The fecal immunochemical test for hemoglobin (FIT) is widely used for colorectal cancer screening but shows limited sensitivity for detecting advanced colorectal adenomas. To overcome this limitation, we evaluated a combined approach using FIT and fecal DNA testing for methylated somatostatin (SST). Fecal samples were collected from healthy subjects, patients with non-advanced adenoma, advanced adenoma, and colorectal cancer. SST methylation levels were quantified using droplet digital PCR after treatment with methylation-sensitive restriction enzymes. Using logistic multivariate analysis, we developed the FAMS (FIT, age, methylated SST) index. The FAMS index improved sensitivity for detecting advanced adenoma compared with FIT alone while maintaining high specificity. Even at higher specificity, the sensitivity of the FAMS index remained higher than that of FIT. These results suggest that the FAMS index can non-invasively enhance early detection of advanced colorectal adenoma and may serve as a promising tool for screening.

Surgical resection is the most effective treatment for gastrointestinal cancer; however, a novel treatment is needed for unresectable cases. We have developed an immunotherapy for gastrointestinal cancers. In 1990, we devised a combination immunotherapy for hepatocellular carcinoma using interleukin-2, OK-432, Adriamycin, cyclophosphamide, and famotidine, which resulted in complete response in 4 out of 24 patients. Subsequently, we developed immune cell therapies for gastrointestinal cancers. Tumor antigen-specific immunity was first reported in 1991. We administered cancer vaccines using epitope peptides derived from oncoantigens for colorectal and pancreatic cancers. As a result, antigen-specific immunity was induced at a high frequency, and cases showing long-term antitumor effects were observed, whereas cases that were ineffective were also observed; therefore, we searched for the cause. A comprehensive analysis of lymphocytes, serum, and tumor tissues revealed the presence of many immunosuppressive factors. Therefore, we developed an immune adjuvant that induces cytotoxic T lymphocytes without fatigue markers and tumor antigen-specific peptides. This vaccine was administered preoperatively in 20 cases of hepatocellular carcinoma; 12 cases showed significant lymphocyte invasion into tumors and tumor necrosis in 6 cases, and these results suggested that hepatocellular carcinoma changed into a hot tumor. This immunotherapy is expected to be useful in combination with checkpoint inhibitors.

Background: Eosinophilic chronic rhinosinusitis (ECRS) is generally characterized by refractory type 2 inflammation along with severe asthma. Although tezepelumab, an anti-thymic stromal lymphopoietin (TSLP) monoclonal antibody, has broad efficacy in severe asthma, its effects on sinonasal inflammation in ECRS remain unclear. In this retrospective observational study, we investigated the radiological and clinical changes in patients with ECRS and severe asthma treated with tezepelumab. Methods: Clinical data, blood eosinophil counts, fractional exhaled nitric oxide (FeNO), total serum IgE levels, lung function, and paranasal sinus CT findings of seven patients with ECRS who received tezepelumab for comorbid severe asthma at a single tertiary care center between 2022 and 2024, were collected. Radiologic severity was assessed using the Lund-Mackay score (LMS) at baseline and ≥6 months after treatment initiation. Results: The median LMS showed a trend toward reduction from baseline to ≥6 months, although this difference did not reach statistical significance (p = 0.051). In contrast, blood eosinophil counts, serum IgE levels, FeNO levels, and the FEV_1/FVC ratio did not show statistically significant changes during the observation period. Conclusion: Tezepelumab therapy showed a trend toward reduction in the Lund-Mackay score in patients with ECRS and severe asthma, although statistical significance was not reached. Systemic biomarkers and lung function remained stable.

Transient receptor potential vanilloid 4 (TRPV4) channels, expressed in presynaptic membranes and astrocytes, have attracted attention as factors involved in epilepsy. We investigated the role of TRPV4 channels as therapeutic targets for epilepsy. We injected Penicillin G (PG) into the cortex of wild-type (WT) mice or TRPV4 KO mice. We also injected RN 1734, a TRPV4 antagonist, before and after PG injection. We recorded epileptic discharges (EDs) and the concentration of extracellular glutamate in the mice. In WT mice, the glutamate concentration increased after PG injection and EDs occurred. In TRPV4 KO mice, the glutamate concentration and beta-band power of the EDs were lower than that in WT mice. When WT mice were administered RN 1734 prior to PG injection, both the glutamate concentration and spike amplitude at late phase following PG injection were comparable to those in TRPV4 KO mice. However, when RN 1734 was administered after the onset of EDs, the alleviation of EDs lasted for only 10 min, and the glutamate concentration did not decrease. RN 1734 modulated EDs in a timing-dependent manner, with significant effects during ongoing EDs and limited effects following pre-treatment. These results indicate that TRPV4 antagonists modulate glutamate dynamics in an activity-dependent manner.

