Immuno-Enzyme Histochemistry of Creatine Phosphokinase

1977 ◽  
Vol 35 ◽  
pp. 446-447
Author(s):  
N. Baba ◽  
E.T. Poe ◽  
J. Scillian ◽  
T. Myser
Keyword(s):  
Creatine Phosphokinase ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Left Ventricular Myocardium ◽  
Rabbit Muscle ◽  
Cytoplasmic Staining ◽  
Heart Muscle Cells ◽  
Diffuse Cytoplasmic Staining ◽  
Strong Staining

With a commercially available rabbit muscle (MM) type isoenzyme of the creatine phosphokinase (CPK) (Worthington Biochemical), anti-CPK antibody was produced in chicken and goat (Figure 1). Fab' fragments of the chicken and goat IgG were isolated and conjugated with a commercially available horseradish peroxidase (HRP) according to the Nakane method (J. Histochem. Cytochem,, 22:1084, 1974).Rabbit hearts were perfused in vivo with freshly prepared 2% paraformaldehyde solution, and the left ventricular myocardium was fixed for 30 minutes. Forty-micrometer-thick frozen sections of the myocardium were incubated with the Fab'-HRP conjugate overnight at 4°C. After a thorough rinse, the sections were stained for the peroxidase reaction.In an electron microscope, diffuse cytoplasmic staining of the heart muscle cells was noted, indicating the diffuse cytosol distribution of CPK (Figure 2). In addition, there was strong staining of the intermembranous and intracristal space of the mitochondria as well as the M-lines of the myofibrils (Figures 3 and 4).

β-blockade abolishes the augmented cardiac tPA release induced by transactivation of heterodimerised bradykinin receptor-2 and β2-adrenergic receptor in vivo

2014 ◽  
Vol 112 (11) ◽  
pp. 951-959 ◽  
Author(s):  
Morten Eriksen ◽  
Arnfinn Ilebekk ◽  
Alessandro Cataliotti ◽  
Cathrine Rein Carlson ◽  
Torstein Lyberg ◽  
...  
Keyword(s):  
Adrenergic Receptor ◽  
Sympathetic Nerves ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Left Ventricular Myocardium ◽  
Adrenergic Stimulation ◽  
Β2 Adrenergic Receptor ◽  
Β Blocker

SummaryBradykinin (BK) receptor-2 (B2R) and β2-adrenergic receptor (β2AR) have been shown to form heterodimers in vitro. However, in vivo proofs of the functional effects of B2R-β2AR heterodimerisation are missing. Both BK and adrenergic stimulation are known inducers of tPA release. Our goal was to demonstrate the existence of B2R-β2AR heterodimerisation in myocardium and to define its functional effect on cardiac release of tPA in vivo. We further investigated the effects of a non-selective β-blocker on this receptor interplay. To investigate functional effects of B2R-β2AR heterodimerisation (i. e. BK transactivation of β2AR) in vivo, we induced serial electrical stimulation of cardiac sympathetic nerves (SS) in normal pigs that underwent concomitant BK infusion. Both SS and BK alone induced increases in cardiac tPA release. Importantly, despite B2R desensitisation, simultaneous BK infusion and SS (BK+SS) was characterised by 2.3 ± 0.3-fold enhanced tPA release compared to SS alone. When β-blockade (propranolol) was introduced prior to BK+SS, tPA release was inhibited. A persistent B2R-β2AR heterodimer was confirmed in BK-stimulated and nonstimulated left ventricular myocardium by immunoprecipitation studies and under non-reducing gel conditions. All together, these results strongly suggest BK transactivation of β2AR leading to enhanced β2AR-mediated release of tPA. Importantly, non-selective β-blockade inhibits both SS-induced release of tPA and the functional effects of B2R-β2AR heterodimerisation in vivo, which may have important clinical implications.

In vivo detection of endogenous acetylcholine release in cat ventricles

1994 ◽  
Vol 266 (3) ◽  
pp. H854-H860 ◽  
Author(s):  
T. Akiyama ◽  
T. Yamazaki ◽  
I. Ninomiya
Keyword(s):  
Nerve Stimulation ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Vagal Nerve ◽  
Vagal Nerve Stimulation ◽  
Left Ventricular Myocardium ◽  
Ach Release ◽  
Vagal Nerves ◽  
Dialysis Technique

