Adenylyl cyclase type V deletion increases basal left ventricular function and reduces left ventricular contractile responsiveness to β–adrenergic stimulation

Abstract

We tested the hypothesis that deletion of adenylyl cyclase type V (AC(V)) would be associated with decreased left ventricular (LV) contractile function and responsiveness to beta-adrenergic receptor (betaAR) stimulation. Absence of cardiac AC(V) expression was confirmed by RT-PCR and immunoblotting in AC(V)-deleted mice (AC(V) (-/-)). Compared to sibling mice with normal amounts of AC(V) (CON), basal and water-soluble forskolin derivative NKH477-stimulated cAMP production was reduced in both LV homogenates and in isolated cardiac myocytes. Basal LV +dP/dt (isolated perfused hearts) was increased (CON: 3,649 +/- 247 mmHg/s; AC(V) (-/-): 4,625 +/- 350 mmHg/s; p = 0.035, n = 10), but the potency of dobutamine on LV +dP/dt was decreased by AC(V) deletion (log EC(50): CON: -6.83 +/- 0.14 M; AC(V) (-/-): -5.99 +/- 0.15 M; p = 0.0007, n = 10). The initial rates of ATP-dependent sarcoplasmic reticulum calcium uptake, assessed in LV homogenates, showed that AC(V) deletion increased SERCA2a affinity for Ca(2+) (log EC(50): CON: -5.94 +/- 0.03 M; AC(V) (-/-): -6.09 +/- 0.02 M; p = 0.001, n = 8). AC(V) deletion is also associated with increased phospholamban phosphorylation, decreased type 1 protein phosphatase catalytic subunit content and activity, and reduced cardiac Galphas protein content. In conclusion, AC(V) deletion has a favorable effect on basal LV function despite reduced cAMP levels. Increased SERCA2a affinity for Ca(2+) and increased phospholamban phosphorylation are contributing factors. However, AC(V) deletion is associated with reduced LV contractile responsiveness to betaAR stimulation, an effect that is associated with reduced Galphas protein content and reduced cAMP generating capacity in cardiac myocytes.

Adenylyl Cyclase Type 6 Deletion Decreases Left Ventricular Function via Impaired Calcium Handling

“…11 The primer pairs used for PCR analysis were designed to cross intron-exon boundaries to eliminate potential contamination of genomic DNA. Details of PCR condition and primer sequences are listed in the online-only Data Supplement.…”

“…Cardiac myocytes were isolated from adult mice as described previously 11 and plated on laminin-coated 25-mm glass coverslips. Cells were loaded with the Ca 2ϩ -sensitive fluorescent indicator Fura-2 AM (3 mol/L) at 37°C for 30 minutes, with 5% CO 2 in the dark.…”

“…Fura-2 fluorescence was detected as 510-nm-wavelength light emission with excitation wavelengths of 340 and 380 nm by use of the digital fluorescence imaging system from Intracellular Imag- cAMP Generation cAMP production in LV homogenates and in isolated cardiac myocytes was measured as described previously. 11,17 Additional experiments to measure cAMP production were also performed with myocardial homogenates from neonatal mice (1 day Activities of intrinsic protein kinase A (PKA) and protein phosphatases 1 (PP1) and 2A (PP2A) were measured as described previously. 11,16 Western blotting was performed as described previously.…”

“…11,17 Additional experiments to measure cAMP production were also performed with myocardial homogenates from neonatal mice (1 day Activities of intrinsic protein kinase A (PKA) and protein phosphatases 1 (PP1) and 2A (PP2A) were measured as described previously. 11,16 Western blotting was performed as described previously. 16 Additional experiments were conducted with LV samples from isolated hearts perfused with dobutamine (10 mol/L, 5 minutes).…”

Pharmacological Stimulation of Type 5 Adenylyl Cyclase Stabilizes Heart Rate Under Both Microgravity and Hypergravity Induced by Parabolic Flight

“…AC5 is responsible for at least one-third of the cardiac AC catalytic activity and plays an important role not only in sympathetic, but also parasympathetic cardiac function and heart rate without affecting Gi-gated K + -channel current, which regulates normal pacing activity, as shown recently by us and others in AC5KO (7,29). Overexpressing AC6 results in maintained basal HR (30), while overexpressing AC5 results in increased basal HR (13,31), suggesting that AC5 may play an important role in regulating HR through the parasympathetic arm of the autonomic nervous system, as well as the sympathetic arm.…”