Leonard G. Meggs

Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats.

To determine the effects of acute myocardial infarction on the regulation of angiotensin II (Ang II) receptors and contractile performance of left and right ventricular myocytes, coronary artery ligation was surgically induced in rats, and Ang II receptor density and affinity and the mechanical properties of surviving muscle cells were examined 1 week later. Physiological determinations of cardiac pump function revealed the presence of ventricular failure, which was associated at the cellular level with a depression in the velocity of myocyte shortening and relengthening, a prolongation of time to peak shortening, and a reduction in the extent of cell shortening. These abnormalities in single-cell function were more prominent in left than in right ventricular myocytes. Cellular hypertrophy was documented by increases in cell length and width, which were also greater in the spared myocytes of the infarcted left ventricle. Reactive hypertrophy was accompanied by a 1.84- and 1.85-fold increase in the density of Ang II receptors on left and right myocytes, respectively. On the other hand, the affinity of Ang II receptors for the radiolabeled antagonist was not altered. However, Ang II-stimulated phosphoinositol turnover was enhanced by 3.7- and 2.5-fold in left and right myocytes, respectively, after infarction. Ventricular myocytes were found to possess the AT1 receptor subtype exclusively. In conclusion, myocardial infarction leads to impairment in the contractile behavior of the remaining cells and to the activation of Ang II receptors and effector pathway associated with these receptors, which may be involved in the reactive growth adaptation of the viable myocytes.

Overexpression of Insulin-Like Growth Factor-1 Attenuates the Myocyte Renin-Angiotensin System in Transgenic Mice

Constitutive overexpression of insulin-like growth factor-1 (IGF-1) in myocytes protects them from apoptosis and interferes with myocyte hypertrophy in the normal and pathological heart. Conversely, angiotensin II (Ang II) triggers cell death and promotes myocyte hypertrophy. Moreover, activation of p53 upregulates the cellular renin-angiotensin system (RAS). Therefore, IGF-1 overexpression in FVB.Igf+/- mice may downregulate the local RAS through the attenuation of p53 and p53-inducible genes. On this basis, p53 DNA binding activity to angiotensinogen (Aogen), bax, and the AT1 receptor was determined in left ventricular myocytes from FVB.Igf-/- and FVB.Igf+/- mice. The quantity of Bax, Bcl-2, Aogen, and AT1 receptor in these cells was evaluated. The presence of Mdm2-p53 complexes was also established. Finally, Ang II levels in myocytes were measured. Upregulation of IGF-1 in myocytes was associated with a protein-to-protein interaction between Mdm2 and p53, which attenuated p53 transcriptional activity for bax, Aogen, and AT1 receptor. Similarly, the amount of Bax, Aogen, and AT1 receptor proteins in these cells decreased. In contrast, the expression of Bcl-2 remained constant. The downregulation of Aogen in myocytes from FVB.Igf+/- mice was characterized by a reduction in Ang II. In conclusion, IGF-1 negatively influences the myocyte RAS through the upregulation of Mdm2 and its binding to p53. This may represent the molecular mechanism responsible for the effects of IGF-1 on cell viability and myocyte hypertrophy in the nonpathological and pathological heart in vivo.

Efficacy of angiotensin-converting enzyme inhibition and AT1 receptor blockade on cardiac pump performance after myocardial infarction in rats

Summary: To determine whether cardiac unloading by inhibition of angiotensin I (AI) to AII conversion by captopril or blockade of the AII receptor (AT1) by losartan was more effective in prevention of the detrimental hemodynamic consequences of myocardial infarction (MI), inhibition of metabolic production of AII by captopril was compared with blockade of AT1 with losartan in Sprague-Dawley rats with large MI. Infarcts were created by surgical occlusion of the left main coronary artery and oral drug therapy initiated immediately and continued until hemodynamic evaluation seven days later. Heart weight was unchanged in untreated infarcted animals, whereas captopril reduced heart weight in control animals and losartan increased heart weight in infarcted animals. Left ventricular (LV) peak systolic blood pressure (SBP) was lower in treated and untreated infarcted animals. Although captopril reduced end-diastolic pressure (EDP) to a greater degree than losartan, all infarcted group showed an increase in this parameter with respect to similarly treated controls. LV peak rates of pressure increase and decay in infarcted hearts were decreased significantly more by captopril than by losartan administration. Captopril also impaired right side cardiac function more than losartan when peak rate of pressure increase was evaluated. Thus, inhibition of the effects of AII during cardiac failure improved but did not normalize cardiac pump performance. Although inhibition of AII production by captopril produced its beneficial effect by reducing diastolic BP (DBP) and SBP, preventing AII binding by administration of losartan appeared to elicit its therapeutic effect by an improvement in myocardial contractility.

Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis.

Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcA/cytochrome-c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome-c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification.

Effect of chronic uremia on the cardiovascular alpha1 receptor

Adrenergic dysfunction in uremia has been well described. Several lines of evidence suggest disorders of blood pressure regulation and myocardial response may occur secondary to adrenergic dysfunction; attenuated pressor response to norepinephrine (NE) in uremia; attenuated chronotropic responses during dialysis induced hypotension. Since the adrenergic receptors are the effector component of the adrenergic nervous system, we have employed the partially nephrectomized uremic rat, to examine the effect of chronic uremia (4-6 weeks) on the binding properties of alpha 1 receptors in rat mesenteric artery and myocardial tissue. The results indicate that moderate levels of uremia alter the binding properties of both the alpha 1 vascular and myocardial receptor.

Chronic nonocclusive coronary artery constriction in rats. Beta-adrenoceptor signal transduction and ventricular failure.

To determine the effects of chronic coronary artery constriction on the relationship between cardiac function and regulation of beta-adrenoceptor signal transduction, the left main coronary artery was narrowed in rats and the animals were killed 5 mo later. An average reduction in coronary luminal diameter of 44% was obtained and this change resulted in an increase in left ventricular end-diastolic pressure and a decrease in positive and negative dP/dt. Significant increases in left and right ventricular weights indicative of global cardiac hypertrophy were observed. Radioligand binding studies of beta-adrenoreceptors, agonist-stimulated adenylate cyclase activity, and ADP ribosylation of 45-kD substrate by cholera toxin were all depressed in the failing left ventricle. In contrast, in the hypertrophic non-failing right ventricle, beta-adrenoreceptor density was preserved and receptor antagonist affinity was increased. In spite of these findings at the receptor level, agonist stimulated cyclic AMP generation was reduced in the right ventricular myocardium. The quantity of the 45-kD substrate was also decreased. In conclusion, longterm nonocclusive coronary artery stenosis of moderate degree has profound detrimental effects on the contractile performance of the heart in association with marked attenuation of adrenergic support mechanisms.

Upregulation of the Vascular Alpha-1 Receptor in Malignant Doca-salt Hypertension

In this investigation we describe regulation of the vascular alpha-1 receptor and functional properties of resistance vessels in malignant hypertensive DOCA-salt rats (DOCA-salt). Uninephrectomized control and DOCA-salt rats were maintained for 6 weeks; microscopic renal morphology provided an index of vascular injury. Radioligand binding studies indicated a striking increase in the density of mesenteric alpha-1 binding sites in DOCA-salt (542 +/- 44 fm/mg) vs. salt control (206 +/- 4 fm/mg) and water control (223 +31 fm/mg) P less than .01. The affinity of the receptor for the radioligand [125I] (+/-) BE 2254 was reduced in DOCA-salt rats. Electrical nerve stimulation and agonist dose response curves were performed on isolated perfused mesenteric arteries. A singular correlation between increased receptor density and vascular responses in DOCA-salt rats could not be demonstrated. The norepinephrine (NE) content of mesenteric arteries was reduced in DOCA-salt (1001 +/- 32 ng/g) vs. water control (1522 +/- 44 ng/g) and saline control (1538 +/- 30 ng/g) P less than .01. Our results indicate, upregulation of the mesenteric alpha-1 receptor occurs in DOCA-salt rats, however, additional factors participate in the vascular response to adrenergic stimulation in this model.

Noncoordinate regulation of alpha-1 adrenoreceptor coupling and reexpression of alpha skeletal actin in myocardial infarction-induced left ventricular failure in rats.

