Robert S. Decker

Myosin-Binding Protein C Phosphorylation, Myofibril Structure, and Contractile Function During Low-Flow Ischemia

Background—Contractile dysfunction develops in the chronically instrumented canine myocardium after bouts of low-flow ischemia and persists after reperfusion. The objective of this study is to identify whether changes in the phosphorylation state of myosin-binding protein C (MyBP-C) are a potential cause of dysfunction. Methods and Results—During low-flow ischemia, MyBP-C is dephosphorylated, and the number of actomyosin cross-bridges in the central core of the sarcomere decreases as thick filaments dissemble from the periphery of the myofibril. During reperfusion, MyBP-C remains dephosphorylated, and its degradation is accelerated. Conclusions—Dephosphorylation of MyBP-C may initiate changes in myofibril thick filament structure that decrease the interaction of myosin heads with actin thin filaments. Limiting the formation of actomyosin cross-bridges may contribute to the contractile dysfunction that is apparent after low-flow ischemia. Breakdown of MyBP-C during reperfusion may prolong myocardial stunning.

Protein synthesis and degradation during starvation-induced cardiac atrophy in rabbits.

To determine the relative importance of protein degradation in the development of starvation-induced cardiac atrophy, in vivo fractional synthetic rates of total cardiac protein, myosin heavy chain, actin, light chain 1, and light chain 2 were measured in fed and fasted rabbits by continuous infusion of [3H] leucine. In addition, the rate of left ventricular protein accumulation and loss were assessed in weight-matched control and fasted rabbits. Rates of total cardiac protein degradation were then estimated as the difference between rates of synthesis and growth. Fasting produced left ventricular atrophy by decreasing the rate of left ventricular protein synthesis (34.8 +/- 1.4, 27.3 +/- 3.0, and 19.3 +/- 1.2 mg/day of left ventricular protein synthesized for 0-, 3-, and 7-day fasted rabbits, respectively). Inhibition of contractile protein synthesis was evident by significant reductions in the fractional synthetic rates of all myofibrillar protein subunits. Although fractional rates of protein degradation increased significantly within 7 days of fasting, actual amounts of left ventricular protein degraded per day were unaffected. Thus, prolonged fasting profoundly inhibits the synthesis of new cardiac protein, including the major protein constituents of the myofibril. Both this inhibition in new protein synthesis as well as a smaller but significant reduction in the average half-lives of cardiac proteins are responsible for atrophy of the heart in response to fasting.

Antirestenotic Effects of a Locally Delivered Caspase Inhibitor in a Balloon Injury Model

Background—The precise role of arterial barotrauma-mediated apoptosis in causing restenosis is unclear. The purpose of this study was to determine if a link exists between angioplasty-mediated medial smooth muscle cell apoptosis and subsequent neointimal hyperplasia. Methods and Results—Bilateral iliac artery angioplasty was performed in 25 male New Zealand White rabbits. Simultaneous with balloon injury, each artery was treated locally with either the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone (ZVAD-fmk) or control. In the acute cohort that was survived to 4 hours (n=10, 7 high dose and 3 low dose), an apoptotic index was calculated using the terminal deoxynucleotidyl TUNEL method. In the intermediate cohort that was survived to 2 weeks (n=5), luminal reendothelialization was measured via CD-31 staining. In the chronic cohort that was survived to 4 weeks (n=10), neointimal area was measured. In the acute cohort, there was a 40% reduction in the apoptotic index with high-dose ZVAD-fmk (P =0.008) and a 33% reduction with low-dose ZVAD-fmk (P =0.08). At 2 weeks, there was no significant difference in the degree of luminal reendothelialization. However, at 4 weeks, there was a 33% (0.33±0.23 versus 0.22±0.20 mm2) (P <0.005) reduction in neointimal area in ZVAD-fmk–treated arteries. Conclusions—The local delivery of ZVAD-fmk during balloon injury inhibits smooth muscle cell apoptosis. This corresponds to a significant reduction in neointimal proliferation seen at 4 weeks without a significant change in the degree of reendothelialization at 2 weeks.

Age-related alterations in cardiac lysosomes

The activities and distributions of several lysosomal enzymes (cathepsin D, β-acetylglucosaminidase, and acid phosphatase) were measured in left ventricular tissue from rats and rabbits of different ages. In both species, the hearts from older animals had significantly higher total activities of cathepsin D. Acid phosphatase activity was similar at all ages tested, and glucosaminidase activity was either unchanged or lower in the older hearts. The proportion of total cathepsin D activity that was nonsedimentable (i.e. not particle-bound) was much greater in older hearts; changes in the nonsedimentable-to-sedimentable distribution were less marked for the other enzymes. Immunohistochemical staining of rabbit cathepsin D with a specific antiserum to the enzyme revealed that age-related increases in cathepsin D occurred most prominently in myocytes rather than interstitial cells. Myocytic organelles that stained positively for the enzyme were larger and more numerous in older hearts, but there was no apparent shift in the general localization of cathepsin D including no significant staining outside lysosomelike particles. The results indicate that major lysosomal changes develop with age, and that the changes are heterogeneous with regard to different lysosomal enzymes. In studies of the effects of experimental interventions on lysosomal function, it is of critical importance always to use age-matched controls.

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.

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.

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.

Angiotensin II and Basic Fibroblast Growth Factor Induce Neonatal Bladder Stromal Cell Mitogenesis

PURPOSE Our aims were to establish primary stromal cell cultures from the neonatal rabbit bladder and investigate the potential mitogenic effects of angiotensin II and basic fibroblast growth factor on these cells. MATERIALS AND METHODS Primary bladder stromal cell cultures were obtained from 3-day-old rabbits, plated at a density of 3 x 10(4) cells per ml. and allowed to grow for 24 hours. Subconfluent cells were growth arrested in serum deficient (0.25% newborn calf serum) or serum-free media for 24 hours and then stimulated with 10(-7) M. angiotensin II or 10 ng./ml. basic fibroblast growth factor for an additional 48 hours. Cell counts and [3H] thymidine incorporation were done to measure cellular proliferation and deoxyribonucleic acid synthesis. RESULTS Angiotensin II and basic fibroblast growth factor each stimulated neonatal bladder stromal cell proliferation and [3H] thymidine incorporation under serum deficient conditions. Angiotensin II provoked an average 26% increase in cell number (p < 0.01) and 35% increase in [3H] thymidine incorporation (p < 0.01) compared to control values. Basic fibroblast growth factor was an even more potent mitogen with a 47% increase in cell number (p < 0.01) and 180% increase in [3H] thymidine incorporation (p < 0.01) compared to controls. In contrast, angiotensin II and basic fibroblast growth factor each failed to have significant stimulatory effects under serum-free conditions. CONCLUSIONS Angiotensin II and basic fibroblast growth factor induce a mitogenic response to neonatal bladder stromal cells in vitro. These mitogenic effects require the presence of serum factors. Whether angiotensin II and basic fibroblast growth factor are involved in the in vivo regulation of bladder growth associated with obstructive uropathy requires further investigation.

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.

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.