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Biomedical subjects

John A McDonald

Publications and source records attributed to John A McDonald.

14 recordsLinked to original sources

Integrin activation in the heart: a link between electrical and contractile dysfunction?

Integrins mechanically link the cytoskeleton to the extracellular matrix in cardiac myocytes and are thereby involved in mechanotransduction. Integrins appear to be necessary for cardiac myocyte hypertrophy. To determine the effect of increased integrin ligation and signaling on adult cardiac function, a heart-specific truncated alpha(5) integrin (gain of function) was conditionally expressed in mice. Four days later, we observed an 80% reduction in amplitude of the QRS complex, profound systolic dysfunction, decreased connexin43, loss of gap junctions, and abnormal intercalated discs. Surprisingly, isolated left ventricular myocytes contracted normally and exhibited normal Ca(2+) transients. This suggested that cell/cell electrical and/or mechanical coupling was disrupted. To distinguish electrical from mechanical coupling deficits, we compared the papillary muscle force generated by electrically stimulated versus rapid cooling contractions in which intracellular Ca(2+) is released without electrical depolarization. Both were decreased in the transgenic muscle. However, electrically stimulated contractions were more significantly reduced than rapid cooling contractures. This suggests a component of cell/cell electrical uncoupling. Optical mapping revealed a loss of the normal elliptical isochronal activation pattern implying a loss of preferential conduction through gap junctions. For the first time, we have shown that integrins can regulate both mechanical and electrical coupling in the adult heart, even in the absence of primary hemodynamic alterations. Furthermore, we demonstrated that unregulated integrin activation leads to both contractile dysfunction and arrhythmias.

Animals↗

Cardiac-specific attenuation of natriuretic peptide A receptor activity accentuates adverse cardiac remodeling and mortality in response to pressure overload.

Atrial (ANP) and brain (BNP) natriuretic peptides are hormones of myocardial cell origin. These hormones bind to the natriuretic peptide A receptor (NPRA) throughout the body, stimulating cGMP production and playing a key role in blood pressure control. Because NPRA receptors are present on cardiomyocytes, we hypothesized that natriuretic peptides may have direct autocrine or paracrine effects on cardiomyocytes or adjacent cardiac cells. Because both natriuretic peptides and NPRA gene expression are upregulated in states of pressure overload, we speculated that the effects of the natriuretic peptides on cardiac structure and function would be most apparent after pressure overload. To attenuate cardiomyocyte NPRA activity, transgenic mice with cardiac specific expression of a dominant-negative (DN-NPRA) mutation (HCAT D 893A) in the NPRA receptor were created. Cardiac structure and function were assessed (avertin anesthesia) in the absence and presence of pressure overload produced by suprarenal aortic banding. In the absence of pressure overload, basal and BNP-stimulated guanylyl cyclase activity assessed in cardiac membrane fractions was reduced. However, systolic blood pressure, myocardial cGMP, log plasma ANP levels, and ventricular structure and function were similar in wild-type (WT-NPRA) and DN-NPRA mice. In the presence of pressure overload, myocardial cGMP levels were reduced, and ventricular hypertrophy, fibrosis, filling pressures, and mortality were increased in DN-NPRA compared with WT-NPRA mice. In addition to their hormonal effects, endogenous natriuretic peptides exert physiologically relevant autocrine and paracrine effects via cardiomyocyte NPRA receptors to modulate cardiac hypertrophy and fibrosis in response to pressure overload.

Animals↗

Effect of fiber orientation on propagation: electrical mapping of genetically altered mouse hearts.

