Overexpression of Na+/Ca2+ exchanger gene attenuates postinfarction myocardial dysfunction

We monitored myocardial function in postinfarcted wild-type (WT) and transgenic (TG) mouse hearts with overexpression of the cardiac Na(+)/Ca(2+) exchanger. Five weeks after infarction, cardiac function was better maintained in TG than WT mice [left ventricular (LV) systolic pressure: WT, 41 +/- 2; TG, 58 +/- 3 mmHg; P < 0.05; maximum rising rate of LV pressure (+dP/dt(max)): WT, 3,750 +/- 346; TG, 5,075 +/- 334 mmHg/s; P < 0.05]. The isometric contractile response to beta-adrenergic stimulation was greater in papillary muscles from TG than WT mice (WT, 13.2 +/- 0.9; TG, 16.3 +/- 1.0 mN/mm(2) at 10(-4) M isoproterenol). The sarcoplasmic reticulum (SR) Ca(2+) content investigated by rapid cooling contractures in papillary muscles was greater in TG than WT mouse hearts. We conclude that myocardial function is better preserved in TG mice 5 wk after infarction, which results from enhanced SR Ca(2+) content via overexpression of the Na(+)/Ca(2+) exchanger.     

Lack of muscarinic regulation of Ca(2+) channels in G(i2)alpha gene knockout mouse hearts

The purpose of the present study was to examine the role of G(i2)alpha in Ca(2+) channel regulation using G(i2)alpha gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the beta-adrenergic agonist isoproterenol (Iso) on cardiac L-type Ca(2+) currents in both 129Sv wild-type (WT) and G(i2)alpha gene knockout (G(i2)alpha-/-) mice. Perfusion with CCh significantly inhibited the Ca(2+) current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca(2+) currents in G(i2)alpha-/- ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated Ca(2+) current in WT cardiomyocytes but not in G(i2)alpha-/- cells. These findings demonstrate that, whereas the Iso-G(s)alpha signal pathway is intact in G(i2)alpha gene knockout mouse hearts, these cells lack the inhibitory regulation of Ca(2+) channels by CCh. Therefore, G(i2)alpha is necessary for the muscarinic regulation of Ca(2+) channels in the mouse heart. Further studies are needed to delineate the possible interaction of G(i) and other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type Ca(2+) current in the heart.     

Cardiac alternans in embryonic mouse ventricles

T-wave alternans, an important arrhythmogenic factor, has recently been described in human fetuses. Here we sought to determine whether alternans can be induced in the embryonic mouse hearts, despite its underdeveloped sarcoplasmic reticulum (SR) and, if so, to analyze the response to pharmacological and autonomic interventions. Immunohistochemistry confirmed minimal sarcoplasmic-endoplasmic reticulum Ca-ATPase 2a expression in embryonic mouse hearts at embryonic day (E) 10.5 to E12.5, compared with neonatal or adult mouse hearts. We optically mapped voltage and/or intracellular Ca (Ca(i)) in 99 embryonic mouse hearts (dual mapping in 64 hearts) at these ages. Under control conditions, ventricular action potential duration (APD) and Ca(i) transient alternans occurred during rapid pacing at an average cycle length of 212 +/- 34 ms in 57% (n = 15/26) of E10.5-E12.5 hearts. Maximum APD restitution slope was steeper in hearts developing alternans than those that did not (2.2 +/- 0.6 vs. 0.8 +/- 0.4; P < 0.001). Disabling SR Ca(i) cycling with thapsigargin plus ryanodine did not significantly reduce alternans incidence (44%, n = 8/18, P = 0.5), whereas isoproterenol (n = 14) increased the incidence to 100% (P < 0.05), coincident with steepening APD restitution slope. Verapamil abolished Ca(i) transients (n = 9). Thapsigargin plus ryanodine had no major effects on Ca(i)-transient amplitude or its half time of recovery in E10.5 hearts, but significantly depressed Ca(i)-transient amplitude (by 47 +/- 8%) and prolonged its half time of recovery (by 18 +/- 3%) in E11.5 and older hearts. Embryonic mouse ventricles can develop cardiac alternans, which generally is well correlated with APD restitution slope and does not depend on fully functional SR Ca(i) cycling.     

