The complex pattern of SMAD signaling in the cardiovascular system.

The SMAD (small mother against decapentaplegic) family comprises transcription factors that function as signal transducers of TGFbeta (transforming growth factor) superfamily members. The number of studies showing expression, activation or involvement of both SMAD and TGFbeta family members in cardiovascular diseases is constantly rising. In this context, the position of SMADs in the diseased heart is particularly interesting because, besides their well-known fibrotic effects, increasing evidence demonstrates direct action of SMADs on cardiomyocytes as well as on the vascular system. In these systems, SMAD proteins are described to have effects on heart development, cell proliferation, cell growth, and apoptosis. As will be discussed in this review, these different consequences of SMAD activation are dependent on different SMAD isoforms, interaction of SMAD with other transcription factors in the particular situation, and modulation of SMAD activity by various kinases. As a result of all these influences, it turns out that activation of SMAD by members of the BMP (bone morphogenetic protein) family, which is a subfamily of the TGFbeta superfamily, is necessary for correct heart development. On the other hand, activation of SMADs by TGFbeta family members results in fibrotic, apoptotic, and anti-hypertrophic processes that are related to a detrimental cardiac remodeling and progression to heart failure.

PKC-independent signal transduction pathways increase SERCA2 expression in adult rat cardiomyocytes.

Catecholamines seem to play a major role in the initial response of the heart to pressure overload. The mechanisms by which alpha(1A)-adrenoceptor stimulation increases protein synthesis and subsequently cell size have been worked out in the past. However, little is known about the functional consequence of this type of hypertrophy. Recent transgenic work seems to indicate an adaptive character of this response, but mechanistic insights have yet to be established. The present study investigates whether chronic (overnight) exposure of cardiomyocytes to phenylephrine, an alpha-adrenoceptor agonist, modifies the expression of calcium-handling proteins and identifies key elements of signal transduction pathways leading to such alterations. Cardiomyocytes exposed to phenylephrine had elevated expression of SR-calcium ATPase (SERCA), but not of the sodium-calcium exchanger (NCX). SERCA induction persisted in the presence of protein kinase C (PKC) inhibitors, but required an increase in diastolic cell calcium levels via activation of the sodium-proton exchanger (NHE) and the reverse mode of the NCX. Downstream of an increase in resting cell calcium concentrations an activation of the calcineurin/NFAT pathway was found to be responsible for SERCA2 induction. Transfection of cardiomyocytes with decoys directed against NFAT activity inhibited the increase in SERCA2 expression. Decoys did not inhibit the concomitant PKC-dependent increase in hypertrophic growth. In the absence of SERCA up-regulation, hypertrophied cardiomyocytes were unable to maintain normal, load-free cell shortening. In conclusion, our data give mechanistic insights into the adaptional process during alpha-adrenoceptor-dependent myocardial hypertrophy.

SMAD proteins are involved in apoptosis induction in ventricular cardiomyocytes.

OBJECTIVE: The transcription factor AP-1 is a mediator of hypertrophic growth and apoptosis in cardiomyocytes. This puts AP-1 in the center of two important processes found in the failing heart and implies that variations (i) in the AP-1 composition itself or (ii) in additional, interacting transcription factors are responsible for the diverse actions of AP-1. To test this hypothesis, we performed studies on isolated ventricular cardiomyocytes of rat under hypertrophy- or apoptosis-inducing conditions. METHODS AND RESULTS: The NO donor SNAP (100 microM), which is a pro-apoptotic stimulus in cardiomyocytes, activated AP-1 within 2 h. c-Jun, JunB and FosB are identified as the main components of this AP-1 complex. This complex formation is identical to the composition of AP-1 found under hypertrophic growth stimulation by phenylephrine (PE, 10 muM). Analysis of other transcription factors able to interact with AP-1 revealed activation of SMAD activity only during stimulation with SNAP to 131+/-9.6% (p < 0.05 vs. control, n = 9). The SMAD complex is formed from SMAD4 and 3. Intracellular scavenging of SMAD proteins by transformation of cardiomyocytes with SMAD decoy oligonucleotides or inhibition of SMAD4 synthesis using SMAD4 antisense oligonucleotides reduced the number of apoptotic cells under stimulation with SNAP from 13.3 +/- 1.2% to control levels (8 +/- 1%, p < 0.05, n = 6). TGFbeta, which is a known stimulator of SMAD proteins, is also shown to stimulate apoptosis in cardiomyocytes. Again, simultaneous activation of AP-1 and SMAD is needed for this apoptosis induction. CONCLUSIONS: In conclusion, AP-1/SMAD signaling has been identified as a common pathway in cardiomyocyte apoptosis. In contrast, SMAD proteins are dispensable for AP-1-mediated hypertrophic growth. This finding characterizes SMAD proteins as potential candidates for proteins that shift AP-1 signaling from hypertrophy to apoptosis.

