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Meng-meng Zheng,* Li-min Shang,* Chun-kai Du,* Lei Zhang, Wen Sun, Zhi-peng Wang, Yi-chen Zhu, Ye Tian Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yi-chen Zhu; Ye Tian, Department of Urology, Capital Medical University, Beijing Friendship Hospital, No. 95 Yong’an Road, Xicheng District, Beijing, 100050, People’s Republic of China, Email zhuyc82@sina.com; tianye166@126.comBackground: Potent immune-suppressive therapy has been demonstrated to increase the risk of infective endocarditis (IE) in renal recipients. Reports of Corynebacterium striatum (C. striatum) endocarditis in renal recipients are scarce, thus limiting understanding of the disease.Case Presentation: We describe a case of native valve endocarditis caused by C. striatum in a 35-year-old male patient. The young man with end-stage renal failure underwent kidney transplantation because of autosomal dominant polycystic kidney disease. Ceftazidime was administered after the surgery according to routine procedures, and the patient was discharged on the 14th day after the surgery without any evidence of infection. The patient experienced fever on the 56th day, and Corynebacterium was cultured from the patient’s blood, in agreement with the results of testing of the donor kidney preservation solution. On the 64th day, multiple thromboses were found in the right external iliac artery, particularly around the anastomotic orifice of the transplanted renal artery. Vegetation was found in the posterior mitral valve tip on the 65th day. The patient had symptoms of persistent angina pectoris and chest tightness and underwent mitral valve replacement and vegetative resection. The patient eventually died. C. striatum was detected in the mitral valve and vegetation tissue with metagenomic next-generation sequencing.Conclusion: C. striatum may cause endocarditis and endanger patients’ lives and thus warrants greater attention. Genotypic assays such as metagenomic next-generation sequencing are demonstrated to be effective in confirming species identity. Adequate anti-infection therapy and early surgery are required after IE is discovered.Keywords: kidney transplantation, infection, endocarditis, Corynebacterium striatum, case report

Purpose: To compare the efficiency and safety between retroperitoneal laparoscopic nephrectomy and traditional open nephrectomy to treat autosomal-dominant polycystic kidney disease before kidney transplantation., Materials and Methods: A total of 57 patients diagnosed with huge autosomal-dominant polycystic kidney disease between 2000 and 2020 at our center were included in this study. Patients were divided into a retroperitoneal laparoscopic (RL; n=23) group and traditional open (TO; n = 34) group. We retrospectively analyzed and compared preoperative and perioperative variables between the two groups., Results: Patients in the RL group showed a longer operation time (201.09±83.76min) compared to patients in the TO group (113.38 ± 51.84min, p < 0.001). The RL group also showed significantly less intraoperative blood loss (p = 0.025) and less intraoperative blood transfusion volume (p = 0.016) compared to the TO group. Meanwhile, time of gastrointestinal function recovery, bed leave, catheter indwelling and postoperative hospitalization in the RL group were 2.13 ± 0.63, 1.30 ± 1.0, 5.22 ± 2.09, 7.35±2.48 days, respectively, which were significantly shorter than the TO group (p < 0.05). Pain degree of patients during the first 48 hours after operation was similar between the RL and TO groups, but the opioid use percentage in the RL group was 8.70% (2/23) and was lower than the 26.47% (9/34) in the TO group (p = 0.022). Meanwhile, 5 and 23 patients exhibited postoperative complications in the RL and TO groups, respectively (p < 0.001)., Conclusion: Both retroperitoneal laparoscopic nephrectomy and traditional open surgery are feasible to treat huge polycystic nephrectomy. However, patients who undergo retroperitoneal laparoscopic nephrectomy experience higher levels of safety and recover more rapidly.

Aim: To develop a split glomerular filtration rate (sGFR) prediction model based on unilateral renal volume parameters using three-dimensional (3D) computed tomography (CT) volumetry., Materials and Methods: Clinical data (age, sex, height, weight, serum creatinine level [sCr], and sGFR measured by 99m Tc-diethylene triamine pentaacetic acid nuclear renal scintigraphy with the double plasma sample method) of 67 healthy renal donors and 111 patients with hydronephrosis admitted from April 2016 to September 2021 were analysed. The split renal parenchymal volume (sRPV) and split renal calyces and pelvis volume (sRCPV) of 67 unilateral donor left kidneys and 111 hydronephrotic kidneys were measured. Statistical analysis of these parameters was performed to develop and validate the sGFR prediction model., Results: sRPV (p<0.001), sRCPV (p=0.012), age (p=0.015), serum creatinine level (p=0.004), and weight (p=0.006) were significantly associated with the measured sGFR and were included in the sGFR prediction formula, which was constructed as: 68.710 + 0.093 × sRPV-0.041 × sRCPV-0.228 × W-0.219 × A-14.432 × sCr (r 2  = 0.416; where A is age, W is weight). The paired difference of internal validation between the measured sGFR (42.34 ± 13.71 ml/min/1.73 m 2 ) and the sGFR estimated by the prediction model (41.46 ± 8.99 ml/min/1.73 m 2 ) was 0.879 ± 11.475 ml/min/1.73 m 2 (p=0.492) with a 95% confidence interval of the mean difference of ±2.54 ml/min/1.73 m 2 ., Conclusion: The proposed model based on sRPV and sRCPV parameters could be used for estimating split renal function of healthy renal donors and patients with hydronephrosis to some extent. Further studies are required to evaluate and rectify the model., (Copyright © 2022 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.)

Objective: To analyze the relationship between the promoter methylation status of Tripartite-motif protein 58 (TRIM58) and its mRNA expression level in acute myeloid leukemia (AML), and to explore the expression and regulation of TRIM58 in AML., Methods: The bisulfite sequencing PCR (BSP) and quantitative real-time PCR (qPCR) technologies were used to detect the promoter methylation status and expression levels of TRIM58 mRNA in primary CD34 + and CD34 - AML cells and the AML cell lines KG1a and K562 were determined., Results: The expression of TRIM58 mRNA in CD34 + cells was down-regulated in 10 AML patients, while that in CD34 - cells was down-regulated in 12 cases. Differences in the promoter methylation level of TRIM58 were statistically significant between AML group and normal control group (P<0.05). Additionally, the expression of TRIM58 mRNA was down-regulated in cell lines KG1a and K562, and up-regulated after decitabine treatment., Conclusion: The down-regulation of mRNA expression of TRIM58 in AML cells may be related to its promoter methylation status.

