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Titin is a giant protein that is in charge of the assembly and passive mechanical properties of the sarcomere. Cardiac titin contains a unique N2B region, which has been proposed to modulate elasticity of the titin filament and to be important for hypertrophy signaling and the ischemic stress response through its binding proteins FHL2 and ␣B-crystallin, respectively. To study the role of the titin N2B region in systole and diastole of the heart, we generated a knockout (KO) mouse deleting only the N2B exon 49 and leaving the remainder of the titin gene intact. The resulting mice survived to adulthood and were fertile. Although KO hearts were small, they produced normal ejection volumes because of an increased ejection fraction. FHL2 protein levels were significantly reduced in the KO mice, a finding consistent with the reduced size of KO hearts. Ultrastructural analysis revealed an increased extension of the remaining spring elements of titin (tandem Ig segments and the PEVK region), which, together with the reduced sarcomere length and increased passive tension derived from skinned cardiomyocyte experiments, translates to diastolic dysfunction as documented by echocardiography. We conclude from our work that the titin N2B region is dispensable for cardiac development and systolic properties but is important to integrate trophic and elastic functions of the heart. The N2B-KO mouse is the first titin-based model of diastolic dysfunction and, considering the high prevalence of diastolic heart failure, it could provide future mechanistic insights into the disease process.cardiac muscle ͉ hypertrophy ͉ mechanics ͉ cardiology ͉ disease T itin forms a continuous filament along the myofibril that determines the elastic properties of cardiac myocytes (for review, see ref. 1). The extensible region of titin is found in the I-band region of the sarcomere and comprises tandemly arranged Ig-like domains and the so-called PEVK region (2). In addition, cardiac titin contains a third extensible region, the N2B element (2), which is absent in skeletal muscle. The N2B region extends greatly toward the upper limit of the physiological sarcomere length of cardiac muscle (3, 4). It has been suggested that this extension reduces the steepness of the passive forcesarcomere length relation, decreasing the likelihood of the unfolding of Ig domains (3). Mutations in the N2B region can lead to dilated or hypertrophic cardiomyopathy, apparently through altered affinity to FHL2, a heart-specific member of the LIM domain gene family (5). To understand the role of the titin N2B region in cardiac function and disease, we have eliminated exon 49, which encodes the N2B region, and investigated its effect on the mechanical and trophical properties of the knockout (KO) heart.

OBJECTIVE: To evaluate and analyze the risk factors for burning mouth syndrome (BMS). METHODS: Eighty-seven consecutive patients with BMS and a randomly selected control group (n = 82) were comprehensively investigated with a self-designed questionnaire, Self-rating Anxiety Scale (SAS) and Self-rating Depression Scale (SDS). A complete blood count and serum sex hormone were also examined in patients with BMS and control subjects. All the data obtained were transferred to a data bank and analyzed statistically in SPSS 11.5 for windows. RESULTS: No statistical difference between the BMS group and the control group was found in blood analyses including white blood cell count, red blood cell count, hemoglobin and platelet count. Among the menopausal or postmenopausal women with BMS, the follicle stimulating hormone (FSH) level was significantly higher, but the estradiol level was significantly lower. The BMS group reported adverse life events more frequently than the control group. Patients with BMS significantly exhibited symptoms of somatization, and both the scores of anxiety, depression in patients with BMS were higher than those of the control group (P < 0.05). A regression equation which included six variables had been established by using logistic regression analysis, indicating that the habit of tongue thrusting, lip sucking, periodontitis, smoking, outcome of recent medication, depression were the principal risk factors, among which tongue thrusting was the most significant. CONCLUSION: Our study indicated that BMS may be of psychological origin, and the measures such as refraining from oral parafunctional activities, removing local irritating factors, stopping smoking, good mental health status could help in the prevention of BMS.