Inhibition of Ca2+-dependent PKC isoforms unmasks ERK-dependent hypertrophic growth evoked by phenylephrine in adult ventricular cardiomyocytes.

OBJECTIVE: The duration of extracellular signal-regulated kinase (ERK) activation and the ERK-dependency of hypertrophic growth differ between stimulation of alpha-adrenoceptors or angiotensin II receptors. As both receptor systems activate different protein kinase C (PKC) isoforms, we hypothesized that PKC isoforms contribute to the specific effect of alpha-adrenoceptor stimulation. METHODS: Isolated adult ventricular cardiomyocytes from rats were used. Different PKC isoforms were inhibited either pharmacologically by six different PKC inhibitors or specifically downregulated by antisense oligonucleotides. ERK activation was determined by phosphorylation relative to total ERK. The rate of protein synthesis was determined by 14C-phenylalanine incorporation. RESULTS: The hypertrophic response of phenylephrine was inhibited in a concentration-dependent fashion by three different inhibitors of Ca2+-independent PKC isoforms (Gö6983, rottlerin, Gö6850), but not by three distinct PKC inhibitors directed preferentially against Ca2+-dependent PKC isoforms (Ro32-0432, HBDDE, Gö6976). Antisense oligonucleotides directed against PKC-alpha, -delta, or -epsilon downregulated their specific isoforms. Their corresponding sense oligonucleotides did not affect PKC isoform expression. The phenylephrine-induced increase in protein synthesis was blocked by antisense oligonucleotides directed against PKC-delta or PKC-epsilon but not PKC-alpha, confirming the pharmacological experiments. Inhibition of Ca2+-dependent PKC isoforms by HBDDE or Gö6976 converted a transient activation of ERK by phenylephrine into a sustained response. Under these conditions, phenylephrine increased protein synthesis in an ERK-dependent way. CONCLUSION: Inhibition of Ca2+-dependent PKC isoforms converts the ERK-independent effect of phenylephrine on protein synthesis into an ERK-dependent induction of protein synthesis. We conclude that co-activation of Ca2+-dependent PKC isoforms by phenylephrine contributes to the specific effect on adult ventricular cardiomyocytes from rat.

Parathyroid hormone-related peptide is induced by stimulation of alpha 1A-adrenoceptors and improves resistance against apoptosis in coronary endothelial cells.

Parathyroid hormone-related peptide (PTHrP) is expressed throughout the vascular system, including coronary endothelial cells. The regulation of endothelial PTHrP expression and the role of PTHrP expression in endothelial cells is not clear. This study investigates the question of whether the stimulation of alpha-adrenergic or angiotensin II receptors increases endothelial expression of PTHrP and whether endogenously expressed PTHrP exerts intracrine effects in coronary endothelial cells. We found that the stimulation of alpha 1A-adrenoceptors, but not that of angiotensin II, increases cellular expression of PTHrP in growing, but not in growth-arrested, coronary endothelial cells. Angiotensin II increases the expression of PTHrP in smooth muscle cells but not in endothelial cells. PTHrP enters the nucleus of endothelial cells at the stadium of confluence, which suggests an intracrine effect of PTHrP. It was further investigated whether the down-regulation of endogenous PTHrP expression by transfection with antisense oligonucleotides alters cell proliferation or apoptosis resistance in growing or nongrowing endothelial cells. Down-regulation of PTHrP did not modify cell proliferation, but it increased the amount of UV-induced apoptosis. An increased expression of PTHrP in cells pretreated with an alpha-adrenoceptor agonist reduced the basal rate of apoptosis and improved resistance against UV-induced apoptosis. These results indicate a novel intracrine effect of PTHrP in coronary endothelial cells that improves cell survival. In endothelial cells, its expression is regulated by alpha-adrenoceptor stimulation in a cell-cycle-dependent and cell-type-specific manner.

