Myocardial Overexpression Research Articles

111Indium-trastuzumab visualises myocardial human epidermal growth factor receptor 2 expression shortly after anthracycline treatment but not during heart failure: A clue to uncover the mechanisms of trastuzumab-related cardiotoxicity

Aim Trastuzumab can induce cardiotoxicity, particularly when combined with anthracyclines. Myocardial human epidermal growth factor receptor 2 (HER2) expression may be transiently upregulated by a compensatory mechanism following cardiac stress. 111In-DTPA-trastuzumab, scintigraphy can detect HER2 positive tumour lesions, however previously, we found myocardial uptake in only 1 of the 15 anthracycline-pre-treated patients with a median of 11 months after the last anthracycline administration. To evaluate whether myocardial HER2 expression is upregulated by anthracycline-induced cardiac stress or in case of heart failure by chronic pressure or volume overload, we performed 111In-DTPA-trastuzumab scans in patients shortly after anthracyclines and with non-anthracycline-related heart failure. Methods Patients within 3 weeks after undergoing 4–6 cycles first-line anthracycline-based chemotherapy and patients with heart failure due to cardiac disease underwent gammacamera imaging 48 and 96 h after 111In-DTPA-trastuzumab intravenously. Results Myocardial 111In-DTPA-trastuzumab uptake was observed in 5 out of 10 anthracycline-treated patients, who all were without symptomatic cardiac dysfunction. None of the 10 heart failure patients showed myocardial uptake. Conclusion Shortly after completion of anthracycline treatment, myocardial HER2 over-expression was detectable in 50% of the patients. 111In-DTPA-trastuzumab scintigraphy after anthracyclines prior to adjuvant trastuzumab potentially identifies patients susceptible for trastuzumab-related cardiotoxicity and thus may facilitate the optimal timing of trastuzumab therapy.

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.

Accelerated Mitochondrial Adenosine Diphosphate/Adenosine Triphosphate Transport Improves Hypertension-Induced Heart Disease

Strong evidence suggests that mitochondrial malfunction, which leads to disturbed energy metabolism and stimulated apoptosis, is a linchpin in the induction and manifestation of cardiac failure. An adequate exchange of ATP and ADP over the inner mitochondrial membrane by the adenine nucleotide translocase (ANT) is thereby essential to guarantee the cellular energy supply. To explore the effect of an ameliorated mitochondrial ATP/ADP transportation on cardiac dysfunction, we generated transgenic rats overexpressing ANT1 in the heart (ANT rats) and crossed them with renin-overexpressing rats (REN rats) suffering from hypertension-induced cardiac insufficiency. Cardiac-specific ANT1 overexpression resulted in a higher ATP/ADP transportation and elevated activities of respiratory chain complexes. Increased ANT activity in double-transgenic (ANT/REN) animals did not influence excessive hypertension seen in REN rats. Hypertension-induced cardiac hypertrophy in the REN rats was prevented by parallel ANT1 overexpression, however, and left ventricular function remarkably improved. The ANT1 overexpression led to a reduction in fibrosis and an improvement in cardiac tissue architecture. Consequently, the survival rate of ANT/REN rats was enhanced. Further investigations into the cardioprotective mechanism of ANT1 overexpression revealed improved mitochondrial structure and function and significantly reduced apoptosis in ANT/REN rats, shown by lowered cytosolic/mitochondrial cytochrome c ratio, reduced caspase 3 level, and prevented DNA degradation. Myocardial ANT1 overexpression protects against hypertension-induced cardiac pathology. Thus, the improvement in mitochondrial function may be a basic principle for new strategies in treating heart disease.

Regulated Overexpression of the A 1 -Adenosine Receptor in Mice Results in Adverse but Reversible Changes in Cardiac Morphology and Function

