Dottorato in MEDICINA SPERIMENTALE

Titolo della tesi: The role of OPA1 and mitochondrial dynamics in the biology of cardiac fibroblasts

Background: Cardiovascular diseases remain the leading cause of mortality worldwide, with heart failure (HF) representing a major clinical burden. A hallmark of HF is pathological cardiac remodeling, a process driven significantly by cardiac fibroblasts (CFs). Upon injury, CFs differentiate into myofibroblasts, leading to excessive extracellular matrix (ECM) deposition and fibrosis. While the role of mitochondria in cardiomyocyte physiology is well-established, their contribution to CF activation and differentiation remains less defined. Specifically, the interplay between mitochondrial dynamics (fission and fusion) and the metabolic reprogramming required for fibroblast activation is not fully understood. Aim: This thesis investigates the role of the mitochondrial fusion protein Optic Atrophy 1 (OPA1) and mitochondrial dynamics in regulating cardiac fibroblast biology. The primary objective is to determine if OPA1-mediated mitochondrial integrity is required for the phenotypic conversion of fibroblasts to myofibroblasts in response to profibrotic stimuli. Methods: Primary cardiac fibroblasts were isolated from C57BL/6J mice via explant culture and stimulated with Transforming Growth Factor β1 (TGFβ1). Mitochondrial morphology was assessed using automated network analysis. To determine the functional requirement of OPA1, RNA interference (siRNA) was employed to silence OPA1 expression. The impact of OPA1 depletion was evaluated using Seahorse extracellular flux analysis for bioenergetics, Next-Generation RNA sequencing (RNA-seq) for transcriptomic profiling, and immunofluorescence/Western blotting for myofibroblast markers (αSMA, procollagen). Additionally, a 3D spheroid culture system was used to assess fibroblast function in a physiologically relevant environment. Results: TGFβ1 stimulation induced a dynamic, biphasic mitochondrial remodeling response in CFs: an initial phase of transient fusion and increased metabolic activity peaking at 16 hours, followed by fission and network fragmentation. This remodeling was accompanied by dynamic fluctuations in OPA1 protein levels, suggesting a feedback mechanism coordinating mitochondrial morphology with the activation state. OPA1 silencing resulted in mitochondrial fragmentation and a significant impairment of bioenergetic capacity, characterized by reduced basal and maximal respiration. Transcriptomic analysis revealed that OPA1 silencing fundamentally alters the transcriptional landscape, preventing the upregulation of genes related to ECM organization, cytoskeletal dynamics, and collagen biosynthesis upon TGFβ1 stimulation. Phenotypically, OPA1-silenced cells exhibited reduced differentiation into myofibroblasts, evidenced by decreased α-smooth muscle actin (αSMA) expression and procollagen production. Furthermore, in the 3D spheroid model, OPA1 depletion impaired spheroid compaction and growth, confirming a defect in activation. Conclusion: Collectively, these findings demonstrate that OPA1-mediated mitochondrial integrity and bioenergetic efficiency are fundamental prerequisites for the initial phase of cardiac fibroblast activation. We propose that OPA1 acts as a metabolic checkpoint; without intact mitochondrial dynamics, CFs fail to mount the necessary metabolic reprogramming required for the reparative response. This study identifies mitochondrial dynamics not merely as a bystander of fibrosis, but as a crucial component of the acute cardiac repair machinery, offering new insights into the mechanisms governing pathological remodeling.