Background: Deterioration of swallowing function markedly impairs quality of life, a problem particularly significant in Japanʼs rapidly aging population. Dysphagia management remains challenging due to limited clinical resources and lack of an organized support system. Objective: To illustrate the effectiveness of multidisciplinary collaboration in facilitating the transition from home parenteral nutrition (HPN) to complete oral intake. Case Presentation: A 66-year-old woman with severe dysphagia secondary to submandibular gland inflammation and prolonged impaired consciousness was maintained on total parenteral nutrition (TPN) during hospitalization because oral intake was deemed impossible. After discharge on HPN, a multidisciplinary home-care team─comprising physicians, nurses, registered dietitians, and certified dysphagia nurses─ initiated structured dietary and swallowing interventions. The patient restored full oral feeding within one year through structured assessment and staged texture advancement. Results: Swallowing ability improved from Repetitive Saliva Swallowing Test (RSST) 0, Modified Water Swallowing Test (MWST) 0 and Kuchikara Taberu Balance Chart (KTBC) 22 to RSST 3, MWST 4 and KTBC 53. Oral intake reached 1200 kcal/day, allowing discontinuation of HPN. Conclusion: Coordinated multidisciplinary collaboration with continuous information sharing between care settings enabled safe restoration of oral intake in a patient previously judged unable to eat orally. Regular reassessment of swallowing function is crucial to identify patients with recovery potential.

This study examined the association between non-invasive liver fibrosis biomarkers and non-alcoholic fatty liver disease (NAFLD) in an apparently healthy population. A matched case-control design was used to analyze 145 pairs of participants with and without NAFLD. Six liver fibrosis indexes were evaluated: Aspartate Aminotransferase to Alanine Aminotransferase Ratio (AAR), Aspartate Aminotransferase to Platelet Ratio Index (APRI), fibrosis index based on four factors (FIB-4), modified FIB-4 (mFIB-4), Forns Index, and Gamma-Glutamyl Transpeptidase to Platelet Ratio (GPR). Adjusted logistic regression analyses showed significant associations between mFIB-4 and Forns Index with NAFLD, highlighting their potential as tools for early detection. These markers demonstrated consistency across multiple analyses, supporting their potential use for screening asymptomatic individuals, especially in resource-limited settings. However, traditional markers like APRI and GPR showed limited utility in this cohort, emphasizing the need for contextual biomarker selection. Future studies should validate these findings across diverse populations and investigate their diagnostic capabilities in prospective cohort studies to
improve early NAFLD detection and intervention.

Background: Less aggressive rectal neuroendocrine tumors (NETs) are mainly located in the submucosal layer. We analyze our clinical experience of endoscopic submucosal dissection (ESD) for rectal NETs. Methods: We experienced 27 consecutive rectal neuroendocrine neoplasms (NENs). In these cases, we retrospectively analyzed our selections and the therapeutic results of endoscopic mucosal resection (EMR) and ESD. Results: We initially used EMR for rectal NETs. However, there was one case of local recurrence with adjacent lymph-node metastasis. This was initially treated by EMR at another hospital 12 years earlier. We changed the resection modality from EMR to ESD, which has been useful for en bloc resection. Thereafter, we performed ESD for 16 cases. En bloc resection was performed in 15 cases. The vertical margin was positive in one case but there was no local residue. In one ESD case, detailed examination of the ESD en bloc specimen clarified lymphovascular invasion and was useful for the decision of additional surgical operation. Conclusions: Based on our experience of this case series of rectal NETs, en bloc resection using ESD is a　promising treatment modality.