Both the A1- and A3-adenosine receptors (ARs) have been implicated in mediating the cardioprotective effects of adenosine. Paradoxically, overexpression of both A1-AR and A3-AR is associated with changes in the cardiac phenotype. To evaluate the temporal relationship between AR signaling and cardiac remodeling, we studied the effects of controlled overexpression of the A1-AR using a cardiac-specific and tetracycline-transactivating factor-regulated promoter. Constitutive A1-AR overexpression caused the development of cardiac dilatation and death within 6 to 12 weeks. These mice developed diminished ventricular function and decreased heart rate. In contrast, when A1-AR expression was delayed until 3 weeks of age, mice remained phenotypically normal at 6 weeks, and >90% of the mice survived at 30 weeks. However, late induction of A1-AR still caused mild cardiomyopathy at older ages (20 weeks) and accelerated cardiac hypertrophy and the development of dilatation after pressure overload. These changes were accompanied by gene expression changes associated with cardiomyopathy and fibrosis and by decreased Akt phosphorylation. Discontinuation of A1-AR induction mitigated cardiac dysfunction and significantly improved survival rate. These data suggest that robust constitutive myocardial A1-AR overexpression induces a dilated cardiomyopathy, whereas delaying A1-AR expression until adulthood ameliorated but did not eliminate the development of cardiac pathology. Thus, the inducible A1-AR transgenic mouse model provides novel insights into the role of adenosine signaling in heart failure and illustrates the potentially deleterious consequences of selective versus nonselective activation of adenosine-signaling pathways in the heart.

Death, Cardiac Dysfunction, and Arrhythmias Are Increased by Calmodulin Kinase II in Calcineurin Cardiomyopathy

Activation of cellular Ca2+ signaling molecules appears to be a fundamental step in the progression of cardiomyopathy and arrhythmias. Myocardial overexpression of the constitutively active Ca2+-dependent phosphatase calcineurin (CAN) causes severe cardiomyopathy marked by left ventricular (LV) dysfunction, arrhythmias, and increased mortality rate, but CAN antagonist drugs primarily reduce hypertrophy without improving LV function or risk of death. We found that activity and expression of a second Ca2+-activated signaling molecule, calmodulin kinase II (CaMKII), were increased in hearts from CAN transgenic mice and that CaMKII-inhibitory drugs improved LV function and suppressed arrhythmias. We devised a genetic approach to "clamp" CaMKII activity in CAN mice to control levels by interbreeding CAN transgenic mice with mice expressing a specific CaMKII inhibitor in cardiomyocytes. We developed transgenic control mice by interbreeding CAN transgenic mice with mice expressing an inactive version of the CaMKII-inhibitory peptide. CAN mice with CaMKII inhibition had reduced risk of death and increased LV and ventricular myocyte function and were less susceptible to arrhythmias. CaMKII inhibition did not reduce transgenic overexpression of CAN or expression of endogenous CaMKII protein or significantly reduce most measures of cardiac hypertrophy. CaMKII is a downstream signal in CAN cardiomyopathy, and increased CaMKII activity contributes to cardiac dysfunction, arrhythmia susceptibility, and longevity during CAN overexpression.

Myocardial HER2 expression with 111In-DTPA-trastuzmab scan in patients shortly after anthracycline treatment

3057 Background: The monoclonal antibody trastuzumab, apart from antitumor effect, can induce cardiotoxicity, particularly when combined with anthracyclines. Myocardial HER2 upregulation may serve, transiently, as a compensatory mechanism induced by cardiac stress. Previously we showed in a xenograft model that 111In-DTPA-trastuzumab scintigraphy can detect HER2 positive lesions (Br J Pharmacol 2004;143:99–106) but that myocardial 111In-DTPA-trastuzumab uptake was found in only 1 of 17 anthracycline-pretreated HER2-positive metastatic breast cancer patients (ESMO 2004#50). This low number may be related to the long interval between anthracycline administration (median 11 months) and performed scan in these patients. To evaluate whether myocardial HER2 expression is induced by anthracyclines, we performed 111In-DTPA-trastuzumab scans in patients shortly after anthracycline treatment. Methods: Patients who completed 4–6 cycles of anthracycline-based chemotherapy (&lt; 3 weeks after last dose) underwent gammacamera imaging 48 and 96 h after iv administration of 150 MBq 111In-DTPA-trastuzumab (5mg). Results: 10 anthracycline-treated patients, 8 as adjuvant breast cancer treatment and 2 for metastatic sarcoma have been enrolled. Myocardial 111In-DTPA-trastuzumab uptake was observed in 5/10 anthracycline-treated patients who all were without symptomatic cardiac dysfunction. Conclusions: Shortly after completion of anthracycline treatment myocardial HER2 overexpression was detectable in 50% of the patients. This may be a transient phenomenon. 111In-DTPA-trastuzumab scan after anthracycline treatment prior to adjuvant trastuzumab may identify patients more susceptible for trastuzumab-induced cardiotoxicity. This important observation may add to optimal timing of trastuzumab therapy i.e. when HER2/neu expression in the heart is negative (again). [Table: see text]

