Depressed heart rate variability and arterial baroreflex in conscious transgenic mice with overexpression of cardiac Gsalpha

Using a continuous recognition memory procedure for spatial location information, rats were given sequential presentation of individual arms on a 12-arm maze. Each arm contained a Froot Loop reinforcement the first time it was presented, and latency to traverse the arm was measured. A subset of the arms were repeated, but did not contain reinforcement. Repeated arms were presented with lags ranging from zero to six (from zero to six different arm presentations occurred between the first and repeated presentation). After completion of acquisition training (significantly longer latencies for repeated arms in comparison with the first presentation of an arm), rats received lesions of the medial or lateral entorhinal cortex, pre- and parasubiculum, or served as sham-operated controls. Based on continued postsurgery training and additional tests, the results indicated that rats with pre- and parasubiculum or pre- and parasubiculum plus medial entorhinal cortex produced sustained impairment in performing the task. Medial or lateral entorhinal cortex and control lesions did not display any sustained deficits. The data suggest that working memory for spatial location information is mediated primarily by the pre- and parasubiculum, but not medial entorhinal and lateral entorhinal cortex.     

Dominant-negative effect of a mutant cardiac troponin T on cardiac structure and function in transgenic mice

Hypertrophic cardiomyopathy (HCM) is a disease of sarcomeric proteins. The mechanism by which mutant sarcomeric proteins cause HCM is unknown. The leading hypothesis proposes that mutant sarcomeric proteins exert a dominant-negative effect on myocyte structure and function. To test this, we produced transgenic mice expressing low levels of normal or mutant human cardiac troponin T (cTnT). We constructed normal (cTnT-Arg92) and mutant (cTnT-Gln92) transgenes, driven by a murine cTnT promoter, and produced three normal and five mutant transgenic lines, which were identified by PCR and Southern blotting. Expression levels of the transgene proteins, detected using a specific antibody, ranged from 1 to 10% of the total cTnT pool. M-mode and Doppler echocardiography showed normal left ventricular dimensions and systolic function, but diastolic dysfunction in the mutant mice evidenced by a 50% reduction in the E/A ratio of mitral inflow velocities. Histological examination showed cardiac myocyte disarray in the mutant mice, which amounted to 1-15% of the total myocardium, and a twofold increase in the myocardial interstitial collagen content. Thus, the mutant cTnT-Gln92, responsible for human HCM, exerted a dominant-negative effect on cardiac structure and function leading to disarray, increased collagen synthesis, and diastolic dysfunction in transgenic mice.     

Mechanical stretch induces hypertrophic responses in cardiac myocytes of angiotensin II type 1a receptor knockout mice

Many lines of evidence have suggested that angiotensin II (AngII) plays an important role in the development of cardiac hypertrophy through AngII type 1 receptor (AT1). To determine whether AngII is indispensable for the development of mechanical stress-induced cardiac hypertrophy, we examined the activity of mitogen-activated protein kinase (MAPK) family and the expression of the c-fos gene as hypertrophic responses after stretching cultured cardiac myocytes of AT1a knockout (KO) mice. When cardiac myocytes were stretched by 20% for 10 min, extracellular signal-regulated protein kinases (ERKs) were strongly activated in KO cardiomyocytes as well as wild type (WT) myocytes. Both basal and stimulated levels of ERKs were higher in cardiomyocytes of KO mice than in those of WT mice. Activation of another member of the MAPK family, p38(MAPK), and expression of the c-fos gene were also induced by stretching cardiac myocytes of both types of mice. An AT1 antagonist attenuated stretch-induced activation of ERKs in WT cardiomyocytes but not in KO cardiomyocytes. Down-regulation of protein kinase C inhibited stretch-induced ERK activation in WT cardiomyocytes, whereas a broad spectrum tyrosine kinase inhibitor (genistein) and selective inhibitors of epidermal growth factor receptor (tyrphostin, AG1478, and B42) suppressed stretch-induced activation of ERKs in KO cardiac myocytes. Epidermal growth factor receptor was phosphorylated at tyrosine residues by stretching cardiac myocytes of KO mice. These results suggest that mechanical stretch could evoke hypertrophic responses in cardiac myocytes that lack the AT1 signaling pathway possibly through tyrosine kinase activation.     

