Background: Kinase oxidation is a critical signalling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, type-1 protein kinase A (PKARIα) can be reversibly oxidized, forming interprotein disulfide bonds within the holoenzyme complex. However, the effect of PKARIα disulfide formation on downstream signaling in the heart, particularly under states of oxidative stress such as ischemia and reperfusion (I/R), remains unexplored. Methods: Atrial tissue obtained from patients before and after cardiopulmonary bypass and reperfusion and left ventricular (LV) tissue from mice subjected to I/R or sham surgery were used to assess PKARIα disulfide formation by immunoblot. To determine the impact of disulfide formation on PKARIα catalytic activity and sub-cellular localization, live-cell fluorescence imaging and stimulated emission depletion super-resolution microscopy were performed in prkar1 knock-out mouse embryonic fibroblasts, neonatal myocytes or adult LV myocytes isolated from 'redox dead' (Cys17Ser) PKARIα knock-in mice and their wild-type littermates. Comparison of intracellular calcium dynamics between genotypes was assessed in fura2-loaded LV myocytes whereas I/R-injury was assessed ex vivo. Results: In both humans and mice, myocardial PKARIα disulfide formation was found to be significantly increased (2-fold in humans, p=0.023; 2.4-fold in mice, p<0.001) in response to I/R in vivo. In mouse LV cardiomyocytes, disulfide-containing PKARIα was not found to impact catalytic activity, but instead led to enhanced A-kinase-anchoring protein (AKAP) binding with preferential localization of the holoenzyme to the lysosome. Redox-dependent regulation of lysosomal two pore channels (TPC) by PKARIα was sufficient to prevent global calcium release from the sarcoplasmic reticulum in LV myocytes, without affecting intrinsic ryanodine receptor leak or phosphorylation. Absence of I/R-induced PKARIα disulfide formation in "redox dead" knock-in mouse hearts resulted in larger infarcts (2-fold, p<0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, p<0.001), which was prevented by administering the lysosomal TPC inhibitor Ned-19 at the time of reperfusion. Conclusions: Disulfide-modification targets PKARIα to the lysosome where it acts as a gatekeeper for TPC-mediated triggering of global calcium release. In the post-ischemic heart, this regulatory mechanism is critical for protecting from extensive injury and offers a novel target for the design of cardioprotective therapeutics.

The ability§ of the heart to adapt to changes in the mechanical environment is critical for normal cardiac physiology. The role of nitric oxide is increasingly recognized as a mediator of mechanical signaling. Produced in the heart by nitric oxide synthases, nitric oxide affects almost all mechano-transduction pathways within the cardiomyocyte, with roles mediating mechano-sensing, mechano-electric feedback (via modulation of ion channel activity), and calcium handling. As more precise experimental techniques for applying mechanical stresses to cells are developed, the role of these forces in cardiomyocyte function can be further understood. Furthermore, specific inhibitors of different nitric oxide synthase isoforms are now available to elucidate the role of these enzymes in mediating mechano-electrical signaling. Understanding of the links between nitric oxide production and mechano-electrical signaling is incomplete, particularly whether mechanically sensitive ion channels are regulated by nitric oxide, and how this affects the cardiac action potential. This is of particular relevance to conditions such as atrial fibrillation and heart failure, in which nitric oxide production is reduced. Dysfunction of the nitric oxide/mechano-electrical signaling pathways are likely to be a feature of cardiac pathology (e.g., atrial fibrillation, cardiomyopathy, and heart failure) and a better understanding of the importance of nitric oxide signaling and its links to mechanical regulation of heart function may advance our understanding of these conditions.

The cytokine interleukin 6 (IL6) is a key mediator of inflammation that contributes to skeletal muscle pathophysiology. IL6 activates target cells by two different mechanisms, the classical and transsignalling pathways. While classical signalling is associated with the anti-inflammatory activities of the cytokine, the IL6 trans-signalling pathway mediates chronic inflammation and is therefore a target for therapeutic intervention. Extracellular vesicles (EVs) are natural, lipid-bound nanoparticles, with potential as targeted delivery vehicles for therapeutic macromolecules. Here, we engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signalling pathway. The potency of the IL6ST decoy receptor EVs was optimized by inclusion of a GCN4 dimerization domain and a peptide sequence derived from syntenin-1 which targets the decoy receptor to EVs. The resulting engineered EVs were able to efficiently inhibit activation of the IL6 transsignalling pathway in reporter cells, while having no effect on the IL6 classical signalling. IL6ST decoy receptor EVs, were also capable of blocking the IL6 trans-signalling pathway in C2C12 myoblasts and myotubes, thereby inhibiting the phosphorylation of STAT3 and partially reversing the antidifferentiation effects observed when treating cells with IL6/IL6R complexes. Treatment of a Duchenne muscular dystrophy mouse model with IL6ST decoy receptor EVs resulted in a reduction in STAT3 phosphorylation in the quadriceps and gastrocnemius muscles of these mice, thereby demonstrating in vivo activity of the decoy receptor EVs as a potential therapy. Taken together, this study reveals the IL6 trans-signalling pathway as a promising therapeutic target in DMD, and demonstrates the therapeutic potential of IL6ST decoy receptor EVs.