Sydänlihassolujen hypertrofia lääkevaikutuksen kohteena

Left ventricular hypertrophy (LVH) is defined as an increase in left ventricular mass. It is initially a coping mechanism by which the heart tries to compensate for the increase in load caused by, for example, hypertension, but it will eventually lead to heart failure. LVH is the result of primarily an increase in cardiac myocyte size, in addition to increased apoptosis and necrosis of cardiac myocytes and fibrosis. Current treatment of LVH is based on a treatment of suspected cause, generally hypertension. Antihypertensive medication has been found to have beneficial effects on LVH. However, antihypertensive drugs can not cure LVH completely, hence other treatment options are needed. To identify new possible drug targets, it is important to increase the inadequate knowledge of the mechanisms and signal transduction pathways mediating LVH. The most relevant stimuli causing hypertrophy are considered to be mechanical stretch, as well as some humoral mediators such as angiotensin II and endothelin 1 (ET-1), to which cardiomyocytes respond through activation of several intracellular signal transduction pathways. As a result, cardiomyocyte gene expression and protein synthesis increase and sarcomeres grow and rearrange, resulting in an increase in cell size. In addition, regulation of calcium, contractile function and energy metabolism of cardiac myocytes change. Numerous intracellular signal mediators interact with each other and can compensate for each other, making it difficult to investigate the significance of individual factors. As important signal mediators are considered to include protein kinase C (PKC) and cardiac transcription factors GATA4 and NKX2-5. In vitro studies of cardiac hypertrophy are usually performed with primary cardiac myocytes isolated from the ventricles of neonatal rats. The H9c2 continuous cell line has been used in some studies as an alternative cell model to reduce the use of laboratory animals. In the experimental part of this thesis, the suitability of H9c2 cells for hypertrophy studies was examined by comparing them to primary cardiac myocytes. In addition, experimental compounds targeted to cardiac transcription factors and PKC were studied by exploring their effects on viability and hypertrophic responses of H9c2 cells and primary cardiac myocytes. The toxicity of the compounds and the effects on cell viability were studied using the lactate dehydrogenase (LDH) assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The hypertrophy responses to cyclic mechanical stretch and ET-1 were primarily assessed by measuring the surface area of cells from fluorescence microscopy images. In addition, the relative expression levels of Nppa and Nppb genes in ET-1 stimulated primary cardiac myocytes were studied by quantitative polymerase chain reaction (qPCR). Both stretching and ET-1 caused an increase in the cell surface area in primary cardiac myocytes but not in H9c2 cells. On this basis, the H9c2 cells respond differently to hypertrophic stimuli than primary cardiac myocytes, and the suitability of H9c2 cell line to hypertrophy studies can therefore be questioned. The compounds targeted to cardiac transcription factors were not cytotoxic at 1-30 µM concentrations, but they also had no significant effect on the hypertrophic responses. In contrast, the PKC compound HMI-1a3 at 30 µM was toxic to primary cardiac myocytes and HMI-1b11 at 30 µM was toxic to H9c2 cells. HMI-1b11 and bryostatin-1 also induced changes in the hypertrophic responses of primary cardiac myocytes, but the significance of these results requires further investigation.Sydämen vasemman kammion hypertrofia (engl. left