[Aging-associated changes in cardiac function and their prevention].

Aging associated changes in cardiac contraction, Ca(2+) homeostasis and their neurohumoral regulations have been studied using rodents. The changes can be understood not only to reduce energy consumption but also to be susceptible to Ca(2+) overload, possibly leading arrhythmia and cell death. N-3 polyunsaturated fatty acid is reported to prevent cardiac Ca(2+) overload. Its content in the cellular membrane is reduced in aged cardiomyocytes, suggesting that the aging-associated change in n-3 fatty acid content is related to the changes in cardiac function. Ingestion balance between n-3 and n-6 fatty acids may affect on the risk of cardiovascular diseases associated with aging.

Overexpression of diacylglycerol kinase zeta inhibits endothelin-1-induced decreases in Ca2+ transients and cell shortening in mouse ventricular myocytes.

Endothelin-1 (ET-1) is released in various cardiovascular disorders including congestive heart failure, and may modulate significantly the disease process by its potent action on vascular and cardiac muscle cell function and gene regulation. In adult mouse ventricular cardiomyocytes loaded with indo-1, ET-1 induced a sustained negative inotropic effect (NIE) in association with decreases in Ca(2+) transients. The ET-1-induced effects on Ca(2+) transients and cell shortening were abolished in diacylglycerol (DAG) kinase zeta-overexpressing mouse ventricular myocytes. A nonselective protein kinase C (PKC) inhibitor, GF109203X, inhibited the ET-1-induced decreases in Ca(2+) transients and cell shortening in concentration-dependent manners, whereas a selective Ca(2+)-dependent PKC inhibitor, Gö6976, did not affect the ET-1-induced effects. A phospholipase Cbeta inhibitor, U73122, and an inhibitor of phospholipase D, C(2)-ceramide, partially, but significantly, attenuated the ET-1-induced effects. Derivatives of the respective inhibitors with no specific effects, U73343 and dihydro-C(2)-ceramide, did not affect the ET-1-induced effects. Taken together, these results indicate that activation of a Ca(2+)-independent PKC isozyme by 1,2-DAG, which is generated by phospholipase Cbeta and phospholipase D activation and inactivated by phosphorylation via DAG kinase, is responsible for the ET-1-induced decreases in Ca(2+) transients and cell shortening in mouse ventricular cardiomyocytes.

Endocardial endothelium-dependent positive inotropy by Ca2+ pump inhibitors: possible involvement of store-operated Ca2+ entry.

Positive inotropy by sarcoplasmic/endoplasmic reticulum Ca(2+) pump inhibitors was found and its mechanisms were analyzed pharmacologically. Thapsigargin and cyclopiazonic acid produced positive inotropy in isolated mouse left atria. The responses were inhibited by pretreatment of the endocardial surface with Triton X-100 or by indomethacin, which suggests that the inotropic responses were mediated by prostaglandin(s) released from the endocardial endothelium as well as acetylcholine-induced positive inotropy. The thapsigargin- and acetylcholine-induced positive inotropy was significantly inhibited by Gd(3+), La(3+) and lavendustin A, a tyrosine kinase inhibitor, but not by Ni(2+) and LOE908, a non-selective cation channel inhibitor. Gd(3+) and lavendustin A had no effect on the exogenously applied PGF(2)alpha-induced positive inotropy. In addition, acetylcholine did not induce any positive inotropy when applied after the application of thapsigargin. These results strongly suggest that thapsigargin- as well as acetylcholine-induced prostaglandin release from endocardial endothelium is mediated by store-operated Ca(2+) entry through Gd(3+)-sensitive channels and activation of tyrosine kinase.

Kv channels contribute to nitric oxide- and atrial natriuretic peptide-induced relaxation of a rat conduit artery.

The role of K(+) channels in nitric oxide (NO)-induced vasorelaxation has been largely investigated in resistance vessels where iberiotoxin-sensitive MaxiK channels play a predominant role. However, the nature of the K(+) channel(s) involved in the relaxation triggered by NO-releasing compounds [nitroglycerin, NTG; NOR 3 [(+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide]] or atrial natriuretic peptide (ANP) in the conduit vessel aorta has remained elusive. We now demonstrate that, in rat aorta, the relaxation due to these vasorelaxants is not affected by the MaxiK channel blocker iberiotoxin (10(-7)-10(-6) M) as was the control vascular bed used (mesenteric artery). The inability of iberiotoxin to prevent NO/ANP-induced aortic relaxations was not due to lower expression of MaxiK in aorta or due to the predominance of iberiotoxin-resistant channels in this conduit vessel. Aortic relaxations were strongly diminished by 4-aminopyridine (4-AP) (> or =5 x 10(-3) M) or by tetraethylammonium (>2 x 10(-3) M) at concentrations known to inhibit voltage-dependent K(+) (K(v)) 2-type channels but not by other K(+) channel inhibitors, glibenclamide, apamin, charybdotoxin, tertiapin, or E-4031 N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl-]carbonyl]phenyl]methanesulfonamide dihydrochloride). Consistent with a role of K(v)2-type channels, K(v) currents in A7r5 aortic myocytes were stimulated by NTG and inhibited by > or =5 x 10(-3) M 4-AP. Furthermore, immunocytochemistry, immunoblot, and real-time polymerase chain reaction analyses confirmed the presence of K(v)2.1 channels in aorta. K(v)2.1 transcripts were approximately 100-fold more abundant than K(v)2.2. Our results support low-affinity 4-AP-sensitive K(v) channels, assembled at least partially by K(v)2.1 subunit, as downstream effectors of NO/ANP-signaling cascade regulating aortic vasorelaxation and further demonstrate vessel-specific K(+) channel involvement in NO/ANP-induced relaxation.