TNF-related apoptosis-inducing ligand and its decoy receptor osteoprotegerin in nonischemic dilated cardiomyopathy

Apoptosis has been attributed an essential role in dilated cardiomyopathy (DCM) recently. We assessed expression of TNF-related apoptosis-inducing ligand (TRAIL) and its decoy receptor osteoprotegerin (OPG) in men with nonischemic DCM, who underwent coronary angiography and endomyocardial biopsy (EMB) after exclusion of coronary artery disease compared to control patients. TRAIL plasma concentrations were elevated in DCM ( p = 0.02 vs. controls), and were positively correlated with left ventricular enddiastolic diameter ( r = 0.15, p = 0.04), whereas OPG plasma levels did not differ between both groups ( p = 0.96). In EMB of DCM patients, TRAIL and OPG protein were detected by immunohistochemistry but not in controls. Furthermore, gene expression in EMB or peripheral blood leukocytes (PBL) of DCM patients assessed by real-time PCR showed an increase of TRAIL mRNA in PBL ( p = 0.01 vs. controls), whereas OPG mRNA was upregulated in endomyocardial specimens ( p < 0.001 vs. controls). In conclusion, myocardial overexpression of antiapoptotic OPG in DCM patients may represent a compensatory mechanism to limit systemic activation of TRAIL in patients with congestive heart disease.

Cardiac Overexpression of the Norepinephrine Transporter Uptake-1 Results in Marked Improvement of Heart Failure

A hyperadrenergic state is one of the key features of human and experimental heart failure. Decreased densities and activities of the presynaptic neuronal norepinephrine (NE) transporter uptake-1 occur both in patients and animal models. It is currently unclear to what extent the reduction of uptake-1 contributes to the deterioration of heart failure. Therefore, we investigated the effects of myocardial overexpression of uptake-1 in both nonfailing rabbit hearts and in an animal model of heart failure. Heart failure was induced in rabbits by rapid ventricular pacing. Adenoviral gene transfer was used to overexpress uptake-1 in the myocardium. Uptake-1 overexpression led to increased NE uptake capacity into the myocardium. In contrast, systemic plasma NE levels in uptake-1-overexpressing failing rabbits (uptake-1-CHF) did not differ from controls. Downregulation of SERCA-2 and beta-adrenergic receptors in the failing myocardium was significantly reversed after uptake-1 overexpression. Uptake-1 overexpression significantly improved left ventricular (LV) diameters (LV end-diastolic diameter: in GCP-overexpressing failing rabbits (GFP-CHF), 17.4+/-0.4 mm; in uptake-1-CHF rabbits, 15.6+/-0.6 mm) and systolic contractility (fractional shortening: GFP-CHF, 20.7+/-0.6%; uptake-1-CHF, 27.3+/-0.7%), as assessed by echocardiography at the end of the heart failure protocol. Intraventricular tip catheter measurements revealed enhanced contractile reserve (dP/dt max with isoproterenol 1.0 microg/kg: GFP-CHF, 6964+/-230 mm Hg/sec; uptake-1-CHF, 7660+/-315 mm Hg/sec) and LV relaxation (dP/dt min with isoproterenol 1.0 microg/kg: GFP-CHF: -3960+/-260 mm Hg/sec; uptake-1-CHF, -4910+/-490 mm Hg/sec). End-diastolic filling pressures (GFP-CHF, 8.5+/-1.2 mm Hg; uptake-1-CHF, 5.6+/-0.7 mm Hg) tended to be lower in uptake-1 overexpressing animals. In summary, local overexpression of uptake-1 in the myocardium results in marked structural and functional improvement of heart failure, thus underlining the importance of uptake-1 as a key protein in heart failure.

