Conditional deficiency of Rho-associated kinases disrupts endothelial cell junctions and impairs respiratory function in adult mice.

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho-associated coiled-coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adult mice. Tamoxifen (TAM)-inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc-CreERT2) were generated and administered a 5-day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double-floxed control mice. On Day 3 post-TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post-TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)-cadherin, ß-catenin, and p120-catenin at cell-cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE-cadherin/ß-catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell-cell adhesion.

Efficacy of Computed Tomography-Based Evaluation of Myocardial Extracellular Volume Combined With Red Flags for Early Screening of Concealed Cardiac Amyloidosis in Patients With Atrial Fibrillation.

BACKGROUND: The prevalence of transthyretin amyloid cardiomyopathy (ATTR-CM) in atrial fibrillation (AF) patients remains unclear. We explored the efficacy of computed tomography-based myocardial extracellular volume (CT-ECV) combined with red flags for the early screening of concealed ATTR-CM in AF patients undergoing catheter ablation.Methods and Results: Patients referred for AF ablation at Oita University Hospital were prescreened using the red-flag signs defined by echocardiographic or electrocardiographic findings, medical history, symptoms, and blood biochemical findings. Myocardial CT-ECV was quantified in red flag-positive patients using routine pre-AF ablation planning cardiac CT with the addition of delayed-phase cardiac CT scans. Patients with high (>35%) ECV were evaluated using technetium pyrophosphate (99 mTc-PYP) scintigraphy. A cardiac biopsy was performed during the planned AF ablation procedure if 99 mTc-PYP scintigraphy was positive. Between June 2022 and June 2023, 342 patients were referred for AF ablation. Sixty-seven (19.6%) patients had at least one of the red-flag signs. Myocardial CT-ECV was evaluated in 57 patients because of contraindications to contrast media, revealing that 16 patients had high CT-ECV. Of these, 6 patients showed a positive 99 mTc-PYP study, and 6 patients were subsequently diagnosed with wild-type ATTR-CM via cardiac biopsy and genetic testing. CONCLUSIONS: CT-ECV combined with red flags could contribute to the systematic early screening of concealed ATTR-CM in AF patients undergoing catheter ablation.

Impact of the COVID-19 Pandemic on the Long-Term Prognosis of Acute Myocardial Infarction in Japan.

Background During the early phase of the coronavirus disease 2019 (COVID-19) pandemic, a global reduction in hospitalizations for acute myocardial infarction (AMI) was observed. Generally, patients experienced increased severity of AMI with delays in time from symptom onset to treatment during the pandemic. However, the impact of the COVID-19 pandemic on in-hospital mortality among patients with AMI remains unclear. This study aimed to compare the long-term prognosis of patients with AMI during the COVID-19 pandemic to that observed in the pre-pandemic period and to evaluate the influence of the COVID-19 pandemic on the prognosis of patients with AMI. Methods We reviewed the data of patients admitted to our hospital for AMI treatment between April 1, 2018, and March 31, 2021. The time from admission to major adverse cardiac events (MACE), as well as the time from admission to all-cause death, were examined between the pandemic period (April 1, 2020, to March 31, 2021) and the pre-pandemic period (April 1, 2018, to March 31, 2020). Results Eighty patients were included in the study, and those admitted during the pandemic exhibited a higher likelihood of advanced age, lower levels of LDL-cholesterol, and a reduced prevalence of hypertension. The 2.5-year MACE-free survival and overall survival rates between the patients during the pre-pandemic and pandemic periods were not significantly different. Conclusion The long-term prognosis of patients with AMI during the COVID-19 pandemic remains unclear. In this study, we reported that the 2.5-year MACE-free survival and overall survival rates of the patients with AMI admitted during the COVID-19 pandemic were not significantly different from those during the pre-pandemic period. The impact of the COVID-19 pandemic on the prognosis of patients with AMI appears to vary according to the study population.

Mitochondrial choline import regulates purine nucleotide pools via SLC25A48.

