AK1 Mouse

What is an AK1 mouse model and why is it important for research?

AK1 mouse models are genetically modified mice with alterations to the Adenylate Kinase 1 gene, which encodes an enzyme that catalyzes the reaction 2ADP ↔ ATP + AMP. These models are crucial for studying energy metabolism, as AK1 extracts additional energy under metabolic stress and promotes energetic homeostasis . There are two primary types:

• AK1 overexpressing (AK1-OE): Mice with increased cardiac-specific expression of AK1

• AK1 knockout (AK1−/−): Mice deficient in the AK1 gene

These models allow researchers to investigate the role of AK1 in numerous physiological processes including cardiac function, skeletal muscle metabolism, and potentially neurological function .

What are the phenotypic characteristics of AK1 knockout mice?

AK1 knockout (AK1−/−) mice exhibit several distinctive characteristics:

• Normal development with no overt phenotypic abnormalities

• Similar body weight and tibial lengths compared to wild-type animals

• Normal baseline ATP and ADP concentrations in resting muscle

• Significantly elevated total AMP content (approximately 0.05 μmol/g wet weight higher than wild-type)

• Normal AMPK phosphorylation at rest, despite higher AMP content

• When subjected to high-energy demands, they show exceptionally elevated ADP accumulation and markedly reduced IMP production

AK1 is the predominant isoform in skeletal muscle, with remaining AK activity in knockout mice being exceptionally low, presumably due to other minor AK isoforms (AK2 and AK3) localized in mitochondria .

How are cardiac-specific AK1 overexpressing mice generated?

The generation of cardiac-specific AK1 overexpressing mice involves several key molecular biology steps:

• The mouse AK1 open reading frame (668 bp) with a hemagglutinin (HA) tag is cloned into SalI and HindIII cloning sites of an αMHC vector

• The αMHC-AK1-HA-polyA fragment is sub-cloned into an integrase-mediated cassette exchange vector (CB92)

• This vector is transfected into embryonic stem cells together with PhiC31 integrase

• Recombinant clones harboring transgenes within the Rosa26 locus are obtained

• These clones are injected into blastocysts to generate chimeras

• Genotyping utilizes a multiplex PCR protocol to detect both the presence of AK1 transgene and zygosity status

Validation of the model includes verification of transgenic expression through measuring transcript levels of both transgenic AK1 and total AK1, with endogenous mRNA levels remaining unchanged .

What are the key considerations when designing experiments with AK1 mouse models?

When designing experiments with AK1 mouse models, researchers should consider:

• Sex-specific differences: Male AK1-OE mice show distinct cardiac phenotypes including mild in vivo dysfunction at baseline with lower LV pressure and impaired relaxation

• Zygosity effects: There is a gene dosing effect in AK1-OE mice, with different expression levels between heterozygotes (WT/AK1) and homozygotes (AK1/AK1)

• Tissue specificity: Expression changes may be tissue-specific. For example, in AK1-OE mice, increased mRNA was found only in left ventricle and atria, not in other tissues examined

• Metabolic state consideration: AK1's role becomes more prominent during metabolic stress, so experimental designs should include both baseline and stress conditions (e.g., ischemia/reperfusion protocols)

• Control selection: Appropriate wild-type controls of similar genetic background are essential (e.g., C57BL/6 × 129/Ola background for AK1−/− studies)

• Activity measurements: Given the presence of other AK isoforms, validation of AK activity levels is important

How does AK1 modification affect AMPK signaling in skeletal muscle?

AK1 modification has significant impacts on AMPK (AMP-activated protein kinase) signaling:

In AK1-deficient mice:

• Contraction-mediated phosphorylation of AMPK is lower in skeletal muscle of AK1−/− mice compared to wild-type controls

• Despite higher total AMP levels in resting AK1−/− muscle, AMPK phosphorylation is not elevated above wild-type levels

• The disconnect between measured AMP and AMPK signaling suggests that changes in total measured AMP do not necessarily reflect alterations in free cytosolic AMP that mediates signaling

In AK1-overexpressing mice:

• Despite 20% higher AMP levels, AMPK is not activated (P = 0.85)

• This suggests complex regulatory mechanisms beyond simple AMP concentration

These findings highlight the nuanced relationship between AK1 activity, AMP levels, and AMPK signaling, which is crucial for understanding energy metabolism regulation .

