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Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The National Medical Research Council (NMRC) Background Additional protection beyond timely reperfusion is still needed as more patients who have survived an acute myocardial infarction (AMI) developed heart failure (HF)1. DJ-1 was recently reported as a cytoprotective protein that preserves mitochondrial complex I activity and subsequently inhibits mitochondrial reactive species (ROS) production2. DJ-1 downregulation dramatically increases the susceptibility to cell death after myocardial infarction in mice. A substantial reduction in total DJ-1 protein levels in left ventricular tissue has been identified in patients at the end-stage of human HF3, suggesting that DJ-1 protects the myocardium against AMI cell death and is essential in the remodelling process post-infarct. Purpose: The translation of cardioprotection to clinical practice has been difficult, and it remains a challenge between the bench and the bedside. Nanotechnology has shown significant improvements in the settings of AMI. Given the protective effect observed with DJ-1, we designed ND-13, a new cell-permeable 13- fragment of the DJ-1 amino acid sequence. Then, we loaded it into nanoparticles (ND-13NPs) to achieve cardioprotective outcomes against IRI. Methods: We tested the efficacy and efficiency of our new ND-13NPs to reduce infarct size in the ex vivo heart perfused IRI model and the in vivo AMI-murine model. Results: Naked ND-13 (20µM) continuously perfused for the first 15 minutes of reperfusion significantly improved LV pressure and systolic function. Afterwards, we tested 60 mg/Kg of the naked peptide injected 5 minutes before reperfusion in the in vivo model. ND-13 reduced 35% of the infarct size (non-treated, 49 ± 6.4% vs. treated, 32 ± 5 %). Fluorescently loaded ND-13NPs were intravenously injected into infarcted mice to assess their distribution in cardiac tissue. The ND-13NPs were abundantly detected in the infarct border and minimally detected in the remote myocardium. 20mg/Kg of ND-13 loaded into NPs reduced 45% the infarct size compared with 60mg/Kg and 20mg/Kg of naked ND-13 (27 ± 6% vs. 32 ± 5% and 44 ± 8%, respectively). We demonstrated that NPs improved the delivery and efficacy of ND-13 in the ischemic heart following AMI. We observed a robust antioxidative effect when the infarcted heart was treated with ND-13NPs (90 ± 1.5% vs 39 ± 9%, respectively). The activation of the myocardial reperfusion injury salvage kinase (RISK) and the survivor activating factor enhancement (SAFE) pathway at reperfusion protects the mitochondria against IRI. Therefore, we addressed whether ND-13NPs impact mitochondrial function. Can ND-13 protect beyond a cardioprotective pathway, or has the threshold of protection already been achieved by activating a direct effect on mitochondria? Conclusion: Intravenously injected ND-13NPs selectively accumulated in the infarct area and protects the myocardium from IRI via the ROS-mitochondria effect. This new drug may potentially bridge the gaps between basic and clinical research. [ABSTRACT FROM AUTHOR]

Coronary artery disease (CAD) is the leading cause of death and disability worldwide, and early and successful restoration of myocardial reperfusion following an ischemic event is the most effective strategy to reduce final infarct size and improve clinical outcome. This process can, however, induce further myocardial damage, namely acute myocardial ischemia-reperfusion injury (IRI) and worsen clinical outcome. Therefore, novel therapeutic strategies are required to protect the myocardium against IRI in patients with CAD. In this regard, the endogenous cardioprotective phenomenon of "ischemic conditioning," in which the heart is put into a protected state by subjecting it to one or more brief nonlethal episodes of ischemia and reperfusion, has the potential to attenuate myocardial injury during acute IRI. Intriguingly, the heart can be protected in this manner by applying the "ischemic conditioning" stimulus to an organ or tissue remote from the heart (termed remote ischemic conditioning or RIC). Furthermore, the discovery that RIC can be noninvasively applied using a blood pressure cuff on the upper arm to induce brief episodes of nonlethal ischemia and reperfusion in the forearm has greatly facilitated the translation of RIC into the clinical arena. Several recently published proof-of-concept clinical studies have reported encouraging results with RIC, and large multicenter randomized clinical trials are now underway to investigate whether this simple noninvasive and virtually cost-free intervention has the potential to improve clinical outcomes in patients with CAD. In this review article, we provide an update of recently published and ongoing clinical trials in the field of RIC. [ABSTRACT FROM AUTHOR]

The last 20 years have witnessed dramatic reductions in cardiovascular risk using 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors ("statins") to lower levels of low-density lipoprotein cholesterol (LDL-C). Using this approach one can achieve a reduction in the risk of major cardiovascular events of 21% for every 1 mmol/l (39 mg/dl) decrease in LDL-C. However, despite intensive therapy with high dose "statins" to lower LDL-C levels below 2.6 mmol/l (100 mg/dl), the risk of a major cardiovascular event in patients with established coronary artery disease remains significant at a level approaching an annual risk of 9%, paving the way for new strategies for reducing the residual cardiovascular risk in this patient group. Early epidemiological studies have identified low levels of high-density lipoprotein cholesterol (HDL-C) (<1.0 mmol/l or 40 mg/dl), a common feature of type 2 diabetes mellitus and the metabolic syndrome, to be an independent determinant of increased cardiovascular risk. The beneficial effects of HDL-C on the cardiovascular system have been attributed to its ability to remove cellular cholesterol, as well as its anti-inflammatory, antioxidant and antithrombotic properties, which act in concert to improve endothelial function and inhibit atherosclerosis, thereby reducing cardiovascular risk. As such, raising HDL-C in patients with aggressively lowered LDL-C provides an additional strategy for addressing the residual cardiovascular risk present in these patients groups. Studies suggest that for every 0.03 mmol/l (1.0 mg/dl) increase in HDL-C, cardiovascular risk is reduced by 2-3%. Raising HDL-C can be achieved by both lifestyle changes and pharmacological means, the former of which include smoking cessation, aerobic exercise, weight loss and dietary manipulation. Therapeutic strategies have included niacin, fibrates, thiazolidinediones and bile acid sequestrants. Newly developed pharmacological agents include apolipoprotein A-I mimetics and the cholesteryl ester transfer protein (CETP) inhibitors, JTT-705 and torcetrapib, the latter of which has been recently withdrawn from clinical testing because of serious adverse effects. Emerging experimental studies investigating the complex pathways of HDL metabolism have identified several new targets for raising HDL-C with new pharmaceutical agents currently in development. For the time being, the long-acting formulations of nicotinic acid remain the most effective and best tolerated pharmacological strategy for raising HDL-C in patients already on statin therapy to control LDL-C. Therefore, raising HDL-C represents an important strategy for reducing residual cardiovascular risk in patients already optimally treated with statins, and should lead to further improvements in clinical outcomes in these patient groups. [ABSTRACT FROM AUTHOR]