Cardiac hypertrophy is widely recognized as a significant risk factor contributing to adverse outcomes in individuals with cardiovascular conditions. The disruption of intracellular calcium ( Ca^{2+} ) balance has been implicated in the development of cardiac hypertrophy, though the precise mechanisms remain poorly understood. In this research, we explored whether hypertrophy induced by pressure overload may arise from the destabilization of the cardiac ryanodine receptor (RyR2) triggered by the dissociation of calmodulin (CaM), leading to subsequent Ca^{2+} leakage. We also assessed whether genetically strengthening the binding affinity between CaM and RyR2 could potentially reverse this process. In the early phases of cardiac hypertrophy caused by pressure overload—when contractile function is still intact—we observed that RyR2 destabilization mediated by reactive oxygen species (ROS) coincides with impaired relaxation. Moreover, stabilizing RyR2 through enhanced CaM binding was found to completely inhibit hypertrophic signaling and improve survival rates. Our findings reveal a crucial connection between RyR2 destabilization and the progression of cardiac hypertrophy.

Blood-brain barrier (BBB) dysfunction found in the multiple sclerosis (MS) cases is generally considered as a consequence of neuroinflammation. In this study we challenge this view by developing and analyzing novel BBB model from MS patients using induced pluripotent stem cells (iPSCs). We differentiated iPSCs into brain microvascular endothelial cell (BMEC)-like cells to establish an in vitro BBB model. We found that BMEC-like cells from MS patients exhibited compromised barrier integrity, characterized by weakened junctions, heightened permeability, and an elevated inflammatory profile when compared to cells from healthy individuals. Notably, the activation of the Wnt/β-catenin signaling pathway led to improvements in barrier function and a reduction in inflammatory responses, indicating potential therapeutic targets for reinforcing BBB stability in MS.

A 70-year-old woman presented with acute fever, impaired consciousness, leukopenia, thrombocytopenia, and right inguinal lymphadenopathy. A lymph node biopsy was diagnosed as diffuse large B-cell lymphoma (DLBCL). However, her symptoms were consistent with severe fever with thrombocytopenia syndrome (SFTS), and RT-PCR for SFTS virus (SFTSV) RNA was positive. The patient’s condition and lymphadenopathy gradually improved with supportive measures and short-term steroid treatment and no lymphadenopathy recurrence was observed. Lymph node pathological examination revealed SFTSV-infected cells, leading to the final diagnosis of necrotizing lymphadenitis associated with SFTS. Careful consideration is required to differentiate necrotizing lymphadenitis associated with SFTS from that associated with DLBCL.

Focal brain cooling (FBC) at 15℃ and transient receptor potential vanilloid 4 (TRPV4) deficiency relieve brain infarction. TRPV4 channels are inactivated by cooling (< 27℃), suggesting that the anti-ischemic effects of FBC include those of TRPV4 inactivation. However, the extent to which TRPV4 inactivation contributes to the anti-ischemic, anti- blood-brain barrier (BBB) disruption, and anti-apoptosis effects of FBC on cerebral infarction remains unclear. We investigated the contribution and mechanisms of RN1734, a TRPV4 antagonist, in FBC for cerebral infarction using TRPV4 knockout and wild-type mice. Focal cerebral infarction was induced by photochemically induced thrombosis. Infarct volume, BBB disruption, and number of apoptotic cells were evaluated. The TRPV4 antagonist or deficiency showed similar anti-ischemic and anti-BBB disruptive effects to those of FBC. Intracerebroventricular injection of RN1734 showed a similar reduction in the number of apoptotic cells to that of FBC. These anti-ischemic and -apoptotic effects were completely inhibited with injection of GSK1016790A, a TRPV4 agonist, immediately before FBC. Our results showed that TRPV4 modulation is the primary factor contributing to the antiischemic effects of FBC, and TRPV4 channel inactivation relieve focal ischemic infarction by relieving BBB disruption and preventing apoptosis. Therefore, FBC treatment improves ischemic stroke through the modulation of TRPV4 channels.

