Heat-hypersensitive mutants of ryanodine receptor type 1 revealed by microscopic heating.

Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca2+ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca2+ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca2+ imaging using the endoplasmic reticulum-targeted fluorescent probes demonstrates that the Ca2+ burst originates from heat-induced Ca2+ release (HICR) through RyR1-mutant channels because of the channels' heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.

Significance of End-Diastolic Forward Flow in Patients With Repaired Tetralogy of Fallot　- Its Interaction With the Left Ventricular Property and End Organ Damage.

BACKGROUND: Although right ventricular (RV) enlargement may affect RV diastolic dysfunction assessed by end-diastolic forward flow (EDFF) in patients with repaired tetralogy of Fallot (TOF), EDFF may also be modified by left ventricular (LV) hemodynamics. We hypothesized that EDFF is affected by LV hemodynamics, not limited to RV diastolic stiffening.Methods and Results: Among 145 consecutive patients with repaired TOF who underwent catheterization, hemodynamic properties in 47 with consistent EDFF and 75 without EDFF were analyzed. Compared with patients without EDFF, those with EDFF had a large RV volume with a high regurgitant fraction. Although cardiac index and central venous pressure (CVP) were similar, contrast injection augmented CVP and LV end-diastolic pressure (EDP) in patients with vs. those without EDFF, suggesting compromised diastolic reserve. In patients with EDFF, the velocity-time integral (VTI) of EDFF was positively correlated with LVEDP and systemic vascular resistance, in addition to RV EDP. EDFF-VTI was correlated with hepatic venous wedge pressure and markers of hepatic dysfunction. Subanalysis of the older (≥6 years) half of the study cohort revealed that EDFF was associated with bi-atrial enlargement independent of RV volume, highlighting the pronounced role of EDFF on the diastolic property in the aged cohort. CONCLUSIONS: EDFF-VTI in patients with repaired TOF reflects RV diastolic dysfunction, affected by the left heart system. EDFF-VTI indicates blood stagnation, which may be attributed to end-organ damage.

Clinical and Sociodemographic Factors Associated With Health-Related Quality of Life in Patients With Adult Congenital Heart Disease　- A Nationwide Cross-Sectional Multicenter Study.

BACKGROUND: Little is known about clinical or sociodemographic factors that influence health-related quality of life (HRQoL) in patients with adult congenital heart disease (ACHD).Methods and Results: We conducted a nationwide prospective cross-sectional multicenter study at 4 large ACHD centers in Japan. From November 2016 to June 2018, we enrolled 1,223 ACHD patients; 1,025 patients had an HRQoL score. Patients completed a questionnaire survey, including sociodemographic characteristics, and the 36-Item Short-Form Health Survey (SF-36). To determine factors associated with HRQoL, correlations between 2 SF-36 summary scores (i.e., physical component score [PCS] and mental component score [MCS]) and other clinical or sociodemographic variables were examined using linear regression analysis. In multivariable analysis, poorer PCS was significantly associated with 11 variables, including older age, higher New York Heart Association class, previous cerebral infarction, being unemployed, and limited participation in physical education classes and sports clubs. Poorer MCS was associated with congenital heart disease of great complexity, being part of a non-sports club, current smoking, and social drinking. Student status and a higher number of family members were positively correlated with MCS. CONCLUSIONS: This study demonstrates that HRQoL in ACHD patients is associated with various clinical and sociodemographic factors. Further studies are needed to clarify whether some of these factors could be targets for future intervention programs to improve HRQoL outcomes.

Chronological T-wave alternation before and after the onset of arrhythmogenic right ventricular cardiomyopathy.

Identification of arrhythmogenic right ventricular cardiomyopathy (ARVC) during childhood is challenging due to the lack of specific ECG manifestation. We report chronological ECG alteration before several years of the ARVC onset in two affected children. Their ECG at the age of 6 years was almost normal for their age, and their chronological ECGs exhibited inversion of T wave in inferior leads, which are typical for ARVC, developed at younger age than that in precordial leads. In addition, the leftmost T-wave inversion in the precordial lead shifted toward the left in our patients, which is a sharp contrast to its physiological transition.