Effects of total replacement of atrial myosin light chain-2 with the ventricular isoform in atrial myocytes of transgenic mice

BACKGROUND: In contrast to their well-known and critical role in excitation-contraction coupling of vascular smooth muscle, the effects of the myosin light chains on cardiomyocyte mechanics are poorly understood. Accordingly, we designed the present experiment to define the cardiac chamber-specific functional effects of the ventricular isoform of the regulatory myosin light chain (MLC2v). METHODS AND RESULTS: Postnatal transgenic cardiac-specific overexpression of MLC2v was achieved by use of the alpha-myosin heavy chain promoter. Enzymatically disaggregated atrial and ventricular mouse myocytes were field-stimulated at multiple frequencies, and mechanical properties and calcium kinetics were studied by use of video edge detection and FURA 2-AM, respectively. MLC2v overexpression resulted in complete replacement of the atrial with the ventricular isoform of the regulatory myosin light chain at the steady-state mRNA and protein levels in the atria of transgenic mice. Mechanical properties of transgenic atrial myocytes were enhanced to the level of ventricular myocytes of control animals in association with modest decreases in the amplitude of the calcium transient. CONCLUSIONS: MLC2v modulates chamber-specific contractility by enhanced calcium sensitivity and/or improved cross-bridge cycling of the thin and thick filaments of the cardiomyocyte.     

Reovirus-induced acute myocarditis in mice correlates with viral RNA synthesis rather than generation of infectious virus in cardiac myocytes

The capacity for different reovirus reassortant viruses to induce acute myocarditis in mice correlates with cytopathogenic effect in primary cultures of murine cardiac myocytes. Multiple viral genes encoding proteins involved in viral RNA synthesis are determinants of this disease. We therefore evaluated the role of viral RNA synthesis in induction of acute myocarditis by infecting primary cultures of cardiac myocytes with a panel of myocarditic and nonmyocarditic viruses and quantitating RNA synthesis. RNA synthesis correlated with induction of myocarditis and with the S1 and M1 reovirus genes. Since one consequence of viral RNA synthesis is generation of infectious virus, we looked next at viral yield from cardiac myocyte cultures. Yield of infectious virus at an early time postinfection or as a final yield from primary infections did not correlate with myocarditis, but instead both correlated with the S1 gene. The S1 gene also determined the fraction of cells infected during primary infections in the culture, which varied dramatically between viruses. Viral yields per infected cell were similar for most myocarditic and nonmyocarditic reoviruses and did not correlate with induction of myocarditis or any reovirus gene. Together, the data provide two insights into reovirus-induced acute myocarditis in mice. First, while the S1 gene. which encodes the viral attachment protein sigma1 (as well as a nonstructural protein, sigma1s, of unknown function) does not determine the myocarditic potential of these viruses, it does determine the efficiency with which they infect cardiac myocytes. Second, while viral RNA synthesis is a determinant of acute myocarditis, this is not due to generation of infectious virus. This finding suggests that some other consequence of viral RNA synthesis, for example, induction of interferon, may determine reovirus-induced acute myocarditis.     

Fusigenic liposome-mediated DNA transfer into cardiac myocytes

Current methods of gene transfer into cultured cardiac myocytes have serious limitations, including low efficiency, toxicity or constraints on DNA insert size. The present study examined the effectiveness of hemagglutinating virus of Japan (HVJ) in promoting liposome-mediated DNA transfer into cultured neonatal rat cardiac myocytes. Fluorescein isothiocyanate-labeled oligonucleotides (F-ODN) or plasmid expression vectors encoding SV40 large T antigen (pActSVT) and beta-galactosidase (pAct beta-gal) were complexed with liposomes and the viral protein coat of HVJ. Plasmid vectors were complexed with the nuclear localizing protein HMG-1 prior to HVJ-liposome encapsulation. Neonatal myocytes were transfected by incubation with HVJ-liposome/DNA complexes on culture day 3 or 7. Using F-ODN, we were able to demonstrate significant uptake of DNA (transfection efficiencies of 80-90%) 1 h after transfection that persisted for 1 week in culture. Interestingly, F-ODN were concentrated in the myocyte nuclei for the first 4 days after transfection. Immunohistochemistry showed that 25-30% of myocytes transfected with either pActSVT or pAct beta-Gal expressed plasmid-encoded protein at 72 h whether they were transfected at day 3 or day 7 of culture, while cells transfected with blank vectors did not. Quantitative beta-galactosidase assays confirmed that the use of HVJ significantly enhanced liposome-mediated transfection. Cell toxicity was not apparent. Gene transfer via intracoronary injection also demonstrated the capacity of HVJ to mediate transfection of rabbit cardiac myocytes in vivo, with F-ODN-dependent fluorescence persisting for up to 1 week. We conclude that HVJ/liposome-mediated transfer is efficient for the transfection of both oligonucleotides and plasmids into cardiac myocytes both in vitro and in vivo, and may provide a new tool for the investigation of cardiac myocyte biology and disease.     

