Excess BMP Signaling in Heterotopic Cartilage Forming in Prg4-null TMJ Discs.

Heterotopic cartilage develops in certain pathologic conditions, including those affecting the human temporomandibular joint (TMJ), but the underlying molecular mechanisms remain obscure. This is in part due to the fact that a reliable animal model of such TMJ diseases is not available. Here, we show that aberrant chondrocyte differentiation and ectopic cartilage formation occur spontaneously in proteoglycan 4 (Prg4) mutant TMJ discs without further invasive procedure. By 2 mo of age, mutant disc cells displayed chondrocyte transdifferentiation, accompanied by strong expression of cartilage master gene Sox9 and matrix genes aggrecan and type II collagen. By 6 mo, heterotopic cartilage had formed in the discs and expressed cartilage hypertrophic markers Runx2 and ColX. The ectopic tissue grew in size over time and exhibited regional mineralization by 12 mo. Bone morphogenetic protein (BMP) signaling was activated with the ectopic chondrogenic cells and chondrocytes, as indicated by phosphorylated Smad 1/5/8 nuclear staining and by elevated expression of Bmp2, Bmpr1b, Bmpr2, and BMP signaling target genes. Likewise, we found that upon treatment with recombinant human BMP 2 in high-density micromass culture, mutant disc cells differentiated into chondrocytes and synthesized cartilage matrix more robustly than control cells. Importantly, a specific kinase inhibitor of BMP receptors drastically attenuated chondrogenesis in recombinant human BMP 2-treated mutant disc cultures. Unexpectedly, we found that Prg4 was expressed at joint-associated sites, including disc/muscle insertion and muscle/bone interface, and all these structures were abnormal in Prg4 mutants. Our data indicate that Prg4 is needed for TMJ disc integrity and function and that its absence leads to ectopic chondrogenesis and cartilage formation in conjunction with abnormal BMP signaling. Our findings imply that the BMP signaling pathway could be a potential therapeutic target for prevention or inhibition of ectopic cartilage formation in TMJ disease.

Lubricin is Required for the Structural Integrity and Post-natal Maintenance of TMJ.

The Proteoglycan 4 (Prg4) product lubricin plays essential roles in boundary lubrication and movement in limb synovial joints, but its roles in temporomandibular joint (TMJ) are unclear. Thus, we characterized the TMJ phenotype in wild-type and Prg4(-/-) mouse littermates over age. As early as 2 weeks of age, mutant mice exhibited hyperplasia in the glenoid fossa articular cartilage, articular disc, and synovial membrane. By 1 month of age, there were fewer condylar superficial tenascin-C/Col1-positive cells and more numerous apoptotic condylar apical cells, while chondroprogenitors displayed higher mitotic activity, and Sox9-, Col2-, and ColX-expressing chondrocyte zones were significantly expanded. Mutant subchondral bone contained numerous Catepsin K-expressing osteoclasts at the chondro-osseous junction, increased invasive marrow cavities, and suboptimal subchondral bone. Mutant glenoid fossa, disc, synovial cells, and condyles displayed higher Hyaluronan synthase 2 expression. Mutant discs also lost their characteristic concave shape, exhibited ectopic chondrocyte differentiation, and occasionally adhered to condylar surfaces. A fibrinoid substance of unclear origin often covered the condylar surface. By 6 months of age, mutant condyles displayed osteoarthritic degradation with apical/mid-zone separation. In sum, lubricin exerts multiple essential direct and indirect roles to preserve TMJ structural and cellular integrity over post-natal life.

Structural and functional analysis of intra-articular interzone tissue in axolotl salamanders.

