Original ArticleMutation analysis of AMP-activated protein kinase subunits in inherited cardiomyopathies: implications for kinase function and disease pathogenesis
Introduction
βFamilial hypertrophic cardiomyopathy (HCM) is one of the most common inherited cardiac disorders with a prevalence of one in 500. It is inherited in an autosomal dominant fashion and in some affected families there may be a high incidence of dysrythmia and sudden death. HCM is diagnosed clinically by the presence of unexplained left ventricular hypertrophy and pathologically by myocyte hypertrophy and disarray [1]. HCM has come to be considered as a “disease of the cardiac sarcomere”, although sarcomeric protein mutations only account for about two-thirds of cases [2], suggesting that other causative genes remain to be identified.
We have hypothesised that in HCM the sarcomeric mutations result in inefficient ATP utilisation leading to an inability to maintain normal cellular ATP availability. Additionally, a number of phenocopies of HCM have been shown to be syndromes associated with abnormal ATP production in the heart [3], [4], [5], [6], [7]. Based on this hypothesis and published genetic mapping data [8], [9], we recently identified disease-causing mutations in a gene, PRKAG2, that encodes the γ2 subunit of AMP-activated protein kinase (AMPK) [10]. Studies by Gollob et al. and Arad et al. have also identified mutations in this gene [11], [12], [13] and have thrown more light on the precise phenotype. Mutations of PRKAG2 (Fig. 1) are associated with variable cardiac hypertrophy, which may be asymmetric and very marked, with a prominent tendency to late dilatation. Histologically, the hypertrophy is not associated with the myocyte disarray that defines sarcomeric HCM, but instead is associated with glycogen deposition [13]. In addition to the progressive muscle phenotype, affected individuals frequently have accessory atrio-ventricular conduction pathways resulting in Wolff–Parkinson–White syndrome (WPW). This congenital electrophysiological phenotype is coupled with progressive conduction disease later in life. Thus, this highly characteristic syndrome shares many features with “classical” HCM, yet is a distinct entity. Our principle interest has been to understand its relevance for disease pathways in cardiac hypertrophy.
AMPK is a key regulator of ATP levels in all tissues and appears to act as a cellular “fuel gauge”, monitoring the cellular AMP/ATP ratio, making critical and continuous adjustments to the relative balance of ATP-consuming and generating metabolic processes. AMPK is activated by cellular stresses (e.g. hypoxia, exercise in muscle) that deplete ATP and consequently increase AMP, which in turn, leads to activation of the kinase by allosteric mechanisms and upstream kinases. Once activated, AMPK switches on catabolic pathways by phosphorylation of metabolic enzymes and potentially also by effects on gene expression, while reducing the activity of many non-essential ATP-consuming processes and, thus, restoring the energetic balance [14], [15].
AMPK exists as a heterotrimeric complex comprising an α catalytic subunit, and β and γ regulatory subunits. In mammals, each subunit is encoded by two or three genes (α1, α2, β1, β2, γ1, γ2 and γ3) (Fig. 2) and 12 heterotrimeric combinations are possible. The α subunit of AMPK contains the kinase domain near the N–terminus, plus a C–terminal auto-inhibitory domain. The β subunit binds both α and γ subunits via the conserved KIS and ASC domains, respectively [14], [15] and has recently been implicated in the binding of AMPK to other molecules, such as glycogen [16], [17]. The γ subunits contain four tandem repeats of a structural module known as a cytathione β-synthase (CBS) domain. The exact function of the CBS domains is uncertain, although it has been suggested that they play a central role in the binding of the adenosine moieties of AMP and ATP, supported by the fact that the archetypal CBS domain of cystathionine β-synthase is allosterically activated by the binding of S–adenosyl-L-methionine [14], [15], [18], [19], [20].
Each subunit isoform of AMPK is expressed in a wide range of tissues, although the levels of expression vary in different tissues. All of the subunit isoforms appear to be expressed in cardiac muscle, leading to a wide range of heterotrimer combinations [14], [15], [18]. This, together with the fact that previous genetic studies of cardiomyopathy have often identified mutations in genes encoding proteins involved in related cellular functions or pathways, led us to investigate whether mutations in AMPK subunit isoforms other than γ2 could cause the HCM/WPW phenotype. In addition, we wished to investigate whether mutations in AMPK result in other phenotypes, such as isolated HCM, isolated dilated cardiomyopathy (DCM) or DCM with evidence of pre-excitation. A mutation screen of all seven AMPK subunit genes was performed in a panel of individuals with these four phenotypes.
Section snippets
Clinical assessment
Family members were ascertained through our clinical practice or the practice of referring clinicians and evaluated by physical examination, electrocardiogram (ECG) and echocardiography, allowing the diagnosis of HCM, HCM/WPW, DCM or DCM/WPW to be made in those clinically affected. Blood was collected from each affected individual and other available family members and DNA was extracted using standard techniques. Informed consent for inclusion in molecular genetic studies was obtained in each
Results
Our panel of patients consisted of 58 unrelated probands selected for different forms of cardiomyopathy. Three had been diagnosed with HCM/WPW and had clinical features similar to those in whom the PRKAG2 mutations had originally been found; four had DCM with associated features also suggesting pre-excitation. These probands had all been diagnosed early in life, with symptomatic dysrhythmias or congestive failure (Table 1); known metabolic or mitochondrial abnormalities that cause childhood
Discussion
Mutations in PRKAG2 were the first in a non-sarcomeric protein encoding gene to be shown to be responsible for a dominant HCM phenotype [10]. Potentially, this might have led onto identification of other components of the kinase complex as new disease genes, just as followed the initial demonstration that HCM was a “disease of the sarcomere” [1]. Recognising that all of the known AMPK subunit isoforms are expressed in cardiac muscle, we undertook a candidate gene study of each of the subunit
Acknowledgements
We would like to thank all the patients for their co-operation. S.M.J.O. is supported by Newton Abraham Studentship in Medical Sciences, University of Oxford. We also acknowledge kind support from the Wellcome Trust and British Heart Foundation.
References (27)
- et al.
The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms
Cell
(2001) - et al.
Hypertrophic cardiomyopathy: a paradigm for myocardial energy depletion
Trend Genet
(2003) - et al.
Is CD36 deficiency an etiology of hereditary hypertrophic cardiomyopathy?
J Mol Cell Cardiol
(1997) - et al.
Molecular cloning, genomic organization, and mapping of PRKAG2, a heart abundant gamma2 subunit of 5′-AMP-activated protein kinase, to human chromosome 7q36
Genomics
(2000) - et al.
A novel domain in AMP-activated protein kinase causes glycogen storage bodies similar to those seen in hereditary cardiac arrhythmias
Curr Biol
(2003) - et al.
AMPK beta subunit targets metabolic stress sensing to glycogen
Curr Biol
(2003) The structure of a domain common to archaebacteria and the homocystinuria disease protein
Trend Biochem Sci
(1997)- et al.
Functional analysis of mutations in the gamma 2 subunit of AMP-activated protein kinase associated with cardiac hypertrophy and Wolff–Parkinson–White syndrome
J Biol Chem
(2002) Glucose repression in yeast
Curr Opin Microbiol
(1999)- et al.
An activating mutation in the gamma1 subunit of the AMP-activated protein kinase
FEBS Lett
(2001)
Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy
Circulation
Maternally inherited hypertrophic cardiomyopathy due to a novel T-to-C transition at nucleotide 9997 in the mitochondrial tRNA (glycine) gene
Am J Hum Genet
Recent advances in the molecular pathogenesis of Friedreich ataxia
Hum Mol Genet
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