Cardiac-specific overexpression of mouse cardiac calsequestrin is associated with depressed cardiovascular function and hypertrophy in transgenic mice

Calsequestrin is a high capacity Ca2+-binding protein in the sarcoplasmic reticulum (SR) lumen. To elucidate the functional role of calsequestrin in vivo, transgenic mice were generated that overexpressed mouse cardiac calsequestrin in the heart. Overexpression (20-fold) of calsequestrin was associated with cardiac hypertrophy and induction of a fetal gene expression program. Isolated transgenic cardiomyocytes exhibited diminished shortening fraction (46%), shortening rate (60%), and relengthening rate (60%). The Ca2+ transient amplitude was also depressed (45%), although the SR Ca2+ storage capacity was augmented, as suggested by caffeine application studies. These alterations were associated with a decrease in L-type Ca2+ current density and prolongation of this channel's inactivation kinetics without changes in Na+-Ca2+ exchanger current density. Furthermore, there were increases in protein levels of SR Ca2+-ATPase, phospholamban, and calreticulin and decreases in FKBP12, without alterations in ryanodine receptor, junctin, and triadin levels in transgenic hearts. Left ventricular function analysis in Langendorff perfused hearts and closed-chest anesthetized mice also indicated depressed rates of contraction and relaxation of transgenic hearts. These findings suggest that calsequestrin overexpression is associated with increases in SR Ca2+ capacity, but decreases in Ca2+-induced SR Ca2+ release, leading to depressed contractility in the mammalian heart.     

Mechanical strain suppresses inducible nitric-oxide synthase in cardiac myocytes

We investigated the effects of precisely controlled mechanical strain on nitric-oxide synthase activity in cultured neonatal rat cardiac myocytes. Incubation of cardiac myocytes for 24 h with 4 ng/ml interleukin-1beta and 100 units/ml interferon-gamma stimulated an increase in nitric oxide production, inducible nitric-oxide synthase (iNOS) mRNA, and iNOS protein. Mechanical strain suppressed nitric oxide production, iNOS mRNA, and iNOS protein stimulated by cytokines in an amplitude-dependent manner. Losartan (1 microM), an angiotensin II type 1 receptor antagonist, weakly inhibited the effect of strain, suggesting that paracrine angiotensin II is not the mediator of the strain effect. In addition, cycloheximide (10 microM), a protein synthesis inhibitor, inhibited the effect of strain by 46%. Transforming growth factor-beta (1 ng/ml) suppressed iNOS mRNA expression, but anti-transforming growth factor-beta antibody (30 microg/ml) did not block the effect of strain. In contrast, staurosporine (100 nM; a nonselective protein kinase inhibitor), calphostin C (1 microM; a selective protein kinase C inhibitor), and pretreatment with phorbol 12-myristate 13-acetate abolished the effect of strain. Genistein (100 microM), a tyrosine kinase inhibitor, partially inhibited the effect of strain. Thus, cyclic mechanical deformation suppresses cytokine-induced iNOS expression in cardiac myocytes, and this effect is mediated at least partially via activation of protein kinase C.     

Hypotonicity stimulates translocation of ICln in neonatal rat cardiac myocytes

When a total shoulder arthroplasty is performed, restoration of the anatomy to near normal is important in order to achieve a stable implant. So as not to sacrifice stability, it is not uncommon for soft tissues to be either over tightened or insufficiently released. This article analyzes the various factors to consider in order to obtain appropriate soft-tissue balancing for a successful total shoulder arthroplasty.     