Choline is an essential nutrient for cellular metabolism, including the biosynthesis of phospholipids, neurotransmitters, and one-carbon metabolism. A critical step of choline catabolism is the mitochondrial import and synthesis of chorine-derived methyl donors, such as betaine. However, the underlying mechanisms and the biological significance of mitochondrial choline catabolism remain insufficiently understood. Here, we report that a mitochondrial inner-membrane protein SLC25A48 controls mitochondrial choline transport and catabolism in vivo. We demonstrate that SLC25A48 is highly expressed in brown adipose tissue and required for whole-body cold tolerance, thermogenesis, and mitochondrial respiration. Mechanistically, choline uptake into the mitochondrial matrix via SLC25A48 facilitates betaine synthesis and one-carbon metabolism. Importantly, cells lacking SLC25A48 exhibited reduced synthesis of purine nucleotides and failed to initiate the G1-to-S phase transition, thereby leading to cell death. Taken together, the present study identified SLC25A48 as a mitochondrial carrier that mediates choline import and plays a critical role in mitochondrial respiratory capacity, purine nucleotide synthesis, and cell survival.

R-R' interval in the left bundle branch block predicts long-term outcomes after cardiac resynchronization therapy by estimating greater mechanical dyssynchrony and viable myocardium.

BACKGROUND: Typical left bundle branch block (LBBB) shows 2 peaks of the R wave, which reflect activation reaching the interventricular septum (R) and posterolateral wall (R') sequentially. OBJECTIVE: The purpose of this study was to investigate the relationship among R-R' interval (RR'), mechanical dyssynchrony, extent of viable myocardium, and long-term outcomes in cardiac resynchronization therapy (CRT) candidates. METHODS: The study enrolled 49 patients (34 men; mean age: 69 ± 11 years) with LBBB who received CRT. The LBBB definition used requires the presence of mid-QRS notching in leads V1, V2, V5, V6, I, and aVL. Baseline evaluations were QRS duration (QRSd) and RR' measured from the 12-lead electrocardiogram; eyeball dyssynchrony (apical rocking and septal flash) and opposing-wall delay by speckle tracking from echocardiography, and extent of viable myocardium assessed by thallium-201 single-photon emission computed tomography. Primary outcomes included the combination of all-cause death and heart failure-related hospitalization. RESULTS: RR' predicted volumetric response better than QRSd (area under the curve 0.73 vs 0.67, respectively). The long RR' group (≥48 ms) revealed more frequent eyeball dyssynchrony and significantly greater radial (SL) and circumferential dyssynchrony (AP and SL) and %viable segment than the short RR' group. In multivariate regression analysis, only RR' ≥48 ms was independently associated with higher event-free survival rates following CRT (hazard ratio 0.21; P = .014). CONCLUSION: These findings suggest that RR' in complete LBBB was associated with mechanical dyssynchrony, extent of viable myocardium, and long-term outcomes following CRT.

Role of polyunsaturated fatty acids in Japanese patients with coronary spastic angina.

BACKGROUND: n-3 polyunsaturated fatty acids (PUFAs) reduce the risk of ischemic heart disease. However, there are few reports of a relationship between n-3 PUFAs and coronary spastic angina (CSA). This study aimed to assess the age-dependent role of serum levels of fatty acid in patients with CSA. METHODS AND RESULTS: We enrolled 406 patients who underwent ergonovine tolerance test (ETT) during coronary angiography for evaluation of CSA. All ETT-positive subjects were diagnosed as having CSA. We categorized the patients by age and results of ETT as follows: (1) young (age ≤ 65 years) CSA-positive (n = 32), (2) young CSA-negative (n = 134), (3) elderly (age > 66 years) CSA-positive (n = 36), and (4) elderly CSA-negative (n = 204) groups. We evaluated the serum levels of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid, and dihomo-gamma-linolenic acid. In the young groups, the serum levels of EPA (64.3 ±â€¯37.7 µg/mL vs. 49.4 ±â€¯28.8 µg/mL, p = 0.015) and DHA (135.7 ±â€¯47.6 µg/mL vs. 117.4 ±â€¯37.6 µg/mL, p = 0.020) were significantly higher in the CSA-positive group than in the CSA-negative group, respectively. However, this was not the case with elderly groups. In the multivariate analysis in young groups, the serum levels of EPA (p = 0.028) and DHA (p = 0.049) were independently associated with the presence of CSA, respectively. CONCLUSION: Our results suggested that the higher serum levels of EPA and/or DHA might be involved in the pathophysiology of CSA in the young population but not in the elderly population.

Association between interatrial septum adiposity and atrial fibrillation: transesophageal echocardiography imaging and autopsy study.