Compromised myocardial energetics in hypertrophied mouse hearts diminish the beneficial effect of overexpressing SERCA2a

The sarcoplasmic reticulum calcium ATPase (SERCA) plays a central role in regulating intracellular Ca(2+) homeostasis and myocardial contractility. Several studies show that improving Ca(2+) handling in hypertrophied rodent hearts by increasing SERCA activity results in enhanced contractile function. This suggests that SERCA is a potential target for gene therapy in cardiac hypertrophy and failure. However, it raises the issue of increased energy cost resulting from a higher ATPase activity. In this study, we determined whether SERCA overexpression alters the energy cost of increasing myocardial contraction in mouse hearts with pressure-overload hypertrophy using (31)P NMR spectroscopy. We isolated and perfused mouse hearts from wild-type (WT) and transgenic (TG) mice overexpressing the cardiac isoform of SERCA (SERCA2a) 8 weeks after ascending aortic constriction (left ventricular hypertrophy (LVH)) or sham operation. We found that overexpressing SERCA2a enhances myocardial contraction and relaxation in normal mouse hearts during inotropic stimulation with isoproterenol. Energy consumption was proportionate to the increase in contractile function. Thus, increasing SERCA2a expression in the normal heart allows an enhanced inotropic response with no compromise in energy supply and demand. However, this advantage was not sustained in LVH hearts in which the energetic status was compromised. Although the overexpression of SERCA2a prevented the down-regulation of SERCA protein in LVH hearts, TG-LVH hearts showed no increase in inotropic response when compared with WT-LVH hearts. Our results suggest that energy supply may be a limiting factor for the benefit of SERCA overexpression in hypertrophied hearts. Thus, strategies combining energetic support with increasing SERCA activity may improve the therapeutic effectiveness for heart failure.     

Human cardiac myosin heavy chain isoforms in fetal and failing adult atria and ventricles

The goal of this study was to test the hypothesis that the relative amounts of the cardiac myosin heavy chain (MHC) isoforms MHC-alpha and MHC-beta change during development and transition to heart failure in the human myocardium. The relative amounts of MHC-alpha and MHC-beta in ventricular and atrial samples from fetal (gestational days 47--110) and nonfailing and failing adult hearts were determined. The majority of the fetal right and left ventricular samples contained small relative amounts of MHC-alpha (mean < 5% of total MHC). There was a small significant decrease in the level of MHC-alpha in the ventricles between 7 and 12 wk of gestation. Fetal atria expressed predominantly MHC-alpha (mean > 95%), with MHC-beta being detected in most samples. The majority of adult nonfailing right and left ventricular samples had detectable levels of MHC-alpha ranging from 1 to 10%. Failing right and left ventricles expressed a significantly lower level of MHC-alpha. MHC-alpha comprised approximately 90% of the total MHC in adult nonfailing left atria, whereas the relative amount of MHC-alpha in the left atria of individuals with dilated or ischemic cardiomyopathy was approximately 50%. The differences in MHC isoform composition between fetal and nonfailing adult atria and between fetal and nonfailing adult ventricles were not statistically significant. We concluded that the MHC isoform compositions of fetal human atria are the same as those of nonfailing adult atria and that the ventricular MHC isoform composition is different between adult nonfailing and failing hearts. Furthermore, the marked alteration in atrial MHC isoform composition, associated with cardiomyopathy, does not represent a regression to a pattern that is uniquely characteristic of the fetal stage.     

The role of reactive oxygen species in the hearts of dystrophin-deficient mdx mice

Duchenne muscular dystrophy (DMD) is caused by deficiency of the cytoskeletal protein dystrophin. Oxidative stress is thought to contribute to the skeletal muscle damage in DMD; however, little is known about the role of oxidative damage in the pathogenesis of the heart failure that occurs in DMD patients. The dystrophin-deficient (mdx) mouse is an animal model of DMD that also lacks dystrophin. The current study investigates the role of the antioxidant N-acetylcysteine (NAC) on mdx cardiomyocyte function, Ca(2+) handling, and the cardiac inflammatory response. Treated mice received 1% NAC in their drinking water for 6 wk. NAC had no effect on wild-type (WT) mice. Immunohistochemistry experiments revealed that mdx mice had increased dihydroethidine (DHE) staining, an indicator of superoxide production; NAC-treatment reduced DHE staining in mdx hearts. NAC treatment attenuated abnormalities in mdx cardiomyocyte Ca(2+) handling. Mdx cardiomyocytes had decreased fractional shortening and decreased Ca(2+) sensitivity; NAC treatment returned mdx fractional shortening to WT values but did not affect the Ca(2+) sensitivity. Immunohistochemistry experiments revealed that mdx hearts had increased levels of collagen type III and the macrophage-specific protein, CD68; NAC-treatment returned collagen type III and CD68 expression close to WT values. Finally, mdx hearts had increased NADPH oxidase activity, suggesting it could be a possible source of increased reactive oxygen species in mdx mice. This study is the first to demonstrate that oxidative damage may be involved in the pathogenesis of the heart failure that occurs in mdx mice. Therapies designed to reduce oxidative damage might be beneficial to DMD patients with heart failure.     

