Roel van der Nagel

MicroRNA-199b targets the nuclear kinase Dyrk1a in an auto-amplification loop promoting calcineurin/NFAT signalling

MicroRNAs (miRs) are a class of single-stranded, non-coding RNAs of about 22 nucleotides in length. Increasing evidence implicates miRs in myocardial disease processes. Here we show that miR-199b is a direct calcineurin/NFAT target gene that increases in expression in mouse and human heart failure, and targets the nuclear NFAT kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a (Dyrk1a), constituting a pathogenic feed forward mechanism that affects calcineurin-responsive gene expression. Mutant mice overexpressing miR-199b, or haploinsufficient for Dyrk1a, are sensitized to calcineurin/NFAT signalling or pressure overload and show stress-induced cardiomegaly through reduced Dyrk1a expression. In vivo inhibition of miR-199b by a specific antagomir normalized Dyrk1a expression, reduced nuclear NFAT activity and caused marked inhibition and even reversal of hypertrophy and fibrosis in mouse models of heart failure. Our results reveal that microRNAs affect cardiac cellular signalling and gene expression, and implicate miR-199b as a therapeutic target in heart failure.

MEF2 Activates a Genetic Program Promoting Chamber Dilation and Contractile Dysfunction in Calcineurin-Induced Heart Failure

Background— Hypertrophic growth, a risk factor for mortality in heart disease, is driven by reprogramming of cardiac gene expression. Although the transcription factor myocyte enhancer factor-2 (MEF2) is a common end point for several hypertrophic pathways, its precise cardiac gene targets and function in cardiac remodeling remain to be elucidated. Methods and Results— We report the existence of synergistic interactions between the nuclear factor of activated T cells and MEF2 transcription factors triggered by calcineurin signaling. To circumvent the embryonic lethality and mitochondrial deficiency associated with germ-line null mutations for MEF2C and MEF2A respectively, we used conditional transgenesis to express a dominant-negative form of MEF2 in the murine postnatal heart and combined this with magnetic resonance imaging to assess MEF2 transcriptional function in Ca2+/calcineurin-induced cardiac remodeling. Surprisingly, end-diastolic and end-systolic ventricular dimensions and contractility were normalized in the presence of severely hypertrophied left ventricular walls on MEF2 inhibition in calcineurin transgenic mice. In line, we generated lines of transgenic mice expressing MEF2A in the heart, which displayed primarily chamber dilation. Microarray profiling indicated that MEF2 promotes a gene profile functioning primarily to or at the nucleus, cytoskeletal and microtubular networks, and mitochondria. Conclusions— These findings assign a novel function to MEF2 transcription factors in the postnatal heart, where they activate a genetic program that minimally affects cardiac growth yet promotes chamber dilation, mechanical dysfunction, and dilated cardiomyopathy.

Downregulation of Apoptosis-Inducing Factor in Harlequin Mutant Mice Sensitizes the Myocardium to Oxidative Stress-Related Cell Death and Pressure Overload-Induced Decompensation

Apoptosis-inducing factor (AIF), or programmed cell death 8 (Pdcd8), is a highly conserved, ubiquitous flavoprotein localized in the mitochondrial intermembrane space. In vivo, AIF provides protection against neuronal apoptosis induced by oxidative stress. Conversely, in vitro, AIF has been demonstrated to have a proapoptotic role when, on induction of the mitochondrial death pathway, AIF translocates to the nucleus where it facilitates chromatin condensation and large scale DNA fragmentation. To determine the role of AIF in myocardial apoptotic processes, we examined cardiomyocytes from an AIF-deficient mouse mutant, Harlequin (Hq). Hq mutant cardiomyocytes demonstrated increased sensitivity to H2O2-induced cell death. Further, Hq hearts subjected to ischemia/reperfusion revealed more cardiac damage and, unlike wild-type mice, the amount of damage increased with the age of the animal. Aortic banding caused enhanced hypertrophy, increased cardiomyocyte apoptotic and necrotic cell death, and accelerated progression toward maladaptive left ventricular remodeling in Hq mutant mice compared with wild-type counterparts. These findings correlated with a reduced capacity of subsarcolemmal mitochondria from Hq mutant hearts to scavenge free radicals. Together, these data demonstrate a critical role for AIF as a cardiac antioxidant in the protection against oxidative stress–induced cell death and development of heart failure induced by pressure overload.

NFATc2 is a necessary mediator of calcineurin-dependent cardiac hypertrophy and heart failure.

