Jacqueline Heger

Growth differentiation factor 15 acts anti-apoptotic and pro-hypertrophic in adult cardiomyocytes.

Growth differentiation factor 15 (GDF15) is induced during heart failure development, and may influence different processes in cardiac remodeling. While its anti‐apoptotic action under conditions of ischemia–reperfusion have been shown, it remained unclear if this is a broadly protective effect applicable to other apoptotic stimuli. Furthermore, effects on cardiac hypertrophy remained obscure. Therefore, we investigated the effects of GDF15 on induction of hypertrophy and apoptosis in ventricular cardiomyocytes. GDF15 (3 ng/ml) enhanced hypertrophic growth of cardiomyocytes as determined by an increase in cell size by 27 ± 5% and rate of protein synthesis by 47 ± 15%. In addition, a time and dose‐dependent increase in SMAD‐binding affinity was found, as well as enhanced phosphorylation of R‐SMAD1. Inhibition of SMADs by transformation of cardiomyocytes with SMAD‐decoy oligonucleotides abolished the hypertrophic growth effect. Specific inhibitors of PI3K (10 µM LY290042 or 10 nM wortmannin) or ERK (10 µM PD98059) also blocked GDF15‐induced hypertrophy and SMAD activation. Apoptosis induction by three different agents, 100 nM angiotensin II, 1 ng/ml TGFβ1, or the NO‐donor SNAP (100 µM) was blocked by addition of GDF15 (3 ng/ml). Scavenging of SMADs by transformation of cardiomyocytes with SMAD‐decoy oligonucleotides abolished the anti‐apoptotic effect of GDF15. In conclusion, GDF15 protects ventricular cardiomyocytes against different apoptotic stimuli and enhances hypertrophic growth. Hypertrophic signaling is thereby mediated via the kinases PI3K and ERK and the transcription factor R‐SMAD1. Thus, GDF15 may influence cardiac remodeling via two different mechanisms, apoptosis protection and induction of hypertrophy. J. Cell. Physiol. 224:120–126, 2010

Inhibition of AP-1 signaling by JDP2 overexpression protects cardiomyocytes against hypertrophy and apoptosis induction

AIMS Expression and activity of the transcription factor AP-1 are enhanced during cardiac remodelling and heart failure progression. In order to test if AP-1 inhibition may limit processes contributing to cardiac remodelling, ventricular cardiomyocytes of mice with cardiac overexpression of the AP-1 inhibitor JDP2 were analysed under stimulation of hypertrophy, apoptosis, or contractile function. METHODS AND RESULTS Three models of JDP2 overexpressing mice were analysed: JDP2 was overexpressed either life-long, for 7 weeks, or 1 week. Then cardiomyocytes were isolated and stimulated with β-adrenoceptor agonist isoprenaline (ISO, 50 nM). This enhanced cross-sectional area and the rate of protein synthesis in WT but not in JDP2 overexpressing cardiomyocytes. To induce apoptosis, cardiomyocytes were stimulated with 3 ng/mL TGFβ1. Again, JDP2 overexpression prevented apoptosis induction compared with WT cells. Determination of contractile function under electrical stimulation at 2 Hz revealed enhancement of cell shortening, and contraction and relaxation velocities under increasing ISO concentrations (0.3-30 nM) in WT cells. This inotropic effect was abrogated in JDP2 overexpression cells. Responsiveness to increased extracellular calcium concentrations was also impaired in JDP2 overexpressing cardiomyocytes. Simultaneously, a reduction of SERCA expression was found in JDP2 mice. CONCLUSION A central role of AP-1 in the induction of hypertrophy and apoptosis in cardiomyocytes is demonstrated. Besides these protective effects of AP-1 inhibition on factors of cardiac remodelling, AP-1-inhibition impairs contractile function. Therefore, AP-1 acts as a double-edged sword that mediates mal-adaptive cardiac remodelling, but is required for maintaining a proper contractile function of cardiomyocytes.

TGFβ receptor activation enhances cardiac apoptosis via SMAD activation and concomitant NO release.

