Involvement of Transient Receptor Potential Vanilloid Channel 2 in the Induction of Lubricin and Suppression of Ectopic Endochondral Ossification in Mouse Articular Cartilage.

OBJECTIVE: Transient receptor potential vanilloid channel 2 (TRPV2) is a Ca2+ -permeable channel and plays a role in mediating intracellular Ca2+ current via mechanical stimuli. This study was undertaken to examine the expression and role of TRPV2 in adult articular cartilage and the development of osteoarthritis (OA). METHODS: We examined TRPV2 expression in mouse and human articular cartilage. We analyzed the development of OA in Col2a1-CreERt2 ;Trpv2fl/fl mice and Trpv2fl/fl littermates in the resection of the medial meniscus and medial collateral ligament model (n = 5 each), the destabilization of the medial meniscus model (n = 5 each), and the aging mouse model (n = 8-9 each). We examined marker protein expression in these joints, Ca2+ influx by mechanical stimuli, and downstream pathways in vitro. RESULTS: TRPV2 was expressed in mouse and human articular cartilage and ectopic ossification lesions. In all mouse models of OA examined, Col2a1-CreERt2 ;Trpv2fl/fl mice were observed to have enhanced degradation of articular cartilage accompanied by decreased expression of lubricin/Prg4, and marked formation of periarticular ectopic ossification. Mechanical stress-induced Ca2+ influx was decreased by Trpv2 knockout (KO). Prg4 induction by fluid-flow shear stress was diminished in Trpv2-KO mouse chondrocytes, and this was mediated by the Ca2+ /calmodulin-dependent protein kinase kinase-cyclic AMP response element binding protein axis. Hypertrophic differentiation was enhanced in Trpv2-KO mouse chondrocytes. Increased activity of calcineurin and nuclear translocation of nuclear factor in activated T cells 1 induced by fluid-flow shear stress or TRP agonist treatment was reversed by Trpv2 knockout. CONCLUSION: Our findings demonstrate regulation of articular cartilage by TRPV2 through Prg4 induction and suppression of ectopic ossification.

A Rare Case of Clival Metastasis in a Patient with Gastric Cancer.

We report a rare case of clival metastasis from gastric cancer. A 73-year-old man with advanced gastric cancer treated with nivolumab as a third-line chemotherapy experienced headache, tongue deviation, and difficulties in speaking clearly. We suspected stroke or brain metastasis, but brain contrast-enhanced magnetic resonance imaging demonstrated a clival mass, diagnosed as clival metastasis from gastric cancer. The tumor could not be identified by plain computed tomography and plain magnetic resonance imaging alone. He received palliative radiotherapy (30 Gy/10 fr); his symptoms improved gradually. Although metastasis from gastric cancer to other organs is common, bone metastases are rare.

Elimination of fukutin reveals cellular and molecular pathomechanisms in muscular dystrophy-associated heart failure.

Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.

TRPV2 is required for mechanical nociception and the stretch-evoked response of primary sensory neurons.

Mechanotransduction plays important roles in many sensory processes, including touch, pain, hearing, and proprioception. However, the molecular mechanisms of mechanical nociception have remained unclear. Here, we showed that elimination of transient receptor potential vanilloid 2 (TRPV2) in mice resulted in the deficit of mechanical nociception due to the lack of mechanosensitivity in a subclass of adult primary sensory neurons (PSNs). The PSN-specific TRPV2-deficient mice showed behavioural impairment of mechanical nociception in tail-pressure and von Frey hair tests, without defects in axonal growth and neuronal composition. Conversely, the mice displayed normal behaviour to noxious heat and non-noxious tactile stimuli. Furthermore, based on the stretch-evoked Ca2+ response of cultured PSNs, we characterised two types of stretch-activated neurons in normal mice; fast-decay high-threshold and slow-decay low-threshold mechanosensitive. The cultured neurons from TRPV2-deficient mice lacked stretch-evoked Ca2+ responses by fast-decay neurons normally activated by high-threshold mechanical stimulation. These results demonstrated that TRPV2 has a critical role in mechanical nociception in the adult somatosensory system.

Effects of induced Na+/Ca2+ exchanger overexpression on the spatial distribution of L-type Ca2+ channels and junctophilin-2 in pressure-overloaded hearts.

