Therapeutic Targets and Personalized Medicine in Cardiac Disease

Despite extensive research that has achieved notable advancements over the last decades, cardiovascular disease (CVD) remains the leading cause of death worldwide, with millions affected around the world [...

Identification of a protein phosphatase-1/phospholamban complex that is regulated by cAMP-dependent phosphorylation.

In human and experimental heart failure, the activity of the type 1 phosphatase is significantly increased, associated with dephosphorylation of phospholamban, inhibition of the sarco(endo)plasmic reticulum Ca(2+) transport ATPase (SERCA2a) and depressed function. In the current study, we investigated the molecular mechanisms controlling protein phosphatase-1 activity. Using recombinant proteins and complementary in vitro binding studies, we identified a multi-protein complex centered on protein phosphatase-1 that includes its muscle specific glycogen-targeting subunit GM and substrate phospholamban. GM interacts directly with phospholamban and this association is mediated by the cytosolic regions of the proteins. Our findings suggest the involvement of GM in mediating formation of the phosphatase-1/GM/phospholamban complex through the direct and independent interactions of GM with both protein phosphatase-1 and phospholamban. Importantly, the protein phosphatase-1/GM/phospholamban complex dissociates upon protein kinase A phosphorylation, indicating its significance in the β-adrenergic signalling axis. Moreover, protein phosphatase-1 activity is regulated by two binding partners, inhibitor-1 and the small heat shock protein 20, Hsp20. Indeed, human genetic variants of inhibitor-1 (G147D) or Hsp20 (P20L) result in reduced binding and inhibition of protein phosphatase-1, suggesting aberrant enzymatic regulation in human carriers. These findings provide insights into the mechanisms underlying fine-tuned regulation of protein phosphatase-1 and its impact on the SERCA2/phospholamban interactome in cardiac function

Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca²⁺ Handling and Arrhythmogenesis

Phospholamban (PLN), a key modulator of Ca2+-homeostasis, inhibits sarcoplasmic reticulum (SR) calcium-ATPase (SERCA2a) and regulates cardiac contractility. The human PLN mutation R14del has been identified in arrhythmogenic cardiomyopathy patients worldwide and is currently extensively investigated. In search of the molecular mechanisms mediating the pathological phenotype, we examined PLN-R14del associations to known PLN-interacting partners. We determined that PLN-R14del interactions to key Ca2+-handling proteins SERCA2a and HS-1-associated protein X-1 (HAX-1) were enhanced, indicating the super-inhibition of SERCA2a’s Ca2+-affinity. Additionally, histidine-rich calcium binding protein (HRC) binding to SERCA2a was increased, suggesting the inhibition of SERCA2a maximal velocity. As phosphorylation relieves the inhibitory effect of PLN on SERCA2a activity, we examined the impact of phosphorylation on the PLN-R14del/SERCA2a interaction. Contrary to PLN-WT, phosphorylation did not affect PLN-R14del binding to SERCA2a, due to a lack of Ser-16 phosphorylation in PLN-R14del. No changes were observed in the subcellular distribution of PLN-R14del or its co-localization to SERCA2a. However, in silico predictions suggest structural perturbations in PLN-R14del that could impact its binding and function. Our findings reveal for the first time that by increased binding to SERCA2a and HAX-1, PLN-R14del acts as an enhanced inhibitor of SERCA2a, causing a cascade of molecular events contributing to impaired Ca2+-homeostasis and arrhythmogenesis. Relieving SERCA2a super-inhibition could offer a promising therapeutic approach for PLN-R14del patients

G_M phosphorylation inhibits its interactions with PLN and PP1.

<p>(A) Blot overlay assays on MBP-G_M (amino acid 382-778) recombinant protein was performed using non-phosphorylated or PKA-phosphorylated GST-PLN protein. Western blot analysis revealed decreased binding of phosphorylated PLN to G_M. (B) Quantification of G_M and PLN binding established a significant reduction upon PLN phosphorylation. n = 6; <i>t</i>-test, two-tailed, *<i>P</i><0.05 vs –PKA. (C) Pull down assays using GST-G_M (amino acid 382-778) recombinant protein and phosphorylated or non-phosphorylated cardiac homogenates illustrated the reduced association of PLN with G_M in the PKA-phosphorylated sample. (D) Blot overlay using phosphorylated or non-phosphorylated MBP-G_M (amino acid 1-386) revealed the lack of PP1 binding to phosphorylated G_M protein.</p

Hsp20 variant (P20L) variant exhibits diminished binding and inhibition of PP1.

<p>(A) SDS-gel stained with Coomassie blue showing purified GST-Hsp20 WT and P20L recombinant proteins. (B) Blot overlay assay with MBP-PP1 protein and immunodetection with MBP antibody demonstrates reduced PP1 binding to P20L protein. Phosphorylation of recombinant Hsp20 proteins by PKA treatment resulted in increased binding of PP1 to Hsp20-WT, however, this effect was not observed for P20L variant. (C) Quantification of PP1 protein binding to WT and P20L under basal and PKA conditions. n = 4; <i>t</i>-test, two-tailed, *<i>P</i><0.05 vs WT (–PKA); #<i>P</i><0.05 vs WT (+PKA). (D) Hsp20 variant P20L presents diminished phosphorylation levels at Ser16. (E) PP1 activity measurements showed reduced inhibition of PP1 by P20L. n = 4; <i>t</i>-test, two-tailed, *<i>P</i><0.05 vs WT (–PKA); #<i>P</i><0.05 vs WT (+PKA). WB: Western blot.</p

Glial responses during epileptogenesis in Mus musculus point to potential therapeutic targets.

The Mesio-Temporal Lobe Epilepsy syndrome is the most common form of intractable epilepsy. It is characterized by recurrence of focal seizures and is often associated with hippocampal sclerosis and drug resistance. We aimed to characterize the molecular changes occurring during the initial stages of epileptogenesis in search of new therapeutic targets for Mesio-Temporal Lobe Epilepsy. We used a mouse model obtained by intra-hippocampal microinjection of kainate and performed hippocampal whole genome expression analysis at 6h, 12h and 24h post-injection, followed by multilevel bioinformatics analysis. We report significant changes in immune and inflammatory responses, neuronal network reorganization processes and glial functions, predominantly initiated during status epilepticus at 12h and persistent after the end of status epilepticus at 24h post-kainate. Upstream regulator analysis highlighted Cyba, Cybb and Vim as central regulators of multiple overexpressed genes implicated in glial responses at 24h. In silico microRNA analysis indicated that miR-9, miR-19b, miR-129, and miR-223 may regulate the expression of glial-associated genes at 24h. Our data support the hypothesis that glial-mediated inflammatory response holds a key role during epileptogenesis, and that microglial cells may participate in the initial process of epileptogenesis through increased ROS production via the NOX complex