Magnesium supplementation prevents angiotensin II-induced myocardial damage and CTGF overexpression

Magnesium deficiency promotes vasoconstriction and myocardial damage. Recent studies provide evidence that Ang II mobilizes intracellular Mg through AT1 receptor-mediated pathways. We tested the hypothesis of whether magnesium supplementation prevents Ang II-induced myocardial damage and induction of the profibrotic connective tissue growth factor (CTGF). Four-week-old double transgenic rats harboring human renin and angiotensinogen genes (dTGR) were given dietary magnesium supplementation (0.6%) for 3 weeks. Control dTGR and normotensive Sprague-Dawley (SD) rats received normal diet (Mg 0.2%). Histopathological, immunohistochemical and mRNA analysis were used to detect the treatment-related effects of dietary magnesium in dTGR. Magnesium (Mg) supplementation decreased blood pressure, ameliorated cardiac hypertrophy, protected against the development of Ang II-induced myocardial damage and increased serum ionized Mg2+ concentration (all variables P < 0.05). There was no difference in serum ionized Mg2+ concentration between dTGR and SD rats. Myocardial connective tissue growth factor (CTGF) mRNA and protein expressions were increased by 300% in dTGR (P < 0.05), especially in areas with myocardial infarctions and vascular inflammation. Magnesium supplementation prevented Ang II-induced myocardial CTGF overexpression (P < 0.05). Magnesium supplementation also improved the therapeutic effects of the calcineurin inhibitor tacrolimus, which produced marked hypomagnesemia when given as monotherapy. Our findings suggest a salutary effect for magnesium supplementation in the treatment of Ang II-induced myocardial complications.

Local myocardial overexpression of growth hormone attenuates postinfarction remodeling and preserves cardiac function

Background Ventricular remodeling with chamber dilation and wall thinning is seen in postinfarction heart failure. Growth hormone induces myocardial hypertrophy when oversecreted. We hypothesized that localized myocardial hypertrophy induced by gene transfer of growth hormone could inhibit remodeling and preserve cardiac function after myocardial infarction. Methods Rats underwent direct intramyocardial injection of adenovirus encoding either human growth hormone (n = 9) or empty null vector as control (n = 9) 3 weeks after ligation of the left anterior descending coronary artery. Analysis of the following was performed 3 weeks after delivery: hemodynamics, ventricular geometry, cardiomyocyte fiber size, and serum growth hormone levels. Results The growth hormone group had significantly better systolic cardiac function as measured by maximum left ventricular pressure (73.6 ± 6.9 mm Hg versus control 63.7 ± 7.8 mm Hg, p < 0.05) and maximum dP/dt (2845 ± 453 mm Hg/s versus 1949 ± 605 mm Hg/s, p < 0.005), and diastolic function as measured by minimum dP/dt (−2,520 ± 402 mm Hg/s versus −1,500 ± 774 mm Hg/s, p < 0.01). Ventricular geometry was preserved in the growth hormone group (ventricular diameter 12.2 ± 0.7 mm versus control 13.1 ± 0.4 mm, p < 0.05; borderzone wall thickness 2.0 ± 0.2 mm versus 1.5 ± 0.1 mm, p < 0.001), and was associated with cardiomyocyte hypertrophy (6.09 ± 0.63 μm versus 4.66 ± 0.55 μm, p < 0.005). Local myocardial expression of growth hormone was confirmed, whereas serum levels were undetectable after 3 weeks. Conclusions Local myocardial overexpression of growth hormone after myocardial infarction resulted in cardiomyocyte hypertrophy, attenuated ventricular remodeling, and improved systolic and diastolic cardiac function. The induction of localized myocardial hypertrophy presents a novel therapeutic approach for the treatment of ischemic heart failure.

Targeted overexpression of growth hormone by adenoviral gene transfer preserves myocardial function and ventricular geometry in ischemic cardiomyopathy

Background. – Post-infarction heart failure is characterized by progressive left ventricular dilatation and wall thinning, with both systolic and diastolic cardiac dysfunction. Human growth hormone (GH) stimulates cardiac hypertrophy when secreted in excess and directly enhances cardiomyocyte contractile function. We hypothesized that local myocardial overexpression of GH could prevent ventricular remodeling and heart failure following myocardial infarction (MI) in rats. Methods and Results. – Rats underwent ligation of the left anterior descending coronary artery with direct intramyocardial injection of adenovirus encoding human GH ( n = 8) or null virus as control ( n = 8). Six weeks following MI, Adeno-GH treated animals had significant preservation of both systolic and diastolic cardiac function compared to Null animals (maximum d P/d t GH 2927 ± 83 vs Null 1622 ± 159 mmHg/sec, p < 0.001; minimum d P/d t –2409 ± 82 vs –1195 ± 179 mmHg/sec, p < 0.01). GH animals had improved ventricular geometry with decreased chamber dilatation (13.2 ± 0.13 vs 14.4±0.15 mm, p < 0.001) and increased wall thickness (2.02 ± 0.10 vs 1.28 ± 0.07 mm, p < 0.001), and this was associated with advantageous myocardial hypertrophy with increased cardiomyocyte fiber size. Local myocardial overexpression of GH protein was seen in Adeno-GH animals, while serum levels of human GH were undetectable after 6 weeks. Conclusions. – Treatment with Adeno-GH following MI resulted in reduced ventricular dilatation, increased local myocardial hypertrophy, and preservation of both systolic and diastolic cardiac function. No significant systemic exposure to growth hormone transgene was observed. The induction of regional hypertrophy is a novel approach to treating heart failure, and may be useful to treat or prevent post-infarction ischemic cardiomyopathy.