Cardiac-specific overexpression of diacylglycerol kinase zeta prevents Gq protein-coupled receptor agonist-induced cardiac hypertrophy in transgenic mice.

BACKGROUND: Diacylglycerol is a lipid second messenger that accumulates in cardiomyocytes when stimulated by Gqalpha protein-coupled receptor (GPCR) agonists such as angiotensin II, phenylephrine, and others. Diacylglycerol functions as a potent activator of protein kinase C (PKC) and is catalyzed by diacylglycerol kinase (DGK) to form phosphatidic acid and inactivated. However, the functional roles of DGK have not been previously examined in the heart. We hypothesized that DGK might prevent GPCR agonist-induced activation of diacylglycerol downstream signaling cascades and subsequent cardiac hypertrophy. METHODS AND RESULTS: To test this hypothesis, we generated transgenic (DGKzeta-TG) mice with cardiac-specific overexpression of DGKzeta. There were no differences in heart size and heart weight between DGKzeta-TG and wild-type littermate mice. The left ventricular function was normal in DGKzeta-TG mice. Continuous administration of subpressor doses of angiotensin II and phenylephrine caused PKC translocation, gene induction of atrial natriuretic factor, and subsequent cardiac hypertrophy in WT mice. However, in DGKzeta-TG mice, neither translocation of PKC nor upregulation of atrial natriuretic factor gene expression was observed after angiotensin II and phenylephrine infusion. Furthermore, in DGKzeta-TG mice, angiotensin II and phenylephrine failed to increase cross-sectional cardiomyocyte areas and heart to body weight ratios. Phenylephrine-induced increases in myocardial diacylglycerol levels were completely blocked in DGKzeta-TG mouse hearts, suggesting that DGKzeta regulated PKC activity by controlling cellular diacylglycerol levels. CONCLUSIONS: These results demonstrated the first evidence that DGKzeta negatively regulated the hypertrophic signaling cascade and resultant cardiac hypertrophy in response to GPCR agonists without detectable adverse effects in in vivo hearts.

Functional and molecular evidence of MaxiK channel beta1 subunit decrease with coronary artery ageing in the rat.

Large-conductance, voltage- and Ca2+ -activated K+ channels (MaxiK, BK) are key regulators of vascular tone. Vascular MaxiK are formed by the pore-forming alpha subunit and the modulatory beta1 subunit, which imprints unique kinetics, Ca2+/voltage sensitivities and pharmacology to the channel. As age progresses, alpha subunit functional expression and protein levels diminish in coronary myocytes. However, whether ageing modifies beta1 subunit expression or the mechanism of alpha subunit reduction is unknown. Thus, we examined functional and pharmacological characteristics of MaxiK, as well as alpha and beta1 transcript levels in coronary myocytes from young and old F344 rats. The mechanism of age-dependent alpha subunit protein reduction involves its transcript downregulation. A corresponding loss of beta1 transcripts was also detected in old myocytes, suggesting a proportional age-dependent decrease of beta1 to alpha subunit protein. Indeed, MaxiK channel properties, defined by coassembly of beta1 and alpha subunits, were equivalent in young versus old, for example in terms of (i) activation kinetics, (ii) sensitivity to Ca2+ levels > 1 microm (iii) dehydrosoyasaponin-I-induced activation, and (iv) iberiotoxin blockade. Consistent with less MaxiK expression/function in older myocytes, the ability of iberiotoxin to contract coronary rings was reduced approximately 50% with ageing confirming our previous findings. 5-Hydroxytryptamine (5-HT) contractile efficacy was reduced by iberiotoxin pretreatment in young > old coronary arteries (explained by larger iberiotoxin-induced contraction and decreased dynamic range for 5-HT contraction in young versus old) with no apparent differences in nitroglycerine-induced relaxation. We propose that the age-related MaxiK reduction involves a parallel decrease of alpha and beta1 functional expression via a transcript downregulatory mechanism; a major impact on basal and possibly stimulated coronary contraction may contribute to altered coronary flow regulation and coronary morbidity in the elderly.

