Effects of mechanical stress and carvedilol in lamin A/C-deficient dilated cardiomyopathy.

RATIONALE: Mutations in the LMNA gene, which encodes the nuclear lamina proteins lamin A and lamin C, are the most common cause of familial dilated cardiomyopathy (DCM). Mechanical stress-induced apoptosis has been proposed as the mechanism underpinning DCM in lamin A/C-deficient hearts, but supporting in vivo evidence has been lacking. OBJECTIVE: Our aim was to study interventions to modify mechanical stress in heterozygous Lmna knockout (Lmna(+/-)) mice. METHODS AND RESULTS: Cardiac structure and function were evaluated before and after exercise training, thoracic aortic constriction, and carvedilol treatment. Lmna(+/-) mice develop adult-onset DCM with relatively more severe disease in males. Lmna(+/-) cardiomyocytes show altered nuclear morphology and perinuclear desmin organization, with enhanced responses to hypo-osmotic stress indicative of cytoskeletal instability. Despite these structural defects that provide a template for mechanical stress-induced damage, young Lmna(+/-) mice subjected to 6 weeks of moderate or strenuous exercise training did not show induction of apoptosis or accelerated DCM. In contrast, regular moderate exercise attenuated DCM development in male Lmna(+/-) mice. Sustained pressure overload generated by thoracic aortic constriction depressed ventricular contraction in young wild-type and Lmna(+/-) mice with no sex or genotype differences in the time-course or severity of response. Treatment of male Lmna(+/-) mice from 12 to 40 weeks with the beta-blocker, carvedilol, prevented the dilatation and contractile dysfunction that was observed in placebo-treated mice. CONCLUSIONS: These data suggest that factors other than mechanical stress-induced apoptosis contribute to DCM and provide the first demonstration that regular moderate exercise and carvedilol can modify disease progression in lamin A/C-deficient hearts.

Nesprin-1 and actin contribute to nuclear and cytoskeletal defects in lamin A/C-deficient cardiomyopathy.

Lamin A/C mutations are the most common cause of familial dilated cardiomyopathy (DCM) but the pathogenetic mechanisms are incompletely understood. Nesprins are spectrin repeat-containing proteins that interact with lamin A/C and are components of the linker-of-nucleoskeleton-and-cytoskeleton (LINC) complex that connects the nuclear envelope to the actin cytoskeleton. Our aim was to determine whether changes in nesprin-1 and actin might contribute to DCM in homozygous Lmna knockout (Lmna(-/-)) mice. Here we find that Lmna(-/-) cardiomyocytes have altered nuclear envelope morphology, disorganization of nesprin-1 and heterogeneity in the distribution of nuclear and cytoskeletal actin. Functional interactions of nesprin-1 with nuclear G-actin and with the cytoskeletal γ-actin, α-cardiac actin and α-smooth muscle actin (α-SMA) isoforms were shown by immunoprecipitation and Western blotting. At 4-6 weeks of age, Lmna(-/-) mice had normal levels of γ-actin and α-cardiac actin, but α-SMA expression was increased by 50%. In contrast to the predominant vascular distribution of α-SMA in WT ventricular sections, α-SMA had a diffuse staining pattern in Lmna(-/-) sections. Osmotic swelling studies showed enhanced radial swelling in Lmna(-/-) cardiomyocytes indicative of cytoskeletal instability. The distensibility of Lmna(-/-) cardiomyocytes with osmotic stress was reduced by addition of α-SMA-specific fusion peptide. Our findings support a model in which uncoupling of the nucleus and cytoskeleton associated with disruption of the LINC complex promotes mechanical instability and defective force transmission in cardiomyocytes. Changes in the distribution and expression patterns of nuclear and cytoskeletal actin suggest that diverse transcriptional and structural defects may also contribute to DCM in Lmna(-/-) mice.

Differential roles for ETS, CREB, and EGR binding sites in mediating VEGF receptor 1 expression in vivo.

Vascular endothelial growth factor receptor 1 (VEGFR1) is a marker for endothelial-specific gene expression. We previously reported that the human VEGFR1 promoter (between -748 and +284) contains information for expression in the intact endothelium of transgenic mice. The objective of this study was to dissect the cis-regulatory elements underlying VEGFR1 promoter activity in vitro and in vivo. In primary endothelial cells, binding sites for E74-like factor 1 (ELF-1; between -49 and -52), cyclic adenosine monophosphate response element binding (CREB; between -74 and -81), and early growth response factor 1/3 (EGR-1/3; between -16 to -25) were shown to play a positive role in gene transcription, whereas a putative E26 transformation-specificsequence (ETS) motif between -36 and -39 had a net negative effect on promoter activity. When targeted to the Hprt locus of mice, mutations of the ELF-1 binding site and the CRE element reduced promoter activity in the embryonic vasculature and resulted in a virtual loss of expression in adult endothelium. Postnatally, the EGR binding site mutant displayed significantly reduced promoter activity in a subset of vascular beds. In contrast, mutation of the -39 ETS site resulted in increased LacZ staining in multiple vascular beds. Together, these results provide new insights into the transcriptional regulatory mechanisms of VEGFR1.

