Distribution and function of sodium channel subtypes in human atrial myocardium.

Voltage-gated sodium channels composed of a pore-forming α subunit and auxiliary ß subunits are responsible for the upstroke of the action potential in cardiac muscle. However, their localization and expression patterns in human myocardium have not yet been clearly defined. We used immunohistochemical methods to define the level of expression and the subcellular localization of sodium channel α and ß subunits in human atrial myocytes. Nav1.2 channels are located in highest density at intercalated disks where ß1 and ß3 subunits are also expressed. Nav1.4 and the predominant Nav1.5 channels are located in a striated pattern on the cell surface at the z-lines together with ß2 subunits. Nav1.1, Nav1.3, and Nav1.6 channels are located in scattered puncta on the cell surface in a pattern similar to ß3 and ß4 subunits. Nav1.5 comprised approximately 88% of the total sodium channel staining, as assessed by quantitative immunohistochemistry. Functional studies using whole cell patch-clamp recording and measurements of contractility in human atrial cells and tissue showed that TTX-sensitive (non-Nav1.5) α subunit isoforms account for up to 27% of total sodium current in human atrium and are required for maximal contractility. Overall, our results show that multiple sodium channel α and ß subunits are differentially localized in subcellular compartments in human atrial myocytes, suggesting that they play distinct roles in initiation and conduction of the action potential and in excitation-contraction coupling. TTX-sensitive sodium channel isoforms, even though expressed at low levels relative to TTX-sensitive Nav1.5, contribute substantially to total cardiac sodium current and are required for normal contractility. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

A collagen α2(I) mutation impairs healing after experimental myocardial infarction.

Collagen breakdown and de novo synthesis are important processes during early wound healing after myocardial infarction (MI). We tested the hypothesis that collagen I, the main constituent of the extracellular matrix, affects wound healing after MI. The osteogenesis imperfecta mouse (OIM), lacking procollagen-α2(I) expression, represents a model of the type III form of the disease in humans. Homozygous (OIM/OIM), heterozygous (OIM/WT), and wild-type (WT/WT) mice were subjected to a permanent myocardial infarction protocol or sham surgery. Baseline functional and geometrical parameters determined by echocardiography did not differ between genotypes. After MI but not after sham surgery, OIM/OIM animals exhibited significantly increased mortality, due to early ventricular rupture between day 3 and 7. Echocardiography at day 1 demonstrated increased left ventricular dilation in OIM/OIM animals. Less collagen I mRNA within the infarct area was found in OIM/OIM animals. At 2 days after MI, MMP-9 expression in the infarct border zone was higher in OIM/OIM than in WT/WT animals. Increased granulocyte infiltration into the infarct border zone occurred in OIM/OIM animals. Neither granulocyte depletion nor MMP inhibition reduced mortality in OIM/OIM animals. In this murine model, deficiency of collagen I leads to a myocardial wound-healing defect. Both structural alterations within pre-existing collagen matrix and impaired collagen de novo expression contribute to a high rate of early myocardial rupture after MI.

Long-term reduction of mortality in the 4-year follow up of tirofiban therapy in elective percutaneous coronary interventions (TOPSTAR) trial.

BACKGROUND: TOPSTAR was a randomized, placebo-controlled trial studying the effects of adding the glycoprotein IIb/IIIa inhibitor tirofiban to conventional treatment with aspirin and clopidogrel in patients undergoing elective percutaneous coronary interventions (PCI). TOPSTAR demonstrated a lower periprocedural troponin release and a reduced 6-month mortality risk following PCI. The present study analyzed the corresponding long-term effects. METHODS: All 96 patients who were initially included were followed for a minimum of 4 years (median follow-up time, 4.3 years). The prespecified endpoints were: 1) all-cause mortality and 2) the combined endpoint of all-cause death, myocardial infarction and target vessel revascularization by intention-to-treat analysis in patients randomly assigned to elective PCI. Survival analyses were carried out using Kaplan-Meier analysis and Cox proportional hazard regression. RESULTS: After 4 years of follow up, no differences were observed between the two groups with respect to medical therapy, NYHA classification and number of reinterventions and target vessel revascularizations. All-cause mortality was still higher in the placebo group (10.9%; 5/46) compared with the tirofiban group (0%; 0/50; Kaplan-Meier log rank = 0.017). The combined endpoint occurred in 21.7% (10/46) in the placebo group versus 8.0% (4/50) in the tirofiban group (Kaplan-Meier log rank = 0.056). CONCLUSIONS: The reduced 6-month mortality risk after elective PCI in the TOPSTAR trial persisted after 4 years of follow up. Even in this relatively small study, periprocedural effective platelet inhibition had a sustained impact on long-term mortality risk.

