Compound Heterozygous KCNQ1 Mutations Causing Recessive Romano-Ward Syndrome: Functional Characterization by Mutant Co-expression.

Next Generation Sequencing has identified many KCNQ1 genetic variants associated with type 1 long QT or Romano-Ward syndrome, most frequently inherited in an autosomal dominant fashion, although recessive forms have been reported. Particularly in the case of missense variants, functional studies of mutants are of aid to establish variant pathogenicity and to understand the mechanistic basis of disease. Two compound heterozygous KCNQ1 mutations (p.A300T and p.P535T) were previously found in a child who suffered sudden death. To provide further insight into the clinical significance and basis for pathogenicity of these variants, different combinations of wildtype, A300T and P535T alleles were co-expressed with the accessory ß-subunit minK in HEK293 cells, to analyze colocalization with the plasma membrane and some biophysical phenotypes of homo and heterotetrameric channels using the patch-clamp technique. A300T homotetrameric channels showed left-shifted activation V1/2 as previously observed in Xenopus oocytes, decreased maximum conductance density, slow rise-time300ms, and a characteristic use-dependent response. A300T slow rise-time300ms and use-dependent response behaved as dominant biophysical traits for all allele combinations. The P535T variant significantly decreased maximum conductance density and Kv7.1-minK-plasma membrane colocalization. P535T/A300T heterotetrameric channels showed decreased colocalization with plasma membrane, slow rise-time300ms and the A300T characteristic use-dependent response. While A300T left shifted activation voltage dependence behaved as a recessive trait when co-expressed with WT alleles, it was dominant when co-expressed with P535T alleles. Conclusions: The combination of P535T/A300T channel biophysical properties is compatible with recessive Romano Ward syndrome. Further analysis of other biophysical traits may identify other mechanisms involved in the pathophysiology of this disease.

Redox signaling in ischemic postconditioning protection involves PKCε and Erk1/2 pathways and converges indirectly in Nrf2 activation.

Ischemic-postconditioning (iPostC) exerts cardioprotection by preserving redox homeostasis in the reperfused heart. This protective effect has been associated with the activation of endogenous antioxidant response driven by transcription factor Nrf2 and with the activation of 'reperfusion injury salvage kinases' (RISK) as PI3K, PKC and Erk1/2. Redox homeostasis is essential for normal cell physiology since reactive oxygen species (ROS) are crucial for processes that involve protein signaling. Thus, it has become clear that not only the perturbation of redox balance to oxidative state is deleterious but also towards a reductive state contributing to pathogenesis of diseases. However, there is still a scarce knowledge about the role of ROS in the cardioprotective signals mediated by RISK in postconditioned hearts. Therefore, we studied the role of ROS as initiator of RISK signaling molecules in iPostC-conferred cardioprotection. With the aim to study the relationship between redox-dependent RISK activation and the downstream activation of the transcription factor Nrf2, we evaluated the effect of redox signaling disruption by the effect of ascorbic acid in iPostC hearts. Our results showed that PKCε and Erk1/2 activation is redox-dependent and that concurs downstream with Nrf2 deficient activation. Besides, using inhibitors we found that neither PI3K nor Erk1/2 are directly related with Nrf2 activation, indicating that these kinases have other targets. We conclude that redox signaling participates in cardioprotection triggered by iPostC through the action of kinase-dependent and -independent mechanisms and concurred with the downstream regulation of Nrf2-mediated antioxidant response to prolonged redox balance during long reperfusion.

Nrf2-regulated antioxidant response is activated by protein kinase C in postconditioned rat hearts.

Postconditioning (PostC) activates endogenous protective mechanisms that contend against reperfusion injury. Nevertheless, although PostC efficiency in both experimental studies and clinical trials has been demonstrated, a complete picture of the interacting mechanisms, particularly the relationship between kinase signaling and redox maintenance, is still lacking. To unravel such association, in this work we focus on the participation of protein kinase C (PKC) and the transcription factor nuclear factor E2-related factor 2 (Nrf2) in the cardioprotective response elicited by PostC. PostC was performed in an in vivo rat model by applying three repetitive cycles of ischemia and reperfusion (10 s each), followed by evaluation of heart function and infarct size measurements. PKC activation and Nrf2 phosphorylation were evaluated after 10 min of reperfusion, whereas Nrf2 activity and the content and activities of Nrf2-regulated antioxidant proteins were evaluated after 60 min of reperfusion in PostC hearts. Maintenance of heart function and diminution in infarct size concurred with PKC activation and Nrf2 phosphorylation. PKC inhibition diminished Nrf2 phosphorylation and transcriptional activity in association with diminished levels and activities of Nrf2-regulated antioxidant proteins. In conclusion, this study proposes that the novel pathway PKC/Nrf2 participates in the long-term protective mechanisms induced by PostC application by maintaining the antioxidant defense system.