Mice with Megakaryocyte-Specific Deletion of TGF-β1 Are Partially Protected From Developing Cardiac Fibrosis and Systolic Dysfunction in a Pressure Overload Model

Abstract

Abstract 362Circulating platelets contain a high concentration of the multifunctional cytokine transforming growth factor-β1 (TGF-β1) in their α-granules and release it as an inactive (latent) complex upon platelet adhesion and/or activation. We recently demonstrated that shear force can activate latent TGF-β1 in vitro, and this mechanism may contribute to the activation of TGF-β1 that we observed in vivo in the carotid arteries following injury and thrombus formation. TGF-β1 is reported to be involved in the development of cardiac fibrosis in both humans and mouse models, but the cellular source(s) of TGF-β1 and its activation mechanism in vivo have not been clearly established. To test the hypothesis that platelet TGF-β1 contributes to cardiac fibrosis, we performed comparative studies of WT mice and gene-targeted animals with a megakaryocyte-specific deletion of TGF-β1 [PF4-Cre/Tgfb1flox (Tgfb1flox)] using the transverse aortic constriction (TAC) model in male C57Bl/6 mice. Both groups also underwent sham surgery as controls. We obtained blood by percutaneous puncture of the LV under ultrasound guidance and plasma samples were prepared by immediate centrifugation at 12,000 g for 5 min. This technique consistently results in plasma TGF-β1 levels in the range of ∼1.0 ng/ml, which are below those previously reported by most investigators.Tgfb1flox mice had 45% lower levels of plasma total TGF-β1 than WT animals, with a median total TGF-β1 level in WT of 1.37 ng/ml (IQR, interquartile range, 1.2–1.6; n=45) compared to 0.76 ng/ml (IQR 0.6–0.9; n=25)] in Tgfb1flox mice (p<0.001). Heart weight/body weight ratios were 42% higher in TAC- (n=15) than in sham- (n=16) operated WT mice (p<0.001) after 4 weeks, but only 11% higher in TAC- (n=13) than sham- (n=12) operated Tgfb1flox mice (p=0.02). The heart weight/body weight ratios correlated with total TGF-β1 levels in WT mice undergoing both sham and TAC surgery (r=0.66; p<0.001), but not in Tgfb1flox mice. Cardiac fibrosis was scored 4 weeks after surgery by an expert veterinary pathologist as 0 for no fibrosis, and 1+, 2+, or 3+ for mild, moderate, and severe fibrosis, respectively. 96% (22/23) of WT mice developed interstitial fibrosis after TAC, with 65% (15/23) developing mild and 30% (7/23) developing moderate (6/23) or severe (1/23) fibrosis. In contrast, only 54% (7/13) of Tgfb1flox mice developed interstitial fibrosis, with 31% (4/13) developing mild and 15% (2/13) developing moderate fibrosis; none developed severe fibrosis (p<0.01). The Tgfb1flox mice also had significantly less perivascular fibrosis than did the WT mice, although the differences were less evident (p=0.03). Cardiac function measured by echocardiography one week after TAC surgery demonstrated that Tgfb1flox mice had better systolic function than WT mice (Table).Table:Cardiac function measurements one week after TAC surgery.WTTgfb1flox†pEF [%]41 [37–48; n=11]56 [48–65; n=11]0.03SV [μl]20 [18–21; n=11]28 [24–33; n=11]0.003FS (%)27 [23–30; n=14]32 [28–37; n=13]0.05EF: ejection fraction; SV: stroke volume; FS: fractional shortening. Data are reported as median [IQR] †Wilcoxon Rank-Sum test. Presurgery values for EF, SV, and FS were similar in WT and Tgfb1flox miceWe conclude that platelet TGF-β1 contributes to the development of cardiac hypertrophy, fibrosis, and systolic dysfunction induced by a high shear, TAC model. These data have important implications for understanding TGF-β1 biology and assessing the role of TGF-β1 in murine models of human diseases. Since shear can dramatically activate TGF-β1 in vitro, it is possible that increased shear force in the TAC mice generates active TGF-β1, which may contribute to the development of cardiac hypertrophy, fibrosis, and systolic dysfunction. Disclosures:No relevant conflicts of interest to declare.