Mammalian γ2 AMPK regulates intrinsic heart rate.

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αßγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.

Chronic Activation of γ2 AMPK Induces Obesity and Reduces ß Cell Function.

Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.

Localisation of AMPK γ subunits in cardiac and skeletal muscles.

The trimeric protein AMP-activated protein kinase (AMPK) is an important sensor of energetic status and cellular stress, and mutations in genes encoding two of the regulatory γ subunits cause inherited disorders of either cardiac or skeletal muscle. AMPKγ2 mutations cause hypertrophic cardiomyopathy with glycogen deposition and conduction abnormalities; mutations in AMPKγ3 result in increased skeletal muscle glycogen. In order to gain further insight into the roles of the different γ subunits in muscle and into possible disease mechanisms, we localised the γ2 and γ3 subunits, along with the more abundant γ1 subunit, by immunofluorescence in cardiomyocytes and skeletal muscle fibres. The predominant cardiac γ2 variant, γ2-3B, gave a striated pattern in cardiomyocytes, aligning with the Z-disk but with punctate staining similar to T-tubule (L-type Ca(2+) channel) and sarcoplasmic reticulum (SERCA2) markers. In skeletal muscle fibres AMPKγ3 localises to the I band, presenting a uniform staining that flanks the Z-disk, also coinciding with the position of Ca(2+) influx in these muscles. The localisation of γ2-3B- and γ3-containing AMPK suggests that these trimers may have similar functions in the different muscles. AMPK containing γ2-3B was detected in oxidative skeletal muscles which had low expression of γ3, confirming that these two regulatory subunits may be co-ordinately regulated in response to metabolic requirements. Compartmentalisation of AMPK complexes is most likely dependent on the regulatory γ subunit and this differential localisation may direct substrate selection and specify particular functional roles.

Embryonic expression of AMPK γ subunits and the identification of a novel γ2 transcript variant in adult heart.

AMP-activated protein kinase (AMPK), the key sensor and regulator of cellular energy status, is a heterotrimeric enzyme with multiple isoforms for each subunit (α1/α 2; ß1/ß2; γ1/γ2/γ3). Mutations in PRKAG2, which encodes the γ2 regulatory subunit, cause a cardiomyopathy characterized by hypertrophy and conduction abnormalities. The two reported PRKAG2 transcript variants, γ2-short and γ2-long (encoding 328 and 569 amino acids respectively), are both widely expressed in adult tissues. We show that both γ2 variants are also expressed during cardiogenesis in mouse embryos; expression of the γ3 isoform was also detected unexpectedly at this stage. As neither γ2 transcript is cardiac specific nor differentially expressed during embryogenesis, it is paradoxical that the disease is largely restricted to the heart. However, a recently annotated γ2 transcript, termed γ2-3B as transcription starts at an alternative exon 3b, has been identified; it is spliced in-frame to exon 4 thus generating a protein of 443 residues in mouse with the first 32 residues being unique. It is increasingly expressed in the developing mouse heart and quantitative PCR analysis established that γ2-3B is the major PRKAG2 transcript (~60%) in human heart. Antibody against the novel N-terminal sequence showed that γ2-3B is predominantly expressed in the heart where it is the most abundant γ2 protein. The abundance of γ2-3B and its tissue specificity indicate that γ2-3B may have non-redundant role in the heart and hence mediate the predominantly cardiac phenotype caused by PRKAG2 mutations.

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.

Subunit composition of AMPK trimers present in the cytokinetic apparatus: Implications for drug target identification.

AMP-activated protein kinase has been shown to be a key regulator of energy homeostasis; it has also been identified as a tumor suppressor and is required for correct cell division and chromosome segregation during mitosis. The enzyme is a heterotrimer, with each subunit having more than one isoform, each encoded by a separate gene (two α, two ß and three γ isoforms). In human endothelial cells, the activated kinase subunit of AMPK in the cytokinetic apparatus is α2, the minority α subunit, which co-localizes with ß2 and γ2. This is the first demonstration of a trimeric complex of AMPK containing the γ2 regulatory subunit becoming selectively activated and being linked to mitotic processes. We also show that α1 and γ1, the predominant AMPK subunits, are almost exclusively localized in the cytoskeleton, while α2 and γ2 are present in all subcellular fractions, including the nuclei. These data suggest that pharmacological interventions targeted to specific AMPK subunit isoforms have the potential to modify selective functions of AMPK.

Urinary magnesium excretion in asthmatic children receiving magnesium supplementation: a randomized, placebo-controlled, double-blind study.

The aims of this study were to establish whether a magnesium (Mg) deficit indicated by a decreased urinary excretion exists and to determine whether 12-week oral Mg supplementation affects the Mg status and bronchodilator use in children with stable bronchial asthma. The effects of long-lasting Mg supplementation were investigated in 89 children 4 to 16 years of age with mild or moderate persistent bronchial asthma in a randomized, double-blind, placebo-controlled, prospective study. Each subject received one capsule of Mg citrate per day (= 7 years: 200 mg, > 7 years: 290 mg) or one capsule of placebo containing 260 mg glucose during 12 weeks. Evaluation was performed at 4-week intervals. Venous blood serum total and free Mg and urine Mg levels were determined at the beginning and end of the 12-week period. Parents recorded the number of bronchodilator doses twice daily. A urinary Mg loss (6.81 +/- 3.9 versus 2.79 +/- 1.39 mmol/day, p = 0.01) was observed in the placebo-treated persistent moderate asthmatics. Bronchodilator use was significantly higher after 8 and 12 weeks in the placebo-treated than in the Mg-treated patients with moderate asthma (31.1 +/- 1.8 versus 29.5 +/- 1.2 puffs per patient/4 weeks, p < 0.05, and 31.0 +/- 2.3 versus 29.3 +/- 0.9 puffs per patient/4 weeks, p < 0.05, respectively). Long-lasting Mg supplementation is clearly of benefit in mildly to moderately asthmatic children and is recommended as a concomitant drug in stable asthma.