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.     

Preserved ventricular contractility in infarcted mouse heart overexpressing beta(2)-adrenergic receptors

Effects of cardiac specific overexpression of beta(2)-adrenergic receptors (beta(2)-AR) on the development of heart failure (HF) were studied in wild-type (WT) and transgenic (TG) mice following myocardial infarction (MI) by coronary artery occlusion. Animals were studied by echocardiography at weeks 7 to 8 and by catheterization at week 9 after surgery. Post-infarct mortality, due to HF or cardiac rupture, was not different among WT mice, and there was no difference in infarct size (IS). Compared with the sham-operated group (all P < 0.01), WT mice with moderate (<36%) and large (>36%) IS developed lung congestion, cardiac hypertrophy, left ventricular (LV) dilatation, elevated LV end-diastolic pressure (LVEDP), and suppressed maximal rate of increase of LV pressure (LV dP/dt(max)) and fractional shortening (FS). Whereas changes in organ weights and echo parameters were similar to those in infarcted WT groups, TG mice had significantly higher levels of LV contractility in both moderate (dP/dt(max) 4,862 +/- 133 vs. 3,694 +/- 191 mmHg/s) and large IS groups (dP/dt(max) 4,556 +/- 252 vs. 3,145 +/- 312 mmHg/s, both P < 0.01). Incidence of pleural effusion (36% vs. 85%, P < 0.05) and LVEDP levels (6 +/- 0.3 vs. 9 +/- 0.8 mmHg, P < 0.05) were also lower in TG than in WT mice with large IS. Thus beta(2)-AR overexpression preserved LV contractility following MI without adverse consequence.     

Sympathetic control of heart rate in nNOS knockout mice

Inhibition of neuronal nitric oxide synthase (nNOS) in cardiac postganglionic sympathetic neurons leads to enhanced cardiac sympathetic responsiveness in normal animals, as well as in animal models of cardiovascular diseases. We used isolated atria from mice with selective genetic disruption of nNOS (nNOS(-/-)) and their wild-type littermates (WT) to investigate whether sympathetic heart rate (HR) responses were dependent on nNOS. Immunohistochemistry was initially used to determine the presence of nNOS in sympathetic [tyrosine hydroxylase (TH) immunoreactive] nerve terminals in the mouse sinoatrial node (SAN). After this, the effects of postganglionic sympathetic nerve stimulation (1-10 Hz) and bath-applied norepinephrine (NE; 10(-8)-10(-4) mol/l) on HR were examined in atria from nNOS(-/-) and WT mice. In the SAN region of WT mice, TH and nNOS immunoreactivity was virtually never colocalized in nerve fibers. nNOS(-/-) atria showed significantly reduced HR responses to sympathetic nerve activation and NE (P < 0.05). Similarly, the positive chronotropic response to the adenylate cyclase activator forskolin (10(-7)-10(-5) mol/l) was attenuated in nNOS(-/-) atria (P < 0.05). Constitutive NOS inhibition with L-nitroarginine (0.1 mmol/l) did not affect the sympathetic HR responses in nNOS(-/-) and WT atria. The paucity of nNOS in the sympathetic innervation of the mouse SAN, in addition to the attenuated HR responses to neuronal and applied NE, indicates that presynaptic sympathetic neuronal NO does not modulate neuronal NE release and SAN pacemaking in this species. It appears that genetic deletion of nNOS results in the inhibition of adrenergic-adenylate cyclase signaling within SAN myocytes.     

The valosin‐containing protein is a novel repressor of cardiomyocyte hypertrophy induced by pressure overload

