Modulation of cardiac AKT and STAT3 signalling in preclinical cancer models and their impact on the heart

BackgroundAdvanced cancer induces fundamental cardiac changes and promotes body wasting and heart failure. We evaluated the impact of cancer on major cardiac signalling pathways, and resulting consequences for the heart.Methods and resultsMetastatic melanoma disease was induced in male C57BL/6 N mice by intraperitoneal injection of the melanoma cell line B16F10 and lead to cardiac atrophy and heart failure. Analyses of key cardiac signalling pathways in left ventricular tissue revealed increased activation of STAT3 and reduced activation of AKT, p38 and ERK1/2. Markers of the ubiquitin proteasomal system (UPS: Atrogin-1) and of mitophagy/autophagy (LC3b, BNIP3) were upregulated. Tumour-bearing C57BL/6 N mice with a cardiomyocyte-specific overexpression of a constitutively active AKT transgene (AKTtg) displayed less cardiac atrophy and dysfunction and normalized Atrogin-1, LC3b and BNIP3 expression while the cardiomyocyte-specific knockout of STAT3 (CKO) had no major effect on these parameters compared to WT.ConclusionCancer alters major cardiac signalling pathways and subsequently the UPS, mitophagy and autophagy. The present study suggests that cancer-induced reduction of cardiomyocyte AKT contributes to these alterations as they were attenuated in tumour-bearing AKTtg mice. In turn, increased cardiomyocyte STAT3 activation appears less relevant, as tumour-induced impairment on the heart was largely similar in CKO and WT mice. Since oncologic therapies frequently target AKT and/or STAT3, their impact on the heart might be different in tumour-bearing mice compared to healthy mice, a feature suggesting to test tumour therapies also in tumour disease models and not only under healthy conditions. This article is part of a Special Issue entitled: Cardiomyocyte biology: new pathways of differentiation and regeneration edited by Marijke Brink, Marcus C. Schaub, and Christian Zuppinger.     

A G‐CSF functionalized scaffold for stem cells seeding: a differentiating device for cardiac purposes

Myocardial infarction and its consequences represent one of the most demanding challenges in cell therapy and regenerative medicine. Transfer of skeletal myoblasts into decompensated hearts has been performed through intramyocardial injection. However, the achievements of both cardiomyocyte differentiation and precise integration of the injected cells into the myocardial wall, in order to augment synchronized contractility and avoid potentially life‐threatening alterations in the electrical conduction of the heart, still remain a major target to be pursued. Recently, granulocytes colony‐stimulating factor (G‐CSF) fuelled the interest of researchers for its direct effect on cardiomyocytes, inhibiting both apoptosis and remodelling in the failing heart and protecting from ventricular arrhythmias through the up‐regulation of connexin 43 (Cx43). We propose a tissue engineering approach concerning the fabrication of an electrospun cardiac graft functionalized with G‐CSF, in order to provide the correct signalling sequence to orientate myoblast differentiation and exert important systemic and local effects, positively modulating the infarction microenvironment. Poly‐(l ‐lactide) electrospun scaffolds were seeded with C2C12 murine skeletal myoblast for 48 hrs. Biological assays demonstrated the induction of Cx43 expression along with morphostructural changes resulting in cell elongation and appearance of cellular junctions resembling the usual cardiomyocyte arrangement at the ultrastructural level. The possibility of fabricating extracellular matrix‐mimicking scaffolds able to promote myoblast pre‐commitment towards myocardiocyte lineage and mitigate the hazardous environment of the damaged myocardium represents an interesting strategy in cardiac tissue engineering.     

Nitrite exerts potent negative inotropy in the isolated heart via eNOS-independent nitric oxide generation and cGMP-PKG pathway activation

The ubiquitous anion nitrite (NO₂⁻) has recently emerged as an endocrine storage form of nitric oxide (NO) and a signalling molecule that mediates a number of biological responses. Although the role of NO in regulating cardiac function has been investigated in depth, the physiological signalling effects of nitrite on cardiac function have only recently been explored. We now show that remarkably low concentrations of nitrite (1 nM) significantly modulate cardiac contractility in isolated and perfused Langendorff rat heart. In particular, nitrite exhibits potent negative inotropic and lusitropic activities as evidenced by a decrease in left ventricular pressure and relaxation, respectively. Furthermore, we demonstrate that the nitrite-dependent effects are mediated by NO formation but independent of NO synthase (NOS) activity. Specifically, nitrite infusion in the Langendorff system produces NO and cGMP/PKG-dependent negative inotropism, as evidenced by the formation of cellular iron-nitrosyl complexes and inhibition of biological effect by NO scavengers and by PKG inhibitors. These data are consistent with the hypothesis that nitrite represents an eNOS-independent source of NO in the heart which modulates cardiac contractility through the NO-cGMP/PKG pathway. The observed high potency of nitrite supports a physiological function of nitrite as a source of cardiomyocyte NO and a fundamental signalling molecule in the heart.     