To detect and monitor endogenous acetylcholine (ACh) release in the in vivo heart, we applied a dialysis technique to the hearts of anesthetized cats. Dialysis probes were implanted in the left ventricular myocardium and were perfused with Krebs-Henseleit solution containing Eserine (10(-4) M) at 3 microliters/min. Dialysate ACh concentration was measured with high-performance liquid chromatography. In four cats, the response to vagal stimulation was studied. Electrical stimulation of efferent vagal nerves (10 Hz) significantly increased dialysate ACh concentration from 596 +/- 118 (control) to 12,210 +/- 1,661 pM. After stimulation, dialysate ACh concentration significantly decreased to 382 +/- 80 pM below control. The influence of ganglionic blocker was determined in six cats. Control vagal nerve stimulation (10 Hz) increased dialysate ACh concentration from 582 +/- 136 to 9,102 +/- 754 pM. Local perfusion of hexamethonium (10(-4) M) did not affect this nerve stimulation-induced ACh increase (8,611 +/- 1,189 pM), and intravenous administration of hexamethonium (20 mg/kg) prevented this increase (340 +/- 88 pM). We examined the response to vagal nerve stimulation at different frequencies in three cats. Vagal nerve stimulation increased dialysate ACh concentration from a control of 588 +/- 211 to 1,227 +/- 195 pM at 2 Hz, 3,946 +/- 1,059 pM at 5 Hz, and 9,366 +/- 1,873 pM at 10 Hz. Dialysate ACh concentration reflects ACh release from postganglionic vagal nerves innervating the left ventricular myocardium; the dialysis technique permits estimation of relative changes in efferent cardiac vagal nerve activity.

Impact of acute and enduring volume overload on mechanotransduction and cytoskeletal integrity of canine left ventricular myocardium

2007 ◽  
Vol 292 (5) ◽  
pp. H2324-H2332 ◽  
Author(s):  
Dirk W. Donker ◽  
Jos G. Maessen ◽  
Fons Verheyen ◽  
Frans C. Ramaekers ◽  
Roel L. H. M. G. Spätjens ◽  
...  
Keyword(s):  
Protein Expression ◽  
Volume Overload ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Left Ventricular Myocardium ◽  
Mechanical Stimuli ◽  
Hypertrophic Growth ◽  
Lim Protein ◽  
Ventricular Mechanics

It is poorly understood how mechanical stimuli influence in vivo myocardial remodeling during chronic hemodynamic overload. Combined quantitation of ventricular mechanics and expression of key proteins involved in mechanotransduction can improve fundamental understanding. Adult anesthetized dogs ( n = 20) were studied at sinus rhythm (SR) and 0, 3, 10, and 35 days of complete atrioventricular block (AVB). Serial left ventricular (LV) myofiber mechanics were measured. Repeated LV biopsies were analyzed for mRNA and/or protein expression of β1D-integrin, melusin, Akt, GSK3β, muscle LIM protein (MLP), four-and-a-half LIM protein 2 (fhl2), desmin, and calpain. Upon AVB, increased ejection strain (0.29 ± 0.01 vs. 0.13 ± 0.02, SR) and end-diastolic stress (4.8 ± 1.1 vs. 2.7 ± 0.4 kPa) dominated mechanical changes. Brain natriuretic peptide plasma levels were correspondingly high (33 ± 4 vs. 19 ± 1 pg/ml, SR). β1D-Integrin protein expression increased chronically after AVB. Melusin was temporarily overexpressed (+33 ± 9%, 3 days AVB vs. SR), followed by elevated ratios of phosphorylated (P)-Akt to Akt and P-GSK3β to GSK3β (+26 ± 6% and +30 ± 8% at 10 days AVB vs. SR). These changes corresponded to peak hypertrophic growth at 3 to 10 days. MLP increased gradually to maxima at chronic AVB (+36 ± 7%). In contrast, fhl2 (−22 ± 3%, 3 days) and desmin (−30 ± 9%, 10 days AVB) transiently declined but recovered at chronic AVB. Calpain protein expression remained unaltered. In conclusion, volume overload after AVB causes a transient compromise of cytoskeletal integrity based, at least partly, on transcriptional downregulation. Subsequent cytoskeletal reorganization coincides with the upregulation of melusin, P-Akt, P-GSK3β, and MLP, indicating a strong drive to compensated hypertrophy.