Hypertension‐induced left ventricular hypertrophy (LVH) is an independent risk factor for heart failure. Regression of LVH has emerged as a major goal in the treatment of hypertensive patients. Here, we tested our hypothesis that the valosin‐containing protein (VCP), an ATPase associate protein, is a novel repressor of cardiomyocyte hypertrophy under the pressure overload stress. Left ventricular hypertrophy (LVH) was determined by echocardiography in 4‐month male spontaneously hypertensive rats (SHRs) vs. age‐matched normotensive Wistar Kyoto (WKY) rats. VCP expression was found to be significantly downregulated in the left ventricle (LV) tissues from SHRs vs. WKY rats. Pressure overload was induced by transverse aortic constriction (TAC) in wild‐type (WT) mice. At the end of 2 weeks, mice with TAC developed significant LVH whereas the cardiac function remained unchanged. A significant reduction of VCP at both the mRNA and protein levels in hypertrophic LV tissue was found in TAC WT mice compared to sham controls. Valosin‐containing protein VCP expression was also observed to be time‐ and dose‐dependently reduced in vitro in isolated neonatal rat cardiomyocytes upon the treatment of angiotensin II. Conversely, transgenic (TG) mice with cardiac‐specific overexpression of VCP showed a significant repression in TAC‐induced LVH vs. litter‐matched WT controls upon 2‐week TAC. TAC‐induced activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling observed in WT mice LVs was also significantly blunted in VCP TG mice. In conclusion, VCP acts as a novel repressor that is able to prevent cardiomyocyte hypertrophy from pressure overload by modulating the mTORC1 signaling pathway.     

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.     

Altered autonomic control in conscious transgenic rabbits with overexpressed cardiac Gsalpha

Both enhanced sympathetic drive and altered autonomic control are involved in the pathogenesis of heart failure. The goal of the present study was to determine the extent to which chronically enhanced sympathetic drive, in the absence of heart failure, alters reflex autonomic control in conscious, transgenic (TG) rabbits with overexpressed cardiac Gsalpha. Nine TG rabbits and seven wild-type (WT) littermates were instrumented with a left ventricular (LV) pressure micromanometer and arterial catheters and studied in the conscious state. Compared with WT rabbits, LV function was enhanced in TG rabbits, as reflected by increased levels of LV dP/dt (5,600 +/- 413 vs. 3,933 +/- 161 mmHg/s). Baseline heart rate was also higher (P < 0.05) in conscious TG (247 +/- 10 beats/min) than in WT (207 +/- 10 beats/min) rabbits and was higher in TG after muscarinic blockade (281 +/- 9 vs. 259 +/- 8 beats/min) or combined beta-adrenergic receptor and muscarinic blockade (251 +/- 6 vs. 225 +/- 9 beats/min). Bradycardia was blunted (P < 0.05), whether induced by intravenous phenylephrine (arterial baroreflex), by cigarette smoke inhalation (nasopharyngeal reflex), or by veratrine administration (Bezold-Jarisch reflex). With veratrine administration, the bradycardia was enhanced in TG for any given decrease in arterial pressure. Thus the chronically enhanced sympathetic drive in TG rabbits with overexpressed cardiac Gsalpha resulted in enhanced LV function and heart rate and impaired reflex autonomic control. The impaired reflex control was generalized, not only affecting the high-pressure arterial baroreflex but also the low-pressure Bezold-Jarisch reflex and the nasopharyngeal reflex.     

Cardiac Fibroblasts Regulate Sympathetic Nerve Sprouting and Neurocardiac Synapse Stability

Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic innervation and neurocardiac synapse stability. They also suggest that neurocardiac synapse functionality relies on a triptych with tight interaction between sympathetic nerve endings, cardiomyocytes and fibroblasts. Deregulations of this triptych may be involved in pathophysiology of cardiac diseases.     

Mechanism of enhanced cardiac function in mice with hypertrophy induced by overexpressed Akt

Transgenic mice with cardiac-specific overexpression of active Akt (TG) not only exhibit hypertrophy but also show enhanced left ventricular (LV) function. In 3-4-month-old TG, heart/body weight was increased by 60% and LV ejection fraction was elevated (84 +/- 2%, p < 0.01) compared with nontransgenic littermates (wild type (WT)) (73 +/- 1%). An increase in isolated ventricular myocyte contractile function (% contraction) in TG compared with WT (6.1 +/- 0.2 versus 3.5 +/- 0.2%, p < 0.01) was associated with increased Fura-2 Ca2+ transients (396 +/- 50 versus 250 +/- 24 nmol/liter, p < 0.05). The rate of relaxation (+dL/dt) was also enhanced in TG (214 +/- 15 versus 98 +/- 18 microm/s, p < 0.01). L-type Ca2+ current (ICa) density was increased in TG compared with WT (-9.0 +/- 0.3 versus 7.2 +/- 0.3 pA/pF, p < 0.01). Sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) protein levels were increased (p < 0.05) by 6.6-fold in TG, which could be recapitulated in vitro by adenovirus-mediated overexpression of Akt in cultured adult ventricular myocytes. Conversely, inhibiting SERCA with either ryanodine or thapsigargin affected myocyte contraction and relaxation and Ca2+ channel kinetics more in TG than in WT. Thus, myocytes from mice with overexpressed Akt demonstrated enhanced contractility and relaxation, Fura-2 Ca2+ transients, and Ca2+ channel currents. Furthermore, increased protein expression of SERCA2a plays an important role in mediating enhanced LV function by Akt. Up-regulation of SERCA2a expression and enhanced LV myocyte contraction and relaxation in Akt-induced hypertrophy is opposite to the down-regulation of SERCA2a and reduced contractile function observed in many other forms of LV hypertrophy.     

