Elimination of AKT in Cardiomyocytes Causes Complex Reorganization of Signal Transduction leading to AMPK Inhibition and Fatal Cardiac Atrophy
Authors: Gödecke Stefanie , Breuling Lukas , Müller Phil‐Torben , Flögel Uli , Heinen Andre , Gödecke Axel 
Published: May 2022

Akt1 and Akt2 are the main protein kinase B (Akt) isoforms expressed in the mammalian heart. Tamoxifen(OHTx) ‐inducible, cardiomyocyte‐specific Akt1/ Akt2 double knockout mice (iCM‐Akt12) show progressive cardiac atrophy and loss of contractile function leading to terminal heart failure 23.9 ± 2 days after first OHTx injection.

TUNEL staining of iCM‐Akt12 hearts revealed that cardiomyocyte apoptosis did not contribute substantially to cardiac atrophy. Rather, cellular atrophy caused the loss of cardiac mass. The cellular area (a), width (w), and length (l) declined between day 9 and day 14 [‐18 (a)/‐18 (w)/‐9% (l)] as compared to WT controls. This size reduction was retarded between d14 and d21 (‐20 (a)/‐24 (w)/‐8% (l). In vivo 31P‐imaging identified a progressive energetic deficiency of iCM‐Akt12 hearts on d15 and d20 after OHTx injection.

Further analyses showed that in the early phase up to day 14 increased autophagic activity and reduced de novo protein synthesis seemed to cause cellular atrophy. As expected, deletion of Akt led to impaired mTORC1 signaling indicated by reduced phosphorylation of S6K, RPS6, 4E‐BP1, all involved in translation. Concomitantly, we measured reduced protein synthesis. Autophagy was increased in iCM‐Akt12 hearts, as shown by increased LC3‐II/I ratio and higher expression of autophagic genes.

From day 14 after KO induction (late phase), protein synthesis appeared to increase and mTORC1 substrates involved in translation (S6K, RPS6 and 4E‐BP1) were higher phosphorylated. Of note, also a higher phosphorylation of the inhibitory mTORC1 target site in the central autophagy kinase Ulk1 (S757) was found. Moreover, autophagy appeared to be disturbed, as p62 accumulated.

Despite increasing energetic depletion, the cellular energy sensor AMPK was not activated in iCM‐Akt12 hearts. Rather, AMPK was inhibited by phosphorylation of the α‐subunit on serine 485/491. The S485/491 phosphorylation has been attributed to several kinases (Akt, PKD1, S6K, PKC, PKA). Akt deletion led to a pronounced hyperactivation of Pdk1 signaling upstream of Akt, causing an activation of protein kinases regulated by Pdk1 (S6K, PKA, PKC, PKD). This was demonstrated by increased phosphorylation of specific kinase substrates and consensus motifs in late iCM‐Akt12 hearts and might promote the AMPK inactivation.

To test to what extent a non‐inhibitable AMPK might attenuate the fatal phenotype of cardiac Akt loss, AAV‐mediated infection of iCM‐Akt12 mice with a phospho‐defective AMPKα2 S491A mutant prior to KO induction was performed. This led to an increased survival and improved heart function, demonstrating that the AMPK inhibition contributes to the lethal phenotype.

In conclusion, loss of Akt signaling leads to a widespread dysregulation of intracellular signal transduction. Whereas enhanced autophagy and inhibition of mTORC1 are direct consequences of Akt deletion, the later reactivation of mTORC1 and inhibition of AMPK causes attenuation of autophagy, increased translation, deceleration of cellular atrophy, worsenes energetic shortage, and aggravates cardiac dysfunction.