The Na+/Ca2+ exchanger 1 (NCX1) is an essential Ca2+ efflux system in cardiomyocytes. Although NCX1 is distributed throughout the sarcolemma, a subpopulation of NCX1 is localized to transverse (T)-tubules. There is growing evidence that T-tubule disorganization is a causal event that shifts the transition from hypertrophy to heart failure (HF). However, the detailed molecular mechanisms have not been clarified. Previously, we showed that induced NCX1 expression in pressure-overloaded hearts attenuates defective excitation-contraction coupling and HF progression. Here, we examined the effects of induced NCX1 overexpression on the spatial distribution of L-type Ca2+ channels (LTCCs) and junctophilin-2 (JP2), a structural protein that connects the T-tubule and sarcoplasmic reticulum membrane, in pressure-overloaded hearts. Quantitative analysis showed that the regularity of NCX1 localization was significantly decreased at 8 weeks after transverse aortic constriction (TAC)-surgery; however, T-tubule organization and the regularities of LTCC and JP2 immunofluorescent signals were maintained at this time point. These observations demonstrated that release of NCX1 from the T-tubule area occurred before the onset of T-tubule disorganization and LTCC and JP2 mislocalization. Moreover, induced NCX1 overexpression at 8 weeks post-TAC not only recovered NCX1 regularity but also prevented the decrease in LTCC and JP2 regularities at 16 weeks post-TAC. These results suggested that NCX1 may play an important role in the proper spatial distribution of LTCC and JP2 in T-tubules in the context of pressure-overloading.

Induced NCX1 overexpression attenuates pressure overload-induced pathological cardiac remodelling.

AIMS: Although increased Na(+)/Ca(2+) exchanger 1 (NCX1) expression is observed during heart failure (HF), the pathological role of NCX1 during the progression of HF remains unclear. We examined alterations of NCX1 expression and activity in hearts after transverse aortic constriction (TAC) surgery and explored whether NCX1 influences pressure overload-induced pathological cardiac remodelling. METHODS AND RESULTS: We generated novel transgenic mice in which NCX1 expression is controlled by a cardiac-specific, doxycycline (DOX)-dependent promoter. In the absence of DOX, TAC surgery caused substantial chamber dilation with a gradual decrease in contractility by 16 weeks. Cardiomyocytes showed a decline in contractility with abnormal Ca(2+) handling during excitation-contraction (E-C) coupling. Reduced NCX1 activity was observed 8 weeks after TAC and was still apparent at 17 weeks. Induced NCX1 overexpression by DOX treatment starting 8 weeks after TAC returned NCX1 activity to pre-TAC levels and prevented chamber dilation with cardiac dysfunction. DOX treatment not only upregulated NCX1 expression in TAC-operated hearts but also returned L-type Ca(2+) channel and sarcoplasmic reticulum (SR) Ca(2+) ATPase expression levels to those in sham-operated hearts. In DOX-treated myocytes, contractility, T-tubule integrity, synchrony of Ca(2+) release from the SR, and Ca(2+) handling during E-C coupling was preserved 16 weeks after TAC surgery. In addition, DOX treatment attenuated the down-regulation of survival signalling and up-regulation of apoptosis signalling 16 weeks after TAC surgery. CONCLUSION: Induced overexpression of NCX1 attenuated pressure overload-induced pathological cardiac remodelling. Thus, maintaining NCX1 activity may be a potential therapeutic strategy for preventing the progression of HF.

Involvement of S1P1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells.

COA-Cl (2Cl-C.OXT-A) is a recently developed adenosine-like nucleic acid analog that promotes angiogenesis via the mitogen-activated protein (MAP) kinases ERK1/2. Endothelial S1P1 receptor plays indispensable roles in developmental angiogenesis. In this study, we examined the functions of S1P1 in COA-Cl-induced angiogenic responses. Antagonists for S1P1, W146, and VPC23019, substantially but still partly inhibited the effects of COA-Cl with regard to ERK1/2 activation and tube formation in cultured human umbilical vein endothelial cells (HUVEC). Antagonists for adenosine A1 receptor and purinergic P2Y1 receptor were without effect. Genetic knockdown of S1P1 with siRNA, but not that of S1P3, attenuated COA-Cl-elicited ERK1/2 responses. The signaling properties of COA-Cl showed significant similarities to those of sphingosine 1-phosphate, an endogenous S1P1 ligand, in that both induced responses sensitive to pertussis toxin (Gα i/o inhibitor), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), (calcium chelator), and PP2 (c-Src tyrosine kinase inhibitor). COA-Cl elevated intracellular Ca(2+) concentration and induced tyrosine phosphorylation of p130Cas, a substrate of c-Src, in HUVEC. COA-Cl displaced [(3)H]S1P in a radioligand-binding competition assay in chem-1 cells overexpressing S1P1. However, COA-Cl activated ERK1/2 in CHO-K1 cells that lack functional S1P1 receptor, suggesting the presence of additional yet-to-be-defined COA-Cl target in these cells. The results thus suggest the major contribution of S1P1 in the angiogenic effects of COA-Cl. However, other mechanism such as that seen in CHO-K1 cells may also be partly involved. Collectively, these findings may lead to refinement of the design of this nucleic acid analog and ultimately to development of small molecule-based therapeutic angiogenesis.