Protective effects of sphingosine-1-phosphate receptor agonist treatment after myocardial ischaemia-reperfusion.

AIMS: Several experimental studies have demonstrated protection against cardiac ischaemia-reperfusion injury achieved by pre-treatment with exogenous sphingosine-1-phosphate (S1P). We tested the hypothesis that pharmacological S1P receptor agonists improve recovery of function when applied with reperfusion. METHODS AND RESULTS: Isolated rat cardiomyocytes were stimulated with exogenous S1P, the selective S1P1 receptor agonist SEW2871, or the S1P1/3 receptor agonist FTY720. Western blot analysis was performed to analyse downstream signalling pathways. Ischaemia-reperfusion studies were conducted in rat cardiomyocytes, isolated Langendorff-perfused rat hearts, and in human myocardial muscle strip preparations to evaluate the effect of S1P receptor agonists on cell death and recovery of mechanical function. All S1P receptor agonists were able to activate Akt. This was associated with transactivation of the epidermal growth factor receptor. In isolated cardiomyocytes, selective stimulation of the S1P1 receptor by SEW2871 induced protection against cell death when administered either before or after ischaemia-reperfusion. In isolated rat hearts, treatment with FTY720 during reperfusion attenuated the rise in left ventricular end-diastolic pressure (LVEDP) and improved the recovery of left ventricular developed pressure without limiting infarct size. However, selective S1P1 receptor stimulation did not improve functional recovery but rather increased LVEDP. Additional experiments employing a human myocardial ischaemia-reperfusion model also demonstrated improved functional recovery induced by FTY720 treatment during reperfusion. CONCLUSION: Pharmacological S1P receptor agonists have distinct effects on ischaemia-reperfusion injury. Their efficacy when applied during reperfusion makes them potential candidates for pharmaceutical postconditioning therapy after cardiac ischaemia.

Evidence for a negative inotropic effect of obesity in human myocardium?

OBJECTIVE: The present study was performed as an attempt to analyze the relationship between body weight and human myocardial performance. As overweight is frequently associated with hypertension, stenosis of epimyocardial coronary arteries and other factors that influence myocardial performance, the experimental model of isolated human atrial myocardium was selected. Atrial contractile performance does neither depend on the extent of stenosis of epicardial coronary arteries nor on the degree of hypertension and its secondary pathology. METHODS: Right atrial muscle preparations (0.5 x 6 mm) of 183 patients undergoing coronary artery bypass surgery were electrically stimulated at optimal length. Active tension (stimulation) and passive resting tension (relaxation) were measured (measurement conditions: 37 degrees C, Krebs-Henseleit solution, optimal length and supramaximal electrical stimulation). The relationship of body weight with the measured parameters was analyzed statistically by using linear regression model and Student's t-test. RESULTS: Active tension (mN/mm2) and passive resting tension (mN/mm(2)) declined significantly with increasing body weight (p < 0.0001). The ratio passive resting tension/active tension correlated significantly with body weight (p < 0.0001). The negative association between body weight and active tension amplitude was more pronounced in women (p < 0.05). The following linear regression was calculated: for men: force = -0.04 x body weight + 8.74 (R = 0.505, p < 0.0001, n = 106); for women: force = -0.08 x body weight + 12.03 (R = 0.717, p < 0.0001, n = 77). CONCLUSION: The experimental data are in accordance with the hypothesis, that obese tissue may exert a direct cardio-depressant effect on electromechanical coupling.

