The precise molecular pathways driving fibrosis and calcification in aortic valve leaflets remain poorly defined. Here, we present the first data indicating a role for caldesmon-1 (CALD1) in calcified aortic valve disease (CAVD) pathogenesis. Analysis of publicly available single-cell RNA sequencing (scRNA-seq) datasets revealed that CALD1 shows prominent upregulation in aortic valve stenosis (AS) cases when compared to normal subjects. Histological examination demonstrated that CALD1 protein expression is elevated in calcified AS valves and co-localises with α-smooth muscle actin (a myofibroblast biomarker) and vimentin, indicating its association with activated valvular interstitial cells (VICs). Bioinformatic analysis showed that CALD1-positive cells predominantly synthesize extracellular matrix components, including COL1A1. Functional experiments using CALD1-depleted VICs revealed that CALD1 is required for maintaining spindle-shaped morphology, actin polymerisation, and proliferative capacity. Moreover, CALD1 loss significantly impaired osteoblast differentiation and attenuated VIC calcification. Bulk RNA-seq combined with pathway analysis demonstrated that CALD1-mediated actin polymerisation positively regulates key osteogenic and valvulopathy-related genes, including RUNX2 and ALPL. Collectively, these findings identify CALD1 as a novel regulator of VIC phenotypic plasticity and osteogenic transition during CAVD progression, providing mechanistic insight and a target for potential AS therapy.

Background Vascular calcification, particularly medial arterial calcification (MAC), considerably affects cardiovascular mortality. Current treatments are limited because of the unclear molecular mechanisms of MAC. This study aimed to establish an MAC mouse model using O-ring-induced transverse aortic constriction (OTAC) and to identify critical genes, pathways, and cellular interactions involved in MAC formation by combining lineage tracing technology and single-cell RNṣA sequencing (scRNA-seq). Methods We developed an OTAC mouse model to mimic MAC. Adult C57BL/6J male mice underwent OTAC, and subsequent analyses were performed at various intervals. Histological and immunohistochemical evaluations were conducted to observe changes in vascular smooth muscle cells (VSMCs). Lineage tracing was used to confirm the origin of osteochondrogenic cells in the tunica media. Additionally, scRNA-seq was performed to capture the dynamic changes in VSMCs and other cell types involved in MAC development. Results Osteochondrogenic cells and the subsequent MAC in OTAC mice were observed using histological evaluations. Immunohistochemistry showed time-dependent decreases in α-SMA expression and increases in SOX9 and RUNX2 expression in VSMCs. Lineage tracing using Myh11CreERT2;ROSA26-EGFP mice confirmed that osteochondrogenic cells originated from contractile VSMCs. scRNA-seq confirmed osteochondrogenic transdifferentiation of VSMCs and highlighted dynamic changes in surrounding cells involved in calcification-related and inflammatory processes. Conclusion The OTAC method enabled a comprehensive understanding of the microenvironmental dynamics during MAC progression at the single-cell level. This powerful model provides a robust platform for future therapeutic intervention research. ![Figure][1] Graphic Abstract ### Competing Interest Statement The authors have declared no competing interest. * cAb : Control antibodies DCs : Dendritic cells DEGs : Differentially expressed genes FC : Fold change FDR : False discovery rate GO : Gene Ontology MAC : Medial arterial calcification nAb Ly6g : neutralizing antibodies OTAC : O-ring-induced transverse aortic constriction RNA- : seq RNA sequencing scRNA- : seq Single-cell RNA sequencing TAA : Thoracic aortic aneurysms TAC : Transverse aortic constriction VSMCs : Vascular smooth muscle cells Basic Research of the Japanese Circulation Society (2024) SENSHIN Medical Research Foundation, https://ror.org/021tj4g88 Japan Arteriosclerosis Prevention Fund, https://ror.org/05wfg6079 Bayer Academic Support, BASJ20230409007 The FOREST (Fusion Oriented REsearch for disruptive Science and Technology) Program, JPMJFR220Q Otsuka Pharmaceutical Academic Research Support, AS2023A000625966 Boehringer Ingelheim Life science Research Support, RS2023A000408937 [1]: pending:yes

