Remote ischemic periconditioning suppresses cardiac sympathetic activation in acute myocardial infarction: a randomized controlled trial.

PURPOSE: Remote ischemic periconditioning (RIPC) has demonstrated cardioprotective effects and improved clinical outcomes as an adjunct to emergent percutaneous coronary intervention (PCI) in patients with ST-elevation myocardial infarction (STEMI). However, whether RIPC affects the cardiac sympathetic nerve activity in patients with STEMI remains unclear. This study investigated the effects of RIPC on cardiac sympathetic nerve activity in patients with STEMI. METHODS: We prospectively assigned patients with STEMI who underwent emergent PCI to receive RIPC or no procedure (control group) upon arrival at the cardiac catheterization laboratory. The primary endpoint was cardiac sympathetic nerve activity assessed through the washout rate (WR) in cardiac 123I-metaiodobenzylguanidine (123I-MIBG) imaging. RESULTS: Patients in the RIPC (n = 62) and control (n = 60) groups had similar demographic and clinical characteristics at baseline. Multivariable linear regression models revealed that the culprit lesion of the left anterior descending artery and hemoglobin level were significantly and independently associated with WR at discharge. WRs of the groups differed insignificantly at discharge. However, the RIPC group (n = 49) showed significantly lower WR than the control group (n = 47) at 1 year after discharge (p = 0.027). In the single-photon emission computed tomography analysis at 1 year after discharge, the RIPC group demonstrated significantly higher late uptake (p = 0.021) and lower WR (p = 0.013) in the nonculprit lesion, with a non-significant decrease in WR for the culprit lesion. CONCLUSION: RIPC can suppress augmented cardiac sympathetic nerve activity in patients with STEMI, particularly in nonculprit lesions.

Improved higher resolution cryo-EM structures reveal the binding modes of hERG channel inhibitors.

During drug discovery, it is crucial to exclude compounds with toxic effects. The human ether-à-go-go-related gene (hERG) channel is essential for maintaining cardiac repolarization and is a critical target in drug safety evaluation due to its role in drug-induced arrhythmias. Inhibition of the hERG channel can lead to severe cardiac issues, including Torsades de Pointes tachycardia. Understanding hERG inhibition mechanisms is essential to avoid these toxicities. Several structural studies have elucidated the interactions between inhibitors and hERG. However, orientation and resolution issues have so far limited detailed insights. Here, we used digitonin to analyze the apo state of hERG, which resolved orientation issues and improved the resolution. We determined the structure of hERG bound to astemizole, showing a clear map in the pore pathway. Using this strategy, we also analyzed the binding modes of E-4031 and pimozide. These insights into inhibitor interactions with hERG may aid safer drug design and enhance cardiac safety.

Extensive ablation for elderly patients with persistent atrial fibrillation: insights from the EARNEST-PVI prospective randomized trial.

BACKGROUND: In patients with persistent atrial fibrillation (AF), extensive ablation for substrate modification, such as linear ablation or complex fractionated atrial electrogram ablation in addition to pulmonary vein isolation (PVI) remains controversial. Previous studies investigating extensive ablation have demonstrated its varying efficacy, suggesting the possible heterogeneity of its efficacy. Aging is a major risk factor for AF and is associated with atrial remodeling. We aimed to compare the efficacy and safety of the extensive ablation strategy compared with PVI alone strategy between young and elderly patients. METHODS: This study is a post-hoc analysis of the multicenter, randomized controlled, noninferiority trial investigating the efficacy and safety of PVI-only (PVI-alone arm) compared with extensive ablation (PVI-plus arm) in patients with persistent AF (EARNEST-PVI trial). We divided the overall population into 2 groups based on age and assessed treatment effects. RESULTS: In the young group (age <65 years, N = 206), there was no significant difference in the recurrence rate between the PVI-alone group and PVI-plus group [hazard ratio (HR): 1.00, 95 % CI: 0.57-1.73, p = 0.987], whereas the recurrence rate was significantly lower in the PVI-plus group compared to the PVI-alone group in the elderly group (age ≥65 years, N = 291) (HR: 0.47, 95 % CI: 0.29-0.76, p = 0.0021) (p for interaction = 0.0446). There were no fatal procedural complications. CONCLUSION: In patients with persistent AF, the extensive ablation strategy was more effective than the PVI-alone strategy in elderly patients, while the effectiveness of both approaches was comparable in young patients. TRIAL REGISTRATION: URL: https://clinicaltrials.gov; Unique identifier: NCT03514693. URL: https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000022454 Unique ID issued by UMIN: UMIN000019449.