Nitric oxide and the enigma of cardiac hypertrophy

In pathological conditions associated with persistent increases in hemodynamic workload (old myocardial infarction, high blood pressure, valvular heart disease), a number of signalling pathways are activated in the heart, all of which promote hypertrophic growth of the heart, characterised at the cellular level by increases in individual cardiac myocyte size. Some of these pathways are required for a successful adaptation to cardiac injury. Other pathways are maladaptive, however, as they lead to progressive contractile dysfunction and heart failure. The free radical gas nitric oxide and natriuretic peptides, both of which are produced in the heart, have emerged as endogenous inhibitors of maladaptive hypertrophy signalling. Overall, it appears that cardiac hypertrophy is controlled by an interplay of pro- and antihypertrophic signalling networks. This delicate balance can tip towards adaptation or heart failure. In the future, patients living with cardiac disease may benefit from therapeutic strategies targeting maladaptive hypertrophy signalling pathways.     

Gene expression profiling on global cDNA arrays gives hints concerning potential signal transduction pathways involved in cardiac fibrosis of renal failure

Cardiac remodelling with interstitial fibrosis in renal failure, which so far is only poorly understood on the molecular level, was investigated in the rat model by a global gene expression profiling analysis. Sprague-Dawley rats were subjected to subtotal nephrectomy (SNX) or sham operation (sham) and followed for 2 and 12 weeks, respectively. Heart-specific gene expression profiling, with RZPD Rat Unigene-1 cDNA arrays containing about 27 000 gene and EST sequences revealed substantial changes in gene expression in SNX compared to sham animals. Motor protein genes, growth and differentiation markers, and extracellular matrix genes were upregulated in SNX rats. Obviously, not only genes involved in cardiomyocyte hypertrophy, but also genes involved in the expansion of non-vascular interstitial tissue are activated very early in animals with renal failure. Together with earlier findings in the SNX model, the present data suggest the hypothesis that the local renin-angiotensin system (RAS) may be activated by at least two pathways: (a) via second messengers and Gproteins (short-term signalling); and (b) via motor proteins, actins and integrins (longterm signalling). The study documents that complex hybridization analysis yields reproducible and promising results of patterns of gene activation pointing to signalling pathways involved in cardiac remodelling in renal failure. The complete array data are available via http://www.rzpd.de/cgi-bin/services/exp/viewExpressionData.pl.cgi.     

Sodium (±)‐5‐bromo‐2‐(α‐hydroxypentyl) benzoate ameliorates pressure overload‐induced cardiac hypertrophy and dysfunction through inhibiting autophagy

Sodium (±)‐5‐bromo‐2‐(a‐hydroxypentyl) benzoate (generic name: brozopine, BZP) has been reported to protect against stroke‐induced brain injury and was approved for Phase II clinical trials for treatment of stroke‐related brain damage by the China Food and Drug Administration (CFDA). However, the role of BZP in cardiac diseases, especially in pressure overload‐induced cardiac hypertrophy and heart failure, remains to be investigated. In the present study, angiotensin II stimulation and transverse aortic constriction were employed to induce cardiomyocyte hypertrophy in vitro and in vivo, respectively, prior to the assessment of myocardial cell autophagy. We observed that BZP administration ameliorated cardiomyocyte hypertrophy and excessive autophagic activity. Further results indicated that AMP‐activated protein kinase (AMPK)‐mediated activation of the mammalian target of rapamycin (mTOR) pathway likely played a role in regulation of autophagy by BZP after Ang II stimulation. The activation of AMPK with metformin reversed the BZP‐induced suppression of autophagy. Finally, for the first time, we demonstrated that BZP could protect the heart from pressure overload‐induced hypertrophy and dysfunction, and this effect is associated with its inhibition of maladaptive cardiomyocyte autophagy through the AMPK‐mTOR signalling pathway. These findings indicated that BZP may serve as a promising compound for treatment of pressure overload‐induced cardiac remodelling and heart failure.     

