Data demonstrating the anti-oxidant role of hemopexin in the heart

The data presented in this article are related to the research article entitled Hemopexin counteracts systolic dysfunction induced by heme-driven oxidative stress (G. Ingoglia, C. M. Sag, N. Rex, L. De Franceschi, F. Vinchi, J. Cimino, S. Petrillo, S. Wagner, K. Kreitmeier, L. Silengo, F. Altruda, L. S. Maier, E. Hirsch, A. Ghigo and E. Tolosano, 2017) [1]. Data show that heme induces reactive oxygen species (ROS) production in primary cardiomyocytes. H9c2 myoblastic cells treated with heme bound to human Hemopexin (Hx) are protected from heme accumulation and oxidative stress. Similarly, the heme-driven oxidative response is reduced in primary cardiomyocytes treated with Hx-heme compared to heme alone. Our in vivo data show that mouse models of hemolytic disorders, β-thalassemic mice and phenylhydrazine-treated mice, have low serum Hx associated to enhanced expression of heme- and oxidative stress responsive genes in the heart. Hx-/- mice do not show signs of heart fibrosis or overt inflammation. For interpretation and discussion of these data, refer to the research article referenced above.

inflammation. For interpretation and discussion of these data, refer to the research article referenced above. &

Value of the data
These data show that the plasma protein hemopexin (Hx) limits heme accumulation within cardiac

cells both in vitro and in vivo
In mice, heme-driven oxidative stress associated to Hx exhaustion can be recovered by the administration of the anti-oxidant α-tocopherol These finding might be exploited in the future for the development of Hx-based drugs able to prevent cardiac heme accumulation and oxidative stress in hemolytic disorders and/or in pathologic conditions associated with heme overload

Data
Data show that heme induced ROS production in primary cardiomyocytes (Fig. 1). Hx limited heme accumulation within H9c2 cell (myoblast cell line) and prevented ROS production. H9c2 cells were treated with heme alone or heme bound to Hx,and heme content, ROS production, the expression of heme-and oxidative stress responsive genes and markers of oxidative stress were evaluated (Fig. 2). These data were confirmed in primary cardiomyocytes isolated from neonatal mice and treated with either heme alone or heme-Hx (Fig. 3) and, indirectly in the heart of Hx -/mice (Fig. 4). Data in Fig. 5 show that the heart of Hx -/mice, despite of heme accumulation and elevated ROS [1], did not show sign of fibrosis and inflammation apart a slight increase in the level of Tumor Necrosis Factor (TNF)α and Interleukin (IL)-6 mRNAs.
In vivo, Hx depletion in mouse models of hemolytic disorders, β-thalassemic mice and phenylhydrazine (PHZ)-treated mice, was associated with heme accumulation and oxidative stress in the heart. Data show that in β-thalassemic mice, low Hx serum level, was associated to increased expression of heme-and oxidative stress responsive genes in the heart (Fig. 6). The same occurred in PHZ-treated mice (Fig. 7). Administration of the anti-oxidant α-tocopherol to PHZ-treated mice normalized the expression of anti-oxidant genes (Fig. 8).

Cells and treatments
H9c2 (ATCC CRL-1446™) cells and primary cardiomyocytes, isolated from neonatal mice were treated with either 10 mM Hx-heme complex or 10 mM heme for 8 hours. Primary adult rat cardiomyocytes were treated with 5 mM heme or vehicle for 15 min. Heme and Hx-heme complex were prepared as described [1].

Gene expression analysis
Total RNA, from cells or tissues, was extracted using Pure Link RNA Mini Kit (Ambion, Life Technologies Italia, Milano, Italy). qRT-PCR was performed on a 7300 Real Time PCR System (Applied Biosystems, Life Technologies Italia). Primers and probes were designed using the ProbeFinder software (http://www.roche-applied-science.com).

Immunohistochemistry and histology
Hearts were processed as described and analyzed by immunohistochemistry with an anti-CD18 antibody (1:100, Biolegend). For collagen quantification, tissue sections were stained with Picrosirius Red and analyzed by Image J program.

Statistical Analysis
Results were expressed as mean 7 SEM. Comparisons between 2 groups were performed with 2sided Welch t tests and among 4 2 groups with 1-or 2-way ANOVA followed by the Bonferroni posttest (GraphPad software Inc, La Jolla, CA). A value of Po0.05 was considered significant.