Microscopic Temperature Control Reveals Cooperative Regulation of Actin-Myosin Interaction by Drebrin E.

Drebrin E is a regulatory protein of intracellular force produced by actomyosin complexes, that is, myosin molecular motors interacting with actin filaments. The expression level of drebrin E in nerve cells decreases as the animal grows, suggesting its pivotal but unclarified role in neuronal development. Here, by applying the microscopic heat pulse method to actomyosin motility assay, the regulatory mechanism is examined from the room temperature up to 37 °C without a thermal denaturing of proteins. We show that the inhibition of actomyosin motility by drebrin E is eliminated immediately and reversibly during heating and depends on drebrin E concentration. The direct observation of quantum dot-labeled drebrin E implies its stable binding to actin filaments during the heat-induced sliding. Our results suggest that drebrin E allosterically modifies the actin filament structure to regulate cooperatively the actomyosin activity at the maintained in vivo body temperature.

Hydrostatic Pressure-Regulated Cellular Calcium Responses.

Hydrostatic pressure control has attracted much attention and presents a still challenging objective from mechanobiological viewpoints. Herein, we reveal the calcium entry processes in HeLa cells by means of hydrostatic pressure spectroscopy. The steady-state fluorescence spectral data comprehensively elucidated the factors controlling the outcomes of the hydrostatic pressure-stimulated calcium entry behavior. The present work leads to a new perspective on ion regulations in living cells and an attractive alternative to conventional mechanostimuli.

Comparing risk factors associated with the late detection of critical congenital heart disease at different facility levels.

AIMS: Critical congenital heart disease (CCHD) requires surgery or catheter intervention within the first year of life; delayed diagnoses result in worsened outcomes. In Japan, there are few reports of delayed CCHD diagnosis. We investigated the diagnoses and factors associated with the late detection of CCHD. METHODS: This retrospective cohort study evaluated 88 CCHD infants admitted to a level IV facility. We compared the late detection rates across facility levels of neonatal care and CCHD characteristics. RESULTS: Critical congenital heart disease was identified prenatally in 46 (52%) infants: early (≤3 days postnatally) in 29 (33%) and late (≥4 days postnatally) in 13 (15%). The oxygen saturation of 27 of 29 infants with early detection and 7 of 13 infants with late detection was measured using pulse oximetry within 3 days postnatally. Factors associated with the late detection of CCHD were lack of clinical recognition of symptoms in five infants, referral to higher-level facilities after discharge in four infants, and definitive diagnosis not confirmed using echocardiography in four infants. The most common factors associated with late detection were referral to higher-level facilities after discharge in level I facilities and definitive diagnosis not confirmed using echocardiography in level II facilities. CONCLUSION: Critical congenital heart disease may require advanced knowledge and echocardiographic techniques for diagnosis. Pulse oximetry and telemedicine should also be incorporated in the diagnostic algorithm. Improvement in these factors might contribute to reducing the late detection of CCHD.

Early Ventricular Septal Defect Closure Prevents the Progression of Aortic Regurgitation: A Long-Term Follow-Up Study.

According to current short-term evidence, ventricular septal defect (VSD) closure should be performed as early as possible after aortic regurgitation (AR) diagnosis in pediatric patients to prevent AR progression. However, long-term follow-up data are lacking. Therefore, our aim was to evaluate the long-term follow-up (≥ 10 years) of patients who underwent VSD closure as early as possible after AR diagnosis and to evaluate whether early VSD closure prevents progression of AR. This was a retrospective cohort study of 42 patients with VSD and AR who had undergone VSD closure at a median age of 2.9 years, with a median waiting period from AR diagnosis to VSD closure of 3.4 months and follow-up of a median 13.1 years (interquartile range 10.0-15.8 years). The preoperative degree of AR was trivial in 25 patients, mild in 15, and moderate in 2. Of the 33 patients followed up for ≥ 10 years, none required aortic valve replacement and there was no incidence of mortality. The degree of AR improved or did not change, except in 1 patient. The size of the VSD (odds ratio [OR] 0.85; 95% confidence interval [CI] 0.62-1.18; p = 0.33), time from diagnosis to surgery (OR 1.00; 95% CI 1.000-1.001; p = 0.657), and age at the time of surgery (OR 1.00; 95% CI 0.998-1.004; p = 0.452) were not predictive of persistent postoperative AR. Therefore, VSD closure performed as early as possible after AR diagnosis could successfully prevent AR progression in patients with less than moderate preoperative AR, eliminating the need for aortic valve replacement and valvuloplasty.