One major intracellular signaling pathway involved in heart failure employs the phosphatase calcineurin and its downstream transcriptional effector nuclear factor of activated T-cells (NFAT). In vivo evidence for the involvement of NFAT factors in heart failure development is still ill defined. Here we reveal that nfatc2 transcripts outnumber those from other nfat genes in the unstimulated heart by severalfold. Transgenic mice with activated calcineurin in the postnatal myocardium crossbred with nfatc2-null mice revealed a significant abrogation of calcineurin-provoked cardiac growth, indicating that NFATc2 plays an important role downstream of calcineurin and validates the original hypothesis that calcineurin mediates myocyte hypertrophy through activation of NFAT transcription factors. In the absence of NFATc2, a clear protection against the geometrical, functional, and molecular deterioration of the myocardium following biomechanical stress was also evident. In contrast, physiological cardiac enlargement in response to voluntary exercise training was not affected in nfatc2-null mice. Combined, these results reveal a major role for the NFATc2 transcription factor in pathological cardiac remodeling and heart failure.

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.

Nfat and miR-25 cooperate to reactivate the transcription factor Hand2 in heart failure

Although aberrant reactivation of embryonic gene programs is intricately linked to pathological heart disease, the transcription factors driving these gene programs remain ill-defined. Here we report that increased calcineurin/Nfat signalling and decreased miR-25 expression integrate to re-express the basic helix-loop-helix (bHLH) transcription factor dHAND (also known as Hand2) in the diseased human and mouse myocardium. In line, mutant mice overexpressing Hand2 in otherwise healthy heart muscle cells developed a phenotype of pathological hypertrophy. Conversely, conditional gene-targeted Hand2 mice demonstrated a marked resistance to pressure-overload-induced hypertrophy, fibrosis, ventricular dysfunction and induction of a fetal gene program. Furthermore, in vivo inhibition of miR-25 by a specific antagomir evoked spontaneous cardiac dysfunction and sensitized the murine myocardium to heart failure in a Hand2-dependent manner. Our results reveal that signalling cascades integrate with microRNAs to induce the expression of the bHLH transcription factor Hand2 in the postnatal mammalian myocardium with impact on embryonic gene programs in heart failure.

Loss of muscle-specific RING-finger 3 predisposes the heart to cardiac rupture after myocardial infarction

RING-finger proteins commonly function as ubiquitin ligases that mediate protein degradation by the ubiquitin-proteasome pathway. Muscle-specific RING-finger (MuRF) proteins are striated muscle-restricted components of the sarcomere that are thought to possess ubiquitin ligase activity. We show that mice lacking MuRF3 display normal cardiac function but are prone to cardiac rupture after acute myocardial infarction. Cardiac rupture is preceded by left ventricular dilation and a severe decrease in cardiac contractility accompanied by myocyte degeneration. Yeast two-hybrid assays revealed four-and-a-half LIM domain (FHL2) and γ-filamin proteins as MuRF3 interaction partners, and biochemical analyses showed these proteins to be targets for degradation by MuRF3. Accordingly, FHL2 and γ-filamin accumulated to abnormal levels in the hearts of mice lacking MuRF3. These findings reveal an important role of MuRF3 in maintaining cardiac integrity and function after acute myocardial infarction and suggest that turnover of FHL2 and γ-filamin contributes to this cardioprotective function of MuRF3.

NF-κB activation is required for adaptive cardiac hypertrophy

AIMS We have previously shown that cardiac-specific inhibition of NF-kappaB attenuates angiotensin II (AngII)-induced left ventricular (LV) hypertrophy in vivo. We now tested whether NF-kappaB inhibition is able to block LV remodelling upon chronic pressure overload and chronic AngII stimulation. METHODS AND RESULTS Cardiac-restricted NF-kappaB inhibition was achieved by expression of a stabilized IkappaBalpha mutant (IkappaBalphaDeltaN) in cells with an active alpha-myosin heavy chain (alphaMHC) promoter employing the Cre/lox technique. Upon low-gradient trans-aortic constriction (TAC, gradient 21 +/- 3 mmHg), hypertrophy was induced in both male and female control mice after 4 weeks. At this time, LV hypertrophy was blocked in transgenic (TG) male but not female mice with NF-kappaB inhibition. Amelioration of LV hypertrophy was associated with activation of NF-kappaB by dihydrotestosterone in isolated neonatal cardiomyocytes. LV remodelling was not attenuated by NF-kappaB inhibition after 8 weeks TAC, demonstrated by decreased fractional shortening (FS) in both control and TG mice irrespective of gender. Similar results were obtained when TAC was performed with higher gradients (48 +/- 4 mmHg). In TG mice, FS dropped to similar low levels over the same time course [FS sham, 29 +/- 1% (mean +/- SEM); FS control + 14 days TAC, 13 +/- 3%; FS TG + 14 days TAC, 9 +/- 5%]. Similarly, LV remodelling was accelerated by NF-kappaB inhibition in an AngII-dependent genetic heart failure model (AT1-R(alphaMHC)) associated with significantly increased cardiac fibrosis in double AT1-R(alphaMHC)/TG mice. CONCLUSION NF-kappaB inhibition attenuates cardiac hypertrophy in a gender-specific manner but does not alter the course of stress-induced LV remodelling, indicating NF-kappaB to be required for adaptive cardiac hypertrophy.