Chronic myeloid leukemia (CML) are initiated and sustained by self-renewing malignant CD34 + stem cells. Extensive efforts have been made to reveal the metabolic signature of the leukemia stem/progenitor cells in genomic, transcriptomic, and metabolomic studies. However, very little proteomic investigation has been conducted and the mechanism regarding at what level the metabolic program was rewired remains poorly understood. Here, using label-free quantitative proteomic profiling, we compared the signature of CD34 + stem/progenitor cells collected from CML individuals with that of healthy donors and observed significant changes in the abundance of enzymes associated with aerobic central carbonate metabolic pathways. Specifically, CML stem/progenitor cells expressed increased tricarboxylic acid cycle (TCA) with decreased glycolytic proteins, accompanying by increased oxidative phosphorylation (OXPHOS) and decreased glycolysis activity. Administration of the well-known OXPHOS inhibitor metformin eradicated CML stem/progenitor cells and re-sensitized CD34 + CML cells to imatinib in vitro and in patient-derived tumor xenograft murine model. However, different from normal CD34 + cells, the abundance and activity of OXPHOS protein were both unexpectedly elevated with endoplasmic reticulum stress induced by metformin in CML CD34 + cells. The four major aberrantly expressed protein sets, in contrast, were downregulated by metformin in CML CD34 + cells. These data challenged the dependency of OXPHOS for CML CD34 + cell survival and underlined the novel mechanism of metformin. More importantly, it suggested a strong rationale for the use of tyrosine kinase inhibitors in combination with metformin in treating CML., (© 2022. The Author(s).)

The clinical therapeutic regimen for acute myeloid leukemia (AML) is not significantly different between adults and children, which is mostly based on IA (idarubicin and cytosine arabinoside) induction chemotherapy. With the rapid development of sequencing technique, people's understandings towards the molecular and biological abnormalities of AML are increasing, diverse AML gene mutation-based targeted drugs have been rapidly developed and applied. In this review, several commonly gene mutations in AML (such as FLT3, NPM1 and C/EBPA) was described, and the therapeutic effects and differences of targeted drugs that used in clinical treatment or had been reported (like tyrosine kinase inhibitor, IDH1 mutation inhibitor and epigenetic modification inhibitor) in child and adult AML patients were summrized.

A high salt intake exacerbates insulin resistance, evoking hypertension due to systemic perivascular inflammation, oxidative-nitrosative stress and endothelial dysfunction. Angiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blockers (ARBs) have been shown to abolish inflammation and redox stress but only partially restore endothelial function in mesenteric vessels. We investigated whether sympatho-adrenal overactivation evokes coronary vascular dysfunction when a high salt intake is combined with insulin resistance in male Goto-Kakizaki (GK) and Wistar rats treated with two different classes of β-blocker or vehicle, utilising synchrotron-based microangiography in vivo. Further, we examined if chronic carvedilol (CAR) treatment preserves nitric oxide (NO)-mediated coronary dilation more than metoprolol (MET). A high salt diet (6% NaCl w/w) exacerbated coronary microvessel endothelial dysfunction and NO-resistance in vehicle-treated GK rats while Wistar rats showed modest impairment. Microvascular dysfunction was associated with elevated expression of myocardial endothelin, inducible NO synthase (NOS) protein and 3-nitrotyrosine (3-NT). Both CAR and MET reduced basal coronary perfusion but restored microvessel endothelium-dependent and -independent dilation indicating a role for sympatho-adrenal overactivation in vehicle-treated rats. While MET treatment reduced myocardial nitrates, only MET treatment completely restored microvessel dilation to dobutamine (DOB) stimulation in the absence of NO and prostanoids (combined inhibition), indicating that MET restored the coronary flow reserve attributable to endothelium-derived hyperpolarisation (EDH). In conclusion, sympatho-adrenal overactivation caused by high salt intake and insulin resistance evoked coronary microvessel endothelial dysfunction and diminished NO sensitivity, which were restored by MET and CAR treatment in spite of ongoing inflammation and oxidative-nitrosative stress presumably caused by uninhibited renin-angiotensin-aldosterone system (RAAS) overactivation., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The treatment of pediatric heart failure is a long-standing unmet medical need. Angiotensin II supports mammalian perinatal circulation by activating cardiac L-type Ca 2+ channels through angiotensin type 1 receptor (AT 1 R) and β-arrestin. TRV027, a β-arrestin-biased AT 1 R agonist, that has been reported to be safe but not effective for adult patients with heart failure, activates the AT 1 R/β-arrestin pathway. We found that TRV027 evokes a long-acting positive inotropic effect specifically on immature cardiac myocytes through the AT 1 R/β-arrestin/L-type Ca 2+ 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., Competing Interests: This work was supported by Grants-in-Aid for Scientific Research to Dr. Kashihara (16K08546) and Dr. Yamada (18K06870), a Grant-in-Aid for Young Scientists to Dr. Kashihara (26860145) and Dr. Kawagishi (19K16488), and a Grant-in-Aid for Research Activity start-up to Dr. Kawagishi (18H06124), all from the Japan Society for the Promotion of Science; grants from the Novartis Foundation (Japan) to Dr. Kashihara and from the Shinshu Public Utility Foundation for Promotion of Medical Sciences to Dr. Kashihara and Dr. Yamada; and scholarship donations to Dr. Yamada from Astellas Pharma, Bayer Yakuhin, Bristol-Myers Squibb, Daiichi Sankyo, and Shionogi and Co. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2020 The Authors.)