Hypothermia and focal brain cooling (FBC) demonstrate neuroprotective effects in ischemic stroke, but their invasiveness limits clinical use. We explored transient receptor potential (TRP) channels as an alternative, focusing on TRP Ankyrin 1 (TRPA1), which operates within the temperature range of FBC. Activation of TRPA1 has been reported to offer neuroprotection, suggesting it may contribute to the effects seen with FBC. We hypothesized that pharmacological activation of TRPA1 could replicate the neuroprotective effects of FBC, providing a less invasive treatment for cerebral infarction. We examined the effects of a TRPA1 agonist and FBC in focal cerebral ischemia induced by photochemically triggered thrombosis in wild-type (WT) and TRPA1 knockout (KO) mice. In WT mice, intracerebroventricular administration of the TRPA1 agonist allyl isothiocyanate reduced infarct size by approximately half, comparable to FBC. TRPA1 KO mice had larger infarcts than WT, but FBC significantly reduced infarct size in both groups. Furthermore, Evans blue extravasation, used to assess the extent of blood-brain barrier disruption, was approximately twice as high in TRPA1KO mice compared to WT mice. These findings underscore the neuroprotective potential of TRPA1 agonists and the increased vulnerability against ischemia with TRPA1 deficiency. However, the neuroprotective effects of TRPA1 activation are likely mediated by a mechanism distinct from that of FBC. Our study suggests TRPA1 channels are crucial for ischemic stroke protection and may offer a novel therapeutic approach.

I, Professor Nobuaki Tanaka, gave a Farewell Lecture on February 29, 2024. First, I talked about the episode that led me to start researching aortic stenosis, and reflected on my own lectures on this topic to students aiming to become medical laboratory scientist. Lastly, I spoke to students majoring in medical laboratory scientist about the importance of practical clinical training.

Purpose: We investigated sex differences in heart rate variability (HRV; a non-invasive indicator of autonomic nervous system) and its frequency-specific responses to whole-body vibration (WBV) at three distinct frequencies among elderly subjects. Methods: Data from 11 males and 13 females were analyzed across four randomized sessions of exposure: WBV at 15, 20, or 25 Hz with a 4 mm peak-to-peak displacement, or control (0 Hz) condition comprising three bouts of 1-minute exposure with 1-minute between-bout rests. HRV measurements were taken before and during the exposure. Results: At baseline, low-frequency power/LF (ms^{2}) were significantly lower in females than males (P < 0.05). During exposure, LF (ms^{2}), high-frequency power (ms^{2}), standard deviation of normal-to-normal intervals, root mean square of successive differences between RR intervals, standard deviation of the Poincaré plot perpendicular to the line-of-identity, and standard deviation of the Poincaré plot along the line-of-identity significantly increased at 20 Hz for males and 25 Hz for females (P < 0.05 to 0.005) compared to respective baselines. Conclusions: Elderly females tend to exhibit reduced autonomic nervous system function compared to males. Furthermore, our results indicate that WBV at 20 Hz for males and 25 Hz for females may be considered beneficial for enhancing HRV in the elderly.

Dementia, a leading cause of death, often initiates with spatial cognitive impairment. Assisting spatial cognition may not only address challenges faced by individuals with spatial cognitive impairment but also facilitate the prevention of dementia. While previous studies have explored voice navigation for the visually impaired, its utility for those without visual impairment remains unexamined. To provide insights for spatial navigation in the elderly, in the present study, we evaluated the usefulness of voice navigation in healthy university students. In a randomized controlled trial, forty students were assigned to either a paper map only group or a paper map and Google Maps voice navigation group and instructed to navigate a predetermined 800-meter route in a park. The effectiveness of voice navigation was evaluated through various means, including time taken to reach the destination, accuracy of the route followed, and participantsʼ experience. The results show that the two groups did not differ in terms of goal completion and time consumed. Nor did they differ in feelings of drowsiness, instability, uneasiness, pleasure, and relaxation. However, participants in the paper map only group demonstrated a decrease in local pain and eyestrains together with improved feelings of vigor at the goal compared to the start, which was absent in participants using both paper map and voice navigation. Although the effectiveness of the voice navigation was not confirmed, our study did provide important insights regarding in what ways voice navigation can be improved. Moreover, we were able to observe mood improvements in participants with a paper map only, which may indicate the effect of physical activity and exposure to the natural environment.

Access to high-quality care at the end-of-life is a fundamental human right; therefore, it is necessary to examine how end-of-life care should be taught in nursing education to prepare for a super-aging society. Japan and Hong Kong are both in the East Asian region and are facing similar super-aging challenges, such as increased mortality and declining birth rates. This paper explores the literature on this topic and discusses how end-of-life care is taught in undergraduate nursing education in both Japan and Hong Kong. A comprehensive literature search was performed using end-of-life care keywords. Subsequently, the authors engaged in a discourse on expert perspectives, insights, and results from the literature. It is necessary to bridge the gap between the desired manner of spending oneʼs final days and the practical reality of considering patientsʼ best interests by referring to the existing policies, laws, guidelines, and frameworks of healthcare systems. This study suggests the importance of understanding patientsʼ views on life, death, values, and cultural backgrounds and educating healthcare personnel to apply these principles flexibly in their practice.

