ligase involved in G0S2 degradation. RNF126-deficient cells exhibited prolonged G0S2 protein turnover and reduced G0S2 ubiquitination. BCL2-associated athanogene 6 (BAG6), involved in the molecular triage of nascent membrane proteins, enhanced RNF126-mediated G0S2 ubiquitination both in vitro and in vivo. Next, we found that Glu-44 in the hydrophobic region of G0S2 acts as a degron necessary for G0S2 polyubiquitination and proteasomal degradation. Because this degron was required for an interaction of G0S2 with BAG6, an alanine-replaced G0S2 mutant (E44A) escaped degradation. In primary cultured cardiomyocytes, both overexpression of the G0S2 E44A mutant and RNF126 knockdown effectively attenuated ATP decline under hypoxic conditions. We conclude that the RNF126/BAG6 complex contributes to G0S2 degradation and that interventions to prevent G0S2 degradation may offer a therapeutic strategy for managing ischemic diseases.

Enhancers regulate gene expressions in a tissue- and pathology-specific manner by altering its activities. Plasma levels of atrial and brain natriuretic peptides, encoded by the Nppa and Nppb, respectively, and synthesized predominantly in cardiomyocytes, vary depending on the severity of heart failure. We previously identified the noncoding conserved region 9 (CR9) element as a putative Nppb enhancer at 22-kb upstream from the Nppb gene. However, its regulatory mechanism remains unknown. Here, we therefore investigated the mechanism of CR9 activation in cardiomyocytes using different kinds of drugs that induce either cardiac hypertrophy or cardiac failure accompanied by natriuretic peptides upregulation. Chronic treatment of mice with either catecholamines or doxorubicin increased CR9 activity during the progression of cardiac hypertrophy to failure, which is accompanied by proportional increases in Nppb expression. Conversely, for cultured cardiomyocytes, doxorubicin decreased CR9 activity and Nppb expression, while catecholamines increased both. However, exposing cultured cardiomyocytes to mechanical loads, such as mechanical stretch or hydrostatic pressure, upregulate CR9 activity and Nppb expression even in the presence of doxorubicin. Furthermore, the enhancement of CR9 activity and Nppa and Nppb expressions by either catecholamines or mechanical loads can be blunted by suppressing mechanosensing and mechanotransduction pathways, such as muscle LIM protein (MLP) or myosin tension. Finally, the CR9 element showed a more robust and cell-specific response to mechanical loads than the -520-bp BNP promoter. We concluded that the CR9 element is a novel enhancer that responds to mechanical loads by upregulating natriuretic peptides expression in cardiomyocytes.

Japanese society faces a low fertility rate and population decline, and the declining population brings about a shortage of labor forces. Therefore, female labor participation is attracting attention. However, cross-sectional data show a positive relationship between the total fertility rate (TFR) and female labor participation in recent years, while the time series data suggest a negative relationship. This may be a puzzle waiting to be solved. This chapter empirically interprets the dynamic relationship between the TFR, female wage, and labor participation from the time series perspective, using Japan’s experience. Generally, the VAR model is used to confirm the dynamic relationship among relevant variables; however, it is not easy to use the VAR model because the variables have a unit root. This study verifies the dynamic relationship using the error correction model (ECM) and Bayesian vector autoregression (BVAR) models. Therefore, both ECM and BVAR models show a similar result. They show that female wages negatively impact the fertility rate, and a similar result was obtained from both ECM and BVAR models. Women’s wages are an opportunity cost of having children, and we reconfirmed this fact in Japan’s experience. Additionally, this means that the compatible situation for women between labor provision and rearing children has been insufficient in Japan. Furthermore, adding the government benefits to young families to the BVAR model shows that this positively affects the TFR by calculating impulse response function. This can be interpreted as the government benefits reducing the opportunity cost of children. Hence, the intervention by the government to improve the TFR is appropriate.