Lack of iNOS impairs endothelial function in endothelin-1 transgenic mice.

BACKGROUND: Endothelin-1 (ET-1) is one of the most potent biologic vasoconstrictors. Nevertheless, transgenic mice overexpressing ET-1 exhibit normal blood pressure. We hypothesized that in states of ET-1 overproduction, the lack of counterregulatory mediators such as nitric oxide (NO), produced by the inducible NO synthase (iNOS), may critically impair endothelial function and may result in blood pressure elevation. METHODS: We generated crossbred animals of ET transgenic mice (ET+/+) and iNOS knockout mice (iNOS-/-) and evaluated blood pressure and endothelial function in these animals. Endothelium-dependent and -independent vascular function was assessed as relaxation/contraction of isolated preconstricted aortic rings to acetylcholine, sodium nitroprusside, and ET-1, alone or in the presence of BQ123 or BQ788. RESULTS: Systolic blood pressure was similar in ET+/+, iNOS-/- and wild-type mice, but was significantly elevated in ET+/+ iNOS-/- crossbred animals versus ET+/+ mice. Maximum endothelium-dependent relaxation was enhanced in ET+/+ mice (95 +/- 5 vs. 78 +/- 5% of preconstriction in wild-type littermates; p < 0.05). Additional knockout of iNOS led to a significant decrease of endothelium-dependent relaxation in combined ET+/+ iNOS-/- animals (75 +/- 6%; p < 0.05 vs. ET+/+ mice). Endothelium-independent relaxation was comparable among all groups. Maximum vascular contraction to ET-1 was reduced in ET+/+ mice (33 +/- 4%), iNOS-/- mice (38 +/- 5%) and ET+/+ iNOS-/- mice (44 +/- 4%) to a similar extent as compared with wild-type littermates (66 +/- 4%; p < 0.05). CONCLUSIONS: Our data show for the first time that in transgenic mice overexpressing human ET-1, additional knockout of iNOS results in impaired endothelium-dependent vasodilatation thus contributing to elevated blood pressure in ET+/+ iNOS-/- animals.

Multi-slice cardiac computed tomography reveals anomalous origin of the right coronary artery between the great arteries.

We report a case of a 72-year-old man whose multi-slice cardiac computed tomography revealed an anomalous origin of the right coronary artery between the great arteries.

Conditional neuronal nitric oxide synthase overexpression impairs myocardial contractility.

The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS(-/-) mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca(2+)-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [(3)H]-l-arginine to [(3)H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17+/-3% decrease of +dp/dt(max) compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca(2+)ATPase and additionally with L-type Ca(2+)- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I(Ca,L) density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca(2+)-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca(2+)-channels and in turn reduces Ca(2+)-transients which accounts for the negative inotropic effect.

The estrogen receptor-alpha agonist 16alpha-LE2 inhibits cardiac hypertrophy and improves hemodynamic function in estrogen-deficient spontaneously hypertensive rats.