Abstract 36: Cardiac Progenitor Cell Lineage Tracing During Embryonic Cardiomyogenesis

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Bingyan J Wang ◽  
Alvin Muliono ◽  
Roberto Alvarez ◽  
Mark A Sussman
Keyword(s):  
Adoptive Transfer ◽  
Connexin 43 ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Lineage Tracing ◽  
Left Ventricular Myocardium ◽  
Post Injection ◽  
Term Survival

Introduction: Stem cell therapy represents great promises to myocardium regeneration. Multipotent c-Kit pos cardiac progenitor cells (CPCs) are able to differentiate into endothelial cells, smooth muscle cells, and cardiomyocytes. However, fundamental knowledge of CPC biology remains incomplete. Studies in rodent myocardial infarction model revealed that CPCs have poor long-term survival and engraftment after adoptive transfer, perhaps due to the severely damaged host environment. Therefore, it is critical to understand how CPCs interface with the recipient environment following transfer in order to enhance their true regenerative potentials. Hypothesis: Adoptively transferred stem cells are thought to survive and engraft best in an environment closely resembling their original habitat. Thus, we hypothesized that the embryonic environment provides the optimal spatiotemporal conditions to promote CPCs engraftment and commitment to cardiac fate. Methods: CPCs isolated from adult mouse hearts were expanded, fluorescence-tagged, and injected into blastocysts at E3.75 and in utero at E15.5. Embryos were analyzed following cardiogenesis by immunofluorescence for presence of CPC-derived tissues. Additionally, CPCs were injected intramyocardially at various stages from P0 to P7, to follow long-term adoptive transfer and assess CPCs lineage commitment. Results and Conclusions: At 48 hours post injection, donor CPCs were found anchoring in blastocoel and trophoblasts at E5.5, and were detected within the host myocardium at E17.5 predominantly at perivascular regions (n=4). Interestingly, CPCs also integrated into aminochorionic sac, indicating a novel non-cardiogenic fate of CPCs (n=5). CPCs injected at P3 stably engrafted into left ventricular myocardium by 14 days post injection (n=4), sharing gap junction proteins (ZO-1, Connexin-43) with neighboring cells. In conclusion, this study provides vivid evidence for the first time of CPC engraftment and survival in vivo under homeostasis during cardiogenesis. Future studies will assess the permissive environmental conditions, which may optimize their use in therapeutic applications, and the cardiogenic potential of CPCs in order to provide fundamental insights on CPCs biology.

scholarly journals Noninvasive measurements of transmural myocardial metabolites using 3-D 31P NMR spectroscopy

2001 ◽  
Vol 280 (1) ◽  
pp. H489-H497 ◽  
Author(s):  
Yong K. Cho ◽  
Hellmut Merkle ◽  
Jianyi Zhang ◽  
Nikolaos V. Tsekos ◽  
Robert J. Bache ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Left Ventricular Myocardium ◽  
31P Nmr Spectroscopy ◽  
Phase Encoding ◽  
Noninvasive Measurements ◽  
Magnetic Field Magnitude

A completely noninvasive three-dimensional (3-D) static magnetic field magnitude spatially localized 31P spectroscopy technique has been developed and applied to study the in vivo canine myocardium at 9.4 T. The technique incorporates both Fourier series windows and selective Fourier transform methods utilizing all three orthogonal gradients for 3-D phase encoding. The number of data acquisitions for each phase-encoding step was weighted according to the Fourier coefficients to define cylindrical voxels. Spatially localized 31P spectra can be generated for voxels of desired location within the field of view as a postprocessing step. The quality of localization was first demonstrated by using a three-compartment phantom. The technique was then applied to in vivo canine models and yielded 31P cardiac spectra with an excellent signal-to-noise ratio. The in vivo validation experiments, using an implanted 2-phosphoenolpyruvate-containing marker, demonstrated that the technique is capable of measuring at least two transmural layers of left ventricular myocardium representing the subepicardium and subendocardium.

scholarly journals Effects of Telmisartan on Myocardial Protein Profiles in Hypertensive Rats

2021 ◽  
Vol 34 (3) ◽  
pp. 299-299
Author(s):  
Yu Feng ◽  
Man-li Zhou ◽  
Jian-zhang Wang ◽  
Jia-qi Zhang ◽  
Shu-le Qian ◽  
...  
Keyword(s):  
Heat Shock ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Left Ventricular Myocardium ◽  
Sterile Water ◽  
Protein Profiles ◽  
Related Protein ◽  
Hypertensive Rats ◽  
Treatment Groups ◽  
Proteasome 26S