BACKGROUND: Whether remote ischaemic preconditioning, an intervention in which brief ischaemia of one tissue or organ protects remote organs from a sustained episode of ischaemia, is beneficial for patients undergoing coronary artery bypass graft surgery is unknown. We did a single-blinded randomised controlled study to establish whether remote ischaemic preconditioning reduces myocardial injury in these patients. METHODS: 57 adult patients undergoing elective coronary artery bypass graft surgery were randomly assigned to either a remote ischaemic preconditioning group (n=27) or to a control group (n=30) after induction of anaesthesia. Remote ischaemic preconditioning consisted of three 5-min cycles of right upper limb ischaemia, induced by an automated cuff-inflator placed on the upper arm and inflated to 200 mm Hg, with an intervening 5 min of reperfusion during which the cuff was deflated. Serum troponin-T concentration was measured before surgery and at 6, 12, 24, 48, and 72 h after surgery. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00397163. FINDINGS: Remote ischaemic preconditioning significantly reduced overall serum troponin-T release at 6, 12, 24, and 48 h after surgery. The total area under the curve was reduced by 43%, from 36.12 microg/L (SD 26.08) in the control group to 20.58 microg/L (9.58) in the remote ischaemic preconditioning group (mean difference 15.55 [SD 5.32]; 95% CI 4.88-26.21; p=0.005). INTERPRETATION: We have shown that adult patients undergoing elective coronary artery bypass graft surgery at a single tertiary centre could benefit from remote ischaemic preconditioning, using transient upper limb ischaemia. [ABSTRACT FROM AUTHOR]

Conflicting evidence exists whether diabetic myocardium can be protected by ischemic preconditioning (IPC). The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is important in IPC. However, components of this cascade have been found to be defective in diabetes. We hypothesize that IPC in diabetic hearts depends on intact signaling through the PI3K-Akt pathway to reduce myocardial injury. Isolated perfused Wistar (normal) and Goto-Kakizaki (diabetic) rat hearts were subjected to 1) 35 min of regional ischemia and 120 min of reperfusion with infarct size determined; 2) preconditioning (IPC) using 5 min of global ischemia followed by 10 min of reperfusion performed one, two, or three times before prolonged ischemia; or 3) determination of Akt phosphorylation after stabilization or after one and three cycles of IPC. In Wistar rats, one, two, and three cycles of IPC reduced infarct size 44.7 +/- 3.8% (P < 0.05), 31.4 +/- 4.9% (P < 0.01), and 34.3 +/- 6.1% (P < 0.01), respectively, compared with controls (60.7 +/- 4.5%). However, in diabetic rats only three cycles of IPC significantly reduced infarction to 20.8 +/- 2.6% from 46.6 +/- 5.2% in controls (P < 0.01), commensurate with significant Akt phosphorylation after three cycles of IPC. To protect the diabetic myocardium, it appears necessary to increase the IPC stimulus to achieve the threshold for cardioprotection and a critical level of Akt phosphorylation to mediate myocardial protection. [ABSTRACT FROM AUTHOR]

Background: Ischaemic heart disease is the major cause of morbidity and mortality worldwide. Novel therapeutic strategies are essential to protect the heart from acute ischaemia-reperfusion injury (IRI). It has been reported that inhibiting mitochondrial fission, protects the heart against cardiomyocyte death during acute IRI. We investigated the mitochondrial fission proteins, MiD49 and MiD51, as novel targets for cardioprotection,Methods and Results: Genetic ablation of both MiD49 and MiD51 in HL-1 cardiac cells (a murine atrial-derived cell line), using shRNAs had the following effects: (1) A significant increase in the proportion of cells expressing predominantly elongated mitochondria (81.5±2.9% MiD49+MiD51 knockdown vs. 49.4±4.9% vector control: n=120 cells/group; P<0.05); (2) It led to a delay in time taken to induce mitochondrial permeability transition pore (MPTP) opening (485±61 seconds MiD49+MiD51 knockdown vs. 233±20 seconds vector control: n=120 cells/group; P<0.05); (3) Cell death following simulated IRI was reduced (27.9±3.0% MiD49+MiD51 knockdown vs. 56.7±2.4% vector control: n=300 cells/group; P<0.05). Interestingly, the individual knockdown of either MiD49 or MiD51 alone had no effect on these parameters. Unexpectedly, the over-expression of either MiD49 or MiD51 had similar beneficial effects: (1) The formation of abnormally elongated (hyperfused) mitochondria with a peri-nuclear distribution; (2) It delayed the induction of MPTP opening (339±23 seconds MiD49 over-expression, 336±19 seconds MiD51 over-expression vs. 208±16 seconds vector control: n=120 cells/group; P<0.05); (3) It reduced cell death following simulated IRI (21.5±3.7% MiD49 over-expression, 20.0±3.7% MiD51 over-expression vs. 52.0±4.6% vector control: n=300; P<0.05). The explanation for this apparent beneficial effect with MiD49 or MiD51 over-expression, may be due to Drp1 sequestration to mitochondria, leading to unopposed abnormal mitochondrial elongation (hyperfusion).Conclusions: Here we show that combined knockdown of the newly discovered mitochondrial fission proteins, MiD49 and MiD51, induced mitochondrial elongation, delayed MPTP opening, and protected cells against simulated acute IRI, implicating MiD49 and MiD51 as novel targets for cardioprotection. Unexpectedly, the over-expression of these fission proteins induced a state of abnormal hyperfusion, delayed MPTP opening, and protected cells against simulated acute IRI. [ABSTRACT FROM AUTHOR]