OBJECTIVE: Cardiac mass increases with age and with declining estradiol serum levels in postmenopausal women. Although the non-selective estrogen receptor-alpha and -beta agonist 17beta-estradiol attenuates cardiac hypertrophy in animal models and in observational studies, it remains unknown whether activation of a specific estrogen receptor subtype (ERalpha or ERbeta) might give similar or divergent results. Therefore, we analyzed myocardial hypertrophy as well as cardiac function and gene expression in ovariectomized, spontaneously hypertensive rats (SHR) treated with the subtype-selective ERalpha agonist 16alpha-LE2 or 17beta-estradiol. METHODS AND RESULTS: Long-term administration of 16alpha-LE2 or 17beta-estradiol did not affect elevated blood pressure, but both agonists efficiently attenuated cardiac hypertrophy and increased cardiac output, left ventricular stroke volume, papillary muscle strip contractility, and cardiac alpha-myosin heavy chain expression. The observed effects of E2 and 16alpha-LE2 were abrogated by the ER antagonist ZM-182780. Improved left ventricular function upon 16alpha-LE2 treatment was also observed in cardiac MRI studies. In contrast to estradiol and 16alpha-LE2, tamoxifen inhibited cardiac hypertrophy but failed to increase alpha-myosin heavy chain expression and cardiac output. CONCLUSIONS: These results support the hypothesis that activation of ERalpha favorably affects cardiac hypertrophy, myocardial contractility, and gene expression in ovariectomized SHR. Further studies are required to determine whether activation ERbeta mediates redundant or divergent effects.

Variable extent of clopidogrel responsiveness in patients after coronary stenting.

Clopidogrel is an effective and specific inhibitor of ADP-induced platelet aggregation. After metabolic activation, the active clopidogrel metabolite irreversibly impairs the human platelet P2Y12 ADP receptor. Gialpha-protein activation and inhibition of vasodilator-stimulated phosphoprotein (VASP) phosphorylation are two key elements of the P2Y12 receptor pathway suitable for quantitation of clopidogrel effects. So far, only limited data exist about a diminished responsiveness to clopidogrel and underlying possible mechanisms. We investigated clopidogrel effects in 57 patients after percutaneous coronary intervention and stent implantation by flow cytometry for the analysis of intracellular VASP phosphorylation. Patients were treated with a 300 mg clopidogrel loading dose, followed by 75 mg/day clopidogrel in combination with 100 mg/day aspirin. Samples were drawn after a median of 5 days of clopidogrel treatment. Considerable differences in the responsiveness to clopidogrel could be observed and it was shown that 17.5% (10/57) of the patients revealed an inadequate responsiveness to clopidogrel despite continuation of clopidogrel intake. Comparable amounts of Gialpha and VASP were found in two clopidogrel low-responding patients as well as in two responding patients. To exclude a molecular defect of P2Y12 ADP receptor, the P2Y12 receptor gene of eight clopidogrel treated patients (seven patients with inadequate responsiveness, one responder) was sequenced. We only found a single silent mutation in exon 2 at position 1828 (GA). We suggest that individual differences in clopidogrel metabolization could cause relevant variations in clopidogrel responsiveness despite the use of a 300 mg clopidogrel loading dose.

Administration of testosterone is associated with a reduced susceptibility to myocardial ischemia.

This study investigated the impact of testosterone on myocardial ischemia-reperfusion injury and corresponding intracellular calcium ([Ca2+]i) metabolism. Nonorchiectomized mature male Wistar rats were randomly assigned to placebo, a single dose of testosterone undecanoate, or 5alpha-dihydrotestosterone. In a further series, orchiectomized rats were treated with placebo. After 2 wk of treatment, the hearts were removed and placed in a Langendorff setup. The isolated, buffer-perfused hearts were subjected to 30 min of no-flow ischemia and 30 min of reperfusion. Recovery of myocardial function was measured by analyzing pre- and postischemic left ventricular (LV) systolic/diastolic pressure and coronary perfusion pressure simultaneously, together with [Ca2+]i handling (aequorin luminescence). Calcium regulatory proteins were analyzed by Western blotting. LV weight/body weight ratio was increased after administration of testosterone vs. orchectomized rats. The recovery of contractile function was improved in testosterone-treated rats: at the end of the reperfusion, LV systolic pressure was higher and end-diastolic pressure was lower in testosterone-treated rats. End-ischemic [Ca2+]i and [Ca2+]i overload upon reperfusion was significantly lower in testosterone vs. orchiectomized rats, too. However, levels of calcium regulatory proteins remained unaffected. In conclusion, administration of testosterone significantly improves recovery from global ischemia. These beneficial effects are associated with an attenuation of reperfusion induced [Ca2+]i overload.

