Adenylyl Cyclases (ACs) are important signal transduction protein molecules and are responsible for production of the secondary messenger, cyclic AMP. Of the 9 isoforms of ACs, ACV and ACVI are the most abundant in the mammalian heart but ACVI expression is lower than ACV in adult rat ventricle. The aim was to overexpress ACVI in order to investigate its role in the regulation of contractility and receptor coupling in adult rat ventricular cardiomyocytes.

The 5 Kb ACVI cDNA was cloned in the sense or antisense orientations into pShuttle. Its entire expression cassette was excised using PI-SceI and I-CeuI and cloned into pAdeno-X for adenovirus packaging. Myocytes were isolated, cultured and transfected for 48 h with adenovirus carrying sense ACVI at 100 MOI (Multiplicity Of Infection), antisense ACVI at 1000 MOI (AS), sense plus antisense (SAS), or a control virus with GFP (Green Fluorescent Protein) at 100 and 1000 MOI. Overexpression of ACVI was detected as a 139 KDa band by Western blotting using rabbit polyclonal anti-mouse ACVI antibodies. Single myocytes were isolated as previously described (Chaudhri et al. 2003). Intracellular cyclic AMP levels were measured using a specific cyclic AMP-antibody competition assay (Amersham Biosciences, Little Chalfont, Buck). Measurement of contraction amplitude (% shortening) of myocytes was as previously described (Gong et al. 2000). Carbachol, an inhibitor of cAMP production was also used to establish whether increased contraction in ACVI myocytes was cyclic AMP-dependent

Basal amplitudes of myocyte contraction were increased in ACVI myocytes compared to GFP (% shortening, GFP vs ACVI: 1.90 ± 1.36 vs 3.91 ± 2.29, P < 0.0001): this increase was reduced by carbachol. AS and SAS myocytes showed no significant difference from GFP myocytes in basal contraction. ACVI overexpression decreased the time to 50% relaxation (GFP = 62.6 ± 24.2 ms (n = 50), ACVI = 45.0 ± 17.2 ms (n = 248), P < 0.0001). ACVI-overexpressing myocytes produced two-fold higher basal cAMP levels than GFP myocytes (GFP = 6.60 ± 0.98, ACVI = 14.2 ± 2.1 fmol/viable cell, n = 4).

Cumulative concentration-response curves to isoprenaline and forskolin were constructed on individual myocytes. ACVI myocytes showed increased amplitudes compared to GFP in response to forskolin but not isoprenaline (a full agonist at ß1- and ß2 AR). The increase in maximum contraction amplitude was significant for forskolin (Emax: GFP vs. ACVI 6.70 ± 1.59% (n=6) vs 9.06 ± 0.69% (n= 14), P < 0.01). Myocytes were pre-incubated for 15 min in either 50 nM ICI 118,551 or 300 nM CGP20712A prior to stimulation of ßARs. Responses to the partial agonist, xamoterol, under ß1AR selective conditions (+ 50 nM ICI 118, 551) were increased in ACVI myocytes. This effect was seen even when the raised basal contraction was subtracted (Emax: GFP vs ACVI: 1.49 ± 0.15 vs 4.06 ± 0.53%; n = 6, P < 0.001). In contrast, isoprenaline-induced contractions under ß2AR selective conditions (+ 300 nM CGP20712A) were lower in ACVI than GFP myocytes.

Overexpression of ACVI constitutively increases myocyte contraction by increasing steady-state cAMP levels. The effect on contraction by agents, such as forskolin, which act through cyclic AMP is therefore increased. The increased response to xamoterol in ACVI myocytes indicated a possible preferential effect on ß1AR responses. This suggests closer coupling of ACVI to ß1ARs than to ß2ARs in adult rat ventricle.

Chaudhri, B. et al. (2003). Mol Cell Biochem 251: 103-09.
Gong, H. et al (2000).Br .J.Pharmacol. 131.3: 594-600.