Infarction alters both the distribution and noradrenergic properties of cardiac sympathetic neurons

Regional changes occur in the sympathetic innervation of the heart after myocardial infarction (MI), including loss of norepinephrine (NE) uptake and depletion of neuronal NE. This apparent denervation is accompanied by increased cardiac NE spillover. One potential explanation for these apparently contradictory findings is that the sympathetic neurons innervating the heart are exposed to environmental stimuli that alter neuronal function. To understand the changes that occur in the innervation of the heart after MI, immunohistochemical, biochemical, and molecular analyses were carried out in the heart and stellate ganglia of control and MI rats. Immunohistochemistry with panneuronal markers revealed extensive denervation in the left ventricle (LV) below the infarct, but sympathetic nerve fibers were retained in the base of the heart. Western blot analysis revealed that tyrosine hydroxylase (TH) expression (normalized to a panneuronal marker) was increased significantly in the base of the heart and in the stellate ganglia but decreased in the LV below the MI. NE transporter (NET) binding sites, normalized to total protein, were unchanged, except in the LV, where [3H]nisoxetine binding was decreased. TH mRNA was increased significantly in the left and right stellate ganglia after MI, while NET mRNA was not. In the base of the heart, increased TH coupled with no change in NET may explain the increase in extracellular NE observed after MI. Coupled with substantial denervation in the LV, these changes likely contribute to the onset of cardiac arrhythmias.     

Regulation of nerve growth factor mRNA levels in developing rat heart ventricle is not altered by sympathectomy

The survival of sympathetic and sensory neurons is known to be controlled by nerve growth factor (NGF) supplied by the targets of innervation, yet little is known about how target NGF synthesis is regulated. We have investigated the pattern of NGF mRNA expression in developing rat heart ventricle using a sensitive RNA blotting procedure. We find that the concentration of NGF mRNA increases steadily from Embryonic Day 17 to peak levels at 10-14 days postnatal and then declines about twofold and stabilizes at the level found in adults. The rise in NGF mRNA concentration correlates with the arrival and differentiation of sympathetic nerve terminals in the heart and the cessation of sympathetic cell death. To assess the role of innervating sympathetic neurons in regulating NGF mRNA expression, neonatal rats were sympathectomized by treatment with 6-hydroxydopamine and heart ventricles were assayed for NGF message. Although this treatment reduced ventricle norepinephrine content by 82%, no significant change in NGF mRNA concentration was observed. These results suggest that the developmental program of NGF mRNA production in the heart is not influenced by innervating sympathetic neurons.     

In vivo left ventricular functional capacity is compromised in cMyBP-C null mice

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated protein that binds tightly to myosin and has a potential role for modulating myocardial contraction. We tested the hypothesis that cMyBP-C 1) contributes to the enhanced in vivo contractile state following beta-adrenergic stimulation and 2) is necessary for myocardial adaptation to chronic increases in afterload. In vivo pressure-volume relations demonstrated that left ventricular (LV) systolic and diastolic function were compromised under basal conditions in cMyBP-C(-/-) compared with WT mice. Moreover, whereas beta-adrenergic treatment significantly improved ejection fraction, peak elastance, and the time to peak elastance in WT mice, these functional indexes remained unchanged in cMyBP-C(-/-) mice. Morphological and functional changes were measured through echocardiography in anesthetized mice following 5 wk of aortic banding. Adaptation to pressure overload was diminished in cMyBP-C(-/-) mice as characterized by a lack of an increase in posterior wall thickness, increased LV diameter, deterioration of fractional shortening, and prolonged isovolumic relaxation time. These results suggest that the absence of cMyBP-C significantly diminishes in vivo LV function and markedly attenuates the increase in LV contractility following beta-adrenergic stimulation or adaptation to pressure overload.     

