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The γ2 subunit of AMP-activated protein kinase regulates mammalian heart rate

ABSTRACT: AMP-activated protein 1 kinase (AMPK), a phylogenetically conserved serine/threonine kinase regarded as a key cellular energy sensor, exists in eukaryotes as a heterotrimer comprising a catalytic α and regulatory β and γ subunits. In humans, activating mutations in the gene encoding the γ2 subunit of AMPK (PRKAG2) display a cardiac phenotype of left ventricular hypertrophy (LVH), conduction system disease, ventricular pre-excitation and increased cardiomyocyte glycogen accumulation. While existing transgenic models have elucidated the pathogenesis of several aspects of the disease5-7, the slow heart rate (sinus bradycardia) – a prominent feature of the disease – remains poorly understood. Here, using gene-targeting to generate mice which recapitulate this bradycardia, we demonstrate that γ2 AMPK activation perturbs fundamental mechanisms that determine sinoatrial pacemaker cell function. Reduction in the sarcolemmal hyperpolarization activated (“funny”) current (If) and damping of ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ releases (LCRs)12,13 contribute to reduced sinoatrial cell spontaneous activity and, ultimately, to a lower heart rate. Pharmacological activation of AMPK reversibly reduces the beating rate of murine pluripotent stem cell-derived induced sinoatrial bodies. In contrast, using a mouse knock-out of γ2 AMPK, which exhibits an increased heart rate, we demonstrate a role for γ2 AMPK in physiological heart rate regulation, including an indispensable role in the bradycardic adaptation to endurance exercise. Through regulating the cardiac pacemaker and thereby heart rate, γ2 AMPK by virtue of its energy-sensing role, is a key physiological determinant of overall cardiac energy homeostasis.

ORGANISM(S): Mus musculus

PROVIDER: GSE73047 | GEO | 2017/09/30

SECONDARY ACCESSION(S): PRJNA296001

REPOSITORIES: GEO

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Project description:Right ventricular free wall (RVFW) pacing results in left ventricular dyssynchrony with early septal shortening followed by late lateral contraction that reciprocally stretches the septum. Dyssynchrony is disadvantageous to cardiac mechano-energetics, yet little is known about its molecular consequences. We tested the hypothesis that dyssynchrony selectively alters regional gene expression in mice, employing a novel miniature implantable cardiac pacemaker. Mice were subjected to 1-week overdrive RVFW pacing (720 min-1, baseline HR 520-620 min-1) to induce dyssynchrony (pacemaker: 3V lithium battery, rate programmable, 0.8 grams, bipolar lead). Electrical capture was confirmed by pulsed-wave Doppler at implantation and terminal study, and dyssynchrony by echocardiography. Gene expression from left ventricular septal and lateral-wall myocardium were assessed by microarray (dual-dye method, Agilent) using oligonucleotide probes and dye swap. Identical analysis was applied to 4 synchronously contracting controls. Of 22,000 genes surveyed, only 18 genes displayed significant (p<0.01) differential expression between septal/lateral walls exceeding 1.5-fold relative to any disparities in synchronous controls. These changes were confirmed by qPCR with excellent correlations. Most (16) of the genes showed greater septal expression. Of particular interest were 7 genes coding proteins involved with stretch responses, matrix remodeling, stem cell differentiation to myocyte lineage, and Purkinje fiber differentiation. One-week cardiac dyssynchrony triggers regional differential expression differences in relatively few select genes. Such analysis using a murine implantable pacemaker should facilitate molecular studies of cardiac dyssynchrony and help elucidate novel mechanisms by which stress/stretch stimuli due to dyssynchrony impact the normal and failing heart. Experiment Overall Design: Left ventricle segments from the mouse heart - septal and lateral, were isolated from 4 dyssynchronous mice that were kept separate. RNA from the synchronous mice were pooled into a control septal and lateral sample. Functional genomic analysis was conducted on these 10 RNA samples with fluorophore reversal, such that each sample was assayed on two different microarrays. Corresponding septal and lateral samples from the same heart were paired on the same microarray to measure the relative differences in gene expression between the different regions of the heart then differences were compared across multiple mice to find overlapping genes that were affected by the pacememaker implantation.