TRPV2 is critical for the maintenance of cardiac structure and function in mice.

The heart has a dynamic compensatory mechanism for haemodynamic stress. However, the molecular details of how mechanical forces are transduced in the heart are unclear. Here we show that the transient receptor potential, vanilloid family type 2 (TRPV2) cation channel is critical for the maintenance of cardiac structure and function. Within 4 days of eliminating TRPV2 from hearts of the adult mice, cardiac function declines severely, with disorganization of the intercalated discs that support mechanical coupling with neighbouring myocytes and myocardial conduction defects. After 9 days, cell shortening and Ca(2+) handling by single myocytes are impaired in TRPV2-deficient hearts. TRPV2-deficient neonatal cardiomyocytes form no intercalated discs and show no extracellular Ca(2+)-dependent intracellular Ca(2+) increase and insulin-like growth factor (IGF-1) secretion in response to stretch stimulation. We further demonstrate that IGF-1 receptor/PI3K/Akt pathway signalling is significantly downregulated in TRPV2-deficient hearts, and that IGF-1 administration partially prevents chamber dilation and impairment in cardiac pump function in these hearts. Our results improve our understanding of the molecular processes underlying the maintenance of cardiac structure and function.

Overexpression of LARGE suppresses muscle regeneration via down-regulation of insulin-like growth factor 1 and aggravates muscular dystrophy in mice.

Several types of muscular dystrophy are caused by defective linkage between α-dystroglycan (α-DG) and laminin. Among these, dystroglycanopathy, including Fukuyama-type congenital muscular dystrophy (FCMD), results from abnormal glycosylation of α-DG. Recent studies have shown that like-acetylglucosaminyltransferase (LARGE) strongly enhances the laminin-binding activity of α-DG. Therefore, restoration of the α-DG-laminin linkage by LARGE is considered one of the most promising possible therapies for muscular dystrophy. In this study, we generated transgenic mice that overexpress LARGE (LARGE Tg) and crossed them with dy(2J) mice and fukutin conditional knockout mice, a model for laminin α2-deficient congenital muscular dystrophy (MDC1A) and FCMD, respectively. Remarkably, in both the strains, the transgenic overexpression of LARGE resulted in an aggravation of muscular dystrophy. Using morphometric analyses, we found that the deterioration of muscle pathology was caused by suppression of muscle regeneration. Overexpression of LARGE in C2C12 cells further demonstrated defects in myotube formation. Interestingly, a decreased expression of insulin-like growth factor 1 (IGF-1) was identified in both LARGE Tg mice and LARGE-overexpressing C2C12 myotubes. Supplementing the C2C12 cells with IGF-1 restored the defective myotube formation. Taken together, our findings indicate that the overexpression of LARGE aggravates muscular dystrophy by suppressing the muscle regeneration and this adverse effect is mediated via reduced expression of IGF-1.

Impaired viability of muscle precursor cells in muscular dystrophy with glycosylation defects and amelioration of its severe phenotype by limited gene expression.

