Extracellular SPARC increases cardiomyocyte contraction during health and disease

Abstract

Secreted protein acidic and rich in cysteine (SPARC) is a non-structural extracellular matrix protein that regulates interactions between the matrix and neighboring cells. In the cardiovascular system, it is expressed by cardiac fibroblasts, endothelial cells, and at lower levels by ventricular cardiomyocytes. SPARC expression levels are increased upon myocardial injury and also during hypertrophy and fibrosis. We have previously shown that SPARC improves cardiac function after myocardial infarction by regulating post-synthetic procollagen processing, however whether SPARC directly affects cardiomyocyte contraction is still unknown. In this study we demonstrate a novel inotropic function for extracellular SPARC in the healthy heart as well as in the diseased state after myocarditis-induced cardiac dysfunction. We demonstrate SPARC presence on the cardiomyocyte membrane where it is co-localized with the integrin-beta1 and the integrin-linked kinase. Moreover, extracellular SPARC directly increases cardiomyocyte cell shortening ex vivo and cardiac function in vivo, both in healthy myocardium and during coxsackie virus-induced cardiac dysfunction. In conclusion, we demonstrate a novel inotropic function for SPARC in the heart, with a potential therapeutic application when myocyte contractile function is diminished such as that caused by a myocarditis-related cardiac injury.

Fig 1. SPARC increases cardiomyocyte contraction through its interaction with integrin-beta1.

A FACS analysis demonstrates increased SPARC staining when cardiomyocytes were isolated and incubated with SPARC ex vivo for 1h, as compared to cells incubated in normal buffer without SPARC. B,C Isolated adult mouse cardiomyocytes displayed higher FS after 1h incubation with SPARC, compared to cells incubated in normal tyrode buffer. D,E Matrices with physiological stiffness were coated with laminin and collagen (Lam + Col) or laminin, collagen and SPARC (Lam + Col + SPARC). SPARC co-localized with collagen on these matrices, but did not affect matrix stiffness. F Adult rat cardiomyocytes were isolated and cultured on these matrices. Cells cultured on SPARC containing matrices displayed higher fractional shortening (FS), compared to cells cultured on matrices coated with L+C alone. G SPARC is present in the membrane fraction, and absent in the cytosolic fraction of isolated cardiomyocytes, as demonstrated by Western Blotting. H SPARC immunoprecipitation (I.P.) demonstrates interaction with integrin-linked kinase (ILK) in LV samples and in isolated cardiomyocytes. I Isolated adult mouse cardiomyocytes were incubated in the presence of SPARC and/or the ILK-inhibitor CPD-22. The increased FS observed in cells incubated with SPARC was abolished in the presence of CPD-22. A-C N = 4 mice and n >4 cells per mouse, D-F N = 3 rats and >20 cells per rat, J N = 3 rats and >4cells per rat, bars panel D 100um, bars panel H 10um, *p<0.05, **p<0.01.

Fig 2. SPARC improves cardiomyocyte function in a mild model of virus-induced heart failure.

A—D A mild VM mouse models is used, where mice are injected with10³ PFU CVB3 intraperitoneally. This results in moderate cardiac inflammation after 1 week), little fibrosis and no cardiomyocyte hypertrophy after 5 weeks. E,F Viral infection caused decreased FS and increased ESD. G Contraction of isolated cardiomyocytes is also compromised after virus-infection. H SPARC is overexpressed with the use of an adenovirus, 2 weeks before mild CVB3 inoculation. I,J 5 weeks after CVB3 injection, higher FS were measured in the SPARC overexpressing group, with no differences in EDD, and slightly smaller ESD. K Isolated myocytes from the SPARC-overexpressing hearts remained their increased shortening capacities as compared to isolated myocytes from control GFP- hearts. L,M There were no differences in the Ca²⁺ transient peak heights, or levels of Akt phosphorylation. A-F n = 11 for sham and n = 13 for VM, G n = 11 for sham and n = 13 for VM and >3 cells per mouse, H-J n = 12 for advGFP group and n = 11 for advSPARC group, K,L n = 12 for advGFP group and n = 11 for advSPARC group and >3cells per mouse, M n = 3 for both groups, bar 1000um for H&E and Sirius Red stainings, 100um for CD45 and laminin stainings, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig 3. SPARC has therapeutic potential in severely virus-induced heart failure.

A—D In the more severe VM model mice are injected with 10⁴ PFU CVB3, which results in severe cardiac inflammation after 1 week, and prominent fibrosis, but no cardiomyocyte hypertrophy after 5 weeks. E, F Viral infection caused severely decreased FS and dilation of the heart. G Mice were infused with SPARC or vehicle for 72h, 5 weeks after high-dose CVB3 inoculation, when dilated cardiomyopathy with severe inflammation and fibrosis had been established. H FS was increased in the SPARC treated group, while FS in the vehicle group continued to decline. I EDD were slightly smaller in the SPARC group prior to treatment, compared to the vehicle group, but did not change due to the SPARC treatment, while in the vehicle group EDD were slightly decreased after 72h. ESD were not different between groups or between time-points. J FS was increased in isolated cardiomyocytes from SPARC-treated mice when compared to cells isolated from vehicle-treated mice. K When cardiomyocytes were isolated from the severely sick, untreated mice, incubation of the cells with SPARC for 1h ex vivo also resulted in increased FS, compared to control cells. L,M Also healthy mice demonstrated higher FS when SPARC was administered for 72 hours, compared to vehicle-administered mice. This resulted in decreased ESD but not decreased end-diastolic diameters EDD in SPARC-administered mice, while diameters did not change in vehicle-administered mice. A-F n = 11 for sham and n = 13 for VM, G-I n = 6 for VM+vehicle and n = 7 for VM+SPARC, J n = 6 for VM+vehicle and n = 7 for VM+SPARC and >3cells per mouse, K n = 13 for both groups and >3cells per mouse, L,M n = 11 for sham+vehicle and n = 8 for sham+SPARC, bar 1000um for H&E and Sirius Red stainings, 100um for CD45 and laminin stainings, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs Sham/Vehicle, #p<0.05 vs SPARC.

Fig 4. Proposed mechanism on how SPARC increases cardiomyocyte contraction.