Mature human induced pluripotent stem cell-derived cardiomyocytes promote angiogenesis through alpha-B crystallin.

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to treat heart diseases; however, the optimal maturity of hiPSC-CMs for effective regenerative medicine remains unclear. We aimed to investigate the benefits of long-term cultured mature hiPSC-CMs in injured rat hearts. METHODS: Cardiomyocytes were differentiated from hiPSCs via monolayer culturing, and the cells were harvested on day 28 or 56 (D28-CMs or D56-CMs, respectively) after differentiation. We transplanted D28-CMs or D56-CMs into the hearts of rat myocardial infarction models and examined cell retention and engraftment via in vivo bioluminescence imaging and histological analysis. We performed transcriptomic sequencing analysis to elucidate the genetic profiles before and after hiPSC-CM transplantation. RESULTS: Upregulated expression of mature sarcomere genes in vitro was observed in D56-CMs compared with D28-CMs. In vivo bioluminescence imaging studies revealed increased bioluminescence intensity of D56-CMs at 8 and 12 weeks post-transplantation. Histological and immunohistochemical analyses showed that D56-CMs promoted engraftment and maturation in the graft area at 12 weeks post-transplantation. Notably, D56-CMs consistently promoted microvessel formation in the graft area from 1 to 12 weeks post-transplantation. Transcriptomic sequencing analysis revealed that compared with the engrafted D28-CMs, the engrafted D56-CMs enriched genes related to blood vessel regulation at 12 weeks post-transplantation. As shown by transcriptomic and western blot analyses, the expression of a small heat shock protein, alpha-B crystallin (CRYAB), was significantly upregulated in D56-CMs compared with D28-CMs. Endothelial cell migration was inhibited by small interfering RNA-mediated knockdown of CRYAB when co-cultured with D56-CMs in vitro. Furthermore, CRYAB overexpression enhanced angiogenesis in the D28-CM grafts at 4 weeks post-transplantation. CONCLUSIONS: Long-term cultured mature hiPSC-CMs promoted engraftment, maturation and angiogenesis post-transplantation in infarcted rat hearts. CRYAB, which was highly expressed in D56-CMs, was identified as an angiogenic factor from mature hiPSC-CMs. This study revealed the benefits of long-term culture, which may enhance the therapeutic potential of hiPSC-CMs.

A reconstituted depolarization-induced Ca2+ release platform for validation of skeletal muscle disease mutations and drug discovery.

In skeletal muscle excitation-contraction (E-C) coupling, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca2+ release (DICR). DICR occurs through the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including ß1a, Stac3 adaptor protein, and junctophilins. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+ indicator, without contaminant of extracellular Ca2+ influx. In these cells, [K+]-dependent Ca2+ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca2+-induced Ca2+ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for the validation of mutations and drug discovery for skeletal muscle diseases.

Expression and Clinical Significance of Spi-B in B-cell Acute Lymphoblastic Leukemia.

Spi-B, a member of the E26 transformation-specific (ETS) family of transcription factors, plays an important role in B cell differentiation. Spi-B also functions in development of diffuse large B-cell lymphoma; thus, we hypothesized that it may participate in leukemogenesis of B-cell acute lymphoblastic leukemia (B-ALL). To test this hypothesis, we first generated an anti-Spi-B monoclonal antibody that recognized Spi-B on formalin-fixed, paraffin-embedded tissue sections. This antibody, designated S28-5, selectively stained B cell nuclei at the pre-plasma cell stage (including centrocytes and centroblasts in germinal centers) and nuclei of plasmacytoid dendritic cells, but not fully differentiated plasma cells, T cells, macrophages, or follicular dendritic cells. Employing S28-5, we then performed immunohistochemical staining of bone marrow aspiration biopsy specimens obtained from B-ALL patients (n=62). Cases that showed stronger nuclear S28-5 signals than T-cell ALL were scored positive. In 26 (42%) of 62 specimens, leukemic cells showed nuclear Spi-B expression, and positivity was associated with patient age at diagnosis, and serum uric acid and creatinine levels. Moreover, Spi-B-positive patients demonstrated significantly shorter overall survival than did Spi-B-negative patients. These results suggest that Spi-B expression may serve as a prognostic indicator of B-ALL.

