San1 Deficiency Leads To Cardiac Dysplasia Due To Excessive R-Loop-Associated Dna Damage And Cardiomyocyte Hypoplasia

Objective:R-loops are naturally occurring transcriptional intermediates containing RNA/DNA hybrids.Excessive R-loops cause genomic instability,DNA damage,and replication stress.Senataxin-associated exonuclease（San1）is a protein that interacts with Senataxin（SETX）,a helicase resolving R-loops.Interestingly,the abundance of R-loops in San1^-/-cells was slightly increased even though the San1 may not directly resolve.CMs in the mammalian heart possess regenerative capacities within 7 days after birth and then become terminally differentiated.DNA damage increases because of oxidative stress derived from the oxygen-rich postnatal environment during the perinatal period,which may trigger cell cycle exit and subsequently impair cardiac development.It remains unknown if R-loops-induced DNA damage plays a role in the heart,especially in the proliferative neonatal cardiomyocytes（CMs）.The purpose of this study was to explore the effects and mechanisms of R-loops aggregation caused by San1 deficiency on the development of newborn heart and the structure and function of adult heart.Methods and results:Firstly,San1^-/-mice were generated using the CRISPR/Cas9technique.The heart weight/body weight ratio of San1^-/-hearts at P1 was slightly decreased and the cardiomyocyte surface area was relatively increased.Immunofluorescence,m RNA and protein levels demonstrated inhibitory proliferative activity of San1^-/-CMs.Transcriptional sequencing data indicated that replication initiation pathway was down-regulated.Serial detection for cardiac structure and function through transthoracic echocardiography revealed ventricular wall thinning and cardiac insufficiency in San1^-/-mice at 12 weeks.Pathological section revealed ventricular wall thinning,reduced heart weight,cardiac hypertrophy,myocardial fibrosis,and cardiac dysfunction.RNAseq results indicated that cardiac hypertrophic genes were up-regulated and cell-cycle relative genes were down-regulated.These data demonstrated that San1 deficiency caused cardiac hypoplasia through inhibitory cardiomyocyte proliferation.Then,DNA damage can lead to checkpoint activation and cell cycle arrest.To further investigate the potential role of San1 in cellular proliferation,a San1-knockout AC16（human cardiomyocyte）cell line was generated utilizing CRISPR-Cas9 editing technology.Through applying Comet assay,immunoblotting,flow cytometry and real-time q PCR,we found that San1 deficiency led to increased DNA damage,checkpoint activation,S/G2 phase arrest and limited proliferative activity.Moreover,San1^-/-cells showed reduced expression of genomic repair protein（XRCC1 and SETX）and proliferative protein（PCNA,Ki67,Cyclin D1 and MCM2-4）.Further experiments demonstrated that the DNA lesions caused by San1 deficiency led to hyperactivation of PARP1,which resulted in accelerated degradation of SETX and XRCC1 via poly（ADP-ribose）-dependent ubiquitination（PARd U）pathway,further aggravating genome instability.Thus,we concluded that San1 deficiency led to reduced proliferation via DNA damage and cell cycle aggregating in S/G2 phase.Finally,since San1 is associated with SETX,which is known to resolve R-loop forming,we next investigated if San1 deficiency would disturb R-loop homeostasis in CMs.Immunofluorescent staining,dot blot assay and DRIP-q PCR,utilizing anti-RNA-DNA hybrids S9.6 antibody,showed that San1 deficiency led to elevated R-loops in both primary and AC16 CMs.RNase H1 is a ribonuclease that recognizes and binds to RNA-DNA hybrids to specifically degrade RNA component within R-loops.Stable transfectants of RNase H1 overexpression were constructed in San1^-/-AC16 CMs.The results showed that RNase H1 overexpression in cultured San1^-/-CMs can effectively eliminate the accumulated R-loops,alleviate DNA damage（response）,PARP1 hyper-activation,checkpoint activation,cell cycle arrest in S/G2 phase,and limited cell proliferation activity.R-loop build-up during the neonatal period of San1^-/-cardiomyocytes leads to increased DNA damage,early cell cycle exit,and CM hypoplasia.Thus,these data suggested that accumulation of R-loops is responsible for replication stress and genomic instability in San1-deleted cells.Conclusion:（1）We reported that San1 encoded a protein that has R-loops metabolic activity,and San1 deletion led to excessive R-loops accumulation and genomic instability.（2）San1 deficiency in cultured CMs results in DNA damage,checkpoint activation,cell cycle aggregating in S/G2 phase and down-regulated cell proliferation.And R-loops is responsible for replication stress and genomic instability in San1-depleted cells.（3）Increased DNA damage in San1^-/-CMs attributes to the impaired CM proliferation.During neonatal heart development,the resulted DNA lesions comproised cell proliferation profiles and eventually led to cadiac morphologic abnormalities and dysfunction.