Low over-expression of TNFalpha in the mouse heart increases contractile performance via TNFR1

TNFalpha is a cytokine wit pleiotropic functions in many organs. In the heart increased TNFalpha levels are not only associated with heart failure, but also, paradoxically, with protection from ischemic damage. To test whether the protective role of TNFalpha in the heart is concentration-dependent, we studied two mouse heart models with low (two- to threefold) over-expression of endogenous TNFalpha: mice deficient in a translational repressor of TNFalpha mRNA, TIA-1(-/-), and mice over-expressing human TNFalpha. Hearts lacking TIA-1 were characterized for their endogenous TNFalpha over-expression during normal Langendorff perfusion. To define which TNFalpha receptor mediates cardiac protection, we also used mice lacking the TNFR1 receptor. Contractile function was assessed in isolated hearts perfused in the isovolumic Langendorff mode during and following global no-flow ischemic stress and in response to varying extracellular [Ca(2+)] to determine their contractile response and Ca(2+) sensitivity. All hearts with low over-expression of TNFalpha, independent of human or murine origin, have improved contractile performance and increased Ca(2+) sensitivity (by 0.2-0.26 pCa). Hearts lacking TNFR1 have contractile performance equal to wild type hearts. Recovery from ischemia was greater in TIA-1(-/-) and was diminished in TNFR1(-/-). Better contractile function in TNFalpha over-expressing hearts is not due to improved cardiac energetics assessed as [ATP] and glucose uptake or to differences in expression of SERCA2a or calmodulin. We suggest that low levels of TNFalpha increase the Ca(2+) sensitivity of the heart via a TNFR1-mediated mechanism.     

Morphological and functional changes in cardiac myocytes isolated from mice overexpressing TNF-alpha

Transgenic (TG) TNF1.6 mice, which cardiac specifically overexpress tumor necrosis factor-alpha (TNF-alpha), exhibit heart failure (HF) and increased mortality, which is markedly higher in young (<20 wk) males (TG-M) than females (TG-F). HF in this model may be partly caused by remodeling of the extracellular matrix and/or structure/function alterations at the single myocyte level. We studied left ventricular (LV) structure and function using echocardiography and LV myocyte morphometry, contractile function, and intracellular Ca(2+) (Ca(i)(2+)) handling using cell edge detection and fura 2 fluorescence, respectively, in 12-wk-old TG-M and TG-F mice and their wild-type (WT) littermates. TG-F mice showed LV hypertrophy without dilatation and only a small reduction of basal fractional shortening (FS) and response to isoproterenol (Iso). TG-M mice showed a large LV dilatation, higher mRNA levels of beta-myosin heavy chain and atrial natriuretic factor versus TG-F mice, reduced FS relative to both WT and TG-F mice, and minimal response to Iso. TG-F and TG-M myocytes were similarly elongated (by approximately 20%). The amplitude of Ca(i)(2+) transients and contractions and the response to Iso were comparable in WT and TG-F myocytes, whereas the time to 50% decline (TD(50%)) of the Ca(i)(2+) transient, an index of the rate of sarcoplasmic reticulum Ca(2+) uptake, was prolonged in TG-F myocytes. In TG-M myocytes, the amplitudes of Ca(i)(2+) transients and contractions were reduced, TD(50%) of the Ca(i)(2+) transient was prolonged, and the inotropic effect of Iso on Ca(i)(2+) transients was reduced approximately twofold versus WT myocytes. Protein expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and phospholamban was unaltered in TG versus WT hearts, suggesting functional origins of impaired Ca(2+) handling in the former. These results indicate that cardiac-specific overexpression of TNF-alpha induces myocyte hypertrophy and gender-dependent alterations in Ca(i)(2+) handling and contractile function, which may at least partly account for changes in LV geometry and in vivo cardiac function in this model.     

