Alice Zemljic-Harpf

Cardiac-Myocyte-Specific Excision of the Vinculin Gene Disrupts Cellular Junctions, Causing Sudden Death or Dilated Cardiomyopathy

ABSTRACT Vinculin is a ubiquitously expressed multiliganded protein that links the actin cytoskeleton to the cell membrane. In myocytes, it is localized in protein complexes which anchor the contractile apparatus to the sarcolemma. Its function in the myocardium remains poorly understood. Therefore, we developed a mouse model with cardiac-myocyte-specific inactivation of the vinculin (Vcl) gene by using Cre-loxP technology. Sudden death was found in 49% of the knockout (cVclKO) mice younger than 3 months of age despite preservation of contractile function. Conscious telemetry documented ventricular tachycardia as the cause of sudden death, while defective myocardial conduction was detected by optical mapping. cVclKO mice that survived through the vulnerable period of sudden death developed dilated cardiomyopathy and died before 6 months of age. Prior to the onset of cardiac dysfunction, ultrastructural analysis of cVclKO heart tissue showed abnormal adherens junctions with dissolution of the intercalated disc structure, expression of the junctional proteins cadherin and β1D integrin were reduced, and the gap junction protein connexin 43 was mislocalized to the lateral myocyte border. This is the first report of tissue-specific inactivation of the Vcl gene and shows that it is required for preservation of normal cell-cell and cell-matrix adhesive structures.

Vinculin–actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth

Vinculin functions as a molecular clutch that organizes leading edge F-actin, generates traction, and promotes focal adhesion formation and turnover but not adhesion growth.

Heterozygous Inactivation of the Vinculin Gene Predisposes to Stress-Induced Cardiomyopathy

Vinculin and its muscle splice variant metavinculin link focal adhesions and cell-to-cell contact sites to the actin cytoskeleton. We hypothesized that normal expression of vinculin isoforms would be essential for integrity of cardiomyocytes and preservation of normal cardiac function. We studied heterozygous vinculin knockout mice (Vin+/-) that develop and breed normally. The Vin+/- mice displayed: 1) a 58% reduction of vinculin and a 63% reduction of metavinculin protein levels versus wild-type littermates; 2) normal basal cardiac function and histology but abnormal electrocardiograms, intercalated disks, and ICD-related protein distribution; 3) increased mortality following acute hemodynamic stress imposed by transverse aortic constriction (TAC); 4) cardiac dysfunction by 6 weeks post-TAC; and 5) misalignment of alpha-actinin containing Z-lines and abnormal myocardial ultrastructure despite preserved cardiac function. Decreased expression of vinculin/metavinculin leads to abnormal myocyte structure without baseline physiological evidence of cardiac dysfunction. These structural changes predispose to stress-induced cardiomyopathy.

Vinculin directly binds zonula occludens-1 and is essential for stabilizing connexin-43-containing gap junctions in cardiac myocytes

ABSTRACT Vinculin (Vcl) links actin filaments to integrin- and cadherin-based cellular junctions. Zonula occludens-1 (ZO-1, also known as TJP1) binds connexin-43 (Cx43, also known as GJA1), cadherin and actin. Vcl and ZO-1 anchor the actin cytoskeleton to the sarcolemma. Given that loss of Vcl from cardiomyocytes causes maldistribution of Cx43 and predisposes cardiomyocyte-specific Vcl-knockout mice with preserved heart function to arrhythmia and sudden death, we hypothesized that Vcl and ZO-1 interact and that loss of this interaction destabilizes gap junctions. We found that Vcl, Cx43 and ZO-1 colocalized at the intercalated disc. Loss of cardiomyocyte Vcl caused parallel loss of ZO-1 from intercalated dics. Vcl co-immunoprecipitated Cx43 and ZO-1, and directly bound ZO-1 in yeast two-hybrid studies. Excision of the Vcl gene in neonatal mouse cardiomyocytes caused a reduction in the amount of Vcl mRNA transcript and protein expression leading to (1) decreased protein expression of Cx43, ZO-1, talin, and &bgr;1D-integrin, (2) reduced PI3K activation, (3) increased activation of Akt, Erk1 and Erk2, and (4) cardiomyocyte necrosis. In summary, this is the first study showing a direct interaction between Vcl and ZO-1 and illustrates how Vcl plays a crucial role in stabilizing gap junctions and myocyte integrity.