OBJECTIVE: Knowledge of mechanisms directing diarthrodial joint development may be useful in understanding joint pathologies and identifying new therapies. We have previously established that axolotl salamanders can fully repair large articular cartilage lesions, which may be due to the presence of an interzone-like tissue in the intra-articular space. Study objectives were to further characterize axolotl diarthrodial joint structure and determine the differentiation potential of interzone-like tissue in a skeletal microenvironment. DESIGN: Diarthrodial joint morphology and expression of aggrecan, brother of CDO (BOC), type I collagen, type II collagen, and growth/differentiation factor 5 (GDF5) were examined in femorotibial joints of sexually mature (>12 months) axolotls. Joint tissue cellularity was evaluated in individuals from 2 to 24 months of age. Chondrogenic potential of the interzone was evaluated by placing interzone-like tissue into 4 mm tibial defects. RESULTS: Cavitation reached completion in the femoroacetabular and humeroradial joints, but an interzone-like tissue was retained in the intra-articular space of distal limb joints. Joint tissue cellularity decreased to 7 months of age and then remained stable. Gene expression patterns of joint markers are broadly similar in developing mammals and mature axolotls. When interzone-like tissue was transplanted into critical size skeletal defects, an accessory joint developed within the defect site. CONCLUSIONS: These experiments indicate that mature axolotl diarthrodial joints are phenotypically similar to developing synovial joints in mammals. Generation of an accessory joint by interzone-like tissue suggests multipotent cellular differentiation potential similar to that of interzone cells in the mammalian fetus. The data support the axolotl as a novel vertebrate model for joint development and repair.

Myofibrillar disruption in hypocontractile myocardium showing perfusion-contraction matches and mismatches.

Chronically instrumented dogs underwent 2- or 5-h regional reductions in coronary flow that were followed, respectively, by balanced reductions in myocardial contraction and O(2) consumption ("hibernation") and persistently reduced contraction despite normal myocardial O(2) consumption ("stunning"). Previously unidentified myofibrillar disruption developed during flow reduction in both experimental models and persisted throughout the duration of reperfusion (2-24 h). Aberrant perinuclear aggregates that resembled thick filaments and stained positively with a monoclonal myosin antibody were present in 34 +/- 3.8% (SE) and 68 +/- 5.9% of "hibernating" and "stunned" subendocardial myocytes in areas subjected to flow reduction and in 16 +/- 2.5% and 44 +/- 7.4% of subendocardial myocytes in remote areas of the same ventricles. Areas of myofibrillar disruption also showed glycogen accretion and unusual heterochromatin clumping adjacent to the inner nuclear envelope. The degrees of flow reduction employed were sufficient to reduce regional myofibrillar creatine kinase activity by 25-35%, but troponin I degradation was not evident. The observed changes may reflect an early, possibly reversible, phase of the myofibrillar loss characteristic of hypocontractile myocardium in patients undergoing revascularization.

Role of angiotensin II in bladder smooth muscle growth and function.

Preliminary studies by our group and others indicate that angiotensin II may have an important role in the cellular regulation of smooth muscle growth and collagen production in the bladder. The exact mechanisms in which angiotensin II elicits its cellular effects are not known. Given the available information thus far, we hypothesize the following (see Figure 2): 1) Outlet obstruction of the bladder causes increased cell stretch/strain which in turn induces the local production of angiotensin II. Angiotensin II may also influence cell stretch/strain via its direct effects on bladder tone. 2) Angiotensin II then acts as a trophic factor in the bladder wall to cause smooth muscle cell hypertrophy/hyperplasia and increased collagen production via an autocrine and/or paracrine pathway. 3) The cellular effect(s) of angiotensin II may be mediated by secondary growth factors such as bFGF and TGFb Much more extensive research is certainly needed to reveal whether some part, or all of this hypothesis is correct. If angiotensin II is indeed active in regulating muscle and collagen changes in the pathologic bladder, then the clinical implications are extremely exciting since numerous pharmacologic agents are now available which can either inhibit angiotensin II production and/or block receptor mediated events. These agents may prove to be extremely useful in the clinical management of the neurogenic bladder in which obstructive changes may be prevented and potentially reversed. Despite this, caution must be exercised with regard to the potential use of any medications which alter the systemic renin-angiotensin system in the pediatric population since some research has suggested that an intact system may be necessary for the normal development of some organs, including the kidney.

Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture.

Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists. Intercellular contact, communication, and transmission of contractile forces between myocytes appears to play a primary role in remodeling the 2-dimensional cell layer into a parallel alignment of elongated myocytes with highly developed intercalated disk-like junctions. This highly differentiated state is very stable, and cultures which achieve this state exhibit significantly greater longevity than more sparsely plated myocytes. These myocytes typically continue beating, and survive from 6 to more than 12 weeks in culture. When this level of contact and differentiation are not achieved, even among beta-adrenergic stimulated myocytes, contractile activity is not sustained, myofibrils atrophy, there is little or no development of junctional complexes, and the period of myocyte viability is typically no more than 5 weeks in vitro.

Captopril (an inhibitor of angiotensin converting enzyme) inhibits obstructive changes in the neonatal rabbit bladder.

OBJECTIVES: To investigate whether angiotensin II has a role in the regulation of bladder smooth muscle growth and function, we developed a model of bladder neck obstruction (BNO) in the neonatal rabbit and investigated the effect of captopril (angiotensin converting enzyme inhibitor) on the obstructive changes in the developing bladder. METHODS: Partial BNO was induced in a group of 2-day-old rabbits (n = 8) by placing a loose 2-0 silk ligature around the vesicourethral junction. A second group of rabbits subjected to the identical partial BNO procedure (n = 8) was given captopril (1 mg/kg/day). Twelve days postobstruction, bladders from these animals, along with paired controls (n = 8), were harvested and assayed for total protein, DNA, and collagen content. RESULTS: Partial BNO resulted in a 170% increase in wet weight (P < 0.05), 132% increase in protein/deoxyribonucleic acid (DNA) ratio (P < 0.05), 75% increase in total DNA (P < 0.05), and 115% increase in total collagen (P < 0.05). When compared with obstructed animals, captopril administration significantly inhibited the increase in total DNA (P < 0.05) and reduced the amount of total collagen (P = 0.054). Examination of histology specimens demonstrated that captopril inhibited the serosal hyperplasia and collagen deposition associated with obstruction. CONCLUSIONS: These data demonstrate that captopril partially inhibits the changes in the neonatal rabbit bladder associated with obstruction, supporting the hypothesis that angiotensin II is involved in the regulation of bladder smooth muscle growth and collagen production.

The effect of angiotensin converting enzyme inhibition and angiotensin II receptor antagonism on obstructed rat bladder.

PURPOSE: Others have demonstrated that inhibition of angiotensin II production partially ameliorates obstructive changes in the neonatal rabbit bladder. We examined the effect of angiotensin II converting enzyme inhibition and receptor antagonism on the obstructed rat bladder. MATERIALS AND METHODS: Three groups of animals were investigated. Partial bladder neck obstruction was created in 23 rats by placing a 2-zero silk ligature around the vesicourethral junction. Eight rats were given untreated tap water, 9 were given water supplemented with 50 mg./kg. of the angiotensin-converting enzyme inhibitor captopril and 6 were given water with 30 mg./kg. of the angiotensin II subtype AT1 receptor antagonist losartan potassium. Eight unobstructed rats served as controls. After 2 weeks of partial outlet obstruction the animals were sacrificed and bladders were harvested. Routine histological evaluation and assays for total protein, deoxyribonucleic acid and collagen content were performed. RESULTS: Histological evaluation revealed that administration of captopril or losartan potassium resulted in a mild decrease in the degree of obstructive bladder changes. Biochemically neither captopril nor losartan potassium caused a significant decrease in the amount of total deoxyribonucleic acid, protein or collagen content per bladder compared to untreated obstructed bladders. CONCLUSIONS: In contrast to previous studies in neonatal rabbits, neither captopril nor losartan potassium significantly ameliorated the histological or biochemical features of partial bladder outlet obstruction in the rat. Further investigation is necessary into species specific differences to understand better the role that angiotensin II may have in mediating the bladder changes of experimentally induced obstruction.