A group of muscular dystrophies, dystroglycanopathy is caused by abnormalities in post-translational modifications of dystroglycan (DG). To understand better the pathophysiological roles of DG modification and to establish effective clinical treatment for dystroglycanopathy, we here generated two distinct conditional knock-out (cKO) mice for fukutin, the first dystroglycanopathy gene identified for Fukuyama congenital muscular dystrophy. The first dystroglycanopathy model-myofiber-selective fukutin-cKO [muscle creatine kinase (MCK)-fukutin-cKO] mice-showed mild muscular dystrophy. Forced exercise experiments in presymptomatic MCK-fukutin-cKO mice revealed that myofiber membrane fragility triggered disease manifestation. The second dystroglycanopathy model-muscle precursor cell (MPC)-selective cKO (Myf5-fukutin-cKO) mice-exhibited more severe phenotypes of muscular dystrophy. Using an isolated MPC culture system, we demonstrated, for the first time, that defects in the fukutin-dependent modification of DG lead to impairment of MPC proliferation, differentiation and muscle regeneration. These results suggest that impaired MPC viability contributes to the pathology of dystroglycanopathy. Since our data suggested that frequent cycles of myofiber degeneration/regeneration accelerate substantial and/or functional loss of MPC, we expected that protection from disease-triggering myofiber degeneration provides therapeutic effects even in mouse models with MPC defects; therefore, we restored fukutin expression in myofibers. Adeno-associated virus (AAV)-mediated rescue of fukutin expression that was limited in myofibers successfully ameliorated the severe pathology even after disease progression. In addition, compared with other gene therapy studies, considerably low AAV titers were associated with therapeutic effects. Together, our findings indicated that fukutin-deficient dystroglycanopathy is a regeneration-defective disorder, and gene therapy is a feasible treatment for the wide range of dystroglycanopathy even after disease progression.

Mechanical stretch increases the proliferation while inhibiting the osteogenic differentiation in dental pulp stem cells.

Dental pulp stem cells (DPSCs), which can differentiate into several types of cells, are subjected to mechanical stress by jaw movement and occlusal forces. In this study, we evaluated how the uniaxial mechanical stretch influences proliferation and differentiation of DPSCs. DPSCs were isolated and cultured from male Sprague-Dawley rats. Cultured DPSCs were identified by surface markers and the differentiation capabilities as adipocytes or osteoblasts. To examine the response to mechanical stress, uniaxial stretch was exposed to cultured DPSCs. We evaluated the impact of stretch on the intracellular signaling, proliferation, osteogenic differentiation, and gene expressions of DPSCs. Stretch increased the phosphorylation of Akt, ERK1/2, and p38 MAP kinase as well as the proliferation of DPSCs. The stretch-induced proliferation of DPSCs was abolished by the inhibition of the ERK pathway. On the other hand, stretch significantly decreased the osteogenic differentiation of DPSCs, but did not affect the adipogenic differentiation. We also confirmed mRNA expressions of osteocalcin and osteopontin were significantly suppressed by stretch. In conclusion, uniaxial stretch increased the proliferation of DPSCs, while suppressing osteogenic differentiation. These results suggest a crucial role of mechanical stretch in the preservation of DPSCs in dentin. Furthermore, mechanical stretch may be a useful tool for increasing the quantity of DPSCs in vitro for regenerative medicine.

[Musculoskeletal rehabilitation and bone. A novel approach to mechanotransduction using cell-adhesion-patterned cells].

Human vascular endothelial cells form the interface between the bloodstream and vessel walls and are continuously subjected to mechanical stimulation. When endothelial cells are stretched cyclically, along one axis, they align perpendicular to the axis of stretch. We previously reported that applying a cyclic, uni-axial strain to cells induced tyrosine phosphorylation of focal adhesion kinase and stimulated mitogen-activated protein kinase. However, it is difficult to quantify and analyze the spatial distribution of tyrosine phosphorylation in these cells, as they form focal adhesions randomly. Recently, we developed a system to overcome this problem by preparing individual, uniform, patterned cells that could be stretched cyclically and uni-axially. In this system we were able to statistically analyze cellular responses in these patterned cells, when subjected to a cyclic, uni-axial strain, using fluorescent microscopy.

Impairment of leukocyte deformability in patients undergoing esophagectomy.