Complications During Continuous Renal Replacement Therapy in Critically Ill Neonates.

Background/ Aims: Owing to practical and technical developments, continuous renal replacement therapy (CRRT) has been administered even in critically ill neonates. In this study, the complications in CRRT for neonates were examined to establish a safe CRRT. METHODS: This retrospective study reviewed the clinical records of neonates who underwent CRRT at our neonatal intensive care unit between 2009 and 2017. RESULTS: Eight neonates with a body weight of 1,462-3,288 g were treated by 70 CRRT sessions with blood priming. Intradialytic hypotension (IDH) was observed in 39 sessions (55.7%), most of which occurred soon after the start of the CRRT. Body temperature decreased in 48 sessions (70.5%), and thrombocytopenia during CRRT occurred 30 times (42.9%). CONCLUSION: Complications during CRRT in neonates comprised IDH at the start of the CRRT, body temperature decline, and thrombocytopenia. These complications need to be analyzed for a safe neonatal CRRT.

Validity of a disposable catheter to drain urine overnight in neurogenic bladder.

BACKGROUND: Overnight catheter drainage (OCD) is introduced to avoid overdistention of the bladder at night-time when clean intermittent catheterization proves ineffective for daytime management of neurogenic bladder. We adopted OCD using disposable silicone no-balloon (DSnB) catheters, with the distal end outside the body opening into diapers. OCD using DSnB catheter, however, had risks of retrograde bacterial contamination. Therefore, in this study, the validity of equipping DSnB catheters with check valves to prevent retrograde bacterial contamination was examined. METHODS: For the in vitro study, excised saline-filled swine bladders were drained using DSnB catheters with or without check valves, and the time required for intravesical pressure to reach 5 cmH2 O was measured. For the in vivo study, in cross-over experiments comparing DSnB catheters with and without check valves, OCD using DSnB catheters for 10 h was performed in rabbits under analgesia. Bacterial growth from urine samples before and after OCD and residual urine volume were examined. RESULTS: For the in vitro experiment, the median drainage time was 368.2 s (range, 88-1,085 s) and 344.7 s (range, 28-840 s) with and without check valves, respectively (n = 6), which was not significantly different. For the in vivo experiment, in cross-over experiments (n = 8) new bacterial growth rates after OCD did not differ, and median residual urine volume was 17.1 mL (range, 0-75 mL) and 1.2 mL (range, 0-5 mL) with and without check valves, respectively (P = 0.055). CONCLUSIONS: Installing a check valve in the DSnB catheter did not decrease new bacterial growth, while tending to increase residual urine volume. DSnB catheters without check valves appear to be better for continuous drainage of urine from bladder.

Fetal intracranial hemorrhage due to maternal subclinical vitamin K deficiency associated with long-term eating disorder.

Vitamin K deficiency in pregnant women causes intracranial hemorrhage (ICH) in fetuses. Fetal ICH frequently causes life-threatening and persistent neurological damage. However, indicators for preventing fetal ICH are not established. Two pregnant women developed long-term eating disorders caused by psychosis. They were administered intravenous fluid and vitamin supplementation, excluding vitamin K. The intracranial low-hypoechoic area on fetal ultrasound was suggestive of fetal ICH due to vitamin K deficiency. Their neonates showed severe developmental delay. Laboratory analysis revealed a normal prothrombin time, but elevated protein induced by vitamin K absence II. Pregnant women who have eating disorders more than 3 weeks could develop fetal ICH due to maternal subclinical vitamin K deficiency. Illness duration and protein induced by vitamin K absence II of pregnant woman may be indicators for vitamin K administration to prevent fetal intracranial hemorrhage.

Successful Management of Fulminant Guillain-Barré Syndrome and Its Complications.