Determination of three‐dimensional ventricular strain distributions in gene‐targeted mice using tagged MRI

A model‐based method for calculating three‐dimensional (3D) cardiac wall strain distributions in the mouse has been developed and tested in a genetically engineered mouse model of dilated cardiomyopathy. Data from MR tagging and harmonic phase (HARP) tracking were used to measure material point displacements, and 3D Lagrangian strains were calculated throughout the entire left ventricle (LV) with a deformable parametric model. A mouse model where cardiomyocytes are specifically made deficient in vinculin (VclKO) were compared to wild‐type (WT) littermates. 3D strain analysis revealed differences in LV wall mechanics between WT and VclKO mice at 8 weeks of age when systolic function had just begun to decline. Most notably, end‐systolic radial strain and torsional shear were reduced in VclKO hearts which contributed to regional mechanical dysfunction. This study demonstrates the feasibility of using MRI tagging methods to detect alterations in 3D myocardial strain distributions in genetically engineered mouse models of cardiovascular disease. Magn Reson Med, 2010.

Vinculin and Talin: Focus on the Myocardium

Cardiomyopathy is a heart muscle disease caused by decreased contractility of the ventricles leading to heart failure and premature death. Multiple conditions like ischemic heart disease (atherosclerosis), hypertension, diabetes, viral infection, alcohol abuse, obesity and genetic mutations can lead to cardiomyopathy. Single gene mutations in sarcomeric proteins, Z-disk-associated proteins, membrane/associated proteins, intermediate filaments, calcium cycle proteins as well as in modifier genes have been linked to cardiomyopathy. Clinical practice guidelines have been formulated by the American Heart Association and the Heart Failure Association of America on how to genetically evaluate patients with cardiomyopathy. To illustrate the concept that alterations in genes cause cardiovascular disease, this review will focus on two membrane-associated proteins, vinculin and talin. We will discuss the general function of vinculin/metavinulin as well as talin1 and talin2, with emphasis on what is understood about their role in the cardiac myocyte and in whole heart.

Novel role for vinculin in ventricular myocyte mechanics and dysfunction.

Vinculin (Vcl) plays a key structural role in ventricular myocytes that, when disrupted, can lead to contractile dysfunction and dilated cardiomyopathy. To investigate the role of Vcl in myocyte and myocardial function, cardiomyocyte-specific Vcl knockout mice (cVclKO) and littermate control wild-type mice were studied with transmission electron microscopy (TEM) and in vivo magnetic resonance imaging (MRI) tagging before the onset of global ventricular dysfunction. MRI revealed significantly decreased systolic strains transverse to the myofiber axis in vivo, but no changes along the muscle fibers or in fiber tension in papillary muscles from heterozygous global Vcl null mice. Myofilament lattice spacing from TEM was significantly greater in cVclKO versus wild-type hearts fixed in the unloaded state. AFM in Vcl heterozygous null mouse myocytes showed a significant decrease in membrane cortical stiffness. A multiscale computational model of ventricular mechanics incorporating cross-bridge geometry and lattice mechanics showed that increased transverse systolic stiffness due to increased lattice spacing may explain the systolic wall strains associated with Vcl deficiency, before the onset of ventricular dysfunction. Loss of cardiac myocyte Vcl may decrease systolic transverse strains in vivo by decreasing membrane cortical tension, which decreases transverse compression of the lattice thereby increasing interfilament spacing and stress transverse to the myofibers.