Rationale: Diabetic heart disease (DHD) is a debilitating manifestation of type 2 diabetes mellitus. Exercise has been proposed as a potential therapy for DHD, although the effectiveness of exercise in preventing or reversing the progression of DHD remains controversial. Cardiac function is critically dependent on the preservation of coronary vascular function., Objective: We aimed to elucidate the effectiveness and mechanisms by which exercise facilitates coronary and cardiac-protection during the onset and progression of DHD., Methods and Results: Diabetic db/db and nondiabetic mice, with or without underlying cardiac dysfunction (16 and 8 weeks old, respectively) were subjected to either moderate-intensity exercise or high-intensity exercise for 8 weeks. Subsequently, synchrotron microangiography, immunohistochemistry, Western blot, and real-time polymerase chain reaction were used to assess time-dependent changes in cardiac and coronary structure and function associated with diabetes mellitus and exercise and determine whether these changes reflect the observed changes in cardiac-enriched and vascular-enriched microRNAs (miRNAs). We show that, if exercise is initiated from 8 weeks of age, both moderate-intensity exercise and high-intensity exercise prevented the onset of coronary and cardiac dysfunction, apoptosis, fibrosis, microvascular rarefaction, and disruption of miRNA signaling, as seen in the nonexercised diabetic mice. Conversely, the cardiovascular benefits of moderate-intensity exercise were absent if the exercise was initiated after the diabetic mice had already established cardiac dysfunction (ie, from 16 weeks of age). The experimental silencing or upregulation of miRNA-126 activity suggests the mechanism underpinning the cardiovascular benefits of exercise were mediated, at least in part, through tissue-specific miRNAs., Conclusions: Our findings provide the first experimental evidence for the critical importance of early exercise intervention in ameliorating the onset and progression of DHD. Our results also suggest that the beneficial effects of exercise are mediated through the normalization of cardiovascular-enriched miRNAs, which are dysregulated in DHD.

Serotonin (5-HT) accumulates in the heart during myocardial ischemia and induces deleterious effects on the cardiomyocytes through receptor-dependent and monoamine oxidase-dependent pathways. We aimed to clarify the involvement of extra-neuronal monoamine transporters in the clearance of 5-HT during ischemia and reperfusion in the heart. Using a microdialysis technique in the anesthetized Wistar rat heart, we monitored myocardial interstitial 5-HT and 5-hydroxyindole acetic acid (5-HIAA) concentration by means of electro-chemical detection coupled with high-performance liquid chromatography (HPLC-ECD). Effects of inhibitors of the plasma membrane monoamine transporter (PMAT) and the organic cation transporter 3 (OCT3) (decynium-22 and corticosterone) on the 5-HT and 5-HIAA concentrations during baseline, coronary occlusion, and reperfusion were investigated. Basal dialysate 5-HT concentration were increased by local administration of decynium-22, but not by corticosterone. Addition of fluoxetine, a serotonin transporter (SERT) inhibitor further increased the 5-HT concentration upon during administration of decynium-22. Decynium-22 elevated the background level of 5-HT during coronary occlusion and maintained 5-HT concentration at a high level during reperfusion. Production of 5-HIAA in the early reperfusion was significantly suppressed by decynium-22. These results indicate that PMAT and SERT independently regulate basal level of interstitial 5-HT, and PMAT plays a more important role in the clearance of 5-HT during reperfusion. These data suggest the involvement of PMAT in the monoamine oxidase-dependent deleterious pathway in the heart., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Ghrelin is a small peptide with important roles in the regulation of appetite, gut motility, glucose homeostasis as well as cardiovascular protection. This review highlights the role that acyl ghrelin plays in maintaining normal endothelial function by maintaining the balance of vasodilator-vasoconstrictor factors, inhibiting inflammatory cytokine production and immune cell recruitment to sites of vascular injury and by promoting angiogenesis., Competing Interests: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review., (© 2019 The authors.)

Adipose tissue is the main energy reserve of the body. When energy is required, adipocyte triglycerides stored in lipid droplets (LDs) are broken down by lipase, and free fatty acids are released to supply the physiological need. Intracellular LDs are active metabolic organelles in mammalian cells, particularly in adipocytes. The present study was aimed to investigate the morphological changes of LDs and the alternation of LD-associated perilipin family proteins during long-term lipolysis stimulated by forskolin. Primary differentiated adipocytes derived from epididymal fat pads of Sprague-Dawley (SD) rats were incubated in the presence or absence of 1 μmol/L forskolin for 24 h. Content of glycerol released to the culture medium was determined by a colorimetric assay and served as an index of lipolysis. Morphological changes of LDs were observed by Nile red staining. The mRNA level of perilipin family genes was detected by quantitative real-time PCR. The protein level and subcellular localization were examined by immunoblotting and immunofluorescence staining, respectively. The results showed that forskolin induced sustained lipolysis in differentiated adipocytes. The morphology of LDs changed in a time-dependent manner. Large clustered LDs became gradually smaller in size and eventually disappeared; in contrast, peripheral micro-LDs increased gradually in number until the cytoplasm was filled with numerous micro-LDs. The protein level of the perilipin family proteins showed obvious alternation. Mature adipocytes physiologically expressed a very low level of Plin2 protein, whereas in adipocytes stimulated with lipolytic forskolin, the protein and mRNA levels of Plin2 were significantly increased, and the increased Plin2 was specifically bound to the surface of LDs. During chronic stimulation of forskolin, the mRNA level of Plin3 was unchanged, but the mRNA levels of Plin1, Plin4 and Plin5 were significantly decreased. These results suggest that the morphology of LDs and perilipin family proteins on the surface of LDs are significantly altered during long-term lipolysis stimulated by forskolin, representing a dynamic process of the remodeling of LDs.

Background: The causal factors underpinning the onset and progression of diabetic heart disease (DHD) remain to be fully elucidated. Myocardial function is critically dependent on optimal coronary blood flow. Considering vascular disease occurs early in diabetes due to endothelial dysfunction, this study aimed to determine whether impaired coronary perfusion contributes to the origins of myocardial dysfunction in DHD, or whether coronary and cardiac dysfunction are independent pathologies associated with diabetes. Methods: Synchrotron radiation microangiography was used to image the coronary circulation of type-2 diabetic db/db and non-diabetic db/+ mice in vivo at 8, 16, and 24 weeks of age. We further assessed vascular function based on the vasodilatory responses to acetylcholine (ACh, 3 μg/kg/min), sodium nitroprusside (SNP, 5 μg/kg/min) and the Rho-kinase inhibitor, fasudil (20 mg/kg, i.v.). Cardiac function was assessed using echocardiography, and cardiac eNOS and ROCK expression were measured using immunohistochemistry. Results: Coronary and cardiac function were normal in 8-week-old diabetic mice. However, by 16 weeks of age, diabetic mice had advanced cardiac dysfunction. In comparison, normal coronary perfusion was preserved in diabetes until 24 weeks of age. Moreover, only the 24-week-old diabetic mice showed clear evidence of advanced coronary vascular dysfunction, based on (i) the absence of a vasodilatory response to ACh, and (ii) an exaggerated vasodilatory response to fasudil. Interestingly, fasudil also restored normal coronary perfusion in the 24-week-old diabetic heart by restoring blood flow to previously constricted vessels (diameter < 100 μm). Importantly, there was a ubiquitous decrease, and increase, in the cardiac expression of eNOS and ROCK, respectively. Conclusion: These results suggest that both cardiac and coronary dysfunction appear to have independent origins associated with diabetes and Rho-kinase pathway may be playing a role in the onset and progression of DHD.