Cytokine receptor gp130 promotes postnatal proliferation of cardiomyocytes required for the normal functional development of the heart.

Mammalian ventricular cardiomyocytes are premature at birth and exhibit substantial phenotypic changes before weaning. Mouse ventricular myocytes undergo cell division several times after birth; however, the regulatory mechanisms and roles of cardiomyocyte division in postnatal heart development remain unclear. Here, we investigated the physiological role of glycoprotein 130 (gp130), the main subunit of multifunctional receptors for the IL-6 family of cytokines, in postnatal cardiomyocyte proliferation. Pharmacological inhibition of gp130 within the first month after birth induced significant systolic dysfunction of the left ventricle in mice. Consistently, mice with postnatal cardiomyocyte-specific gp130 depletion exhibited impaired left ventricular contractility compared with control mice. In these mice, cardiomyocytes exhibited a moderately decreased size and dramatically inhibited proliferation in the left ventricle but not in the right ventricle. Stereological analysis revealed that this change significantly decreased the number of cardiomyocytes in the left ventricle. Furthermore, IL-6 was mainly responsible for promoting ventricular cardiomyocyte proliferation by activating the JAK/STAT3 pathway. Taken together, the IL-6/gp130/JAK/STAT3 axis plays a crucial role in the physiological postnatal proliferation and hypertrophy of left ventricular cardiomyocytes to ensure normal cardiac functional development.NEW & NOTEWORTHY Although cardiomyocytes undergo proliferation in the early postnatal period, the regulatory mechanisms and physiological importance of this process have not been clarified. We found that the pharmacological and genetic depletion of gp130 in preweaning mice resulted in significant impairment of cardiomyocyte proliferation, thinning of the myocardium, and systolic dysfunction of the left but not right ventricle by perturbing JAK/STAT3 signaling. Thus, the IL-6/gp130/JAK/STAT3 axis is crucial for the postnatal functional development of the left ventricle.

[Regulation of localization and function of L-type calcium channels by junctophilins].

Striated muscle L-type calcium channels (LTCC) are localized specifically to the junctional membrane (JM) where the sarcolemma is closely apposed to the sarcoplasmic reticulum. Although this allocation of LTCC is critical for efficient excitation-contraction coupling in striated muscles, its underlying molecular mechanism has not been clarified. Junctophilins (JPs) stabilize the structure of JM by bridging the sarcolemmal and SR membranes. In addition, immunoprecipitation and pull-down assay revealed that the proximal C-terminus of CaV1.1 subunits directly binds to both JP1 and JP2, indicating that JPs might also directly recruit and hold LTCC in JM. Indeed, expression of a JP1 mutant lacking its C-terminus including the transmembrane domain in mouse skeletal muscles exerted a dominant-negative effect on endogenous JPs by impairing LTCC-RyR coupling at triads and reducing contractile force. To investigate a role of cardiac JP2 in a similar strategy, we injected adeno-associated virus vector expressing a C-terminus lacking JP2 mutant (JP2Δ427) driven by a cardiac troponin T promoter into C57BL/6 mice. Echocardiography recorded 4 weeks after the viral injection showed that the fractional shortening in JP2Δ427 group was significantly decreased compared to that of the control group. Calcium transient of isolated ventricular myocytes was significantly decreased by JP2Δ427 expression. Immunocytochemistry showed that JP2Δ427 recruited LTCC to the surface sarcolemma from T-tubules. Taken together, expression of C-terminus lacking JP mutants down-regulated contractile force by impairing ECC of skeletal and cardiac myocytes. Thus, the physical binding between LTCC and JP is essential for contraction of striated muscles.

NRSF-GNAO1 Pathway Contributes to the Regulation of Cardiac Ca2+ Homeostasis.