A long-term decline trend in the TFR has been observed in almost all the OECD countries, although there are differences in the country-level or speed of change. Many factors cause the decline in the TFR. However, there are common factors in OECD countries, despite many different social and economic backgrounds. Thus, this chapter explores these common factors using panel econometrics analysis. Furthermore, this research is distinguished from the past studies by other researchers as it confirms the effectiveness of government policy tools in improving TFR. This research uses balanced panel data for OECD countries from 1986 to 2017 and prepares several different balanced panel datasets. To explore all factors that determine birth behavior is almost impossible because several variables are related to the TFR. Therefore, we chose four kinds of variables: labor market, macroeconomic environments, political tools, and demographic conditions. The empirical results using the basic dataset are not statistically significant in a fixed-effect model with more explanatory variables. However, while the female labor participation (FLP) rate correlates positively with TFR in the simple pooling model, the female unemployment rate (FUR) negatively impacts TFR, being statistically significant in all cases. As for the macroeconomic environment, the GDP per capita affects TFR negatively, and on the other side the economic growth rate positively affects the TFR. It can be interpreted that the expectation for better economic circumstances in the future improves the decision to have children. Furthermore, while the coefficient of the length of maternity and parental leave has negative signs, social expenditure has a positive influence on the TFR. Lastly, the estimated coefficient of the demographic variable, that is, the young female population ratio to the total population and the ratio of a child born out of wedlock, is positive. Additionally, we estimated the dynamic panel model. However, the lagged variable of TFR explains the current TFR. Additionally, we estimate using the AR model. It is required to show the time series characteristics to deal with such series. More concretely, stationarity is a necessary condition to estimate the time series model. Confirming the unit root test for main variables, we concluded that those variables contain a unit root, and there is no cointegration among them. We confirmed the same conclusions using different variables, as in the level variables.

This chapter examines the background of the disparity in the TFR at the municipality level. The large regional disparity in the TFR is driven by the differences in child-related costs, the work/life balance between employment and child-rearing for young families, the existence of a young population, and the policies of municipalities. Among these factors, we consider the relationship between the TFR and population density in particular. In economic studies of birth behavior, direct children-related and opportunity costs are important factors affecting the birth behavior of young families. Furthermore, those costs are heavily determined by the place of residence. It is often reported that the TFR is relatively low in municipalities with high population density. Urban areas with high population density are incompatible for young families raising children and working, and rarely provide nursery facilities. Additionally, the high housing cost generates suburbanization and leads to long-distance commuting. Hence, population density is a proxy variable for children-related costs. Additionally, the rate of female labor force participation is used as a proxy for an environment in which employment and child-rearing are compatible and flexible for female workers and young families. We also adopt the ratio of young women to the total population to represent the age structure in the municipality and variables such as child welfare expenses, nursery school capacity, and the number of children on the waiting list for nursery school to account for the municipality’s fertility rate improvement policy. Using the TFR data in 2015, we examined the relationship between the TFR and socioeconomic variables. Furthermore, we prepared pooled data, including the TFR and socioeconomic variables, adding 2010 data to 2015 data. A three-period dataset including data in 2015, 2010, and 2005 is also used in the empirical analysis. We find that the coefficient of population density is negative and that of the female labor force participation rate is positive, and both are statistically significant. These findings suggest that children-related costs negatively influence the TFR and the compatible environment has a positive influence. Finally, a large young population and the government’s policy for raising the TFR positively affect the TFR. In contrast, an increase in the number of children on the waiting list for nursery school negatively affects the TFR.

