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8-bromo-cGMP reduces the myofilament response to Ca2+ in intact cardiac myocytes
A M Shah1, H A Spurgeon, S J Sollott
1Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224.
Insights
Cyclic guanosine monophosphate (cGMP) may influence heart muscle relaxation and diastolic tone. This study found that cGMP reduces the myofilament response to calcium, likely through cGMP-dependent protein kinase.
Area of Science:
- Cardiovascular Physiology
- Molecular Cardiology
- Cellular Biology
Background:
- The precise role of cyclic guanosine monophosphate (cGMP) in regulating myocardial contraction remains unclear.
- Emerging evidence suggests nitric oxide-mediated increases in cGMP impact cardiac contractility.
Purpose of the Study:
- To investigate the direct effects of 8-bromo-cGMP (8bcGMP) on adult rat ventricular myocyte contraction and intracellular calcium handling.
- To elucidate the signaling pathways involved in cGMP's modulation of cardiac function.
Main Methods:
- Isolated adult rat ventricular myocytes were treated with 8bcGMP (50 µmol/L).
- Myocyte contraction (cell shortening) and intracellular Ca2+ transients (using Indo-1 fluorescence) were measured simultaneously.
- cGMP-dependent protein kinase inhibition (KT5823) and beta-adrenergic stimulation (isoproterenol) were used to probe signaling pathways.
Main Results:
- 8bcGMP significantly reduced myocyte twitch amplitude and hastened relaxation, while increasing diastolic cell length.
- No significant effects were observed on shortening velocity or the kinetics of intracellular Ca2+ transients.
- Analysis revealed a reduced myofilament responsiveness to Ca2+ in the presence of 8bcGMP.
- These effects were attenuated by KT5823 and isoproterenol, suggesting involvement of cGMP-dependent protein kinase.
Conclusions:
- cGMP likely modulates myocardial relaxation and diastolic tone by decreasing the myofilament sensitivity to Ca2+.
- This modulation is probably mediated by the cGMP-dependent protein kinase signaling pathway.
Abstract:
The role of cGMP in myocardial contraction is not established. Recent reports suggest that nitric oxide, released by endothelial cells or within myocytes, modifies myocardial contraction by raising cGMP. We studied the effects of 8-bromo-cGMP (8bcGMP, 50 mumol/L) on contraction (cell shortening) and simultaneous intracellular Ca2+ transients (indo 1 fluorescence ratio) in intact adult rat ventricular myocytes (0.5 Hz and 25 degrees C) 8bcGMP reduced myocyte twitch amplitude and time to peak shortening (-19.6 +/- 4.2% and -17.6 +/- 1.3%, respectively) and increased steady-state diastolic cell length (+0.6 +/- 0.1 microns, mean +/- SEM, n = 8; all P < .05) but had no effect on shortening velocity, systolic or diastolic fluorescence ratio, or time to peak fluorescence ratio (all P = NS). In 7 of 13 myocytes, this negative inotropic effect was preceded by a transient positive inotropic effect, with small increases in twitch amplitude, shortening velocity, and cytosolic Ca2+ transient. Analysis of 8bcGMP effects on both the dynamic and steady-state relation between cell shortening and intracellular Ca2+ (during twitch contraction and tetanic contraction, respectively) indicated reduction in the myofilament response to Ca2+ in all cases. These 8bcGMP effects were inhibited by KT5823 (1 mumol/L), an inhibitor of cGMP-dependent protein kinase, or by the presence of isoproterenol (3 nmol/L). 8bcGMP had no effect on cytosolic pH in cells (n = 4) loaded with the fluorescent probe carboxyseminaphthorhodafluor-1. These data indicate that cGMP may modulate myocardial relaxation and diastolic tone by reducing the relative myofilament response to Ca2+, probably via cGMP-dependent protein kinase.