A 3-year-old girl presented with muscle weakness of her limbs and trunk 6 days after developing symptoms of common cold. Two days later, she experienced respiratory arrest with a Glasgow Coma Scale score of 3, necessitating endotracheal intubation. Therefore, she was transferred to our hospital with suspected acute encephalopathy. Although no abnormalities were observed on brain and spinal magnetic resonance imaging and electroencephalography, peripheral nerve conduction velocity tests failed to evoke motor and sensory nerve action potentials. Thus, we gave a diagnosis of fulminant Guillain-Barré syndrome and initiated immunoglobulin therapy. On day 3 of admission, she developed sinus tachycardia that induced circulatory failure and oliguria, which was successfully treated with landiolol. Subsequently, we performed plasmapheresis followed by immunoglobulin and steroid pulse therapies. She was weaned off the mechanical ventilator by day 20 of admission, was ambulatory by day 44, and had completely recovered without any adverse sequelae by day 55. In conclusion, landiolol was effective for treating acute sinus tachycardia-induced circulatory failure and played a key role in saving the life of this patient.

Successful cord blood transplantation in an adult acute lymphoblastic leukemia patient with congenital heart disease.

Recent advances in surgical corrections and supportive care for congenital heart disease have resulted in increasing numbers of adult survivors who may develop hematological malignancies. Treatments including chemotherapy for such patients may cause serious hemodynamic or cardiac complications, especially in those receiving stem cell transplantation. We present a 29-year-old woman with acute lymphoblastic leukemia and congenital heart disease. She had been diagnosed with pulmonary atresia with an intact ventricular septum at birth, and the anomaly was surgically corrected according to the Fontan technique at age 9 years. Her induction chemotherapy required modifications due to poor cardiac status with Fontan circulation. However, after surgical procedures including total cavopulmonary connection and aortic valve replacement at first complete remission, her cardiac status was significantly improved. Subsequently, she underwent cord blood stem cell transplantation at the third complete remission. She required intensive supportive care for circulatory failure as a pre-engraftment immune reaction and stage III acute graft versus host disease of the gut, but recovered from these complications. She was discharged on day 239, and remained in complete remission at 1-year post-transplantation.

Directional bleb formation in spherical cells under temperature gradient.

Living cells sense absolute temperature and temporal changes in temperature using biological thermosensors such as ion channels. Here, we reveal, to our knowledge, a novel mechanism of sensing spatial temperature gradients within single cells. Spherical mitotic cells form directional membrane extensions (polar blebs) under sharp temperature gradients (≥∼0.065°C µm(-1); 1.3°C temperature difference within a cell), which are created by local heating with a focused 1455-nm laser beam under an optical microscope. On the other hand, multiple nondirectional blebs are formed under gradual temperature gradients or uniform heating. During heating, the distribution of actomyosin complexes becomes inhomogeneous due to a break in the symmetry of its contractile force, highlighting the role of the actomyosin complex as a sensor of local temperature gradients.

High-frequency sarcomeric auto-oscillations induced by heating in living neonatal cardiomyocytes of the rat.

In the present study, we investigated the effects of infra-red laser irradiation on sarcomere dynamics in living neonatal cardiomyocytes of the rat. A rapid increase in temperature to >~38 °C induced [Ca(2+)]i-independent high-frequency (~5-10 Hz) sarcomeric auto-oscillations (Hyperthermal Sarcomeric Oscillations; HSOs). In myocytes with the intact sarcoplasmic reticular functions, HSOs coexisted with [Ca(2+)]i-dependent spontaneous beating in the same sarcomeres, with markedly varying frequencies (~10 and ~1 Hz for the former and latter, respectively). HSOs likewise occurred following blockade of the sarcoplasmic reticular functions, with the amplitude becoming larger and the frequency lower in a time-dependent manner. The present findings suggest that in the mammalian heart, sarcomeres spontaneously oscillate at higher frequencies than the sinus rhythm at temperatures slightly above the physiologically relevant levels.

Successful Atrial Septal Defect Closure Subsequent to Medical Pulmonary Preconditioning in an Infant With Severe Pulmonary Hypertension Associated With Bronchopulmonary Dysplasia.