A case of biatrial tachycardia involving the intercaval bundle with assumed dual loop reentry.

Epicardial connections provided the anatomical substrate for the biatrial reentry circuit. The connections between the right atrium and right pulmonary vein were called "intercaval bundle," and there are few reports of atrial flutter related to this bundle. We present a case of a biatrial tachycardia, involving the intercaval bundle.

Extensive ablation for persistent atrial fibrillation patients with mitral regurgitation: Insights from the EARNEST-PVI prospective randomized trial.

BACKGROUND: Extensive ablation in addition to pulmonary vein isolation (PVI) in patients with persistent atrial fibrillation (AF) has not yielded consistent results, indicating diversity in their efficacy. Mitral regurgitation (MR) associated with AF may indicate a higher prevalence of arrhythmogenic substrate, suggesting potential benefits of extensive ablation for these patients. METHODS: This post-hoc analysis of the EARNEST-PVI trial compared PVI alone versus an extensive ablation strategy (PVI-plus) in persistent AF patients, stratified by MR presence. The primary endpoint of the study was the recurrence of AF. The secondary endpoints included death, cerebral infarction, and procedure-related complications. RESULTS: The trial included 495 eligible patients divided into MR and non-MR groups. The MR group consisted of 192 patients (89 in the PVI-alone arm and 103 in the PVI-plus arm), while the non-MR group had 303 patients (158 in the PVI-alone arm and 145 in the PVI-plus arm). In the non-MR group, recurrence rates were similar between PVI-alone and PVI-plus arms (Log-rank P = 0.47, Hazard ratio = 0.85 [95%CI: 0.54-1.33], P = 0.472). However, in the MR group, PVI-plus was significantly more effective in preventing AF recurrence (Log-rank P = 0.0014, Hazard ratio = 0.40 [95%CI: 0.22-0.72], P = 0.0021). No significant differences were observed in secondary endpoints between the two arms. CONCLUSIONS: For persistent AF patients with mild or greater MR, receiving PVI-plus was superior to PVI-alone in preventing AF recurrence. Conversely, for patients without MR, the effectiveness of extensive ablation was not demonstrated. These findings suggest tailoring ablation strategies based on MR presence can lead to better outcomes in AF management.

Cyclic Sesquiterpene-Flavanone [4+2] Hybrids, Syzygioblanes A-C, Found in an Indonesian Traditional Medicine, "Jampu Salo" (Syzygium oblanceolatum).

A plant used in an Indonesian traditional herbal medicine as a diabetes treatment and known locally as "Jampu Salo" was collected on Sulawesi Island, Indonesia. It was identified as Syzygium oblanceolatum (C. B. Rob.) Merr. (Myrtaceae) and found for the first time in Sulawesi; it was previously reported only in the eastern Philippines and Borneo. A phytochemical study of S. oblanceolatum led to the isolation of three unprecedented meroterpenoids, syzygioblanes A-C (1-3, respectively). These compounds might be biosynthesized through [4+2] cycloaddition of various germacrane-based cyclic sesquiterpenoids with the flavone desmethoxymatteucinol to form a spiro skeleton. The unique and complex structures were elucidated by microcrystal electron diffraction analysis in addition to general analytical techniques such as high-resolution mass spectrometry, various nuclear magnetic resonance methods, and infrared spectroscopy. Synchrotron X-ray diffraction and calculations of electronic circular dichroism spectra helped to determine the absolute configurations. The newly isolated compounds exhibited collateral sensitivity to more strongly inhibit the growth of a multidrug resistant tumor cell line compared to a chemosensitive tumor cell line.

Appropriate Selection of Substrate Ablation for Persistent Atrial Fibrillation Using Intraprocedural Assessment.