Rationale: The giant protein titin plays key roles in myofilament assembly and determines the passive mechanical properties of the sarcomere. The cardiac titin molecule has 2 mayor elastic elements, the N2B and the PEVK region. Both have been suggested to determine the elastic properties of the heart with loss of function data only available for the N2B region. Objective: The purpose of this study was to investigate the contribution of titin's proline-glutamate-valine-lysine (PEVK) region to biomechanics and growth of the heart. Methods and Results: We removed a portion of the PEVK segment (exons 219 to 225; 282 aa) that corresponds to the PEVK element of N2B titin, the main cardiac titin isoform. Adult homozygous PEVK knockout (KO) mice developed diastolic dysfunction, as determined by pressure-volume loops, echocardiography, isolated heart experiments, and muscle mechanics. Immunoelectron microscopy revealed increased strain of the N2B element, a spring region retained in the PEVK-KO. Interestingly, the PEVK-KO mice had hypertrophied hearts with an induction of the hypertrophy and fetal gene response that includes upregulation of FHL proteins. This contrasts the cardiac atrophy phenotype with decreased FHL2 levels that result from the deletion of the N2B element. Key Words: diastole Ⅲ connectin Ⅲ hypertrophy Ⅲ compliance Ⅲ FHL T itin is the largest protein in mammals and forms a continuous elastic filament along the myofibril (reviewed in 1 ). Because of its enormous size, titin is a prominent target for mutations that give rise to diseases such as familial dilated cardiomyopathy and muscular dystrophy. 2,3 Titin's extensible region resides in the I-band of the sarcomere and consists of immunoglobulin (Ig)-like domains arranged in tandem, the heart specific N2B element, and the prolineglutamate-valine-lysine (PEVK) element. 4 The PEVK element is thought to function as a largely unfolded polypeptide that extends at low force levels and that thereby provides an important source of elasticity at physiological sarcomere lengths. [5][6][7] Unlike the 1-exon heart specific N2B element, the titin gene contains 112 PEVK exons that are differentially expressed between muscle types. 8 Of these PEVK exons, 219 to 225 are expressed in the so-called N2B titin isoform, that constitutes the dominant cardiac isoform in the left ventricle of a wide range of species, including rodents and human. 9 Here we generated a mouse deficient in titin's exons 219 to 225 that results in a deletion of the c-terminal PEVK region (282 aa) and determined its role in cardiac function using echocardiography, in vivo pressure-volume loops, isolated heart physiology, muscle mechanics, immunoelectron microscopy, and expression analysis. We investigated the hypertrophy phenotype and studied members of the four-and-a-half LIM family involved in atrophy/hypertrophy signaling-FHL1 and FHL2. 10,11 Our results reveal the strong effect of the PEVK element on diastolic function but also that the role of the PEVK extends beyond that of a mechanical spr...

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Many studies demonstrate that accumulation of reactive aldehydes plays an important role in cellular oxidative injury and aldehyde dehydrogenase 2 (ALDH2)-mediated detoxification of reactive aldehydes is thought as an endogenous protective mechanism against cell injury. This study was performed to explore whether Alda-1, a newly identified ALDH2 activator, was able to protect brain against ischemia/reperfusion injury through clearance of reactive aldehydes. In a rat model of focal cerebral ischemia/reperfusion injury, neurological function, infarct volume, cellular apoptosis, mortality, ALDH2 activity and protein expression, contents of 4-hydroxy-2-nonenal (4-HNE), and malondialdehyde (MDA) were determined. The results showed that ischemia/reperfusion treatment led to increase in neurological deficit score, infarct volume, cellular apoptosis, and mortality accompanied by the elevated levels of reactive aldehydes (4-HNE and MDA). There was no significant change in ALDH2 activity and protein expression. Alda-1 treatment at both dosages (15 mg/kg × 2 or 50 mg/kg × 2, i.g.) was able to increase the activity of ALDH2 and decrease the accumulation of reactive aldehydes concomitantly with the improvement of brain injury (decrease in infarct volume, cellular apoptosis, and mortality) and neurological function (decrease in neurological deficit score). However, Alda-1 treatment did not affect ALDH2 protein expression. Our results suggest that the protective effect of Alda-1 on cerebral ischemia/reperfusion injury is related to ALDH2 activation and clearance of reactive aldehydes.