The organ of Corti is an auditory organ located in the cochlea, comprising hair cells (HCs) and other supporting cells. Cellular shape changes of HCs are important for the development of auditory epithelia and hearing function. It was previously observed that HCs and inner sulcus cells (ISCs) demonstrate cellular shape changes similar to the apical constriction of the neural epithelia. Apical constriction is induced via actomyosin cable contraction in the apical junctional complex and necessary for the physiological function of the epithelium. Actomyosin cable contraction is mainly regulated by myosin regulatory light chain (MRLC) phosphorylation by myosin light chain kinase (MLCK). However, MRLC and MLCK isoforms expressed in HCs and ISCs are unknown. Hence, we investigated the expression patterns and roles of MRLCs and MLCKs in HCs. Droplet digital PCR revealed that HCs expressed MYL12A/B and MYL9, which are non-muscle MRLC and smooth muscle MLCK (smMLCK), respectively. Immunofluorescence staining throughout the organ of Corti demonstrated that only MYL12 was expressed in the apical portion of HCs, whereas MYL12 and MYL9 were expressed on ISCs. In addition, purified MYL12B was phosphorylated by smMLCK in vitro, and the harvested HCs contained phosphorylated MYL12. Furthermore, accompanied by the expansion of the cell area of outer HCs, MYL12 phosphorylation was reduced by ML-7, which is an inhibitor of smMLCK. In conclusion, MYL12 phosphorylation by smMLCK contributed to the apical constriction-like cellular shape change of HCs possibly relating to the development of auditory epithelia and hearing function.

AMP‐activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP‐170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell–cell junction present between cardiomyocytes. A contractile inhibitor, MYK‐461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK‐461, suggesting that the localization of AMPK is regulated by mechanical stress. Time‐lapse imaging analysis revealed that the inhibition of CLIP‐170 Ser‐311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK‐461 increased the individual cell area of cardiomyocytes in CLIP‐170 phosphorylation‐dependent manner. Moreover, heart‐specific CLIP‐170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP‐170 at the intercalated disks., Mechanical stress elicited by the heart contractility promotes AMPK localization to the intercalated discs, where AMPK regulates microtubule dynamics and cardiomyocyte function.

Background Biphenotypic sinonasal sarcoma (BSNS) is a rare sarcoma with neural and myogenic pathological features. Histopathologically, BSNS has been regarded as a low-malignant tumor with favorable prognosis. Herein, we report a case of BSNS with a seemingly high-grade malignancy and an extremely acute exacerbation. Case description A 64-year-old woman was diagnosed with BSNS and underwent surgical extraction of a tumor twice. Two months later after the second operation, recurrence occurred, with intracranial tumor extension as well as infiltrations around the carotid artery. Two cycles of chemotherapy with doxorubicin and ifosfamide were administered, which significantly reduced the tumor volume. Subsequently, a treatment with oral pazopanib was administered. No tumor progression was observed in the next 2 and a half months, followed by local progression. Discussion Our case seems to be entirely different from those described in previous reports on BSNS and should be regarded clinically as an extremely high malignant tumor. This high malignancy involved the high proliferative activity and invasiveness of critical organs such as in the intracranial area. AI therapy consisted of a regimen with doxorubicin and ifosfamide showed anti-tumor effects, as well as a temporary inhibition of tumor progression was achieved with pazopanib. Conclusion AI therapy and pazopanib might be considered as a potential postoperative adjuvant therapy and second-line therapy for severe BSNS.

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 °C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 ± 0.10 mol/min/mol kinase and the average Km was around 0.5 USA µM at 25 °C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.

SummaryAMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated discs in the heart, a specific cell-cell junction present between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of AMPK at the intercalated discs, and the effect was reversed by the removal of MYK-461, suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated discs. Interestingly, MYK-461 increased the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. Moreover, heart-specific CLIP-170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP-170 at the intercalated discs.

Summary Introduction Bone defect reconstruction in orbital wall fractures with absorbable alloplastic such as the unsintered hydroxyapatite and poly L-lactide composite (u-HA/PLLA) system is gaining popularity. The u-HA/PLLA material has osteoconductive and osteosynthetic properties. However, quantitative, long-term outcome data after the use of u-HA/PLLA for orbital wall fractures are lacking. Patients and Methods We retrospectively analyzed 115 patients who underwent surgical repair of orbital wall fractures with a u-HA/PLLA sheet from 2011 to 2016. A chart review was performed, and the time-dependent changes at fracture sites were assessed by imaging. The immediate postoperative and the latest follow-up bony orbital volumes of the affected side were compared. Results Seventy patients were eligible for this study (mean age, 44.6 ± 22.1 years; 48 men and 22 women; mean follow-up period, 29.7 ± 12.8 months). Except for one case of hematoma, there were no postoperative wound complications. Of the 70 patients, 10 had postoperative diplopia and 2 had enophthalmos; these conditions were presumably caused by the extension and severity of the fracture. Satisfactory reduction in the entire orbital wall, without pathological changes, was demonstrated. There were no significant differences in the mean bony orbital volumes of the affected side immediately after surgery (24.774 ± 3.092 cm3) and at the latest follow-up (24.749 ± 3.205 cm3) (p = 0.756). Conclusion The u-HA/PLLA sheet is useful for orbital wall fracture reconstruction because of its desirable handling characteristics, initial mechanical strength, long-term maintenance of structural stability, radiopacity, and few associated complications. Future randomized controlled trials need to be performed to compare u-HA/PLLA with other conventional materials.