ventricular hypertrophy, LVH) tarkoittaa vasemman kammion massan kasvua. Se on alun perin sopeutumiskeino, jolla sydän yrittää kompensoida siihen kohdistuvaa esimerkiksi kohonneen verenpaineen aiheuttamaa lisääntynyttä kuormitusta, mutta se johtaa lopulta sydämen vajaatoimintaan. LVH on seurausta sydänlihassolujen eli kardiomyosyyttien koon kasvusta, minkä lisäksi niiden apoptoosi ja nekroosi sekä sydämen fibroosi lisääntyvät. Nykyinen LVH:n hoito perustuu epäillyn syyn, yleensä kohonneen verenpaineen, hoitoon. Verenpainelääkkeillä on havaittu suotuisia vaikutuksia LVH:aan, mutta niillä ei ole voitu estää hypertrofiaa täysin, joten muitakin hoitovaihtoehtoja kaivataan. LVH:n puutteellisesti tunnettujen syntymekanismien ja signaalinvälitysreittien selvittäminen on tärkeää uusien mahdollisten lääkevaikutuskohteiden löytämiseksi. Keskeisimpinä hypertrofiaa aiheuttavina ärsykkeinä pidetään mekaanista venytystä sekä joitakin humoraalisia välittäjäaineita, kuten angiotensiini II:ta ja endoteliini-1:tä (ET-1), joihin kardiomyosyytit reagoivat aktivoimalla useita solunsisäisiä signaalinvälitysreittejä. Tämän seurauksena kardiomyosyyttien geeniekspressio ja proteiinisynteesi lisääntyvät ja sarkomeerit kasvavat ja järjestäytyvät uudelleen, mikä johtaa solujen koon kasvuun. Lisäksi sydänlihassolujen kalsiumin säätely, supistustoiminta ja energia-aineenvaihdunta muuttuvat. Lukuisat solunsisäiset signaalinvälittäjät vuorovaikuttavat keskenään sekä voivat kompensoida toisiaan, mikä vaikeuttaa eri tekijöiden merkitysten tutkimista. Tärkeinä signaalinvälittäjinä pidetään muun muassa proteiinikinaasi C:tä (PKC) sekä sydämen transkriptiotekijöitä GATA4:ää ja NKX2-5:tä. Sydämen hypertrofian in vitro -tutkimuksia tehdään yleensä vastasyntyneen rotan sydämestä eristetyillä primäärisydänlihassoluilla. Vaihtoehtoisena menetelmänä on joissakin tutkimuksissa käytetty jatkuvaa H9c2-solulinjaa, minkä avulla on pyritty vähentämään koe-eläinten käyttöä. Tämän pro gradu -tutkielman kokeellisessa osassa tutkittiin H9c2-solujen soveltuvuutta hypertrofiatutkimuksiin vertaamalla niitä primäärisydänlihassoluihin. Lisäksi tutkittiin uusien kokeellisten PKC:hen ja sydämen transkriptiotekijöihin vaikuttavien yhdisteiden vaikutuksia H9c2-solujen ja primäärisydänlihassolujen elinkykyyn ja hypertrofiavasteisiin. Yhdisteiden toksisuutta ja vaikutusta solujen elinkykyyn tutkittiin laktaattidehydrogenaasitestillä (LDH-testi) ja 3-(4,5-dimetyyli-2-tiatsolyyli)-2,5-difenyylitetratsoliumbromidi-testillä (MTT-testi). Mekaanisen syklisen venytyksen ja ET-1:n aiheuttamia hypertrofiavasteita arvioitiin ensisijaisesti solujen pinta-alan määrityksellä fluoresenssivärjätyistä soluista. Lisäksi ET-1-stimuloitujen primäärisydänlihassolujen hypertrofiaan liitettyjen Nppa- ja Nppb-geenien ekspressiota mitattiin kvantitatiivisella polymeraasiketjureaktiolla (engl. quantitative polymerase chain reaction, qPCR). Sekä venytys että ET-1 aiheuttivat primäärisydänlihassoluissa pinta-alan kasvun, jota ei havaittu H9c2-soluissa. Tämän perusteella H9c2-solujen vaste hypertrofiaa aiheuttaviin stimulaatioihin ei vastaa primäärikardiomyosyyteillä saatavaa vastetta ja solulinjan soveltuvuutta hypertrofiatutkimukseen voidaan siksi pitää kyseenalaisena. Tutkitut transkriptiotekijöihin vaikuttavat yhdisteet eivät 1-30 µM pitoisuuksilla olleet juuri sytotoksisia, mutta niillä ei myöskään havaittu merkittävää vaikutusta hypertrofiavasteisiin. Sen sijaan PKC:hen vaikuttavista yhdisteistä 30 µM HMI-1a3 oli toksinen primäärikardiomyosyyteille ja 30 µM HMI-1b11 H9c2-soluille. HMI-1b11 ja bryostatiini-1 aiheuttivat myös muutoksia primäärisydänlihassolujen hypertrofiavasteisiin, mutta tulosten merkityksen selvittäminen vaatii vielä lisää tutkimuksia