Combined reduction of intercellular coupling and membrane excitability differentially affects transverse and longitudinal cardiac conduction

AIMS Reduced excitability and gap junction expression are commonly found in electrically remodelled diseased hearts, but their contribution to slow conduction and arrhythmias is unclear. In this study, we have investigated the effect of isolated and combined reductions in membrane excitability and intercellular coupling on impulse propagation and arrhythmogeneity in genetically modified mice. METHODS AND RESULTS Cx43 and Scn5a(1798insD/+) heterozygous (HZ) mice were crossbred to create a mixed offspring: wild-type (WT, n = 15), Cx43 HZ (n = 14), Scn5a(1798insD/+) (Scn5a) HZ (n = 17), and Cx43/Scn5a(1798insD/+) (Cx43/Scn5a) HZ (n = 15) mice. After ECG recording, epicardial activation mapping (208 recording sites) was performed on Langendorff-perfused hearts. Arrhythmia inducibility was tested by one to three premature stimuli and burst pacing. Conduction velocity longitudinal (CV(L)) and transverse (CV(T)) to fibre orientation and dispersion of conduction were determined during S1-S1 pacing (150 ms). Connexin43 (Cx43) and sodium channel Nav1.5 protein expression and myocardial tissue collagen content were determined by immunohistology. Compared with WT animals, P, QRS, and QTc intervals were prolonged in Scn5a HZ and Cx43/Scn5a HZ, but not in Cx43 HZ animals. Scn5a HZ mice showed decreased CV(L) in right ventricle (RV) but not in left ventricle compared with WT. In the RV of Cx43/Scn5a HZ, CV(T) was reduced, but CV(L) was not different from WT. Arrhythmia inducibility was low and not increased in either single- or double-mutant mice. CONCLUSION Reduction of both electrical coupling and excitability results in normal conduction velocity parallel to fibre orientation but in pronounced conduction slowing transverse to fibre orientation in RV only, although this does not affect arrhythmogeneity.

Reduced Cx43 expression triggers increased fibrosis due to enhanced fibroblast activity.

Background— Arrhythmogenic ventricular remodeling is hallmarked by both reduced gap junction expression and increased collagen deposition. We hypothesized that reduced connexin43 (Cx43) expression is responsible for enhanced fibrosis in the remodeled heart, resulting in an arrhythmogenic substrate. Therefore, we investigated the effect of normal or reduced Cx43 expression on the formation of fibrosis in a physiological (aging) and pathophysiological (transverse aortic constriction [TAC]) mouse model. Methods and Results— The Cx43fl/fl and Cx43CreER(T)/fl mice were aged 18 to 21 months or, at the age of 3 months, either TAC or sham operated and euthanized after 16 weeks. Epicardial activation mapping of the right and left ventricles was performed on Langendorff perfused hearts. Sustained ventricular arrhythmias were induced in 0 of 11 aged Cx43fl/fl and 10 of 15 Cx43Cre-ER(T)/fl mice (P<0.01). Cx43 expression was reduced by half in aged Cx43CreER(T)/fl compared with aged Cx43fl/fl mice, whereas collagen deposition was significantly increased from 1.1±0.2% to 7.4±1.3%. Aged Cx43CreER(T)/fl mice with arrhythmias had significantly higher levels of fibrosis and conduction heterogeneity than aged Cx43CreER(T)/fl mice without arrhythmias. The TAC operation significantly increased fibrosis in control compared with sham (4.0±1.2% versus 0.4±0.06%), but this increase was significantly higher in Cx43CreER(T)/fl mice (10.8±1.4%). Discoidin domain receptor 2 expression was unchanged, but procollagen peptide I and III expression and collagen type 1&agr;2 mRNA levels were higher in TAC–operated Cx43HZ mice. Conclusions— Reduced cellular coupling results in more excessive collagen deposition during aging or pressure overload in mice due to enhanced fibroblast activity, leading to increased conduction in homogeneity and proarrhythmia.