Effects of two Gbetagamma-binding proteins--N-terminally truncated phosducin and beta-adrenergic receptor kinase C terminus (betaARKct)--in heart failure.

Myocardial overexpression of the C-terminus of beta-adrenergic receptor kinase (betaARKct) has been shown to result in a positive inotropic effect or an improvement of survival in heart failure. However, it is not clear whether this beneficial effect is mainly because of dominant-negative inhibition of betaARK1, and a consecutive resensitization of beta-adrenergic receptors (betaAR), or rather due to inhibition of other Gbetagamma-mediated effects. In this study, we tested whether overexpression of N-terminally truncated phosducin (nt-del-phosducin), another Gbetagamma-binding protein that does not resensitize betaARs owing to simultaneous inhibition of GDP release from Galpha subunits, shows the same effects as betaARKct. Adenoviral gene transfer was used to express nt-del-phosducin and betaARKct in isolated ventricular cardiomyocytes and in myocardium of rabbits, which suffered from heart failure because of rapid ventricular pacing. BetaAR-stimulated cAMP formation was increased by betaARKct, but not by nt-del-phosducin, whereas both proteins inhibited Gbetagamma-mediated effects. Both transgenes also increased contractility of normal and failing isolated cardiomyocytes and improved contractility in rabbits with heart failure after gene transfer in vivo. In conclusion, overexpression of nt-del-phosducin enhances the contractility of cardiomyocytes to the same extent as betaARKct, suggesting that the effects of betaARKct might be owing to inhibition of Gbetagamma rather than to betaAR resensitization.

Heat Shock Protein 72 Enhances Manganese Superoxide Dismutase Activity During Myocardial Ischemia-Reperfusion Injury, Associated With Mitochondrial Protection and Apoptosis Reduction

Heat shock protein 72 (HSP72) is known to provide myocardial protection against ischemia-reperfusion injury by its chaperoning function. Target molecules of this effect are presumed to include not only structural proteins but also other self-preservation proteins. The details, however, remain unknown. Manganese superoxide dismutase (Mn-SOD) is an enzyme that preserves mitochondria, a key organelle for cellular respiration, from reperfusion injury and limits mitochondria-related apoptosis. We hypothesized that Mn-SOD would play a role in HSP72-mediated cardioprotection. Rat hearts were transfected with human HSP72 by intra-coronary infusion of Hemagglutinating Virus of Japan-liposome, resulting in global myocardial overexpression of HSP72. After ischemia-reperfusion injury, cardiac function (left ventricular systolic pressure, maximum dP/dt, minimum dP/dt, and coronary flow) was improved in the HSP72-transfected hearts compared with control-transfected ones, corresponding with less leakage of creatine kinase and mitochondrial aspartate aminotransferase. Postischemic Mn-SOD content and activity in the HSP72-transfected hearts were enhanced in comparison with the controls (content: 96.9+/-4.1 versus 85.5+/-2.5% to the preischemic level, P=0.038; activity: 93.9+/-2.2 versus 82.2+/-3.7%, P=0.022), associated with improved mitochondrial respiratory function (postischemic percent respiratory control index; NAD(+)-linked: 81.3+/-3.8 versus 18.5+/-4.4%; FAD-linked: 71.8+/-5.5 versus 20.7+/-5.3%, P<0.001). In addition, incidence of postischemic cardiomyocyte apoptosis was attenuated in the HSP72-transfected hearts (4.0+/-1.1 versus 10.3+/-3.3%, P=0.036), correlating with an increased Bcl-2 level and reduced up-regulation of caspase-3. These data suggest that the enhanced Mn-SOD activity during ischemia-reperfusion injury, which is associated with mitochondrial protection and apoptosis reduction, is a possible mechanism of HSP72-induced cardioprotection.

