Extracellular SPARC improves cardiomyocyte contraction during health and disease

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 in 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 improves 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.

the specific agent(s) for 1 hour and then cell shortening was measured as described 167 above. 168 Amsterdam, the Netherlands) in combination with an indentation probe with a stiffness 189 of 1 N/m and a tip radius of 44 um (Optics 11, Amsterdam, the Netherlands). The gel's 190

In vitro experiments with adult rat cardiac myocytes
Young's modulus was determined by averaging 9 individual measurements. 191

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Cardiac tissue was processed and histochemical and immunohistochemical analyses were 193 performed as previously described [22][23][24] , and all morphometric analyses were done 194 on sections with myocyte in cross-sectional images. Hematoxylin and eosin -stained 195 performed using a microscope (Leitz DMRXE; Leica), and QWin morphometry software 202 (Leica). All analyses were performed according to standard operating procedures. 203

Immunostaining of isolated cardiac myocytes 204
Adult cardiac myocytes were isolated from healthy mice as described, fixed in 2% PFA in 205 PBS for 10 min, incubated in 50mM glycine for 30 min to remove auto-fluorescence 206 caused by PFA at 488nm, and subsequently stained for SPARC (polyclonal goat antibody, 207 R&D systems, AF492, 5μg/ml) overnight at 4°C. The next day cells were initially incubated 208 with a secondary donkey-anti goat-alexa 488 labeled antibody for 90min. at room 209 temperature and some cells were subsequently stained for integrin beta1 (monoclonal 210 rat antibody, BD, 553715, 0.5μg/ml) for 4 hours at room temperature and afterwards 211 incubated with a secondary goat-anti rat-alexa 568 labeled antibody for 90min. at room 212 temperature. Cells were visualized with confocal microscopy on a Zeiss LSM700 213 microscope (Leica) using the Zen software (Leica), or analyzed using a BD FACSAria III flow 214 cytometer (Becton Dickinson (BD), San Jose, CA) and FlowJo software (Ashland, Oregon). 215

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Adult cardiac myocytes were isolated from healthy mice as described, incubated in lysis 226 buffer, containing (mM): 5 TrisHCl, 5 NaCl, 2 EDTA, 1 CaCl2, 1MgCl2, 2 DTT and pH was 227 adjusted to 7.4. Phosphatase inhibitors (2%, Sigma, P044 and P5726) and protease 228 inhibitors (4%, Roche, 11697498001) were added to the buffer, and cells were incubated 229 overnight at 4°C. The next day, the cell suspension was centrifuged for 1 hour at 4°C and 230 supernatant was collected as cytoplasmic fraction, the pellet was dissolved in lysis buffer 231 and collected as the membrane fraction. Student t-test for 2 groups or ANOVA, followed by a Bonferroni post hoc test for more 262 groups was used in most of the comparisons when groups passed the normality test.
When the standard deviation of two groups significantly differed, a Mann-Whitney test 264 for 2 groups or a Kruskal-Wallis test, followed by a Dunn's post hoc test for more groups, 265 was used. A paired Student's t test was used to analyze baseline and follow-up 266 echocardiographic measurements, a Wilcoxon test was used when data did not pass 267 normality test. A two-sided p-value of ≤ 0.05 was considered statistically significant.  Figure 1G). SPARC, a known collagen-281 binding protein, co-localized with collagen on the matrices ( Figure 1D) and importantly, 282 the presence of SPARC did not alter matrix stiffness ( Figure 1E). Next, we isolated 283 stimulated at 0.5, 1 and 2 Hz, rat cardiomyocytes cultured on SPARC-containing matrices 285 demonstrated an increased cardiomyocyte shortening at all frequencies when compared 286 to cells cultured on matrices without SPARC ( Figure 1F), while, once again, both TTP and 287 RT50 were unchanged (Supplementary Figure 1C and D). 288 289 Using Western Blot, we demonstrate SPARC presence in the membrane fraction, yet 290 absence in the cytosolic fraction of isolated cardiomyocytes ( Figure 1G). Using 291 immunostaining and confocal microscopy we confirmed SPARC's presence on the 292 membrane of the cardiac myocyte, where it co-localizes with integrin-beta1 ( Figure 1H). 293 Furthermore, immuno-precipitation demonstrated an interaction of SPARC with integrin-294 linked kinase (ILK) in both whole LV samples and in isolated cardiomyocytes ( Figure 1I). 295