Translocation of 60S ribosomal subunit in spreading cardiac myocytes

Cardiac myocytes in culture undergo considerable structural reorganization. The remodeling of the myofibrils and the nonmyofibrillar cytoskeleton that occurs in the spreading cardiac myocytes resembles the cellular features observed in the hypertrophying heart. In this study we examined the distribution of the large 60S ribosomal subunit in freshly isolated cardiac myocytes and during the course of attachment and spreading in culture. Initially, anti-60S immunolabeling was scattered widely throughout the sarcoplasm of the dissociated cardiac myocytes. After attachment to the substrate, the 60S ribosomal subunit attained wide sarcoplasmic localization before a sarcomere-related staining pattern appeared in the spreading cell. Double labeling experiments with alpha-actinin confirmed co-localization of the 60S ribosomal subunit with nascent and mature myofibrils. These findings demonstrate that translocation of the 60S ribosomal subunit coincides with the cytoskeletal reorganization taking place in these cells. Moreover, the close association between the myofibrils indicates a particular role for the ribosomes in maintenance and growth of the contractile apparatus. (J Histochem Cytochem 46:963-969, 1998)     

Islet regeneration in IFNgamma transgenic mice

The adult pancreas is a developmentally stable organ with limited mitotic activity. This minimal mitotic activity proves critical in insulin-dependent diabetes mellitus (IDDM) since the pathogenesis is characterized by a selective and permanent destruction of islet beta cells. The IFNgamma transgenic mouse model of islet regeneration elucidates the differentiation pathway involved in the regeneration of functional beta cells from ductal precursors and reveals the functional plasticity of the adult pancreas.     

Spark-to-wave transition: saltatory transmission of calcium waves in cardiac myocytes

In order to investigate a possible regional specificity of activation asymmetries of different parts of the cortex and the stability of these asymmetries across time and conditions, EEG was recorded during rest and stimulation conditions in a large sample of right-handed university students (n = 60). Recordings were made in two sessions (interval between sessions 2-4 weeks). In the first session two rest periods were conducted (interval 30 min) and one under stimulation. In the second session EEG was again recorded at rest. Reliability analyses show that the long-term stability of orbitofrontal asymmetries is markedly lower than that of dorsolateral, temporal, and parietal scores. Intercorrelations of EEG asymmetries at different electrode positions suggest that anterior and posterior EEG asymmetries are largely independent measures of cortical laterality. Both this partial independence of activation asymmetries and the differences in temporal stability underscore the significance of the anterior-posterior dimension in laterality research and may be one reason for several contradictory observations in studies on brain laterality.     

Extent of radial sarcomere coupling revealed in passively stretched cardiac myocytes

We report a simple method, the PinPoint assay, for detecting and identifying single-base variations (polymorphisms) at specific locations within DNA sequences. An oligonucleotide primer is annealed to the target DNA immediately upstream of the polymorphic site and is extended by a single base in the presence of all four dideoxynucleotide triphosphates and a thermostable DNA polymerase. The extension products are desalted, concentrated, and subjected to delayed-extraction MALDI-TOF mass spectrometry. The base at the polymorphic site is identified by the mass added onto the primer. Heterozygous targets produce two mass-resolved species that represent the addition of both bases complementary to those at the polymorphic site. The assay is suitable for double-stranded PCR products without purification or strand separation. More than one primer can be simultaneously extended and then mass-analyzed. The mass spectrometric method thus shows promise for high-volume diagnostic or genotyping applications.     

Structural remodeling and mechanical dysfunction of cardiac myocytes in heart failure

End stage heart failure due to ischemic (ICM) or dilated (DCM) cardiomyopathy is characterized by a dilated, relatively thin-walled ventricle. The hypothesis has been proposed that the structural basis of ventricular expansion is due to side-to-side slippage of myocytes within the wall. Although this represents one potential mechanism for the observed phenomena of chamber dilatation and subsequent wall thinning, the degree of slippage claimed is not necessarily in harmony with the magnitude of chamber enlargement and mural thinning. Moreover, sarcomere extension was not examined in the base to the apical regions of the heart, leaving open the question as to the role of changes in resting sarcomere length in acute chamber dilatation. In this regard, an alternative etiology for the detrimental cardiac architectural rearrangement seen in dilated failure can be supplied by postulating the occurrence of maladaptive remodeling of cardiac myocyte morphology. In this model, myocytes increase in length by an increase in the number of sarcomeres in series, thus increasing chamber diameter in an attempt to maintain cardiac output. However, these cells do not enlarge to any significant degree in the transverse diameter preventing the heart from developing adequate force. This hypothesis is supported by recent evidence from patients with ICM and DCM indicating that myocyte lengthening alone could account for all the dilatation observed. Furthermore, it appears that the thinning of the ventricular wall in failure is due to inadequate transverse growth of cardiac myocytes coupled with scattered myocyte cell loss throughout the ventricular wall.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active signaling by Neu in transgenic mice