Hesperetin protects against cardiac remodelling induced by pressure overload in mice

Cardiac remodelling is a major determinant of heart failure (HF) and is characterised by cardiac hypertrophy, fibrosis, oxidative stress and myocytes apoptosis. Hesperetin, which belongs to the flavonoid subgroup of citrus flavonoids, is the main flavonoid in oranges and possesses multiple pharmacological properties. However, its role in cardiac remodelling remains unknown. We determined the effect of hesperetin on cardiac hypertrophy, fibrosis and heart function using an aortic banding (AB) mouse. Male, 8–10-week-old, wild-type C57 mice with or without oral hesperetin administration were subjected to AB or a sham operation. Our data demonstrated that hesperetin protected against cardiac hypertrophy, fibrosis and dysfunction induced by AB, as assessed by heart weigh/body weight, lung weight/body weight, heart weight/tibia length, echocardiographic and haemodynamic parameters, histological analysis, and gene expression of hypertrophic and fibrotic markers. Also, hesperetin attenuated oxidative stress and myocytes apoptosis induced by AB. The inhibitory effect of hesperetin on cardiac remodelling was mediated by blocking PKCα/βII-AKT, JNK and TGFβ1-Smad signalling pathways. In conclusion, we found that the orange flavonoid hesperetin protected against cardiac remodelling induced by pressure overload via inhibiting cardiac hypertrophy, fibrosis, oxidative stress and myocytes apoptosis. These findings suggest a potential therapeutic drug for cardiac remodelling and HF.     

Thyroid hormone and myocardial ischaemia

Thyroid hormone has various effects on the cardiovascular system and its effects on cardiac contractility, heart rhythm and vascular function has long been recognized. However, new evidence is emerged on the importance of thyroid hormone in the response of the myocardium to ischaemic stress and cardiac remodelling following myocardial infarction. Based on this new information, this review highlights the role of thyroid hormone in myocardial ischaemia and cardiac remodelling, the possible underlying mechanisms and the potential therapeutic implications. Thyroid hormone or analogs may prove new therapeutic agents for treating ischaemic heart disease.     

Troponin I phosphorylation in human myocardium in health and disease

Cardiac troponin I (cTnI) is well known as a biomarker for the diagnosis of myocardial damage. However, because of its central role in the regulation of contraction and relaxation in heart muscle, cTnI may also be a potential target for the treatment of heart failure. Studies in rodent models of cardiac disease and human heart samples showed altered phosphorylation at various sites on cTnI (i.e. site-specific phosphorylation). This is caused by altered expression and/or activity of kinases and phosphatases during heart failure development. It is not known whether these (transient) alterations in cTnI phosphorylation are beneficial or detrimental. Knowledge of the effects of site-specific cTnI phosphorylation on cardiomyocyte contractility is therefore of utmost importance for the development of new therapeutic strategies in patients with heart failure. In this review we focus on the role of cTnI phosphorylation in the healthy heart upon activation of the beta-adrenergic receptor pathway (as occurs during increased stress and exercise) and as a modulator of the Frank-Starling mechanism. Moreover, we provide an overview of recent studies which aimed to reveal the functional consequences of changes in cTnI phosphorylation in cardiac disease.     

Talin Is Required Continuously for Cardiomyocyte Remodeling during Heart Growth in Drosophila

Mechanotransduction of tension can govern the remodeling of cardiomyocytes during growth or cardiomyopathy. Tension is signaled through the integrin adhesion complexes found at muscle insertions and costameres but the relative importance of signalling during cardiomyocyte growth versus remodelling has not been assessed. Employing the Drosophila cardiomyocyte as a genetically amenable model, we depleted the levels of Talin, a central component of the integrin adhesion complex, at different stages of heart growth and remodeling. We demonstrate a continuous requirement for Talin during heart growth to maintain the one-to-one apposition of myofibril ends between cardiomyocytes. Retracted myofibrils cannot regenerate appositions to adjacent cells after restoration of normal Talin expression, and the resulting deficit reduces heart contraction and lifespan. Reduction of Talin during heart remodeling after hatching or during metamorphosis results in pervasive degeneration of cell contacts, myofibril length and number, for which restored Talin expression is insufficient for regeneration. Resultant dilated cardiomyopathy results in a fibrillating heart with poor rhythmicity. Cardiomyocytes have poor capacity to regenerate deficits in myofibril orientation and insertion, despite an ongoing capacity to remodel integrin based adhesions.     