BACKGROUND: It has not been fully elucidated which patients with persistent atrial fibrillation (PerAF) should undergo substrate ablation plus pulmonary vein isolation (PVI). This study aimed to identify PerAF patients who required substrate ablation using intraprocedural assessment of the baseline rhythm and the origin of atrial fibrillation (AF) triggers. METHODS AND RESULTS: This was a post hoc subanalysis using extended data of the EARNEST-PVI trial, a prospective multicenter randomized trial comparing PVI-alone and PVI-plus (i.e., PVI with added catheter ablation) arms. We divided 492 patients into 4 groups according to baseline rhythm and the location of AF triggers before PVI: Group A (n=22), sinus rhythm with pulmonary vein (PV)-specific AF triggers (defined as reproducible AF initiation from PVs only); Group B (n=211), AF with PV-specific AF triggers; Group C (n=94), sinus rhythm with no PV-specific AF trigger; Group D (n=165), AF with no PV-specific AF trigger. Among the 4 groups, only in Group D (AF at baseline and no PV-specific AF triggers) was arrhythmia-free survival significantly lower in the PVI-alone than PVI-plus arm (P=0.032; hazard ratio 1.68; 95% confidence interval 1.04-2.70). CONCLUSIONS: Patients with sinus rhythm or PV-specific AF triggers did not receive any benefit from substrate ablation, whereas patients with AF and no PV-specific AF trigger benefited from substrate ablation.

Cryo-EM structure of P-glycoprotein bound to triple elacridar inhibitor molecules.

P-glycoprotein (P-gp) is an ATP-binding cassette transporter known for its roles in expelling xenobiotic compounds from cells and contributing to cellular drug resistance through multidrug efflux. This mechanism is particularly problematic in cancer cells, where it diminishes the therapeutic efficacy of anticancer drugs. P-gp inhibitors, such as elacridar, have been developed to circumvent the decrease in drug efficacy due to P-gp efflux. An earlier study reported the cryo-EM structure of human P-gp-Fab (MRK-16) complex bound by two elacridar molecules, at a resolution of 3.6 Å. In this study, we have obtained a higher resolution (2.5 Å) structure of the P-gp- Fab (UIC2) complex bound by three elacridar molecules. This finding, which exposes a larger space for compound-binding sites than previously acknowledged, has significant implications for the development of more selective inhibitors and enhances our understanding of the compound recognition mechanism of P-gp.

The prognostic impact of follow-up cardiac metaiodobenzylguanidine imaging on left ventricular trajectory after acute decompensation.

AIMS: Cardiac metaiodobenzylguanidine (MIBG) imaging provides prognostic information in patients with heart failure (HF). Recently, the trajectory of left ventricular ejection fraction (LVEF) has been a focus in patients with reduced LVEF admitted for acute decompensated HF (ADHF). We sought to investigate the prognostic value of follow-up cardiac MIBG imaging in ADHF patients with reduced LVEF in relation to LVEF trajectory. METHODS AND RESULTS: We prospectively studied 145 ADHF patients with a reduced LVEF of <40%. The cardiac MIBG heart-to-mediastinum ratio (the late HMR) was measured on the delayed image at discharge and at the 6-month follow-up (6FUP). At 6 months after discharge, 54 (37%) patients had complete recovery of LVEF ≥50% (HFcorEF), and 43 (30%) patients had partial recovery of LVEF 40-50% (HFparEF), while the remaining 48 (33%) patients had no functional recovery of LVEF (HFnorEF). The late HMR at the 6FUP in HFcorEF patients was significantly greater than that in HFparEF and HFnorEF patients. During a follow-up period of 4.3 ± 2.6 years, 43 patients had cardiac events, defined as a composite of readmission for worsening HF and cardiac death. Patients with a lower late HMR at the 6FUP had a greater risk of cardiac events than those with a higher late HMR at the 6FUP in the group with recovered LVEF, especially HFparEF, which was not observed in the HFnorEF subgroup. CONCLUSION: Follow-up MIBG imaging after discharge could provide additional prognostic information in ADHF patients with recovered left ventricular function.

Duration of atrial fibrillation persistence: Implications for recurrence risk after catheter ablation and efficacy of additional substrate ablation.