Recent clinical evidence has suggested that interatrial septal (IAS) adiposity contributes to atrial fibrillation (AF). The present study aimed to confirm the usefulness of transesophageal echocardiography (TEE) to estimate IAS adiposity in patients with AF. The histological IAS analysis based on autopsy samples sought to clarify characteristics that underlie the contribution of IAS adiposity to AF. The imaging study analyzed the TEE results in patients with AF (n = 184) in comparison with transthoracic echocardiography (TTE) and computed tomography (CT) results. The autopsy study histologically analyzed IAS in subjects with (n = 5) and without (n = 5) history of AF. In the imaging study, the ratio of interatrial septum adipose tissue (IAS-AT) volume per epicardial adipose tissue (EpAT) volume was greater in patients with persistent AF compared (PerAF) to those with paroxysmal AF (PAF). Multivariable analysis revealed that both TEE-assessed IAS thickness and TTE-assessed left atrial dimension were predicted by CT-assessed IAS-AT volume. In the autopsy study, the histologically-assessed IAS section thickness was greater in the AF group than that in the non-AF group and was positively correlated with the IAS-AT area percentage. In addition, the size of adipocytes in IAS-AT was smaller, compared to EpAT and subcutaneous adipose tissue (SAT). IAS-AT infiltrated into the IAS myocardium, as if adipose tissue split the myocardium (designated as myocardial splitting by IAS-AT). The number of island-like myocardium pieces as a result of myocardial splitting by IAS-AT was greater in the AF group than in the non-AF group and was positively correlated with the IAS-AT area percentage. The present imaging study confirmed the usefulness of TEE to estimate IAS adiposity in patients with AF without radiation exposure. The autopsy study suggested that the myocardial splitting by IAS-AT may contribute to atrial cardiomyopathy leading to AF.

Role of epicardial adipose tissue in human atrial fibrillation.

A recent meta-analysis among which four reports were conducted in Japan demonstrated that epicardial adipose tissue (EAT) is closely associated with an increased risk of atrial fibrillation (AF) recurrence after catheter ablation. We previously investigated the role of EAT in AF in humans. Left atrial (LA) appendage samples were obtained from AF patients during cardiovascular surgery. Histologically, the severity of fibrotic EAT remodeling was associated with LA myocardial fibrosis. Total collagen in the LA myocardium (i.e., LA myocardial fibrosis) was positively correlated with proinflammatory and profibrotic cytokines/chemokines, including interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α, in EAT. Human peri-LA EAT and abdominal subcutaneous adipose tissue (SAT) were obtained by autopsy. EAT- or SAT-derived conditioned medium was applied to the rat LA epicardial surface using an organo-culture system. EAT-conditioned medium induced atrial fibrosis in organo-cultured rat atrium. The profibrotic effect of EAT was greater than that of SAT. The fibrotic area of the organo-cultured rat atrium treated with EAT from patients with AF was greater than in patients without AF. Treatment with human recombinant angiopoietin-like protein 2 (Angptl2) induced fibrosis in organo-cultured rat atrium, which was suppressed by concomitant treatment with anti-Angptl2 antibody. Finally, we attempted to detect fibrotic EAT remodeling on computed tomography (CT) images, which demonstrated that the percent change in EAT fat attenuation was positively correlated with EAT fibrosis. Based on these findings, we conclude that the percent change in EAT fat attenuation determined using CT non-invasively detects EAT remodeling.

Lipolysis-derived linoleic acid drives beige fat progenitor cell proliferation.

De novo beige adipocyte biogenesis involves the proliferation of progenitor cells in white adipose tissue (WAT); however, what regulates this process remains unclear. Here, we report that in mouse models but also in human tissues, WAT lipolysis-derived linoleic acid triggers beige progenitor cell proliferation following cold acclimation, ß3-adrenoceptor activation, and burn injury. A subset of adipocyte progenitors, as marked by cell surface markers PDGFRα or Sca1 and CD81, harbored cristae-rich mitochondria and actively imported linoleic acid via a fatty acid transporter CD36. Linoleic acid not only was oxidized as fuel in the mitochondria but also was utilized for the synthesis of arachidonic acid-derived signaling entities such as prostaglandin D2. Oral supplementation of linoleic acid was sufficient to stimulate beige progenitor cell proliferation, even under thermoneutral conditions, in a CD36-dependent manner. Together, this study provides mechanistic insights into how diverse pathophysiological stimuli, such as cold and burn injury, promote de novo beige fat biogenesis.

Post-translational control of beige fat biogenesis by PRDM16 stabilization.