Background: A mitochondrial specific deacetylase, Sirtuin-3 has been reported to regulate oxidative phosphorylation, the activity of Cyclophilin D (a key component of the mitochondrial permeability transition pore, MPTP), and the ROS scavenger MnSOD. We hypothesised that Sirtuin-3 could be a potential therapeutic target for cardio-protection based its ability to prevent MPTP formation and inhibit ROS generation.Methods and Results: In HL-1 cells (a murine cardiac cell line), over-expression of Sirtuin-3 reduced cell death following simulated ischemia-reperfusion injury (assessed by propidium iodide staining). Futhermore, Sirtuin-3 over-expression reduced MPTP formation (assessed by ROS-induced mitochondrial depolarization), and induced mitochondrial fusion (assessed by 3 blinded investigators and the PEG fusion assay). The catalytically inactive mutant form of Sirtuin-3 failed to mediate any of these beneficial effects. To investigate the role of endogenous Sirtuin-3 in the adult heart, Sirtuin-3 (whole body) KO mice and WT littermates were subjected to in vivo cardiac ischemia (30 min) followed by 24 hrs reperfusion. myocardial infarct (MI) size was determined as a percentage of area at risk. Interestingly, no differences in MI size were observed between WT and KO mice under fed conditions. However, overnight fasting (to induce Sirtuin-3 expression and activity) resulted in a smaller MI size in the Sirtuin-3 KO when compared to WT mice.Conclusions: We report that the role of Sirtuin-3 in cardio-protection differs between the HL-1 cardiac cell line and the adult heart. In the HL-1 cell, Sirtuin-3 over-expression had beneficial effects against acute IRI, suggesting that activating Sirtuin-3 in this cell-line may be cardio-protective. In contrast, fasted mice deficient in Sirtuin-3 had smaller MI following IRI, suggesting that inhibiting Sirtuin-3 in the fasted adult heart may be cardio-protective. This finding may have clinical implications in patients who are fasted before surgery. [ABSTRACT FROM AUTHOR]

Despite optimal therapy, the morbidity and mortality of patients presenting with an acute myocardial infarction (MI) remain significant. Extracellular RNA (eRNA), exposed after cell damage, serves as cofactor for coagulation proteases and cytokines thereby promoting their procoagulant and proinflammatory functions in vivo. Following myocardial ischemia/reperfusion (I/R) in mice or I/R induced in the isolated Langendorff heart, increased eRNA levels were found together with cell injury markers. Likewise, eRNA was released from cardiomyocytes under hypoxia and subsequently induced tumor-necrosis-factor-a (TNF-α) liberation by activation of TNF-α converting enzyme (TACE) and provoked cardiomyocyte death. Conversely, TNF-a promoted eRNA release especially under hypoxia, feeding a vicious cell damaging cycle during I/R. Administration of RNase1 or TAPI (TACE-inhibitor) prevented cell death and myocardial infarction. Likewise, RNase1 significantly reduced I/R-mediated energy exhaustion, opening of mitochondrial permeability transition pores (mPTP) as well as oxidative damage in cardiomyocytes. Together, RNase1 as well as inhibition of TACE provide novel therapeutic regimen to interfere with the adverse eRNA-TNF-a interplay and significantly reduce or prevent the pathological outcome of ischemic heart injury. [ABSTRACT FROM PUBLISHER]

Purpose: Novel therapeutic strategies are required to protect the heart against acute ischaemia-reperfusion injury (IRI). Assessing their cardioprotective efficacy requires measurement of infarct size (IS) and the "area-at-risk (AAR). T2-weighted cardiac magnetic resonance imaging (cMR) can delineate the AAR in vivo by detecting areas of oedema following IRI. However, clinical studies suggest that some cardioprotective interventions can reduce the extent of oedema measured by T2-weighted cMR, thereby affecting AAR measurement by this cMR method. We sought to investigate the validity of T2-mapping cMR for in vivo AAR quantification in hearts protected by ischaemic preconditioning (IPC).Methods: B6SV129 mice were subjected to in vivo acute IRI comprising LAD ligation for 30 minutes followed by 72 hours reperfusion. IPC-treated animals were subjected to a standard cardioprotective protocol of 5 minutes ischaemia and 5 minutes reperfusion prior to the main ischaemic insult. Following 3 days reperfusion, mice underwent cMR consisting of late-gadolinium enhancement and T2-mapping for assessment of infarct size and putative AAR, respectively. Immediately following cMR, hearts were excised and subjected to histological staining using triphenyl tetrazolium chloride and Evan's blue for infarct size and AAR, respectively. Assessment of cMR quantifications was performed by comparison with equivalent histological staining of left ventricular myocardial slices for infarct size (IS/LV%) and AAR (AAR/LV%).Results: cMR T2-maps of the myocardium in control and IPC-treated mice showed clearly defined regions of elevated T2-signal in spatially distinct regions of the myocardium. Comparison of T2-maps and histological staining for each slice demonstrated good agreement in the spatial localisation of AAR judged by T2-cMR and histology. Quantification of AAR from T2-maps showed no significant difference to AAR quantified by ‘gold-standard’ histological staining in control mice (AAR/LV% control: T2-cMR 60.4±2.4; versus histology 64.3±2.5: n=6, p>0.05). Importantly, T2-cMR significantly underestimated AAR in in IPC treated mice (AAR/LV% IPC: T2-cMR 46.0±13.2; versus histology 59.8±7.8: n=10, *p<0.05).Conclusions: Although the T2-mapping cMR protocol developed in here permits accurate quantification of myocardial area-at-risk in control mice following IRI; crucially, IPC significantly reduced the area of oedema and thus invalidated AAR measurement by T2-cMR in this model in the presence of IPC. Further investigation of the validity of T2-cMR for in vivo AAR assessment is now required for other therapeutic interventions. [ABSTRACT FROM AUTHOR]

BACKGROUND: Myocardial infarction (MI) can be readily assessed using late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR). Inversion recovery (IR) sequences provide the highest contrast between enhanced infarct areas and healthy myocardium. Applying such methods to small animals is challenging due to rapid respiratory and cardiac rates relative to T1 relaxation. METHODS: Here we present a fast and robust protocol for assessing LGE in small animals using a multi-slice IR gradient echo sequence for efficient assessment of LGE. An additional Look-Locker sequence was used to assess the optimum inversion point on an individual basis and to determine most appropriate gating points for both rat and mouse. The technique was applied to two preclinical scenarios: i) an acute (2 hour) reperfused model of MI in rats and ii) mice 2 days following non-reperfused MI. RESULTS: LGE images from all animals revealed clear areas of enhancement allowing for easy volume segmentation. Typical inversion times required to null healthy myocardium in rats were between 300-450 ms equivalent to 2-3 R-waves and ~330 ms in mice, typically 3 R-waves following inversion. Data from rats was also validated against triphenyltetrazolium chloride staining and revealed close agreement for infarct size. CONCLUSION: The LGE protocol presented provides a reliable method for acquiring images of high contrast and quality without excessive scan times, enabling higher throughput in experimental studies requiring reliable assessment of MI. [ABSTRACT FROM AUTHOR]