Electrophysiological consequences of human IKs channel expression in adult murine heart

We expressed human delayed rectifier K(+) cardiac current (I(Ks)) channels in the murine heart, which lacks native I(Ks), to determine their electrophysiological role. Mice expressing human I(Ks) channels were anesthetized, and an electrocardiogram and monophasic action potentials (MAP) recorded from the left ventricle. Sinus rate was not different between wild-type mice (WT) and transgenic mice (TG). Infusion of isoproterenol accelerated WT heart rate but not TG. Lack of TG sinus rate responsiveness may have resulted from accumulated outward current in I(Ks) channels in sinus node. Ventricular MAP duration of TG mice to 50% repolarization (APD(50)) during ventricular pacing was shorter than WT, likely resulting from outward current through I(Ks) channels. TG APD(50) showed enhanced responsiveness (shortening) to isoproterenol compared with WT. Ventricular tachyarrhythmias were initiated in TG mice by programmed stimulation but not in WT and were accelerated by isoproterenol. I(Ks) channels impart beta-adrenergic sensitivity to the ventricles and may be responsible for ventricular tachyarrhythmias.     

Modified sympathetic nerve system activity with overexpression of the voltage-dependent calcium channel beta3 subunit

N-type voltage-dependent calcium channels (VDCCs) play determining roles in calcium entry at sympathetic nerve terminals and trigger the release of the neurotransmitter norepinephrine. The accessory beta3 subunit of these channels preferentially forms N-type channels with a pore-forming CaV2.2 subunit. To examine its role in sympathetic nerve regulation, we established a beta3-overexpressing transgenic (beta3-Tg) mouse line. In these mice, we analyzed cardiovascular functions such as electrocardiography, blood pressure, echocardiography, and isovolumic contraction of the left ventricle with a Langendorff apparatus. Furthermore, we compared the cardiac function with that of beta3-null and CaV2.2 (alpha1B)-null mice. The beta3-Tg mice showed increased expression of the beta3 subunit, resulting in increased amounts of CaV2.2 in supracervical ganglion (SCG) neurons. The beta3-Tg mice had increased heart rate and enhanced sensitivity to N-type channel-specific blockers in electrocardiography, blood pressure, and echocardiography. In contrast, cardiac atria of the beta3-Tg mice revealed normal contractility to isoproterenol. Furthermore, their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through VDCCs. Langendorff heart perfusion analysis revealed enhanced sensitivity to electric field stimulation in the beta3-Tg mice, whereas beta3-null and Cav2.2-null showed decreased responsiveness. The plasma epinephrine and norepinephrine levels in the beta3-Tg mice were significantly increased in the basal state, indicating enhanced sympathetic tone. Electrophysiological analysis in SCG neurons of beta3-Tg mice revealed increased calcium channel currents, especially N- and L-type currents. These results identify a determining role for the beta3 subunit in the N-type channel population in SCG and a major role in sympathetic nerve regulation.     

Cardiac dilatation and pump dysfunction without intrinsic myocardial systolic failure following chronic beta-adrenoreceptor activation