压力负荷性心衰小鼠左心室跨壁L-型钙电流的变化

为了观察正常和心衰时心内膜下和心外膜下心肌细胞L-型钙电流(ICa-L)的差别,我们采用主动脉弓狭窄的方法建立小鼠压力超负荷性心衰模型,采用全细胞膜片钳技术记录了正常、主动脉狭窄(band)及假手术对照(sham)组动物左心室游离壁内、外膜下心肌细胞的动作电位时程(action potential duration,APD)和ICa-L。结果显示:(1)与sham组同龄的正常小鼠左心室心内膜下细胞动作电位复极达90％的时程(APD90)为(38．2±6．44)ms,较心外膜下细胞的APD90(15.67±5.31)ms明显延长,二者的比值约为2．5:1;内膜下细胞和外膜下细胞ICa-L密度没有差异,峰电流密度分别为(-2.7±0.49)pA/pF和(-2.54±0．53)pA/pF;(2)Band组内、外膜下细胞的动作电位复极达50％的时程(APD50)、APD90均较sham组显著延长,尤以内膜下细胞延长突出,分别较sham组延长了400％和360％,内、外膜下细胞APD90的比值约为4.2:1;(3)与sham组相比, band组内膜下细胞ICa-L密度显著减小,在+10 mV～+40 mV的4个电压下分别降低了20．2％、21．4％、21.6％和25.7％(P< 0．01),但其激活电位、峰电位和翻转电位没有改变;band组外膜下细胞的ICa-L密度与同期sham组相比无明显变化;band组钙通道激活、失活及复活的动力学特征与sham组相比没有改变。以上结果提示,生理状态下小鼠左心室内、外膜下细胞ICa-L密度不存在明显差别,提示ICa-L与APD跨壁异质性的产生无关;心衰时左心室内、外膜下细胞APD明显延长,以内膜下细胞延长尤为突出,内膜下细胞ICa-L密度明显减少,而外膜下细胞ICa-L密度无明显改变,这种ICa-L的非同步变化在心衰时可能起到对抗APD延长、减少复极离散度的有益作用。     

Cellular and molecular determinants of altered Ca2+ handling in the failing rabbit heart: primary defects in SR Ca2+ uptake and release mechanisms

Myocytes from the failing myocardium exhibit depressed and prolonged intracellular Ca(2+) concentration ([Ca(2+)](i)) transients that are, in part, responsible for contractile dysfunction and unstable repolarization. To better understand the molecular basis of the aberrant Ca(2+) handling in heart failure (HF), we studied the rabbit pacing tachycardia HF model. Induction of HF was associated with action potential (AP) duration prolongation that was especially pronounced at low stimulation frequencies. L-type calcium channel current (I(Ca,L)) density (-0.964 +/- 0.172 vs. -0.745 +/- 0.128 pA/pF at +10 mV) and Na(+)/Ca(2+) exchanger (NCX) currents (2.1 +/- 0.8 vs. 2.3 +/- 0.8 pA/pF at +30 mV) were not different in myocytes from control and failing hearts. The amplitude of peak [Ca(2+)](i) was depressed (at +10 mV, 0.72 +/- 0.07 and 0.56 +/- 0.04 microM in normal and failing hearts, respectively; P < 0.05), with slowed rates of decay and reduced Ca(2+) spark amplitudes (P < 0.0001) in myocytes isolated from failing vs. control hearts. Inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a revealed a greater reliance on NCX to remove cytosolic Ca(2+) in myocytes isolated from failing vs. control hearts (P < 0.05). mRNA levels of the alpha(1C)-subunit, ryanodine receptor (RyR), and NCX were unchanged from controls, while SERCA2a and phospholamban (PLB) were significantly downregulated in failing vs. control hearts (P < 0.05). alpha(1C) protein levels were unchanged, RyR, SERCA2a, and PLB were significantly downregulated (P < 0.05), while NCX protein was significantly upregulated (P < 0.05). These results support a prominent role for the sarcoplasmic reticulum (SR) in the pathogenesis of HF, in which abnormal SR Ca(2+) uptake and release synergistically contribute to the depressed [Ca(2+)](i) and the altered AP profile phenotype.     