BACKGROUND: Epicardial potentials reveal the strong effects of fiber anisotropy, rotation, imbrication, and coupling on propagation in the intact heart. From the patterns of the surface potentials, we can obtain information about the local fiber orientation, anisotropy, the transmural fiber rotation, and which direction the wave front is traveling through the wall. In this study, lessons learned from epicardial potential mapping of large hearts were applied to studies conducted in genetically altered mouse hearts. METHODS: An inducible model of the overexpression of a gain-of-function alpha5 integrin (cytoplasmic domain truncation) was created in mouse. After 3 days of administration of doxycycline, the animals exhibited an altered electrical phenotype of markedly reduced amplitude of the QRS complex on the surface electrocardiogram. Epicardial potentials were recorded from Langendorff-perfused mouse hearts with alpha5 integrin gain-of-function mutations and from wild-type (WT) control hearts. A cylindrical electrode array consisting of 184 sites with 1-mm uniform interelectrode spacing was placed around the heart, and unipolar electrograms were recorded during atrial and ventricular stimulation at different basic cycle lengths. RESULTS: The total ventricular activation time for the transgenic animals was greater than that of the WT hearts for atrial and ventricular pacing locations. The isopotential maps from the mutated hearts showed a loss of anisotropy, as revealed by the more rounded and less elliptically shaped wave fronts seen immediately after epicardial point stimulation when compared with WT hearts. The weaker potential maxima in the mutated hearts did not exhibit the normal expansion and rotation associated with an advancing wave front in a normal heart, suggesting abnormalities in myocyte coupling in these hearts. Isopotential maps provided additional information about fiber architecture from the electric field that was not obtained from optical recordings alone. These findings provided a phenotypic characterization and specific insights into the mechanisms of the electrical abnormalities associated with altered integrin signaling in cardiac myocytes.

Action Potentials↗

Doxycycline inducible expression of SERCA2a improves calcium handling and reverts cardiac dysfunction in pressure overload-induced cardiac hypertrophy.

Delayed cardiac relaxation in failing hearts has been attributed to reduced activity and/or expression of sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a). Although constitutive overexpression of SERCA2a has proven effective in preventing cardiac dysfunction, it is unclear whether increasing SERCA2a expression in hearts with preexisting hypertrophy will be therapeutic. To test this hypothesis, we generated a binary transgenic (BTG) system that allows tetracycline-inducible, cardiac-specific SERCA2a expression. In this system (tet-on SERCA2a), a FLAG-tagged SERCA2a transgene is expressed in the presence of doxycycline (Dox) but not in the absence of Dox (2.3-fold more mRNA, 45% more SERCA2a protein). Calcium transients measured in isolated cardiac myocytes from nonbanded Dox-treated BTG mice showed an accelerated calcium decline and an increased systolic Ca2+ peak. Sarcoplasmic reticulum (SR) calcium loading was increased by 45% in BTG mice. In the presence of pressure overload (aortic banding), echocardiographic analysis revealed that expression of SERCA2a-FLAG caused an improvement in fractional shortening. SERCA2a-FLAG expression alleviated the resultant cardiac dysfunction. This was illustrated by an increase in the rate of decline of the calcium transient. Cell shortening and SR calcium loading were also improved in cardiac myocytes isolated from banded BTG mice after SERCA2a overexpression. In conclusion, we generated a novel transgenic mouse that conditionally overexpresses SERCA2a. This model is suitable for both long- and short-term studies of the effects of controlled SERCA2a expression on cardiac function. In addition, inducible overexpression of SERCA2a improved cardiac function and calcium handling in mice with established contractile dysfunction.

Animals↗

Cardiac-specific induction of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha promotes mitochondrial biogenesis and reversible cardiomyopathy in a developmental stage-dependent manner.

Recent evidence has identified the peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) as a regulator of cardiac energy metabolism and mitochondrial biogenesis. We describe the development of a transgenic system that permits inducible, cardiac-specific overexpression of PGC-1alpha. Expression of the PGC-1alpha transgene in this system (tet-on PGC-1alpha) is cardiac-specific in the presence of doxycycline (dox) and is not leaky in the absence of dox. Overexpression of PGC-1alpha in tet-on PGC-1alpha mice during the neonatal stages leads to a dramatic increase in cardiac mitochondrial number and size coincident with upregulation of gene markers associated with mitochondrial biogenesis. In contrast, overexpression of PGC-1alpha in the hearts of adult mice leads to a modest increase in mitochondrial number, derangements of mitochondrial ultrastructure, and development of cardiomyopathy. The cardiomyopathy in adult tet-on PGC-1alpha mice is characterized by an increase in ventricular mass and chamber dilatation. Surprisingly, removal of dox and cessation of PGC-1alpha overexpression in adult mice results in complete reversal of cardiac dysfunction within 4 weeks. These results indicate that PGC-1alpha drives mitochondrial biogenesis in a developmental stage-dependent manner permissive during the neonatal period. This unique murine model should prove useful for the study of the molecular regulatory programs governing mitochondrial biogenesis and characterization of the relationship between mitochondrial dysfunction and cardiomyopathy and as a general model of inducible, reversible cardiomyopathy.

