Autocrine/Paracrine Determinants of Strain-activated Brain Natriuretic Peptide Gene Expression in Cultured Cardiac Myocytes

Abstract

The application of mechanical strain leads to activation of human brain natriuretic peptide gene promoter activity, a marker of hypertrophy, in cultured neonatal rat ventricular myocytes. We have used a combination of transient transfection analysis and reverse transcriptase-polymerase chain reaction to examine the role of locally produced factors in contributing to this activation. Conditioned media from strained, but not static, cultures led to a dose-dependent increase in human brain natriuretic peptide gene promoter activity. This increase was completely blocked by losartan or BQ-123, implying a role for angiotensin and endothelin as autocrine/paracrine mediators of the response to strain. Inclusion of the same antagonists in the cultures themselves led to only partial inhibition (ϳ60%), whereas inclusion of exogenous endothelin or angiotensin II resulted in amplification of the strain response. Angiotensin II and endothelin appear to be arrayed in series in the regulatory circuitry; the angiotensin response was blocked by BQ-123, whereas the endothelin response was unaffected by losartan. Mechanical strain was also shown to stimulate expression of the endogenous angiotensinogen, angiotensin-converting enzyme, and endothelin genes in this system. Collectively, these data indicate that locally generated angiotensin II and endothelin, acting in series, play an important autocrine/paracrine role in mediating strain-dependent activation of cardiac-specific gene expression.Application of mechanical strain, or passive stretch, to myocardial cells in culture results in a series of phenotypic changes that closely resemble those that occur with myocyte hypertrophy in vivo. This includes activation of the immediate early gene family (e.g. c-fos, c-jun, c-myc, and egr-1), increased expression of the fetal gene program (e.g. atrial natriuretic peptide, ␣ skeletal actin, and ␤-myosin heavy chain), and increased protein synthesis (1, 2).A number of recent studies have suggested that activation of autocrine/paracrine regulatory mechanisms in the myocardium may play an important, if not dominant, role in determining the myocyte response to hemodynamic load. Specific local regulators invoked as participating in this process include angiotensin II (AII) 1 (3-6), endothelin (ET) (7-9), transforming growth factor ␤ (TGF-␤) (10, 11), fibroblast growth factor (11, 12), myotrophin (13), and cardiotrophin (14, 15), among others. Sadoshima et al. (3) suggested that locally produced angiotensin, stored in and secreted from the cardiac myocyte in vitro, plays a dominant role in effecting the response to mechanical strain. Losartan, an angiotensin type 1 (AT1) receptor antagonist, blocked strain-dependent increases in c-fos, ␣ skeletal actin and atrial natriuretic factor gene expression in their system. Yamazaki et al. (4,5) confirmed that AII plays a significant role in determining the response to strain. In their hands, the induction of MAP kinase, MAP kinase kinase, Raf-1 kinase, or protein synthesis by strain was suppressed, altho...

Endothelin-Dependent and -Independent Components of Strain-Activated Brain Natriuretic Peptide Gene Transcription Require Extracellular Signal Regulated Kinase and p38 Mitogen-Activated Protein Kinase

“…11 In the present study, we used exogenous ET as a surrogate of the autocrine/paracrine system to probe the signaling mechanism or mechanisms responsible for this component of the strain response. As shown in Figure 1A, ET effected a time-dependent increment in p38 MAPK activity that peaked at 15 minutes.…”

“…10 This stimulation appears to be derived in part from a direct effect on the cardiac myocyte and in part from an autocrine/paracrine pathway that involves the sequential generation of angiotensin II and endothelin (ET). 11 From a mechanotransduction standpoint, strain has been shown to activate ERKs, JNKs, and p38 MAPKs in these myocyte cultures, [7][8][9][10] with the latter (ie, p38 MAPK) accounting for Ϸ50% of the composite hBNP promoter response. 12 This activity depends on interaction of the transcription factor nuclear factor (NF)-B with 3 shear stress response element (SSRE)-like structures in the proximal hBNP promoter.…”

Thioredoxin-Interacting Protein Controls Cardiac Hypertrophy Through Regulation of Thioredoxin Activity

“…16 Realtime PCR showed that mechanical strain (8%, 1 Hz, 6 hours) induced BNP mRNA expression in cardiomyocytes in AdGFP-infected cells (2.6Ϯ0.6-fold, nϭ4 to 5 normalized to ␤-tubulin expression, PϽ0.05, Figure 1C). There was a trend toward an increase in BNP gene expression with mechanical strain when cells overexpressed thioredoxin, although this did not reach statistical significance (Pϭ0.09).…”

Load-induced focal adhesion kinase activation in the myocardium: role of stretch and contractile activity

“…Activation of the FAK multicomponent signaling complex induced by increases in mechanical input to myocardial cells might be mediated by a number of factors, namely, the engagement and activation of cytoskeletal proteins associated with the cytoplasmic domains of integrins, release of autocrine/paracrine factors, or stretch-induced activation of ion channels (6,17,21,28,40). However, studies in cells in culture showing the strict dependence of FAK activation by mechanical stimuli on integrin engagement and cytoskeletal integrity favor the hypothesis that the integrin/cytoskeletal complex is the dominant mechanism of FAK activation in cells subjected to mechanical stress (31,35).…”