What is vinculin needed for in platelets

Summary.  Background: Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F‐actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin’s function in platelets is unknown. Objective: To determine whether vinculin is required for the functions of platelets and their major integrin, αIIbβ3. Methods: The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4–Cre mice. Platelet and integrin functions were studied in vivo and ex vivo. Results: Vinculin was undetectable in platelets from Vcl fl/fl Cre+ mice, as determined by immunoblotting and fluorescence microscopy. Vinculin‐deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on αIIbβ3 with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin‐deficient platelets displayed normal agonist‐induced fibrinogen binding to αIIbβ3, aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin‐deficient platelets adhered to immobilized fibrinogen or collagen normally, under both static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin‐deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to those in wild‐type littermates in response to carotid artery injury with FeCl3. Conclusion: Despite promoting membrane cytoskeleton integrity when mechanical force is applied to αIIbβ3, vinculin is not required for the traditional functions of αIIbβ3 or the platelet actin cytoskeleton.

Long-term atorvastatin treatment leads to alterations in behavior, cognition, and hippocampal biochemistry.

Membrane/lipid rafts (MLR) are plasmalemmal microdomains that are essential for neuronal signaling and synaptic development/stabilization. Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in the biosynthesis of mevalonic, a precursor to cholesterol via the mevalonate pathway. Because there has been controversy over the effects of statins on neuronal and cognitive function, we investigated the impact of long-term atorvastatin treatment (5mg/kg/d for 7 months by oral gavage) on behavior, cognition, and brain biochemistry in mice. We hypothesized that long-term statin treatment would alter lipid rafts and cognitive function. Atorvastatin treatment resulted in behavioral deficits as measured in paradigms for basic exploration (open field activity) and cognitive function (Barnes maze, startle response) without impairment in global motor function (Rotor Rod). Furthermore, significant changes in MLR-associated proteins (syntaxin-1α and synaptophysin) and a global change of post-synaptic density protein-95 (PSD95) were observed. The observed decreases in the MLR-localized pre-synaptic vesicle proteins syntaxin-1α and synaptophysin suggest a molecular mechanism for the statin-associated impairment of cognitive function that was observed and that has been suggested by the clinical literature.

Electrophysiology and metabolism of caveolin-3-overexpressing mice

Caveolin-3 (Cav-3) plays a critical role in organizing signaling molecules and ion channels involved in cardiac conduction and metabolism. Mutations in Cav-3 are implicated in cardiac conduction abnormalities and myopathies. Additionally, cardiac-specific overexpression of Cav-3 (Cav-3 OE) is protective against ischemic and hypertensive injury, suggesting a potential role for Cav-3 in basal cardiac electrophysiology and metabolism involved in stress adaptation. We hypothesized that overexpression of Cav-3 may alter baseline cardiac conduction and metabolism. We examined: (1) ECG telemetry recordings at baseline and during pharmacological interventions, (2) ion channels involved in cardiac conduction with immunoblotting and computational modeling, and (3) baseline metabolism in Cav-3 OE and transgene-negative littermate control mice. Cav-3 OE mice had decreased heart rates, prolonged PR intervals, and shortened QTc intervals with no difference in activity compared to control mice. Dobutamine or propranolol did not cause significant changes between experimental groups in maximal (dobutamine) or minimal (propranolol) heart rate. Cav-3 OE mice had an overall lower chronotropic response to atropine. The expression of Kv1.4 and Kv4.3 channels, Nav1.5 channels, and connexin 43 were increased in Cav-3 OE mice. A computational model integrating the immunoblotting results indicated shortened action potential duration in Cav-3 OE mice linking the change in channel expression to the observed electrophysiology phenotype. Metabolic profiling showed no gross differences in VO2, VCO2, respiratory exchange ratio, heat generation, and feeding or drinking. In conclusion, Cav-3 OE mice have changes in ECG intervals, heart rates, and cardiac ion channel expression. These findings give novel mechanistic insights into previously reported Cav-3 dependent cardioprotection.