Increased prostaglandin-D2 in male but not female STAT3-deficient hearts shifts cardiac progenitor cells from endothelial to white adipocyte differentiation

Cardiac levels of the signal transducer and activator of transcription factor-3 (STAT3) decline with age, and male but not female mice with a cardiomyocyte-specific STAT3 deficiency (CKO) display premature age-related heart failure associated with reduced cardiac capillary density. In the present study isolated male and female CKO-cardiomyocytes exhibit increased prostaglandin (PG)-generating cyclooxygenase-2 (COX-2) expression. The PG-degrading hydroxyprostaglandin-dehydrogenase-15 (HPGD) expression is only reduced in male cardiomyocytes, which is associated with increased PGD2 secretion from isolated male but not female CKO-cardiomyocytes. Reduced HPGD expression in male cardiomyocytes derive from impaired androgen-receptor-(AR)-signaling due to loss of its co-factor STAT3. Elevated PGD2 secretion in males is associated with increased white adipocyte accumulation in aged male but not female hearts. Adipocyte differentiation is enhanced in isolated SCA-1+-cardiac-progenitor-cells (CPC) from young male CKO-mice compared to the adipocyte differentiation of male wildtype (WT)-CPC and CPC isolated from female mice. Epigenetic analysis in freshly isolated male CKO-CPC display hypermethylation in pro-angiogenic genes (Fgfr2, Epas1) and hypomethylation in the white adipocyte differentiation gene Zfp423 associated with upregulated ZFP423 expression and a shift from endothelial to white adipocyte differentiation compared to WT-CPC. The expression of the histone-methyltransferase EZH2 is reduced in male CKO-CPC compared to male WT-CPC whereas no differences in the EZH2 expression in female CPC were observed. Clonally expanded CPC can differentiate into endothelial cells or into adipocytes depending on the differentiation conditions. ZFP423 overexpression is sufficient to induce white adipocyte differentiation of clonal CPC. In isolated WT-CPC, PGD2 stimulation reduces the expression of EZH2 thereby upregulating ZFP423 expression and promoting white adipocyte differentiation. Thus, cardiomyocyte STAT3-deficiency leads to age-related and sex-specific cardiac remodeling and failure in part due to sex-specific alterations in PGD2 secretion and subsequent epigenetic impairment of the differentiation potential of CPC. Causally involved is the impaired AR signaling in absence of STAT3, which reduces the expression of the PG degrading enzyme HPGD.

162 that the AR is expressed by HL-1 cardiomyocytes (27). We observed that HPGD expression 163 was significantly lower in control HL-1-STAT3-KD-CM compared to HL-1-ctrl-CM (Figs 2F and 164 2H). Testosterone stimulation markedly induced HPGD in HL-1-ctrl-CM (+6816%), which was 165 attenuated in HL-1-STAT3-KD-CM (+299%) (Fig 2F). In turn, COX-2 was higher in HL-1-166 STAT3-KD-CM without an additional effect of testosterone treatment compared to HL-1-ctrl-167 CM (Fig 2G). Treatment with estrogen did not influence HPGD or COX-2 expression of HL-1-  267 We previously reported that cardiomyocyte-specific STAT3-deficiency in male but not female 268 mice leads to age-related heart failure (14, 17). Here, we provide evidence that impaired AR 269 receptor signaling caused by STAT3 deficiency leads to a higher secretion of PGD 2 by male 270 but not female CKO-CM (Fig 7). PGD 2 subsequently impairs the vascular regeneration 271 potential of CPC by inducing an epigenetic shift from endothelial to white adipocyte 272 differentiation and thereby leads to a degradation of capillaries and an increase in WAT 273 deposits in hearts from aging male but not female CKO mice (Fig 7). These data shed light on 274 sex-specific age-related remodeling processes that contribute to age-related heart failure in 275 males.

