Laura Zelarayan

Beta-Catenin downregulation attenuates ischemic cardiac remodeling through enhanced resident precursor cell differentiation.

We analyzed the effect of conditional, αMHC-dependent genetic β-catenin depletion and stabilization on cardiac remodeling following experimental infarct. β-Catenin depletion significantly improved 4-week survival and left ventricular (LV) function (fractional shortening: CTΔex3–6: 24 ± 1.9%; β-catΔex3–6: 30.2 ± 1.6%, P < 0.001). β-Catenin stabilization had opposite effects. No significant changes in adult cardiomyocyte survival or hypertrophy were observed in either transgenic line. Associated with the functional improvement, LV scar cellularity was altered: β-catenin-depleted mice showed a marked subendocardial and subepicardial layer of small cTnTpos cardiomyocytes associated with increased expression of cardiac lineage markers Tbx5 and GATA4. Using a Cre-dependent lacZ reporter gene, we identified a noncardiomyocyte cell population affected by αMHC-driven gene recombination localized to these tissue compartments at baseline. These cells were found to be cardiac progenitor cells since they coexpressed markers of proliferation (Ki67) and the cardiomyocyte lineage (αMHC, GATA4, Tbx5) but not cardiac Troponin T (cTnT). The cell population overlaps in part with both the previously described c-kitpos and stem cell antigen-1 (Sca-1)pos precursor cell population but not with the Islet-1pos precursor cell pool. An in vitro coculture assay of highly enriched (>95%) Sca-1pos cardiac precursor cells from β-catenin-depleted mice compared to cells isolated from control littermate demonstrated increased differentiation toward α-actinpos and cTnTpos cardiomyocytes after 10 days (CTΔex3–6: 38.0 ± 1.0% α-actinpos; β-catΔex3–6: 49.9 ± 2.4% α-actinpos, P < 0.001). We conclude that β-catenin depletion attenuates postinfarct LV remodeling in part through increased differentiation of GATA4pos/Sca-1pos resident cardiac progenitor cells.

Krueppel-like factor 15 regulates Wnt/β-catenin transcription and controls cardiac progenitor cell fate in the postnatal heart

Wnt/β‐catenin signalling controls adult heart remodelling in part via regulation of cardiac progenitor cell (CPC) differentiation. An enhanced understanding of mechanisms controlling CPC biology might facilitate the development of new therapeutic strategies in heart failure. We identified and characterized a novel cardiac interaction between Krueppel‐like factor 15 and components of the Wnt/β‐catenin pathway leading to inhibition of transcription. In vitro mutation, reporter assays and co‐localization analyses revealed that KLF15 requires both the C‐terminus, necessary for nuclear localization, and a minimal N‐terminal regulatory region to inhibit transcription. In line with this, functional Klf15 knock‐out mice exhibited cardiac β‐catenin transcriptional activation along with functional cardiac deterioration in normal homeostasis and upon hypertrophy. We further provide in vivo and in vitro evidences for preferential endothelial lineage differentiation of CPCs upon KLF15 deletion. Via inhibition of β‐catenin transcription, KLF15 controls CPC homeostasis in the adult heart similar to embryonic cardiogenesis. This knowledge may provide a tool for reactivation of this apparently dormant CPC population in the adult heart and thus be an attractive approach to enhance endogenous cardiac repair.

Erythropoietin Responsive Cardiomyogenic Cells Contribute to Heart Repair Post Myocardial Infarction

The role of erythropoietin (Epo) in myocardial repair after infarction remains inconclusive. We observed high Epo receptor (EPOR) expression in cardiac progenitor cells (CPCs). Therefore, we aimed to characterize these cells and elucidate their contribution to myocardial regeneration on Epo stimulation. High EPOR expression was detected during murine embryonic heart development followed by a marked decrease until adulthood. EPOR‐positive cells in the adult heart were identified in a CPC‐enriched cell population and showed coexpression of stem, mesenchymal, endothelial, and cardiomyogenic cell markers. We focused on the population coexpressing early (TBX5, NKX2.5) and definitive (myosin heavy chain [MHC], cardiac Troponin T [cTNT]) cardiomyocyte markers. Epo increased their proliferation and thus were designated as Epo‐responsive MHC expressing cells (EMCs). In vitro, EMCs proliferated and partially differentiated toward cardiomyocyte‐like cells. Repetitive Epo administration in mice with myocardial infarction (cumulative dose 4 IU/g) resulted in an increase in cardiac EMCs and cTNT‐positive cells in the infarcted area. This was further accompanied by a significant preservation of cardiac function when compared with control mice. Our study characterized an EPO‐responsive MHC‐expressing cell population in the adult heart. Repetitive, moderate‐dose Epo treatment enhanced the proliferation of EMCs resulting in preservation of post‐ischemic cardiac function. Stem Cells 2014;32:2480–2491