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To investigate whether this SPARC-induced increased cardiomyocyte cell shortening is 297 through ILK-signaling, we incubated cells in the presence of SPARC and/or the ILK-298 inhibitor CPD-22. Importantly, the SPARC-induced increased cardiomyocyte cell 299 shortening is blunted in the presence of the ILK-inhibitor ( Figure 1J). These results indicate 300 that SPARC increases cardiomyocyte cell shortening, at least in part, through ILK signaling. 301 Notably, CPD-22 alone did not affect cardiomyocyte shortening ( Figure 1J).  (ESD) ( Figure 2F). Importantly, fractional shortening of isolated cardiomyocytes was also 326 compromised in this model ( Figure 2G). Under field stimulation conditions, these cardiomyocytes did not display a prolonged TTP, but RT50 values were significantly 328 increased ( Supplementary Figure 2A and B). Importantly, myocyte cross-sectional area, the amount of fibrosis, collagen cross-linking, 339 and the number of CD45 positive cells in the heart did not differ between the 2 groups, 5 340 weeks after CVB3 injection (Table 1). Still, increased fractional shortening was 341 demonstrated by isolated cardiomyocytes from SPARC-overexpressing animals as 342 compared to isolated cardiomyocytes from control GFP overexpressing animals ( Figure  343 2K), indicating a protective, or positive inotropic effect of SPARC at the level of the 344 cardiomyocyte. Notably, no effect on contraction or relaxation times was observed 345 (Supplementary Figure 2E and F). Furthermore, despite SPARC being a Ca 2+ -binding 346 protein, we could not find indications that SPARC influenced Ca 2+ -handling, as there were 347 no differences in the Ca 2+ transient peak heights (Figure 2L), TTP or RT50 (Supplementary 348 Figure 3G and H) of these isolated myocytes. Moreover, we did not find any differences in Akt phosphorylation between LV samples from both groups, which is known to increase 350 intracellular Ca 2+ -availability and enhance contraction [26], in LV samples from both 351 groups, as shown by Western Blotting (Figure 2M), further supporting no immediate role 352 for SPARC in Ca 2+ -handling. Taken together, these data demonstrate a protective effect 353 of SPARC on cardiomyocyte function prior to the establishment of virus-induced heart 354 failure. Furthermore, these data indicate that SPARC affects filament sensitivity to Ca 2+ 355 rather than altering Ca 2+ handling within the cell.  Figure 3G). We found an increased FS in the SPARC treated group, while FS in 369 the vehicle group continued to decline ( Figure 3H). End diastolic diameter (EDD) was 370 slightly smaller in the SPARC group prior to treatment, compared to the vehicle group.
However, EDD did not change due to the SPARC treatment, while in the vehicle group 372 EDD were slightly decreased after 72h. ESD, on the other hand, was not different between 373 groups or time-points ( Figure 3I). Moreover, myocyte cross-sectional area and the 374 amount of CD45 positive cells in hearts did not differ between the 2 groups (Table 2). Yet, 375 while the amount of fibrosis did not differ, collagen cross-linking was increased in the 376 SPARC-treated group as compared to the vehicle group (Table 2)  Finally, when we infused healthy adult mice with SPARC or vehicle for 72h, we also found 388 increased FS compared to baseline measurements and compared to vehicle-mice ( Figure  389 3L). SPARC administration caused decreased end-systolic diameters (ESD), but not end-390 diastolic diameters (EDD), while diameters did not change in hearts of vehicle-mice 391 ( Figure 3M) Again, SPARC administration did not affect cardiomyocyte hypertrophy, the 392 amount of fibrosis, collagen cross-linking, or the amount of CD45 cells (Table 3)

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To our knowledge this study is the first to demonstrate a direct role for a non-structural 395 matrix protein on cardiomyocyte contraction, via interactions of this protein with 396 intracellular effectors. Our previous study on SPARC in myocardial infarction suggested a 397 previously unexplored potential inotropic function for SPARC in the heart. Here, we 398 demonstrated increased cardiac contraction when SPARC was overexpressed, not only in 399 infarcted mice, but also in sham-operated mice [5] yet how SPARC might directly affected 400 cardiomyocyte contraction remained undetermined. Therefore, in this study we aimed to 401 explore the role of SPARC on cardiomyocyte contractility using various ex vivo and in vivo 402 models. We have shown that extracellular SPARC increases cardiomyocyte contraction, 403 during both health and disease, possibly by interacting with the integrin-beta1-ILK 404 complex on the cardiomyocyte membrane. Not only is SPARC able to prevent a decrease 405 in cardiac function, but it is also able to rescue myocytes that are already compromised 406 through viral infection. These data highlight the potential of SPARC as a therapy in VM 407 and potentially in other disease states where cardiac function is equally compromised.   Our working hypothesis is that SPARC interacts with integrin-beta 1 and ILK on the 643 cardiomyocyte membrane. This results in increased ILK signaling, blocking myosin light 644 chain phosphatase (MLCP), and in this way increasing MLC phosphorylation and thus 645 contraction. 646 Table 1