Objectives This study aimed to investigate the utility of the RAND/UCLA appropriateness method (RAM) in validating expert consensus-based multiple-choice questions (MCQs) on electrocardiogram (ECG).Methods According to the RAM user's manual, nine panelists comprising various experts who routinely handle ECGs were asked to reach a consensus in three phases: a preparatory phase (round 0), an online test phase (round 1), and a face-to-face expert panel meeting (round 2). In round 0, the objectives and future timeline of the study were elucidated to the nine expert panelists with a summary of relevant literature. In round 1, 100 ECG questions prepared by two skilled cardiologists were answered, and the success rate was calculated by dividing the number of correct answers by 9. Furthermore, the questions were stratified into "Appropriate," "Discussion," or "Inappropriate" according to the median score and interquartile range (IQR) of appropriateness rating by nine panelists. In round 2, the validity of the 100 ECG questions was discussed in an expert panel meeting according to the results of round 1 and finally reassessed as "Appropriate," "Candidate," "Revision," and "Defer."Results In round 1 results, the average success rate of the nine experts was 0.89. Using the median score and IQR, 54 questions were classified as " Discussion." In the expert panel meeting in round 2, 23% of the original 100 questions was ultimately deemed inappropriate, although they had been prepared by two skilled cardiologists. Most of the 46 questions categorized as "Appropriate" using the median score and IQR in round 1 were considered "Appropriate" even after round 2 (44/46, 95.7%).Conclusions The use of the median score and IQR allowed for a more objective determination of question validity. The RAM may help select appropriate questions, contributing to the preparation of higher-quality tests.

Objective: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a key mediator of inflammation and plays an important role in the pathogenesis of atherosclerosis. Both inflammation and atherosclerosis have been proposed as contributors to abdominal aortic aneurysm (AAA) pathogenesis. However, the role of LOX-1 in AAA pathogenesis remains unknown. We aimed to investigate effects of Olr1, which encodes LOX-1, deletion on angiotensin II (Ang II)-induced AAA in apolipoprotein E knockout (ApoE KO) mice to determine whether LOX-1 deficiency mitigates AAA development. Design and method: Either Ang II (1.44 mg/kg/day) or saline (control) was administered continuously through a subcutaneously implanted osmotic pump for 4 weeks in the following 8-week-old male mice: Apoe-/-Olr1+/+ mice (Ang II, n = 40; saline, n = 10), Apoe-/-Olr1+/- mice (Ang II, n = 30; saline, n = 9), and Apoe-/-Olr1-/- mice (Ang II, n = 39; saline, n = 9). Results: Ang II infusion raised systolic blood pressure by tail-cuff methods in all three groups. Serial non-invasive assessment with ultrasound demonstrated that Ang II gradually increased the maximum area of the suprarenal abdominal aortas leading to AAAs. Although the incidence and expansion rate of AAA were comparable among genotypes, Olr1 deletion significantly increased the severity and rupture rate of AAAs (p = 0.01; incidence of type IV and ruptured AAA was 25% in Apoe-/-Olr1-/- mice vs 5% in Apoe-/-Olr1+/+ mice). Since atherosclerosis is considered a major contributor to the development of AAAs, the extent of the atheroma burden localized in aneurysmal lesions was evaluated by Oil Red O staining. Olr1 deletion did not decrease atherosclerosis formation in the aneurysmal wall. Interestingly, further histopathological analysis revealed that aneurysmal lesions in LOX-1-deficient mice had fewer fibroblasts and myofibroblasts, as well as thinner adventitial collagen, although the degree of elastin fragmentation or disruption was similar between LOX-1-deficient and control mice. These in vivo data suggested that adventitial fibroblasts play a pivotal role in the increased severity of AAA in LOX-1-deficient mice. To further determine the effects of Olr1 deletion on fibroblast differentiation, fibroblasts were isolated from the adventitia of abdominal aortas harvested from 8-week-old Apoe-/-Olr1+/+ and Apoe-/-Olr1-/- mice, and then cultured with Ang II stimulation. An in vitro study confirmed that Olr1 deletion completely abolished the Ang II-induced proliferative activity in the aortic adventitial fibroblasts (p = 0.02). Conclusions: Olr1 deletion may not mitigate aneurysm development but rather increase the vulnerability to rupture by suppressing adventitial fibroblast proliferation and collagen.