Transgenic mice engineered to overexpress the HER-2/neu/erbB-2 protooncogene under the control of a mammary-specific promoter develop mammary tumors and are a model for human breast cancer. Signal transduction by Neu was examined in situ in the tumors of these transgenic mice. This was accomplished using the PN2A monoclonal antibody, which recognizes Neu only in the phosphorylated, and therefore actively signaling, state. Immunohistochemistry using PN2A demonstrated that Neu actively signals in the tumors of Neu transgenic mice. Expression of Neu was always accompanied by co-overexpression of the endogenous epidermal growth factor receptor. Qualitatively similar results were found in mammary tumors from mice bitransgenic for the neu and transforming growth factor-alpha genes (both driven by the mouse mammary tumor virus promoter). Early mammary lesions demonstrated distinctive patterns of Neu activation relative to expression levels. Overexpression and activation were separable both temporally and spatially. These results refine the multi-step model for the role of Neu in mammary neoplasia and establish phosphorylation-state specific antibodies as a powerful tool for investigating tumor progression.     

Osteochondrodysplasia occurring in transgenic mice expressing interferon-gamma

Arterial thrombi are primarily composed of platelets. Platelets are bound to injured endothelial cells, sub-endothelial matrices, and other platelets by a range of adhesive proteins. Some of these reactions are governed by shear forces. The role of adhesive proteins in the pathogenesis of arterial thrombosis is not fully understood. The aim of this study was to examine the involvement of von Willebrand factor (vWF), fibrinogen (Fg), and fibronectin (FN) in the formation of microvascular thrombi in vivo using a helium-neon laser-induced thrombosis method. Transmission electron microscopy demonstrated that laser irradiation resulted in platelet-rich thrombosis in arterioles and venules, and revealed that this occurred in the absence of endothelial denudation. The mean wall shear rates in mesenteric arterioles and venules were 641 +/- 40 and 280 +/- 20 s-1, respectively. Shear rates increased approximately fivefold in arterioles and tenfold in venules during the formation of occlusive thrombi. Antibody to vWF inhibited thrombosis in arterioles and venules. Antibodies to Fg and FN inhibited thrombosis in venules but not in arterioles. These results confirm that vWF, Fg and FN were involved in thrombogenesis in vivo and demonstrated that significantly higher shear rates were required for the reactions involving vWF than those involving either Fg or FN.     

Age-associated alterations in calcium current and its modulation in cardiac myocytes

Stimulatory effects of several types of adjuvants on secondary antibody response to inactivated Newcastle disease virus (iNDV) were examined in chickens. For this purpose, animals were primed with iNDV without adjuvant resulting in a low but significant antibody response, boosted with iNDV plus adjuvant 3 weeks later, and analysed for specific antibody titres in serum 3 weeks after the booster. Water-in-mineral oil emulsion (W/O) caused significant increase in antibody titres measured in an indirect enzyme-linked immunosorbent (ELISA), haemagglutination inhibition (HI), and virus neutralisation (VN) assay. The adjuvants tested included three oil-in-water emulsions (i.e. mineral oil-in-water, sulpholipo(SL)-Ficoll400/squalane-in-water and sulpholipo-cyclodextrin/squalane-in-water), three negatively-charged polymers with high molecular weight (i.e. polyacrylate, polystyrenesulphonate and sulpho(S)-Ficoll400) and two surface-active agents (i.e. dimethyldioctadecylammonium bromide (DDA) and Quil A). These adjuvants enhanced significantly the secondary immune response but none reached the titre obtained with W/O. Combinations of adjuvants with distinct physicochemical properties, i.e. polyacrylate and DDA revealed only slight, beneficial effects. We concluded that the various types of adjuvants tested can stimulate secondary immune responses in primed animals but that W/O is superior.     

Lessons from TGF-beta transgenic mice

Aberrant expression of TGF-beta and/or receptor/signaling function is present in a wide variety of disease processes. Overexpression of TGF-beta isoforms in transgenic mice using tissue-specific promoters has provided model systems to study the effects of increased activity of TGF-beta in the intact organism. We will review the pertinent features of some of these models, and discuss new insights provided by these studies into regulation and role of TGF-beta in health and disease.