Impaired deformability might contribute to the accumulation of activated leukocytes within pulmonary microcapillaries, leading to acute lung injury. The purpose of our study was to investigate changes in leukocyte deformability during periods of inflammation after esophagectomy. The study group comprised 20 patients who underwent esophagectomy. Changes in leukocyte deformability were investigated by examining filtration through a silicon microchannel, which simulated human pulmonary microcapillaries. Changes in the neutrophil cytoskeleton were investigated by measuring neutrophil F-actin assembly. The severity of patient clinical outcome was evaluated by the lung injury score. Leukocyte filtration through the microchannel was significantly weaker in esophagectomy patients than in healthy subjects (p<0.01). After esophagectomy, filtration was further impaired compared with preoperative values (p<0.05). The neutrophil F-actin content was higher in patients than in controls (p<0.01), and increased after esophagectomy compared with preoperative values (p<0.01). We concluded that circulating leukocytes showed reduced deformability and appeared to be sequestered within microcapillaries after esophagectomy. Changes in neutrophil cytoskeleton were considered to be responsible for the reduced deformability. Leukocyte accumulation within pulmonary microcapillaries might be related to the pathogenesis of lung injury after esophagectomy.

Dominant-negative inhibition of Ca2+ influx via TRPV2 ameliorates muscular dystrophy in animal models.

Muscular dystrophy is a severe degenerative disorder of skeletal muscle characterized by progressive muscle weakness. One subgroup of this disease is caused by a defect in the gene encoding one of the components of the dystrophin-glycoprotein complex, resulting in a significant disruption of membrane integrity and/or stability and, consequently, a sustained increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)). In the present study, we demonstrate that muscular dystrophy is ameliorated in two animal models, dystrophin-deficient mdx mice and delta-sarcoglycan-deficient BIO14.6 hamsters by dominant-negative inhibition of the transient receptor potential cation channel, TRPV2, a principal candidate for Ca(2+)-entry pathways. When transgenic (Tg) mice expressing a TRPV2 mutant in muscle were crossed with mdx mice, the [Ca(2+)](i) increase in muscle fibers was reduced by dominant-negative inhibition of endogenous TRPV2. Furthermore, histological, biochemical and physiological indices characterizing dystrophic pathology, such as an increased number of central nuclei and fiber size variability/fibrosis/apoptosis, elevated serum creatine kinase levels, and reduced muscle performance, were all ameliorated in the mdx/Tg mice. Similar beneficial effects were also observed in the muscles of BIO14.6 hamsters infected with adenovirus carrying mutant TRPV2. We propose that TRPV2 is a principal Ca(2+)-entry route leading to a sustained [Ca(2+)](i) increase and muscle degeneration, and that it is a promising therapeutic target for the treatment of muscular dystrophy.

A rapid microfluidic switching system for analysis at the single cellular level.

Analysis of cellular responses to chemicals at high spatiotemporal resolution is required for precise understanding of intracellular signal transduction. Here, we demonstrated a novel method for applying different solutions to a part of or all of a cell at high spatiotemporal resolution. We fabricated a microfluidic device using polydimethylsiloxane, and the sharp interface between the two solution streams flowing in the channel was used for the application of different solutions. We constructed a computer-controlled system to control the interface movement precisely, rapidly, and reproducibly during positioning, and spatial and temporal resolutions attained were 1.6 mum and 189 ms, respectively. We then applied the present system to the analysis of intracellular responses to chemicals. We were able to measure [Ca (2+)] (i) increases within 500 ms, when one laminar stream covered a part of the cell. This method can be used as a generic platform to investigate responses against drugs at the single cell level.

Analysis of cyclic-stretching responses using cell-adhesion-patterned cells.

Human vascular endothelial cells form the interface between the bloodstream and vessel walls and are continuously subjected to mechanical stimulation. When endothelial cells are stretched cyclically, along one axis, they align perpendicular to the axis of stretch. We previously reported that applying a cyclic, uni-axial strain to cells induced tyrosine phosphorylation of focal adhesion kinase and stimulated mitogen-activated protein kinase. However, it is difficult to quantify and analyze the spatial distribution of tyrosine phosphorylation in these cells, as they form focal adhesions randomly. In this study, we developed a system to overcome this problem by preparing individual, uniform, patterned cells that could be stretched cyclically and uni-axially. We constructed polydimethylsiloxane stretch chambers and used microcontact printing technology to imprint a pattern of 2 microm fibronectin dots (10 lines x 10 columns in a 38 microm square) before seeding them with human umbilical vein endothelial cells (HUVEC). We found that most HUVEC attached to the patterned dots after 2h and were similar in size and morphology, based on phase-contrast microscopy. In this system we were able to statistically analyze tyrosine phosphorylation and actin polymerization in these patterned cells, when subjected to a cyclic, uni-axial strain, using fluorescent microscopy.