BACKGROUND: The optimal duration of atrial fibrillation (AF) persistence for predicting poor outcomes after catheter ablation of long-standing AF (LsAF) and the best ablation strategy for these patients remain unclear. OBJECTIVE: We aimed to assess the impact of the duration of AF persistence on outcomes after catheter ablation of AF. METHODS: We analyzed the Efficacy of Pulmonary Vein Isolation Alone in Patients with Persistent Atrial Fibrillation (EARNEST-PVI) trial data comparing pulmonary vein isolation (PVI) alone (PVI-alone) with additional linear ablation or defragmentation (PVI-plus) in persistent AF (PerAF). Patients who received catheter ablation by contact force-sensing catheter were enrolled in the study. In patients with LsAF, the optimal cutoff duration of AF persistence was evaluated. With use of the threshold, patients with LsAF were divided into 2 groups and compared with PerAF <1 year for arrhythmia-free survival after a 3-month blanking period. RESULTS: The optimal cutoff duration was 2.4 years. Of 458 patients, arrhythmia-free survival rates for LsAF 1-2.4 years were comparable to those of PerAF (hazard ratio [HR], 1.01; 95% CI, 0.67-1.52). However, LsAF >2.4 years had a higher recurrence risk than PerAF (HR, 2.22; 95% CI, 1.42-3.47). In LsAF >2.4 years, the PVI-plus strategy showed advantages over the PVI-alone strategy (HR, 0.36; 95% CI, 0.14-0.89). However, the interaction effect between LsAF 1-2.4 years and LsAF >2.4 years did not reach statistical significance (P = .116). CONCLUSION: Whereas LsAF 1-2.4 years has similar outcomes to those of PerAF, LsAF >2.4 years was linked to higher arrhythmia recurrence risks. For LsAF >2.4 years, the PVI-plus strategy showed a potential to be superior to the PVI-alone strategy.

Uplift modeling to identify patients who require extensive catheter ablation procedures among patients with persistent atrial fibrillation.

Identifying patients who would benefit from extensive catheter ablation along with pulmonary vein isolation (PVI) among those with persistent atrial fibrillation (AF) has been a subject of controversy. The objective of this study was to apply uplift modeling, a machine learning method for analyzing individual causal effect, to identify such patients in the EARNEST-PVI trial, a randomized trial in patients with persistent AF. We developed 16 uplift models using different machine learning algorithms, and determined that the best performing model was adaptive boosting using Qini coefficients. The optimal uplift score threshold was 0.0124. Among patients with an uplift score ≥ 0.0124, those who underwent extensive catheter ablation (PVI-plus) showed a significantly lower recurrence rate of AF compared to those who received only PVI (PVI-alone) (HR 0.40; 95% CI 0.19-0.84; P-value = 0.015). In contrast, among patients with an uplift score < 0.0124, recurrence of AF did not significantly differ between PVI-plus and PVI-alone (HR 1.17; 95% CI 0.57-2.39; P-value = 0.661). By employing uplift modeling, we could effectively identify a subset of patients with persistent AF who would benefit from PVI-plus. This model could be valuable in stratifying patients with persistent AF who need extensive catheter ablation before the procedure.

The clinical relevance of mild cognitive impairment in acute heart failure: A comparison with cognitive impairment.

BACKGROUND: Although mild cognitive impairment (MCI) has received much attention as a precursor of dementia, its prognostic role has not been fully clarified in patients with heart failure (HF). METHODS AND RESULTS: We studied 274 patients admitted for acute decompensated HF. Cognitive function was evaluated using Mini Mental State Examination (MMSE). According to the previous definition, MMSE of 0-23, 24-27, and 28-30 were classified as CI (n = 132), MCI (n = 81), and normal cognitive function (n = 61). The primary endpoint was cardiac events, defined as the composite of unplanned HF hospitalization and cardiovascular mortality. During a mean follow-up period of 4.9 ±â€¯3.1 years, 145 patients experienced cardiac events. Multivariable logistic regression analysis showed that hypertension (p = 0.043), low cardiac index (p = 0.022), and low serum albumin level (p = 0.041) had a significant association with cognitive abnormalities. Both CI and MCI were significantly associated with cardiac events after Cox multivariable adjustment [CI: p = 0.001, adjusted HR 2.66 (1.48-4.77); MCI: p = 0.025, adjusted HR 1.90 (1.09-3.31), normal cognitive function group: reference]. Patients with MCI had a significantly higher risk of unplanned HF hospitalization [p = 0.033, adjusted HR 1.91 (1.05-3.47)], but not all-cause mortality (p = 0.533) or cardiovascular mortality (p = 0.920), while CI was significantly associated with all-cause mortality (p = 0.025) and cardiovascular mortality (p = 0.036). CONCLUSION: Even MCI had a significant risk of cardiac events in patients with acute decompensated HF. This risk was mainly derived from unplanned HF hospitalization.

A Rare Case of a Common Inferior Pulmonary Vein Presumed to Be a Remnant of the Common Pulmonary Vein.

