970 8-Bromo-cGMP Reduces the Myofilament Response to Ca 21 in Intact Cardiac Myocytes

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 grmol/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°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 gm, 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 he role of cGMP in regulating myocardial contraction has been controversial. ' Myocardial cGMP is known to be elevated by cholinergic stimulation,2 but this effect has not been clearly correlated with inotropic activity. This is perhaps not surprising, since cholinergic agonists have several cellular actions that may be independent of cGMP, eg, phosphatidylinositol hydrolysis and modulation of K' channels.3 Other studies have either reported depression or no change in myocardial peak isometric force after the addition of exogenous cGMP or lipid-soluble analogues.4-7 In some cases, negative inotropic effects have been observed only when preparations have been prestimulated with cAMP-elevating interventions,8 suggesting interaction between cGMPand cAMP-mediated mechanisms. Recent studies by two groups suggest that elevation of cGMP results in relatively subtle contractile effects influencing predominantly myocardial relaxation: the onset of relaxation occurs earlier with a consequent slight reduction in peak force but with relatively little or no effect on the early systolic phase of contraction, eg, no change in the rate of force development or in maximal unloaded shortening velocity.9-11 These effects, observed both in ferret and cat Received July 1, 1993; accepted December 22, 1993. From the Laboratory of Cardiovascular Science (A.M.S., H.A.S., S.J.S., A.T., E.G.L.), Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Md; and the Department of Cardiology (A.M.S.), University of Wales College of Medicine, Heath Park, Cardiff, UK. Correspondence to Dr A.M. Shah, Department of Cardiology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK, or Laboratory of Cardiovascular Science, GRC/NIA, National Institutes of Health, 4940 Eastern Ave, Baltimore, MD 21224. 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 Ca 2+ in all cases. These 8bcGMP effects were inhibited by KT5823 (1 jumol/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. (Circ Res. 1994; 74:970-978.)