BACKGROUND: During the development of heart failure, a fetal cardiac gene program is reactivated and accelerates pathological cardiac remodeling. We previously reported that a transcriptional repressor, NRSF (neuron restrictive silencer factor), suppresses the fetal cardiac gene program, thereby maintaining cardiac integrity. The underlying molecular mechanisms remain to be determined, however. METHODS: We aim to elucidate molecular mechanisms by which NRSF maintains normal cardiac function. We generated cardiac-specific NRSF knockout mice and analyzed cardiac gene expression profiles in those mice and mice cardiac-specifically expressing a dominant-negative NRSF mutant. RESULTS: We found that cardiac expression of Gαo, an inhibitory G protein encoded in humans by GNAO1, is transcriptionally regulated by NRSF and is increased in the ventricles of several mouse models of heart failure. Genetic knockdown of Gnao1 ameliorated the cardiac dysfunction and prolonged survival rates in these mouse heart failure models. Conversely, cardiac-specific overexpression of GNAO1 in mice was sufficient to induce cardiac dysfunction. Mechanistically, we observed that increasing Gαo expression increased surface sarcolemmal L-type Ca2+ channel activity, activated CaMKII (calcium/calmodulin-dependent kinase-II) signaling, and impaired Ca2+ handling in ventricular myocytes, which led to cardiac dysfunction. CONCLUSIONS: These findings shed light on a novel function of Gαo in the regulation of cardiac Ca2+ homeostasis and systolic function and suggest Gαo may be an effective therapeutic target for the treatment of heart failure.

Detection of 2-Hydroxyglutarate by 3.0-Tesla Magnetic Resonance Spectroscopy in Gliomas with Rare IDH Mutations: Making Sense of "False-Positive" Cases.

We have previously published a study on the reliable detection of 2-hydroxyglutarate (2HG) in lower-grade gliomas by magnetic resonance spectroscopy (MRS). In this short article, we re-evaluated five glioma cases originally assessed as isocitrate dehydrogenase (IDH) wildtype, which showed a high accumulation of 2HG, and were thought to be false-positives. A new primer was used for the detection of IDH2 mutation by Sanger sequencing. Adequate tissue for DNA analysis was available in 4 out of 5 cases. We found rare IDH2 mutations in two cases, with IDH2 R172W mutation in one case and IDH2 R172K mutation in another case. Both cases had very small mutant peaks, suggesting that the tumor volume was low in the tumor samples. Thus, the specificity of MRS for detecting IDH1/2 mutations was higher (81.3%) than that originally reported (72.2%). The detection of 2HG by MRS can aid in the diagnosis of rare, non-IDH1-R132H IDH1 and IDH2 mutations in gliomas.

Adrenomedullin-RAMP2 and -RAMP3 Systems Regulate Cardiac Homeostasis during Cardiovascular Stress.

Adrenomedullin (AM) is a peptide hormone with multiple physiological functions, which are regulated by its receptor activity-modifying proteins, RAMP2 and RAMP3. We previously reported that AM or RAMP2 knockout (KO) (AM-/-, RAMP2-/-) is embryonically lethal in mice, whereas RAMP3-/- mice are apparently normal. AM, RAMP2, and RAMP3 are all highly expressed in the heart; however, their functions there are not fully understood. Here, we analyzed the pathophysiological functions of the AM-RAMP2 and AM-RAMP3 systems in hearts subjected to cardiovascular stress. Cardiomyocyte-specific RAMP2-/- (C-RAMP2-/-) and RAMP3-/- showed no apparent heart failure at base line. After 1 week of transverse aortic constriction (TAC), however, C-RAMP2-/- exhibited significant cardiac hypertrophy, decreased ejection fraction, and increased fibrosis compared with wild-type mice. Both dP/dtmax and dP/dtmin were significantly reduced in C-RAMP2-/-, indicating reduced ventricular contractility and relaxation. Exposing C-RAMP2-/- cardiomyocytes to isoproterenol enhanced their hypertrophy and oxidative stress compared with wild-type cells. C-RAMP2-/- cardiomyocytes also contained fewer viable mitochondria and showed reduced mitochondrial membrane potential and respiratory capacity. RAMP3-/- also showed reduced systolic function and enhanced fibrosis after TAC, but those only became apparent after 4 weeks. A reduction in cardiac lymphatic vessels was the characteristic feature in RAMP3-/-. These observations indicate the AM-RAMP2 system is necessary for early adaptation to cardiovascular stress through regulation of cardiac mitochondria. AM-RAMP3 is necessary for later adaptation through regulation of lymphatic vessels. The AM-RAMP2 and AM-RAMP3 systems thus play separate critical roles in the maintenance of cardiovascular homeostasis against cardiovascular stress.