Genetic Background Defines the Regulation of Postnatal Cardiac Growth by 17β-Estradiol Through a β-Catenin Mechanism

Estrogen regulates several biological processes in health and disease. Specifically, estrogen exerts antihypertrophic effects in the diseased heart. However, its role in the healthy heart remains elusive. Our initial aim was to identify the effects of 17β-estradiol (E2) on cardiac morphology and global gene expression in the healthy mouse heart. Two-month-old C57BL/6J mice were ovariectomized and treated with E2 or vehicle for 3 months. We report that E2 induced physiological hypertrophic growth in the healthy C57BL/6J mouse heart characterized by an increase in nuclear β-catenin. Hypothesizing that β-catenin mediates these effects of E2, we employed a model of cardiac β-catenin deletion. Our surprising finding is that E2 had the opposite effects in wild-type littermates, which were actually on the C57BL/6N background. Notably, E2 exerted no significant effect in hearts of mice with depleted β-catenin. We further demonstrate an E2-dependent increase in glycogen synthase kinase 3β (GSK3β) phosphorylation and endosomal markers in C57BL/6J but not C57BL/6N mice. Together, these findings indicate an E2-driven inhibition of GSK3β and consequent activation of β-catenin in C57BL/6J mice, whereas the opposite occurs in C57BL/6N mice. In conclusion, E2 exerts divergent effects on postnatal cardiac growth in mice with distinct genetic backgrounds modulating members of the GSK3β/β-catenin cascade.

Hepoxilin A3 protects β-cells from apoptosis in contrast to its precursor, 12-hydroperoxyeicosatetraenoic acid

Pancreatic β-cells have a deficit of scavenging enzymes such as catalase (Cat) and glutathione peroxidase (GPx) and therefore are susceptible to oxidative stress and apoptosis. Our previous work showed that, in the absence of cytosolic GPx in insulinoma RINm5F cells, an intrinsic activity of 12 lipoxygenase (12(S)-LOX) converts 12S-hydroperoxyeicosatetraenoic acid (12(S)-HpETE) to the bioactive epoxide hepoxilin A(3) (HXA(3)). The aim of the present study was to investigate the effect of HXA(3) on apoptosis as compared to its precursor 12(S)-HpETE and shed light upon the underlying pathways. In contrast to 12(S)-HpETE, which induced apoptosis via the extrinsic pathway, we found HXA(3) not only to prevent it but also to promote cell proliferation. In particular, HXA(3) suppressed the pro-apoptotic BAX and upregulated the anti-apoptotic Bcl-2. Moreover, HXA(3) induced the anti-apoptotic 12(S)-LOX by recruiting heat shock protein 90 (HSP90), another anti-apoptotic protein. Finally, a co-chaperone protein of HSP90, protein phosphatase 5 (PP5), was upregulated by HXA(3), which counteracted oxidative stress-induced apoptosis by dephosphorylating and thus inactivating apoptosis signal-regulating kinase 1 (ASK1). Taken together, these findings suggest that HXA(3) protects insulinoma cells from oxidative stress and, via multiple signaling pathways, prevents them from undergoing apoptosis.

