To investigate the local effects of angiotensin II on the heart, we created a mouse model with 100-fold normal cardiac angiotensin-converting enzyme (ACE), but no ACE expression in kidney or vascular endothelium. This was achieved by placing the endogenous ACE gene under the control of the ␣-myosin heavy chain promoter using targeted homologous recombination. These mice, called ACE 8/8, have cardiac angiotensin II levels that are 4.3-fold those of wild-type mice. Despite near normal blood pressure and a normal renal function, ACE 8/8 mice have a high incidence of sudden death. Both histological analysis and in vivo catheterization of the heart showed normal ventricular size and function. In contrast, both the left and right atria were three times normal size. ECG analysis showed atrial fibrillation and cardiac block. In conclusion, increased local production of angiotensin II in the heart is not sufficient to induce ventricular hypertrophy or fibrosis. Instead, it leads to atrial morphological changes, cardiac arrhythmia, and sudden death. The renin-angiotensin system (RAS) is a key regulator of blood pressure and electrolyte homeostasis. A critical component of this system is angiotensin-converting enzyme (ACE), which produces the eight amino acid peptide angiotensin II, the effector molecule of the RAS. 1 ACE is a zinc metallopeptidase located on the cell surface of endothelium. In this location, ACE produces angiotensin II adjacent to vascular smooth muscle, a critical target organ for this vasoconstrictor. ACE is also produced by a variety of other tissues including renal tubular epithelium, activated macrophages, proximal gut epithelium, and areas of the brain. Endothelium and these other tissues make the isozyme of ACE, termed somatic ACE, which consists of two catalytic domains that are independently capable of producing angiotensin II. Studies of knockout mice established that somatic ACE influences blood pressure and other cardiovascular functions. 2,3 In contrast, within the testis, developing male germ cells produce a different ACE isozyme called testis ACE, which plays an important role in normal male reproduction. 4 In addition to regulating normal physiology, substantial evidence suggests that the RAS plays an important role in disease, including heart disease. 5 Genetic studies reported a link between somatic ACE polymorphisms and the incidence of cardiac hypertrophy, sudden cardiac death, and acute coronary events. 6 This is consistent with the clinical effectiveness of ACE inhibitors in treating heart failure. 7 The beneficial effects of ACE inhibitors may not be solely the result of blood pressure reduction since other antihypertensive drugs do not produce the same effect. Rather, ACE may directly influence heart function through the local production of angiotensin II. Studies have found that angiotensinogen, renin, and ACE exist in the heart, implying that local generation of angiotensin II

Given the central position of the focal adhesion complex, both physically in coupling integrins to the interstitium and biochemically in providing an upstream site for anabolic signal generation, we asked whether the recruitment of non-receptor tyrosine kinases to the cytoskeleton might be a mechanism whereby cellular loading could activate growth regulatory signals responsible for cardiac hypertrophy. Analysis revealed cytoskeletal association of c-Src, FAK, and ␤3-integrin, but no Fyn, in the pressure-overloaded right ventricle. This association was seen as early as 4 h after right ventricular pressure overloading, increased through 48 h, and reverted to normal in 1 week. Cytoskeletal binding of nonreceptor tyrosine kinases was synchronous with tyrosine phosphorylation of several cytoskeletal proteins, including c-Src. Examination of cytoskeleton-bound cSrc revealed that a significant portion of the tyrosine phosphorylation was not at the Tyr-527 site and therefore presumably was at the Tyr-416 site. Thus, these studies strongly suggest that non-receptor tyrosine kinases, in particular c-Src, may play a critical role in hypertrophic growth regulation by their association with cytoskeletal structures, possibly via load activation of integrin-mediated signaling.