Most eukaryotic cells generate adenosine triphosphate (ATP) through the oxidative phosphorylation system (OXPHOS) to support cellular activities. In cultured cell-based experiments, we recently identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS, and showed that G0s2 protects cultured cardiomyocytes from hypoxia. In this study, we examined the in vivo protective role of G0s2 against hypoxia by generating both loss-of-function and gain-of-function models of g0s2 in zebrafish. Zebrafish harboring transcription activator-like effector nuclease (TALEN)-mediated knockout of g0s2 lost hypoxic tolerance. Conversely, cardiomyocyte-specific transgenic zebrafish hearts exhibited strong tolerance against hypoxia. To clarify the mechanism by which G0s2 protects cardiac function under hypoxia, we introduced a mitochondrially targeted FRET-based ATP biosensor into zebrafish heart to visualize ATP dynamics in in vivo beating hearts. In addition, we employed a mosaic overexpression model of g0s2 to compare the contraction and ATP dynamics between g0s2-expressing and non-expressing cardiomyocytes, side-by-side within the same heart. These techniques revealed that g0s2-expressing cardiomyocyte populations exhibited preserved contractility coupled with maintained intra-mitochondrial ATP concentrations even under hypoxic condition. Collectively, these results demonstrate that G0s2 provides ischemic tolerance in vivo by maintaining ATP production, and therefore represents a promising therapeutic target for hypoxia-related diseases.

Oxidative phosphorylation generates most of the ATP in respiring cells. ATP is an essential energy source, especially in cardiomyocytes because of their continuous contraction and relaxation. Previously, we reported that G(0)/G(1) switch gene 2 (G0S2) positively regulates mitochondrial ATP production by interacting with F(O)F(1)-ATP synthase. G0S2 overexpression mitigates ATP decline in cardiomyocytes and strongly increases their hypoxic tolerance during ischemia. Here, we show that G0S2 protein undergoes proteasomal degradation via a cytosolic molecular triage system and that inhibiting this process increases mitochondrial ATP production in hypoxia. First, we performed screening with a library of siRNAs targeting ubiquitin-related genes and identified RING finger protein 126 (RNF126) as an E3 ligase involved in G0S2 degradation. RNF126-deficient cells exhibited prolonged G0S2 protein turnover and reduced G0S2 ubiquitination. BCL2-associated athanogene 6 (BAG6), involved in the molecular triage of nascent membrane proteins, enhanced RNF126-mediated G0S2 ubiquitination both in vitro and in vivo. Next, we found that Glu-44 in the hydrophobic region of G0S2 acts as a degron necessary for G0S2 polyubiquitination and proteasomal degradation. Because this degron was required for an interaction of G0S2 with BAG6, an alanine-replaced G0S2 mutant (E44A) escaped degradation. In primary cultured cardiomyocytes, both overexpression of the G0S2 E44A mutant and RNF126 knockdown effectively attenuated ATP decline under hypoxic conditions. We conclude that the RNF126/BAG6 complex contributes to G0S2 degradation and that interventions to prevent G0S2 degradation may offer a therapeutic strategy for managing ischemic diseases.