Histologic evaluations revealed excessive accumulations of macrophages and absence of fibroblastic interstitial cells in explanted bioprosthetic valves. Comprehensive gene and protein expression analysis and histology unveiled an accumulation of fibrinogen and plasminogen, an activator of infiltrated macrophages, from degenerated valve surfaces in the interstitial spaces. These pathologies were completely reproduced in a goat model replaced with an autologous pericardium-derived aortic valve. Further preclinical animal experiments using goats demonstrated that preventing infiltration of macrophages and circulating proteins by increasing collagen density and leaflet strength is an effective treatment option.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a key mediator of inflammation and plays an important role in the pathogenesis of atherosclerosis. Conversely, LOX-1 deficiency has been shown to decrease inflammation and atherosclerosis, both of which have been proposed to contribute to abdominal aortic aneurysm (AAA) pathogenesis. However, the role of LOX-1 in AAA pathogenesis remains unknown. Here, we investigated the effects of Olr1 (which encodes LOX-1) deletion on angiotensin II (Ang II)-induced AAA in apolipoprotein E knockout (ApoE KO) mice to determine whether LOX-1 deficiency mitigates AAA development. To accomplish this, we used serial, non-invasive ultrasound assessment, which revealed that the incidence and expansion rate of AAA were similar regardless of Olr1 deletion. However, Olr1 deletion significantly increased severe AAAs, including ruptured AAAs resulting in death. Oil Red O staining of the harvested aortas showed that the extent of atheroma burden localized in aneurysmal lesions did not differ between LOX-1-deficient and control mice, suggesting that Olr1 deletion did not decrease atheroma burden in the aneurysmal wall. Further histopathological analysis revealed that aneurysmal lesions in LOX-1-deficient mice had fewer fibroblasts and myofibroblasts, as well as thinner adventitial collagen, although the degree of elastin fragmentation or disruption was similar between LOX-1-deficient and control mice. An in vitro study confirmed that the proliferation of adventitial fibroblasts collected from LOX-1-deficient mice was significantly attenuated despite Ang II stimulation. In conclusion, Olr1 deletion may not mitigate aneurysm development but rather increases the vulnerability of rupture by suppressing adventitial fibroblast proliferation and collagen synthesis.

Pulmonary artery intimal sarcomas (PAIS) are often misdiagnosed as pulmonary embolisms (PE) as their clinical findings and imaging findings are similar. However, given the clinical outcome of both diseases is different in its prognosis, accurate and rapid diagnosis is mandatory. This is a case report of a histologically-proven PAIS which was initially treated as a PE. The color-coded iodine map using dual-energy computed tomography (dual-energy CT iodine map) well reflected the distribution of the tumor consistent with 18fluoro-2-deoxyglucose-uptake region using positron emission tomography/CT. This case demonstrates the potential of using dual-energy CT iodine map to differentiate PAIS from PE.

Suture-based transverse aortic constriction (TAC) in mice is one of the most frequently used experimental models for cardiac pressure overload-induced heart failure. However, the incidence of heart failure in the conventional TAC depends on the operator's skill. To optimize and simplify this method, we proposed O-ring-induced transverse aortic constriction (OTAC) in mice. C57BL/6J mice were subjected to OTAC, in which an o-ring was applied to the transverse aorta (between the brachiocephalic artery and the left common carotid artery) and tied with a triple knot. We used different inner diameters of o-rings were 0.50 and 0.45 mm. Pressure overload by OTAC promoted left ventricular (LV) hypertrophy. OTAC also increased lung weight, indicating severe pulmonary congestion. Echocardiographic findings revealed that both OTAC groups developed LV hypertrophy within one week after the procedure and gradually reduced LV fractional shortening. In addition, significant elevations in gene expression related to heart failure, LV hypertrophy, and LV fibrosis were observed in the LV of OTAC mice. We demonstrated the OTAC method, which is a simple and effective cardiac pressure overload method in mice. This method will efficiently help us understand heart failure (HF) mechanisms with reduced LV ejection fraction (HFrEF) and cardiac hypertrophy.