Mutations in genes encoding components of the sarcomere cause cardiomyopathy, which is often associated with abnormal Ca 2+ sensitivity of muscle contraction. We have previously shown that a heart-specific myosin light chain phosphatase small subunit (hHS-M 21 ) increases the Ca 2+ sensitivity of muscle contraction. The aim of the present study was to investigate the function of hHS-M 21 in vivo and the causative role of abnormal Ca 2+ sensitivity in cardiomyopathy. We generated transgenic mice with cardiac-specific overexpression of hHS-M 21 . We confirmed that hHS-M 21 increased the Ca 2+ sensitivity of cardiac muscle contraction in vivo, which was not followed by an increased phosphorylation of myosin light chain 2 isoforms. hHS-M 21 transgenic mice developed severe systolic dysfunction with myocardial fibrosis and degeneration of cardiomyocytes in association with sinus bradycardia and atrioventricular conduction defect. The contractile dysfunction and cardiac fibrosis were improved by treatment with the Rho kinase inhibitor fasudil. Our findings suggested that the overexpression of hHS-M 21 results in cardiac dysfunction and conduction disturbance via non-myosin light chain 2 phosphorylation-dependent regulation. NEW & NOTEWORTHY The present study is the first to develop mice with transgenic overexpression of a heart-specific myosin light chain phosphatase small subunit (hHS-M 21 ) and to examine the effects of hHS-M 21 on cardiac function. Elevation of hHS-M 21 induced heart failure with myocardial fibrosis and degeneration of cardiomyocytes accompanied by supraventricular arrhythmias.

The aim of this study was to evaluate cardiac vagal nerve activity and identify the abnormality of cardiac vagal control in heart failure caused by dilated cardiomyopathy (DCM) using a knock-in mouse model with a ΔK210 mutation in the cardiac troponin T gene. The effects of electrical stimulation of the cervical vagal nerve at 5 and 10Hz (peripheral vagal control) and α 2 -adrennoceptor stimulation by intravenous medetomidine at 0.1mg/kg (central vagal control) were examined in wild-type (WT) mice and DCM mice. Microdialysis technique was applied to the left ventricular myocardium of anesthetized mice and myocardial interstitial acetylcholine (ACh) levels were measured by HPLC as an index of ACh release from cardiac vagal nerve endings. Electrical vagal nerve stimulation increased cardiac interval and myocardial interstitial ACh level in both WT and DCM mice, and these responses did not differ between WT and DCM mice. In contrast, intravenous medetomidine increased cardiac interval and myocardial interstitial ACh level in both WT and DCM mice, but the responses of cardiac interval and myocardial interstitial ACh level were significantly suppressed in DCM mice compared to WT mice. Medetomidine did not affect the myocardial interstitial ACh response induced by vagal nerve stimulation in WT mice. In this mouse model of DCM, peripheral vagal control including ACh release from vagal nerve endings and the postsynaptic response of pacemaker cells was preserved, but central vagal control through α 2 -adrenoceptors was impaired., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aim of this study was to examine the accumulation of serotonin (5-HT) and degradation of 5-HT taken up into cells in the ischemic region during myocardial ischemia-reperfusion. Using microdialysis technique in anesthetized rats, we monitored myocardial interstitial levels of 5-HT and its metabolite produced by monoamine oxidase (MAO), 5-hydroxyindole acetic acid (5-HIAA), during 30-min coronary occlusion followed by 45-min reperfusion, and investigated the effects of local administration of the MAO inhibitor pargyline and the 5-HT uptake inhibitor fluoxetine. In the vehicle group, the dialysate 5-HT concentration increased from 1.3 ± 0.2 nM at baseline to 29.6 ± 2.8 nM at 22.5-30 min of occlusion, but the dialysate 5-HIAA concentration did not change from baseline (9.9 ± 1.1 nM). Upon reperfusion, the dialysate 5-HT concentration increased further to a peak (34.2 ± 4.2 nM) at 0-7.5 min and then declined. The dialysate 5-HIAA concentration increased to 31.9 ± 5.2 nM at 7.5-15 min of reperfusion and maintained this high level until 45 min. Pargyline markedly suppressed the increase in dialysate 5-HIAA concentration after reperfusion and increased the averaged dialysate 5-HT concentration during the reperfusion period. Fluoxetine suppressed the increase in dialysate 5-HT concentration during occlusion but did not change dialysate 5-HT or 5-HIAA concentration after reperfusion. During ischemia, 5-HT secreted from ischemic tissues accumulates but 5-HT degradation by MAO is suppressed. After reperfusion, degradation of 5-HT taken up into cells is enhanced and contributes to the clearance of accumulated 5-HT. This degradation following cellular uptake is dependent on MAO activity but not the fluoxetine-sensitive uptake transporter., New & Noteworthy: By monitoring myocardial interstitial levels of 5-HT and its metabolite, 5-hydroxyindole acetic acid, we investigated 5-HT kinetics during myocardial ischemia-reperfusion. 5-HT accumulates but 5-HT degradation is suppressed during ischemia. After reperfusion, 5-HT degradation is enhanced and this degradation is dependent on monoamine oxidase activity but not fluoxetine-sensitive uptake transporter., (Copyright © 2017 the American Physiological Society.)

