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Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Medical Research Council (NMRC) Background / Introduction There are currently no treatments for directly improving diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF). Improving myocardial relaxation via manipulating sarcomere function has great potential to unveil novel targets for treating HFpEF. Mavacamten, a small molecule inhibitor of myosin ATPase, has been developed through drug screening as a treatment for hypertrophic cardiomyopathy (HCM), and it is currently being tested in HFpEF patients. Interestingly, emerging evidence suggests that mavacamten may not only modulate contractility but may also modulate myocardial relaxation. Purpose The aim of this study was to investigate whether mavacamten directly modulates sarcomere mechanical properties to improve myocardial relaxation in a mouse HFpEF model. Methods Eight to ten weeks old adult male C57Bl/6 mice were fed with (1) standard diet or (2) high-fat diet (HFD) + L-NAME to induce HFpEF (n=10 mice/group). Cardiac function was assessed by transthoracic two-dimensional echocardiography at baseline and after 15 weeks. Mouse myofibrils (n=10-15 per animals) were obtained from control and HFpEF mice using a triton X-100-based skinning method. The contractile function of the sarcomere with or without the presence of mavacamten were investigated using a myofibril mechanical system. General myofibril mechanical parameters measured included resting and maximal tension (RT, FMAX, mN/mm2), rate constant of tension development (kACT, S-1), duration of linear relaxation phase (tLIN, mSec) and rate constant of linear and exponential phase relaxation (linear and exponential kREL, S-1). Furthermore, force production as a function of the bathing Ca2+ concentration (pCa 4.5-9.0) was measured to determine the Ca2+ sensitivity of the myofibrils. Results As previously reported, C57Bl/6 mice fed with HFD + L-NAME developed cardiac hypertrophy, diastolic dysfunction, exercise intolerance despite having preserved systolic function at 15 weeks. Mechanical analysis showed myofibrils isolated from HFpEF mice has mildly reduced force generation, significantly higher stiffness and elevated Ca2+ sensitivity, compared with control animals. Ex vivo treatment of mavacamten completely normalised Ca2+ sensitivity of the myofibrils from HFpEF animals. Interestingly, mavacamten treatment further accelerated the rate of linear phase relaxation (linear kREL). Conclusion This is the first study to characterise in detail the mechanical properties of myofibrils in a mouse HFpEF model. We demonstrated myosin ATPase inhibition using mavacamten could normalise elevated Ca2+ sensitivity as well as facilitate relaxation kinetics at the sarcomere level in HFpEF. These findings position mavacamten to be a potential therapeutic intervention for improving diastolic function in patients with HFpEF. [ABSTRACT FROM AUTHOR]

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]

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: 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]