Fibroblast-Specific Proteo-Transcriptomes Reveal Distinct Fibrotic Signatures of Human Sinoatrial Node in Non-Failing and Failing Hearts.

Background: Up to fifty percent of the adult human sinoatrial node (SAN), is composed of dense connective tissue. Cardiac diseases including heart failure (HF) may further increase fibrosis within the SAN pacemaker complex, leading to impaired automaticity and conduction of electrical activity to the atria. However, unlike the role of cardiac fibroblasts in pathological fibrotic remodeling and tissue repair, nothing is known about fibroblasts that maintain the inherently fibrotic SAN environment. Methods: Intact SAN pacemaker complex was dissected from cardioplegically arrested explanted non-failing (non-HF, n=22; 48.7±3.1y.o,) and HF human hearts (n=16; 54.9±2.6y.o.). Connective tissue content was quantified from Masson's trichrome stained head-center and center-tail SAN sections. Expression of extracellular matrix (ECM) proteins, including Collagens 1, 3A1, cartilage intermediate layer protein 1 (CILP1) and periostin, fibroblast and myofibroblast numbers were quantified by in situ and in vitro immunolabeling. Fibroblasts from the central intramural SAN pacemaker compartment (~10x5x2 mm3) and right atria (RA) were isolated, cultured, passaged once, and treated ±transforming growth factor beta-1 (TGFβ1) and subjected to comprehensive high-throughput next-generation sequencing of whole transcriptome, microRNA and proteomic analyses. Results: Intranodal fibrotic content was significantly higher in SAN pacemaker complex from HF vs non-HF hearts (57.7±2.6% vs 44.0±1.2% p<0.0001). Proliferating phosphorylated histone3+/vimentin+/CD31- fibroblasts were higher in HF SAN. Vimentin+/alpha smooth muscle actin+/CD31- myofibroblasts along with increased interstitial periostin expression were found only in HF SAN. RNA sequencing and proteomic analyses identified unique differences in mRNA, long non-coding RNA, microRNA and proteomic profiles between non-HF and HF SAN and RA fibroblasts, and TGFβ1-induced myofibroblasts. Specifically, proteins and signaling pathways associated with ECM flexibility, stiffness, focal adhesion and metabolism were altered in HF SAN fibroblasts compared to non-HF SAN. Conclusions: This study revealed increased SAN-specific fibrosis with presence of myofibroblasts, CILP1 and periostin-positive interstitial fibrosis only in HF vs non-HF human hearts. Comprehensive proteo-transcriptomic profiles of SAN fibroblasts identified upregulation of genes and proteins promoting stiffer SAN ECM in HF hearts. Fibroblast-specific profiles generated by our proteo-transcriptomic analyses of the human SAN, provide a comprehensive framework for future studies to investigate the role of SAN-specific fibrosis in cardiac rhythm regulation and arrhythmias.