Background: Cardiocerebral infarction (CCI), which is concomitant with acute myocardial infarction (AMI) and acute ischemic stroke (AIS), is a rare but severe presentation. However, there are few data on CCI, and the treatment options are uncertain. We investigated the characteristics and outcomes of CCI compared with AMI or AIS alone., Methods: We performed a retrospective cohort study of 120 531 patients with AMI and AIS from the national stroke and AMI registries in Singapore. Patients were categorized into AMI only, AIS only, synchronous CCI (same-day), and metachronous CCI (within 1 week). The primary outcome was all-cause mortality, and the secondary outcome was cardiovascular mortality. The mortality risks were compared using Cox regression. Multivariable models were adjusted for baseline demographics, clinical variables, and treatment for AMI or AIS., Results: Of 127 919 patients identified, 120 531 (94.2%) were included; 74 219 (61.6%) patients had AMI only, 44 721 (37.1%) had AIS only, 625 (0.5%) had synchronous CCI, and 966 (0.8%) had metachronous CCI. The mean age was 67.7 (SD, 14.0) years. Synchronous and metachronous CCI had a higher risk of 30-day mortality (synchronous: adjusted HR [aHR], 2.41 [95% CI, 1.77-3.28]; metachronous: aHR, 2.80 [95% CI, 2.11-3.73]) than AMI only and AIS only (synchronous: aHR, 2.90 [95% CI, 1.87-4.51]; metachronous: aHR, 4.36 [95% CI, 3.03-6.27]). The risk of cardiovascular mortality was higher in synchronous and metachronous CCI than AMI (synchronous: aHR, 3.03 [95% CI, 2.15-4.28]; metachronous: aHR, 3.41 [95% CI, 2.50-4.65]) or AIS only (synchronous: aHR, 2.58 [95% CI, 1.52-4.36]; metachronous: aHR, 4.52 [95% CI, 2.95-6.92]). In synchronous CCI, AMI was less likely to be managed with PCI and secondary prevention medications ( P <0.001) compared with AMI only., Conclusions: Synchronous CCI occurred in 1 in 200 cases of AIS and AMI. Synchronous and metachronous CCI had higher mortality than AMI or AIS alone.

Background: Given the rapidly growing burden of cardiovascular disease (CVD) in Asia, this study forecasts the CVD burden and associated risk factors in Asia from 2025 to 2050., Methods: Data from the Global Burden of Disease 2019 study was used to construct regression models predicting prevalence, mortality, and disability-adjusted life years (DALYs) attributed to CVD and risk factors in Asia in the coming decades., Findings: Between 2025 and 2050, crude cardiovascular mortality is expected to rise 91.2% despite a 23.0% decrease in the age-standardised cardiovascular mortality rate (ASMR). Ischaemic heart disease (115 deaths per 100,000 population) and stroke (63 deaths per 100,000 population) will remain leading drivers of ASMR in 2050. Central Asia will have the highest ASMR (676 deaths per 100,000 population), more than three-fold that of Asia overall (186 deaths per 100,000 population), while high-income Asia sub-regions will incur an ASMR of 22 deaths per 100,000 in 2050. High systolic blood pressure will contribute the highest ASMR throughout Asia (105 deaths per 100,000 population), except in Central Asia where high fasting plasma glucose will dominate (546 deaths per 100,000 population)., Interpretation: This forecast forewarns an almost doubling in crude cardiovascular mortality by 2050 in Asia, with marked heterogeneity across sub-regions. Atherosclerotic diseases will continue to dominate, while high systolic blood pressure will be the leading risk factor., Funding: This was supported by the NUHS Seed Fund (NUHSRO/2022/058/RO5+6/Seed-Mar/03), National Medical Research Council Research Training Fellowship (MH 095:003/008-303), National University of Singapore Yong Loo Lin School of Medicine's Junior Academic Fellowship Scheme, NUHS Clinician Scientist Program (NCSP2.0/2024/NUHS/NCWS) and the CArdiovascular DiseasE National Collaborative Enterprise (CADENCE) National Clinical Translational Program (MOH-001277-01)., Competing Interests: S.T has received research grant support from Janssen, Merck, American Heart Association (USA) and the National Medical Research Council (Singapore). She has received speaker and/or consulting fees from AstraZeneca, Eli Lilly, Boehringer-Ingelheim, Merck, Novo Nordisk, Abbott, Medtronic, Dexcom, MIMS, Janssen, Novartis and Sanofi. She is also a shareholder of a health technology company (NOVI Health). D.J.H. has received consultant fees from Faraday Pharmaceuticals Inc. and Boehringer Ingelheim International GmbH, honoraria from Servier, and research funding from Astra Zeneca, Merck Sharp & Dohme Corp and Novonordisk. DJH is supported by the Duke-NUS Signature Research Programme funded by the Ministry of Health, Singapore Ministry of Health's National Medical Research Council under its Singapore Translational Research Investigator Award (MOH-STaR21jun-0003), Centre Grant scheme (NMRC CG21APR1006), and Collaborative Centre Grant scheme (NMRC/CG21APRC006). L.B. was supported by the National Medical Research Council (NMRC) of Singapore Centre Grant (Program for Transforming and Evaluating Outcomes in Cardiometabolic disease [PROTECT], Grant number: CG21APR1006), the National Medical Research Counsil (NMRC) of Singapore Transitional Award Grant (Improving Obstructive Coronary Artery Disease and Cardiovascular Risk Prediction Using Deep Learning Analysis on Coronary Artery Calcium Imaging, Grant number: TA21nov-0001), and the National Medical Research Council (NMRC) of Singapore Collaborative Centre Grant (CCG) (IMplementing a PArtnership for Cardiovacular Trials-2 [IMPACT-2] Program, Grant number: NMRC/CG2/001a/2021-NHCS). L.Z is supported by National Medical Research Council under its Clinician-Innovator Award Senior Investigator (CIASI24jan-0001) and Health Service Research Award (MOH-000358), SingHealth Duke-NUS Academic Medical Centre AM strategic fund award (07 FY2023 HTP P2 15-A2) and Industry Alignment Fund Pre-Positioning (IAFPP award) (H20c6a0035). S.L.L. is supported by the National Medical Research Council Transitional Award; she has received research grants from National University Health System, National Kidney Foundation of Singapore and Singapore Heart Foundation. She is an Associate Editor of Resuscitation and Guest Editor of Resuscitation Plus. M.Y.C. receives speaker's fees and research grants from Astra Zeneca, Abbott Technologies and Boston Scientific. N.W.S.C has received research grant support from NUHS Seed Fund (NUHSRO/2022/RO5+6/Seed-Mar/03), National Medical Research Council Research Training Fellowship (MH 095:003/008-303), National University of Singapore Yong Loo Lin School of Medicine's Junior Academic Fellowship Scheme, and the NUHS Clinician Scientist Program (NCSP2.0/2024/NUHS/NCWS)., (© 2024 The Author(s).)