Calcium/Calmodulin-Dependent Protein Kinase II Activity Persists During Chronic β-Adrenoceptor Blockade in Experimental and Human Heart Failure

Background— Considerable evidence suggests that calcium/calmodulin-dependent protein kinase II (CaMKII) overactivity plays a crucial role in the pathophysiology of heart failure (HF), a condition characterized by excessive &bgr;-adrenoceptor (&bgr;-AR) stimulation. Recent studies indicate a significant cross talk between &bgr;-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental &bgr;-AR signaling in HF. In this study, we investigated the effect of chronic &bgr;-AR blocker treatment on CaMKII activity in human and experimental HF. Methods and Results— Immunoblot analysis of myocardium from end-stage HF patients (n=12) and non-HF subjects undergoing cardiac surgery (n=12) treated with &bgr;-AR blockers revealed no difference in CaMKII activity when compared with non–&bgr;-AR blocker–treated patients. CaMKII activity was judged by analysis of CaMKII expression, autophosphorylation, and oxidation and by investigating the phosphorylation status of CaMKII downstream targets. To further evaluate these findings, CaMKII&dgr;C transgenic mice were treated with the &bgr;1-AR blocker metoprolol (270 mg/kg*d). Metoprolol significantly reduced transgene-associated mortality (n≥29; P<0.001), attenuated the development of cardiac hypertrophy (−14±6% heart weight/tibia length; P<0.05), and strongly reduced ventricular arrhythmias (−70±22% premature ventricular contractions; P<0.05). On a molecular level, metoprolol expectedly decreased protein kinase A–dependent phospholamban and ryanodine receptor 2 phosphorylation (−42±9% for P-phospholamban-S16 and −22±7% for P-ryanodine receptor 2-S2808; P<0.05). However, this was paralled neither by a reduction in CaMKII autophosphorylation, oxidation, and substrate binding nor a change in the phosphorylation of CaMKII downstream target proteins (n≥11). The lack of CaMKII modulation by &bgr;-AR blocker treatment was confirmed in healthy wild-type mice receiving metoprolol. Conclusions— Chronic &bgr;-AR blocker therapy in patients and in a mouse model of CaMKII-induced HF is not associated with a change in CaMKII activity. Thus, our data suggest that the molecular effects of &bgr;-AR blockers are not based on a modulation of CaMKII. Directly targeting CaMKII may, therefore, further improve HF therapy in addition to &bgr;-AR blockade.Background Considerable evidence suggests that CaMKII overactivity plays a crucial role in the pathophysiology of heart failure (HF), a condition characterized by excessive β-adrenoceptor (β-AR) stimulation. Recent studies indicate a significant crosstalk between β-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental β-AR signaling in HF. In this study we investigated the effect of chronic β-AR blocker treatment on CaMKII activity in human and experimental HF.

Treatment-Sensitive Premature Renal and Heart Senescence in Hypertension

Hypertension-induced renal and heart failure account for a large proportion of chronic disease burden in the elderly. Antihypertensive therapy may halt the progression of disease. Preventing organ damage has emerged as a primary target for new approaches to treat hypertension. Signaling pathways affected by hypertension but not necessarily involved in blood pressure regulation itself have been identified as attractive new targets. Already in the early 1960s, a cellular stopwatch was described, which induced a growth arrest of normal somatic cells after several rounds of cell division in culture. In contrast, cancer cells were found to proliferate unlimited, implicating cellular senescence as an important molecular mechanism of protection against cancer. The molecular and cellular pathways controlling senescence have since been identified. The 3 “Hayflick factors” recording the proliferative history of cells and tissues are telomere shortening, accumulation of damaged DNA plus chromosomal damage, as well as derepression of the INK4a/ARF genomic locus. The molecular pathways mediating senescence, namely, telomere shortening and expression of p16 INK4a through stress and aberrant signaling–induced senescence are dissociated, indicating independent pathways.1 On a cellular level, a picture is emerging that tissue senescence is not only about the differentiated cells having a limited life span but also, and possibly even more important, reduced regenerative …

The Four and a Half LIM‐Domain 2 Controls Early Cardiac Cell Commitment and Expansion Via Regulating β‐Catenin‐Dependent Transcription