What metabolic alterations are observed in AK1 overexpressing mouse hearts?

AK1 overexpressing mouse hearts display several significant metabolic alterations:

• Enzyme activities: 31% higher AK1 activity, with unchanged total creatine kinase and citrate synthase activities

• Nucleotide levels:

  • Significantly raised creatine

  • Unaltered total adenine nucleotides

  • 20% higher AMP levels (P = 0.05)

• Metabolite profile: 1H-NMR revealed significant differences in LV metabolite levels with:

  • Elevated aspartate, tyrosine, sphingomyelin, and cholesterol

  • Significantly lower taurine and triglycerides

• Structural changes: 19% higher LV weight in male AK1-OE mice due to higher tissue water content in the absence of hypertrophy or fibrosis

These metabolic changes suggest that AK1 overexpression has broad effects beyond just adenine nucleotide metabolism, affecting various metabolic pathways and cardiac composition.

How are AK1 mouse models used to study ischemia/reperfusion injury?

AK1 mouse models provide valuable tools for studying ischemia/reperfusion (I/R) injury mechanisms:

These models help researchers understand the complex balance between ATP preservation, AMP signaling, and adenosine production during cardiac stress conditions .

What is the relationship between NTRK1, AMPK, and mitophagy in neurological research using mouse models?

While not directly related to AK1 mouse models, research on NTRK1 knockdown mice demonstrates the importance of AMPK signaling pathways that AK1 may influence:

• NTRK1 knockdown induces mouse hippocampal neuronal damage through suppression of mitophagy via inactivating the AMPK/ULK1/FUNDC1 pathway

• NTRK1 knockdown attenuates ATP production, mitochondrial membrane potential, and mitophagy

• Pre-treatment with the AMPK activator O304 can abrogate the suppression of mitophagy and the promotion of neuronal damage induced by NTRK1 silencing

This research highlights how AMPK signaling (which can be influenced by AK1 activity through AMP generation) plays crucial roles in mitochondrial quality control and neuronal protection . Cross-application of findings between AK1 and NTRK1 models may provide insights into metabolic regulation of neuronal function.

How should researchers interpret contradictory metabolic data from AK1 mouse models?

When faced with seemingly contradictory data from AK1 mouse models, researchers should consider:

• Compartmentalization of nucleotides: The vast majority of AMP measured from muscle extracts is bound or restricted to compartments where it is not metabolically active

• Free vs. total nucleotide levels: Changes in measured total AMP do not necessarily reflect alterations in free cytosolic AMP that participates in signaling

• Equilibrium considerations: When interpreting AMP levels, consider whether creatine kinase and AK are in equilibrium

• Tissue-specific effects: AK1 modifications may have different impacts across tissues; findings in cardiac tissue may not apply to skeletal muscle

• Compensatory mechanisms: Other AK isoforms (e.g., mitochondrial AK2, AK3) may partially compensate for AK1 deficiency or respond to overexpression

• Methodological differences: Different extraction and measurement techniques may yield varying results for adenine nucleotides; standardized protocols are essential

What research methodologies are most appropriate for studying AK1 mouse models?

When studying AK1 mouse models, researchers should consider these methodological approaches:

• Quantitative research approaches:

  • Controlled experiments with precise manipulation of variables to establish cause-effect relationships

  • Between-subjects or within-subjects designs depending on the research question

  • Detailed documentation of experimental tools, techniques, and procedures

• Enzyme activity assays:

  • Measurement of AK1 activity, total creatine kinase, and citrate synthase activities

  • Compare activities across genotypes to confirm phenotypes

• Metabolite profiling:

  • 1H-NMR for comprehensive metabolite analysis

  • HPLC for adenine nucleotide measurements

  • Consider both baseline and stressed conditions

• Functional assessments:

  • In vivo cardiac function assessment

  • Ex vivo ischemia/reperfusion protocols

  • Measurement of contractile parameters

• Molecular biology techniques:

  • RT-PCR for mRNA expression analysis

  • Western blotting for protein expression and phosphorylation status

  • Multiplex PCR for genotyping

These methodological considerations are crucial for obtaining reliable and meaningful data from AK1 mouse models in research settings.