Adenosine Triphosphate↗

Monitoring change in the three-dimensional shape of the human left ventricle.

BACKGROUND: Characterizing left ventricular (LV) remodeling after myocardial infarction or LV shape change resulting from LV shape-restoration operation can yield valuable prognostic information. However, current methods measure only global parameters of LV shape. METHODS: We developed and validated a method for measuring change in regional LV shape by aligning a patient's follow-up 3-dimensional LV surface reconstruction to baseline surface. We tested the diagnostic power of 6 distance functions to detect a known shape deformation. To create the test data, the LV endocardial surface of a control subject was reconstructed using 3-dimensional echocardiographic techniques. The surface was deformed 9 different ways to model LV dilation (3 different locations and severities). Normal shape variability was defined from 18 serial studies of 6 control subjects. The severity of regional dilation was computed as the orthogonal distance between the aligned baseline and deformed LV surfaces. Deformation was quantified according to regional location using the 16-segment map of the LV. RESULTS: Normal LV shape variability was 3.38 mm. The LV deformations ranged from 2.95 to 8.02 mm. Gaussian distance function produced the highest accuracy for measuring deformation distances (P <.005 by analysis of variance). In addition, the gaussian function correctly identified the location of the maximum deformation in 6 of the 9 distorted surfaces. In the 3 remaining surfaces, the gaussian alignment selected an adjacent basal segment with a similar deformation distance (mean error: 0.2 +/- 0.17 mm). The gaussian function's accuracy in pinpointing the deformation equaled or exceeded the performance of the other 5 functions tested. CONCLUSION: This new method of aligning 3-dimensional LV surfaces in space facilitates detecting, measuring, and localizing regional shape change in the human LV independent of anatomic landmarks or geometric references. Potential applications include quantitative monitoring of change in regional LV shape after a pathologic process and/or surgical procedure to document efficacy of treatment and to assess prognosis.

Echocardiography, Three-Dimensional↗

VEGF modulates early heart valve formation.

Although hypoxic and/or nutritional insults during gestation are believed to contribute to congenital heart defects, the mechanisms responsible for these anomalies are not understood. Given the role vascular endothelial growth factor (VEGF) plays in response to hypoxia, it is a likely candidate for mediating deleterious effects of embryonic hypoxia. The ectopic or overproduction of endogenous factors such as VEGF may contribute to specific heart defects. Here we compared hypoxia-induced precocious production of VEGF during early heart valve development to normal VEGF production. Mouse prevalvular cardiac endocardial cushions were explanted onto hydrated type I collagen gels under normoxic or hypoxic conditions. The extent of transformation of cardiac endothelium into mesenchyme was inversely correlated with the levels of VEGF during the various culture conditions. A soluble VEGF antagonist confirmed that endogenous production of VEGF was specific for blocking normal cushion mesenchyme formation. We further demonstrated that E10.5 endocardium retains the ability to transform into cardiac mesenchyme in the absence of endogenous VEGF.

Animals↗

Hyaluronan synthases in normal and regenerating joint cartilage.

Repair of full thickness joint cartilage defects is within reach of routine clinical practice. The quality of regenerating hyaline cartilage, however, is difficult to assess. Synthesis of an extracellular matrix with high hyaluronan content is crucial for its metabolic and functional properties. We studied hyaluronan synthase (HAS) expression in knee joints of adult sheep as a novel cellular marker for chondrocyte function. Six house-bred Merino sheep (age 4-6 years) underwent two-stage surgery of their femoral condyles for autologous chondrocyte transplantation (ACT). First, cells were isolated from biopsies and expanded in vitro for 2 weeks using standard culture techniques. In a second session, three defects were made and either left untreated, covered with periosteal flap alone, or in combination with chondrocyte suspensions injected under the flaps. After 16 weeks, biopsies were taken from the operated knees at the defect sites and from the untreated condyle. Specimens were processed for safranin O and electron microscopy, and for immunofluorescence using three different polyclonal anti-HAS antibodies recognizing one or all of the three known mammalian HAS. Control and regenerating tissues were compared regarding their morphology and the expression of HAS, in relation to collagens type I and II, and adult cartilage proteoglycan core protein. In comparison with untreated defects or with periosteal flap alone, ACT provided a neocartilage with better-differentiated morphology. In healthy joint cartilage, about 50% of the chondrocytes expressed HAS, independent of antibody. Following ACT, a higher density of chondrocytes was observed, of which more than 75% expressed HAS, whereas the regenerates without ACT showed a lower density of HAS-expressing cells. We propose to use HAS immunofluorescence as an additional marker of matrix synthesis by chondrocytes and joint cartilage regeneration.

