The findings suggest that NOX-derived ROS results in increased mito-ROS. Whereas short-term increase in mito-ROS was counteracted by MnSOD, long-term increase in ROS resulted in nitrotyrosine-mediated inactivation of MnSOD, leading to unchecked increase in mito-ROS and loss of Δψm followed by inhibition of endothelial function and proliferation.

Increases in protein kinase C (PKC) are associated with diminished cardiac function, but the contribution of downstream myofilament phosphorylation is debated in human and animal models of heart failure. The current experiments evaluated PKC isoform expression, downstream cardiac troponin I (cTnI) S44 phosphorylation (p-S44), and contractile function in failing (F) human myocardium, and in rat models of cardiac dysfunction caused by pressure overload and aging. In F human myocardium, elevated PKCα expression and cTnI p-S44 developed before ventricular assist device implantation. Circulatory support partially reduced PKCα expression and cTnI p-S44 levels and improved cellular contractile function. Gene transfer of dominant negative PKCα (PKCαDN) into F human myocytes also improved contractile function and reduced cTnI p-S44. Heightened cTnI phosphorylation of the analogous residue accompanied reduced myocyte contractile function in a rat model of pressure overload and in aged Fischer 344 × Brown Norway F1 rats (≥26 mo). Together, these results indicate PKC-targeted cTnI p-S44 accompanies cardiac cellular dysfunction in human and animal models. Interfering with PKCα activity reduces downstream cTnI p-S44 levels and partially restores function, suggesting cTnI p-S44 may be a useful target to improve contractile function in the future.

Introduction Endomyocardial biopsy (EMB)-based traditional microscopy remains the gold standard for the detection of cardiac allograft rejection, despite its limitation of inherent subjectivity leading to inter-reader variability. Alternative techniques now exist to surveil for allograft injury and classify rejection. Donor-derived cell-free DNA (dd-cfDNA) testing is now a validated blood-based assay used to surveil for allograft injury. The molecular microscope diagnostic system (MMDx) utilizes intragraft rejection-associated transcripts (RATs) to classify allograft rejection and identify injury. The use of dd-cfDNA and MMDx together provides objective molecular insight into allograft injury and rejection. The aim of this study was to measure the diagnostic agreement between dd-cfDNA and MMDx and assess the relationship between dd-cfDNA and MMDx-derived RATs which may provide further insight into the pathophysiology of allograft rejection and injury. Methods: This is a retrospective observational study of 186 endomyocardial biopsy (EMB) evaluated with traditional microscopy and MMDx. All samples were paired with dd-cfDNA from peripheral blood prior to EMB (up to 1 month). Diagnostic agreement between traditional microscopy, MMDx, and dd-cfDNA (threshold of 0.20%) were compared for assessment of allograft injury. In addition, the relationship between dd-cfDNA and individual RAT expression levels from MMDx was evaluated. Results MMDx characterized allograft tissue as no rejection (NR) (64.5%), antibody-mediated rejection (ABMR) (25.8%), T-cell-mediated rejection (TCMR) (4.8%), and mixed ABMR/ TCMR (4.8%). For the diagnosis of any type of rejection (TCMR, ABMR, and mixed rejection), there was substantial agreement between MMDx and dd-cfDNA (74.7% agreement). All transcript clusters (group of gene sets designated by MMDx) and individual transcripts considered abnormal from MMDx had significantly elevated dd-cfDNA. In addition, a positive correlation between dd-cfDNA levels and certain MMDx-derived RATs was observed. Tissue transcript clusters correlated with dd-cfDNA scores, including DSAST, GRIT, HT1, QCMAT and S4. For individual transcripts, tissue ROBO4 was significantly correlated with dd-cfDNA in both non-rejection and rejection as assessed by MMDx. Conclusion: Collectively, we have shown substantial diagnostic agreement between dd-cfDNA and MMDx. Furthermore, based on the findings presented, we postulate a common pathway between the release of dd-cfDNA and ROBO4 (a vascular endothelial-specific gene that stabilizes the vasculature) in the setting of AMR, which may provide a mechanistic rationale for observed elevations in dd-cfDNA in AMR, compared to ACR.