Comparison of the electromechanical responsiveness of alpha-1-adrenoceptor stimulation in ventricles of normal and cardiomyopathic hamsters

Alterations in alpha(1)-adrenoceptor (alpha(1)AR) density and related signal transduction proteins were reported in cardiomyopathic hearts in the failing stage. The electromechanical modification of alpha(1)-adrenergic stimulation in the failing heart is unclear. The present study compares the alpha(1)AR-stimulated electromechanical response in failing ventricles of genetically cardiomyopathic BIO 14.6 hamsters (280-320 days old) with that in age-matched normal Syrian hamsters. The action potential was recorded with a conventional microelectrode technique, and twitch force was measured with a transducer. In the presence of propranolol, phenylephrine increased the contraction and prolonged the action potential duration (APD) to similar values in ventricles of both strains, despite a prolonged basal APD in cardiomyopathic ventricles. The positive inotropism stimulated by phenylephrine was inhibited by staurosporine, and was potentiated by 4 beta-phorbol-12,13-dibutyrate (PDBu) in both strains. The maximum positive inotropic effect of phenylephrine in PDBu-treated ventricles of normal hamsters was significantly greater than that in BIO 14.6 hamsters. The effects of phenylephrine on the ventricular force-frequency relationship and on the mechanical restitution in both normal and BIO 14.6 strain hamsters were examined. The uniform negative force-frequency relationship and the altered mechanical restitution reveal a defect of intracellular Ca(2+) handling in cardiomyopathic BIO 14.6 hamsters. alpha(1)-Adrenergic modulation cannot convert the defective properties in the model of the failing heart. Nevertheless, phenylephrine decreased post-rest potentiation in short rest periods, and enhanced post-rest decay after longer resting periods. The results indicate that alpha(1)-adrenergic action enhances a gradual loss of Ca(2+) from the sarcoplasmic reticulum, although its action in prolonging the APD can indirectly increase the influx of Ca(2+).     

Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure

It is currently unclear whether left ventricular (LV) myofilament function is depressed in experimental LV hypertrophy (LVH) or congestive heart failure (CHF). To address this issue, we studied pressure overload-induced LV hypertrophy (POLVH) and myocardial infarction-elicited congestive heart failure (MICHF) in rats. LV myocytes were isolated from control, POLVH, and MICHF hearts by mechanical homogenization, skinned with Triton, and attached to micropipettes that projected from a sensitive force transducer and high-speed motor. A subset of cells was treated with either unphosphorylated, recombinant cardiac troponin (cTn) or cTn purified from either control or failing ventricles. LV myofilament function was characterized by the force-[Ca(2+)] relation yielding Ca(2+)-saturated maximal force (F(max)), myofilament Ca(2+) sensitivity (EC(50)), and cooperativity (Hill coefficient, n(H)) parameters. POLVH was associated with a 35% reduction in F(max) and 36% increase in EC(50). Similarly, MICHF resulted in a 42% reduction in F(max) and a 30% increase in EC(50). Incorporation of recombinant cTn or purified control cTn into failing cells restored myofilament Ca(2+) sensitivity toward levels observed in control cells. In contrast, integration of cTn purified from failing ventricles into control myocytes increased EC(50) to levels observed in failing myocytes. The F(max) parameter was not markedly affected by troponin exchange. cTnI phosphorylation was increased in both POLVH and MICHF left ventricles. We conclude that depressed myofilament Ca(2+) sensitivity in experimental LVH and CHF is due, in part, to a decreased functional role of cTn that likely involves augmented phosphorylation of cTnI.     