An adult feline right ventricular pressure overload (RVPO) model was used to examine the two S6 kinase (S6K) isoforms, p70 S6K and p85 S6K , that are involved in translational and transcriptional activation. Biochemical and confocal microscopy analyses at the level of the cardiocyte revealed that p70 S6K is present predominantly in the cytosol, substantially activated in 1-h RVPO (>12 fold), and phosphorylated in the pseudosubstrate domain at the Ser-411, Thr-421, and Ser-424 sites. p85 S6K , which was localized exclusively in the nucleus, showed activation subsequent to p70 S6K , with a sustained increase in phosphorylation for up to 48 h of RVPO at equivalent sites of p70 S6K , Thr-421 and Ser-424, but not at Ser-411. Neither isoform translocated between the cytosol and the nucleus. Further studies to determine potential upstream elements of S6K activation revealed: (i) similar time course of activation for protein kinase C isoforms (␣, ␥, and ⑀) and c-Raf, (ii) absence of accompanying phosphatidylinositol 3-kinase activation, (iii) activation of c-Src subsequent to p70 S6K , and (iv) similar changes in adult cardiocytes after treatment with 12-O-tetradecanoylphorbol-13-acetate. Thus, these studies suggest that a protein kinase C-mediated pathway couples pressure overload to growth induction via differential activation of S6K isoforms in cardiac hypertrophy.Hypertrophic cardiac growth occurs by an increase in cellular mass via a relatively coordinate increase in cellular protein content (1). In the terminally differentiated adult cardiac muscle cell, translational mechanisms have a key role in regulating the rate of protein synthesis during hypertrophic growth. Phosphorylation of the carboxyl terminus of 40 S ribosomal S6 protein (S6 protein) has been shown to regulate protein synthesis rate by mediating protein translational initiation and elongation (2), and a growing number of studies show strong correlation between translational efficiency and S6 protein phosphorylation (3-5). Two distinct families of S6 kinases, p90Rsk (ribosomal S6 kinase) and p70/85 S6 kinase (S6K), 1 appear to be involved in phosphorylation of the S6 protein (2, 6, 7). However, S6K is suggested to be primarily responsible for the S6 protein phosphorylation upon stimulation of cells with insulin and a variety of mitogenic stimuli (8 -10). S6K consists of two isoforms transcribed by a single gene due to alternative splicing: a 70-kDa cytoplasmic isoform (p70 S6K ) and a 85-kDa nuclear isoform (p85 S6K ) with an additional 23 residues at the amino terminus consisting of a putative nuclear targeting sequence (11). Both isoforms are known to phosphorylate the S6 protein and mediate the translation of polypyrimidine tract mRNA, including transcripts of ribosomal proteins and translational elongation factors (12). In addition to playing a role in protein translational control, nuclear p85 S6K has been shown to be important for transcriptional activation (13), increased DNA synthesis, and G 1 to S phase transition (14).For activation, both is...

(RAS) system activation is associated with an increased risk of sudden death. Previously, we used cardiac-restricted angiotensin-converting enzyme (ACE) overexpression to construct a mouse model of RAS activation. These ACE 8/8 mice die prematurely and abruptly. Here, we have investigated cardiac electrophysiological abnormalities that may contribute to early mortality in this model. In ACE 8/8 mice, surface ECG voltages are reduced. Intracardiac electrograms showed atrial and ventricular potential amplitudes of 11% and 24% compared with matched wild-type (WT) controls. The atrioventricular (AV), atrioHisian (AH), and Hisian-ventricular (HV) intervals were prolonged 2.8-, 2.6-, and 3.9-fold, respectively, in ACE 8/8 vs. WT mice. Various degrees of AV nodal block were present only in ACE 8/8 mice. Intracardiac electrophysiology studies demonstrated that WT and heterozygote (HZ) mice were noninducible, whereas 83% of ACE 8/8 mice demonstrated ventricular tachycardia with burst pacing. Atrial connexin 40 (Cx40) and connexin 43 (Cx43) protein levels, ventricular Cx43 protein level, atrial and ventricular Cx40 mRNA abundances, ventricular Cx43 mRNA abundance, and atrial and ventricular cardiac Na ϩ channel (Scn5a) mRNA abundances were reduced in ACE 8/8 compared with WT mice. ACE 8/8 mice demonstrated ventricular Cx43 dephosphorylation. Atrial and ventricular L-type Ca 2ϩ channel, Kv4.2 K ϩ channel ␣-subunit, and Cx45 mRNA abundances and the peak ventricular Na ϩ current did not differ between the groups. In isolated heart preparations, a connexin blocker, 1-heptanol (0.5 mM), produced an electrophysiological phenotype similar to that seen in ACE 8/8 mice. Therefore, cardiac-specific ACE overexpression resulted in changes in connexins consistent with the phenotype of low-voltage electrical activity, conduction defects, and induced ventricular arrhythmia. These results may help explain the increased risk of arrhythmia in states of RAS activation such as heart failure.peptidyl-dipeptidase A; angiotensin II; heart block ARRHYTHMIC SUDDEN DEATH is a common terminal event in various cardiomyopathies and end-stage heart failure. Upregulation of the renin-angiotensin system (RAS) has been implicated in risk of sudden death in these conditions. A critical component of this system is angiotensin-converting enzyme (ACE), which produces the eight-amino acid peptide angiotensin II (ANG II), a major effector peptide of the RAS. In humans, increased ANG II levels are associated with an increased risk of arrhythmia (2), which is reduced by use of ACE inhibitors or ANG II receptor blockers (4,13,20,23,27,30,49).A number of ion-handling protein changes have been posited to underlie the increase in risk of arrhythmia in states of RAS activation, and ANG II is known to act on a number of these proteins (3, 41). For example, ANG II has been implicated in Na ϩ -K ϩ pump regulation (24). Furthermore, ANG II inhibits the Ca 2ϩ -activated K ϩ current in vascular smooth muscle cells (51) and the delayed rectifier K ϩ currents in heart and...