The multiphasic regulation of the Wnt/β‐catenin canonical pathway is essential for cardiogenesis in vivo and in vitro. To achieve tight regulation of the Wnt/β‐catenin signaling, tissue‐ and cell‐specific coactivators and repressors need to be recruited. The identification of such factors may help to elucidate mechanisms leading to enhanced cardiac differentiation efficiency in vitro as well as promote regeneration in vivo. Using a yeast‐two‐hybrid screen, we identified four‐and‐a‐half‐LIM‐domain 2 (FHL2) as a cardiac‐specific β‐catenin interaction partner and activator of Wnt/β‐catenin‐dependent transcription. We analyzed the role of this interaction for early cardiogenesis in an in vitro model by making use of embryoid body cultures from mouse embryonic stem cells (ESCs). In this model, stable FHL2 gain‐of‐function promoted mesodermal cell formation and cell proliferation while arresting cardiac differentiation in an early cardiogenic mesodermal progenitor state. Mechanistically, FHL2 overexpression enhanced nuclear accumulation of β‐catenin and activated Wnt/β‐catenin‐dependent transcription leading to sustained upregulation of the early cardiogenic gene Igfbp5. In an alternative P19 cell model, transient FHL2 overexpression led to early activation of Wnt/β‐catenin‐dependent transcription, but not sustained high‐level of Igfbp5 expression. This resulted in enhanced cardiogenesis. We propose that early Wnt/β‐catenin‐dependent transcriptional activation mediated by FHL2 is important for the transition to and expansion of early cardiogenic mesodermal cells. Collectively, our findings offer mechanistic insight into the early cardiogenic code and may be further exploited to enhance cardiac progenitor cell activity in vitro and in vivo. STEM CELLS 2013;31:928–940

Wnt Signaling Molecules in Left Ventricular Remodeling Focus on Dishevelled 1

Under acute or chronic stresses, the adult heart undergoes a remodeling process that involves cardiomyocyte hypertrophy accompanied by apoptosis, necrosis, and fibrosis that lead to impaired cardiac contractility. The role of endogenous regeneration in this process is currently under investigation. Sustained deleterious stimuli will lead to a decompensated form of hypertrophy often culminating in heart failure.1 This form of hypertrophy is often referred to as “maladaptive.” When dealing with hypertrophy, it appears important to distinguish between the term being used on the cellular and molecular level (enlargement of individual cardiomyocytes and re-expression of fetal/embryonic genes) and the organ level (increased heart weight, left ventricular wall thickness, and functional diastolic and systolic impairment). In our view, these processes are certainly linked but not identical. Hypertrophy on the organ levels summarizes several independent cellular and molecular processes (see below), where cardiomyocyte growth is not necessarily the most important. Independent of its origin, cardiac hypertrophy is associated with alterations in cardiac geometry, mass, architecture, and function controlled by a complex network of interconnected and abundant signal-transduction pathways.2 New signaling molecules are emerging as possible targets to specifically attenuate maladaptive hypertrophy. Pathological, stress-induced growth of cardiomyocytes was shown to depend on Wnt/β-catenin nuclear signaling rather than its adhesive function in cell adhesion. However, the specificity of the cell type and the molecular mechanisms governing the Wnt signaling–dependent changes are currently unknown.3 In this issue of the Hypertension , the study by Malekar et al4 provides new evidences concerning the …

RALT Specifically Halts Maladaptive Cardiac Hypertrophy A New Kid on the Block

The adult heart enlarges and increases in weight in response to ischemic stress and hypertension, a process termed cardiac hypertrophy. However, the heart also increases in size after regular physiological exercise. Cardiac hypertrophy is recognized as an important process in the development of heart failure. Initial results pointed to a clear signaling pattern of physiological hypertrophy after training versus pathological hypertrophy after damage. Although the signaling mechanisms involved have been studied for more than a decade, there is no clear distinction at the molecular level of physiological and pathological hypertrophy.1 Rather, the balance between signaling pathways preventing deterioration and pathways activated in association with preserving left ventricular function is regarded as critical. Accordingly, the terms adaptive and maladaptive were introduced to reflect these processes, respectively.2 A potential treatment avenue could be to selectively identify pathways involved in maladaptive hypertrophy that could be blocked. Alternatively, enhancing signaling pathways specifically involved in adaptive hypertrophy might allow us to conserve left ventricular function. Cai et al have identified a new target in cardiac hypertrophy: the receptor-associated late transducer (RALT).3 RALT appears to be a specific antagonist of maladaptive hypertrophy (Figure). Mechanistically, RALT functions as a negative regulator of epidermal growth factor receptor-mediated signaling not only in the heart but in other organs as well. The investigators used angiotensin II and isoproterenol as stimuli for stress-induced, maladaptive left ventricular remodeling in transgenic mice overexpressing RALT under the control of the α-myosin heavy …