Kinin B1 receptor participates in the control of cardiac function in mice

The kinins have an important role in control of the cardiovascular system. They have been associated with protective effects in the heart tissue. Kinins act through stimulation of two 7-transmembrane G protein-coupled receptors, denoted B(1) and B(2) receptors. However, the physiological relevance of B(1) receptor in the heart has not been clearly established. Using B(1) kinin receptor gene knock-out mice we tested the hypothesis that the B(1) receptor plays an important role in the control of baseline cardiac function. We examined the functional aspects of the intact heart and also in the isolated cardiomyocytes to study intracellular Ca(2+) cycling by using confocal microscopy and whole-cell voltage clamp techniques. We measured heart rate, diastolic and systolic tension, contraction and relaxation rates and, coronary perfusion pressure. Whole-cell voltage clamp was performed to measure L-type Ca(2+) current (I(Ca,L)). The hearts from B(1)(-/-) mice showed smaller systolic tension. The average values for WT and B(1)(-/-) mice were 2.6+/-0.04 g vs. 1.6+/-0.08 g, respectively. This result can be explained, at least in part, by the decrease in the Ca(2+) transient (3.1+/-0.06 vs. 3.4+/-0.09 for B(1)(-/-) and WT, respectively). There was an increase in I(Ca,L) at depolarized membrane potentials. Interestingly, the inactivation kinetics of I(Ca,L) was statistically different between the groups. The coronary perfusion pressure was higher in the hearts from B(1)(-/-) mice indicating an increase in coronary resistance. This result can be explained by the significant reduction of eNOS (NOS-3) expression in the aorta of B(1)(-/-) mice. Collectively, our results demonstrate that B(1) receptor exerts a fundamental role in the mammalian cardiac function.     

Decrease in the density of t-tubular L-type Ca2+ channel currents in failing ventricular myocytes

In some forms of cardiac hypertrophy and failure, the gain of Ca(2+)-induced Ca(2+) release [CICR; i.e., the amount of Ca(2+) released from the sarcoplasmic reticulum normalized to Ca(2+) influx through L-type Ca(2+) channels (LTCCs)] decreases despite the normal whole cell LTCC current density, ryanodine receptor number, and sarcoplasmic reticulum Ca(2+) content. This decrease in CICR gain has been proposed to arise from a change in dyad architecture or derangement of the t-tubular (TT) structure. However, the activity of surface sarcolemmal LTCCs has been reported to increase despite the unaltered whole cell LTCC current density in failing human ventricular myocytes, indicating that the "decreased CICR gain" may reflect a decrease in the TT LTCC current density in heart failure. Thus, we analyzed LTCC currents of failing ventricular myocytes of mice chronically treated with isoproterenol (Iso). Although Iso-treated mice exhibited intact t-tubules and normal LTCC subunit expression, acute occlusion of t-tubules of isolated ventricular myocytes with osmotic shock (detubulation) revealed that the TT LTCC current density was halved in Iso-treated versus control myocytes. Pharmacological analysis indicated that kinases other than PKA or Ca(2+)/calmodulin-dependent protein kinase II insufficiently activated, whereas protein phosphatase 1/2A excessively suppressed, TT LTCCs in Iso-treated versus control myocytes. These results indicate that excessive β-adrenergic stimulation causes the decrease in TT LTCC current density by altering the regulation of TT LTCCs by protein kinases and phosphatases in heart failure. This phenomenon might underlie the decreased CICR gain in heart failure.     

Calcium-dependent arrhythmias in transgenic mice with heart failure

Transgenic mice overexpressing the inflammatory cytokine tumor necrosis factor (TNF)-alpha (TNF-alpha mice) in the heart develop a progressive heart failure syndrome characterized by biventricular dilatation, decreased ejection fraction, atrial and ventricular arrhythmias on ambulatory telemetry monitoring, and decreased survival compared with nontransgenic littermates. Programmed stimulation in vitro with single extra beats elicits reentrant ventricular arrhythmias in TNF-alpha (n = 12 of 13 hearts) but not in control hearts. We performed optical mapping of voltage and Ca(2+) in isolated perfused ventricles of TNF-alpha mice to study the mechanisms that lead to the initiation and maintenance of the arrhythmias. When compared with controls, hearts from TNF-alpha mice have prolonged of action potential durations (action potential duration at 90% repolarization: 23 +/- 2 ms, n = 7, vs. 18 +/- 1 ms, n = 5; P < 0.05), no increased dispersion of refractoriness between apex and base, elevated diastolic and depressed systolic [Ca(2+)], and prolonged Ca(2+) transients (72 +/- 6 ms, n = 10, vs. 54 +/- 5 ms, n = 8; P < 0.01). Premature beats have diminished action potential amplitudes and conduct in a slow, heterogeneous manner. Lowering extracellular [Ca(2+)] normalizes conduction and prevents inducible arrhythmias. Thus both action potential prolongation and abnormal Ca(2+) handling may contribute to the initiation of reentrant arrhythmias in this heart failure model by mechanisms distinct from enhanced dispersion of refractoriness or triggered activity.     