A 74-year-old woman with an implanted physiological DDD pacemaker visited our department complaining of palpitations due to atrial fibrillation (AF). Catheter ablation therapy for AF was scheduled. Preoperative multidetector computed tomography showed that the inferior pulmonary vein (PV) was a common trunk, and the left and right superior PVs branched from the center of the left atrial roof. In addition, mapping of the left atrium before AF ablation revealed no potential in either the inferior PV or common trunk. We performed left and right superior PV and posterior wall isolation. After ablation, AF was not observed on pacemaker recordings.

Human leukocyte antigen-DQ risk heterodimeric haplotypes of left ventricular dysfunction in cardiac sarcoidosis: an autoimmune view of its role.

Cardiac sarcoidosis (CS) is the scarring of heart muscles by autoimmunity, leading to heart abnormalities and patients with sarcoidosis with cardiac involvements have poor prognoses. Due to the small number of patients, it is difficult to stratify all patients of CS by human leukocyte antigen (HLA) analysis. We focused on the structure of antigen-recognizing pockets in heterodimeric HLA-class II, in addition to DNA sequences, and extracted high-affinity combinations of antigenic epitopes from candidate autoantigen proteins and HLA. Four HLA heterodimer-haplotypes (DQA1*05:03/05:05/05:06/05:08-DQB1*03:01) were identified in 10 of 68 cases. Nine of the 10 patients had low left ventricular ejection fraction (< 50%). Fourteen amino-acid sequences constituting four HLA anchor pockets encoded by the HLA haplotypes were all common, suggesting DQA1*05:0X-DQB1*03:01 exhibit one group of heterodimeric haplotypes. The heterodimeric haplotypes recognized eight epitopes from different proteins. Assuming that autoimmune mechanisms might be activated by molecular mimicry, we searched for bacterial species having peptide sequences homologous to the eight epitopes. Within the peptide epitopes form the SLC25A4 and DSG2, high-homology sequences were found in Cutibacterium acnes and Mycobacterium tuberculosis, respectively. In this study, we detected the risk heterodimeric haplotypes of ventricular dysfunction in CS by searching for high-affinity HLA-class II and antigenic epitopes from candidate cardiac proteins.

The two-step cargo recognition mechanism of heterotrimeric kinesin.

Kinesin-driven intracellular transport is essential for various cell biological events and thus plays a crucial role in many pathological processes. However, little is known about the molecular basis of the specific and dynamic cargo-binding mechanism of kinesins. Here, an integrated structural analysis of the KIF3/KAP3 and KIF3/KAP3-APC complexes unveils the mechanism by which KIF3/KAP3 can dynamically grasp APC in a two-step manner, which suggests kinesin-cargo recognition dynamics composed of cargo loading, locking, and release. Our finding is the first demonstration of the two-step cargo recognition and stabilization mechanism of kinesins, which provides novel insights into the intracellular trafficking machinery.

Long-Term Impact of Additional Ablation After Pulmonary Vein Isolation: Results From EARNEST-PVI Trial.

Background An optimal strategy for left atrial ablation in addition to pulmonary vein isolation (PVI) in patients with persistent atrial fibrillation (AF) has not been determined. Methods and Results We conducted an extended follow-up of the multicenter randomized controlled EARNEST-PVI (Efficacy of Pulmonary Vein Isolation Alone in Patients With Persistent Atrial Fibrillation) trial, which compared 12-month rhythm outcomes in patients with persistent AF between patients randomized to a PVI-alone strategy (n=248) or PVI-plus strategy (n=248; PVI followed by left atrial additional ablation, including linear ablation or ablation targeting areas with complex fractionated electrograms). The present study extended the follow-up period to 3 years after enrollment. Outcomes were compared not only between randomly allocated groups but also between on-treatment groups categorized by actually created ablation lesions. Recurrence rate of AF or atrial tachycardia (AT) was lower in the randomly allocated to PVI-plus group than the PVI-alone group (29.0% versus 37.5%, P=0.036). On-treatment analysis revealed that patients with PVI+linear ablation (n=205) demonstrated a lower AF/AT recurrence rate than those with PVI only (26.3% versus 37.8%, P=0.007). In contrast, patients with PVI+complex fractionated electrograms ablation (n=37) had an AF/AT recurrence rate comparable to that of patients with PVI only (40.5% versus 37.8%, P=0.76). At second ablation in 126 patients with AF/AT recurrence, ATs excluding common atrial flutter were more frequent in patients with PVI+linear ablation than in those with PVI only (32.6% versus 5.7%, P<0.0001). Conclusions Left atrial ablation in addition to PVI was efficacious during 3-year follow-up. Linear ablation was superior to other ablation strategies but may increase iatrogenic ATs. Registration URL: http://www.umin.ac.jp/ctr/index-j.htm; Unique identifier: UMIN000019449.