To determine the effects of myocardial infarction-induced left ventricular failure on the regulation of surface alpha-1 adrenoreceptors and signal transduction, large infarcts were produced in rats and the animals killed seven days later. After the documentation of impaired left ventricular pump performance, radioligand binding studies of the alpha-1 adrenoreceptor, norepinephrine-stimulated phosphoinositol turnover, and ADP ribosylation of 41 kD substrate by pertussis toxin were examined in the hypertrophying unaffected myocardium. Moreover, the expression of sarcomeric actin isoforms was analyzed by Northern blots and hybridization with specific oligonucleotide probes. Alpha-1 adrenoreceptor density was found not to be altered in membranes obtained from the spared left ventricular tissue, whereas phosphoinositol turnover was increased 3.1-fold in the viable myocytes of infarcted hearts. Furthermore, pertussis toxin substrate was augmented 2.5-fold in membranes prepared from the surviving left ventricular myocardium. Finally, an upregulation of the skeletal actin isoform was detected in the tissue of the failing left ventricle. In conclusion, the possibility is raised that in the presence of severe myocardial dysfunction and ongoing reactive hypertrophy, effector pathways linked to the alpha-1 adrenoreceptor may stimulate the myocyte hypertrophic response which would tend to normalize cardiac hemodynamics. The reexpression of alpha skeletal actin may be a molecular indicator of the persistance of an overload on the myocardium.

HIV gene expression deactivates redox-sensitive stress response program in mouse tubular cells both in vitro and in vivo

Human immunodeficiency virus (HIV)-1 has been reported to cause tubular cell injury both in in vivo and in vitro studies. In the present study, we evaluated the role of oxidative stress in the induction of apoptosis in HIV gene expressing mouse tubular cells in in vivo (Tg26, a transgenic mouse model of HIV-associated nephropathy) and in vitro (tubular cells were transduced with pNL4-3: ΔG/P-GFP, VSV.G psueudo typed virus) studies. Although Tg26 mice showed enhanced tubular cell reactive oxygen species (ROS) generation and apoptosis, renal tissue did not display a robust antioxidant response in the form of enhanced free radical scavenger (MnSOD/catalase) expression. Tg26 mice not only showed enhanced tubular cell expression of phospho-p66ShcA but also displayed nuclear Foxo3a translocation to the cytoplasm. These findings indicated deactivation of tubular cell Foxo3A-dependent redox-sensitive stress response program (RSSRP) in Tg26 mice. In in vitro studies, NL4-3 (pNL4-3: ΔG/P-GFP, VSV.G pseudotyped virus)-transduced mouse proximal tubular cells (NL4-3/MPTEC) displayed enhanced phosphorylation of p66ShcA. NL4-3/MPTECs also displayed greater (P < 0.01) ROS generation when compared with empty vector-transduced tubular cells; however, both diminution of p66ShcA and N-acetyl cysteine attenuated NL4-3-induced tubular cell ROS generation as well as apoptosis. In addition, both antioxidants and free radical scavengers partially inhibited HIV-induced tubular cell apoptosis. NL4-3/MPTEC displayed deactivation of RSSRP in the form of enhanced phosphorylation of Foxo3A and attenuated expression of superoxide dismutase (SOD) and catalase. Since both SOD and catalase were able to provide protection against HIV-1-induced tubular cell apoptosis, it suggests that HIV-1-induced proapoptotic effect may be a consequence of the deactivated RSSRP.

Null mutations at the p66 and bradykinin 2 receptor loci induce divergent phenotypes in the diabetic kidney

Candidate genes have been identified that confer increased risk for diabetic glomerulosclerosis (DG). Mice heterozygous for the Akita (Ins2(+/C96Y)) diabetogenic mutation with a second mutation introduced at the bradykinin 2 receptor (B2R(-/-)) locus express a disease phenotype that approximates human DG. Src homology 2 domain transforming protein 1 (p66) controls mitochondrial metabolism and cellular responses to oxidative stress, aging, and apoptosis. We generated p66-null Akita mice to test whether inactivating mutations at the p66 locus will rescue kidneys of Akita mice from disease-causing mutations at the Ins2 and B2R loci. Here we show null mutations at the p66 and B2R loci interact with the Akita (Ins2(+/C96Y)) mutation, independently and in combination, inducing divergent phenotypes in the kidney. The B2R(-/-) mutation induces detrimental phenotypes, as judged by increased systemic and renal levels of oxidative stress, histology, and urine albumin excretion, whereas the p66-null mutation confers a powerful protection phenotype. To elucidate the mechanism(s) of the protection phenotype, we turned to our in vitro system. Experiments with cultured podocytes revealed previously unrecognized cross talk between p66 and the redox-sensitive transcription factor p53 that controls hyperglycemia-induced ROS metabolism, transcription of p53 target genes (angiotensinogen, angiotensin II type-1 receptor, and bax), angiotensin II generation, and apoptosis. RNA-interference targeting p66 inhibits all of the above. Finally, protein levels of p53 target genes were upregulated in kidneys of Akita mice but unchanged in p66-null Akita mice. Taken together, p66 is a potential molecular target for therapeutic intervention in DG.