Myocardial overexpression of the cardiac b-adrenergic receptor kinase-1 inhibitor (BARKI) delay the development of cardiomyopathy induced by myocardial expression of monocyte chemo-tactic protein-1 (MCP-1)

Myocardial overexpression of the cardiac b-adrenergic receptor kinase-1 inhibitor (BARKI) delay the development of cardiomyopathy induced by myocardial expression of monocyte chemo-tactic protein-1 (MCP-1)

Regional overexpression of insulin-like growth factor-I and transforming growth factor-beta1 in the myocardium of patients with hypertrophic obstructive cardiomyopathy.

Hypertrophic obstructive cardiomyopathy has been proposed to be the result of gene mutations of contractile proteins. However, we have previously shown significant elevation of insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) at the messenger RNA, protein, and receptor levels in patients with hypertrophic obstructive cardiomyopathy when compared with myocardium from patients without this disorder. We hypothesized that this growth factor overexpression is a regional phenomenon. To test this hypothesis, we compared levels of IGF-I and TGF-beta1 in hypertrophic and nonhypertrophic myocardium within the same group of patients with hypertrophic obstructive cardiomyopathy. Two biopsy specimens were obtained from each patient undergoing septal myectomy for severely symptomatic hypertrophic obstructive cardiomyopathy, from hypertrophied septum and from nonhypertrophied myocardium (8 patients in total). Clinical data were prospectively recorded. Messenger RNA levels for growth factor were quantified by means of multiplex reverse transcriptase-polymerase chain reaction, expressed as a densitometric ratio of growth factor/glyceraldehyde-3-phosphate dehydrogenase. Protein levels were quantified by means of chemiluminescent slot blot analysis. Growth factor proteins were used to generate a standard curve. IGF-I messenger RNA and protein levels in hypertrophic myocardium were 2.6 and 2.9 times greater, respectively, than in nonhypertrophic myocardium of the same patients (both P <.01). TGF-beta 1 messenger RNA and protein levels in the hypertrophic myocardium were 2.5 and 2.8 times greater, respectively, than the levels in the nonhypertrophied myocardium (both P <.01). There was a significant correlation between the IGF-I protein ratio (hypertrophic/nonhypertrophic myocardium) and the inducible left ventricular outflow tract gradients measured at cardiac catheterization (r = 0.77, P =.025). Myocardial overexpression of IGF-I and TGF-beta1 is a regional phenomenon in patients with hypertrophic obstructive cardiomyopathy and is likely involved in the pathogenesis of the disorder.

Adrenergic regulation of cardiac myocyte apoptosis.

The direct effects of catecholamines on cardiac myocytes may contribute to both normal physiologic adaptation and pathologic remodeling, and may be associated with cellular hypertrophy, apoptosis, and alterations in contractile function. Norepinephrine (NE) signals via alpha- and beta-adrenergic receptors (AR) that are coupled to G-proteins. Pharmacologic studies of cardiac myocytes in vitro demonstrate that stimulation of beta1-AR induces apoptosis which is cAMP-dependent and involves the voltage-dependent calcium influx channel. In contrast, stimulation of beta2-AR exerts an anti-apoptotic effect which appears to be mediated by a pertussis toxin-sensitive G protein. Stimulation of alpha1-AR causes myocyte hypertrophy and may exert an anti-apoptotic action. In transgenic mice, myocardial overexpression of either beta1-AR or G(alpha)s is associated with myocyte apoptosis and the development of dilated cardiomyopathy. Myocardial overexpression of beta2-AR at low levels results in improved cardiac function, whereas expression at high levels leads to dilated cardiomyopathy. Overexpression of wildtype alpha1B-AR does not result in apoptosis, whereas overexpression of G(alpha)q results in myocyte hypertrophy and/or apoptosis depending on the level of expression. Differential activation of the members of the mitogen-activated protein kinase (MAPK) superfamily and production of reactive oxygen species appear to play a key role in mediating the actions of adrenergic pathways on myocyte apoptosis and hypertrophy. This review summarizes current knowledge about the molecular and cellular mechanisms involved in the regulation of cardiac myocyte apoptosis via stimulation of adrenergic receptors and their coupled effector pathways.

Transgenic myocardial overexpression of fibroblast growth factor-1 increases coronary artery density and branching.