Transcriptomics reveal stretched human pluripotent stem cell-derived cardiomyocytes as an advantageous hypertrophy model

Peer reviewe

Pharmacological protein kinase C modulators reveal a pro-hypertrophic role for novel protein kinase C isoforms in human induced pluripotent stem cell-derived cardiomyocytes

Background: Hypertrophy of cardiomyocytes (CMs) is initially a compensatory mechanism to cardiac overload, but when prolonged, it leads to maladaptive myocardial remodeling, impairing cardiac function and causing heart failure. A key signaling molecule involved in cardiac hypertrophy is protein kinase C (PKC). However, the role of different PKC isoforms in mediating the hypertrophic response remains controversial. Both classical (cPKC) and novel (nPKC) isoforms have been suggested to play a critical role in rodents, whereas the role of PKC in hypertrophy of human CMs remains to be determined. Here, we aimed to investigate the effects of two different types of PKC activators, the isophthalate derivative HMI-1b11 and bryostatin-1, on CM hypertrophy and to elucidate the role of cPKCs and nPKCs in endothelin-1 (ET-1)-induced hypertrophy in vitro. Methods and Results: We used neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of pharmacological PKC modulators and ET-1. We used quantitative reverse transcription PCR to quantify hypertrophic gene expression and high-content analysis (HCA) to investigate CM morphology. In both cell types, ET-1, PKC activation (bryostatin-1 and HMI-1b11) and inhibition of cPKCs (Gö6976) increased hypertrophic gene expression. In NRVMs, these treatments also induced a hypertrophic phenotype as measured by increased recognition, intensity and area of α-actinin and F-actin fibers. Inhibition of all PKC isoforms with Gö6983 inhibited PKC agonist-induced hypertrophy, but could not fully block ET-1-induced hypertrophy. The mitogen-activated kinase kinase 1/2 inhibitor U0126 inhibited PKC agonist-induced hypertrophy fully and ET-1-induced hypertrophy partially. While ET-1 induced a clear increase in the percentage of pro-B-type natriuretic peptide-positive hiPSC-CMs, none of the phenotypic parameters used in HCA directly correlated with gene expression changes or with phenotypic changes observed in NRVMs. Conclusions: This work shows similar hypertrophic responses to PKC modulators in NRVMs and hiPSC-CMs. Pharmacological PKC activation induces CM hypertrophy via activation of novel PKC isoforms. This pro-hypertrophic effect of PKC activators should be considered when developing PKC-targeted compounds for e.g. cancer or Alzheimer’s disease. Furthermore, this study provides further evidence on distinct PKC-independent mechanisms of ET-1-induced hypertrophy both in NRVMs and hiPSC-CMs.Peer reviewe

Application of Human Induced Pluripotent Stem Cell Technology for Cardiovascular Regenerative Pharmacology

Cardiovascular diseases are one of the leading causes of mortality in the western world. Myocardial infarction is among the most prevalent and results in significant cell loss within the myocardium. Similarly, numerous drugs have been identified as having cardiotoxic side effects. The adult human heart is however unable to instigate an effective repair mechanism and regenerate the myocardium in response to such damage. This is in large part due to the withdrawal of cardiomyocytes (CMs) from the cell cycle. Thus, identifying, screening, and developing agents that could enhance the proliferative capacity of CMs holds great potential in cardiac regeneration. Human induced pluripotent stem cells (hiPSCs) and their cardiovascular derivatives are excellent tools in the search for such agents. This chapter outlines state-of-the art techniques for the two-dimensional differentiation and attainment of hiPSC-derived CMs and endothelial cells (ECs). Bioreactor systems and three-dimensional spheroids derived from hiPSC-cardiovascular derivatives are explored as platforms for drug discovery before focusing on relevant assays that can be employed to assess cell proliferation and viability.Peer reviewe

Conventional rigid 2D substrates cause complex contractile signals in monolayers of human induced pluripotent stem cell derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in monolayers interact mechanically via cell-cell and cell-substrate adhesion. Spatiotemporal features of contraction were analysed in hiPSC-CM monolayers (1) attached to glass or plastic (Young's modulus (E) >1 GPa), (2) detached (substrate-free) and (3) attached to a flexible collagen hydrogel (E = 22 kPa). The effects of isoprenaline on contraction were compared between rigid and flexible substrates. To clarify the underlying mechanisms, further gene expression and computational studies were performed. HiPSC-CM monolayers exhibited multiphasic contractile profiles on rigid surfaces in contrast to hydrogels, substrate-free cultures or single cells where only simple twitch-like time-courses were observed. Isoprenaline did not change the contraction profile on either surface, but its lusitropic and chronotropic effects were greater in hydrogel compared with glass. There was no significant difference between stiff and flexible substrates in regard to expression of the stress-activated genes NPPA and NPPB. A computational model of cell clusters demonstrated similar complex contractile interactions on stiff substrates as a consequence of cell-to-cell functional heterogeneity. Rigid biomaterial surfaces give rise to unphysiological, multiphasic contractions in hi PSC-CM monolayers. Flexible substrates are necessary for normal twitch-like contractility kinetics and interpretation of inotropic interventions.Peer reviewe

In vitro Evaluation of the Therapeutic Effects of Dual-Drug Loaded Spermine-Acetalated Dextran Nanoparticles Coated with Tannic Acid for Cardiac Applications