The slow flow phenomenon is a critical complication during percutaneous coronary intervention (PCI) that leads to poor outcomes. We aimed to evaluate the mechanisms underlying the slow flow phenomenon using intravascular ultrasound (IVUS). We retrospectively analyzed IVUS data from 62 lesions in 58 consecutive patients who experienced the slow flow phenomenon during PCI. IVUS was performed immediately after the development of the slow flow phenomenon to assess its cause. No IVUS-based evidence of mechanical obstruction was categorized as distal embolization. Distal embolization was observed in 46 lesions (74%). The slow flow phenomenon occurred in all these lesions after stent implantation. In addition to distal embolization, three different mechanisms underlying the induction of the slow flow phenomenon due to mechanical obstructions (16 lesions, 26%) were detected on IVUS, namely, medial dissection/hematoma (6 lesions), intimal flap (6 lesions), and thrombus obstruction (4 lesions). Most mechanical obstructions (13 lesions, 81%) could not be identified by angiography alone. In 15/16 lesions (94%) with mechanical obstruction, deteriorated flow improved immediately after balloon dilatation or bail-out stent implantation, although intracoronary vasodilator administration could not reestablish coronary flow. The frequency of mechanical obstruction as the cause of the slow flow phenomenon was relatively high. Given the difficulty in angiographical differentiation, IVUS-guided management of slow flow may be a useful strategy.

Abstract Aims Cardiac amyloidosis (CA) is an infiltrative myocardial disease that occasionally mimics hypertrophic cardiomyopathy (HCM). The aim of this study is to investigate the discriminatory ability of visual assessment of left atrial (LA) function between CA and HCM on echocardiography. Methods and results In total, 93 patients with cardiac magnetic resonance imaging (CMR)‐confirmed HCM and 34 with cardiac biopsy‐confirmed CA were retrospectively assessed. LA dilatation was assessed via echocardiography in an apical four‐chamber view. Visual assessment was performed to identify LA dilatation grade (preserved = 1, abnormal = 2, and restricted = 3) based on the extent of outward expansion in the LA reservoir phase. Regarding the reproducibility of visually assessing LA dilatation grade, the kappa values between intra‐ and inter‐observer measurements were 0.82 and 0.70, respectively. Of 127 participants, 57 (45%), 42 (33%), and 28 (22%) presented with LA dilatation Grades 1, 2, and 3, respectively. All 57 patients with preserved LA dilatation (Grade 1) had HCM, and 20 of 28 patients (71%) with restricted LA dilatation (Grade 3) presented with CA. Patients with CA had a higher LA dilatation grade than those with HCM (P < 0.01). LA emptying fraction and reservoir strain were also quantitatively evaluated. The area under the curves of LA dilatation grade (0.88) and LA emptying fraction (0.88) for differentiation of these two diseases were higher than that of LA reservoir strain (0.73) (P < 0.01, respectively). During follow‐up, nine patients with HCM and 16 with CA experienced cardiac event (cardiac death or hospitalization due to heart failure). In Kaplan–Meier analysis including both groups of HCM and CA, the incidence of cardiac events was higher in patients with restricted LA dilatation than in those with preserved or abnormal LA dilatation (log‐rank test, P < 0.01). Conclusions Restricted LA dilatation is an indicator for the diagnosis of CA. Further, visual assessment of abnormal LA motion may facilitate diagnosis in patients with CA and high‐risk patients with HCM.

performed without complications (Figure D). Cervical lymph node biopsy diagnosed the mass as a diffuse large B-cell lymphoma (Figure E,F). After chemotherapy, CT revealed marked reduction of the cardiac mass with improvement of the SSS (Figure G,H). Although her condition was temporarily stable, she died due to tumor relapse 10 months after initial admission. A leadless pacemaker should be considered as a viable alternative to a conventional transvenous pacemaker for unusual clinical scenarios.

Supplemental Digital Content is available in the text.

