Abstract
Cardiac-directed expression of AC6 has pronounced favorable effects on cardiac function possibly not linked with cAMP production. To determine rigorously whether cAMP generation is required for the beneficial effects of increased AC6 expression, we generated a catalytically inactive AC6 mutant (AC6mut) that has markedly diminished cAMP generating capacity by replacing aspartic acid with alanine at position 426 in the C1 domain (catalytic region) of AC6. Gene transfer of AC6 or AC6mut (adenovirus-mediated) in adult rat cardiac myocytes resulted in similar expression levels and intracellular distribution, but AC6mut expression was associated with marked reduction in cAMP production. Despite marked reduction in cAMP generation, AC6mut influenced intracellular signaling events similarly to that observed after expression of catalytically intact AC6. For example, both AC6 and AC6mut reduced phenylephrine-induced cardiac myocyte hypertrophy and apoptosis (p < 0.001), expression of cardiac ankyrin repeat protein (p < 0.01), and phospholamban (p < 0.05). AC6mut expression, similar to its catalytically intact cohort, was associated with increased Ca2+ transients in cardiac myocytes after isoproterenol stimulation. Many of the biological effects of AC6 expression are replicated by a catalytically inactive AC6 mutant, indicating that the mechanisms for these effects do not require increased cAMP generation.
Footnotes
This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants 5P01-HL066941, HL081741, HL088426-01]; a Department of Veterans Affairs merit grant; and American Heart Association Beginning Grant-In-Aid Awards [0765064Y, 0865147F].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.067298.
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ABBREVIATIONS:
- AC
- adenylyl cyclase
- AC6
- adenylyl cyclase type 6
- AC6mut
- adenylyl cyclase type 6 mutant
- PHLPP2
- pleckstrin homology domain leucine-rich repeat protein phosphatase 2
- Ad.AC6
- E1-deleted adenoviruses encoding murine AC6
- Ad.AC6mut
- E1-deleted adenoviruses encoding murine AC6 mutant
- PE
- phenylephrine
- TUNEL
- terminal deoxynucleotidyl transferase dUTP nick-end labeling
- qRT-PCR
- quantitative reverse transcription-polymerase chain reaction
- BNP
- B-type natriuretic peptide
- CARP
- cardiac ankyrin repeat protein
- PLB
- phospholamban
- TnI
- troponin I
- cTnI
- cardiac troponin-I
- CamKII
- calcium/calmodulin-dependent protein kinase II
- ANF
- atrial natriuretic factor
- LV
- left ventricular
- SR
- sarcoplasmic reticulum
- ANOVA
- analysis of variance.
- Received July 1, 2010.
- Accepted December 1, 2010.
- U.S. Government work not protected by U.S. copyright
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