Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Background Cardiovascular issues associated with diabetes, such as diabetic cardiomyopathy (DCM), can lead to heart failure. DCM is etiologically related to myocardial inflammation and can stem from a complex interplay of different cell types. Cardiomyocyte as an active mediator of the inflammatory response is an emerging concept with limited mechanistic understanding. Purpose We aimed to address the knowledge gap of cardiomyocyte endoplasmic reticulum (ER) dysfunction-mediated macrophage response and provide functional evidence for the therapeutic feasibility of managing inflammatory paracrine signals in response to diet-induced metabolic stress. Methods In vivo mouse model of high fat high sucrose diet-induced diabetes, cardiomyocyte-specific p21-activated kinase 2 (PAK2) knockout model, echocardiography, histology, 3D imaging, qPCR, co-culture of H9c2 culturing medium and bone-marrow derived macrophages, immunoblotting, macrophage isolation from myocardium, flow cytometry and AAV9-gene therapy. Results In a time-course study, diet-induced diabetic mice demonstrated an association between cardiac ER stress and sustained myocardial inflammation, with a maladaptive shift in myocardial ER stress response over time. Furthermore, as a cardiac ER dysfunction model, mice with cardiac-specific PAK2 deletion exhibited heightened myocardial inflammatory response in diabetes. Using human and mice diabetic heart samples, we show that ER stress-induced CCAAT/enhancer-binding protein homologous protein (CHOP) is a novel transcriptional regulator of high mobility group box-1 (HMGB1). Cardiac stress-induced active release of HMGB1 facilitated M1 macrophage polarization, and aggravated myocardial inflammatory signatures. Therapeutically, sequestering the extracellular HMGB1 using Glycyrrhizin conferred cardioprotection through its anti-inflammatory action. Also, as functional evidence, we showed that un-mitigated cardiac ER response due to PAK2 loss under diabetes may account as a barrier for leveraging the anti-inflammatory potential of Vildagliptin. Conclusion Collectively, we introduce an ER stress-mediated cardiomyocyte-macrophage link, altering the macrophage response in the myocardium, thereby providing insight into therapeutic prospects for diabetes-associated cardiac dysfunction.

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Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Background Heart failure with preserved ejection fraction (HFpEF) is the most predominant form of heart failure (HF) worldwide, one which confers substantial mortality and has no efficacious treatments. Lipid mishandling greatly contributes to the pathophysiology of HFpEF, nevertheless, the key mechanistic features regulating cardiac lipid overload remain poorly understood. EDEM2 plays a crucial role in the ER-associated degradation (ERAD) pathway. Recent studies show that ERAD-associated proteins are implicated in cellular lipid homeostasis, however, the role of EDEM2 is cardiac lipid metabolism remains poorly understood. Here, we investigate the mechanisms by which EDEM2 regulates cardiomyocyte lipid metabolism and cardiac dysfunction in HFpEF. Methods In vivo two-hit disease model of HFpEF, Adeno-associated virus serotype 9 (AAV9)-mediated cardiac-specific gene overexpression, tail-cuff blood pressure recordings, echocardiography, histology and qPCR. Results Under HFpEF inducing conditions, AAV9-Gfp injected mice (as controls) displayed an increased E/A ratio and IVRT both of which are indicative of diastolic dysfunction. Conversely, cardiac-specific EDEM2 overexpression prevented diastolic dysfunction in mice subjected to HFpEF conditions. Systolic function remained unchanged between groups. Interestingly, EDEM2 overexpression reduced myocardial neutral lipid accumulation and prevented severe oxidative stress and cardiac pathological remodeling. More importantly, although genes participating in fatty acid uptake and lipid droplets synthesis in the heart were comparable between AAV9-Gfp and AAV9-EDEM2 groups, those involved in lipolysis and fatty acid oxidation were upregulated in mice with cardiac-specific EDEM2 overexpression. Conclusion Cardiomyocyte-specific EDEM2 overexpression mitigates myocardial steatosis in a two-hit murine model of HFpEF, likely through enhancing fatty acid utilisation in the myocardium.

Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): British Heart Foundation Introduction The understanding of stress-induced cardiac hypertrophy remains vital for preventing and better treatment of heart failure. Studies of the p-21activated kinase (PAK) family describe the cardioprotective roles of PAK1 and PAK2 proteins; however, the function of PAK3 is not known. Unlike the first two, the expression of PAK3 increases in failing hearts, suggesting it has distinct regulatory pathways and functions. Purpose Elucidate the role of PAK3 during the cardiac stress response and its contribution to the development of cardiac hypertrophy and dysfunction. Methods A cardiac-specific PAK3 overexpression mouse model was established by administering an AAV9 vector carrying a PAK3 coding sequence regulated by the Troponin promoter. Acute cardiac stress consisted of two consecutive isoprenaline injections, while the long-term experiment employed subcutaneous minipumps for constant release over two weeks. Cardiac function was assessed by echocardiography, cardiac morphology through histological staining, and protein expression by western blot. For the in vitro experiments, an adenoviral vector was used for overexpression on neonatal rat ventricular myocytes, and starvation was used to induce stress. The tandem mCherry-Gfp-LC3 reporter was used to detect autophagic flux by immunofluorescence and flow cytometry. Cell death was evaluated by measuring lactate dehydrogenase release (LDH) and using the Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay. Results After only two days of isoprenaline-induced stress, cardiac PAK3 overexpression in mice resulted in a reduced fractional shortening and enlarged ventricular chamber diameter. Histological analysis showed a rise in interstitial fibrosis and apoptotic cardiomyocytes. Increasing the exposure time to cardiac stress to two weeks aggravated the alterations detected in the acute model. Western blot revealed an mTOR overactivation, possibly through the disassembly of the TSC1 complex, accompanied by the alteration of autophagic markers. In vitro experiments confirmed that PAK3 overexpression led to basal mTOR and autophagosome formation activation but with an impaired flux. One hour of starvation induced LDH release and cardiomyocyte apoptosis, worsened by PAK3 overexpression. These effects were recovered by the MTOR-independent autophagy inducer ABT-737 in vitro and by the recently produced MSL-7. Conclusion Contrary to the proteins in the family, PAK3 overexpression is damaging for cardiac function. It dysregulates the autophagic process and renders the cardiomyocytes unable to sustain acute stress. The administration of the autophagy inducer MSL-7 improved muscle tolerance to stress and preserved function showing its therapeutic properties in stress-induced cardiac hypertrophy. The rapidity with which PAK3 overexpression induced systolic dysfunction portrays the importance of further studying its function in cardiac health and disease.

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Background In clinics, metabolic stress-induced cardiomyopathy is featured as diastolic/systolic dysfunction, at least partially resulting from impairment of metabolic flexibility in the myocardium, which leads to lipotoxicity and oxidative stress accompanied with cardiomyocytes death. P21-activated kinases (Paks) are a family of serine/threonine kinases, involved in cell survival, proliferation and cytoskeleton remodelling. In spite of the cardioprotective functions of Pak1 and Pak2 in the myocardium, the role of cardiac Pak3 is unexplored. Purpose The study is to investigate the function of cardiac Pak3 on myocardial steatosis in response to diet-induced metabolic stress, which will provide proof-of-concept evidence for the therapeutic potential for alleviating cardiac dysfunction by targeting lipotoxicity in the myocardium. Methods Pak3 overexpression in the myocardium was achieved by tail vein injection of adeno associated virus (AAV9) delivery of troponin T promoter-driven Pak3 on C57BL/6J mice. The mice were fed with high fat high sucrose diet, the metabolic profiles and cardiac function were measured at 4-week intervals. In addition, cardiac morphological and cellular changes were assessed, including cell size, fibrosis formation, cell death, lipid accumulation and lipid peroxidation. In addition, the transcript levels of the genes either participating lipid metabolism or regulating oxidative stress were also assessed by qPCR. Results First, we detected that Pak3 expression and activation was increased in the myocardium from human with obese or diabetes. Consistently, the augmented Pak3 was also detected in the failing heart from C57BL/6J mice, ob/ob and db/db mice. Next, in a time-course study, the mice with AAV9-delivery cardiac Pak3 overexpression were vulnerable to cardiac dysfunction upon diet-induced metabolic stress. Pak3 overexpression did not alter cardiomyocyte size; however, more fibrosis and cell death in the myocardium were detected due to Pak3 enhancement. More importantly, myocardial lipid accumulation and profound lipid peroxidation were determined by immunefluorescent staining of Perilipin 5 (PLIN5) and 4-Hydroxynonenal (4-NHE), respectively. Finally, the abnormal expression of the genes involved in lipid synthesis and fatty acid oxidation was detected in Pak3-overexpressed myocardium under metabolic stress, whereas the levels of anti-oxidative stress genes were decreased. Conclusion Altogether, the study has demonstrated that Pak3 over-activation makes the heart more susceptible to myocardial lipotoxicity, which accelerates cardiac steatosis and dysfunction in the fact of metabolic stress.