Aggrecans↗

Temporal and distinct TGFbeta ligand requirements during mouse and avian endocardial cushion morphogenesis.

The formation of endocardial cushions in the atrioventricular (AV) canal of the rudimentary heart requires epithelial-to-mesenchymal cell transformation (EMT). This is a complex developmental process regulated by multiple extracellular signals and transduction pathways. A collagen gel assay, long used to examine endocardial cushion development in avian models, is now being employed to investigate genetically engineered mouse models with abnormal heart morphogenesis. In this study, we determine interspecies variations for avian and mouse cultured endocardial cushion explants. Considering these observed morphologic differences, we also define the temporal requirements for TGFbeta2 and TGFbeta3 during mouse endocardial cushion morphogenesis. TGFbeta2 and TGFbeta3 blocking antibodies inhibit endothelial cell activation and transformation, respectively, in avian explants. In contrast, neutralizing TGFbeta2 inhibits cell transformation in the mouse, while TGFbeta3 antibodies have no effect on activation or transformation events. This functional requirement for TGFbeta2 is concomitant with expression of TGFbeta2, but not TGFbeta3, within mouse endocardial cushions at a time coincident with transformation. Thus, both TGFbeta2 and TGFbeta3 appear necessary for the full morphogenetic program of EMT in the chick, but only TGFbeta2 is expressed and obligatory for mammalian endocardial cushion cell transformation.

Actins↗

Heart-valve mesenchyme formation is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors.

Heart septation and valve malformations constitute the most common anatomical birth defects. These structures arise from the endocardial cushions within the atrioventricular canal (AVC) through dynamic interactions between cushion cells and the extracellular matrix (termed cardiac jelly). Transformation of endothelial cells to mesenchymal cells is essential for the proper development of the AVC and subsequent septation and valve formation. Atrioventricular septal defects can result from incomplete endocardial cushion morphogenesis. We show that hyaluronan-deficient AVC explants from Has2(-/-) embryos, which normally lack mesenchyme formation, are rescued by heregulin treatment, which restores phosphorylation of ErbB2 and ErbB3. These events were blocked using a soluble ErbB3 molecule, as well as with an inhibitor of ErbB2, herstatin. We show further that ErbB3 is activated during hyaluronan treatment of Has2(-/-) explants. These data provide a link between extracellular matrix-hyaluronan and ErbB receptor activation during development of early heart-valve and septal mesenchyme.

Animals↗

Three-dimensional echocardiographic measurement of left and right ventricular mass and volume: in vitro validation.

INTRODUCTION: Three-dimensional (3D) echocardiography has been shown to offer highly accurate measurements of left ventricular (LV) volume and mass. The present study evaluated the accuracy of 3D surface reconstruction by the piecewise smooth subdivision method in measuring volume and mass not only in the LV but also in the more complexly shaped right ventricle (RV). METHODS: 3D echo scans were obtained of in vitro LV's (n = 15) and RVs (n = 10). From digitized images, ventricular borders were traced and used in surface reconstructions. Mass and volume determined from the reconstructions were compared to true volume and mass determined prior to imaging. Additionally casts of two RVs were made and laser-scanned. Distances between the laser-identified points on the RV surface and the corresponding 3D echo reconstructions were measured. RESULTS: 3D LV volume agreed well with the true volume (y = 0.99x + 1.73, r = 0.99, SEE = 3.35 ml, p < 0.0001), as did 3D LV mass (y = 0.99x - 4.71, r = 0.99, SEE = 9.85 g, p < 0.0001). 3D RV volume overestimated true volume (y = 1.11x + 1.77, r = 0.99, SEE = 3.36 ml, p < 0.001) by 6.23+/-3.70 ml (p < 0.0001). 3D mass agreed well with RV mass (y = 0.78x + 17.32, r2 = 0.93, SEE = 3.54 g, p < 0.0001). 3D echo reconstructions matched the laser-scanned RV closely with residual distances of 1.1+/-0.9 and 1.4+/-1.2 mm, respectively. CONCLUSIONS: 3D echo using freehand scanning combined with surface reconstruction by the piecewise smooth subdivision surface method enables accurate determination of LV mass and volume, of RV mass and volume, and of the RV's complex shape.