Remodelling of gap junctions and connexin expression in heart disease

Different combinations and relative quantities of three connexins-connexin43, connexin40 and connexin45-are expressed in different subsets of cardiomyocyte. In the healthy heart, gap junctions assembled from these different connexin combinations form the cell-to-cell pathways for the precisely orchestrated patterns of current flow that govern the normal heart rhythm. Remodelling of gap junction organization and connexin expression is a conspicuous feature of human heart disease in which there is an arrhythmic tendency. This remodelling may take the form of structural remodelling, involving disturbances in the distribution of gap junctions (i.e., disruption of the normal ordered pathways for cell-to-cell conduction), and remodelling of connexin expression, involving alteration in the amount or type of connexin(s) present. Most notable among quantitative alterations in connexin expression is a reduction in ventricular connexin43 levels in human congestive heart failure. By correlating data from studies in experimental animal models, gap junction and connexin remodelling emerges as a factor to be considered in understanding the pro-arrhythmic substrate characteristic of many forms of heart disease. However, our knowledge of the functional correlates of the specific patterns of multiple connexin expression found in different regions of the heart in health and disease remains rudimentary, and the development of new experimental cell models heralds advances in this area over the next few years.     

Functional modulation of cardiac form through regionally confined cell shape changes

Developing organs acquire a specific three-dimensional form that ensures their normal function. Cardiac function, for example, depends upon properly shaped chambers that emerge from a primitive heart tube. The cellular mechanisms that control chamber shape are not yet understood. Here, we demonstrate that chamber morphology develops via changes in cell morphology, and we determine key regulatory influences on this process. Focusing on the development of the ventricular chamber in zebrafish, we show that cardiomyocyte cell shape changes underlie the formation of characteristic chamber curvatures. In particular, cardiomyocyte elongation occurs within a confined area that forms the ventricular outer curvature. Because cardiac contractility and blood flow begin before chambers emerge, cardiac function has the potential to influence chamber curvature formation. Employing zebrafish mutants with functional deficiencies, we find that blood flow and contractility independently regulate cell shape changes in the emerging ventricle. Reduction of circulation limits the extent of cardiomyocyte elongation; in contrast, disruption of sarcomere formation releases limitations on cardiomyocyte dimensions. Thus, the acquisition of normal cardiomyocyte morphology requires a balance between extrinsic and intrinsic physical forces. Together, these data establish regionally confined cell shape change as a cellular mechanism for chamber emergence and as a link in the relationship between form and function during organ morphogenesis.     

PKM2 promotes angiotensin-II-induced cardiac remodelling by activating TGF-β/Smad2/3 and Jak2/Stat3 pathways through oxidative stress

Hypertensive cardiac remodelling is a common cause of heart failure. However, the molecular mechanisms regulating cardiac remodelling remain unclear. Pyruvate kinase isozyme type M2 (PKM2) is a key regulator of the processes of glycolysis and oxidative phosphorylation, but the roles in cardiac remodelling remain unknown. In the present study, we found that PKM2 was enhanced in angiotensin II (Ang II)-treated cardiac fibroblasts and hypertensive mouse hearts. Suppression of PKM2 by shikonin alleviated cardiomyocyte hypertrophy and fibrosis in Ang-II-induced cardiac remodelling in vivo. Furthermore, inhibition of PKM2 markedly attenuated the function of cardiac fibroblasts including proliferation, migration and collagen synthesis in vitro. Mechanistically, suppression of PKM2 inhibited cardiac remodelling by suppressing TGF-β/Smad2/3, Jak2/Stat3 signalling pathways and oxidative stress. Together, this study suggests that PKM2 is an aggravator in Ang-II-mediated cardiac remodelling. The negative modulation of PKM2 may provide a promising therapeutic approach for hypertensive cardiac remodelling.     

Myocardial calcium signalling and arrhythmia pathogenesis

Myocardial calcium signalling is a vital component of the normal physiological function of the heart. Key amongst the many roles calcium plays is its use as the primary signalling component of excitation-contraction coupling, the intracellular process that links cardiomyocyte depolarisation to contraction. Defective cellular calcium handling, due to abnormalities of the various components which mediate and control excitation-contraction coupling, is widely recognised as a significant patho-physiological event in the contractile dysfunction of the failing heart. In addition, similar defects also appear to be increasingly recognised as mediators of certain forms of cardiac arrhythmias. Such defects include single gene defects in excitation-contraction coupling components that lead to inherited sudden death arrhythmia syndromes. Alternatively, arrhythmogenesis occurring within the context of acquired cardiac disease, in particular heart failure, also appears to be highly dependent on abnormal calcium homeostasis. In this article we review the defects in cardiomyocyte calcium homeostasis that lead to particular pro-arrhythmogenic phenomena and discuss recent insights gained into a variety of inherited and acquired arrhythmia syndromes that appear to involve defective calcium signalling as a central component of their patho-physiology. Potential opportunities for new anti arrhythmic therapeutic strategies based on these recent insights are also discussed.     