Transforming growth factor β (TGFβ) expression is induced in the myocardium during transition from compensated hypertrophy to heart failure. In cardiomyocytes, stimulation with TGFβ results in restricted contractile function and enhanced apoptosis. Nitric oxide (NO) also induces apoptosis and influences cardiac function. Therefore, we wanted to know whether NO is causally involved in TGFβ‐induced apoptosis. In isolated ventricular cardiomyocytes of adult rat incubation with TGFβ1 increased NO release which was inhibited by NOS inhibitor ETU but not with iNOS inhibitor (1400 W) or nNOS inhibitor (TFA). In addition, TGFβ‐induced apoptosis was blocked with ETU and ODQ, but not with 1400 W or TFA. The consequent assumption that endothelial NOS is involved in TGFβ‐induced NO formation and apoptosis was supported by increased phosphorylation of eNOS at serine 1177 and by the fact that TGFβ did not increase NO release in eNOS KO mice. Furthermore, TGFβ‐induced apoptosis, NO formation, SMAD binding activity and SMAD2 phosphorylation were blocked by a TGFβ receptor antagonist, but only apoptosis and NO formation could be blocked with ETU. Expression of SMAD7 was increased after TGFβ stimulation and blocked with TGFβ receptor antagonist but not after blocking NO synthase with ETU. Conclusion: In cardiomyocytes TGFβ‐induced apoptosis is mediated via TGFβ receptor activation that concomitantly activates SMAD transcription factors and the eNOS/NO/sGC pathway. Both of these pathways are needed for apoptosis induction by TGFβ. This reveals a new pathway of cardiac NO release and identifies NO as a possible contributor to heart failure progression mediated by TGFβ. J. Cell. Physiol. 226: 2683–2690, 2011.

Controlling cardiomyocyte length: the role of renin and PPAR-γ

AIMS Renin and peroxisome proliferator-activated receptor (PPAR-γ) interact directly with cardiomyocytes and influence protein synthesis. We investigated their effects and interaction on the size of cardiomyocytes. METHODS AND RESULTS Effects of renin and PPAR-γ activation were studied in cultured adult rat ventricular cardiomyocytes, transgenic mice with a cardiomyocyte-restricted knockout of PPAR-γ, and transgenic rats overexpressing renin, TGR(mRen2)27. The length and width of cardiomyocytes were analysed 24 h after administration of factors. Renin caused an unexpected effect on the length of cardiomyocytes that was inhibited by mannose-6-phosphate and monensin, but not by administration of glucose-6-phosphate. Endothelin-1 used as a classical pro-hypertrophic agonist increased cell width but not cell length. Renin caused an activation of p38 and p42/44 mitogen-activated protein (MAP) kinases. The latter activation was impaired by mannose-6-phosphate. Inhibition of p42/44 but not of p38 MAP kinase activation attenuated the effect of renin on cell length. In contrast, activation of PPAR-γ reduced cell length. Feeding wild-type mice with pioglitazone, a PPAR-γ agonist, reduced cell length. Cardiomyocytes isolated from PPAR-γ knockout mice were longer, and their length was not affected by pioglitazone. Cardiomyocytes isolated from TGR(mRen2)27 rats were longer than those of non-transgenic littermates. Cell length was reduced by feeding these mice with pioglitazone. Pioglitazone affected cell length independent of blood pressure. CONCLUSION The length of cardiomyocytes is controlled by the activation of cardiac-specific mannose-6-phosphate/insulin-like growth factor II receptors and activation of PPAR-γ. This type of cell size modification differs from that of any other known pro-hypertrophic agonists.

Interaction between Connexin 43 and nitric oxide synthase in mice heart mitochondria

Connexin 43 (Cx43), which is highly expressed in the heart and especially in cardiomyocytes, interferes with the expression of nitric oxide synthase (NOS) isoforms. Conversely, Cx43 gene expression is down‐regulated by nitric oxide derived from the inducible NOS. Thus, a complex interplay between Cx43 and NOS expression appears to exist. As cardiac mitochondria are supposed to contain a NOS, we now investigated the expression of NOS isoforms and the nitric oxide production rate in isolated mitochondria of wild‐type and Cx43‐deficient (Cx43Cre‐ER(T)/fl) mice hearts. Mitochondria were isolated from hearts using differential centrifugation and purified via Percoll gradient ultracentrifugation. Isolated mitochondria were stained with an antibody against the mitochondrial marker protein adenine‐nucleotide‐translocator (ANT) in combination with either a neuronal NOS (nNOS) or an inducible NOS (iNOS) antibody and analysed using confocal laser scanning microscopy. The nitric oxide formation was quantified in purified mitochondria using the oxyhaemoglobin assay. Co‐localization of predominantly nNOS (nNOS: 93 ± 4.1%; iNOS: 24.6 ± 7.5%) with ANT was detected in isolated mitochondria of wild‐type mice. In contrast, iNOS expression was increased in Cx43Cre‐ER(T)/fl mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43Cre‐ER(T)/fl mitochondria (0.14 ± 0.02 nmol/min./mg protein) in comparison to wild‐type mitochondria (0.24 ± 0.02 nmol/min./mg). These are the first data demonstrating, that a reduced mitochondrial Cx43 content is associated with a switch of the mitochondrial NOS isoform and the respective mitochondrial rate of nitric oxide formation.