Summary Objective This study aims at selecting recipient vessels for free flap following intra-arterial chemoradiotherapy. At present, many centers combine intra-arterial chemotherapy and concomitant radiotherapy for treating head and neck cancer with favorable results. However, some patients develop recurrent, residual disease/complications after completing treatment protocols and thus require free-flap surgery. The feasibility of microsurgery following intra-arterial chemoradiotherapy remains unclear. Study design and methods We retrospectively analyzed 730 patients who underwent free-flap surgery for managing treatment-related complications/defects following head and neck surgery from January 1999 to February 2012. All patients were categorized into two groups according to the presence (group A)/absence (group B) of a history of intra-arterial chemotherapy. Microvascular revision rates were compared between the groups. In group A, the correlation between catheterization sites and recipient vessels used for microsurgery was assessed. Results A total of 769 free flaps were created in 730 patients and comprised 36 and 733 flaps in groups A and B, respectively. Microvascular revision rates were 2.8% (1/36) and 4.2% (31/733) in groups A and B, respectively, with no significant difference observed between the groups. No statistically significant difference was observed when only cases of arterial thrombosis were included. Vessels used for recipient anastomoses were not directly exposed to intra-arterially injected agents. Conclusion Preoperative intra-arterial chemoradiotherapy does not significantly increase the risk of microvascular revisions, particularly artery-related revisions, if vessels selectively catheterized and perfused with chemotherapeutic agents during intra-arterial chemotherapy are avoided as recipient vessels.

Background: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. Methods: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. Results: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5 ) to form the acetylcholine-activated potassium channel ( I KACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5 , suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of I KACh channel function by increasing the basal current, even in the absence of m 2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective I KACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. Conclusions: The I KACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant I KACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective I KACh channel blocker. Thus, the I KACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the I KACh channel.

Dipeptidyl peptidase 4 (DPP4) inhibitors suppress the metabolism of the potent antihyperglycemic hormone glucagon-like peptide-1 (GLP-1). DPP4 was recently shown to provide cardioprotection through a reduction of infarct size, but the mechanism for this remains elusive. Known interactions between DPP4 and adenosine deaminase (ADA) suggest an involvement of adenosine signaling in DPP4 inhibitor-mediated cardioprotection. We tested whether the protective mechanism of the DPP4 inhibitor alogliptin against myocardial ischemia-reperfusion injury involves GLP-1- and/or adenosine-dependent signaling in canine hearts. In anesthetized dogs, the coronary artery was occluded for 90 min followed by reperfusion for 6 h. A 4-day pretreatment with alogliptin reduced the infarct size from 43.1 ± 2.5% to 17.1 ± 5.0% without affecting collateral flow and hemodynamic parameters, indicating a potent antinecrotic effect. Alogliptin also suppressed apoptosis as demonstrated by the following analysis: 1) reduction in the Bax-to-Bcl2 ratio; 2) cytochrome c release, 3) an increase in Bad phosphorylation in the cytosolic fraction; and 4) terminal deoxynucleotidyl transferase dUTP nick end labeling assay. This DPP4 inhibitor did not affect blood ADA activity or adenosine concentrations. In contrast, the nonselective adenosine receptor blocker 8-( p-sulfophenyl)theophylline (8SPT) completely blunted the effect of alogliptin. Alogliptin did not affect Erk1/2 phosphorylation, but it did stimulate phosphorylation of Akt, glycogen synthase kinase-3β, and cAMP response element-binding protein (CREB). Only 8SPT prevented alogliptin-induced CREB phosphorylation. In conclusion, the DPP4 inhibitor alogliptin suppresses ischemia-reperfusion injury via adenosine receptor- and CREB-dependent signaling pathways.