Benign paroxysmal positional vertigo (BPPV) is the most common vertigo disease and is more likely to occur in perimenopausal women, suggesting an association with osteoporosis. Since otoconia are primarily composed of calcium carbonate, abnormal calcium metabolism may lead to otoconia dislocation. However, the detailed mechanism is currently unknown. In this study, we investigated the effects of drugs (cadmium and dexamethasone) that cause abnormal calcium metabolism on otolith formation in zebrafish larvae. Here, otolith size was clearly reduced in the cadmium group, and the calcium content of the larvae was also markedly reduced. In contrast, in the dexamethasone group, which also had a lower calcium content than the control group, otolith size increased. Our results suggest that, as in bone, calcium metabolism influences the repeated dissolution and recrystallization of otoliths and maintains homeostasis in response to calcium concentrations in the endolymphatic fluid.

Objective: The objective was to explore the potential existence and nature of the relationship between serum of uric acid (SUA) and serum uric acid to serum creatinine ratio (SUA/SCr) with reduced estimated glomerular filtration rate (eGFR) in patients with non-alcoholic fatty liver disease (NAFLD). Methods: A cross-sectional study was conducted among apparently healthy subjects with NAFLD (n=485). The association between tertiles of SUA and SUA/SCr with reduced eGFR (n=56) were investigated after adjustments for potentially relevant confounders. Also, the diagnostic performances of SUA and SUA/SCr were evaluated with the receiver operating characteristic (ROC) curve analysis. Results: In the adjusted models, SUA showed a significant positive association with reduced eGFR in the highest tertile (OR 5.65, 95% CI 2.48-12.86, p<0.001), and SUA/SCr, in the lowest tertile (4.21, 95% CI 1.76-10.07, p=0.001). The ROC curve analysis did not reveal any significant difference between the corresponding values of area under the curve for SUA and SUA/SCr (0.70 and 0.67, respectively; p=0.521). Conclusions: We revealed significant and independent associations of elevated SUA and reduced SUA/SCr with kidney function decline in NAFLD. However, the clinical utility of these two biomarkers seemed to be limited for the mentioned purpose and needs further investigations.

Vestibular hair cells are susceptible to damage from various stimuli such as infections, ischemia, and certain therapeutic drugs, including aminoglycoside antibiotics and the antineoplastic agent cisplatin. In mammals, damage to the vestibular hair cells is permanent. This study aimed to evaluate the protective effects of nobiletin (NOB) against aminoglycoside-induced hair cell death using utricles collected from adult mice. The utricles removed from CBA/N mice were assigned to eight groups according to the dose of NOB and the administration or not of neomycin. Hair cells in the utricles were counted by double labeling with calmodulin and calbindin. NOB inhibited hair cell death in utricles exposed to neomycin. The protective effect of NOB on hair cells in the utricles was also suggested to have resulted from the inhibition of the production and accumulation of 4-hydroxy-2-nonenal, the final product of lipid peroxide aldehyde. NOB suppressed neomycin-induced hair cell death. The principle of hair cell protection from aminoglycoside-induced hair cell death suggests that NOB inhibits reactive oxygen species formation in the utricles exposed to neomycin.

This study investigated the potential of adalimumab (ADA), a monoclonal antibody targeting TNF-alfa, to protect the inner ear from intense sound exposure, given that inflammatory cytokines, including TNF-alfa, are linked to hearing loss in acoustic disorders. In this study, adalimumab was administered to mice, and its effect on the inner ear was assessed. We examined the translocation of ADA to the inner ear and its ototoxicity and impact on acoustic exposure. The results showed that adalimumab partially reached the cochlea after administration but increased the susceptibility to acoustic exposure, resulting in higher hair cell loss in the inner ear. While TNF-alfa had been considered a potential therapeutic target, the results suggested that excessive TNF-alfa suppression could harm the inner ear. We acknowledged some limitations, such as the use of adalimumab instead of an anti-mouse TNF-alfa antibody and the need to explore the suppression of other cytokines for better inner ear protection. In conclusion, adalimumab administration was found to increase the inner earʼs susceptibility to acoustic exposure, potentially leading to more significant hair cell damage, possibly due to excessive TNF-alfa suppression