The characterization of aortic valve interstitial cells (VICs) cultured under optimal conditions is essential for understanding the molecular mechanisms underlying aortic valve stenosis. Here, we propose 2% hypoxia as an optimum VIC culture condition. Leaflets harvested from patients with aortic valve regurgitation were digested using collagenase and VICs were cultured under the 2% hypoxic condition. A significant increase in VIC growth was observed in 2% hypoxia (hypo-VICs), compared to normoxia (normo-VICs). RNA-sequencing revealed that downregulation of oxidative stress-marker genes (such as superoxide dismutase) and upregulation of cell cycle accelerators (such as cyclins) occurred in hypo-VICs. Accumulation of reactive oxygen species was observed in normo-VICs, indicating that low oxygen tension can avoid oxidative stress with cell-cycle arrest. Further mRNA quantifications revealed significant upregulation of several mesenchymal and hematopoietic progenitor markers, including CD34, in hypo-VICs. The stemness of hypo-VICs was confirmed using osteoblast differentiation assays, indicating that hypoxic culture is beneficial for maintaining growth and stemness, as well as for avoiding senescence via oxidative stress. The availability of hypoxic culture was also demonstrated in the molecular screening using proteomics. Therefore, hypoxic culture can be helpful for the identification of therapeutic targets and the evaluation of VIC molecular functions in vitro.

Perioperative management of pregnant women with heart failure is difficult. Management of anesthesia in pregnant women is especially difficult because all of the currently available choices present challenges. We report a patient with peripartum cardiomyopathy (PPCM) who required an emergent cesarean section and discuss the possible tactics for managing anesthesia. A 40-year-old primipara with severe cardiac and respiratory failure required an emergent cesarean section at 39+1 gestational weeks. Her left ventricular ejection fraction was between 10% and 15%, and she had orthopnea. General anesthesia was planned after inserting sheaths for percutaneous cardiopulmonary support from the femoral artery and vein. However, when the patient was asked to lie down on the operation bed, she panicked and resisted because of labor pain and dyspnea. Therefore, anesthesia was induced instead of the initial plan. Finally, we successfully managed the anesthesia and delivered the newborn. There are no alternatives to general anesthesia in patients with PPCM presenting with orthopnea. Anesthesia induction in the supine position is impossible in such patients owing to dyspnea. Anesthesia should be started with light sedation in the sitting position, and ketamine or low-dose remifentanil may be an option to maintain maternal hemodynamics and prevent neonatal asphyxia.

Abstract Aims Traditional criteria for heart transplantation by cardiopulmonary exercise testing (CPX) include peak oxygen uptake (VO2) < 14 mL/kg/min. Reaching a sufficient exercise load is challenging for patients with refractory heart failure (HF) because of their exercise intolerance. Recently, a substantial impact of right ventricular (RV) dysfunction was highlighted on urgent heart transplantation and mortality. This study aims to investigate the impact of RV contractile reserve, assessed by low‐load exercise stress echocardiography (ESE), on exercise intolerance defined as peak VO2 < 14 mL/kg/min, in patients with HF. Methods and results We prospectively examined 67 consecutive patients hospitalized for HF who underwent ESE and CPX under a stabilized HF condition. Although low‐load ESE was defined as 25 W load exercise, an increment in RV systolic (s′) velocity was regarded as the preservation of RV contractile reserve. All patients completed low‐load ESE. During low‐load ESE, the variation in RV s′ velocity significantly correlated with peak VO2 (r = 0.787, P < 0.001). The change in RV s′ velocity during low‐load ESE accurately identified patients with peak VO2 < 14 mL/kg/min (area under the curve, 0.95; sensitivity, 92%; specificity, 85%). The intraclass correlation coefficient for intra‐observer and inter‐observer agreement for the change in RV s′ velocity was 0.96 (95% confidence interval, 0.88–0.99, P < 0.001) and 0.86 (95% confidence interval, 0.64–0.95, P < 0.001), respectively. The RV‐to‐pulmonary circulation (PC) coupling, which was assessed by the slope of the relationship between RV s′ velocity and pulmonary artery systolic pressure at rest and low‐load exercise, was worse in the low‐peak VO2 group (<14 mL/kg/min) than the preserved‐peak VO2 group (≥14 mL/kg/min). Conclusions The change in RV s′ velocity during low‐load ESE could estimate the exercise capacity in HF patients. The assessments of RV contractile reserve and RV‐to‐PC coupling could be clinically beneficial to distinguish high‐risk HF patients.

