Introduction Follistatin-like 3 (FSTL3) is a secreted protein that has been suggested to play an important role in modulating cardiac remodeling and hypertrophy. In this study, we aim to determine whether: 1) FSTL3 is incrementally elevated in patients with HF vs those with other cardiovascular disease (CVD); and 2) increased FSTL3 is associated with 2 or more hospital admissions due to major adverse CV events (MACE) within 1 year. Methods and results We measured circulating levels of FSTL3 using commercially available ELISA (R&D systems) in a total of n=696 patients. FSTL3 levels were compared between: 1) healthy-aging volunteers with no prior major CVD (n=267, age 67±6 years) and 2) patients admitted to cardiology unit for various CVD (n=429, age 66±14 years); among those (n=178, age 68±13 years) had HF. Patients with HF had 2-fold higher FSTL3 levels vs healthy age-matched controls vs those with other CVD (p<0.001). Occurrences of MACE were recorded up to 1 year for patients admitted to cardiology unit. On univariate analyses, patient with 2 or more MACE within 1 year (n=91, 27%) had significant elevated FSTL3 levels (P=0.003), is associated with older age (P<0.005). On multivariate analysis, high FSTL3 levels (P=0.034) is an independent predictor of 2 or more MACE admissions within 1 year after adjusting for age, clinical comorbidities and medications. Conclusions FSTL3 is incrementally increased in patients with HF and is associated with poorer prognosis. Elevated FSTL3 levels is associated with increased risks of cardiac hospital readmissions for patients with multiple CV morbidities. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NSW Ministry of Health EMC Fellowship (Australia)

Background Coronary artery spasm (CAS) is known to occur commonly even in a “pure culture”, but little is known regarding the precipitation of the symptomatic crises which characterize this disorder. There have been recent reports of the occurrence of plaque erosion and intracoronary thrombosis during CAS crises. We have recently shown that (1) the anti-aggregatory effects of nitric oxide (NO) are impaired in CAS patient and (2) release into plasma of the endothelial glycocalyx component syndecan-1 (SD-1) occurs during acute episodes of CAS, suggesting glycocalyx erosion by inflammatory enzyme release. Objective In the current study, we sought to determine whether this phenomenon is accompanied by damage to circulating platelets and whether activation of mast cells may represent a possible precipitant. Methods In patients with acute episodes and chronic phases of CAS (n=10), as well as normal subjects (n=12), plasma concentrations of SD-1, of platelet-derived microparticles (PMPs), of the mast cell enzyme tryptase and of malondialdehyde (MDA), a measure of oxidative stress, were evaluated. Results The results are summarized in the table. Symptomatic crises were associated with substantial elevation of SD-1 concentrations and also those of PMPs and of tryptase, relative to chronic status. However, MDA concentrations did not vary significantly during acute episodes. Impact of acute exacerbation of CAS on markers of glycocalyx shedding (SD-1), platelet activation (PMP counts), mast cell activation (tryptase) and oxidative stress (MDA) Parameter Acute CAS Chronic CAS p* Normal subjects** SD-1 (μg/L) 50.3±3.9 14.3±4.7 <0.0001 12.7±2.4 PMPs (counts) 24200±8100 4800±1010 0.02 10400±2900 Tryptase (μg/L) 4.4±0.4 4.1±0.4 0.03 <12.0 MDA (μM) 3.2±0.2 3.1±0.1 NS 3.09±0.15 *Acute vs. chronic; **provided for reference only. Conclusion (1) The combination of SD-1 release and formation of PMPs suggests that CAS crises reflect both glycocalyx “shedding” and platelet activation/apoptosis, a combination which would facilitate coronary thrombosis. (2) CAS crises are associated with mast cell activation, which may contribute to the above vascular/platelet damage.

Background Coronary artery spasm (CAS) represents a major cause of patient morbidity, with variable clinical response to prophylaxis with calcium antagonists and generally poor symptomatic relief with organic nitrates. The precipitation of CAS with acetylcholine may reflect impaired nitric oxide (NO) release and/or signalling. We have recently demonstrated that platelets from patients with CAS exhibit markedly impaired anti-aggregatory responses to the NO donor sodium nitroprusside (SNP) (“NO resistance”). Purpose In the current experiments we sought to determine whether N-acetylcysteine (NAC), which is known to potentiate haemodynamic responses to organic nitrates, reverses NO resistance in platelets from CAS patients. Methods Patients with CAS were studied during acute (n=11) and chronic (n=24) phases of symptoms. NAC (10 g/24 hours) was infused together with low dose NTG (2.5 μg/min) in patients presenting with acute exacerbations, and platelets were studied ex vivo. In blood samples taken from chronic CAS patients, in vitro studies were performed to evaluate the possible role of H2S release (via cysteine formation) from NAC in putative potentiation of NO effect. Results (1) In acute patients, NTG/NAC infusion resulted in increases in platelet response to SNP (p=0.003); (2) In vitro studies showed that incubation with NAC or the H2S donor NaHS potentiated SNP responses (Figure 1A); (3) Effects of NAC were reversed by co-incubation with aminooxyacetic acid (AOAA) and D, L-propargylglycine (PAG), inhibitors of enzymatic cysteine bioconversion to release H2S (Figure 1B). Figure 1 Conclusion CAS-associated impairment of platelet NO signaling reflects a deficiency of the H2S/NO interaction, and can be reversed using exogenous H2S donors, including NAC.