Background: The administration of intravenous cangrelor at reperfusion achieves faster onset of platelet P2Y12 inhibition than oral ticagrelor and has been shown to reduce myocardial infarction (MI) size in the preclinical setting. We hypothesized that the administration of cangrelor at reperfusion will reduce MI size and prevent microvascular obstruction in patients with ST-segment-elevation MI undergoing primary percutaneous coronary intervention., Methods: This was a phase 2, multicenter, randomized, double-blind, placebo-controlled clinical trial conducted between November 2017 to November 2021 in 6 cardiac centers in Singapore. Patients were randomized to receive either cangrelor or placebo initiated before the primary percutaneous coronary intervention procedure on top of oral ticagrelor. The key exclusion criteria included presenting <6 hours of symptom onset; previous MI and stroke or transient ischemic attack; on concomitant oral anticoagulants; and a contraindication for cardiovascular magnetic resonance. The primary efficacy end point was acute MI size by cardiovascular magnetic resonance within the first week expressed as percentage of the left ventricle mass (%LVmass). Microvascular obstruction was identified as areas of dark core of hypoenhancement within areas of late gadolinium enhancement. The primary safety end point was Bleeding Academic Research Consortium-defined major bleeding in the first 48 hours. Continuous variables were compared by Mann-Whitney U test (reported as median [first quartile-third quartile]), and categorical variables were compared by Fisher exact test. A 2-sided P <0.05 was considered statistically significant., Results: Of 209 recruited patients, 164 patients (78%) completed the acute cardiovascular magnetic resonance scan. There were no significant differences in acute MI size (placebo, 14.9% [7.3-22.6] %LVmass versus cangrelor, 16.3 [9.9-24.4] %LVmass; P =0.40) or the incidence (placebo, 48% versus cangrelor, 47%; P =0.99) and extent of microvascular obstruction (placebo, 1.63 [0.60-4.65] %LVmass versus cangrelor, 1.18 [0.53-3.37] %LVmass; P =0.46) between placebo and cangrelor despite a 2-fold decrease in platelet reactivity with cangrelor. There were no Bleeding Academic Research Consortium-defined major bleeding events in either group in the first 48 hours., Conclusions: Cangrelor administered at the time of primary percutaneous coronary intervention did not reduce acute MI size or prevent microvascular obstruction in patients with ST-segment-elevation MI given oral ticagrelor despite a significant reduction of platelet reactivity during the percutaneous coronary intervention procedure., Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03102723., Competing Interests: L.T. is on the Astra Zeneca international advisory board of management of adverse events with the new antibody drug conjugate T-DXd in Asian patients with metastatic breast cancer, Roche Singapore immunotherapy in early stage NSCLC patient journey advisory board. L.T. has received a Philips speaker honorarium in kind and a Siemens Healthineers speaker honorarium. Y.K.K. has received research funding from Amgen, Astra Zeneca, Abbott Vascular, Bayer, Boston Scientific, Shockwave Medical, and Novartis (via institution); consulting fees from Abbott Vascular, Medtronic, Novartis, and Peijia Medical; and speaker fees from Shockwave Medical, Abbott Vascular, Boston Scientific, Medtronic, Alvimedica, Biotronik, Orbus Neich, Amgen, Novartis, Astra Zeneca, Microport, Terumo, and Omnicare. Y.K.K. is also cofounder and owns equity in Trisail, for which OrbusNeich is an investor. D.J.H. has received consultant fees from Faraday Pharmaceuticals Inc and Boehringer Ingelheim International GmbH, honoraria from Servier, and research funding from Astra Zeneca and Merck Sharp & Dohme Corp. C.Y.C. has received speaker fees from Novartis and consultancy fees from Boston Scientific and Philips. The other authors report no conflicts.

