Co-regulation And Function Of Popdc1 And Popdc3 Transcriptional Output In The Developing Heart

Heart disease is a leading cause of human mortality.Arrhythmia is one of the most common heart diseases associated with an abnormal conduction system.Sick sinus syndrome,Brugada syndrome,and atrial fibrillation all belong to the category of arrhythmia,among which sick sinus syndrome is the most frequently-occurring one.Popeye domain containing（POPDC）proteins are the effector protein of second messenger c AMP and have been demonstrated to be essential elements in hear rate control.Deletion of Popdc1 or Popdc2 in mice leads to stress-induced sick sinus syndrome.There is ample evidence that Popdc1/Popdc3 are co-regulated.For example,it has been reported that POPDC1 and POPDC3 are downregulated or hypermethylated in the failing human heart or gastric and colorectal tumor specimen,which may be associated with the pathogenesis of these diseased.A hypothesis proposed that the expression of Popdc1 and Popdc3 is co-regulated by a common transcriptional machinery.However,this has yet been proved.Dissecting the regulatory mechanism of POPDC protein transcriptional output is a key step toward understanding the pathology,prevention,and even therapeutical treatment of such heart diseases.To dissect the regulatory mechanism of Popdc1/3 transcriptional output,we analyzed the Ch IP data sets of SHOX2,ISLET1,TBX3,NKX2-5,GATA4,HAND2,TBX5,and MEF2A and identified a common binding peak located about 100 kb upstream of Popdc1and 60 kb upstream of Popdc3 with the deposition of active enhancer markers H3K4me1and H3K27ac at the same peak site.Meanwhile,these transcription factors all showed binding peaks at the promotor site of Popdc1 and Popdc3.Hi-C analysis revealed that the binding site and Popdc1 and Popdc3 are in the same TAD with the signature of physical contact.sc RNA-Seq data indicate co-existence of eight transcription factors including SHOX2,ISLET1,TBX3,NKX2-5,GATA4,HAND2,TBX5,and MEF2A with Popdc1and Popdc3 in pacemaker cells of sinoatrial node and that of five transcription factors including NKX2-5,GATA4,HAND2,TBX5,and MEF2A with Popdc1 and Popdc3 in working cardiomyocyte of atria and ventricle.We hypothesized that this binding site co-occupied by multiple transcription factors could be an enhancer that co-regulate the expression of Popdc1 and Popdc3 transcriptional output and named as Popdc1/3^{d EN}.To validate the enhancer characteristic of Popdc1/3^{d EN},we performed the analysis of enhancer activity in vitro and in vivo.The luciferase reporter assay by transfection of HL-1 cell line with p GL4.23-Popdc1/3^{d EN} plasmids and the assay of Hsp68-Lac Z-Popdc1/3^{d EN} transgenic mice both demonstrated the specific enhancer activity of Popdc1/3^{d EN} in heart cells and tissues.3C experiments revealed the physical contact of Popdc1/3^{d EN} with the promoters of Popdc1 and Popdc3 in wildtypes but not in Popdc1/3^{d EN} knockout mutants.Immunostaining confirmed the significant downregulation of Popdc1 and Popdc3 expression in the sinoatrial node,atria,and ventricle of Popdc1/3^{d EN-/-}mice.To investigate the biological significance of co-regulation of Popdc1 and Popdc3 in the heart,the ergology of Popdc1/3^{d EN-/-}heart was examined.ECG analysis revealed severe sinus node dysfunction with bradycardia,abnormal P wave,and atrioventricular block in freely roaming mutant mice,exhibiting symptoms of sick sinoatrial node syndrome in humans.These symptoms became more severe in an age-dependent manner.The ECG recordings further indicated arrhythmia accompanied with extension of PP interval,PR interval,and RR interval in addition to symptoms of sick sinoatrial node syndrome under freely roaming condition in the mutant mice when they were under stress condition induced by injection of ISO.These symptoms also became more severe in an age-dependent manner.Histological examination found that,although the atria and ventricle of the mutant heart exhibited no obvious abnormality,but the sinus node was malformed,exhibiting a smaller volume with loss of tail tissue,and significantly enlarged right atria in 12-month and older mice.sc RNA-Seq transcriptome analysis found the downregulation of Popdc1 and Popdc3 induced by deletion of Popdc1/3^{d EN} enhancer results in the extensive downregulated expression of heart rate-associated genes in the pacemaker cells in the mutants,which is the major cause of sick sinus node syndrome.In addition,deletion of Popdc1/3^{d EN} also leads to the downregulation of muscle contraction associated gene expression in cardiomyocytes of atria and ventricle in the mutant.Differentiation trajectory by pseudotime analysis revealed the mostly severe functional impairments were found in pacemaker cells,then in atrial cardiomyocytes,and just slightly in ventricular cardiomyocytes,which are consistent with the histological results.In summary,our study dissects the co-regulatory mechanism of Popdc1 and Popdc3transcriptional output and identifies its biological significance,further demonstrating the essential role of POPDC protein in control of heart rate.Disfunction of sinoatrial node induced by deficiency of NKX2-5,SHOX2,TBX3,and ISLET1 could be the consequence of POPDC1 and POPDC3 downregulation.Our findings provide additional evidence for understanding of the pathogenesis of sick sinus node syndrome and further emphasize the importance of non-coding regulatory elements in susceptibility and occurrence of heart diseases.