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277 PGD 2 is generated by multiple enzymatic steps from arachidonic acid and COX-2 is a rate 278 limiting enzyme in this process (34). COX-2 is elevated in CM from female and male CKO 279 mice, an observation that contrasts with findings showing that STAT3 is a direct transcription 280 factor of COX-2 in ischemic preconditioning, where activation of cardiac STAT3 upregulates 281 COX-2 in the heart (35). However, in our study with young male and female mice canonical 282 STAT3 signaling is not activated suggesting that inactive STAT3 acts either directly or 283 indirectly as a negative regulator of COX-2, a feature that will be explored in future studies.
284 PGs are also regulated by the PG degrading enzyme HPGD (36) and here we observed lower 285 expression of HPGD in male CKO-CM but not in female CKO-CM. In this context it has been 286 shown that the AR can be activated by different ligands including interleukin (IL)-6 and 287 testosterone (37). The AR is expressed in cardiomyocytes from various species including 288 mouse and human (26, 38, 39) and STAT3 acts as a positive co-factor for AR signaling by 289 directly interacting with amino acids 234-558 in the N-terminal domain of the AR (24). The AR 290 is expressed on HL-1 cells and indeed, HPGD expression is lower in HL-1-STAT3-KD-CM 291 compared to ctrl HL-1-CM. Moreover, testosterone induced a marked increase in HPGD in ctrl 292 HL-1-CM, which was blunted in HL-1-STAT3-KD-CM supporting the notion that impaired AR-293 signaling under low STAT3 condition leads to lower expression of HPGD and with this explains 14 294 the sex-specific increase in PGD 2 secretion in male but not female cardiomyocytes from CKO 295 mice (Fig 7).

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We demonstrated that PGD 2 is able to induce white adipocyte differentiation in WT-CPC. In 313 Finally, and in line with the sex-specific difference in PGD 2 production, CPC isolated from 314 female CKO hearts did neither exhibit increased ZFP423 expression nor enhanced adipocyte 315 differentiation. 316 BMP2, BMP4 and ZFP521 are known regulators of ZFP423 (30, 31) and ZFP521 suppresses 317 the adipocyte lineage by direct transcriptional repression of ZFP423 (42). In the present study, 318 expression of ZFP521, BMP2 and BMP4 were not altered in male CKO-hearts or in freshly 319 isolated male CKO-CPC compared with male WT hearts or WT-CPC, suggesting that these 320 factors are not responsible for the ZFP423 regulation in male CKO-CPC. In turn, PGD 2 and its 321 non-enzymatically generated metabolite 15-deoxy-delta(12,14)-prostaglandin-J 2 (PGJ 2 ) were 15 322 reported to decrease EZH2 mRNA and protein expression (43). EZH2 is a subunit of PRC2 323 and functions as a histone methyltransferase that is able to control CpG methylation through 324 direct physical contact with DNA methyltransferases (DNMTs) (44). Reduced EZH2 levels in 325 the Zfp423 promoter are associated with lower histone and DNA methylation and higher 326 expression of the Zfp423 gene, which predisposes fetal progenitor cells to adipogenic 327 differentiation (45). Indeed, in freshly isolated male CKO-CPC the EZH2 expression was 328 reduced and several DMRs were hypomethylated in the ZFP423 gene compared to male WT-329 CPC. Moreover, PGD 2 stimulation of WT-CPC reduced EZH2 expression and increased 330 ZFP423 expression supporting the notion that PGD 2 alters the epigenetic signature of CPC 331 towards adipocyte differentiation. This observation extends previous studies reporting that 332 PGD 2 suppresses lipolysis in adipocytes and is associated with insulin resistance and body 333 weight gain (46). This novel aspect of PGD 2 is interesting with regard to the controversial roles 334 of PGD 2 documented for the cardiovascular system. In fact, it has been shown that PGD 2 has 335 protective features for example in I/R injury (47) but may also have adverse effects, for 336 example by inducing cardiomyocyte death (48) and/or vasoconstriction (49). 347 Previous data reported reduced STAT3 in failing human hearts (15) and the present study 348 reveals significantly lower HPGD expression in LV tissue from male patients with terminal heart 349 failure. Furthermore, PGD 2 induced ZFP423 expression and adipocyte differentiation in human 16 350 iPSC. Therefore, a similar mechanism as described for male mice may also exists in humans 351 and may explain sex-specific development of heart failure.

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In conclusion, age-related reduction of cardiomyocyte STAT3 leads to impaired AR receptor 353 signaling and subsequent reduced expression of the PG degrading enzyme HPGD (Fig 7).