While atrial septal defect (ASD) may contribute to right ventricular decompression in patients with severe pulmonary hypertension (PH), the pulmonary vasculature might be compromised by increased pulmonary blood flow, even though pulmonary vasodilators successfully reduce resistance. ASD closure is a treatment option that may ameliorate PH symptoms associated with bronchopulmonary dysplasia (BPD) in infants. However, the feasibility of ASD closure is obscure in patients with BPD-PH causing right-to-left shunting. Here, we present an eight-month-old girl with ASD complicated by BPD-PH, in which the pulmonary pressure exceeded the systemic pressure; the ASD was successfully closed after pulmonary preconditioning with dexamethasone and high-dose diuretics. Our patient was delivered as the third baby in triplets at a gestational age of 25 weeks, with a birth weight of 344 g. She was diagnosed with BPD at three months of age (37 weeks of postmenstrual age) with a body weight of 1.4 kg. Mild pulmonary hypertension was identified at the age of five months, and oral sildenafil was initiated. While her atrial septal defect was small at the time of PH diagnosis, it became hemodynamically significant when she grew up to 3.4 kg of body weight, at seven months after birth. Her estimated right ventricular pressure was apparently more than the systemic pressure, and oxygen saturation fluctuated between 82% and 97% under oxygen supplementation due to bidirectional interatrial shunt with predominant right-to-left shunting. Pulmonary preconditioning lowered the estimated right ventricular pressure to almost equal the systemic pressure and elevated arterial oxygen saturation while also suppressing right-to-left shunting. Cardiac catheterization after preconditioning revealed a ratio of pulmonary blood pressure to systemic blood pressure ratio (Pp/Ps) of 0.9, pulmonary resistance of 7.3 WU-m2, and a pulmonary to systemic blood flow ratio (Qp/Qs) of 1.3 (approximately 1.0 in the normal circulation without significant shunt), with the cardiac index of 2.8 L/min/m2. The acute pulmonary vasoreactivity test against the combination of 20 ppm nitric oxide and 100% oxygen was negative, although the patient had consistently high pulmonary flow with makeshift improvements after preconditioning. Despite the high pulmonary resistance even after preconditioning, aggressive ASD closure was performed so that pulmonary flow could be consistently suppressed regardless of the pulmonary condition. Her Pp/Ps under 100% oxygen with 20 ppm nitric oxide was 0.7 immediately after closure. After two years of follow-up, her estimated right ventricular pressure was less than half of the systemic pressure with the use of three pulmonary vasodilators, including sildenafil, macitentan, and beraprost. A strategy to temporarily improve PH and respiratory status aimed at ASD closure could be a treatment option for the effective use of multiple pulmonary vasodilators, by which intensive treatment of BPD can be achieved.

Genetic background of catecholaminergic polymorphic ventricular tachycardia in Japan.

BACKGROUND: The genetic background of catecholaminergic polymorphic ventricular tachycardia (CPVT) has been extensively investigated for the last decade in Western countries, but it remains unstudied in the Asian population. METHODS AND RESULTS: In 50 Japanese probands from unrelated families who satisfied clinical criteria for CPVT, genetic testing was conducted in all exons on 3 CPVT-related genes: cardiac ryanodine receptor 2 (RYR2), calsequestrin 2 (CASQ2) and inward rectifier potassium channel 2 (KCNJ2), and the clinical features between RYR2-genotyped and -non-genotyped patient groups were compared. Genetic and clinical evaluation was also done in 46 family members. In the genetic screening, 28 (18 novel) RYR2 (56.0%), 1 compound heterozygous CASQ2 (2.0%) and 1 KCNJ2 (2.0%) mutation carriers were identified. In the RYR2 mutation-positive group, the frequency of bidirectional ventricular tachycardia and the use of ß-blockers were significantly higher than in the mutation-negative group. In contrast, there was no significant difference in supraventricular arrhythmias between the 2 groups. With regard to disease penetrance, the number of family members of RYR2-genotyped probands with a clinical diagnosis of CPVT was high. CONCLUSIONS: Thirty gene mutation carriers were found for 3 genes in 50 probands clinically diagnosed as having CPVT. The penetrance of CPVT phenotype was significantly higher in RYR2 mutation carriers, thus RYR2 gene screening in CPVT patients would be indispensable to prevent unexpected cardiac sudden death of young family members.