Aim: Pathophysiological differences between patients with heart failure with preserved (HFpEF) and reduced (HFrEF) ejection fraction (EF) remain unclear. Therefore we used a phenomics approach, integrating selected proteomics data with patient characteristics and cardiac structural and functional parameters, to get insight into differential pathophysiological mechanisms and identify potential treatment targets., Methods and Results: We report data from a representative subcohort of the prospective Singapore Heart Failure Outcomes and Phenotypes (SHOP), including patients with HFrEF (EF <40%, n = 217), HFpEF (EF ≥50%, n = 213), and age- and sex-matched controls without HF (n = 216). We measured 92 biomarkers using a proximity extension assay and assessed cardiac structure and function in all participants using echocardiography. We used multi-block projection to latent structure analysis to integrate clinical, echocardiographic, and biomarker variables. Candidate biomarker targets were cross-referenced with small-molecule and drug databases. The total cohort had a median age of 65 years (interquartile range 60-71), and 50% were women. Protein profiles strongly discriminated patients with HFrEF (area under the curve [AUC] = 0.89) and HFpEF (AUC = 0.94) from controls. Phenomics analyses identified unique druggable inflammatory markers in HFpEF from the tumour necrosis factor receptor superfamily (TNFRSF), which were positively associated with hypertension, diabetes, and increased posterior and relative wall thickness. In HFrEF, interleukin (IL)-8 and IL-6 were possible targets related to lower EF and worsening renal function., Conclusion: We identified pathophysiological mechanisms related to increased cardiac wall thickness parameters and potentially druggable inflammatory markers from the TNFRSF in HFpEF., (© 2024 The Authors. European Journal of Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Background and Aims: New treatments are needed to prevent neointimal hyperplasia that contributes to post-angioplasty and stent restenosis in patients with coronary artery disease (CAD) and peripheral arterial disease (PAD). We investigated whether modulating mitochondrial function using mitochondrial division inhibitor-1 (Mdivi-1) could reduce post-vascular injury neointimal hyperplasia by metabolic reprogramming of macrophages from a pro-inflammatory to anti-inflammatory phenotype., Methods and Results: In vivo Mdivi-1 treatment of Apoe -/- mice fed a high-fat diet and subjected to carotid-wire injury decreased neointimal hyperplasia by 68%, reduced numbers of plaque vascular smooth muscle cells and pro-inflammatory M1-like macrophages, and decreased plaque inflammation, endothelial activation, and apoptosis, when compared to control. Mdivi-1 treatment of human THP-1 macrophages shifted polarization from a pro-inflammatory M1-like to an anti-inflammatory M2-like phenotype, reduced monocyte chemotaxis and migration to CCL2 and macrophage colony stimulating factor (M-CSF) and decreased secretion of pro-inflammatory mediators. Finally, treatment of pro-inflammatory M1-type-macrophages with Mdivi-1 metabolically reprogrammed them to an anti-inflammatory M2-like phenotype by inhibiting oxidative phosphorylation and attenuating the increase in succinate levels and correcting the decreased levels of arginine and citrulline., Conclusions: We report that treatment with Mdivi-1 inhibits post-vascular injury neointimal hyperplasia by metabolic reprogramming macrophages towards an anti-inflammatory phenotype thereby highlighting the therapeutic potential of Mdivi-1 for preventing neointimal hyperplasia and restenosis following angioplasty and stenting in CAD and PAD patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This study aim to investigate if remote intensive coaching for the first 6 months post-AMI will improve adherence to the twice-a-day antiplatelet medication, ticagrelor. Between July 8, 2015, to March 29, 2019, AMI patients were randomly assigned to remote intensive management (RIM) or standard care (SC). RIM participants underwent 6 months of weekly then two-weekly consultations to review medication side effects and medication adherence coaching by a centralized nurse practitioner team, whereas SC participants received usual cardiologist face-to-face consultations. Adherence to ticagrelor were determined using pill counting and serial platelet reactivity measurements for 12 months. A total of 149 (49.5%) of participants were randomized to RIM and 152 (50.5%) to SC. Adherence to ticagrelor was similar between RIM and SC group at 1 month (94.4 ± 0.7% vs. 93.6±14.7%, p = 0.537), 6 months (91.0±14.6% vs. 90.6±14.8%, p = 0.832) and 12 months (87.4±17.0% vs. 89.8±12.5%, p = 0.688). There was also no significant difference in platelet reactivity between the RIM and SC groups at 1 month (251AU*min [212-328] vs. 267AU*min [208-351], p = 0.399), 6 months (239AU*min [165-308] vs. 235AU*min [171-346], p = 0.610) and 12 months (249AU*min [177-432] vs. 259AU*min [182-360], p = 0.678). Sensitivity analysis did not demonstrate any association of ticagrelor adherence with bleeding events and major adverse cardiovascular events. RIM, comprising 6 months of intensive coaching by nurse practitioners, did not improve adherence to the twice-a-day medication ticagrelor compared with SC among patients with AMI. A gradual decline in ticagrelor adherence over 12 months was observed despite 6 months of intensive coaching., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

There remains an unmet need to identify novel therapeutic strategies capable of protecting the myocardium against the detrimental effects of acute ischemia-reperfusion injury (IRI), to reduce myocardial infarct (MI) size and prevent the onset of heart failure (HF) following acute myocardial infarction (AMI). In this regard, perturbations in mitochondrial morphology with an imbalance in mitochondrial fusion and fission can disrupt mitochondrial metabolism, calcium homeostasis, and reactive oxygen species production, factors which are all known to be critical determinants of cardiomyocyte death following acute myocardial IRI. As such, therapeutic approaches directed at preserving the morphology and functionality of mitochondria may provide an important strategy for cardioprotection. In this article, we provide an overview of the alterations in mitochondrial morphology which occur in response to acute myocardial IRI, and highlight the emerging therapeutic strategies for targeting mitochondrial shape to preserve mitochondrial function which have the future therapeutic potential to improve health outcomes in patients presenting with AMI., (© 2023. The Author(s).)

The present review summarizes the beneficial and detrimental roles of reactive oxygen species in myocardial ischemia/reperfusion injury and cardioprotection. In the first part, the continued need for cardioprotection beyond that by rapid reperfusion of acute myocardial infarction is emphasized. Then, pathomechanisms of myocardial ischemia/reperfusion to the myocardium and the coronary circulation and the different modes of cell death in myocardial infarction are characterized. Different mechanical and pharmacological interventions to protect the ischemic/reperfused myocardium in elective percutaneous coronary interventions and coronary artery bypass grafting, in acute myocardial infarction and in cardiotoxicity from cancer therapy are detailed. The second part keeps the focus on ROS providing a comprehensive overview of molecular and cellular mechanisms involved in ischemia/reperfusion injury. Starting from mitochondria as the main sources and targets of ROS in ischemic/reperfused myocardium, a complex network of cellular and extracellular processes is discussed, including relationships with Ca 2+ homeostasis, thiol group redox balance, hydrogen sulfide modulation, cross-talk with NAPDH oxidases, exosomes, cytokines and growth factors. While mechanistic insights are needed to improve our current therapeutic approaches, advancements in knowledge of ROS-mediated processes indicate that detrimental facets of oxidative stress are opposed by ROS requirement for physiological and protective reactions. This inevitable contrast is likely to underlie unsuccessful clinical trials and limits the development of novel cardioprotective interventions simply based upon ROS removal., Competing Interests: Declaration of competing interest PF is the founder and CEO of Pharmahungary Group, a group of R&D companies. AMS is an adviser to Forcefield Therapeutics and CYTE – Global Network for Clinical Research and sits on the Board of Heqet Therapeutics. CM served as an advisor to Amgen, Boehringer Ingelheim, Bristol Myers Squibb, NovoNordisk and Servier and received speaker honoraria from AstraZeneca, Bayer, Bristol Myers Squibb, Boehringer Ingelheim, Berlin Chemie, Novartis and NovoNordisk. No other author had an interest to declare., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background Patients with prior cancer are at increased risk of acute coronary syndrome (ACS) with poorer post-ACS outcomes. We aimed to ascertain if the Global Registry of Acute Coronary Events (GRACE) score accurately predicts mortality risk among patients with ACS and prior cancer. Methods We linked nationwide ACS and cancer registries from 2007 to 2018 in Singapore. A total of 24,529 eligible patients had in-hospital and 1-year all-cause mortality risk calculated using the GRACE score (2471 prior cancer; 22,058 no cancer). Results Patients with prior cancer had two-fold higher all-cause mortality compared to patients without cancer (in-hospital: 22.8% versus 10.3%, p < 0.001; 1-year: 49.0% vs. 18.7%, p < 0.001). Cardiovascular mortality did not differ between groups (in-hospital: 5.2% vs. 4.8%, p = 0.346; 1-year: 6.9% vs. 6.1%, p = 0.12). The area under the receiver operating characteristic curve of the GRACE score for prediction of all-cause mortality was less for prior cancer (in-hospital: 0.64 vs. 0.80, p < 0.001; 1-year: 0.66 vs. 0.83, p < 0.001). Among patients with prior cancer and a high-risk GRACE score > 140, in-hospital revascularization was not associated with lower cardiovascular mortality than without in-hospital revascularization (6.7% vs. 7.6%, p = 0.50). Conclusions The GRACE score performs poorly in risk stratification of patients with prior cancer and ACS.

