Alpha1 catalytic subunit of AMPK modulates contractile function of cardiomyocytes through phosphorylation of troponin I.

AIMS: The specific role of AMPKα1 or AMPKα2 in mediating cardiomyocyte contractile function remains elusive. The present study investigated how AMPK activation modulates the contractility of isolated cardiomyocytes. MAIN METHODS: Mechanical properties and intracellular Ca(2+) properties were measured in isolated cardiomyocytes. The stress signaling was evaluated using western blot and immunoprecipitation analysis. KEY FINDINGS: AMPK activator, A-769662 induced maximal velocity of shortening (+dL/dt) and relengthening (-dL/dt), peak height and peak shortening (PS) amplitude in both WT and AMPKα2 KO cardiomyocytes, but did not affect time-to-90% relengthening (TR90). AMPK KD cardiomyocytes demonstrated contractile dysfunction compared with cardiomyocytes from WT and AMPKα2 KO hearts. However, the rise of intracellular Ca(2+) levels as well as intracellular ATP levels has no significant difference among WT, AMPKα2 KO and AMPK KD groups with and without the presence of A-769662. Besides, WT, AMPKα2 KO and AMPK KD group displayed a phosphorylated AMPK and downstream acetyl-CoA carboxylase (ACC) phosphorylation. Interestingly, A-769662 also triggered troponin I (cTnI) phosphorylation at Ser(149) site which is related to contractility of cardiomyocytes. Furthermore, the immunoprecipitation analysis revealed that AMPKα1 of cardiomyocytes was phosphorylated by A-769662. SIGNIFICANCE: This is the first study illustrating that activation of AMPK plays a significant role in mediating the contractile function of cardiomyocytes using transgenic animal models. AMPK activator facilitates the contractility of cardiomyocytes via activating AMPKα1 catalytic subunit. The phosphorylation of cTnI by AMPK could be a factor attributing to the regulation of contractility of cardiomyocytes.

AMP-activated protein kinase phosphorylates cardiac troponin I and alters contractility of murine ventricular myocytes.

RATIONALE: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory γ2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. OBJECTIVE: The γ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of γ2 may be involved in conferring substrate specificity or in determining intracellular localization. METHODS AND RESULTS: A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of γ2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca(2+) sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca(2+) transient and prolonged relaxation despite shortening the time constant of Ca(2+) transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca(2+) sensitivity. CONCLUSIONS: We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.

Generation and functional characterization of knock-in mice harboring the cardiac troponin I-R21C mutation associated with hypertrophic cardiomyopathy.

The R21C substitution in cardiac troponin I (cTnI) is the only identified mutation within its unique N-terminal extension that is associated with hypertrophic cardiomyopathy (HCM) in man. Particularly, this mutation is located in the consensus sequence for ß-adrenergic-activated protein kinase A (PKA)-mediated phosphorylation. The mechanisms by which this mutation leads to heart disease are still unclear. Therefore, we generated cTnI knock-in mouse models carrying an R21C mutation to evaluate the resultant functional consequences. Measuring the in vivo levels of incorporated mutant and WT cTnI, and their basal phosphorylation levels by top-down mass spectrometry demonstrated: 1) a dominant-negative effect such that, the R21C+/- hearts incorporated 24.9% of the mutant cTnI within the myofilament; and 2) the R21C mutation abolished the in vivo phosphorylation of Ser(23)/Ser(24) in the mutant cTnI. Adult heterozygous (R21C+/-) and homozygous (R21C+/+) mutant mice activated the fetal gene program and developed a remarkable degree of cardiac hypertrophy and fibrosis. Investigation of cardiac skinned fibers isolated from WT and heterozygous mice revealed that the WT cTnI was completely phosphorylated at Ser(23)/Ser(24) unless the mice were pre-treated with propranolol. After propranolol treatment (-PKA), the pCa-tension relationships of all three mice (i.e. WT, R21C+/-, and R21C+/+) were essentially the same. However, after treatment with propranolol and PKA, the R21C cTnI mutation reduced (R21C+/-) or abolished (R21C+/+) the well known decrease in the Ca(2+) sensitivity of tension that accompanies Ser(23)/Ser(24) cTnI phosphorylation. Altogether, the combined effects of the R21C mutation appear to contribute toward the development of HCM and suggest that another physiological role for the phosphorylation of Ser(23)/Ser(24) in cTnI is to prevent cardiac hypertrophy.