TGF-β1 improves cardiac performance via up-regulation of laminin receptor 37/67 in adult ventricular cardiomyocytes

TGF-β1 plays an important role in cardiac fibrosis, apoptosis, induction of hypertrophy and contractile dysfunction. This study investigates whether TGF-β1 plays a role in laminin receptor 37/67 (37/67 LR)-dependent regulation of cardiac performance. Therefore, isolated adult cardiomyocytes were stimulated with TGF-β1, the expression of the 37/67 LR was determined and cell shortening was investigated on cells attached to a non-specific, serum-based attachment substrate or to specific, laminin-coated dishes. The role of the MAP kinases in TGF-β1-dependent induction of the 37/67 LR was examined by addition of PD98059, SB202190 and SP600125. Finally, the expression of receptor mRNA was investigated in transgenic mice constitutively over-expressing TGF-β1 and the relationship to distress score and lung wet weight-to-body weight was analysed. TGF-β1 induced a significant increase of the 37/67 LR mRNA and protein expression. The cytokine induced p38 MAP kinase and JNK, but not ERK. Inhibition of either p38 MAP kinase or JNK attenuated the TGF-β1-dependent increase in 37/67 LR expression. TGF-β1 induced a loss of cell shortening in cells attached to a non-specific substrate, but not in cells on a pre-coated laminin matrix. Inhibition of JNK attenuated the protective effect of laminin receptor up-regulation on cardiac performance. Inhibition of p38 MAP kinase attenuated the depressive effect of TGF-β1 on basal cell shortening. In transgenic mice over-expressing TGF-β1 a strong induction of laminin receptor expression attenuated the severeness of the mice’ symptoms. This study shows a new and protective role of TGF-β1-dependent up-regulation of the 37/67 LR in cardiomyocytes in cardiac remodelling with increased laminin expression.

SMAD‐proteins as a molecular switch from hypertrophy to apoptosis induction in adult ventricular cardiomyocytes

Heart failure development goes along with a transition from hypertrophic growth to apoptosis induction. In adult cardiomyocytes SMAD proteins are only activated under apoptotic, but not under hypertrophic conditions and are increased at the transition to heart failure. Therefore, SMADs could be candidates that turn the balance from hypertrophic growth to apoptosis resulting in heart failure development. To test this hypothesis we infected isolated rat ventricular cardiomyocytes with adenovirus encoding SMAD4 (AdSMAD4) and investigated the impact of SMAD4 overexpression on the development of apoptosis and hypertrophy under stimulation with phenylephrine (PE). Infection of cardiomyocytes with AdSMAD4 significantly enhanced SMAD‐binding activity while apoptosis after 24 and 36 h infection did not rise. But when SMAD4 overexpressing cardiomyocytes were incubated with PE (10 µM), the number of apoptotic cells increased (Ctrl: 94.97 ± 6.91%; PE: 102.48 ± 4.78% vs. AdSMAD4 + PE: 118.64 ± 3.28%). Furthermore expression of caspase 3 as well as bax/bcl2 ratio increased in SMAD4 overexpressing, PE‐stimulated cardiomyocytes. In addition, the effects of SMAD4 overexpression on PE‐induced hypertrophic growth were analyzed. Protein synthesis 36 h after AdSMAD4 infection was comparable to control cells, whereas the increase in protein synthesis stimulated by phyenylephrine was significantly reduced in SMAD4 overexpressing cells (134.28 ± 10.02% vs. 100.57 ± 8.86%). SMAD4 triggers the transition from hypertrophy to apoptosis in ventricular cardiomyocytes. Since SMADs are increased under several pathophysiological conditions in the heart, it can be assumed that it triggers apoptosis induction and therefore contributes to negative remodeling and heart failure progression. J. Cell. Physiol. 220: 515–523, 2009.

Molecular switches under TGFβ signalling during progression from cardiac hypertrophy to heart failure.

Cardiac hypertrophy is a mechanism to compensate for increased cardiac work load, that is, after myocardial infarction or upon pressure overload. However, in the long run cardiac hypertrophy is a prevailing risk factor for the development of heart failure. During pathological remodelling processes leading to heart failure, decompensated hypertrophy, death of cardiomyocytes by apoptosis or necroptosis and fibrosis as well as a progressive dysfunction of cardiomyocytes are apparent. Interestingly, the induction of hypertrophy, cell death or fibrosis is mediated by similar signalling pathways. Therefore, tiny changes in the signalling cascade are able to switch physiological cardiac remodelling to the development of heart failure. In the present review, we will describe examples of these molecular switches that change compensated hypertrophy to the development of heart failure and will focus on the importance of the signalling cascades of the TGFβ superfamily in this process. In this context, potential therapeutic targets for pharmacological interventions that could attenuate the progression of heart failure will be discussed.

