Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure

Abstract

Rationale In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. Objective This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. Methods and Results We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is due to an enhanced β2 adrenergic receptor (β2AR) signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2AR signal by preventing PKA signal access to the myofilaments, and to restore contractile response to adrenergic stimulation. Conclusions In hypertrophic rabbit myocytes, selectively enhanced β2AR signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2AR signaling and restore myocyte contractility in response to β-adrenergic stimulation.

T-tubule remodelling disturbs localized β2-adrenergic signalling in rat ventricular myocytes during the progression of heart failure

“…Hence, we assume Cav-3 dissociation to be partially responsible for the reorientation of T-tubules into the longitudinal direction. A recent publication studied HF in rabbit after ∼7.1 months of aortic insufficiency and constriction 38 . In this rabbit model a Cav-3 protein reduction was shown together with an increased β 2 AR signal in the myofilament compartment of cardiomyocytes and unaltered signalling levels at the sarcoplasmic reticulum (SR) and plasma membrane.…”

Arrhythmogenic Mechanisms in Heart Failure: Linking β-Adrenergic Stimulation, Stretch, and Calcium

“…Interestingly, an additional study utilizing a rabbit model, also showed that reintroduction of caveolin-3 was able to normalizes β-adrenergic-induced contractile responses in HF myocytes, while also showing that in HF β2-induced signaling gains access to myofilament which may contribute to abnormal PKA phosphorylation of troponin I and contractile dysfunction ( Barbagallo et al, 2016 ). In addition, work from the Sacconi group has shown that while cells from HF myocytes respond to β-adrenergic stimulation, this is not the case at the T-tubules that do not conduct APs, where the alterations seen in response may be caused by a lack of electrical activity.…”

Axelrod Symposium 2019: Phosphoproteomic Analysis of G-Protein–Coupled Pathways

“…Genetically encoded cAMP and PKA-activity sensors can be engineered to localise at defined cellular sites by introducing a unique targeting sequence (DiPilato et al, 2004;Di Benedetto et al, 2008;Herget et al, 2008;Di Benedetto et al, 2013;Sprenger et al, 2015;Barbagallo et al, 2016;Surdo et al, 2017). Targeting the same cAMP FRET reporter to the plasma membrane and the mitochondria revealed differential dynamics of cAMP signalling in these two compartments.…”

“…We are only beginning to understand the components of signalosomes in different cell types and cellular compartments (Maurice et al, 2014). We predict that combining advanced imaging methods, such as live cell fluorescence microscopy using targeted reporters (Barbagallo et al, 2016;Surdo et al, 2017) and super-resolution imaging (Ni et al, 2018), with proteomics approaches will be instrumental in identifying additional signalling components and novel signalosomes.…”