MITOCHONDRIAL COMPLEX 1 ACTIVITY IN MYOCARDIAL-SPECIFIC CHF1 DEFICIENT MICE

Jeffrey Gordon11/23/2010

CHF1 – A Brief Overview

• CHF1 is a basic-HLH transcription repressor which plays an important role in cardiovascular development

• CHF1 deficient mice are more susceptible to heart failure

• CHF1 myocardial transgenic mice are less susceptible to heart failure

My Project

• Hypothesis: differences of mitochondrial complex 1 activity may explain the phenotypic difference in susceptibility to heart failure in the myocardial specific CHF1 deficient mouse model

• Why investigate complex 1?

• Who cares / to what end?

In heart failure, decreased mitochondrial content and overall mitochondrial dysfunction exist

There are increased RNA transcripts of complex 1 in CHF1 transgenic mice

Preliminary data suggests decreased mitochondrial respiration in CHF1 deficient mice

Complex I is a manageable target

Why Test Complex 1 ?

Mitochondria

Mitochondria generate most of a cell’s ATP.

Mitochondria have a more broad function Made of 4 complexes and ATP synthase

http://en.wikipedia.org/wiki/File:ETC_electron_transport_chain.svg

Complex I

Largest complex – 45 different polypeptides

Redox reaction –

NADH + H(+) + ubiquinone (Q)

NAD(+) + dihydroubiquinone (QH2)

Who Cares / to what end?

Further characterize the mechanism responsible for increased susceptibility to heart failure of CHF1 deficient mice

Further understand mitochondria, which may offer therapeutic targets in the future

Overview of My Project

1. Breed wild-type controls and myocardial-specific CHF1 deficient mice

2. Isolate left ventricular mitochondrial tissue

3. Measure complex 1 activity via spectrophotometry

4. Normalize this to total mitochondrial activity

5. Compare the two groups

Spectrophotometry for Complex 1 Beer - Lambert Law Measure light absorbance of a material

to calculate concentration. For complex 1:

NADH + H(+) + ubiquinone (Q)

NAD(+) + dihydroubiquinone (QH2)

http://www.adinstruments.com/solutions/experiments/labchart_experiments/full.php?exp_id=187&section_id=13&name_id=245&template=teaching

Results

Genotype C1 activity C1/CSF/F aMHC-Cre 639.34 1.602F/F aMHC-Cre 567.73 1.836F/F aMHC-Cre 255.73 2.178F/F aMHC-Cre 506.35 1.61F/F aMHC-Cre 673.43 1.757F/F 427.93 1.932F/F 603.53 2.168F/F 499.54 2.055F/F 368.26 1.921F/F 334.16 1.802F/F 339.27 1.857F/F 521.7 2.3F/F 663.21 1.985F/F 579.24 1.428

-C1 = complex 1. CS = citrate synthase. -C1 Activity measured in nm/ (min *mg)

Results

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Complex 1 Activity

CHF1 DeficientWT

P Value: 0.314564499

Conclusions

Complex 1 activity is not significantly different between wild-type and myocardial specific CHF-1 deficient mice

Thus, differences in complex 1 are unlikely to explain the increased susceptibility to heart failure between these groups

Future Avenues

CHF1 deficient mice have abnormal with calcium signaling and apoptosis. Further studies into mitochondrial dysfunction may lead to better understandings of these mechanisms.