Regulation of cardiac myofilament function by 5'-AMP activated protein kinase (AMPK)

AMPK is a serine/threonine protein kinase that becomes activated when cellular AMP:ATP ratios rise, thereby serving as a key regulator of cellular energetics. Among the known target proteins phosphorylated by AMPK are catabolic and anabolic enzymes, but little is known about its ability to regulate the cardiac contractile apparatus. This study demonstrates that cTnI, a subunit of the cardiac troponin (cTn) complex, is readily phosphorylated by AMPK.;Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex in cardiac muscle. Two of the three subunits, cTnI and cTnT, are subject to post-translational modifications such as proteolysis and phosphorylation. To obtain a global view of the biochemical state of cTn in native tissue, we performed high resolution top-down mass spectrometry of cTn heterotrimers from healthy adult rat hearts. High resolution top-down mass spectrometry revealed naturally occurring single amino acid sequence variants co-existing within a single rat heart.;To study the effects of AMPK on cardiac contracile function, the kinase domain of AMPK was expressed and purified. Using a combination of recombinant mouse cTnI with candidate sites mutated to Alanine, screening of synthetic peptides mimicking selected regions of cTnI, phosphospecific antibodies and high resolution mass spectrometry analysis of synthetic peptide substrates, Ser22 and Ser149 were identified as the targeted sites by AMPK. Top-down MS analysis of purified rat cTn complexes confirmed that Ser149 and the PKA sites were phosphorylated by AMPK. Time course of phosphorylation experiments revealed that Ser149 is the preferred site. Experiments with skinned myocytes determined that AMPK can phosphorylate cTnI while assembled in the myofilament and that Ser 149 was a target site under these conditions.;To determine the physiological consequences of AMPK-mediated phosphorylation of cTnI, myofilament contractile function was studied in skinned and intact cardiomyocytes. In addition, the phosphorylation pattern of cTnI in the exercise-trained state was examined, as exercise is associated with both activation of AMPK. Finally, fast skeletal troponin was found to be a good substrate for AMPK making it a possibly useful model to study the physiological consequences of phosphorylating Ser149 independent of the phosphorylation of Ser22.