Antiinflammatory effects of the ETS factor ERG in endothelial cells are mediated through transcriptional repression of the interleukin-8 gene.

ERG (Ets-related gene) is an ETS transcription factor that has recently been shown to regulate a number of endothelial cell (EC)-restricted genes including VE-cadherin, von Willebrand factor, endoglin, and intercellular adhesion molecule-2. Our preliminary data demonstrate that unlike other ETS factors, ERG exhibits a highly EC-restricted pattern of expression in cultured primary cells and several adult mouse tissues including the heart, lung, and brain. In response to inflammatory stimuli, such as tumor necrosis factor-alpha, we observed a marked reduction of ERG expression in ECs. To further define the role of ERG in the regulation of normal EC function, we used RNA interference to knock down ERG. Microarray analysis of RNA derived from ERG small interfering RNA- or tumor necrosis factor-alpha-treated human umbilical vein (HUV)ECs revealed significant overlap (P<0.01) in the genes that are up- or downregulated. Of particular interest to us was a significant change in expression of interleukin (IL)-8 at both protein and RNA levels. Exposure of ECs to tumor necrosis factor-alpha is known to be associated with increased neutrophil attachment. We observed that knockdown of ERG in HUVECs is similarly associated with increased neutrophil attachment compared to control small interfering RNA-treated cells. This enhanced adhesion could be blocked with IL-8 neutralizing or IL-8 receptor blocking antibodies. ERG can inhibit the activity of the IL-8 promoter in a dose dependent manner. Direct binding of ERG to the IL-8 promoter in ECs was confirmed by chromatin immunoprecipitation. In summary, our findings support a role for ERG in promoting antiinflammatory effects in ECs through repression of inflammatory genes such as IL-8.

Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, KCa1.1, in Sinus Node Function and Arrhythmia Risk.

BACKGROUND: KCNMA1 encodes the α-subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. METHODS: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of KCa1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. RESULTS: A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. CONCLUSIONS: Our data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that KCa1.1 loss of function may predispose to AF.

A GABP-binding element in the Robo4 promoter is necessary for endothelial expression in vivo.

We recently demonstrated that the 3-kb 5'-flanking region of the human ROBO4 gene directs endothelial cell-specific expression in vitro and in vivo. Moreover, a GA-binding protein (GABP)-binding motif at -119 was necessary for mediating promoter activity in vitro. The goal of the present study was to confirm the functional relevance of the -119 GABP-binding site in vivo. To that end, the Hprt locus of mice was targeted with a Robo4-LacZ transgenic cassette in which the GABP site was mutated. In other studies, the GABP mutation was introduced into the endogenous mouse Robo4 locus in which LacZ was knocked-in. Compared with their respective controls, the mutant promoters displayed a significant reduction in activity in embryoid bodies, embryos, and adult animals. Together, these data provide strong support for the role of the GABP-binding motif in mediating Robo4 expression in the intact endothelium.

ERG is required for the differentiation of embryonic stem cells along the endothelial lineage.

BACKGROUND: The molecular mechanisms that govern stem cell differentiation along the endothelial lineage remain largely unknown. Ets related gene (ERG) has recently been shown to participate in the transcriptional regulation of a number of endothelial specific genes including VE-cadherin (CD144), endoglin, and von Willebrand's Factor (vWF). The specific role of the ETS factor ERG during endothelial differentiation has not been evaluated. RESULTS: ERG expression and function were evaluated during the differentiation of embryonic stem cells into embryoid bodies (EB). The results of our study demonstrate that ERG is first expressed in a subpopulation of vascular endothelial growth factor receptor 2 (VEGF-R2) expressing cells that also express VE-cadherin. During ES cell differentiation, ERG expression remains restricted to cells of the endothelial lineage that eventually coalesce into primitive vascular structures within embryoid bodies. ERG also exhibits an endothelial cell (EC)-restricted pattern during embryogenesis. To further define the role of ERG during ES cell differentiation, we used a knockdown strategy to inhibit ERG expression. Delivery of three independent shRNA led to 70-85% reductions in ERG expression during ES cell differentiation compared to no change with control shRNA. ERG knockdown was associated with a marked reduction in the number of ECs, the expression of EC-restricted genes, and the formation of vascular structures. CONCLUSION: The ETS factor ERG appears to be a critical regulator of EC differentiation.

Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144) during endothelial differentiation.

BACKGROUND: Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation. METHODS: Mouse embryonic stem (ES) cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2+ cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2+ stem cells expressing the endothelial-specific marker CD144 (VE-cadherin) was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs) formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages. RESULTS: Microarray analysis of >45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2+, CD41+, and CD144+ and VEGF-R2-, CD41-, and CD144-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation. CONCLUSION: The isolation of CD144+ cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that are distinct from hematopoiesis.

Endocardial TRPC-6 Channels Act as Atrial Mechanosensors and Load-Dependent Modulators of Endocardial/Myocardial Cross-Talk.