Endothelial S1pr1 regulates pressure overload‐induced cardiac remodelling through AKT‐eNOS pathway

Cardiac vascular microenvironment is crucial for cardiac remodelling during the process of heart failure. Sphingosine 1‐phosphate (S1P) tightly regulates vascular homeostasis via its receptor, S1pr1. We therefore hypothesize that endothelial S1pr1 might be involved in pathological cardiac remodelling. In this study, heart failure was induced by transverse aortic constriction (TAC) operation. S1pr1 expression is significantly increased in microvascular endothelial cells (ECs) of post‐TAC hearts. Endothelial‐specific deletion of S1pr1 significantly aggravated cardiac dysfunction and deteriorated cardiac hypertrophy and fibrosis in myocardium. In vitro experiments demonstrated that S1P/S1pr1 praxis activated AKT/eNOS signalling pathway, leading to more production of nitric oxide (NO), which is an essential cardiac protective factor. Inhibition of AKT/eNOS pathway reversed the inhibitory effect of EC‐S1pr1‐overexpression on angiotensin II (AngII)‐induced cardiomyocyte (CM) hypertrophy, as well as on TGF‐β‐mediated cardiac fibroblast proliferation and transformation towards myofibroblasts. Finally, pharmacological activation of S1pr1 ameliorated TAC‐induced cardiac hypertrophy and fibrosis, leading to an improvement in cardiac function. Together, our results suggest that EC‐S1pr1 might prevent the development of pressure overload‐induced heart failure via AKT/eNOS pathway, and thus pharmacological activation of S1pr1 or EC‐targeting S1pr1‐AKT‐eNOS pathway could provide a future novel therapy to improve cardiac function during heart failure development.     

Sympathetic mechanism of regulating heart function in acute transitory coronary insufficiency of different duration

Experiments on 102 outbred white male rats with experimental acute transitory coronary insufficiency (ATCI) were carried out to show a consistent phasic time course of the functioning of the sympathetic cardiac activity regulation mechanism. The sympathetic neuroeffector influence on the myocardium with ATCI depends to a large measure on the intensity of the neurotransmitter biosynthesis and function of cardiomyocyte adrenoreceptors. It is suggested that the time couse of heart contractility and rhythm under ATCI is related to changes in the pre- and post-synaptic component of the sympathetic regulation mechanism of cardiac function. It is also assumed that cardiomyocyte receptors might exert a substantial influence on the possibility and development of arrhythmias. The advantages of pharmacological correction (as compared with the blockade) of sympathetic neuroeffector influences on the myocardium under ATCI are emphasized.     

Fibroblast Growth Factor Receptor 1 Signaling in Adult Cardiomyocytes Increases Contractility and Results in a Hypertrophic Cardiomyopathy

Fibroblast growth factors (FGFs) and their receptors are highly conserved signaling molecules that have been implicated in postnatal cardiac remodeling. However, it is not known whether cardiomyocyte-expressed FGF receptors are necessary or sufficient for ventricular remodeling in the adult heart. To determine whether cardiomyocytes were competent to respond to an activated FGF receptor, and to determine if this signal would result in the development of hypertrophy, we engineered a doxycycline (DOX)-inducible, cardiomyocyte-specific, constitutively active FGF receptor mouse model (αMHC-rtTA, TRE-caFgfr1-myc). Echocardiographic and hemodynamic analysis indicated that acute expression of caFGFR1 rapidly and directly increased cardiac contractility, while chronic expression resulted in significant hypertrophy with preservation of systolic function. Subsequent histologic analysis showed increased cardiomyocyte cross-sectional area and regions of myocyte disarray and fibrosis, classic features of hypertrophic cardiomyopathy (HCM). Analysis of downstream pathways revealed a lack of clear activation of classical FGF-mediated signaling pathways, but did demonstrate a reduction in Serca2 expression and troponin I phosphorylation. Isolated ventricular myocytes showed enhanced contractility and reduced relaxation, an effect that was partially reversed by inhibition of actin-myosin interactions. We conclude that adult cardiomyocytes are competent to transduce FGF signaling and that FGF signaling is sufficient to promote increased cardiomyocyte contractility in vitro and in vivo through enhanced intrinsic actin-myosin interactions. Long-term, FGFR overexpression results in HCM with a dynamic outflow tract obstruction, and may serve as a unique model of HCM.