Compelling evidence shows that brown and beige adipose tissue are protective against metabolic diseases1,2. PR domain-containing 16 (PRDM16) is a dominant activator of the biogenesis of beige adipocytes by forming a complex with transcriptional and epigenetic factors and is therefore an attractive target for improving metabolic health3-8. However, a lack of knowledge surrounding the regulation of PRDM16 protein expression hampered us from selectively targeting this transcriptional pathway. Here we identify CUL2-APPBP2 as the ubiquitin E3 ligase that determines PRDM16 protein stability by catalysing its polyubiquitination. Inhibition of CUL2-APPBP2 sufficiently extended the half-life of PRDM16 protein and promoted beige adipocyte biogenesis. By contrast, elevated CUL2-APPBP2 expression was found in aged adipose tissues and repressed adipocyte thermogenesis by degrading PRDM16 protein. Importantly, extended PRDM16 protein stability by adipocyte-specific deletion of CUL2-APPBP2 counteracted diet-induced obesity, glucose intolerance, insulin resistance and dyslipidaemia in mice. These results offer a cell-autonomous route to selectively activate the PRDM16 pathway in adipose tissues.

Reduction of bleeding complications on puncture site after percutaneous coronary intervention using a 6.5-French sheathless guiding catheter.

BACKGROUND: Reducing complications at the puncture site after percutaneous coronary intervention (PCI) is important. The diameter of a 6.5-French (Fr) sheathless guiding catheter (GC) is smaller by approximately 2-Fr compared to a 6-Fr conventional sheath. In the present study, we investigated the post-PCI puncture site complications of a transradial approach in each gender while using a 6.5-Fr sheathless GC. METHODS AND RESULTS: Our study consisted of 332 patients who underwent transradial coronary intervention (TRI) between August 2017 and July 2019. We classified the patients into either the 6.5-Fr sheathless GC (Asahi, Intecc, Aichi, Japan) Group (Sheathless group: n = 182 males, 58 females) or the 6-Fr sheathed GC Group (Sheathed group: n = 150 males, 36 females). We determined the complications at the puncture site: oozing, subcutaneous hemorrhage, formation of hematoma, pseudoaneurysms, and peripheral neuropathy. The body mass index of the patients was greater in the sheathless GC group compared to the sheathed GC group (24.5 ± 3.5 kg/m2 vs. 23.6 ± 3.7 kg/m2, p = 0.02). In males, there was no significant difference in the complication rate at the puncture site between the sheathless GC and sheathed GC groups (19.3% vs. 18.6%, p = 0.88). However, the complication rate at the puncture site in females was higher in the sheathed GC group than in the sheathless GC group (36% vs. 15.5%, p = 0.02). A multiple logistic regression analysis revealed that the use of a 6.5-Fr sheathless GC independently reduced the complications in female patients (p = 0.006). CONCLUSION: The use of the 6.5-Fr sheathless GC system in a transradial approach reduced the complications at the puncture site in female patients. The 6.5-Fr sheathless GC system may be a safe option for them compared to the conventional sheath system.

Suppression of acute heart failure rehospitalization by biventricular pacing in wide QRS and mid-range ejection fraction.

We present a 66-year-old male patient with heart failure, mid-range ejection fraction and QRS widening suffering from recurrent hospitalization due to acute heart failure. We measured intra-cardiac pressure by cardiac catheterization to clearly demonstrate the augmentation of afterload by a vasoconstricting drug induced increase of left ventricular end-diastolic blood pressure and pulmonary capillary wedge pressure with pulmonary arterial V-wave augmentation (indicator of worsening of mitral regurgitation). Because the patient was considered as refractory to optimal medication, cardiac resynchronization therapy (CRT) was performed. After CRT implantation, these factors were improved, and the patient has not experienced recurrent hospitalization for >2 years.

Detection of fibrotic remodeling of epicardial adipose tissue in patients with atrial fibrillation: Imaging approach based on histological observation.

BACKGROUND: Fibrotic remodeling of epicardial adipose tissue (EAT) is crucial for proinflammatory atrial myocardial fibrosis, which leads to atrial fibrillation (AF). OBJECTIVES: We tested the hypothesis that the ratio of central to marginal adipocyte diameter in EAT represents its fibrotic remodeling. Based on a similar concept, we also tested whether the percent (%) change in EAT fat attenuation determined using computed tomographic (CT) images can detect this remodeling. METHODS: Left atrial appendages were obtained from 76 consecutive AF patients during cardiovascular surgery. EAT in the central area (central EAT: C-EAT) and that adjacent to the atrial myocardium (Marginal EAT: M-EAT) were evaluated histologically. CT images for all of the 76 patients were also analyzed. RESULTS: The adipocyte diameter was smaller, fibrotic remodeling of EAT (EAT fibrosis) was more severe, and infiltration of macrophages and myofibroblasts was more extensive in M-EAT than in C-EAT. EAT fibrosis was positively correlated with adipocyte diameter in C-EAT and negatively correlated in M-EAT, resulting in a positive correlation between EAT fibrosis and the ratio of central to marginal adipocyte diameter (C/M diameter ratio; r = 0.73, P < .01). The C/M diameter ratio was greater in patients with persistent AF than in those with paroxysmal AF. CT images demonstrated that the %change in EAT fat attenuation was positively correlated with EAT fibrosis. CONCLUSION: Our results suggest that the central-to-marginal adipocyte diameter ratio is tightly associated with fibrotic remodeling of EAT. In addition, the %change in EAT fat attenuation determined using CT imaging can detect remodeling noninvasively.