Regulation of adult cardiocyte growth: effects of active and passive mechanical loading.

Fluctuations in hemodynamic load have been documented to modulate contractile protein turnover and myofibrillar structure in the heart; however, the relative importance of active and passive loading in regulating adult cardiocyte growth remains unresolved. To address this issue at the cellular level, adult feline cardiocytes were cultured either on Silastic membranes or plastic surfaces. Cardiocyte-laden membranes were stretched 10% of their rest length to enhance passive loading, whereas heart cells cultured on plastic or Silastic were field stimulated at 1 Hz to mimic active loading. Turnover of contractile proteins and structural integrity of the contractile-cytoskeletal apparatus were monitored for periods ranging from 4 to 72 h. Active and passive loading elevated contractile protein synthesis nearly equally (approximately 50%) and promoted the attachment of remodeled myofibrils to vinculin-positive focal contacts and/or costameres during the first 24 h of loading. Thereafter, rates of contractile protein synthesis returned to control values in passively stretched heart cells but remained elevated in field-stimulated cultures. The fractional rate of growth was increased significantly (approximately 8%/day) in electrically paced cells, whereas in passively stretched cardiocytes the growth rate rose only modestly (approximately 2%/day). Changes in the rate of myocyte growth appeared more closely correlated with the development of focal contacts and myofibril remodeling than with changes in myofibrillar protein turnover per se. 2,3-Butanedione monoxime, nifedipine, and, to a lesser extent, ryanodine blocked field-stimulated contractile protein synthesis and myofibrillar remodeling but had no impact on protein turnover or myofibril reassembly in passively loaded cardiocytes. The results of these experiments imply that both active and passive loading stimulate contractile protein turnover and myofibril remodeling, but the generation of active tension accelerates cardiocyte growth to a greater extent than passive loading. Furthermore, pharmacological interventions suggest that unique pathways may mediate these cellular events in actively and passively loaded adult cardiocytes.

Some growth factors stimulate cultured adult rabbit ventricular myocyte hypertrophy in the absence of mechanical loading.

Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-beta 1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with beta-MHC synthesis being elevated and alpha-MHC synthesis being suppressed. However, the other growth factors were able to modestly stimulate the rate of DNA synthesis in this preparation. Bromodeoxyuridine labeling revealed that these growth factors increased DNA synthesis in myocytes and nonmyocytes alike, but the heart cells displayed neither karyokinesis or cytokinesis. In contrast, cocultures of cardiac myocytes and nonmyocytes and nonmyocyte-conditioned culture medium failed to enhance the rate of cardiac MHC synthesis or its accumulation, implying that quiescent heart cells do not respond to "conditioning" by cardiac nonmyocytes. These findings demonstrated that insulin and the IGFs promote passively loaded cultured adult rabbit heart cells to hypertrophy but suggest that other growth factors tested may be limited in this regard.

Electrophysiology of adult cat cardiac ventricular myocytes: changes during primary culture.

To investigate the nature of electrophysiological changes in adult cat cardiac ventricular myocytes that may occur when cells are maintained in primary culture for 1-2 wk, the electrophysiology of cells freshly isolated from collagenase-perfused hearts (day 0 controls) was compared with that of cells maintained in primary culture for up to 14 days 1) on a two-dimensional (2D) surface (laminin-coated coverslips), which allowed for changes in cellular morphology, or 2) in a three-dimensional (3D) alginate matrix, which minimized changes in cell shape. Action potentials and whole cell ionic currents were recorded using a conventional whole cell patch technique. Whereas cellular resting potential and the depth of the "notch" terminating phase 1 were diminished relative to controls in 2D- and 3D-cultured cells, the action potential duration and the incidence of early afterdepolarizations (EADs) were increased relative to controls in 2D- but not in 3D-cultured cells. Corresponding alterations in whole cell ionic currents included a 40% reduction in inwardly rectifying K current (IK1) conductance (GK1) and a 90% reduction in transient outward K current (Ito) conductance (Gto) in 2D- and 3D-cultured cells relative to day 0 controls and a 50% increase in L-type Ca current (ICa-L) conductance (GCa-L) in 2D-cultured cells relative to 3D-cultured cells and day 0 controls. The reduction in Gto in long-term culture was half-maximal by days 7 and 8 and could not be attributed to reduced Ito availability, involvement of a noninactivating Ito, the cell culture procedure itself, or the presence of serum in the culture media. Gto was larger in day 0 cells from a heart with right ventricular hypertrophy than in day 0 normal control cells and was reduced subsequent to placement of cells in 3D culture for 19 days. The results suggest that long-term culture and change in cellular morphology can affect the electrophysiology of cardiac ventricular myocytes.