Transgenic overexpression of the adenine nucleotide translocase 1 protects cardiomyocytes against TGFβ1-induced apoptosis by stabilization of the mitochondrial permeability transition pore

AIMS Since adenine nucleotide translocase 1 (ANT1) overexpression improved cardiac function in rats with activated renin-angiotensin system (RAS) and angiotensin II is known to enhance transforming growth factor β (TGFβ) signaling in cardiomyocytes, we assumed that ANT1 might modulate the classical TGFβ/SMAD pathway. We therefore investigated whether the cardioprotective effect of ANT1 overexpression suppresses TGFβ(1)-induced apoptosis, whether mitochondrial permeability transition pore (MPTP) regulation is involved, and SMAD signaling pathway is affected. METHODS AND RESULTS Ventricular cardiomyocytes isolated from wild-type (WT) and ANT1 transgenic rats were treated with the apoptosis-inducing agent TGFβ(1) (1 ng/ml). TGFβ(1) treatment of WT cells enhanced the number of apoptotic cells by 31.8 ± 11.7% (p<0.01 vs. WT) measured by chromatin condensation. Apoptosis was blocked by 1μM cyclosporine A and by ANT1 overexpression. The protecting effect of ANT1 overexpression on TGFβ(1)-induced apoptosis was verified by reduced caspase 3/7 activity and increased Bcl-2 expression. In addition, TGFβ(1) decreased mitochondrial membrane potential as measured by JC-1 staining by 18.0 ± 3.7% in WT cardiomyocytes, but only by 7.2 ± 2.8% (p<0.05 vs. WT) in ANT1 cardiomyocytes. Cyclosporine A also attenuated the decline in mitochondrial membrane potential under TGFβ(1) in WT cardiomyocytes. Determination of MPTP opening by Calcein assay in isolated cardiomyocytes and calcium retention assay in isolated mitochondria revealed a reduced open probability of MPTP after ANT1 overexpression. In addition to the effects of ANT1 on MPTP opening we investigated if ANT1 may interfere with the classical TGFβ signaling pathway. Interestingly, ANT1-transgenic cardiomyocytes expressed less TGFβ receptor II than WT cells. However, SMAD2 phosphorylation was already enhanced without TGFβ(1) stimulation in these cells. Although no additional increase in SMAD2 phosphorylation was detectable after TGFβ(1) treatment, SMAD signaling was still responsive to TGFβ(1) indicated by an upregulation of SMAD7, a TGFβ(1) target protein. CONCLUSION Heart-specific overexpression of ANT1 leads to a reduced apoptotic response to TGFβ(1) by preservation of the mitochondrial membrane potential, resistance to MPTP opening and altered TGFβ signaling.

Transgenic overexpression of heart-specific adenine nucleotide translocase 1 positively affects contractile function in cardiomyocytes.

Background/Aims: The adenine nucleotide translocase (ANT) exchanges ATP and ADP over the inner mitochondrial membrane, supplying the cells with energy. Interestingly, myocardial ANT1 overexpression preserves cardiac structure and function under pathophysiological conditions. To ascertain whether the contractile system is directly affected by increased ANT1 expression, we analyzed cell morphology, contraction and relaxation parameters of ANT1 transgenic (ANT1-TG) cardiomyocytes, myofibrillar protein expression, and Ca2+ handling in ANT1-TG rat hearts. Results: ANT1-TG cardiomyoycytes displayed an elevation in cell volume (52.6±12.0%; p<0.0001) in comparison to wildtype (WT) cells. Concurrently, contractile function in ANT1-TG cells was significantly increased, measured by a decline in time to peak contraction (TTP) and RT50, the time from peak contraction to 50% relaxation, during stimulation with 0.5, 1, and 2 Hz. Quantification of myofibrillar proteins exhibited a marked increase in total cardiac myosin heavy chain (51.8±12.8%) (p<0.03), beta myosin heavy chain (22.9±5.0%; p<0.03), actin (23.8±8.8%; p<0.05), and troponin I (51.5±13.7%; p<0.01). Regarding intracellular Ca2+ handling, ANT1-TGs revealed a significant elevation in sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a) protein level (22.2±4.7%; p<0.01) associated with increased Ca2+ uptake into the SR (34%; p<0.01). Moreover, the plasmalemmal Ca2+ ATPase (PMCA) indicated advanced protein expression (23.8±4.8%; p<0.01), whereas the protein amount of the Na+/Ca2+ exchanger was not altered in ANT1 overexpressing hearts. Conclusion: These data reveal a close association of elevated mitochondrial ATP/ADP transportation via ANT1 with increased contractile function. Furthermore, the ANT1-TGs exhibit an elevation in SR Ca2+ transport that contributes to increased cardiac work, which may protect the heart under pathophysiological conditions.