Fibroblast growth factor (FGF)-1 plays important roles during myocardial and coronary morphogenesis. FGF-1 is also involved in the physiological response of the adult heart against ischemia, which includes cardiomyocyte protection and vascular growth. In the present study, we have generated transgenic mice with specific myocardial overexpression of the gene. Transgene expression was verified by Northern blot, and increased FGF-1 protein content was assessed by Western blot and immunoconfocal microscopy. Anatomic, histomorphological, and ultrastructural analyses revealed no major morphological or developmental abnormalities of transgenic hearts. Capillary density was unaltered, whereas the density of coronary arteries, especially arterioles, was significantly increased, as was the number of branches of the main coronary arteries. In addition, the coronary flow was significantly enhanced in transgenic mice ex vivo. These differences in the anatomic pattern of the coronary vasculature are established during the second month of postnatal life. The present findings demonstrate an important role of FGF-1 in the differentiation and growth of the coronary system and suggest that it is a key regulatory molecule of the differentiation of the arterial system.

Intracoronary Adenovirus-Mediated Delivery and Overexpression of the β 2 -Adrenergic Receptor in the Heart

Genetic modulation of ventricular function may offer a novel therapeutic strategy for patients with congestive heart failure. Myocardial overexpression of beta(2)-adrenergic receptors (beta(2)ARs) has been shown to enhance contractility in transgenic mice and reverse signaling abnormalities found in failing cardiomyocytes in culture. In this study, we sought to determine the feasibility and in vivo consequences of delivering an adenovirus containing the human beta(2)AR cDNA to ventricular myocardium via catheter-mediated subselective intracoronary delivery. Rabbits underwent percutaneous subselective catheterization of either the left or right coronary artery and infusion of adenoviral vectors containing either a marker transgene (Adeno-betaGal) or the beta(2)AR (Adeno-beta(2)AR). Ventricular function was assessed before catheterization and 3 to 6 days after gene delivery. Both left circumflex- and right coronary artery-mediated delivery of Adeno-beta(2)AR resulted in approximately 10-fold overexpression in a chamber-specific manner. Delivery of Adeno-betaGal did not alter in vivo left ventricular (LV) systolic function, whereas overexpression of beta(2)ARs in the LV improved global LV contractility, as measured by dP/dt(max), at baseline and in response to isoproterenol at both 3 and 6 days after gene delivery. Percutaneous adenovirus-mediated intracoronary delivery of a potentially therapeutic transgene is feasible, and acute global LV function can be enhanced by LV-specific overexpression of the beta(2)AR. Thus, genetic modulation to enhance the function of the heart may represent a novel therapeutic strategy for congestive heart failure and can be viewed as molecular ventricular assistance.

Carvedilol and Lacidipine Prevent Cardiac Hypertrophy and Endothelin-1 Gene Overexpression After Aortic Banding

Carvedilol and lacidipine have been shown to exert cardioprotective effects in rat models of chronic hypertension. We investigated their effects in an acute model of pressure overload produced by suprarenal aortic constriction, in which enhanced myocardial production of endothelin-1 could play a crucial role. In the absence of drug treatment, after 1 week, aortic banding provoked an increase in carotid pressure associated with left ventricular hypertrophy (29%; P<0.01). These changes were accompanied by increased myocardial expression of preproendothelin-1 (2.5 times; P<0.05) and skeletal alpha-actin (3.6 times; P<0.05), but the expression of cardiac alpha-actin was not modified. Oral administration of carvedilol at a dose of 30 mg. kg(-1). d(-1) to rats with aortic banding normalized carotid pressure and left ventricular weight as well as preproendothelin-1 and skeletal alpha-actin gene expression. Carvedilol at a lower dose (7.5 mg x kg(-1) x d(-1)) and lacidipine 1 mg x kg(-1) x d(-1) had only moderate and nonsignificant effects on carotid pressure but largely prevented left ventricular hypertrophy (P<0.01) and preproendothelin-1 overexpression (P<0.05). Labetalol (60 mg x kg(-1) x d(-1)) tended to exert similar effects but insignificantly. These results show that the antihypertrophic properties of carvedilol and lacidipine are partly independent of their antihypertensive effects and may be related to their ability to blunt myocardial preproendothelin-1 overexpression. Moreover, carvedilol at a dose of 7.5 mg x kg(-1) x d(-1) did not prevent myocardial overexpression of skeletal alpha-actin, which suggests that, in this model, reexpression of a fetal gene can be activated by pressure overload independently of cardiac hypertrophy.

In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.

The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.