Function and clustered expression of MaxiK channels in cerebral myocytes remain intact with aging.

The incidence of stroke increases significantly in the aging population where stroke related deaths boost at >75 years and survivors are often permanently disabled. Aging is known to decrease cerebral blood flow likely due to an increase in arterial tone. Although MaxiK channels are key regulators of cerebral arterial tone their pattern of expression and function in cerebral blood vessels during aging is unknown. Using specific antibodies against the alpha-subunit of MaxiK channels and current recordings, we now demonstrate that in aging cerebral myocytes, MaxiK channels remain healthy. Furthermore, we show for the first time that in the vasculature, MaxiK channels are expressed in clusters. Clusters have an estimated radius of approximately 200 nm in young rats (3-5 month old Fisher 344 rats) which remains normal in old (25-30 month rats) cerebral myocytes. Consistent with a healthy MaxiK channel expression in old cerebral arteries, MaxiK current density, kinetics and Ca(2+) sensitivity were practically identical in young and old myocytes. Sensitivity to nanomolar concentrations of dehydrosoyasaponin-I that activates channels formed by alpha and beta subunits is also the same in young and old myocytes. These results demonstrate that MaxiK channels maintain normal expression during cerebral aging which is in sharp contrast to our previous finding of loss of expression in aging coronary arteries. It seems therefore, that cerebral myocytes have developed a protective anti-aging mechanism leading to the continued expression of MaxiK channels.

Inhibition of agonist-induced positive inotropy by a selective Rho-associated kinase inhibitor, Y-27632.

We examined the effect of Y-27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclo-hexanecarboxamide), a selective Rho-associated kinase (ROCK) inhibitor, on agonist-induced inotropy in isolated mouse left atria. Endothelin-1, angiotensin-II, and prostaglandin F(2)(alpha) (PGF(2)(alpha)) produced positive inotropy, which was significantly attenuated by Y-27632 (100 microM). On the other hand, isoproterenol-induced positive inotropy was not attenuated by the drug. These results provide the first evidence that the Rho/ROCK pathway is involved in endothelin-1-, angiotensin-II-, and PGF(2)(alpha)-induced positive inotropy, but not in beta-adrenoceptor-mediated positive inotropy.

Pharmacological evidence for involvement of phospholipase D, protein kinase C, and sodium-calcium exchanger in alpha-adrenoceptor-mediated negative inotropy in adult mouse ventricle.

The intracellular signalling pathway for alpha-adrenoceptor-mediated negative inotropy was studied pharmacologically in isolated adult mouse ventricle. The negative inotropy was inhibited by GF-109203X, a nonselective protein kinase C inhibitor. Phorbol 12-myristate 13-acetate also produced sustained negative inotropy, which was inhibited by KB-R7943, a Na(+)/Ca(2+) exchanger inhibitor. The alpha-adrenoceptor-mediated negative inotropy was augmented by RHC-80267, a diacylglycerol lipase inhibitor, but was inhibited either by C(2)-ceramide, a phospholipase D inhibitor, and high concentration of propranolol (50 micro M), which inhibits phosphatidate phosphohydrolase. The inotropy was not affected by U-73122, a phospholipase C inhibitor. Lavendustin-A, a tyrosine kinase inhibitor, also inhibited the negative inotropy. These findings suggest that alpha-adrenoceptor-mediated negative inotropy in adult mouse ventricle is mediated by activation of tyrosine kinase, the phospholipase D-phosphatidate phosphohydrolase pathway, and protein kinase C.

Possible involvement of prostaglandins F(2alpha) and D(2) in acetylcholine-induced positive inotropy in isolated mouse left atria.

The inotropic action of prostaglandins PGF(2alpha), PGD(2) and PGE(2) on isolated mouse left atria was characterized and compared with the positive inotropic action of acetylcholine, which has previously been shown to be mediated by prostaglandins released from the endocardial endothelium. PGF(2alpha), PGD(2) and PGE(2) produced positive inotropic responses; the time course of the change in contractile force induced by PGF(2alpha) and PGD(2) was about the same as that by acetylcholine, while that by PGE(2) was slower. Fluprostenol and sulprostone, FP and EP receptor agonists, respectively, had positive inotropic effects while BW-245C, a DP receptor agonist, had no effect. AH-6809, a DP receptor antagonist, had no inhibitory effect on the positive inotropic response to PGD(2). Dimethylamiloride, an inhibitor of Na(+)/H(+) exchange, inhibited the positive inotropic response to PGF(2alpha), PGD(2) and acetylcholine, but not PGE(2). Fluorometric pH measurement with carboxy-SNARF-1-loaded atrial myocytes revealed no change in intracellular pH on application of PGF(2alpha). PGF(2alpha) and PGD(2) significantly prolonged the duration of the atrial action potential while PGE(2) had no significant effect. These findings suggest that prostaglandins induce positive inotropic response in mouse atria through FP and EP receptor stimulation and that the former mechanism mediates in part the positive inotropic response to acetylcholine.