Glycogen synthase kinase-3β (GSK-3β) plays a central role in both cardiac physiology and pathology. Herein we want to clarify the role of GSK-3β in familial dilated cardiomyopathy. We generated a mouse model carrying a heterozygous knockout mutation of GSK-3β (GSK-3β(+/-) KO), together with a ΔK210 knockin mutation in cardiac troponin T (ΔK210 cTnT KI), which was proved to be one of the genetic causes of familial dilated cardiomyopathy (DCM). GSK-3β(+/-) KO prevented the slow and rapid deterioration in left ventricular systolic function accompanying heart failure (HF) in DCM mice with heterozygous and homozygous ΔK210 cTnT KI mutations, respectively. GSK-3β(+/-) KO also prevented cardiac enlargement, myocardial fibrosis, and cardiomyocyte apoptosis and markedly reduced the expression of cardiac β-myosin heavy chain isoform, indicative of HF, in DCM mice with homozygous ΔK210 cTnT KI mutation. GSK-3β(+/-) KO also extended the life span of these DCM mice. This study suggests that the inhibition of GSK-3β is cardioprotective in familial DCM associated with ΔK210 cTnT mutation., (Copyright © 2016 the American Physiological Society.)

To elucidate the involvement of monoamine oxidase (MAO) in hydroxyl radical production and cardiomyocyte injury during ischemia as well as after reperfusion, we applied microdialysis technique to the heart of anesthetized rats. Dialysate samples were collected during 30 min of induced ischemia followed by 60 min of reperfusion. We monitored dialysate 3,4-dihydrobenzoic acid (3,4-DHBA) concentration as an index of hydroxyl radical production using a trapping agent (4-hydroxybenzoic acid), and dialysate myoglobin concentration as an index of cardiomyocyte injury in the ischemic region. The effect of local administration of a MAO inhibitor, pargyline, was investigated. Dialysate 3,4-DHBA concentration increased from 1.9 ± 0.5 nM at baseline to 3.5 ± 0.7 nM at 20-30 min of occlusion. After reperfusion, dialysate 3,4-DHBA concentration further increased reaching a maximum (4.5 ± 0.3 nM) at 20-30 min after reperfusion, and stabilized thereafter. Pargyline suppressed the averaged increase in dialysate 3,4-DHBA concentration by ∼72% during occlusion and by ∼67% during reperfusion. Dialysate myoglobin concentration increased from 235 ± 60 ng/ml at baseline to 1309 ± 298 ng/ml at 20-30 min after occlusion. After reperfusion, dialysate myoglobin concentration further increased reaching a peak (5833 ± 1017 ng/ml) at 10-20 min after reperfusion, and then declined. Pargyline reduced the averaged dialysate myoglobin concentration by ∼56% during occlusion and by ∼41% during reperfusion. MAO plays a significant role in hydroxyl radical production and cardiomyocyte injury during ischemia as well as after reperfusion.

Background: Impaired actin-myosin cross-bridge (CB) dynamics correlate with impaired left ventricular (LV) function in early diabetic cardiomyopathy (DCM). Elevated expression and activity of Rho kinase (ROCK) contributes to the development of DCM. ROCK targets several sarcomeric proteins including myosin light chain 2, myosin binding protein-C (MyBP-C), troponin I (TnI) and troponin T that all have important roles in regulating CB dynamics and contractility of the myocardium. Our aim was to examine if chronic ROCK inhibition prevents impaired CB dynamics and LV dysfunction in a rat model of early diabetes, and whether these changes are associated with changes in myofilament phosphorylation state., Methods: Seven days post-diabetes induction (65 mg/kg ip, streptozotocin), diabetic rats received the ROCK inhibitor, fasudil (10 mg/kg/day ip) or vehicle for 14 days. Rats underwent cardiac catheterization to assess LV function simultaneous with X-ray diffraction using synchrotron radiation to assess in situ CB dynamics., Results: Compared to controls, diabetic rats developed mild systolic and diastolic dysfunction, which was attenuated by fasudil. End-diastolic and systolic myosin proximity to actin filaments were significantly reduced in diabetic rats (P < 0.05). In all rats there was an inverse correlation between ROCK1 expression and the extension of myosin CB in diastole, with the lowest ROCK expression in control and fasudil-treated diabetic rats. In diabetic and fasudil-treated diabetic rats changes in relative phosphorylation of TnI and MyBP-C were not significant from controls., Conclusions: Our results demonstrate a clear role for ROCK in the development of LV dysfunction and impaired CB dynamics in early DCM.

Aims: The aim of this study was to elucidate myocardial interstitial serotonin (5-HT) kinetics in the heart, including 5-HT reuptake and enzymatic degradation to 5-hydroxyindole acetic acid (5-HIAA) via monoamine oxidase (MAO)., Main Methods: Using microdialysis technique in anesthetized rats, we simultaneously monitored myocardial interstitial levels of 5-HT and its major metabolite, 5-HIAA, in the left ventricle and examined the effects of local administration of a MAO inhibitor, pargyline, or a 5-HT uptake inhibitor, fluoxetine., Key Findings: Pargyline increased dialysate 5-HT concentration from 1.8±0.3 at baseline to 3.9±0.5nM but decreased dialysate 5-HIAA concentration from 20.7±1.0 at baseline to 15.8±1.4nM at 60-80min of administration. Fluoxetine increased dialysate 5-HT concentration from 1.9±0.4 at baseline to 6.5±0.9nM at 60-80min of administration, but did not change dialysate 5-HIAA concentration. Local administration of ADP (100mM) increased dialysate 5-HT and 5-HIAA concentrations. Pargyline did not affect ADP-induced increase in dialysate 5-HT concentration but suppressed ADP-induced increase in dialysate 5-HIAA concentration during 60min of ADP administration. Fluoxetine increased dialysate 5-HT concentration at 40-60min of ADP administration, but did not affect ADP-induced increase in dialysate 5-HIAA concentration., Significance: Simultaneous monitoring of myocardial interstitial 5-HT and 5-HIAA levels provides valuable information on 5-HT kinetics including reuptake and enzymatic degradation by MAO, which play a role in the regulation of myocardial interstitial 5-HT levels at baseline and when 5-HT levels are elevated., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Although ghrelin has been demonstrated to improve cardiac function in heart failure, its therapeutic efficacy on the life expectancy remains unknown. We aim to examine whether ghrelin can improve the life survival in heart failure using a mouse model of inherited dilated cardiomyopathy (DCM) caused by a deletion mutation ΔK210 in cardiac troponin T (cTnT). From 30 days of age, ghrelin (150 μg/kg) was administered subcutaneously to DCM mice once daily, control mice received saline only. The survival rates were compared between the two groups for 30 days. After 30-day treatment, functional and morphological measurements were conducted. Ghrelin-treated DCM mice had significantly prolonged life spans compared with saline-treated control DCM mice. Echocardiography showed that ghrelin reduced left ventricular (LV) end-diastolic dimensions and increased LV ejection fraction. Moreover, histoanatomical data revealed that ghrelin decreased the heart-to-body weight ratio, prevented cardiac remodeling and fibrosis, and markedly decreased the expression of brain natriuretic peptide. Telemetry recording and heart rate variability analysis showed that ghrelin suppressed the excessive cardiac sympathetic nerve activity (CSNA) and recovered the cardiac parasympathetic nerve activity. These results suggest that ghrelin has therapeutic benefits for survival as well as for the cardiac function and remodeling in heart failure probably through suppression of CSNA and recovery of cardiac parasympathetic nerve activity.