ß-Arrestin-Biased AT1 Agonist TRV027 Causes a Neonatal-Specific Sustained Positive Inotropic Effect Without Increasing Heart Rate.

The treatment of pediatric heart failure is a long-standing unmet medical need. Angiotensin II supports mammalian perinatal circulation by activating cardiac L-type Ca2+ channels through angiotensin type 1 receptor (AT1R) and ß-arrestin. TRV027, a ß-arrestin-biased AT1R agonist, that has been reported to be safe but not effective for adult patients with heart failure, activates the AT1R/ß-arrestin pathway. We found that TRV027 evokes a long-acting positive inotropic effect specifically on immature cardiac myocytes through the AT1R/ß-arrestin/L-type Ca2+ channel pathway with minimum effect on heart rate, oxygen consumption, reactive oxygen species production, and aldosterone secretion. Thus, TRV027 could be utilized as a valuable drug specific for pediatric heart failure.

Skull diploë is rich in aquaporin-4.

Aquaporin-4 (AQP4) is a water conducting membrane integral protein channel which is widely expressed in the astrocyte system of the brain. During the development of the AQP4 positron emission tomography (PET) imaging agent [11C]TGN-020 (N-(1,3,4-thiadiazol-2-yl)pyridine-3-[11C]-carboxamide), significant radioligand uptake was observed in the skull, where there was no known distribution of any aquaporin family proteins. Herein we confirmed via a newly developed method for bone-tissue immunohistology, a hitherto unrecognized distribution of AQP4, and not AQP1, in the skull. Other bony structures, by contrast, showed virtually no uptake of [11C]TGN-020, and likewise, do not express either AQP4 or AQP1. Immunohistological analysis demonstrated that the AQP4 expression in the skull is restricted to the diploë. Consequently, we suspect AQP4 plays a pivotal role in the formation and maintenance of yellow marrow and the diploë. However, elucidating the exact nature of that role will require further studies.

Evolutionary Elongation of the Time Window of Integration in Auditory Cortex: Macaque vs. Human Comparison of the Effects of Sound Duration on Auditory Evoked Potentials.

The auditory cortex integrates auditory information over time to obtain neural representations of sound events, the time scale of which critically affects perception. This work investigated the species differences in the time scale of integration by comparing humans and monkeys regarding how their scalp-recorded cortical auditory evoked potentials (CAEPs) decrease in amplitude as stimulus duration is shortened from 100 ms (or longer) to 2 ms. Cortical circuits tuned to processing sounds at short time scales would continue to produce large CAEPs to brief sounds whereas those tuned to longer time scales would produce diminished responses. Four peaks were identified in the CAEPs and labeled P1, N1, P2, and N2 in humans and mP1, mN1, mP2, and mN2 in monkeys. In humans, the N1 diminished in amplitude as sound duration was decreased, consistent with the previously described temporal integration window of N1 (>50 ms). In macaques, by contrast, the mN1 was unaffected by sound duration, and it was clearly elicited by even the briefest sounds. Brief sounds also elicited significant mN2 in the macaque, but not the human N2. Regarding earlier latencies, both P1 (humans) and mP1 (macaques) were elicited at their full amplitudes even by the briefest sounds. These findings suggest an elongation of the time scale of late stages of human auditory cortical processing, as reflected by N1/mN1 and later CAEP components. Longer time scales of integration would allow neural representations of complex auditory features that characterize speech and music.

Automaticity of pitch class-color synesthesia as revealed by a Stroop-like effect.