Alteration of the sodium current in cat cardiac ventricular myocytes during primary culture.

To determine the response of cardiac Na current (INa) in adult cardiac ventricular myocytes to culture, single isolated ventricular myocytes from collagenase-perfused adult cat hearts were placed in primary culture for up to 2 wk on a two-dimensional (2D) surface (laminin-coated coverslips), which allowed the morphology of the myocytes to change markedly, or in a three-dimensional matrix (3D) of alginate, in which cell shape changed only minimally. Action potentials and INa were recorded from groups of 1) freshly isolated myocytes serving as the control (day 0),2) cells maintained in 2D culture for 9-14 days (2D, day 9-14), and 3) cells cultured in alginate for 9-14 days (3D, day 9-14) with use of a conventional whole cell patch technique. Maximal upstroke velocity (Vmax) of the action potential was reduced by approximately 50% in 2D- and 3D-cultured cells relative to controls. INa in 2D- and 3D-cultured cells was strikingly different from that in control myocytes. Half-maximal voltage (V 1/2) for the chord conductance-voltage relationship was shifted approximately 15 mV negatively to that for controls in 2D- and 3D-cultured cells. INa steady-state availability curve also shifted negatively relative to controls in 2D- and 3D-cultured myocytes, but the magnitude of this shift (approximately 16-20 mV) was greater than that for the chord conductance-voltage curve.(ABSTRACT TRUNCATED AT 250 WORDS)

Contractile activity and cell-cell contact regulate myofibrillar organization in cultured cardiac myocytes.

Adult feline ventricular myocytes cultured on a laminin-coated substratum reestablish intercellular junctions, yet disassemble their myofibrils. Immunofluorescence microscopy reveals that these non-beating heart cells lack vinculin-positive focal adhesions; moreover, intercellular junctions are also devoid of vinculin. When these quiescent myocytes are stimulated to contract with the beta-adrenergic agonist, isoproterenol, extensive vinculin-positive focal adhesions and intercellular junctions emerge. If solitary myocytes are stimulated to beat, an elaborate series of vinculin-positive focal adhesions develop which appear to parallel the reassembly of myofibrils. In cultures where neighboring myocytes reestablish cell-cell contact, myofibrils appear to reassemble from the fascia adherens rather than focal contacts. Activation of beating is accompanied by a significant reduction in the rate of total and cytoskeletal protein synthesis; in fact, myofibrillar reassembly, redevelopment of focal adhesions and fascia adherens junctions require no protein synthesis for at least 24 h, implying the existence of an assembly competent pool of cytoskeletal proteins. Maturation of the fasciae adherens and the appearance of vinculin within Z-line/costameres, does require de novo synthesis of new cytoskeletal proteins. Changes in cytoskeletal protein turnover appear dependent on beta agonist-induced cAMP production, but myofibrillar reassembly is a cAMP-independent event. Such observations suggest that mechanical forces, in the guise of contractile activity, regulate vinculin distribution and myofibrillar order in cultured adult feline heart cells.

Catecholamines modulate protein turnover in cultured, quiescent rabbit cardiac myocytes.