X-ray and Electron Diffraction Observations of Steric Zipper Interactions in Metal-Induced Peptide Cross-ß Nanostructures.

The steric zipper is a common hydrophobic packing structure of peptide side chains that forms between two adjacent ß-sheet layers in amyloid and related fibrils. Although previous studies have revealed that peptide fragments derived from native protein sequences exhibit steric zipper structures, their de novo designs have rarely been studied. Herein, steric zipper structures were artificially constructed in the crystalline state by metal-induced folding and assembly of tetrapeptide fragments Boc-3pa-X1-3pa-X2-OMe (3pa: ß-(3-pyridyl)-l-alanine; X1 and X2: hydrophobic amino acids). Crystallographic studies revealed two types of packing structures, interdigitation and hydrophobic contact, that result in a class 1 steric zipper geometry when the X1 and X2 residues contain alkyl side chains. Furthermore, a class 3 steric zipper geometry was also observed for the first time among any reported steric zippers when using tetrapeptide fragments with (X1, X2) = (Thr, Thr) and (Phe, Leu). The system could also be extended to a knob-hole-type zipper using a pentapeptide sequence.

Effect of Alkali Metal Cations on the Unilamellar Vesicle of a Squalene Bearing 18-Crown-6.

Squalene bearing 18-crown-6 was synthesized and formed unilamellar vesicles with a membrane thickness of about 6 nm and a diameter of about 0.32 µm. In the wake of the recognition of alkali metal cations, squalene unilamellar vesicles become larger as multilamellar vesicles or smaller while maintaining unilamellar vesicles depending on cations.

Uphill energy transfer mechanism for photosynthesis in an Antarctic alga.

Prasiola crispa, an aerial green alga, forms layered colonies under the severe terrestrial conditions of Antarctica. Since only far-red light is available at a deep layer of the colony, P. crispa has evolved a molecular system for photosystem II (PSII) excitation using far-red light with uphill energy transfer. However, the molecular basis underlying this system remains elusive. Here, we purified a light-harvesting chlorophyll (Chl)-binding protein complex from P. crispa (Pc-frLHC) that excites PSII with far-red light and revealed its ring-shaped structure with undecameric 11-fold symmetry at 3.13 Šresolution. The primary structure suggests that Pc-frLHC evolved from LHCI rather than LHCII. The circular arrangement of the Pc-frLHC subunits is unique among eukaryote LHCs and forms unprecedented Chl pentamers at every subunit‒subunit interface near the excitation energy exit sites. The Chl pentamers probably contribute to far-red light absorption. Pc-frLHC's unique Chl arrangement likely promotes PSII excitation with entropy-driven uphill excitation energy transfer.

Reversible Assembly of an Artificial Protein Nanocage Using Alkaline Earth Metal Ions.

Protein nanocages are of increasing interest for use as drug capsules, but the encapsulation and release of drug molecules at appropriate times require the reversible association and dissociation of the nanocages. One promising approach to addressing this challenge is the design of metal-dependent associating proteins. Such designed proteins typically have Cys or His residues at the protein surface for connecting the associating proteins through metal-ion coordination. However, Cys and His residues favor interactions with soft and borderline metal ions, such as Au+ and Zn2+, classified by the hard and soft acids and bases concept, restricting the types of metal ions available to drive association. Here, we show the alkaline earth (AE) metal-dependent association of the recently designed artificial protein nanocage TIP60, which is composed of 60-mer fusion proteins. The introduction of a Glu (hard base) mutation to the fusion protein (K67E mutant) prevented the formation of the 60-mer but formed the expected cage structure in the presence of Ca, Sr, or Ba ions (hard acids). Cryogenic electron microscopy (cryo-EM) analysis indicated a Ba ion at the interface of the subunits. Furthermore, we demonstrated the encapsulation and release of single-stranded DNA molecules using this system. Our results provide insights into the design of AE metal-dependent association and dissociation mechanisms for proteins.