Transcriptomic and proteomic analysis of unloaded normal hearts in rats: insights into barriers to bridge to recovery in LVAD therapy

Background Functional recovery leading to successful LVAD explantation occurs in only 1–2% of cases globally. While the severity of the underlying disease is a primary limitation, myocardial atrophy and fibrosis induced by mechanical unloading are considered significant biological barriers that hinder potential recovery. However, the molecular mechanisms underlying this "unloading-induced maladaptation" remain poorly understood, and effective preventive strategies are yet to be established. Purpose This study aimed to investigate the effects of unloading through unbiased transcriptomic and proteomic analyses in a rat model of heterotopic heart and lung transplantation. Methods We established an LV unloading model by performing heterotopic heart-lung transplantation from 9-week-old male inbred LEW rats into the abdominal aorta of syngeneic recipients (n=5). Untransplanted hearts served as controls (n=6). The LV function was assessed by ex-vivo Langendorff perfusion system under constant LV loading conditions after 2 weeks of unloading. Cardiomyocyte area and fibrosis were assessed using hematoxylin-eosin and Masson's trichrome staining. Unpaired t test was used for the comparison and the difference between means was indicated with 95% CI. Transcriptomic (RNA sequencing) and proteomic analyses were performed using LV tissue. Differentially expressed genes and proteins shared between both datasets were subjected to Gene Ontology (GO) pathway analysis. Subsequently, protein-protein interaction analysis and hub gene identification were performed using STRING and the Cytoscape cytoHubba plugin, respectively. Results After 2 weeks of LV unloading, significant functional deterioration (difference in rate pressure product: RPP, −6,641; 95%CI [−11,776, −1,506] bpm·mmHg), myocardial atrophy (difference in myocyte size, −91; 95%CI [−109, −73] μm2), and fibrosis (difference in percent area, ＋3.9%; 95%CI [2.1, 5.7]) were observed. Multiomics integration identified 30 commonly upregulated factors, with the collagen-containing extracellular matrix (ECM) pathway showing the highest enrichment. Top 3 hub genes were Thbs1 (Thrombospondin-1), Col3a1 (Collagen Type III Alpha 1), and Postn (Periostin) —frequently appear together as a signature of an activated ECM. Conversely, 11 commonly downregulated factors were identified, with Tfrc (Transferrin Receptor) emerging as the top hub gene related to iron/heme homeostasis. Conclusions LV unloading induced significant LV dysfunction accompanied by myocardial atrophy and fibrosis. Our multiomics approach identified ECM turnover and iron/heme homeostasis as critical biological signatures of this process. We considered that if unloading exerts maladaptive effects even in structurally normal hearts, such effects are likely to be more pronounced in diseased myocardium subjected to LVAD support. This study presents potential targets for overcoming barriers to functional recovery in LVAD therapy.