Matricellular protein CCN1 promotes collagen alignment and scar integrity after myocardial infarction

Members of the cellular communication network family (CCN) of matricellular proteins, like CCN1, have long been implicated in the regulation of cellular processes underlying wound healing, tissue fibrogenesis, and collagen dynamics. While many studies suggest antifibrotic actions for CCN1 in the adult heart through the promotion of myofibroblast senescence, they largely relied on exogenous supplementation strategies in in vivo models of cardiac injury where its expression is already induced—which may confound interpretation of its function in this process. The objective of this study was to interrogate the role of the endogenous protein on fibroblast function, collagen structural dynamics, and its associated impact on cardiac fibrosis after myocardial infarction (MI). Here, we employed CCN1 loss-of-function methodologies, including both in vitro siRNA-mediated depletion and in vivo fibroblast-specific knockout mice to assess the role of the endogenous protein on cardiac fibroblast fibrotic signaling, and its involvement in acute scar formation after MI. In vitro depletion of CCN1 reduced cardiac fibroblast senescence and proliferation. Although depletion of CCN1 decreased the expression of collagen processing and stabilization enzymes (i.e., P4HA1, PLOD1, and PLOD2), it did not inhibit myofibroblast induction or type I collagen synthesis. Alone, fibroblast-specific removal of CCN1 did not negatively impact ventricular performance or myocardial collagen content but did contribute to disorganization of collagen fibrils and increased matrix compliance. Similarly, Ccn1 ablated animals subjected to MI showed no discernible alterations in cardiac structure or function one week after permanent coronary artery ligation, but exhibited marked increases in incidence of mortality and cardiac rupture. Consistent with our findings that CCN1 depletion does not assuage myofibroblast conversion or type I collagen synthesis in vitro, Ccn1 knockout animals revealed no measurable differences in collagen scar width or mass compared to controls; however, detailed structural analyses via SHG and TEM of scar regions revealed marked alterations in their scar collagen topography—exhibiting changes in numerous macro- and micro-level collagen architectural attributes. Specifically, Ccn1 knockout mice displayed heightened ECM structural complexity in post-MI scar regions, including diminished local alignment and heightened tortuosity of collagen fibers, as well as reduced organizational coherency, packing, and size of collagen fibrils. Associated with these changes in ECM topography with the loss of CCN1 were reductions in fibroblast-matrix interactions, as evidenced by reduced fibroblast nuclear and cellular deformation in vivo and reduced focal-adhesion formation in vitro; findings that ultimately suggest CCN1's ability to influence fibroblast-led collagen alignment may in part be credited to its capacity to augment fibroblast-matrix interactions. These findings underscore the pivotal role of endogenous CCN1 in the scar formation process occurring after MI, directing the appropriate arrangement of the extracellular matrix's collagenous components in the maturing scar—shaping the mechanical properties that support its structural stability. While this suggests an adaptive role for CCN1 in regulating collagen structural attributes crucial for supporting scar integrity post MI, the long-term protracted expression of CCN1 holds maladaptive implications, potentially diminishing collagen structural complexity and compliance in non-infarct regions. The cellular communication network (CCN) family of matricellular proteins, including CCN1, plays a critical role in regulating cellular processes essential for wound healing, tissue fibrogenesis, and collagen dynamics. However, previous studies predominantly relied on exogenous supplementation strategies in in vivo models of cardiac injury, potentially confounding interpretations of CCN1′s function in these processes. This study aimed to investigate the endogenous protein's role in fibroblast function, collagen structural dynamics, and its impact on cardiac fibrosis following myocardial infarction (MI). Employing CCN1 loss-of-function approaches, including in vitro siRNA-mediated depletion and in vivo fibroblast-specific knockout mice, we assessed CCN1′s influence on cardiac fibroblast fibrotic signaling and acute scar formation post-MI. In vitro CCN1 depletion reduced cardiac fibroblast senescence and proliferation, as well as decreased the expression of enzymes crucial for collagen processing and stabilization. In vivo fibroblast-specific CCN1 removal did not impair ventricular performance or alter myocardial collagen content but led to collagen fibril disorganization and increased matrix compliance. Ccn1 knockout animals exhibited elevated mortality and cardiac rupture post-MI, with no significant differences in collagen scar width or mass compared to wildtype controls. Yet, detailed structural analyses revealed alterations in scar collagen topography, including increased ECM structural complexity and diminished collagen alignment. These changes correlated with reduced fibroblast-matrix interactions, suggesting CCN1′s role in influencing collagen alignment through augmenting these interactions. Endogenous CCN1 plays a pivotal role in scar formation post-MI by orchestrating the arrangement of collagenous components in the maturing scar, thereby shaping its mechanical properties and structural stability. While CCN1′s adaptive role in regulating collagen structural attributes crucial for scar integrity is evident, prolonged expression may lead to diminished collagen structural complexity and compliance in non-infarct regions, highlighting potential maladaptive implications in the long-term.