Factors associated with bleeding events during catheter ablation with uninterrupted periprocedural edoxaban for atrial fibrillation: a subanalysis of the KYU-RABLE study.

BACKGROUND: Data are limited on patient background characteristics associated with catheter ablation (CA)-related bleeding events in Japanese patients with non-valvular atrial fibrillation receiving uninterrupted periprocedural edoxaban. This subanalysis of the KYU-RABLE study focused on univariate and multivariate analyses to identify correlations between bleeding events and baseline patient demographics and CA-related characteristics. METHODS: Patients with non-valvular atrial fibrillation (NVAF) enrolled from the KYU-RABLE study were included in the study. We performed univariate and multivariate analyses to investigate the correlation of major, minor, and clinically relevant non-major bleeding events with the patient baseline data at enrollment, and with CA procedures. RESULTS: A total of 513 NVAF patients were included in the full analysis set. Univariate analysis showed that the incidence of the bleeding events was higher in patients with HAS-BLED score ≥ 3 compared with those with a score < 3 (odds ratio [OR]: 9.48, 95% CI: 2.36-38.01; p = 0.002), in those with creatinine clearance (CrCL) ≤50 mL/min compared with those with CrCL > 50 mL/min (OR: 10.59, 95% CI: 3.65-30.79; p < 0.0001), and in those receiving edoxaban 30 mg compared with those receiving edoxaban 60 mg (OR: 3.49, 95% CI: 1.18-10.38; p = 0.025). Multivariate analysis showed that HAS-BLED score ≥ 3 (OR: 7.93, 95% CI: 1.66-37.88; p = 0.0094) and CrCl ≤ 50 mL/min (OR: 7.78, 95% CI: 2.17-27.90; p = 0.0016) were significant predictors of bleeding events among KYU-RABLE patients. CONCLUSIONS: These predictors of CA-related bleeding events may allow informed decision-making and better AF patient selection for CA with uninterrupted periprocedural edoxaban. TRIAL REGISTRATION: KYU-RABLE, UMIN000029693 . Registered 1 December 2017.

Distinctively different predictors for long-term outcomes between responders and nonresponders who underwent cardiac resynchronization therapy.

BACKGROUND: It is common to develop heart failure (HF) events even in respondents to cardiac resynchronization therapy (CRT) during a long-term observation period. We investigated the predictors for long-term outcome in responders in comparison with nonresponders in patients diagnosed with HF along with implanted CRT. METHODS: We enrolled 133 consecutive patients (mean age, 70 ± 10 years; 72 males) implanted with CRT from April 2010 to July 2019. Accurate follow-up information (mean follow-up period, 983 ± 801 days) was obtained from 66 responders and 53 nonresponders. RESULTS: Kaplan-Meier event-free curves showed that major adverse cerebral and cardiovascular event (MACCE)-free ratio was significantly lower as the stage of renal function progresses (log rank, 19.5; P < .0001). The baseline estimated glomerular filtration rate (e-GFR) before CRT was not significantly different between nonresponders and responders. The e-GFR after judgment of CRT response was lower in patients with MACCEs than those without. Cox proportional hazards regression analysis revealed that low baseline e-GFR before CRT and after judgment of CRT response was closely related with MACCEs in responders, but not in nonresponders. The survival rate in responders without MACCEs assessed using Kaplan-Meier analysis was significantly larger in the preserved e-GFR (baseline value before CRT, >44 mL/min/1.73 m2) group than in the depressed group (log rank, 20.29; P < .0001). CONCLUSION: We demonstrate that the factors for MACCEs during long follow-up periods were distinctively different between responders and nonresponders. Patients with depressed e-GFRs are suggested to have poor prognosis even if they are responders to CRT.