Project description:Right ventricular free wall (RVFW) pacing results in left ventricular dyssynchrony with early septal shortening followed by late lateral contraction that reciprocally stretches the septum. Dyssynchrony is disadvantageous to cardiac mechano-energetics, yet little is known about its molecular consequences. We tested the hypothesis that dyssynchrony selectively alters regional gene expression in mice, employing a novel miniature implantable cardiac pacemaker. Mice were subjected to 1-week overdrive RVFW pacing (720 min-1, baseline HR 520-620 min-1) to induce dyssynchrony (pacemaker: 3V lithium battery, rate programmable, 0.8 grams, bipolar lead). Electrical capture was confirmed by pulsed-wave Doppler at implantation and terminal study, and dyssynchrony by echocardiography. Gene expression from left ventricular septal and lateral-wall myocardium were assessed by microarray (dual-dye method, Agilent) using oligonucleotide probes and dye swap. Identical analysis was applied to 4 synchronously contracting controls. Of 22,000 genes surveyed, only 18 genes displayed significant (p<0.01) differential expression between septal/lateral walls exceeding 1.5-fold relative to any disparities in synchronous controls. These changes were confirmed by qPCR with excellent correlations. Most (16) of the genes showed greater septal expression. Of particular interest were 7 genes coding proteins involved with stretch responses, matrix remodeling, stem cell differentiation to myocyte lineage, and Purkinje fiber differentiation. One-week cardiac dyssynchrony triggers regional differential expression differences in relatively few select genes. Such analysis using a murine implantable pacemaker should facilitate molecular studies of cardiac dyssynchrony and help elucidate novel mechanisms by which stress/stretch stimuli due to dyssynchrony impact the normal and failing heart. Keywords: Murine cardiac dyssynchrony and differential gene expression, Agilent, microarray, pacing

Project description:to study the effect of induced expression of TBX3 within the atrium of the heart Background: Treatment of congenital or acquired disorders of the sinus node or atrioventricular node requires insight into the molecular mechanisms for the development and homeostasis of these pacemaker tissues. In the developing heart, transcription factor TBX3 is required for pacemaker and conduction system development. Here, we explore the role of TBX3 in the adult heart and investigate whether TBX3 is able to reprogram terminally differentiated working cardiomyocytes into pacemaker cells. This would be an attractive approach in biological pacemaker formation. Methods and results: TBX3 expression was ectopically induced in cardiomyocytes of adult transgenic mice. Expression analysis revealed an efficient switch from the working myocardial expression profile to that of the pacemaker myocardium. This included suppression of genes encoding gap junction subunits (Cx40, Cx43), the cardiac Na+ channel (NaV1.5; INa) and inwardly rectifying K+ ion channels (Kir-genes; IK1). Concordantly, we observed conduction slowing in these hearts, and reductions in INa and IK1 in cardiomyocytes isolated from these hearts. The reduction in IK1 resulted in a more depolarized maximum diastolic potential, thus enabling spontaneous diastolic depolarization. Neither ectopic pacemaker activity nor pacemaker current, If, were observed. Lentiviral expression of TBX3 in ventricular cardiomyocytes resulted in conduction slowing and development of heterogeneous phenotypes, including depolarized and spontaneously active cardiomyocytes. Conclusions: TBX3 partially reprograms terminally differentiated working cardiomyocytes into pacemaker-like cells and induces important pacemaker properties. The ability of TBX3 to reduce intercellular coupling to overcome current-to-load mismatch and the ability to reduce IK1 density to enable diastolic depolarization, are very promising TBX3 characteristics for biological pacemaker formation strategies. 5 TBX3 expressing left atrial appendage samples (Tamoxifen-treated Myh6MCM;CTBX3 adult male mice) and 6 controls ((Tamoxifen-treated Myh6MCM adult male mice)

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The γ2 subunit of AMP-activated protein kinase regulates mammalian heart rate

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