Cytokine response after percutaneous coronary intervention in stable angina: effect of selective glycoprotein IIb/IIIa receptor antagonism.

BACKGROUND: It is unclear whether modulation of inflammatory markers by glycoprotein IIb/IIIa receptor inhibition after percutaneous coronary intervention (PCI) is caused by an interaction with the alpha(v)beta3 and alpha(M)beta2 receptor or it correlates with ischemic events during PCI. This study investigates the inflammatory profile after elective, nonacute PCI and whether and how administration of the glycoprotein IIb/IIIa receptor antagonist tirofiban modulates the postinterventional inflammatory myocardial response. METHODS: The time course of inflammatory parameters (C-reactive protein [CRP], interleukin-1 [IL-1], interleukin-6 [IL-6], and tumor necrosis factor alpha [TNF-alpha]) of patients receiving peri- and postinterventional placebo (n = 46) or tirofiban infusion (n = 50) was analyzed by use of enzyme-linked immuno assays. Samples were collected before and 30 minutes, 2.5 hours, 6.5 hours, 12 hours, 24 hours, and 48 hours after elective PCI. RESULTS: Among the inflammatory markers analyzed, TNF-alpha, IL-6, and CRP levels increased significantly. However, the latter markers followed individual time courses in patients given placebo and patients treated with tirofiban after PCI, compared with pre-PCI levels (P <.01), with no significant differences between the placebo and tirofiban-treated groups. However, by subgroup analysis, significant differences were revealed in TNF-alpha, IL-6, and CRP levels of patients who were troponin T-positive versus patients who were troponin T-negative after PCI. CONCLUSIONS: The administration of the selective glycoprotein IIb/IIIa receptor antagonist tirofiban has no direct impact on the inflammatory profile after elective PCI. Change of the inflammatory profile was only related to the presence or absence of postinterventional troponin.

Cannabinoids acting on CB1 receptors decrease contractile performance in human atrial muscle.

Cannabinoids elicit hypotension mainly via activated CB(1) receptors and show complex cardiovascular actions. Effects on human heart muscle have not been studied yet. Isolated human atrial heart muscle preparations were stimulated by electrical field with 1 Hz to contract isometrically at optimal length and were challenged with the endogenous cannabinoid arachidonyl ethanolamide (anandamide), the metabolically stable analogue R-methanandamide, and the potent synthetic CB(1) receptor agonist HU-210. Anandamide dose-dependently decreased systolic force (82.2 +/- 4.8% and 60.8 +/- 6.8% of maximal systolic force for 0.1 and 1 microM, respectively, P < 0.05). The selective CB(1) receptor antagonist AM-251 (1 microM, P < 0.05), but not the CB(2) receptor antagonist, AM-630 (1 microM), the nitric oxide synthase inhibitor N omega-nitro-l-arginine methyl ester (l-NAME) (500 microM), or the cyclooxygenase inhibitor indomethacin (100 microM), prevented the effect. Contrary to indomethacin, l-NAME alone showed negative inotropic effects (72.1 +/- 3.54%, P < 0.001). The R-methanandamide (1 microM: 50.4 +/- 3.5%, P < 0.001) and HU-210 (1 microM: 60.1 +/- 3.8%, P < 0.001) had similar negative inotropic effects. The existence of CB(1) receptors on heart muscle was verified using Western blot analysis and immunofluorescence staining. The conclusion is that anandamide, R-methanandamide, and HU-210 decrease contractile performance in human atrial muscle via CB(1) receptors.

Increased myocardial oxygen consumption by TNF-alpha is mediated by a sphingosine signaling pathway.