There is no direct evidence to indicate that pump dysfunction in a dilated chamber reflects the impact of chamber dilatation rather than the degree of intrinsic systolic failure resulting from myocardial damage. In the present study, we explored the relative roles of intrinsic myocardial systolic dysfunction and chamber dilatation as mediators of left ventricular (LV) pump dysfunction. Administration of isoproterenol, a beta-adrenoreceptor agonist, for 3 mo to rats (0.1 mg.kg(-1).day(-1)) resulted in LV pump dysfunction as evidenced by a reduced LV endocardial fractional shortening (echocardiography) and a decrease in the slope of the LV systolic pressure-volume relation (isolated heart preparations). Although chronic beta-adrenoreceptor activation induced cardiomyocyte damage (deoxynucleotidyl transferase-mediated dUTP nick-end labeling) as well as beta(1)- and beta(2)-adrenoreceptor inotropic downregulation (attenuated contractile responses to dobutamine and salbutamol), these changes failed to translate into alterations in intrinsic myocardial contractility. Indeed, LV midwall fractional shortening (echocardiography) and the slope of the LV systolic stress-strain relation (isolated heart preparations) were unchanged. A normal intrinsic myocardial systolic function, despite the presence of cardiomyocyte damage and beta-adrenoreceptor inotropic downregulation, was ascribed to marked increases in myocardial norepinephrine release, to upregulation of alpha-adrenoreceptor-mediated contractile effects as determined by phenylephrine responsiveness, and to compensatory LV hypertrophy. LV pump failure was attributed to LV dilatation, as evidenced by increased LV internal dimensions (echocardiography), and a right shift and increased volume intercept of the LV diastolic pressure-volume relation. In conclusion, chronic sympathetic stimulation, despite reducing beta-adrenoreceptor-mediated inotropic responses and promoting myocyte apoptosis, may nevertheless induce pump dysfunction primarily through LV dilatation, rather than intrinsic myocardial systolic failure.     

Overexpression of eNOS in brain stem reduces enhanced sympathetic drive in mice with myocardial infarction

Reduced nitric oxide (NO) in the brain might contribute to enhanced sympathetic drive in heart failure (HF). The aim of this study was to determine whether increased NO production induced by local overexpression of endothelial NO synthase (eNOS) in the nucleus tractus solitarius (NTS) of the brain stem reduces the enhanced sympathetic drive in mice with HF. Myocardial infarction (MI) was induced in mice by ligating the left coronary artery. MI mice exhibited left ventricular dilatation and a reduced left ventricular ejection fraction. Urinary norepinephrine excretion in MI mice was greater than that in sham-operated mice, indicating that sympathetic drive was enhanced in this model. Thus this model has features that are typical of HF. Western blot analysis and immunohistochemical staining for neuronal NOS (nNOS) indicated that nNOS protein expression was significantly reduced in the brain stem of MI mice. MI mice had a significantly smaller increase in blood pressure evoked by intracisternal injection of N(G)-monomethyl-L-arginine than sham-operated mice. Adenoviral vectors encoding either eNOS (AdeNOS) or beta-galactosidase (Adbeta gal) were transfected into the NTS to examine the effect of increased NO production in the NTS on the enhanced sympathetic drive in HF. After the gene transfer, urinary norepinephrine excretion was reduced in AdeNOS-transfected MI mice but not in Adbeta gal-transfected MI mice. These results indicate that nNOS expression in the brain stem, especially in the NTS, is reduced in the MI mouse model of HF, and increased NO production induced by overexpression of eNOS in the NTS attenuates the enhanced sympathetic drive in this model.     

Ventricular-specific expression of angiotensin II type 2 receptors causes dilated cardiomyopathy and heart failure in transgenic mice

The angiotensin II type 2 (AT2) receptor is upregulated in the left ventricle in heart failure, but its pathophysiological roles in vivo are not understood. In the present study, AT2 receptors were expressed in transgenic (TG) mice using the ventricular-specific myosin light-chain (MLC-2v) promoter. In TG compared with nontransgenic (NTG) mice, in vivo left ventricular (LV) systolic pressure and peak +dP/dt were depressed while LV diastolic pressure was elevated (P < 0.05). Echocardiography showed severely depressed LV fractional shortening, increased systolic and diastolic dimensions, and wall thinning (P < 0.05). Confocal and electron microscopy studies revealed an increase in the size of myocytes and interstitial spaces as well as an increase in interstitial collagen, disruption of the Z-band, and changes in cytochrome c localization. The changes were most prominent in the highest-expressing TG line, which implies a dose-response relationship. AT2 overexpression was also directly associated with the increase of phosphorylated protein levels of PKC-alpha, PKC-beta, and p70S6 kinase. These data demonstrate that ventricular myocyte-specific expression of AT2 receptors promotes the development of dilated cardiomyopathy and heart failure in vivo.     