Pitch classes (e.g., do, re, and mi) in music evoke color sensations in pitch class-color synesthesia, which is a recently described form of synesthesia in musicians. The synesthetic color sensations were confirmed to be consistent over an extended time interval, fulfilling a widely-accepted criterion for the authenticity of synesthesia. However, it remains unclear whether the color sensations occurred automatically (i.e., without voluntary effort), which is another defining property of synesthesia. We utilized the Stroop paradigm to investigate this issue in 10 pitch class-color synesthetes. Participants were visually presented with pitch class names in font colors that were either congruent or incongruent with the participants' own color sensations. The speed for reporting the font color was slower when it was incongruent with the synesthetic sensation than when it was congruent. The finding verifies the authenticity of pitch class-color synesthesia by demonstrating that the color sensations occur automatically, even when unnecessary.

Visualizing the Distribution of Matrix Metalloproteinases in Ischemic Brain Using In Vivo 19F-Magnetic Resonance Spectroscopic Imaging.

Matrix metalloproteinases (MMPs) damage the neurovascular unit, promote the blood-brain barrier (BBB) disruption following ischemic stroke, and play essential roles in hemorrhagic transformation (HT), which is one of the most severe side effects of thrombolytic therapy. However, no biomarkers have presently been identified that can be used to track changes in the distribution of MMPs in the brain. Here, we developed a new 19F-molecular ligand, TGF-019, for visualizing the distribution of MMPs in vivo using 19F-magnetic resonance spectroscopic imaging (19F-MRSI). We demonstrated TGF-019 has sufficient sensitivity for the specific MMPs suspected in evoking HT during ischemic stroke, i.e., MMP2, MMP9, and MMP3. We then utilized it to assess those MMPs at 22 to 24 hours after experimental focal cerebral ischemia on MMP2-null mice, as well as wild-type mice with and without the systemic administration of the recombinant tissue plasminogen activator (rt-PA). The 19F-MRSI of TGN-019-administered mice showed high signal intensity within ischemic lesions that correlated with total MMP2 and MMP9 activity, which was confirmed by zymographic analysis of ischemic tissues. Based on the results of this study, 19F-MRSI following TGN-019 administration can be used to assess potential therapeutic strategies for ischemic stroke.

Fluid Dynamics Inside the Brain Barrier: Current Concept of Interstitial Flow, Glymphatic Flow, and Cerebrospinal Fluid Circulation in the Brain.

The discovery of the water specific channel, aquaporin, and abundant expression of its isoform, aquaporin-4 (AQP-4), on astrocyte endfeet brought about significant advancements in the understanding of brain fluid dynamics. The brain is protected by barriers preventing free access of systemic fluid. The same barrier system, however, also isolates brain interstitial fluid from the hydro-dynamic effect of the systemic circulation. The systolic force of the heart, an essential factor for proper systemic interstitial fluid circulation, cannot be propagated to the interstitial fluid compartment of the brain. Without a proper alternative mechanism, brain interstitial fluid would stay stagnant. Water influx into the peri-capillary Virchow-Robin space (VRS) through the astrocyte AQP-4 system compensates for this hydrodynamic shortage essential for interstitial flow, introducing the condition virtually identical to systemic circulation, which by virtue of its fenestrated capillaries creates appropriate interstitial fluid motion. Interstitial flow in peri-arterial VRS constitutes an essential part of the clearance system for ß-amyloid, whereas interstitial flow in peri-venous VRS creates bulk interstitial fluid flow, which, together with the choroid plexus, creates the necessary ventricular cerebrospinal fluid (CSF) volume for proper CSF circulation.

Developmental abnormalities of the brain exposed to childhood maltreatment detected by diffusion tensor imaging.