Enhanced Na+/H+ exchange activity contributes to the pathogenesis of muscular dystrophy via involvement of P2 receptors.

A subset of muscular dystrophy is caused by genetic defects in dystrophin-associated glycoprotein complex. Using two animal models (BIO14.6 hamsters and mdx mice), we found that Na(+)/H(+) exchanger (NHE) inhibitors prevented muscle degeneration. NHE activity was constitutively enhanced in BIO myotubes, as evidenced by the elevated intracellular pH and enhanced (22)Na(+) influx, with activation of putative upstream kinases ERK42/44. NHE inhibitor significantly reduced the increases in baseline intracellular Ca(2+) as well as Na(+) concentration and stretch-induced damage, suggesting that Na(+)(i)-dependent Ca(2+)overload via the Na(+)/Ca(2+) exchanger may cause muscle damage. Furthermore, ATP was found to be released continuously from BIO myotubes in a manner further stimulated by stretching and that the P2 receptor antagonists reduce the enhanced NHE activity and dystrophic muscle damage. These observations suggest that autocrine ATP release may be primarily involved in genesis of abnormal ionic homeostasis in dystrophic muscles and that Na(+)-dependent ion exchangers play a critical pathological role in muscular dystrophy.

Regulation of the cardiac Na+/Ca2+ exchanger by calcineurin and protein kinase C.

Na+/Ca2+ exchanger (NCX) activity is markedly inhibited in hypertrophic neonatal rat cardiomyocytes subjected to chronic phenylephrine treatment. This inhibition is reversed partially and independently by acute inhibition of calcineurin and protein kinase C (PKC) activities. Similar NCX inhibition occurs in CCL39 cells expressing cloned wild-type NCX1, when they are infected with adenoviral vectors carrying activated calcineurin A and then treated acutely with phorbol myristoyl acetate or protein phosphatase-1 inhibitors. The data obtained with these cells suggest that calcineurin activity, PKCalpha-mediated NCX1 phosphorylation, and the central loop of NCX1 (possibly its beta1 repeat) are required for the observed NCX inhibition. We observe partial inhibition of NCX activity independent of NCX1 phosphorylation when CCL39 cells are infected with activated calcineurin A but not further treated with phorbol myristoyl acetate or phosphatase inhibitors. Calcineurin thus appears to downregulate NCX activity via two independent mechanisms, one involving NCX1 phosphorylation and the other not involving NCX1 phosphorylation. These data indicate the existence of a novel regulatory mechanism for NCX1 involving calcineurin and PKC, which may be important in cardiac pathology.

Phosphorylation of Na+/Ca2+ exchanger in TAB-induced cardiac hypertrophy.

Both protein kinase Calpha-dependent Na+/Ca2+ exchanger1 (NCX1) phosphorylation and calcineurin activity are required for the depression of NCX activity observed in chronically phenylephrine (PE)-treated hypertrophic neonatal rat cardiomyocytes. In this study, we explored the possibility that the same changes occur in vivo hypertrophy. In the hypertrophic hearts of thoracic aortic-banded (TAB) mice, NCX1 phosphorylation increased significantly compared with control hearts. Furthermore, the TAB-induced cardiac hypertrophy was much less prominent in transgenic mice overexpressing an NCX1 mutant having defective phosphorylation sites. These data suggest that the phosphorylation status of NCX1 may play an important role in the pathogenesis of load-induced cardiac hypertrophy.

Heart slice NMR.

Nuclear magnetic resonance (NMR) spectroscopy of the heart is normally carried out using whole heart preparations under coronary perfusion. In such preparations, either radical changes in ionic composition of the perfusate or applications of numerous drugs would affect coronary microcirculation. This report communicates the first (31)P NMR spectroscopy study using a heart slice preparation (left ventricular slices) superfused with extracellular medium. The ratio of phosphocreatine concentration to ATP concentration was approximately 2.1. Also, intracellular pH and Mg(2+) concentration ([Mg(2+)](i)), estimated from the chemical shifts of inorganic phosphate and ATP, were comparable with those under retrograde perfusion. [Mg(2+)](i) was significantly increased by the removal of extracellular Na(+), supporting the essential role of Na(+)-coupled Mg(2+) transport in Mg(2+) homeostasis of the heart. Heart slice preparation could also be used to evaluate the potency of cardiac drugs, regardless of their possible effects on coronary microcirculation.