Right ventricular (RV) dysfunction and its linked arrhythmias play a crucial role in determining the prognosis of pulmonary arterial hypertension (PAH). Our paper aimed to explore the potential protective effects of direct pharmacological intervention in the RV muscle using dantrolene (DAN), a stabilizer of the cardiac ryanodine receptor (RyR2), against RV dysfunction and arrhythmia in a rat model of monocrotaline (MCT)-induced PAH. To induce PAH, male 8-week-old Sprague-Dawley rats received MCT injections. The study also assessed the induction of ventricular tachycardia (VT) by catecholamines, examining RyR2-mediated Ca^{2+} release properties in isolated cardiomyocytes. Additionally, a pulmonary artery-banding model was established to evaluate the independent effects of chronic pressure overload on RV morphology and function. In the MCT-induced PAH rat model, findings revealed RV hypertrophy, dilation, and functional decline, resulting in 0% survival rate two months post-MCT induction. Conversely, chronic DAN treatment demonstrated improvements in these RV parameters and an 80% increase in survival. Furthermore, chronic DAN treatment prevented the dissociation of calmodulin from RyR2, inhibiting Ca^{2+} sparks and spontaneous Ca^{2+} transients in MCT-induced hypertrophied RV cardiomyocytes. Epinephrine induced VT in over 50% of rats with MCT-induced PAH, while chronic DAN treatment achieved complete suppression of VT. The paper concludes that stabilizing RyR2 with DAN holds promise as a novel therapeutic approach against the development of RV dysfunction and fatal arrhythmias associated with PAH.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in the cardiac type 2 ryanodine receptor (RyR2). Using knock-in mouse (KI) model (R2474S/+), we previously reported that a single point mutation within the RyR2 sensitized the channel to agonists, primarily mediated by defective inter-domain interaction within the RyR2 and subsequent dissociation of calmodulin (CaM) from the RyR2. Here, we examined whether CPVT can be genetically rescued by enhancing the binding affinity of CaM to the RyR2. We first determined whether there was a possible amino-acid substitution within the CaM-binding domain in the RyR2 (3584-3603) that can enhance its binding affinity to CaM, and found that V3599K substitution showed the highest binding affinity of CaM to CaM-binding domain. Hence, we generated a heterozygous KI mouse model (V3599K/+) with a single amino acid substitution in the CaM-binding domain of the RyR2, and crossbred it with the heterozygous CPVT –associated R2474S/+ KI mouse to obtain a double heterozygous R2474S/V3599K KI mouse model. The CPVT phenotypes, bidirectional or polymorphic ventricular tachycardia, were inhibited in the R2474S/V3599K mice. Thus, enhancement of the CaM binding affinity of the RyR2 is essential to prevent CPVT-associated arrhythmogenesis.

To investigate whether dantrolene (DAN), cardiac ryanodine receptor (RyR2) stabilizer, improves impaired diastolic function in an early pressure-overloaded hypertrophied heart, pressure-overload hypertrophy was induced by transverse aortic constriction (TAC) in mice. Wild-type (WT) mice were divided into four groups: sham-operated mice (Sham), sham-operated mice treated with DAN (DAN+Sham), TAC mice (TAC), and TAC mice treated with DAN (DAN+TAC). The mice were then followed up for 2 weeks. Left ventricular (LV) hypertrophy was induced in TAC, but not DAN+TAC mice, 2 weeks after TAC. There were no differences in LV fractional shortening among the four groups. Catheter tip micromanometer showed that the time constant of LV pressure decay, an index of diastolic function, was significantly prolonged in TAC but not in DAN+TAC mice. Diastolic function was significantly impaired in TAC, but not in DAN+TAC mice as determined by cell shortening and Ca^{2+} transients. An increase in diastolic Ca^{2+} leakage and a decrease in calmodulin (CaM) binding affinity to RyR2 were observed in TAC mice, while diastolic Ca^{2+} leakage improved in DAN+TAC mice. Thus, DAN prevented the progression of hypertrophy and improved the impairment of LV relaxation by inhibiting diastolic Ca^{2+} leakage through RyR2 and the dissociation of CaM from RyR2.