Fibronectin is a master organizer of extracellular matrices (ECMs) and promotes the assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its secretion by osteoblasts, chondrocytes, and mesenchymal cells. Spondylometaphyseal dysplasias (SMDs) comprise a diverse group of skeletal dysplasias and often manifest as short stature, growth-plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of ''corner fractures'' at metaphyses, we identified three individuals with fibronectin (FN1) variants affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotype. The severe scoliosis in most individuals and rare developmental coxa vara distinguish individuals with FN1 mutations from those with classical Sutcliffe-type SMD. To study functional consequences of these FN1 mutations on the protein level, we introduced three disease-associated missense variants (p.Cys87Phe [c.260G>T], p.Tyr240Asp [c.718T>G], and p.Cys260Gly [c.778T>G]) into a recombinant secreted N-terminal 70 kDa fragment (rF70K) and the full-length fibronectin (rFN). The wild-type rF70K and rFN were secreted into the culture medium, whereas all mutant proteins were either not secreted or secreted at significantly lower amounts. Immunofluorescence analysis demonstrated increased intracellular retention of the mutant proteins. In summary, FN1 mutations that cause defective fibronectin secretion are found in SMD, and we thus provide additional evidence for a critical function of fibronectin in cartilage and bone.

In hearts overexpressing vascular endothelial growth factor B (VEGFB), besides its known angiogenic response, multiple regulatory mechanisms lowered coronary LPL. This was accompanied by limited cardiac lipid metabolite accumulation with an augmentation of cardiac insulin action. Our data for the first time links VEGFB to coronary LPL in regulation of cardiac metabolism. VEGFB may be cardioprotective in metabolic disorders like diabetes.

HSPG consist of a core protein to which several linear heparan sulphate (HS) side chains are linked. Heparanase (Hpa) is exceptional in its ability to degrade HS. This property is used to regulate cardiac metabolism by releasing LPL, for its onward movement to the vascular lumen to hydrolyze lipoprotein-TG for FA provision to the heart. We hypothesized that another function of Hpa is its ability to promote cardiac hypertrophy. Hearts from mice with global overexpression of Hpa appeared larger, had higher heart weight/tibia length ratios, and exhibited posterior wall thickening. This evidence of cardiac hypertrophy was not reflected in alterations in cardiac function; ejection fraction, fractional shortening, and stroke volume were largely unaffected. This suggested a physiological rather than pathological hypertrophy, and is linked to a role for Hpa in regulation of arterial structure and mechanics. Hearts from animals with cardiac-specific overexpression of VEGFB also released greater amounts of Hpa, and this coincided with a hypertrophic phenotype. Heart function in these VEGFB-Tg rats remained similar to wild-type, and was likely a consequence of the expanded coronary vasculature and metabolic reprogramming that favoured glucose utilization. Rats made diabetic with STZ did not show any changes in cardiac Hpa gene or protein expression. Nevertheless, immediately after retrograde perfusion, the diabetic hearts released significantly greater amounts of Hpa with evidence of relative cardiac hypertrophy. This cardiac hypertrophy in diabetic rats was pathological with ventricular wall thinning and reduced cardiac function. Our data imply that Hpa can stimulate cardiac hypertrophy that remains physiological when there is associated development of coronary vasculature with metabolic flexibility. With diabetes and the occurrence of microangiopathy along with metabolic inflexibility, hypertrophy progresses towards being pathological, leading to heart failure. Disclosure C.Lee: None. R.Shang: None. B.Hussein: None. B.Rodrigues: None. Funding Canadian Institutes of Health Research (PJT178134); Heart and Stroke Foundation of Canada (G190026493)

VEGFB is known to up-regulate FA delivery through its effect on EC FA transporters. In its absence, intracellular lipid accumulation was reduced. Paradoxically, cardiac-specific overexpression of VEGFB demonstrated a lowered heparin-releasable lipoprotein lipase (LPL) activity with reduced FA utilization in vivo. Currently, the mechanism behind this in vivo metabolic effect of VEGFB on cardiac LPL is unclear. In transgenic (Tg) hearts, the vectorial transfer of LPL from myocytes to the vascular lumen is obstructed, resulting in LPL buildup within cardiomyocytes. Given that insulin is known to inhibit LPL translocation and thus FA metabolism in the heart, this observation is likely a secondary effect of VEGFB on vascular development with its associated augmentation of insulin delivery. We tested whether lowering of insulin by fasting would validate our hypothesis. WT fasted hearts enhanced their heparin-releasable LPL activity in an attempt to switch substrate utilization to FA. Intriguingly, this increase in LPL activity following fasting was even more dramatic in Tg hearts. Thus, with insulin deficiency following fasting, VEGFB works unimpeded to facilitate LPL movement. This effect occurred through the activation of p38 MAPK. PET imaging using 18FTHA revealed augmented LPL-derived FA and impaired FDG glucose uptake in the Tg fasted heart. Additionally, this increased delivery of LPL-derived FA correlated to augmented mitochondrial oxidation determined by high resolution oxygraphy. We examined whether this property of VEGFB on FA uptake and oxidation could be useful following diabetes, with its attendant loss of metabolic flexibility. Unexpectedly, in Tg hearts, diabetes inhibited myocyte VEGFB gene expression and protein secretion together with its downstream receptor signaling. Our data suggests that following diabetes, loss of VEGFB action may contribute towards the metabolic inflexibility, lipotoxicity and diabetic cardiomyopathy. Disclosure R.Shang: None. C.Lee: None. B.Hussein: None. B.Rodrigues: None. Funding Canadian Institutes of Health Research (PJT180558); Diabetes Canada (OG3215585BR)

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