Objective: This study aimed to investigate brain developmental alterations in individuals exposed to childhood maltreatment (CM) with dissociative experiences and motor coordination symptoms using diffusion tensor imaging on a 3Tesla (3T) magnetic resonance (MR) system. Methods: Five individuals exposed to CM who manifest behavioral and developmental problems with dissociative experiences and motor coordination symptoms (age range: 14-18 years; all female), as well as seven age- and gender-matched normal control individuals, participated in the study using a 3T MR system. Diffusion characteristics, as indexed by fractional anisotropy (FA), were assessed for cerebral white matter structures. A preliminary whole brain analysis was performed complementary to an anatomically guided region of interest (ROI) analysis. Results: In individuals exposed to CM, scattered decreases in FA were detected in multiple brain regions over the frontoparietal and temporal areas in the whole brain map. ROI analysis subsequently identified significant decreases in FA (p < 0.05) in the right parietal white matter area as well as in the right prefrontal, bilateral premotor, bilateral orbitofrontal, and temporal white matter areas in CM-exposed individuals compared to that in controls. Conclusion: The observed altered diffusion characteristics indicate attendant developmental abnormalities within the white matter structures, which are associated with the observed clinical and behavioral patterns including dissociative experiences and coordination symptoms in individuals exposed to CM. The study provides objective evidence regarding the effects of CM on brain microstructure.

Prostanoid EP4 Receptor-Mediated Augmentation of I h Currents in Aß Dorsal Root Ganglion Neurons Underlies Neuropathic Pain.

An injury of the somatosensory system causes neuropathic pain, which is usually refractory to conventional analgesics, thus warranting the development of novel drugs against this kind of pain. The mechanism of neuropathic pain in rats that had undergone left L5 spinal nerve transection was analyzed. Ten days after surgery, these rats acquired neuropathic pain. The patch-clamp technique was used on the isolated bilateral L5 dorsal root ganglion neurons. The current-clamped neurons on the ipsilateral side exhibited significantly higher excitability than those on the contralateral side. However, only neurons with diameters of 40-50 µm on the ipsilateral side exhibited significantly larger voltage sags in response to hyperpolarizing current pulses than those on the contralateral side. Under the voltage clamp, only these neurons on the ipsilateral side showed a significantly larger density of an inward current at < -80 mV [hyperpolarization-activated nonselective cation (I h) current] with a rightward-shifted activation curve than that on the contralateral side. Ivabradine-an I h current inhibitor-inhibited I h currents in these neurons on both sides in a similar concentration-dependent manner, with an IC50 value of ∼3 µM. Moreover, the oral administration of ivabradine significantly alleviated the neuropathic pain on the ipsilateral side. An inhibitor of adenylyl cyclase or an antagonist of prostanoid EP4 receptors (CJ-023423) inhibited ipsilateral, but not contralateral I h, currents in these neurons. Furthermore, the intrathecal administration of CJ-023423 significantly attenuated neuropathic pain on the ipsilateral side. Thus, ivabradine and/or CJ-023423 may be a lead compound for the development of novel therapeutics against neuropathic pain.

Absolute pitch is not necessary for pitch class-color synesthesia.

Sounds evoke color sensations in sound-color synesthesia. Recently, we showed that pitch classes (do, re, mi, etc.) have rainbow hues and that colors are linked to the names of pitch classes rather than to their sounds in 15 subjects who had "pitch class-color synesthesia." However, all synesthetes in our previous study had high levels of absolute pitch (AP); therefore the effects of AP on the condition remained unclear. The present study investigated 18 additional pitch class-color synesthetes who had no or lower levels of AP, and confirmed the generality of the above findings. Furthermore, behavioral experiments indicated a two-step process underlying color sensations: pitches are first associated with their pitch class names, and then the pitch class names evoke color sensations. Two separable brain functions underlie pitch-to-color conversion in pitch class-color synesthesia: a musical function of pitch class identification, and the synesthetic association between pitch class and color.

Physical interaction of junctophilin and the CaV1.1 C terminus is crucial for skeletal muscle contraction.

Close physical association of CaV1.1 L-type calcium channels (LTCCs) at the sarcolemmal junctional membrane (JM) with ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) is crucial for excitation-contraction coupling (ECC) in skeletal muscle. However, the molecular mechanism underlying the JM targeting of LTCCs is unexplored. Junctophilin 1 (JP1) and JP2 stabilize the JM by bridging the sarcolemmal and SR membranes. Here, we examined the roles of JPs in localization and function of LTCCs. Knockdown of JP1 or JP2 in cultured myotubes inhibited LTCC clustering at the JM and suppressed evoked Ca2+ transients without disrupting JM structure. Coimmunoprecipitation and GST pull-down assays demonstrated that JPs physically interacted with 12-aa residues in the proximal C terminus of the CaV1.1. A JP1 mutant lacking the C terminus including the transmembrane domain (JP1ΔCT) interacted with the sarcolemmal/T-tubule membrane but not the SR membrane. Expression of this mutant in adult mouse muscles in vivo exerted a dominant-negative effect on endogenous JPs, impairing LTCC-RyR coupling at triads without disrupting JM morphology, and substantially reducing Ca2+ transients without affecting SR Ca2+ content. Moreover, the contractile force of the JP1ΔCT-expressed muscle was dramatically reduced compared with the control. Taken together, JPs recruit LTCCs to the JM through physical interaction and ensure robust ECC at triads in skeletal muscle.

