Stereochemical requirements for the mineralocorticoid receptor antagonist activity of dihydropyridines.

A number of known 1,4-dihydropyridine CCBs were identified as having comparable potency to the steroidal MR antagonist eplerenone. Chiral resolution of mebudipine revealed that MR and CCB activity reside in opposite enantiomers. Small molecule X-ray crystal structures showed that the C4 stereochemistry of optimized selective MR analogues, e.g. 5, is consistent with MR-active mebudipine. Molecular modeling supports a binding pose consistent with that previously proposed for DHP diesters.

Palmitate attenuates myocardial contractility through augmentation of repolarizing Kv currents.

There is considerable evidence to support a role for lipotoxicity in the development of diabetic cardiomyopathy, although the molecular links between enhanced saturated fatty acid uptake/metabolism and impaired cardiac function are poorly understood. In the present study, the effects of acute exposure to the saturated fatty acid, palmitate, on myocardial contractility and excitability were examined directly. Exposure of isolated (adult mouse) ventricular myocytes to palmitate, complexed to bovine serum albumin (palmitate:BSA) as in blood, rapidly reduced (by 54+/-4%) mean (+/-SEM) unloaded fractional cell shortening. The amplitudes of intracellular Ca(2+) transients decreased in parallel. Current-clamp recordings revealed that exposure to palmitate:BSA markedly shortened action potential durations at 20%, 50%, and 90% repolarization. These effects were reversible and were occluded when the K(+) in the recording pipettes was replaced with Cs(+), suggesting a direct effect on repolarizing K(+) currents. Indeed, voltage-clamp recordings revealed that palmitate:BSA reversibly and selectively increased peak outward voltage-gated K(+) (Kv) current amplitudes by 20+/-2%, whereas inwardly rectifying K(+) (Kir) currents and voltage-gated Ca(2+) currents were unaffected. Further analyses revealed that the individual Kv current components I(to,f), I(K,slow) and I(ss), were all increased (by 12+/-2%, 37+/-4%, and 34+/-4%, respectively) in cells exposed to palmitate:BSA. Consistent with effects on both components of I(K,slow) (I(K,slow1) and I(K,slow)(2)) the magnitude of the palmitate-induced increase was attenuated in ventricular myocytes isolated from animals in which the Kv1.5 (I(K,slow)(1)) or the Kv2.1 (I(K,slow)(2)) locus was disrupted and I(K,slow)(1) or I(K,slow2) is eliminated. Both the enhancement of I(K,slow) and the negative inotropic effect of palmitate:BSA were reduced in the presence of the Kv1.5 selective channel blocker, diphenyl phosphine oxide-1 (DPO-1).Taken together, these results suggest that elevations in circulating saturated free fatty acids, as occurs in diabetes, can directly augment repolarizing myocardial Kv currents and impair excitation-contraction coupling.

Ca2+-independent alterations in diastolic sarcomere length and relaxation kinetics in a mouse model of lipotoxic diabetic cardiomyopathy.

Previous studies demonstrated increased fatty acid uptake and metabolism in MHC-FATP transgenic mice that overexpress fatty acid transport protein (FATP)1 in the heart under the control of the alpha-myosin heavy chain (alpha-MHC) promoter. Doppler tissue imaging and hemodynamic measurements revealed diastolic dysfunction, in the absence of changes in systolic function. The experiments here directly test the hypothesis that the diastolic dysfunction in MHC-FATP mice reflects impaired ventricular myocyte contractile function. In vitro imaging of isolated adult MHC-FATP ventricular myocytes revealed that mean diastolic sarcomere length is significantly (P<0.01) shorter than in wild-type (WT) cells (1.79+/-0.01 versus 1.84+/-0.01 microm). In addition, the relaxation rate (dL/dt) is significantly (P<0.05) slower in MHC-FATP than WT myocytes (1.58+/-0.09 versus 1.92+/-0.13 microm/s), whereas both fractional shortening and contraction rates are not different. Application of 40 mmol/L 2,3-butadionemonoxime (a nonspecific ATPase inhibitor that relaxes actin-myosin interactions) increased diastolic sarcomere length in both WT and MHC-FATP myocytes to the same length, suggesting that MHC-FATP myocytes are partially activated at rest. Direct measurements of intracellular Ca(2+) revealed that diastolic [Ca(2+)](i) is unchanged in MHC-FATP myocytes and the rate of calcium removal is unexpectedly faster in MHC-FATP than WT myocytes. Moreover, diastolic sarcomere length in MHC-FATP and WT myocytes was unaffected by removal of extracellular Ca(2+) or by buffering of intracellular Ca(2+) with the Ca(2+) chelator BAPTA (100 micromol/L), indicating that elevated intracellular Ca(2+) does not underlie impaired diastolic function in MHC-FATP ventricular myocytes. Functional assessment of skinned myocytes, however, revealed that myofilament Ca(2+) sensitivity is markedly increased in MHC-FATP, compared with WT, ventricular cells. In addition, biochemical experiments demonstrated increased expression of the beta-MHC isoform in MHC-FATP, compared with WT ventricles, which likely contributes to the slower relaxation rate observed in MHC-FATP myocytes. Collectively, these data demonstrate that derangements in lipid metabolism in MHC-FATP ventricles, which are similar to those observed in the diabetic heart, result in impaired diastolic function that primarily reflects changes in myofilament function, rather than altered Ca(2+) cycling.