Cytochrome c oxidase (CcO) is the only enzyme that uses oxygen to produce a proton gradient for ATP production during mitochondrial oxidative phosphorylation. Although CcO activity increases in response to hypoxia, the underlying regulatory mechanism remains elusive. By screening for hypoxia-inducible genes in cardiomyocytes, we identified hypoxia inducible domain family, member 1A (Higd1a) as a positive regulator of CcO. Recombinant Higd1a directly integrated into highly purified CcO and increased its activity. Resonance Raman analysis revealed that Higd1a caused structural changes around heme a, the active center that drives the proton pump. Using a mitochondria-targeted ATP biosensor, we showed that knockdown of endogenous Higd1a reduced oxygen consumption and subsequent mitochondrial ATP synthesis, leading to increased cell death in response to hypoxia; all of these phenotypes were rescued by exogenous Higd1a. These results suggest that Higd1a is a previously unidentified regulatory component of CcO, and represents a therapeutic target for diseases associated with reduced CcO activity.

The oxidative phosphorylation (OXPHOS) system generates most of the ATP in respiring cells. ATP-depleting conditions, such as hypoxia, trigger responses that promote ATP production. However, how OXPHOS is regulated during hypoxia has yet to be elucidated. In this study, selective measurement of intramitochondrial ATP levels identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS. A mitochondria-targeted, FRET-based ATP biosensor enabled us to assess OXPHOS activity in living cells. Mitochondria-targeted, FRET-based ATP biosensor and ATP production assay in a semiintact cell system revealed that G0s2 increases mitochondrial ATP production. The expression of G0s2 was rapidly and transiently induced by hypoxic stimuli, and G0s2 interacts with OXPHOS complex V (FoF1-ATP synthase). Furthermore, physiological enhancement of G0s2 expression prevented cells from ATP depletion and induced a cellular tolerance for hypoxic stress. These results show that G0s2 positively regulates OXPHOS activity by interacting with FoF1-ATP synthase, which causes an increase in ATP production in response to hypoxic stress and protects cells from a critical energy crisis. These findings contribute to the understanding of a unique stress response to energy depletion. Additionally, this study shows the importance of assessing intramitochondrial ATP levels to evaluate OXPHOS activity in living cells.

Aim: Recurrence rates of oral cancer following primary treatment have been reported in the range of 25–48%. However, salvage therapy remains a critical challenge to improving outcomes. Here, we investigated prognostic factors and outcomes for salvage surgery in patients with recurrent oral tongue squamous cell carcinoma (OTSCC). Methods: We retrospectively reviewed patients who were referred to Aichi Cancer Center, Japan, for the treatment of recurrent OTSCC. All patients included in the present study had undergone salvage surgery. Data to identify the predictive value of prognostic factors were available from 69 patients. Prognostic factors were assessed using Cox’s proportional hazards regression analysis. Differences in overall survival between groups of patients were assessed by the log–rank test. Results: In all, 36 patients (52%) developed second recurrence or died, of which 21 (58%) occurred within 12 months of salvage surgery. Univariate analysis indicated that survival was significantly worse in patients with recurrent stage III or IV tumors, two or more positive cervical lymph nodes, levels IV or V positive cervical lymph nodes, extracapsular spread (ECS) of positive cervical lymph nodes and a disease-free interval from initial treatment of less than 12 months. On multivariate analysis, ECS was an independent prognostic factor for overall survival after salvage surgery. Conclusion: Neck status at the time of salvage surgery, particularly ECS is a significant prognostic factor for surgical salvage. Survival was also influenced by the stage of the recurrent tumor and disease-free interval, suggesting that the biological features of recurrent tumors might impact on prognosis.

Increased expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) is frequently observed in certain cancers such as ovarian and breast cancers, and this protein is a desirable target for drug delivery by a drug delivery system (DDS). In the present study, we developed novel immunoliposomes targeting HB-EGF for cancer therapy. The immunoliposomes significantly associated with Vero-H cells overexpressing HB-EGF compared with their binding to wild-type Vero cells, whereas liposomes without modification by the antibody did not associate with either type of cells. Moreover, enhanced uptake of the immunoliposomes into Vero-H cells was observed as well as that into MDA-MB-231 human breast cancer cells, which are known to highly express HB-EGF. These results suggest that HB-EGF mediates the binding and uptake of the immunoliposomes in HB-EGF-expressing cells. Next, we determined the therapeutic effect of these immunoliposomes encapsulating an anticancer drug on tumor-bearing mice. For this purpose, we prepared doxorubicin (DOX)-encapsulated immunoliposomes and injected them intravenously into mice bearing MDA-MB-231 cancer cells. As a result, these DOX-encapsulated immunoliposomes suppressed not only tumor progression but also tumor regression. In conclusion, our results indicate that anti-HB-EGF antibody-modified liposomes could be a useful DDS carrier for the treatment of HB-EGF-expressing cancers.

