Cardiac ischemia-reperfusion injury induces ROS-dependent loss of PKA regulatory subunit RIα

Abstract

Type I PKA regulatory α-subunit (RIα; encoded by the Prkar1a gene) serves as the predominant inhibitor protein of the catalytic subunit of cAMP-dependent protein kinase (PKAc). However, recent evidence suggests that PKA signaling can be initiated by cAMP-independent events, especially within the context of cellular oxidative stress such as ischemia-reperfusion (I/R) injury. We determined whether RIα is actively involved in the regulation of PKA activity via reactive oxygen species (ROS)-dependent mechanisms during I/R stress in the heart. Induction of ex vivo global I/R injury in mouse hearts selectively downregulated RIα protein expression, whereas RII subunit expression appears to remain unaltered. Cardiac myocyte cell culture models were used to determine that oxidant stimulus (i.e., H2O2) alone is sufficient to induce RIα protein downregulation. Transient increase of RIα expression (via adenoviral overexpression) negatively affects cell survival and function upon oxidative stress as measured by increased induction of apoptosis and decreased mitochondrial respiration. Furthermore, analysis of mitochondrial subcellular fractions in heart tissue showed that PKA-associated proteins are enriched in subsarcolemmal mitochondria (SSM) fractions and that loss of RIα is most pronounced at SSM upon I/R injury. These data were supported via electron microscopy in A-kinase anchoring protein 1 (AKAP1)-knockout mice, where loss of AKAP1 expression leads to aberrant mitochondrial morphology manifested in SSM but not interfibrillar mitochondria. Thus, we conclude that modification of RIα via ROS-dependent mechanisms induced by I/R injury has the potential to sensitize PKA signaling in the cell without the direct use of the canonical cAMP-dependent activation pathway. NEW & NOTEWORTHY We uncovered a previously undescribed phenomenon involving oxidation-induced activation of PKA signaling in the progression of cardiac ischemia-reperfusion injury. Type I PKA regulatory subunit RIα, but not type II PKA regulatory subunits, is dynamically regulated by oxidative stress to trigger the activation of the catalytic subunit of PKA in cardiac myocytes. This effect may play a critical role in the regulation of subsarcolemmal mitochondria function upon the induction of ischemic injury in the heart.

Coronary Endothelium No‐Reflow Injury Is Associated with ROS‐Modified Mitochondrial Fission through the JNK‐Drp1 Signaling Pathway

“…Since carbon dioxide in the blood stream cannot be quickly removed, the pH in endothelial cells significantly decreases [53]. As the pH decreases, massive hydrogen ions within endothelial cells promote the opening of Na + /H + channels, which in turn promotes sodium translocation into the cytoplasm [54]. Subsequently, abnormal sodium stimulates sodium-calcium exchange channels that trigger intracellular calcium overload, which leads to coronary spasm and blunted coronary artery relaxation [55].…”

Detection of Discordant Peptide Quantities in Shotgun Proteomics Data by Peptide Correlation Analysis (PeCorA)

“…This peptide was detected with and without the oxidation, but only the oxidized peptide was increased due to LPS stimulation. PKA R1α is thought to be regulated by oxidative stress [20][21][22] , and the finding that LPS can induce this site-specific oxidation may prove to be yet another mechanism by which cellular metabolism is tuned. This site M331 is on the surface of second nucleotide binding domain (CNB-B) near the cAMP binding site 23 , and could regulate cAMP binding or protein-protein interactions ( Figure 3B).…”

A-Kinase Anchoring Protein 1: Emerging Roles in Regulating Mitochondrial Form and Function in Health and Disease

“…Despite the loss of AKAP1 under this condition, there was an observed increase in the activity of the catalytic subunit of PKA (PKAc), presumably due to loss of the inhibitory RIα subunit. Indeed, overexpression of RIα increased apoptosis following cardiac ischemia/reperfusion injury [28].…”

“…The AKAP1 knockout mouse was generated by deletion of the first coding exon that includes the mitochondrial targeting sequence and the PKA binding site [22]. Even though the AKAP1 knockout mouse was generated over a decade ago [22], a recent flurry of publications has described the use of the AKAP1 gene deletion to tease out the function of AKAP1 in a variety of tissues [23][24][25][26][27][28]. As we begin to discuss the specific roles of AKAP1 in mitochondrial form and function, it is worth noting how the dynamic changes in mitochondrial morphology impact function.…”

“…Looking into the future, generating a tissue-specific AKAP1 KO mouse could help us to further dissect the roles of AKAP1 in different diseases. In cardiovascular diseases, hyperoxia-induced lung injury and ischemia-induced neurodegeneration, AKAP1 has been shown to support cell survival [23][24][25]27,28,50]. However, all these studies described the loss of AKAP1 as being detrimental.…”