Aging, ion channel expression, and vascular function.

Cardiovascular disease remains the leading cause of death in the United States, and aging is one of the main risk factors for its development. Coronary arteries nurture the heart, but as age progresses, they suffer changes that make them stiffer, thicker, and with higher spontaneous contractile activity. Even in the absence of pathological atherosclerotic lesions, these changes make the coronary arteries at risk for vasospasm and the individual at risk for myocardial ischemia and heart failure. Thus, knowledge of the molecular mechanisms involved in the vascular physiology, disease, and aging of the coronary circulation is required to develop strategies to preserve the quality of life of an increasingly aging population. One of the key factors that regulate coronary arterial tone is the activity of K+ channels in the vascular smooth muscle cells (SMCs). In particular, voltage-dependent and Ca(2+)-activated K+ (BKCa) channels, which are abundant in the coronary SMCs, are targets of vasoconstrictors and vasorelaxants, and play a key role in determining arterial tone and diameter. Aging induces a reduction in the density of the alpha-subunit of BKCa channels in coronary smooth muscle, lowers baseline endothelial release of the relaxant nitric oxide (NO), and increases the response to endothelial constrictor factors and K+. Thus, aging induces the remodeling of important proteins involved in the excitability and contractility of the coronary circulation. Altogether, these changes increase the risk of coronary artery vasospasm, myocardial ischemia, and infarct in the elderly.

Coupling of c-Src to large conductance voltage- and Ca2+-activated K+ channels as a new mechanism of agonist-induced vasoconstriction.

The voltage-dependent and Ca(2+)-activated K(+) channel (MaxiK, BK) and the cellular proto-oncogene pp60(c-Src) (c-Src) are abundant proteins in vascular smooth muscle. The role of MaxiK channels as a vasorelaxing force is well established, but their role in vasoconstriction is unclear. Because Src participates in regulating vasoconstriction, we investigated whether c-Src inhibits MaxiK as a mechanism for agonist-induced vasoconstriction. Functional experiments in human and rat show that inhibitors of Src (Lavendustin A, PP2) but not inactive compounds (Lavendustin B, PP3) induce a pronounced relaxation of coronary or aortic smooth muscle precontracted with 5-hydroxytriptamine, phenylephrine, or Angiotensin II. Iberiotoxin, a MaxiK blocker, antagonizes the relaxation induced by Lavendustin A or PP2, indicating that c-Src inhibits the Iberiotoxin-sensitive component, likely MaxiK channels. In agreement, coronary muscle MaxiK currents were enhanced by Lavendustin A. To investigate the molecular mechanism of c-Src action on MaxiK channels, we transiently expressed its alpha subunit, hSlo, with or without c-Src in HEK293T cells. The voltage sensitivity of hSlo was right-shifted by approximately 16 mV. hSlo inhibition by c-Src is due to channel direct phosphorylation because: (i) excised patches exposed to protein tyrosine phosphatase (CD45) resulted in a partial reversal of the inhibitory effect by approximately 10 mV, and (ii) immunoprecipitated hSlo channels were recognized by an anti-phosphotyrosine Ab. Furthermore, coexpression of hSlo and c-Src demonstrate a striking colocalization in HEK293T cells. We propose that MaxiK channels via direct c-Src-dependent phosphorylation play a significant role supporting vasoconstriction after activation of G protein-coupled receptors by vasoactive substances and neurotransmitters.

Effect of SEA0400, a novel inhibitor of sodium-calcium exchanger, on myocardial ionic currents.

The effects of 2-[4-[(2,5-difluorophenyl) methoxy]phenoxy]-5-ethoxyaniline (SEA0400), a newly synthesized Na(+)-Ca(2+) exchanger (NCX) inhibitor, on the NCX current and other membrane currents were examined in isolated guinea-pig ventricular myocytes and compared with those of 2-[2-[4-(4-nitrobenzyloxy) phenyl]ethyl]isothiourea (KB-R7943). SEA0400 concentration-dependently inhibited the NCX current with a 10 fold higher potency than that of KB-R7943; 1 microM SEA0400 and 10 microM KB-R7943 inhibited the NCX current by more than 80%. KB-R7943, at 10 microM, inhibited the sodium current, L-type calcium current, delayed rectifier potassium current and inwardly rectifying potassium current by more than 50%, but SEA0400 (1 microM) had no significant effect on these currents. These results indicate that SEA0400 is a potent and highly selective inhibitor of NCX, and would be a powerful tool for further studies on the role of NCX in the heart and the therapeutic potential of its inhibition.