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Truncating variants in TTN (TTNtv), coding for the largest structural protein in the sarcomere, contribute to the largest portion of familial and ambulatory dilated cardiomyopathy (DCM). TTN haploinsufficiency caused by TTNtv is suggested as the disease mechanism. However, it is unclear whether TTN insufficiency causes DCM. Moreover, it is unknown whether modulation of downstream pathways serves as a therapeutic strategy for DCM caused by TTN insufficiency. Here, we show that reduction of cardiac Ttn expression by adeno-associated virus mediated shRNA (Ttn shRNA) generated DCM in mouse, demonstrating impaired cardiac performance, enlarged left ventricle (LV) and reduced LV wall thickness. A screen of 10 dysregulated and selected genes identified that Yin Yang 1 (Yy1) significantly suppressed DCM caused by Ttn shRNA. Gene profiling by RNAseq showed Yy1 modulated cell growth related genes. Ttn insufficiency activated cardiomyocyte cell cycle reentry by upregulating of Ccnd1 and Ccnd2. Cardiomyocytes activated by Ttn insufficiency did not advance to S phase by EdU incorporation assay. Yy1 promoted cardiomyocyte cell cycle by further enhancing Ccnd1 and Ccnd2 and increasing DNA replication without undergoing cell division. Importantly, upregulation of Ccnd1 and Ccnd2 suppressed DCM caused by Ttn insufficiency. Our findings demonstrate that DCM caused by Ttn insufficiency can be treated by therapeutically promoting cardiac cell cycle.

Rationale: Pathogenic variations in the lamin gene ( LMNA ) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA -related DCM. Objective: To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 ( Yy1 ) in suppressing Lmna DCM. Methods and Results: We developed a Lmna DCM mouse model induced by cardiac-specific Lmna short hairpin RNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf . Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGF-β (transforming growth factor-β)/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGFβ/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts. Conclusions: Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.

The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and is implicated in cardiac pathology, the contribution of its cytoplasmic domain to post-infarct myocardial injury and adverse left ventricular (LV) remodeling remains unknown.Methods: Myocardial infarction was induced in wild-type mice or mice lacking the TF cytoplasmic domain (TF increment CT) by occlusion of the left anterior descending coronary artery. Heart function was monitored with echocardiography. Heart tissue was collected at different time-points for histological, molecular and flow cytometry analysis.Results: Compared with wild-type mice, TF increment CT had a higher survival rate during a 28-day follow-up after myocardial infarction. Among surviving mice, TF increment CT mice had better cardiac function and less LV remodeling than wild-type mice. The overall improvement of post-infarct cardiac performance in TF increment CT mice, as revealed by speckle-tracking strain analysis, was attributed to reduced myocardial deformation in the peri-infarct region. Histological analysis demonstrated that TF increment CT hearts had in the infarct area greater proliferation of myofibroblasts and better scar formation. Compared with wild-type hearts, infarcted TF increment CT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1) -Rac1 axis. In particular, infarcted TF increment CT hearts displayed markedly lower ratios of inflammatory M1 macrophages and reparative M2 macrophages (M1/M2). In vitro experiment with primary macrophages demonstrated that deletion of the TF cytoplasmic domain inhibited macrophage polarization toward the M1 phenotype. Furthermore, infarcted TF increment CT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Finally, the overall cardioprotective effects observed in TF increment CT mice could be abolished by subcutaneously infusing a cocktail of PAR1-activating peptide and PAR2-inhibiting peptide via osmotic minipumps.Conclusions: Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation.

Introduction: The mammalian hearts including humans have a limited regenerative capacity. Activation of proliferation or related signaling pathways has been shown to reinstate cell renewal capacity of cardiomyocytes (CM) which is believed to ameliorate heart failure (HF). However, whether new CM are generated underlying cardiac regeneration in adult mammalian hearts upon damage is elusive. It is also unknown whether and by what mechanisms activation of CM proliferation benefits HF besides cardiac regeneration. Hypothesis: The protective role of cardiac cell cycle activation for damaged hearts could be independent of heart regeneration. Methods and Results: We show that cardiac specific activation of YAP1 , the terminal effector of the Hippo signaling pathway, suppressed HF and cardiac fibrosis induced by Lmna insufficiency ( Lmna HF). Although activation of YAP1 promoted G1 and S phase of cardiac cell cycle in disease hearts, CM did not undergo mitotic phase, a critical step for heart regeneration. RNAseq followed by signaling pathway analysis and validation showed that Fanconi anemia (FA) DNA repair pathway was activated by YAP1 . Cardiac DNA damage in Lmna HF was suppressed by YAP1 activation. Importantly, reduction of Fancd2 compromised the suppressive role of YAP1 in Lmna HF. Mechanistically, YAP1 promoted mono-ubiquitination of FANCD2 (ub-FANCD2) through promoting cell cycle. Conclusions: Our findings demonstrate that promoting cardiac DNA repair becomes a new therapeutic strategy against HF.

RATIONALE: Pathogenic variations in the lamin gene (LMNA) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA-related DCM. OBJECTIVE: To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 (Yy1) in suppressing Lmna DCM. METHODS AND RESULTS: We developed a Lmna DCM mouse model induced by cardiac specific Lmna shRNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf. Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGFβ/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGFβ/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts. CONCLUSIONS: Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.

Introduction: Cardiac Lmna insufficiency causes dilated cardiomyopathy (DCM) coupled with cardiac fibrosis and inflammation. TGFβ family members are key mediators for cardiac performance, cardiac fibrosis and inflammation. However, how TGFβ family members are regulated in heart is unknown. Hypothesis: Modulation of downstream pathways could be served as a therapeutic strategy. Methods and Results: We demonstrate that regulation of Bmp7 and Ctgf suppresses Lmna DCM and cardiac fibrosis. Upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, downregulation of Ctgf inhibited TGFβ/Smad signaling pathway in DCM hearts. Co-modulation of Bmp7 and Ctgf reduced infiltration of T cells in hearts. A screen of cardiac related factors identified Yy1 enhanced Bmp7 promoter activity and prevent the upregulation of Ctgf promoter activity. Yy1 suppressed Lmna DCM by inducing Bmp7 expression and reduced Ctgf upregulation in cardiomyocytes. Importantly, knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. Conclusions: Our findings demonstrate that DCM caused by LMNA insufficiency can be treated with co-modulation of Bmp7 and Ctgf .