Received June 16, 2020; accepted June 16, 2020; J-STAGE Advance Publication released online July 30, 2020 Time for primary review: 1 day Department of Cardiology, Pulmonology, Hypertension and Nephrology, Ehime University Graduate School of Medicine, Toon (Y.N., J.A., S. Inaba, K.N., S. Ikeda, O.Y.); Department of Cardiology, Nihon Medical School Tama Nagayama Hospital, Tokyo (O.I.), Japan Mailing address: Jun Aono, MD, PhD, Department of Cardiology, Pulmonology, Hypertension and Nephrology, Ehime University Graduate School of Medicine, Toon 791-0295, Japan. E-mail: junaono0616@gmail.com All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: cr@j-circ.or.jp ISSN-2434-0790 Visualization of Cardiac Perforation During Micra Transcatheter Leadless Pacemaker Implantation ― A Lesson From Cadaver Training ―

The management of systemic artery aneurysms secondary to Kawasaki disease (KD) in adults remains a therapeutic challenge. KD guidelines recommend the use of anticoagulation therapy with warfarin in addition to antiplatelet therapy when a giant coronary aneurysm or a history of thrombosis is documented. However, long-term use of warfarin presents several concerns. This case reports acute thrombotic occlusion due to the giant arterial aneurysm in an adult KD. A surgical resection of the aneurysm was performed because of recurrent thrombotic events, despite anticoagulant therapy with warfarin. Pathological examinations revealed a layered thrombus with inflammation in the aneurysm and Factor Xa expression mainly in newly formed thrombus. This study provides an insight into the anticoagulation therapy for cardiovascular sequelae after KD.

The Impella (Abiomed, Danvers, MA, USA) is a novel percutaneous heart pump device for left ventricular (LV) assistance; however, LV thrombus is a notable contraindication for this device. Contrast computed tomography assessment is useful for detecting LV thrombus and preventing thromboembolism in patients recommended for Impella use. .

Objective: Vascular calcification is a critical health concern in patients with diabetes and kidney dysfunction. Since vascular calcification is linked to systemic mineral imbalance such as hyperphosphatemia, lowering serum phosphorus concentrations with nutritional therapy and administration of oral phosphate binders has been a major therapy for preventing vascular calcification. However, a decrease in serum phosphorus concentrations alone does not completely suppress vascular calcification. To determine a mode of enhanced suppression of vascular calcification, we examined whether, an anti-diabetic drug, canagliflogin (Sodium-glucose co-transporter 2 inhibitors: SGLT2 inhibitors), inhibited progressive kidney injury and vascular calcification. Methods and Results: At 9 weeks of age, diabetes was induced in male Wistar rats by intraperitoneal administration of streptozotocin (STZ), and one week later CKD was induced by removal of the left kidney. One week after the surgery, rats were fed a high phosphate diet (2.0% wt/wt)) and intraperitoneal administration of paricalcitol (3x a week), and were administered 10 mg/kg/day either canagliflogin or vehicle for a duration of 2 weeks. At termination, blood and urine were collected, and renal and thoraco-abdominal aortas were dissected. Plasma concentrations of urea nitrogen (BUN), creatinine (Cre), calcium, and phosphate were measured. Immunostaining was performed to evaluate kidney injury. Vascular calcification in aortas was evaluated by Von Kossa and Alizarin Red S staining, and measurement of calcium content. STZ induced diabetes and nephrectomy that provoked kidney damage with renal tubular injury, renal-enlargement, and vascular calcification in aortas. Inhibition of SGLT2 by canagliflozin suppressed renal tubular injury, renal-enlargement, and vascular calcification. Conclusions: Our results support that selective inhibition of SGLT2 receptor improved the progression of kidney injury and reduced vascular calcification in diabetic and chronic kidney disease related conditions.