We report the case of an 87-year-old woman with sternal bone metastasis and multiple lung metastases from thyroid cancer who underwent hemithyroidectomy, then noticed anterior chest swelling. Chest CT showed a mass destroying the upper sternum. As the anterior chest mass grew, CT showed an increasingly large tumor, suggesting expansion to the anterior mediastinum. The size of multiple lung nodules, however, was unchanged from the previous scan. Suspecting the tumor of causing the woman's discomfort, we partially resected the sternum and reconstructed the sternal defect using a major myocutaneous pectoral flap. She has had no physical or pulmonary impairment since.

我々の施設では，上顎癌に対する切除は，上顎全摘以上がほとんどである。これに対して整容的再建を行う上で，我々が重要視している点は，眼球温存例での眼球の支持と頬骨隆起の再建である。これら切除を要する進行癌では，術後照射例が多く，チタンメッシュを使うと，その周囲での照射の影響が強く出るため，使用を控えている。これに代わり，最近ではラクトソーブ®や筋膜で眼球を支持している。まだ短期結果だが，眼球は支持されている。頬骨隆起の再建では，上顎全摘以上では，硬性再建無しで整容的結果を出すのは難しく，遊離肋軟骨や血管柄付き骨を移植し，良好な形態が得られた。しかし，術後照射のある症例に，一期的血管柄付き骨移植を行うか否かは，未だ議論中の問題である。二期再建では，この制限が無く，整容的再建を追求できる。術後照射のある上顎全摘以上に対する整容的再建は，今後も大きな課題である。

AMP-activated protein kinase (AMPK) is an energy-sensing Ser/Thr protein kinase originally shown to be regulated by AMP. AMPK is activated by various cellular stresses that inhibit ATP production or stimulate ATP consumption. In addition to its role in metabolism, AMPK has recently been reported to reshape cells by regulating cell polarity and division. However, the downstream targets of AMPK that participate in these functions have not been fully identified. Here, we show that phosphorylation of the microtubule plus end protein CLIP-170 by AMPK is required for microtubule dynamics and the regulation of directional cell migration. Both inhibition of AMPK and expression of a non-phosphorylatable CLIP-170 mutant resulted in prolonged and enhanced accumulation of CLIP-170 at microtubule tips, and slower tubulin polymerization. Furthermore, inhibition of AMPK impaired microtubule stabilization and perturbed directional cell migration. All of these phenotypes were rescued by expression of a phosphomimetic CLIP-170 mutant. Our results demonstrate, therefore, that AMPK controls basic cellular functions by regulating microtubule dynamics through CLIP-170 phosphorylation.

Approaches to pterygopalatine and infratemporal fossa lesions are numerous because they are relatively surgically inaccessible. We report 9 cases of pterygopalatine and infratemporal fossa lesions. We treated 2 subjects with benign schwannoma using transmaxillar sinus and orbitozygomatic approaches. We used resection without facial skin incision, assuming that presurgical intraarterial embolization and navigational use was important in removing tumors safety with minimal invasion. One case involved lower infratemporal fossa adenoma. We used a mandibular swing for en block removal. We treated 3 with recurrent palatal carcinoma using maxillary swing. In 2 with lesions extending laterally to the parapharyngeal space, mandibular swing was added to maxillary swing. We treated 1 with recurrent chordoma in the infratemporal fossa using maxillary swing to preserve postoperative function and facial form. We treated 2 with massive infratemporal fossa tumors using an infratemporal approach. One of these 2 had a massive tumor invading the maxilla, necessitating maxillectomy with a Weber incision. We concluded that surgical approaches with wide access to safety must be used to resect tumors completely in those with malignancies