Aims: We have previously demonstrated that propyl gallate has a Ca(2+) sensitizing effect on the force generation in membrane-permeabilized (skinned) cardiac muscle fibers. However, in vivo beneficial effects of propyl gallate as a novel Ca(2+) sensitizer remain uncertain. In the present study, we aim to explore in vivo effects of propyl gallate., Main Methods: We compared effects of propyl gallate on ex vivo intact cardiac muscle fibers and in vivo hearts in healthy mice with those of pimobendan, a clinically used Ca(2+) sensitizer. The therapeutic effect of propyl gallate was investigated using a mouse model of dilated cardiomyopathy (DCM) with reduced myofilament Ca(2+) sensitivity due to a deletion mutation ΔK210 in cardiac troponin T., Key Findings: Propyl gallate, as well as pimobendan, showed a positive inotropic effect. Propyl gallate slightly increased the blood pressure without changing the heart rate in healthy mice, whereas pimobendan decreased the blood pressure probably through vasodilation via inhibition of phosphodiesterase and increased the heart rate. Propyl gallate prevented cardiac remodeling and systolic dysfunction and significantly improved the life-expectancy of knock-in mouse model of DCM with reduced myofilament Ca(2+) sensitivity due to a mutation in cardiac troponin T. On the other hand, gallate, a similarly strong antioxidant polyphenol lacking Ca(2+) sensitizing action, had no beneficial effects on the DCM mice., Significance: These results suggest that propyl gallate might be useful for the treatment of inherited DCM caused by a reduction in the myofilament Ca(2+) sensitivity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aim: Calpain activation has a putative role in ischaemia-reperfusion injury of cardiomyocytes. This study clarified the in vivo contribution of calpain to disruption of cardiomyocyte sarcolemma during ischaemia and after reperfusion in anaesthetized rats., Methods: Using a microdialysis technique in the hearts of anaesthetized rats, we investigated the effects of the calpain inhibitors on myocardial interstitial myoglobin level in the ischaemic region during coronary occlusion and after reperfusion. The calpain inhibitors were administered locally via a dialysis probe. Two durations of coronary occlusion were tested., Results: Thirty-minute coronary occlusion: dialysate myoglobin concentration increased markedly from 385 ± 46 ng mL(-1) at baseline to 3701 ± 527 ng mL(-1) at 20-30 min of occlusion. After reperfusion, dialysate myoglobin concentration further increased, reaching a peak (12 296 ± 1564 ng mL(-1) ) at 10-20 min post-reperfusion and then declined gradually. The calpain inhibitors, MDL-28170 and SNJ-1945 did not change dialysate myoglobin concentration during occlusion but attenuated the increase after reperfusion to 6826 ± 1227 and 8130 ± 938 ng mL(-1) at 10-20 min post-reperfusion (P < 0.05), respectively. Ninety-minute coronary occlusion: dialysate myoglobin concentration increased from 516 ± 33 ng mL(-1) at baseline to 5463 ± 387 ng mL(-1) at 80-90 min after occlusion. After reperfusion, there was no significant increase in dialysate myoglobin concentration. MDL-28170 did not affect dialysate myoglobin concentration during occlusion or after reperfusion., Conclusion: Calpain contributes to sarcolemmal disruption immediately after reperfusion following 30-min coronary occlusion, but has little effects during ischaemia and after reperfusion in 90-min coronary occlusion., (© 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

We applied a microdialysis technique to the left ventricular myocardium of anesthetized mice and tried to monitor acetylcholine (ACh) release from cardiac vagal nerves. Transection of bilateral cervical vagal nerves decreased dialysate ACh concentration. Electrical stimulation of the left cervical vagal nerve increased dialysate ACh concentration in proportion to the frequency of stimulation. Intravenous administration of hexamethonium, prevented the increase in dialysate ACh concentration during vagal nerve stimulation, indicating that ACh in the dialysate primarily reflects ACh released from post-ganglionic cardiac vagal nerves. Microdialysis permits monitoring of ACh release from post-ganglionic cardiac vagal nerves that are most likely to be innervating the left ventricle in mice., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Aim: Although deleterious effects of serotonin (5-HT) have been demonstrated during myocardial ischaemia-reperfusion, little information is available on myocardial interstitial 5-HT kinetics. This study evaluated the contribution of 5-HT reuptake and degradation to myocardial interstitial 5-HT levels during ischaemia-reperfusion., Methods: Using microdialysis technique in anaesthetized rabbits, we monitored myocardial interstitial 5-HT levels in the ischaemic region during ischaemia (30 min) followed by reperfusion (60 min) and investigated the effects of local infusion of fluoxetine, a 5-HT uptake inhibitor, and/or pargyline, a monoamine oxidase inhibitor., Results: In vehicle control, dialysate 5-HT concentration increased gradually from 16 ± 3 at baseline to 85 ± 18 nM during 20-30 min of ischaemia. Dialysate 5-HT concentration further increased to 236 ± 47 nM at 0-10 min of reperfusion and then began to decline. Averaged 5-HT concentration was 61 ± 11 during ischaemia and 113 ± 13 nM during reperfusion. Fluoxetine elevated dialysate 5-HT level at baseline and at 10-30 min of reperfusion; it increased averaged dialysate 5-HT concentration by approx. 304% during reperfusion compared to control. Pargyline elevated averaged dialysate 5-HT concentration during ischaemia by approx. 243% and that during reperfusion by approx. 250% compared to control. The changes in dialysate 5-HT concentration by fluoxetine + pargyline were similar to those of fluoxetine alone., Conclusion: The 5-HT reuptake function plays an important role in the clearance of myocardial interstitial 5-HT during reperfusion. When 5-HT reuptake function is intact, degradation of 5-HT by monoamine oxidase contributes to reduce myocardial interstitial 5-HT level throughout ischaemia-reperfusion., (© 2012 The Authors Acta Physiologica © 2012 Scandinavian Physiological Society.)