Myocardial infarction results in a massive loss of cardiomyocytes (CMs). Unfortunately, current therapies are unsuccessful in replacing lost CMs, and thus, there is an urgent need for innovative approaches. Here, a nanosystem based on spermine-acetalated dextran (AcDXSp) and encapsulating two drug compounds able to stimulate in vitro CMs proliferation is developed. The nanosystem is coated by deposition of a film constituted by tannic acid (TA) and Fe3+ ions. The coating with TA increases the retention of the nanocarrier in cell co-cultures of CMs and fibroblasts stimulated with transforming growth factor (TGF)-β, due to the high affinity of TA for components of the cardiac extracellular matrix. The system exhibits biocompatibility toward primary CMs and induces their proliferation, as indicated by the two-fold increase of CMs in the active cell cycle. At the same time, the presence of TA synergistically helps contrasting fibrosis by reducing profibrotic genes expression, such as collagen 1 and osteopontin, by approximately 80% compared to the control. Overall, the developed nanosystem demonstrates the capability to stimulate CMs proliferation and reduce fibrosis, showing potential benefits for future in vivo applications.Peer reviewe

Discovery of Small Molecules Targeting the Synergy of Cardiac Transcription Factors GATA4 and NKX2-5

Transcription factors are pivotal regulators of gene transcription, and many diseases are associated with the deregulation of transcriptional networks. In the heart, the transcription factors GATA4 and NKX2-5 are required for cardiogenesis. GATA4 and NKX2-5 interact physically, and the activation of GATA4, in cooperation with NKX2-5, is essential for stretch-induced cardiomyocyte hypertrophy. Here, we report the identification of four small molecule families that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy. A fragment-based screening, reporter gene assay, and pharmacophore search were utilized for the small molecule screening, identification, and optimization. The compounds modulated the hypertrophic agonist-induced cardiac gene expression. The most potent hit compound, N-[4-(diethylamino)phenyl]-5-methyl-3-phenylisoxazole-4-carboxamide (3, IC50 = 3 mu M), exhibited no activity on the protein kinases involved in the regulation of GATA4 phosphorylation. The identified and chemically and biologically characterized active compound, and its derivatives may provide a novel class of small molecules for modulating heart regeneration.Peer reviewe

Application of Human Induced Pluripotent Stem Cell Technology for Cardiovascular Regenerative Pharmacology

Cardiovascular diseases are one of the leading causes of mortality in the western world. Myocardial infarction is among the most prevalent and results in significant cell loss within the myocardium. Similarly, numerous drugs have been identified as having cardiotoxic side effects. The adult human heart is however unable to instigate an effective repair mechanism and regenerate the myocardium in response to such damage. This is in large part due to the withdrawal of cardiomyocytes (CMs) from the cell cycle. Thus, identifying, screening, and developing agents that could enhance the proliferative capacity of CMs holds great potential in cardiac regeneration. Human induced pluripotent stem cells (hiPSCs) and their cardiovascular derivatives are excellent tools in the search for such agents. This chapter outlines state-of-the art techniques for the two-dimensional differentiation and attainment of hiPSC-derived CMs and endothelial cells (ECs). Bioreactor systems and three-dimensional spheroids derived from hiPSC-cardiovascular derivatives are explored as platforms for drug discovery before focusing on relevant assays that can be employed to assess cell proliferation and viability.Peer reviewe

Stem cells are the most sensitive screening tool to identify toxicity of GATA4-targeted novel small-molecule compounds

Safety assessment of drug candidates in numerous in vitro and experimental animal models is expensive, time consuming and animal intensive. More thorough toxicity profiling already in the early drug discovery projects using human cell models, which more closely resemble the physiological cell types, would help to decrease drug development costs. In this study we aimed to compare different cardiac and stem cell models for in vitro toxicity testing and to elucidate structure-toxicity relationships of novel compounds targeting the cardiac transcription factor GATA4. By screening the effects of eight compounds at concentrations ranging from 10 nM up to 30 A mu M on the viability of eight different cell types, we identified significant cell type- and structure-dependent toxicity profiles. We further characterized two compounds in more detail using high-content analysis. The results highlight the importance of cell type selection for toxicity screening and indicate that stem cells represent the most sensitive screening model, which can detect toxicity that may otherwise remain unnoticed. Furthermore, our structure-toxicity analysis reveals a characteristic dihedral angle in the GATA4-targeted compounds that causes stem cell toxicity and thus helps to direct further drug development efforts towards non-toxic derivatives.Peer reviewe