Remote ischemic conditioning (RIC) has been investigated as a promising, safe, and well-tolerated nonpharmacological therapy for cardio-cerebrovascular disease over the past 3 decades; variable results have been found when it is used in cerebrovascular versus cardiovascular disease. For patients with cardiovascular disease, milestone studies suggest that the roles of RIC may be limited. Recently, however, 2 large trials investigating RIC in patients with cerebrovascular disease found promising results, which may reignite the field's research prospects after its setbacks in the cardiovascular field. This perspectives article highlights several important clinical trials of RIC in the cardio-cerebrovascular disease and describes the many challenges of RIC in clinical translation. Finally, based on the available evidence, several promising research directions such as chronic RIC, early initiation in target population, improvement of compliance, better understanding of dosing, and identification of specific biomarkers are proposed and should be investigated before RIC can become applied into clinical practice for patient benefit., Competing Interests: Disclosures Dr Hess disclosed a patent for use of MAPC in neurological diseases, which has been issued and now is licensed by Athersys, Inc. The other authors report no conflicts.

Peripheral artery disease (PAD) is a common and debilitating condition characterized by the narrowing of the limb arteries, primarily due to atherosclerosis. Non-invasive multi-modality imaging approaches using computed tomography (CT), magnetic resonance imaging (MRI), and nuclear imaging have emerged as valuable tools for assessing PAD atheromatous plaques and vessel walls. This review provides an overview of these different imaging techniques, their advantages, limitations, and recent advancements. In addition, this review highlights the importance of molecular markers, including those related to inflammation, endothelial dysfunction, and oxidative stress, in PAD pathophysiology. The potential of integrating molecular and imaging markers for an improved understanding of PAD is also discussed. Despite the promise of this integrative approach, there remain several challenges, including technical limitations in imaging modalities and the need for novel molecular marker discovery and validation. Addressing these challenges and embracing future directions in the field will be essential for maximizing the potential of molecular and imaging markers for improving PAD patient outcomes.

Introduction: Obesity is an important risk factor for acute myocardial infarction (AMI), but the interplay between metabolic health and obesity on AMI mortality has been controversial. In this study, we aimed to elucidate the risk of short- and long-term all-cause mortality by obesity and metabolic health in AMI patients using data from a multi-ethnic national AMI registry., Methods: A total of 73,382 AMI patients from the national Singapore Myocardial Infarction Registry (SMIR) were included. These patients were classified into four groups based on the presence or absence of metabolic diseases, diabetes mellitus, hyperlipidaemia, and hypertension, and obesity: (1) metabolically-healthy-normal-weight (MHN); (2) metabolically-healthy-obese (MHO); (3) metabolically-unhealthy-normal-weight (MUN); and (4) metabolically-unhealthy-obese (MUO)., Results: MHO patients had reduced unadjusted risk of all-cause in-hospital, 30-day, 1-year, 2-year, and 5-year mortality following the initial MI event. However, after adjusting for potential confounders, the protective effect from MHO on post-AMI mortality was lost. Furthermore, there was no reduced risk of recurrent MI or stroke within 1-year from onset of AMI by the MHO status. However, the risk of 1-year mortality was higher in female and Malay AMI patients with MHO compared to MHN even after adjusting for confounders., Conclusion: In AMI patients with or without metabolic diseases, the presence of obesity did not affect mortality. The exception to this finding were female and Malay MHO who had worse long-term AMI mortality outcomes when compared to MHN suggesting that the presence of obesity in female and Malay patients may confer worsened outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Sia, Ko, Zheng, Ho, Foo, Foo, Lim, Liew, Chai, Yeo, Yip, Chua, Chan, Tan, Bulluck and Hausenloy.)

Objective: Cut-offs for high-sensitivity troponin (hs-Tn) elevations to define prognostically significant peri-operative myocardial injury (PMI) in cardiac surgery is not well-established. We evaluated the associations between peri-operative high-sensitivity troponin T (hs-TnT) elevations and 1-year all-cause mortality in patients undergoing cardiac surgery., Methods: The prognostic significance of baseline hs-TnT and various thresholds for post-operative hs-TnT elevation at different time-points on 1-year all-cause mortality following cardiac surgery were assessed after adjusting for baseline hs-TnT and EuroSCORE in a post-hoc analysis of the ERICCA trial., Results: 1206 patients met the inclusion criteria. Baseline elevation in hs-TnT >x1 99th percentile upper reference limit (URL) was significantly associated with 1-year all-cause mortality (adjusted hazard ratio 1.90, 95% confidence interval 1.15-3.13). In the subgroup with normal baseline hs-TnT ( n = 517), elevation in hs-TnT at all post-operative time points was associated with higher 1-year mortality, reaching statistical significance for elevations above: ≥100 × URL at 6 h; ≥50 × URL at 12 and 24 h; ≥35 × URL at 48 h; and ≥30 × URL at 72 h post-surgery. Elevation in hs-TnT at 24 h ≥ 50 × URL had the optimal sensitivity and specificity (73% and 75% respectively). When the whole cohort of patients was analysed, including those with abnormal baseline hs-TnT (up to 10 × URL), the same threshold had optimal sensitivity and specificity (66% and 70%)., Conclusions: Both baseline and post-operative hs-TnT elevations are independently associated with 1-year all-cause mortality in patients undergoing cardiac surgery. The optimal threshold to define a prognostically significant PMI in our study was ≥50 × URL elevation in hs-TnT at 24 h., Competing Interests: The authors declare no conflict of interest.