When rabbit ventricular myocytes were cultured for 1 wk and then exposed to alpha- and/or beta-adrenergic agonists, such nonbeating heart cell preparations disclosed increased protein-to-DNA ratios and elevated RNA content, indicative of cellular hypertrophy. Norepinephrine, isoproterenol, and phenylephrine provoked hypertrophy with norepinephrine eliciting a greater response than isoproterenol or phenylephrine. Specific alpha- and beta-antagonists blocked growth by inhibiting catecholamine-induced changes in protein turnover. Each catecholamine enhanced the fractional rate of protein synthesis within 48 h; however, changes in growth rates appeared to be modulated, in part, by alterations in protein degradation. Even though rates of total protein and actin synthesis resembled values measured in vivo, myosin heavy chain fractional rate of synthesis was only 22% of in vivo levels. Double label immunofluorescence microscopy further illustrated that catecholamine treatment accelerated myofibrillar disruption in these quiescent heart cells. These observations suggested that in the absence of beating, neurohumoral modulation of contractile protein turnover was not associated with the maintenance of myofibrillar integrity even though catecholamines induced cellular hypertrophy.

Cultured adult cardiac myocytes maintain protein synthetic capacity of intact adult hearts.

Previous studies have shown that the rates of protein synthesis observed in embryonic and neonatal heart cells in culture are as much as nine times greater than the rates of synthesis observed in the intact adult heart either in situ or in isolated perfusion studies. This study addressed whether adult cardiomyocytes in long-term culture maintain the protein synthetic capacity of the adult myocardium or, rather, whether the protein synthetic capacity expands or falls as adult cardiac myocytes progress in culture. Protein synthesis was evaluated in isolated adult feline cardiomyocytes maintained in serum and insulin-supplemented medium for up to 53 days in vitro. With the use of both pulse- and saturation-labeling techniques it was determined that the rate of protein synthesis in adult cardiomyocytes was maintained at a level very close to that observed in the intact heart for over 1 mo in culture. Saturation-labeling studies indicate a fractional rate of protein synthesis at 6.1%/day and an absolute synthesis rate of 1,300 nmol leucine incorporated.g protein-1.h-1. Pulse-labeling studies revealed an initial increase in protein synthesis rates during adaptation to culture and a further increase after activation of beating and cellular hypertrophy.

Evaluation of multiple parameters of HIV-1 replication cycle in testing of AIDS drugs in vitro.

Evaluation of the activities of antiretroviral agents and an immunoregulatory compound has been made using two models of HIV-1 infection and three measurements of virus expression. Acute infection of Jurkat cells or chronic/inducible infection in U1.1 cells was monitored at multiple time points after drug treatment. The 50% effective concentrations (EC50) of the HIV-1 inhibitors suramin, 3'-azido-3'-deoxythymidine (AZT), and 2',3'-dideoxycytidine, as measured by HIV-1 RNA hybridization in Jurkat cells two days after infection, were comparable to EC50 values obtained in parallel measurements of extracellular p24 levels and percent HIV-1 IF-positive cells. However, these measurements diverged: at seven days after infection the EC50 of AZT was greater than 10 microM when intracellular HIV-1 RNA was assayed, 0.2 microM by IF, and 0.03 microM by p24 assay. Human thymic humoral factor displayed no direct inductive activity in chronic HIV-1 infection in U1.1 cells, while phorbol ester and lymphocyte supernatants induced all parameters. These observations warrant care when interpreting results of only a single assay and suggest that definitive assay of HIV-1 infection requires measurements of multiple parameters of virus expression.

Hypertrophy of isolated adult feline heart cells following beta-adrenergic-induced beating.