Inherited or non-inherited dilated cardiomyopathy (DCM) patients develop varied disease phenotypes leading to death after developing congestive heart failure (HF) or sudden death with mild or no overt HF symptoms, suggesting that environmental and/or genetic factors may modify the disease phenotype of DCM. In this study, we sought to explore unknown genetic factors affecting the disease phenotype of monogenic inherited human DCM. Knock-in mice bearing a sarcomeric protein mutation that causes DCM were created on different genetic backgrounds; BALB/c and C57Bl/6. DCM mice on the BALB/c background showed cardiac enlargement and systolic dysfunction and developed congestive HF before died. In contrast, DCM mice on the C57Bl/6 background developed no overt HF symptoms and died suddenly, although they showed considerable cardiac enlargement and systolic dysfunction. BALB/c mice have brain serotonin dysfunction due to a single nucleotide polymorphism (SNP) in tryptophan hydroxylase 2 (TPH2). Brain serotonin dysfunction plays a critical role in depression and anxiety and BALB/c mice exhibit depression- and anxiety-related behaviors. Since depression is common and associated with poor prognosis in HF patients, we examined therapeutic effects of anti-depression drug paroxetine and anti-anxiety drug buspirone that could improve the brain serotonin function in mice. Both drugs reduced cardiac enlargement and improved systolic dysfunction and symptoms of severe congestive HF in DCM mice on the BALB/c background. These results strongly suggest that genetic backgrounds involving brain serotonin dysfunction, such as TPH2 gene SNP, may play an important role in the development of congestive HF in DCM., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background and Purpose: Catechins, biologically active polyphenols in green tea, are known to have a protective effect against cardiovascular diseases. In this study, we investigated direct actions of green tea catechins on cardiac muscle function to explore their uses as potential drugs for cardiac muscle disease., Experimental Approach: The effects of catechins were systematically investigated on the force-pCa relationship in skinned cardiac muscle fibres to determine their direct effects on cardiac myofilament contractility. The mechanisms of action of effective catechins were investigated using troponin exchange techniques, quartz crystal microbalance, nuclear magnetic resonance and a transgenic mouse model., Key Results: (-)-Epicatechin-3-gallate (ECg) and (-)-epigallocatechin-3-gallate (EGCg), but not their stereoismers (-)-catechin-3-gallate and (-)-gallocatechin-3-gallate, decreased cardiac myofilament Ca(2+) sensitivity probably through its interaction with cardiac troponin C. EGCg restored cardiac output in isolated working hearts by improving diastolic dysfunction caused by increased myofilament Ca(2+) sensitivity in a mouse model of hypertrophic cardiomyopathy., Conclusions and Implications: The green tea catechins, ECg and EGCg, are Ca(2+) desensitizers acting through binding to cardiac troponin C. These compounds might be useful compounds for the development of therapeutic agents to treat the hypertrophic cardiomyopathy caused by increased Ca(2+) sensitivity of cardiac myofilaments., (© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.)

Aims: Thyroid hormone (TH) has prominent effects on the heart, and hyperthyroidism is occasionally found to be a cause of dilated cardiomyopathy (DCM). We aim to explore the potential role of TH in the pathogenesis of DCM., Methods and Results: The pathophysiological role of TH in the heart was investigated using a knock-in mouse model of inherited DCM with a deletion mutation DeltaK210 in the cardiac troponin T gene. Serum tri-iodothyronine (T(3)) levels showed no significant difference between wild-type (WT) and DCM mice, whereas cardiac T(3) levels in DCM mice were significantly higher than those in WT mice. Type 2 iodothyronine deiodinase (Dio2), which produces T(3) from thyroxin, was up-regulated in the DCM mice hearts. The cAMP levels were increased in DCM mice hearts, suggesting that transcriptional up-regulation of Dio2 gene is mediated through the evolutionarily conserved cAMP-response element site in its promoter. Propylthiouracil (PTU), an anti-thyroid drug, prevented the hypertrophic remodelling of the heart in DCM mice and improved their cardiac function and life expectancy. Akt and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation increased in the DCM mice hearts and PTU treatment significantly reduced the phosphorylation levels, strongly suggesting that Dio2 up-regulation is involved in cardiac remodelling in DCM through activating the TH-signalling pathways involving Akt and p38 MAPK. Dio2 gene expression was also markedly up-regulated in the mice hearts developing similar eccentric hypertrophy after myocardial infarction., Conclusion: Local hyperthyroidism via transcriptional up-regulation of the Dio2 gene may be an important underlying mechanism for the hypertrophic cardiac remodelling in DCM.