Recent studies have uncovered that accumulation of glutamate after ischaemic stroke is closely associated with the down-regulation of glutamate transporter-1 (GLT-1) expression, suggesting that GLT-1 expression critically controls glutamate accumulation and the abnormal glutamate transport-elicited neuronal cell excitotoxicity in patients with ischaemic stroke. However, it remains unknown how GLT-1 expression is regulated under ischaemic stroke conditions. In the present study, we screened the expression of nine brain-specific or brain-enriched miRNAs in a focal cerebral ischaemia/reperfusion (I/R) injury rat model, which showed glutamate accumulation and down-regulated GLT-1 expression as expected, and revealed that the miR-107 level was elevated in both brain tissue and plasma in the model. Next, we examined the functional relationship of miR-107 with GLT-1 expression in a nerve cell hypoxia/reoxygenation (H/R) injury model. H/R treatment increased apoptosis of the nerve cells concomitant with glutamate accumulation, miR-107 elevation and suppressed GLT-1 expression, mimicking our in vivo findings in the cerebral I/R injury rat model in vitro. Co-treating the cells with an miR-107 inhibitor blocked all of the effects, demonstrating that miR-107 functions to inhibit GLT-1 expression and elevate glutamate accumulation. To extend these animal and cell-based studies to clinical patients, we measured the plasma levels of miR-107 and glutamate, and observed that both miR-107 and glutamate were elevated in patients with ischaemic stroke. On the basis of these observations, we conclude that elevated miR-107 expression after ischaemic stroke accounts, at least partially, for glutamate accumulation through suppression of GLT-1 expression. Our findings also highlight that the plasma level of miR-107 may serve as a novel biomarker for monitoring excitotoxicity in patients with ischaemic stroke.

Background-Titin is a giant protein crucial for the assembly and elasticity of the sarcomere. Recently, titin has been linked to signal transduction through its kinase domain, which has been proposed to sense mechanical load. We developed a knockout in which expression of M-line-deficient titin can be induced in adult mice and investigated the role of the titin kinase region in cardiac function. Methods and Results-Isolated heart experiments revealed that in titin M-line-deficient mice, the contractile response to ␤-adrenergic agonists and extracellular calcium is reduced. However, the Ca 2ϩ sensitivity and cooperativity of activation of skinned cardiac muscle were unchanged. In knockout mice, calcium transients showed a reduced rate of calcium uptake, and expression analysis showed reduced levels of calmodulin, phospholamban, and SERCA2. Ultimately, knockout mice developed cardiac hypertrophy and heart failure, which involves protein kinase C signal transduction but not the mitogen-activated protein kinase pathway. Conclusions-The titin kinase region emerges as a regulator of contractile function through effects on calcium handling and hypertrophy through protein kinase signal transduction. These novel functions of titin might provide a rationale for future therapeutic approaches to attenuate or reverse symptoms of heart failure. Key Words: cardiac output Ⅲ cardiomyopathy Ⅲ cells Ⅲ genes Ⅲ heart failure Ⅲ mechanics Ⅲ models, animal H eart failure is one of the main causes of mortality in the developed world. It has various underlying causes such as cardiomyopathy, coronary artery disease, hypertension, and diabetes, which lead to reduced contractility (systolic dysfunction) or altered relaxation (diastolic dysfunction). 1-3 Among the genetic defects that lead to heart failure are mutations in the sarcomeric protein titin, which cause dilated or hypertrophic cardiomyopathy, depending on the underlying defect. 4 -6 Titin, a giant sarcomeric protein that contributes to the diastolic properties of the heart through its elastic domains, 7-9 is involved in signal transduction through its kinase region. 10,11 Although the elastic properties of titin have been studied extensively, 12-16 its signaling functions are less well understood. The titin kinase (TK) domain has been proposed to regulate protein expression in striated muscle in a strain-dependent manner via its substrates nbr1 and p62. 17 TK is encoded in M-line exon 1, together with binding sites for proteins such as the ubiquitin ligase MuRF-1, 18 the adaptor protein FHL2/DRAL, which binds both signaling proteins and metabolic enzymes, 19 and calmodulin, which has been shown to regulate TK activity in vitro. 20 Clinical Perspective p 751To study the kinase region in vivo, we have previously used a conditional knockout (KO) approach to selectively delete this region (M-line exon 1 and M-line exon 2) in striated muscle using the MCKcre transgene. 21 Although this approach results in live offspring, neonatal mice develop cardiomyopathy and skeletal muscle wasting...