Animals↗

A lethal perinatal cardiac phenotype resulting from altered integrin function in cardiomyocytes.

BACKGROUND: Integrins are heterodimeric receptors that couple the extracellular matrix to intracellular signaling pathways and the cyoskeleton. Integrins are strain transducers and candidates for modulators or effectors of cardiac hypertrophy. METHODS: To begin to probe this function, we have transgenically expressed a chimeric protein that alters integrin function in cardiomyocytes. The transgene (Tac-beta(1D)) consists of the biologically inert extracellular and transmembrane domain of the interleukin-2 receptor alpha subunit (Tac) fused to the cytoplasmic tail of the human beta(1D) integrin driven by the cardiac alpha-myosin heavy chain promoter. Transgene expression results in a severe, usually fatal, perinatal cardiac phenotype, characterized by initial electrocardiographic abnormalities followed by extensive myocyte loss, macrophage infiltration, and replacement fibrosis. RESULTS: Expression of Tac-beta(1D) resulted in displacement of endogenous beta(1D) integrin from Z-lines and T-tubules, decreased expression of endogenous beta(1D), and disrupted the fibronectin pericellular matrix. These results are consistent with an essential role for beta(1) integrins in maintenance of cardiomyocyte viability and interaction with extracellular matrix. CONCLUSION: The appearance of conduction abnormalities before morphologic changes suggests that integrins are important in the development or maintenance of the conducting system of the heart.

Animals↗

Method for three-dimensional data registration from disparate imaging modalities in the NOGA Myocardial Viability Trial.

Region-by-region comparison of data concerning left ventricular (LV) status is difficult to perform quantitatively if the data was acquired from disparate imaging modalities. We validated a method for comparing measurements obtained by electromechanical mapping (EMM) catheter with dobutamine stress echocardiography (DSE) via biplane contrast ventriculography, with the assistance of three-dimensional (3-D) echocardiographic data. The ventriculograms were traced and the borders were used to reconstruct the LV in 3-D with the aid of a database of 3-D echocardiographic studies. The 3-D LV was oriented to the EMM data based on the body coordinates and then manually scaled and translated to fit. The EMM data were mapped to the 3-D surface. The 3-D surface was divided into the 16 regions defined for echocardiographic assessment. The mean EMM value for local linear shortening, a parameter of function, was computed in each segment. The EMM and semiquantitative echocardiographic assessments of regional myocardial function were compared by segment, and the volume of the 3-D LV was compared with the volume computed from the ventriculogram. The volume of the 3-D surface correlated closely with that of the ventriculogram (r = 0.97, SEE = 27.4 ml) but with a significant overestimation of 63 +/- 35 ml. There was a highly significant (p < 0.0001) agreement in regional function between EMM and echo. Local linear shortening correlated significantly (p < 0.0001) with echocardiographic severity of wall motion, averaging 9.5 +/- 6.5, 8.1 +/- 5.4, 5.9 +/- 4.8, and 6.2 +/- 3.3 in segments read as normal, hypokinetic, akinetic, and dyskinetic, respectively. The method presented is valid for comparing cardiac parameters derived from disparate image data on a region-by-region basis by employing anatomic landmarks on 3-D reconstructions of the LV endocardial surface.

Body Surface Potential Mapping↗

Hyaluronan: genetic insights into the complex biology of a simple polysaccharide.

It is appropriate that this review should appear in a volume dedicated to Mert Bernfield. Much of my interest in the cell biology of the extracellular matrix, particularly during development, echoes Mert's pioneering studies. His kind but provocative questioning during meetings is especially missed. The glycosaminoglycan hyaluronan is ubiquitous, and is especially abundant during embryogenesis. Hydrated matrices rich in hyaluronan expand the extracellular space, facilitating cell migration. The viscoelastic properties of hyaluronan are also essential for proper function of cartilage and joints. Recent understanding of hyaluronan biology has benefited from the identification of genes encoding hyaluronan synthases and hyaluronidases, genetic analysis of the roles of hyaluronan during development, elucidation of the biochemical mechanisms of hyaluronan synthesis, and by studies of human genetics and tumors. This review focuses on recent studies utilizing hyaluronan-deficient, gene targeted mice with null alleles for the principal source of hyaluronan during mid-gestation, hyaluronan synthase-2 (has-2).

Animals↗