Aims: Extensive clinical studies have demonstrated that beta-adrenoceptor blocking agents (beta-blockers) are beneficial in the treatment of chronic heart failure, which is due to various aetiologies, including idiopathic dilated cardiomyopathy (DCM) and ischaemic heart disease. However, little is known about the therapeutic efficacy of beta-blockers in the treatment of the inherited form of DCM, of which causative mutations have recently been identified in various genes, including those encoding cardiac sarcomeric proteins. Using a mouse model of inherited DCM with a troponin mutation, we aim to study the treatment benefits of beta-blockers., Methods and Results: Three different types of beta-blockers, carvedilol, metoprolol, and atenolol, were orally administered to a knock-in mouse model of inherited DCM with a deletion mutation DeltaK210 in the cardiac troponin T gene (TNNT2). Therapeutic effects were examined on the basis of survival and myocardial remodelling. The lipophilic beta(1)-selective beta-blocker metoprolol was found to prevent cardiac dysfunction and remodelling and extend the survival of knock-in mice. Conversely, both the non-selective beta-blocker carvedilol and the hydrophilic beta(1)-selective beta-blocker atenolol had no beneficial effects on survival and myocardial remodelling in this mouse model of inherited DCM., Conclusion: The highly lipophilic beta(1)-selective beta-blocker metoprolol, known to prevent ventricular fibrillation via central nervous system-mediated vagal activation, may be especially beneficial to DCM patients showing a family history of frequent sudden cardiac death, such as those with a deletion mutation DeltaK210 in the TNNT2 gene.

Aims: We carried out a screening of survival factors released from cells exposed to simulated ischaemia and reperfusion (sI/R) using the embryonic rat heart-derived cell line, H9c2 cells, and examined the physiological role of the identified factor., Method and Results: The culture medium supernatant of H9c2 cells exposed to sI/R was separated by column chromatography and the fractions examined for survival activity. The protein with survival activity was identified by mass spectrometry, and its physiological role was examined in the models of ischaemia. Cell survival activity was detected in at least three fractions of the cell supernatant collected during sI/R and subjected to a series of column chromatographic steps. Among the proteins measured by mass spectrometry and western blotting, a p36 protein identified as a glycolytic enzyme, lactate dehydrogenase muscle subunit (M-LDH), showed strong survival activity. H(2)O(2)-induced intracellular calcium overload in H9c2 cells and irregular Ca(2+) transients in adult rat cardiomyocytes were both found to be inhibited by pretreatment with M-LDH. M-LDH also lowered the frequency and amplitude of early afterdepolarizations induced by H(2)O(2) in adult rat cardiomyocytes and suppressed the ischaemia-reperfusion-induced reduction of cardiac output from mouse working heart preparations. M-LDH was found to increase the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), which plays a role in H9c2 cell survival., Conclusion: M-LDH released from cardiomyocytes after hypoxia and reoxygenation has a role in protecting the heart from oxidative stress-induced injury through an intracellular signal transduction pathway involving ERK1/2.

We created knock-in mice in which a deletion of 3 base pairs coding for K210 in cardiac troponin (cTn)T found in familial dilated cardiomyopathy patients was introduced into endogenous genes. Membrane-permeabilized cardiac muscle fibers from mutant mice showed significantly lower Ca(2+) sensitivity in force generation than those from wild-type mice. Peak amplitude of Ca(2+) transient in cardiomyocytes was increased in mutant mice, and maximum isometric force produced by intact cardiac muscle fibers of mutant mice was not significantly different from that of wild-type mice, suggesting that Ca(2+) transient was augmented to compensate for decreased myofilament Ca(2+) sensitivity. Nevertheless, mutant mice developed marked cardiac enlargement, heart failure, and frequent sudden death recapitulating the phenotypes of dilated cardiomyopathy patients, indicating that global functional defect of the heart attributable to decreased myofilament Ca(2+) sensitivity could not be fully compensated by only increasing the intracellular Ca(2+) transient. We found that a positive inotropic agent, pimobendan, which directly increases myofilament Ca(2+) sensitivity, had profound effects of preventing cardiac enlargement, heart failure, and sudden death. These results verify the hypothesis that Ca(2+) desensitization of cardiac myofilament is the absolute cause of the pathogenesis of dilated cardiomyopathy associated with this mutation and strongly suggest that Ca(2+) sensitizers are beneficial for the treatment of dilated cardiomyopathy patients affected by sarcomeric regulatory protein mutations.

Exogenous H(2)O(2) pretreatment significantly increased the endogenous H(2)O(2) content and calmodulin activity in maize seedlings, alleviated rapid loss of calmodulin activity, and raised the survival rate of maize seedlings under heat stress (Figs. 1, 4). The formation of H(2)O(2)-induced heat tolerance in maize seedlings was strengthened by exogenous Ca(2+) treatment, and weakened by Ca(2+) chelator EGTA, plasma-membrame-channel blocker La(3+), intracellular channel blocker ruthenium red, calmodulin antagonists chlorpromazine and trifluoperazine treatment (Figs. 1, 2). These results confirmed that Ca(2+) and calmodulin are involved in the regulation of H(2)O(2)-induced heat tolerance in maize seedlings.

A missense mutation R141W in the strong tropomyosin-binding region of cardiac troponin T (cTnT) has recently been reported to cause dilated cardiomyopathy (DCM), following the first report of a DCM-causing deletion mutation DeltaK210. To clarify the molecular mechanism for the pathogenesis of DCM caused by this novel mutation in cTnT gene, functional analyses were made on the recombinant human cTnT mutant proteins. Exchanging human wild-type and mutant cTnTs into rabbit skinned cardiac muscle fibers revealed that R141W mutation resulted in a decrease in the Ca(2+) sensitivity of force generation, as in the case of DeltaK210 mutation lying outside the strong tropomyosin-binding region. In contrast, a missense mutation R94L in the vicinity of the strong tropomyosin-binding region associated with hypertrophic cardiomyopathy (HCM) resulted in an increase in the Ca(2+) sensitivity of force generation, as in the case of the other HCM-causing mutations in cTnT reported previously. An assay using a quartz-crystal microbalance (a very sensitive mass-measuring device) revealed that R141W mutation increased the affinity of cTnT for alpha-tropomyosin by approximately three times, whereas an HCM-causing mutation DeltaE160 in the strong tropomyosin-binding region, as well as DeltaK210 and R94L mutations, had no effects on the interaction between cTnT and alpha-tropomyosin. Since cTnT has an important role in structurally integrating cardiac troponin I (cTnI) into the thin filaments via its two-way interactions with cTnI and tropomyosin, the present results suggest that R141W mutation in the strong tropomyosin-binding region in cTnT strengthens the integrity of cTnI in the thin filament by stabilizing the interaction between cTnT and tropomyosin, which might allow cTnI to inhibit the thin filament more effectively, leading to a Ca(2+) desensitization.