Aquaporin-4 facilitator TGN-073 promotes interstitial fluid circulation within the blood-brain barrier: [17O]H2O JJVCPE MRI study.

The blood-brain barrier (BBB), which imposes significant water permeability restriction, effectively isolates the brain from the systemic circulation. Seemingly paradoxical, the abundance of aquaporin-4 (AQP-4) on the inside of the BBB strongly indicates the presence of unique water dynamics essential for brain function. On the basis of the highly specific localization of AQP-4, namely, astrocyte end feet at the glia limitans externa and pericapillary Virchow-Robin space, we hypothesized that the AQP-4 system serves as an interstitial fluid circulator, moving interstitial fluid from the glia limitans externa to pericapillary Virchow-Robin space to ensure proper glymphatic flow draining into the cerebrospinal fluid. The hypothesis was tested directly using the AQP-4 facilitator TGN-073 developed in our laboratory, and [O]H2O JJ vicinal coupling proton exchange MRI, a method capable of tracing water molecules delivered into the blood circulation. The results unambiguously showed that facilitation of AQP-4 by TGN-073 increased turnover of interstitial fluid through the system, resulting in a significant reduction in [O]H2O contents of cortex with normal flux into the cerebrospinal fluid. The study further suggested that in addition to providing the necessary water for proper glymphatic flow, the AQP-4 system produces a water gradient within the interstitial space promoting circulation of interstitial fluid within the BBB.

PDGF-induced migration of synthetic vascular smooth muscle cells through c-Src-activated L-type Ca2+ channels with full-length CaV1.2 C-terminus.

In atherosclerosis, vascular smooth muscle cells (VSMC) migrate from the media toward the intima of the arteries in response to cytokines, such as platelet-derived growth factor (PDGF). However, molecular mechanism underlying the PDGF-induced migration of VSMCs remains unclear. The migration of rat aorta-derived synthetic VSMCs, A7r5, in response to PDGF was potently inhibited by a CaV1.2 channel inhibitor, nifedipine, and a Src family tyrosine kinase (SFK)/Abl inhibitor, bosutinib, in a less-than-additive manner. PDGF significantly increased CaV1.2 channel currents without altering CaV1.2 protein expression levels in A7r5 cells. This reaction was inhibited by C-terminal Src kinase, a selective inhibitor of SFKs. In contractile VSMCs, the C-terminus of CaV1.2 is proteolytically cleaved into proximal and distal C-termini (PCT and DCT, respectively). Clipped DCT is noncovalently reassociated with PCT to autoinhibit the channel activity. Conversely, in synthetic A7r5 cells, full-length CaV1.2 (CaV1.2FL) is expressed much more abundantly than truncated CaV1.2. In a heterologous expression system, c-Src activated CaV1.2 channels composed of CaV1.2FL but not truncated CaV1.2 (CaV1.2Δ1763) or CaV1.2Δ1763 plus clipped DCT. Further, c-Src enhanced the coupling efficiency between the voltage-sensing domain and activation gate of CaV1.2FL channels by phosphorylating Tyr1709 and Tyr1758 in PCT. Compared with CaV1.2Δ1763, c-Src could more efficiently bind to and phosphorylate CaV1.2FL irrespective of the presence or absence of clipped DCT. Therefore, in atherosclerotic lesions, phenotypic switching of VSMCs may facilitate pro-migratory effects of PDGF on VSMCs by suppressing posttranslational CaV1.2 modifications.