Atrial myocardium is the predominant inotropic target of adrenomedullin in the human heart

Adrenomedullin (ADM) is an endogenous peptide with favorable hemodynamic effects in vivo. In this study, we characterized the direct functional effects of ADM in isolated preparations from human atria and ventricles. In electrically stimulated human nonfailing right atrial trabeculae, ADM (0.0001-1 micromol/l) increased force of contraction in a concentration-dependent manner, with a maximal increase by 35 +/- 8% (at 1 micromol/l; P < 0.05). The positive inotropic effect was accompanied by a disproportionate increase in calcium transients assessed by aequorin light emission [by 76 +/- 20%; force/light ratio (DeltaF/DeltaL) 0.58 +/- 0.15]. In contrast, elevation of extracellular calcium (from 2.5 to 3.2 mmol/l) proportionally increased force and aequorin light emission (DeltaF/DeltaL 1.0 +/- 0.1; P < 0.05 vs. ADM). Consistent with a cAMP-dependent mechanism, ADM (1 micromol/l) increased atrial cAMP levels by 90 +/- 12%, and its inotropic effects could be blocked by the protein kinase A (PKA) inhibitor H-89. ADM also exerted positive inotropic effects in failing atrial myocardium and in nonfailing and failing ventricular myocardium. The inotropic response was significantly weaker in ventricular vs. atrial myocardium and in failing vs. nonfailing myocardium. In conclusion, ADM exerts Ca(2+)-dependent positive inotropic effects in human atrial and less-pronounced effects in ventricular myocardium. The inotropic effects are related to increased cAMP levels and stimulation of PKA. In heart failure, the responsiveness to ADM is reduced in atria and ventricles.     

Autonomic regulation of calcium cycling in developing embryonic mouse hearts

In the present study, we combined optical Ca(2+) imaging with immunocytochemistry studies to characterize autonomic regulation of Ca(2+) cycling during early development in isolated embryonic mouse hearts. At embryonic days 9.5-11.5 (E9.5-E11.5), the Ca(2+) transient originated in the superior portion of the right atrium, propagated rapidly through both atria, slowly through the atrio-ventricular (AV) ring, and rapidly through both ventricles. Isoproterenol (ISO) significantly increased heart rate, increased Ca(2+) transient amplitude, rate of rise (RR) and a rate of decay, and shortened AV conduction time, indicating the presence of functional beta-adrenergic receptors. The muscarinic agonist carbachol (CCh) had no effects until 1 day later at E10.5. Both beta1-adrenergic and M2 muscarinic receptors were detected in ventricular muscle sections by immunochemistry at E10.5. Growing nerves, labeled using growth-associated protein 43 antibodies, were detected at the E14.5 stage, but not at E10.5, whereas mature sympathetic nerves, detected by tyrosine hydroxylase (TH) labeling, were not yet present at E14.5. These results demonstrate that functional regulation of Ca(2+) cycling by beta-adrenergic receptors occurs earliest in developing embryonic mouse hearts, followed a day later by muscarinic receptor responsiveness, with autonomic innervation developing later. These results define the functional and structural sequence of autonomic regulation of Ca(2+) transient in the embryonic mouse heart.     

Cardiac arrhythmia mechanisms in rats with heart failure induced by pulmonary hypertension

Pulmonary hypertension provokes right heart failure and arrhythmias. Better understanding of the mechanisms underlying these arrhythmias is needed to facilitate new therapeutic approaches for the hypertensive, failing right ventricle (RV). The aim of our study was to identify the mechanisms generating arrhythmias in a model of RV failure induced by pulmonary hypertension. Rats were injected with monocrotaline to induce either RV hypertrophy or failure or with saline (control). ECGs were measured in conscious, unrestrained animals by telemetry. In isolated hearts, electrical activity was measured by optical mapping and myofiber orientation by diffusion tensor-MRI. Sarcoplasmic reticular Ca(2+) handling was studied in single myocytes. Compared with control animals, the T-wave of the ECG was prolonged and in three of seven heart failure animals, prominent T-wave alternans occurred. Discordant action potential (AP) alternans occurred in isolated failing hearts and Ca(2+) transient alternans in failing myocytes. In failing hearts, AP duration and dispersion were increased; conduction velocity and AP restitution were steeper. The latter was intrinsic to failing single myocytes. Failing hearts had greater fiber angle disarray; this correlated with AP duration. Failing myocytes had reduced sarco(endo)plasmic reticular Ca(2+)-ATPase activity, increased sarcoplasmic reticular Ca(2+)-release fraction, and increased Ca(2+) spark leak. In hypertrophied hearts and myocytes, dysfunctional adaptation had begun, but alternans did not develop. We conclude that increased electrical and structural heterogeneity and dysfunctional sarcoplasmic reticular Ca(2+) handling increased the probability of alternans, a proarrhythmic predictor of sudden cardiac death. These mechanisms are potential therapeutic targets for the correction of arrhythmias in hypertensive, failing RVs.     

