Organization Of Cardiomyocytes Research Articles

Regulation of the Cell Surface Expression and Function of Angiotensin II Type 1 Receptor by Rab1-mediated Endoplasmic Reticulum-to-Golgi Transport in Cardiac Myocytes

Rab1 GTPase coordinates vesicle-mediated protein transport specifically from the endoplasmic reticulum (ER) to the Golgi apparatus. We recently demonstrated that Rab1 is involved in the export of angiotensin II (Ang II) type 1 receptor (AT1R) to the cell surface in HEK293 cells and that transgenic mice overexpressing Rab1 in the myocardium develop cardiac hypertrophy. To expand these studies, we determined in this report whether the modification of Rab1-mediated ER-to-Golgi transport can alter the cell surface expression and function of endogenous AT1R and AT1R-mediated hypertrophic growth in primary cultures of neonatal rat ventricular myocytes. Adenovirus-mediated gene transfer of wild-type Rab1 (Rab1WT) significantly increased cell surface expression of endogenous AT1R in neonatal cardiomyocytes, whereas the dominant-negative mutant Rab1N124I had the opposite effect. Brefeldin A treatment blocked the Rab1WT-induced increase in AT1R cell surface expression. Fluorescence analysis of the subcellular localization of AT1R revealed that Rab1 regulated AT1R transport specifically from the ER to the Golgi in HL-1 cardiomyocytes. Consistent with their effects on AT1R export, Rab1WT and Rab1N124I differentially modified the AT1R-mediated activation of ERK1/2 and its upstream kinase MEK1. More importantly, adenovirus-mediated expression of Rab1N124I markedly attenuated the Ang II-stimulated hypertrophic growth as measured by protein synthesis, cell size, and sarcomeric organization in neonatal cardiomyocytes. In contrast, Rab1WT expression augmented the Ang II-mediated hypertrophic response. These data strongly indicate that AT1R function in cardiomyocytes can be modulated through manipulating AT1R traffic from the ER to the Golgi and provide the first evidence implicating the ER-to-Golgi transport as a regulatory site for control of cardiomyocyte growth.

Mutations in the motor domain modulate myosin activity and myofibril organization.

We have investigated the functional impact on cardiac myofibril organization and myosin motor activity of point mutations associated with familial hypertrophic cardiomyopathies (FHC). Embryonic chicken cardiomyocytes were transfected with vectors encoding green fluorescent protein (GFP) fused to a striated muscle myosin heavy chain (GFP-myosin). Within 24 hours of transfection, the GFP-myosin is found co-assembled with the endogenous myosin in striated myofibrils. The wild-type GFP-myosin had no effect on the organization of the contractile cytoskeleton of the cardiomyocytes. However, expression of myosin with the R403Q FHC mutation resulted in a small but significant decrease in myofibril organization, and the R453C and G584R mutations caused a more dramatic increase in myofibril disarray. The embryonic cardiomyocytes beat spontaneously in culture and this was not affected by expression of the wild-type or mutant GFP-myosin. For the biochemical analysis of myosin motor activity, replication defective adenovirus was used to express the wild-type and mutant GFP-myosin in C2C12 myotubes. The R403Q mutation enhanced actin filament velocity but had no effect on the myosin duty ratio. The R453C and G584R mutations impaired actin filament movement and both increased the duty ratio. The effects of these mutations on myosin motor activity correlate with changes in myofibril organization of live cardiomyocytes. Thus, mutations associated with hypertrophic cardiomyopathies that alter myosin motor activity can also impair myofibril organization.

Nkx2.5 homeoprotein regulates expression of gap junction protein connexin 43 and sarcomere organization in postnatal cardiomyocytes

Nkx2.5, an evolutionarily conserved homeodomain containing transcription factor, is one of the earliest cardiogenic markers. Although its expression continues through adulthood, its function in adult cardiomyocytes is not well understood. To examine the effect of Nkx2.5 in terminal differentiated postnatal cardiomyocytes, we generated transgenic mice expressing either wild-type Nkx2.5 (TG-wild), a putative transcriptionally active mutant (carboxyl-terminus deletion mutant: TG-ΔC) or a DNA non-binding point mutant of Nkx2.5 (TG-I183P) under α-myosin heavy chain promoter. Most TG-wild and TG-ΔC mice died before 4 months of age with heart failure associated with conduction abnormalities. Cardiomyocytes expressing wild-type Nkx2.5 or a putative transcriptionally active mutant (ΔC) had dramatically reduced expression of connexin 43 and changed sarcomere structure. Wild-type Nkx2.5 adenovirus-infected adult cardiomyocytes demonstrated connexin 43 downregulation as early as 16 h after infection, indicating that connexin 43 downregulation is due to Nkx2.5 overexpression but not due to heart failure phenotype in vivo. These studies indicate that overexpression of Nkx2.5 in terminally differentiated cardiomyocytes dramatically alters cardiac cell structure and function.