Ischemic heart disease (IHD) is the leading cause of heart failure (HF) and is a significant cause of morbidity and mortality globally. An ischemic event induces cardiomyocyte death, and the ability for the adult heart to repair itself is challenged by the limited proliferative capacity of resident cardiomyocytes. Intriguingly, changes in metabolic substrate utilisation at birth coincide with the terminal differentiation and reduced proliferation of cardiomyocytes, which argues for a role of cardiac metabolism in heart regeneration. As such, strategies aimed at modulating this metabolism-proliferation axis could, in theory, promote heart regeneration in the setting of IHD. However, the lack of mechanistic understanding of these cellular processes has made it challenging to develop therapeutic modalities that can effectively promote regeneration. Here, we review the role of metabolic substrates and mitochondria in heart regeneration, and discuss potential targets aimed at promoting cardiomyocyte cell cycle re-entry. While advances in cardiovascular therapies have reduced IHD-related deaths, this has resulted in a substantial increase in HF cases. A comprehensive understanding of the interplay between cardiac metabolism and heart regeneration could facilitate the discovery of novel therapeutic targets to repair the damaged heart and reduce risk of HF in patients with IHD.

Background: Exploratory studies have shown that remote ischemic conditioning (RIC) has the potential to lower blood pressure (BP). We investigated whether chronic RIC reduces BP for hypertension., Methods: This is a multicenter, randomized, double-blind, parallel-controlled trial. Patients with an office BP of 130/80 to 160/100 mm Hg and a 24-hour average BP ≥125/75 mm Hg not on antihypertensive medications were recruited. After a 1-week compliance screening phase, they were randomly assigned in a 1:1 ratio to receive RIC or sham RIC twice daily for 4 weeks. The primary efficacy outcome was the change in 24-hour average systolic BP from baseline to 4 weeks. Safety events were assessed over the study period., Results: Ninety-five participants were randomly allocated to the RIC (n=49) and sham RIC (n=46) groups. In the intention-to-treat analysis, the reduction in 24-hour average systolic BP was greater in the RIC group than the sham RIC group (-4.6±9.5 versus -0.9±6.8 mm Hg; baseline-adjusted between-group mean difference: -3.6 mm Hg [95% CI, -6.9 to -0.3 mm Hg]; adjusted P =0.035). The per-protocol analysis showed that 24-hour average systolic BP reduced -5.9±8.6 mm Hg in the RIC group and -0.7±6.7 mm Hg in the sham RIC group (baseline-adjusted between-group mean difference: -5.2 mm Hg [95% CI, -8.5 to -1.9 mm Hg]; adjusted P =0.002). No major adverse events were reported in both groups., Conclusions: RIC is safe in patients with mild hypertension and may lower BP in the absence of antihypertensive medications. However, the effects of RIC on clinical outcomes in these patients require further investigation., Registration: URL: https://www., Clinicaltrials: gov; Unique identifier: NCT04915313., Competing Interests: Disclosures The patent of the RIC device (patent no. ZL200820123637.X) used in this study belongs to Xuanwu Hospital Capital Medical University.

Patients with Type 2 diabetes mellitus (T2DM) frequently exhibit a distinctive cardiac phenotype known as diabetic cardiomyopathy. Cardiac complications associated with T2DM include cardiac inflammation, hypertrophy, fibrosis, and diastolic dysfunction in the early stages of the disease, which can progress to systolic dysfunction and heart failure. Effective therapeutic options for diabetic cardiomyopathy are limited and often have conflicting results. The lack of effective treatments for diabetic cardiomyopathy is due in part, to our poor understanding of the disease development and progression, as well as a lack of robust and valid preclinical human models that can accurately recapitulate the pathophysiology of the human heart. In addition to cardiomyocytes, the heart contains a heterogeneous population of non-myocytes including fibroblasts, vascular cells, autonomic neurons, and immune cells. These cardiac non-myocytes play important roles in cardiac homeostasis and disease, yet the effect of hyperglycaemia and hyperlipidaemia on these cell types is often overlooked in preclinical models of diabetic cardiomyopathy. The advent of human-induced pluripotent stem cells provides a new paradigm in which to model diabetic cardiomyopathy as they can be differentiated into all cell types in the human heart. This review will discuss the roles of cardiac non-myocytes and their dynamic intercellular interactions in the pathogenesis of diabetic cardiomyopathy. We will also discuss the use of sodium-glucose cotransporter 2 inhibitors as a therapy for diabetic cardiomyopathy and their known impacts on non-myocytes. These developments will no doubt facilitate the discovery of novel treatment targets for preventing the onset and progression of diabetic cardiomyopathy., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Purposes: The study aimed to optimize diffusion-weighted imaging (DWI) image acquisition and analysis protocols in calf muscles by investigating the effects of different model-fitting methods, image quality, and use of high b-value and constraints on parameters of interest (POIs). The optimized modeling methods were used to select the optimal combinations of b-values, which will allow shorter acquisition time while achieving the same reliability as that obtained using 16 b-values., Methods: Test-retest baseline and high-quality DWI images of ten healthy volunteers were acquired on a 3T MR scanner, using 16 b-values, including a high b-value of 1200 s/mm 2 , and structural T1-weighted images for calf muscle delineation. Three and six different fitting methods were used to derive ADC from monoexponential (ME) model and D d , f p, and D p from intravoxel incoherent motion (IVIM) model, with or without the high b-value. The optimized ME and IVIM models were then used to determine the optimal combinations of b-values, obtainable with the least number of b-values, using the selection criteria of coefficient of variance (CV) ≤10% for all POIs., Results: The find minimum multivariate algorithm was more flexible and yielded smaller fitting errors. The 2-steps fitting method, with fixed D d , performed the best for IVIM model. The inclusion of high b-value reduced outliers, while constraints improved 2-steps fitting only., Conclusions: The optimal numbers of b-values for ME and IVIM models were nine and six b-values respectively. Test-retest reliability analyses showed that only ADC and D d were reliable for calf diffusion evaluation, with CVs of 7.22% and 4.09%., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Derek J Hausenloy reports financial support was provided by National Medical Research Council. Derek J Hausenloy reports financial support was provided by National Research Foundation. Cheryl Pei Ling Lian reports financial support was provided by Singapore Institute of Technology (SIT)., (Copyright © 2023 Elsevier Ltd. All rights reserved.)