Mechanoelectrical feedback may increase arrhythmia susceptibility, but the molecular mechanisms are incompletely understood. This study showed that mechanical stretch altered the localization, protein levels, and function of the cation-selective transient receptor potential channel (TRPC)-6 in atrial endocardial cells in humans, pigs, and mice. In endocardial/myocardial cross-talk studies, addition of media from porcine atrial endocardium (AE) cells altered the calcium (Ca2+) transient characteristics of human-induced pluripotent stem cell-derived cardiomyocytes. These changes did not occur with media from stretched AE cells. Our data suggested that endocardial TRPC-6-dependent paracrine signaling may modulate myocardial Ca2+ homeostasis under basal conditions and protect against stretch-induced atrial arrhythmias.

Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension.

Mechanosensitive ion channels are force-transducing enzymes that couple mechanical stimuli to ion flux. Understanding the gating mechanism of mechanosensitive channels is challenging because the stimulus seen by the channel reflects forces shared between the membrane, cytoskeleton and extracellular matrix. Here we examine whether the mechanosensitive channel PIEZO1 is activated by force-transmission through the bilayer. To achieve this, we generate HEK293 cell membrane blebs largely free of cytoskeleton. Using the bacterial channel MscL, we calibrate the bilayer tension demonstrating that activation of MscL in blebs is identical to that in reconstituted bilayers. Utilizing a novel PIEZO1-GFP fusion, we then show PIEZO1 is activated by bilayer tension in bleb membranes, gating at lower pressures indicative of removal of the cortical cytoskeleton and the mechanoprotection it provides. Thus, PIEZO1 channels must sense force directly transmitted through the bilayer.

Irreversible triggers for hypertrophic cardiomyopathy are established in the early postnatal period.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein genes, and left ventricular hypertrophy (LVH) develops as an adaptive response to sarcomere dysfunction. It remains unclear whether persistent expression of the mutant gene is required for LVH or whether early gene expression acts as an immutable inductive trigger. OBJECTIVES: The aim of this study was to use a regulatable murine model of HCM to study the reversibility of pathological LVH. METHODS: The authors generated a double-transgenic mouse model, tTAxαMHCR403Q, in which expression of the HCM-causing Arg403Gln mutation in the α-myosin heavy chain (MHC) gene is inhibited by doxycycline administration. Cardiac structure and function were evaluated in groups of mice that received doxycycline for varying periods from 0 to 40 weeks of age. RESULTS: Untreated tTAxαMHCR403Q mice showed increased left ventricular (LV) mass, contractile dysfunction, myofibrillar disarray, and fibrosis. In contrast, mice treated with doxycycline from conception to 6 weeks had markedly less LVH and fibrosis at 40 weeks. Transgene inhibition from 6 weeks reduced fibrosis but did not prevent LVH or functional changes. There were no differences in LV parameters at 40 weeks between mice with transgene inhibition from 20 weeks and mice with continuous transgene expression. CONCLUSIONS: These findings highlight the critical role of the early postnatal period in HCM pathogenesis and suggest that mutant sarcomeres manifest irreversible cardiomyocyte defects that induce LVH. In HCM, mutation-silencing therapies are likely to be ineffective for hypertrophy regression and would have to be administered very early in life to prevent hypertrophy development.

Regulation of murine cardiac contractility by activation of α(1A)-adrenergic receptor-operated Ca(2+) entry.

AIMS: Sympathetic regulation of cardiac contractility is mediated in part by α(1)-adrenergic receptors (ARs), and the α(1A)-subtype has been implicated in the pathogenesis of cardiac hypertrophy. However, little is known about α(1A)-AR signalling pathways in ventricular myocardium. The aim of this study was to determine the signalling pathway that mediates α(1A)-AR-coupled cardiac contractility. METHODS AND RESULTS: Using a transgenic model of enhanced cardiac α(1A)-AR expression and signalling (α(1A)-H mice), we identified a receptor-coupled signalling pathway that enhances Ca(2+) entry and increases contractility. This pathway involves α(1A)-AR-activated translocation of Snapin and the transient receptor potential canonical 6 (TRPC6) channel to the plasma membrane. In ventricular cardiomyocytes from α(1A)-H and their non-transgenic littermates (or WTs), stimulation with α(1A)-AR-specific agonists resulted in increased [Ca(2+)](i), which was dose-related and proportional to the level of α(1A)-AR expression. Blockade of TRPC6 inhibited the α(1A)-AR-mediated increase in [Ca(2+)](i) and contractility. External Ca(2+) entry, underlying the [Ca(2+)](i) increase, was not due to store-operated Ca(2+) entry but to a receptor-operated mechanism of Ca(2+) entry resulting from α(1A)-AR activation. CONCLUSION: These findings indicate that Ca(2+) entry via the α(1A)-AR-Snapin-TRPC6-pathway plays an important role in physiological regulation of cardiac contractility and may be an important target for augmenting cardiac performance.