Simulation of the undiseased human cardiac ventricular action potential: model formulation and experimental validation

Cellular electrophysiology experiments, important for understanding cardiac arrhythmia mechanisms, are usually performed with channels expressed in non myocytes, or with non-human myocytes. Differences between cell types and species affect results. Thus, an accurate model for the undiseased human ventricular action potential (AP) which reproduces a broad range of physiological behaviors is needed. Such a model requires extensive experimental data, but essential elements have been unavailable. Here, we develop a human ventricular AP model using new undiseased human ventricular data: Ca(2+) versus voltage dependent inactivation of L-type Ca(2+) current (I(CaL)); kinetics for the transient outward, rapid delayed rectifier (I(Kr)), Na(+)/Ca(2+) exchange (I(NaCa)), and inward rectifier currents; AP recordings at all physiological cycle lengths; and rate dependence and restitution of AP duration (APD) with and without a variety of specific channel blockers. Simulated APs reproduced the experimental AP morphology, APD rate dependence, and restitution. Using undiseased human mRNA and protein data, models for different transmural cell types were developed. Experiments for rate dependence of Ca(2+) (including peak and decay) and intracellular sodium ([Na(+)](i)) in undiseased human myocytes were quantitatively reproduced by the model. Early afterdepolarizations were induced by I(Kr) block during slow pacing, and AP and Ca(2+) alternans appeared at rates >200 bpm, as observed in the nonfailing human ventricle. Ca(2+)/calmodulin-dependent protein kinase II (CaMK) modulated rate dependence of Ca(2+) cycling. I(NaCa) linked Ca(2+) alternation to AP alternans. CaMK suppression or SERCA upregulation eliminated alternans. Steady state APD rate dependence was caused primarily by changes in [Na(+)](i), via its modulation of the electrogenic Na(+)/K(+) ATPase current. At fast pacing rates, late Na(+) current and I(CaL) were also contributors. APD shortening during restitution was primarily dependent on reduced late Na(+) and I(CaL) currents due to inactivation at short diastolic intervals, with additional contribution from elevated I(Kr) due to incomplete deactivation.     

KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

KCNE2 functions as an auxiliary subunit in voltage-gated K and HCN channels in the heart. Genetic variations in KCNE2 have been linked to long QT syndrome. The underlying mechanisms are not entirely clear. One of the issues is whether KCNE2 protein is expressed in ventricles. We use adenovirus-mediated genetic manipulations of adult cardiac myocytes to validate two antibodies (termed Ab1 and Ab2) for their ability to detect native KCNE2 in the heart. Ab1 faithfully detects native KCNE2 proteins in spontaneously hypertensive rat and guinea pig hearts. In both cases, KCNE2 protein is more abundant in ventricles than in atria. In both ventricular and atrial myocytes, KCNE2 protein is preferentially distributed on the cell surface. Ab1 can detect a prominent KCNE2 band in human ventricular muscle from nonfailing hearts. The band intensity is much fainter in atria and in failing ventricles. Ab2 specifically detects S98 phosphorylated KCNE2. Through exploring the functional significance of S98 phosphorylation, we uncover a novel mechanism by which KCNE2 modulates the human ether-a-go-go related gene (hERG) current amplitude: by accelerating hERG protein degradation and thus reducing the hERG protein level on the cell surface. S98 phosphorylation appears to be required for this modulation, so that S98 dephosphorylation leads to an increase in hERG/rapid delayed rectifier current amplitude. Our data confirm that KCNE2 protein is expressed in the ventricles of human and animal models. Furthermore, KCNE2 can modulate its partner channel function not only by altering channel conductance and/or gating kinetics, but also by affecting protein stability.