Overexpression of glutathione peroxidase attenuates myocardial remodeling and preserves diastolic function in diabetic heart

Oxidative stress plays an important role in the structural and functional abnormalities of diabetic heart. Glutathione peroxidase (GSHPx) is a critical antioxidant enzyme that removes H(2)O(2) in both the cytosol and mitochondia. We hypothesized that the overexpression of GSHPx gene could attenuate left ventricular (LV) remodeling in diabetes mellitus (DM). We induced DM by injection of streptozotocin (160 mg/kg ip) in male GSHPx transgenic mice (TG+DM) and nontransgenic wildtype littermates (WT+DM). GSHPx activity was higher in the hearts of TG mice compared with WT mice, with no significant changes in other antioxidant enzymes. LV thiobarbituric acid-reactive substances measured in TG+DM at 8 wk were significantly lower than those in WT+DM (58 +/- 3 vs. 71 +/- 5 nmol/g, P < 0.05). Heart rate and aortic blood pressure were comparable between groups. Systolic function was preserved normal in WT+DM and TG+DM mice. In contrast, diastolic function was impaired in WT+DM and was improved in TG+DM as assessed by the deceleration time of peak velocity of transmitral diastolic flow and the time needed for relaxation of 50% maximal LV pressure to baseline value (tau; 13.5 +/- 1.2 vs. 8.9 +/- 0.7 ms, P < 0.01). The TG+DM values were comparable with those of WT+Control (tau; 7.8 +/- 0.2 ms). Improvement of LV diastolic function was accompanied by the attenuation of myocyte hypertrophy, interstitial fibrosis, and apoptosis. Overexpression of GSHPx gene ameliorated LV remodeling and diastolic dysfunction in DM. Therapies designed to interfere with oxidative stress might be beneficial to prevent cardiac abnormalities in DM.     

Overexpression of APOC1 in obob mice leads to hepatic steatosis and severe hepatic insulin resistance

Obese obob mice with strong overexpression of the human apolipoprotein C1 (APOC1) exhibit excessive free fatty acid (FFA) and triglyceride (TG) levels and severely reduced body weight (due to the absence of subcutaneous adipose tissue) and skin abnormalities. To evaluate the effects of APOC1 overexpression on hepatic and peripheral insulin sensitivity in a less-extreme model, we generated obob mice with mild overexpression of APOC1 (obob/APOC1(+/-)) and performed hyperinsulinemic clamp analysis. Compared with obob littermates, obob/APOC1(+/-) mice showed reduced body weight (-25%) and increased plasma levels of TG (+632%), total cholesterol (+134%), FFA (+65%), glucose (+73%), and insulin (+49%). Hyperinsulinemic clamp analysis revealed severe whole-body and hepatic insulin resistance in obob/APOC1(+/-) mice and, in addition, increased hepatic uptake of FFA and hepatic TG content. Treatment of obob/APOC1(+/-) mice with rosiglitazone strongly improved whole-body insulin sensitivity as well as hepatic insulin sensitivity, despite a further increase of hepatic fatty acid (FA) uptake and a panlobular increase of hepatic TG accumulation. We conclude that overexpression of APOC1 prevents rosiglitazone-induced peripheral FA uptake leading to severe hepatic steatosis. Interestingly, despite rosiglitazone-induced hepatic steatosis, hepatic insulin sensitivity improves dramatically. We hypothesize that the different hepatic fat accumulation and/or decrease in FA intermediates has a major effect on the insulin sensitivity of the liver.     

Calcitonin gene-related peptide and substance P contribute to reduced blood pressure in sympathectomized rats

CGRP and substance P (SP) are produced in dorsal root ganglia (DRG) sensory neurons and modulate vascular tone. Sympathetic and sensory nerves compete for NGF, a potent stimulator of CGRP and SP, and it has been suggested that sympathetic hyperinnervation in spontaneously hypertensive rats may reduce the availability of NGF to sensory nerves, thus reducing CGRP and SP. The purpose of this study was to determine whether destruction of peripheral sympathetic nerves in normal rats would increase the availability of NGF for sensory neurons and enhance expression of CGRP and SP. Sympathectomy was produced in rats by guanethidine sulfate administration. Control rats received saline. Sympathectomized rats displayed reductions in blood pressure (BP) and atria norepinephrine levels, whereas NGF levels in the DRG, spleen, and ventricles were increased. Sympathectomy also enhanced CGRP and SP mRNA and peptide content in DRG. Administration of CGRP and SP receptor antagonists increased the BP in sympathectomized rats but not in the controls. Thus sympathectomy enhances sensory neuron CGRP and SP expression that contributes to the BP reduction.