Catecholamine-induced beating and myocardial hypertrophy were evaluated in isolated adult feline cardiomyocytes maintained in culture for up to 30 days. Adult feline cardiomyocytes were used in this study because they displayed several unique characteristics that facilitated assessment of factors regulating cardiomyocyte hypertrophy in vitro. These characteristics included the following. 1) A single heart provides a high yield of 20-40 x 10(6) calcium-tolerant rod-shaped myocytes. 2) In culture, isolated adult feline cardiomyocytes maintain a stable population of differentiated myocytes that could be maintained without the dramatic loss of cell number, DNA content, or cell structure seen in adult rat cardiomyocyte cultures. 3) Cultured feline cardiomyocytes remained quiescent in culture unless appropriately stimulated to begin beating. 4) Sustained regular beating activity could be readily initiated up to 3 wk in culture by addition of 1 x 10(-5) M isoproterenol, other beta-adrenergic agonists, or agents known to elevate adenosine 3',5'-cyclic monophosphate. Beating could be maintained indefinitely in the presence of isoproterenol, but ceased upon removal of isoproterenol from the medium. Initiation of beating in 7-day-old cultures resulted in a profound restructuring of cardiomyocyte morphology compared with quiescent cultures. Beating heart cells were 66% larger with increased protein content, and they had significantly greater development of striated myofibrillar structure than quiescent myocytes at the same age in culture. We conclude that maintenance of an organized myofibrillar structure in cultured adult cardiac myocytes requires activation of intrinsic beating. Cardiomyocyte hypertrophy also develops following beta-adrenergic activation of beating, but it is unclear whether beating per se is required for inducing hypertrophy in isolated adult cardiomyocytes in vitro.

Cell shape and organization of the contractile apparatus in cultured adult cardiac myocytes.

The isolation and culture of adult cardiac myocytes has proved to be an ideal model system to explore myocardial biology at the cellular level. A major criticism of this model, however, has been that organ-specific characteristics such as cell shape and subcellular structural organization cannot be retained in vitro for prolonged periods of time. Encasing freshly isolated myocytes in a matrix of calcium alginate enables one to maintain the rod-like, three-dimensional (3D) shape of the cultured myocyte. Such preparations more closely resemble their in vivo counterparts with respect to the organization of the contractile apparatus, the transverse tubular system and the sarcoplasmic reticulum than do heart cells cultured on a two-dimensional (2D) plastic surface. Stereologic measurements reveal that myofibrillar volume density (VvMYF) decreases in both non-beating preparations over a 2-week interval, but VvMYF is conserved in cells cultured in an alginate matrix when compared to those myocytes maintained on a laminin-coated substratum. The present observations suggest that in the absence of contractile function myofibrillar atrophy appears responsible for the decline in VvMYF in alginate (3D) preparations, whereas atrophy and subcellular remodelling probably mediate the myofibrillar loss and reorganization that develops when adult heart cells are cultured on a 2D surface.

Morphometric evaluation of the contractile apparatus in primary cultures of rabbit cardiac myocytes.

Rabbit cardiac myocytes remain quiescent for more than 1 month when cultured at low density. During this period, myofibrillar volume density declines sixfold as myofibrils are disassembled or degraded and are replaced by actin and alpha-actinin-positive, myosin-negative structures that resemble myofibrils but lack thick filaments. Such structures are termed minute myofibrils. The length of the sarcomeres in these altered myofibrils is significantly less than length values obtained from freshly isolated heart cells or from contracting myocytes. A number of high density cultures develop spontaneous, synchronous contraction during the second week of culture. Myofibrillar volume density is stabilized when beating begins, and no further decline is observed in the succeeding weeks of culture. Such contracting myocytes display myofibrils typical of normal heart with no visible evidence of minute myofibrils. The volume density of the transverse tubular system also declines significantly in both beating and nonbeating myocytes, and its reduction appears more closely correlated with cell spreading than with beating per se. No quantitative changes in volume density of mitochondria or sarcoplasmic reticulum could be documented, but the structural organization of the sarcoplasmic reticulum seems to be greatly influenced by the physiological state of the heart cell. The present observations document the importance of mechanical factors in regulating the integrity of the contractile apparatus in cardiac myocytes and emphasize the utility of the cultured heart cell to directly investigate structure-function relations in individual myocytes.