Abstract Background To investigate the effects of telmisartan on the protein profiles of the left ventricular myocardium in spontaneously hypertensive rats (SHR). Methods Sixteen SHR were randomly divided into control and telmisartan treatment groups. Rats were treated with sterile water (10 ml/kg) or telmisartan (4.33 mg/kg) by gavage for 12 weeks. Wistar-Kyoto (WKY) rats treated with sterile water (10 ml/kg) as controls. At the end of 12 weeks of control or telmisartan treatment, rats were sacrificed, and hearts were collected for protein preparations, isotope labeling, and mass spectrometric analysis. Results In total, there were 23 differentially expressed proteins in the left ventricular myocardium between control and telmisartan treatment groups in SHR. Compared with the telmisartan group, the upregulated proteins in the SHR were dual-specificity mitogen-activated protein kinase kinase 3-like, transgelin, and haptoglobin subtype 2. The downregulated proteins in the SHR were as follows: von Willebrand factor (fragment), kininogen 1, small ribonucleoprotein-related protein, fibrinogen beta chain, protein mass 3 (fragment), proteasome 26s, heat shock protein 27-related protein 1, tenascin X, fibronectin subtype 2, transferrin receptor protein, platelets 1, cathepsin L1, complement factor B, isoform CRA_b, fibrinogen isomer, immunoglobulin heavy chain (γ polypeptide), and α 1 antiprotease. Conclusions Telmisartan differentially regulates myocardial protein expression in hypertensive rats including heat shock protein 27, fibrinogen, fibronectin, proteasome 26s and transgelin, as well as proteins in biochemical, metabolic, and signal transduction pathways. These changes in protein expression may contribute to the antihypertrophic effects of telmisartan in hypertension.

scholarly journals Remodeling of t-system and proteins underlying excitation-contraction coupling in aging versus failing human heart

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yankun Lyu ◽  
Vipin K. Verma ◽  
Younjee Lee ◽  
Iosif Taleb ◽  
Rachit Badolia ◽  
...  
Keyword(s):  
Heart Function ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Left Ventricular Myocardium ◽  
Excitation Contraction Coupling ◽  
Transverse Tubular System ◽  
Contraction Coupling ◽  
Senescent Phenotype ◽  
Cellular Microstructure ◽  
T System

AbstractIt is well established that the aging heart progressively remodels towards a senescent phenotype, but alterations of cellular microstructure and their differences to chronic heart failure (HF) associated remodeling remain ill-defined. Here, we show that the transverse tubular system (t-system) and proteins underlying excitation-contraction coupling in cardiomyocytes are characteristically remodeled with age. We shed light on mechanisms of this remodeling and identified similarities and differences to chronic HF. Using left ventricular myocardium from donors and HF patients with ages between 19 and 75 years, we established a library of 3D reconstructions of the t-system as well as ryanodine receptor (RyR) and junctophilin 2 (JPH2) clusters. Aging was characterized by t-system alterations and sarcolemmal dissociation of RyR clusters. This remodeling was less pronounced than in HF and accompanied by major alterations of JPH2 arrangement. Our study indicates that targeting sarcolemmal association of JPH2 might ameliorate age-associated deficiencies of heart function.

scholarly journals Myxomatous Mitral Valve Disease with Mitral Valve Prolapse and Mitral Annular Disjunction: Clinical and Functional Significance of the Coincidence

2021 ◽  
Vol 8 (2) ◽  
pp. 9
Author(s):  
Nina C. Wunderlich ◽  
Siew Yen Ho ◽  
Nir Flint ◽  
Robert J. Siegel
Keyword(s):  
Mitral Valve ◽  
Mitral Valve Disease ◽  
Morphological Changes ◽  
Morphological Characteristics ◽  
Mitral Annulus ◽  
Ventricular Myocardium ◽  
Left Ventricular ◽  
Valve Disease ◽  
Left Ventricular Myocardium ◽  
Basal Portion

The morphological changes that occur in myxomatous mitral valve disease (MMVD) involve various components, ultimately leading to the impairment of mitral valve (MV) function. In this context, intrinsic mitral annular abnormalities are increasingly recognized, such as a mitral annular disjunction (MAD), a specific anatomical abnormality whereby there is a distinct separation between the mitral annulus and the left atrial wall and the basal portion of the posterolateral left ventricular myocardium. In recent years, several studies have suggested that MAD contributes to myxomatous degeneration of the mitral leaflets, and there is growing evidence that MAD is associated with ventricular arrhythmias and sudden cardiac death. In this review, the morphological characteristics of MAD and imaging tools for diagnosis will be described, and the clinical and functional aspects of the coincidence of MAD and myxomatous MVP will be discussed.

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