The present study investigated the effect of tumor necrosis factor (TNF)-alpha on myocardial energy metabolism as estimated by myocardial oxygen consumption (MVo(2)). MVo(2) of electrically stimulated isolated trabeculae of right ventricular Wistar rat myocardium was analyzed using a Clark-type oxygen probe. After the initial data collection in the absence of TNF-alpha, measurements were repeated after TNF-alpha stimulation. In separate experiments, pretreatment with the nitric oxide (NO) synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) or the ceramidase inhibitor n-oleoylethanolamine (NOE) was done to investigate NO/sphingosine-related effects. TNF-alpha impaired myocardial economy at increasing stimulation frequencies without altering baseline MVo(2). Incubation with TNF-alpha in the presence of l-NAME further impaired myocardial economy. NOE preincubation abrogated the TNF-alpha effect on myocardial economy. Moreover, the negative inotropic effect of TNF-alpha was observed in NOE-pretreated but not l-NAME-pretreated muscle fibers. Exogenous sphingosine mimicked the TNF-alpha effect on mechanics and energetics. We conclude that TNF-alpha impairs the economy of chemomechanical energy transduction primarily through a sphingosine-mediated pathway.

Functional properties and [Ca(2+)](i) metabolism of creatine kinase--KO mice myocardium.

One major function of the creatine kinase system is to maintain energy demand of myofibrillar contraction processes. Loss of the CK-system led to adaptations in skeletal muscle. To analyze the impact on myocardial function contractile parameters and intracellular calcium metabolism of transgenic mice lacking mitochondrial CK (ScCKmit(-/-)) alone or both mitochondrial and cytoplasmic ScCK (CK(-/-)) were investigated compared to wild type at various workload conditions using isolated intact muscle fibers. Force development at baseline conditions, force-frequency relationship (60-600/min), and rapid frequency switch (60-600/min) were unaltered in myocardium of transgenic mice compared to wild type. Intracellular calcium metabolism revealed unchanged amplitude of the intracellular calcium transients (ICT), refilling of the sarcoplasmic reticulum (calcium reuptake, post-rest behavior) in the ScCKmit(-/-) and CK(-/-) mice. The results demonstrate the effectiveness of myocardial energy-recruiting compensatory mechanisms at baseline as well as under stress conditions in CK depleted myocardium of transgenic mice.

Effect of additional temporary glycoprotein IIb/IIIa receptor inhibition on troponin release in elective percutaneous coronary interventions after pretreatment with aspirin and clopidogrel (TOPSTAR trial).

OBJECTIVES: The Troponin in Planned PTCA/Stent Implantation With or Without Administration of the Glycoprotein IIb/IIIa Receptor Antagonist Tirofiban (TOPSTAR) trial investigated: 1) the amount of troponin T (TnT) release after nonacute, elective percutaneous coronary intervention (PCI) in patients pretreated with aspirin and clopidogrel; and 2) the effect of additional glycoprotein (GP) IIb/IIIa receptor inhibiton on postinterventional TnT release. BACKGROUND: No data are available yet as to whether additional administration of a GP IIb/IIIa receptor antagonist might be beneficial in patients undergoing elective PCI already pretreated with aspirin and clopidogrel. METHODS: After bolus application of the study medication (tirofiban [T] or placebo [P]), PCI was performed followed by an 18-h continuous infusion of T/P. Primary end point of the study was incidence and amount of TnT release after elective PCI after 24 h. RESULTS: A total of 12 h after PCI troponin release was detected in 63% of the patients receiving P and in 40% of the patients receiving T (p < 0.05), after 24 h in 69% (P) and 48% (T) (p < 0.05) and after 48 h in 74% (P) versus 58% (T) (p < 0.08) of the patients. No differences were observed regarding major bleeding, intracranial bleeding or nonhemorrhagic strokes. After nine months a reduction of combined death/myocardial infarction/target vessel revascularization could be observed in the tirofiban group ([T] 2.3% vs. [P] 13.04%, p < 0.05). CONCLUSIONS: Troponin T release occurs after successful intervention in 74% of the patients undergoing elective PCI after 48 h even after pretreatment with aspirin and clopidogrel. The GP IIb/IIIa receptor antagonist tirofiban is able to decrease the incidence of troponin release significantly in this patient population.