Cardiotoxic effects of the antiarrhythmic rhythmidazol and their correction by suphan, befol, and their combinations

The antiarrhythmic rhythmidazol produces a cardiotoxic effect that can be corrected by suphan, befol, and their combinations, as evidenced by normalization of ultrastructural organization of cardiomyocytes and myocardial oxygen consumption by these drugs.

SEM/TEM investigation of rat cardiac subcellular alterations induced by changing duration of noise stress.

Many studies have shown that loud noise is an environmental stressor. Noise exposure causes changes at different levels in living beings; the cardiovascular system, considered to be the most affected apparatus, has received much attention in both animals and humans. The present study investigated the effect of changing duration of noise stimulation on rat myocardium to determine whether the responses could be related to time. The study was performed on young albino male rats. Three groups of animals were exposed to white noise (100 dBA) for 1, 6, and 12 hr, respectively, and samples of right atrium and ventricle were processed for ultrastructural examination. The subcellular organization of cardiomyocytes was examined by transmission and scanning electron microscopy, the latter being carried out with the modified osmium maceration method. After 1 hr of noise exposure, atrial and ventricular cardiomyocytes did not present significant alterations. After 6 hr, changes were observed in atrial tissue at the mitochondrial level, whereas the ventricular structure appeared unaffected. After 12 hr of treatment, in addition to the mitochondrial alterations, changes in atrial and ventricular myocardium, consisting of areas of enlargement in intercalated disc membranes and decreased density in sarcoplasm, were detected. The methods we applied for the visualization of cellular organelles allowed us to demonstrate that atrial tissue is primarily involved in stress response, especially at the mitochondrial level; a more prolonged time of exposure seems to be necessary for ventricular involvement.

Isolation and preparation of single mouse cardiomyocytes for confocal microscopy

Myofibril degeneration is a common hallmark of cardiomyopathies leading to cardiac failure. However, information regarding myofibrillar organization in histologic analysis of sectioned tissue is limited by both large size and variable orientation of the cardiomyocytes. In order to observe individual cardiomyocytes from a single mouse heart, we have developed a new procedure for ‘trapping’ dissociated cardiomyocytes on a polyester mesh. Cells can then be visualized by fluorescence confocal microscopy and three-dimensional reconstruction of cells can be performed without distortion of intracellular organization resulting from cytofugation. Since the cells are trapped between the mesh and coverslip, the mesh is not seen in reconstructed images. Sarcomeric protein organization in cardiomyocytes can now be conveniently studied using this procedure. Furthermore, individual atrial or ventricular cardiomyocytes are easily identified in the preparation and decreased expression of α-myosin heavy chain protein expression is apparent in propythiouracil (PTU) treated mice. This procedure will be a valuable asset in the analysis of intracellular changes occurring within the hearts of cardiomyopathic mice.

Mechanical stimulation of organogenic cardiomyocyte growth in vitro.

Adherent cultures of neonatal rat cardiomyocytes were subjected to progressive, unidirectional lengthening for 2-4 days in serum-containing medium. This mechanical stretch (25% increase in initial length each day) simulates the eccentric mechanical load placed on in vivo heart cells by increases in postnatal blood pressure and volume. The in vitro mechanical stimuli initiated a number of morphological alterations in the confluent cardiomyocyte population which were similar to those occurring during in vivo heart growth. These include cardiomyocyte organization into parallel arrays of rod-shaped cells, increased cardiomyocyte binucleation, and cardiomyocyte hypertrophy by longitudinal cell growth. Stretch stimulated DNA synthesis in the noncardiomyocyte population but not in the cardiomyocytes. Myosin heavy chain (MHC) content increased 62% over 4 days of stretch and included increased accumulation of both fetal beta-MHC and adult alpha-MHC isoforms. This new model of stretch-induced cardiomyocyte hypertrophy may assist in examining some of the complex mechanogenic growth processes that occur in the rapidly enlarging neonatal heart.

Histostereologic analysis of the parenchyma and stroma of the atrophied myocardium in starvation

The reduced functional load during starvation or protein deficiency leads to reduced protein synthesis in heart muscle [11-13, 15]. Inhibition of protein metabolism associated with interruption of the supply of structural material with the food causes delay in the renewal of contractile proteins, and the unsynchronized changes in the ultrastructural organization of cardiomyocytes are connected with this [3, 4]. The principal structural manifestation of inhibition of protein synthesis when the functional load is reduced is atrophy of the myocardium [2, 6, 9]. Quantitative morphological investigations of the myocardium during starvation have revealed considerable changes in the architectonics of the cardiomyocytes during their atrophy [3, 7]. However, the character and direction of relations between the parenchyma and connective tissue in heart muscle during starvation have not been adequately studied.