Inhibition of apoptotic responses after ischemic stress in isolated hearts and cardiomyocytes.

Recent findings on the induction of anti-apoptotic gene expression in ischemic/reperfused hearts encouraged us to investigate whether ischemic/reperfused hearts may be protected against apoptosis induction. To analyze this hypothesis we performed studies on isolated perfused hearts of rat. For apoptosis induction, hearts were perfused with the NO donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 10 microM) for 30 minutes. Four hours thereafter apoptosis was detected by DNA laddering and TUNEL assay. Under normoperfusion SNAP induced 5.5 +/- 1.4 TUNEL-positive myocytes per tissue section (vs. 1.8 +/- 0.5 in controls). But when hearts were subjected to 20 minutes of no flow ischemia, which was sufficient for energy depletion of the hearts without inducing severe necrotic or apoptotic cell death, reperfusion in the presence of SNAP did not induce apoptosis. To analyze if this mode of protection is a property of the cardiomyocytes, we performed corresponding experiments on ventricular cardiomyocytes of rat. Again, under normoxic conditions SNAP (100 (microM) increased the number of TUNEL-positive cells to 12.6 +/- 4.9 % (vs. 5.4 +/- 0.7 % in controls). But when SNAP was added after 3 h of simulated ischemia, which was sufficient for energy depletion of the cells without inducing apoptotic cell death, the number of apoptotic cells did not increase. The ischemia-induced protection of hearts and cardiomyocytes goes along with an increased expression of several anti-apoptotic genes, mainly of the bcl-2 family. This indicates that ischemic conditions induce an anti-apoptotic gene program in cardiomyocytes, which may also be responsible for the observed anti-apoptotic actions in the intact ischemic/reperfused myocardium.

Neuropeptide Y modifies the hypertrophic response of adult ventricular cardiomyocytes to norepinephrine.

OBJECTIVE: The hypertrophic response of adult rat cardiomyocytes to norepinephrine via alpha-adrenoceptor stimulation is limited by an inhibitory cross-talk of simultaneously beta-adrenoceptor stimulation. On the other hand, neuropeptide Y (NPY), known to be co-secreted with norepinephrine from intramural nerve endings of the heart, exerts an anti-beta-adrenergic effect. Therefore, it should be expected that NPY enhances the hypertrophic response to norepinephrine. This hypothesis was addressed in the present study. METHODS: Isolated adult ventricular cardiomyocytes from rats were used. As parameters of hypertrophic growth we investigated cell volume, cross-sectional area, protein mass. Protein and RNA synthesis were determined by incorporation of [(14)C]phenylalanine or [(14)C]uridine, respectively. RESULTS: Norepinephrine (1 micromol/l) did not significantly increase protein or RNA synthesis. In co-presence of NPY (100 nmol/l), however, norepinephrine increased protein synthesis by 44% and RNA synthesis by 18%. Under the same conditions, NPY enhanced the effect of norepinephrine on cell volume from +6.4 to +18.2%, its effect on cross-sectional area from +16 to +23%, and increased the protein/DNA ratio from 32.5 to 35.6 mg/mg. In parallel, norepinephrine caused a translocation of PKC-alpha and PKC-delta into the particular fractions and this effect of norepinephrine was also enhanced by co-presence of NPY. In contrast, NPY did not enhance ERK-activation caused by norepinephrine. CONCLUSION: Our study indicates the anti-beta-adrenergic effect of NPY is sufficient to modulate the hypertrophic response of adult ventricular cardiomyocytes to norepinephrine. The results suggest that the hypertrophic effect of norepinephrine via alpha-adrenoceptor stimulation can be modulated by co-release of NPY from intramural nerve endings.