Marlene L. 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.

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.

The dynamic role of cardiac myosin binding protein-C during ischemia

Cardiac myosin binding protein C (cMyBP-C) is a myofibrillar protein important for normal myocardial contractility and stability. In mutated form it can cause cardiomyopathy and heart failure. cMyBP-C appears to have separate regions for different functions. Three phosphorylation sites near the N terminus modulate contractility by their effect on both the kinetics of contraction and the binding site of the N-terminus. The C terminal region binds to myosin rods and stabilizes thick filament structure. The aim of the study reported here was to test whether cMyBPC is important in producing the structural and functional changes that result from ischemia/reperfusion. In this study the sequential changes in cMyBP-C, contractility, and thick filament structure following dephosphorylation of cMyBP-C associated with ischemia and reperfusion have been studied in biopsied specimens from chronically instrumented dogs. One and two dimensional electrophoresis, electron microscopy and immunocytochemistry with multiple antibodies generated against different domains in cMyBP-C have been used to follow structural changes in cMyBP-C. Ischemia produced dephosphorylation of cMyBP-C. Subsequent reperfusion released the dephosphorylated cMyBP-C from myofibrils and activated proteolysis of the cytoplasmic cMyBP-C. This in turn leads to increased vulnerability of cMyBP-C to proteolysis and increased degradation of thick filaments. The state of cMyBP-C appears to be closely related to phosphorylation and dephosphorylation of serine 282. In the absence of the stabilizing action of cMyBP-C either as a consequence of genetic mutation or dephosphorylation, premature degradation of thick filaments occurs and is accompanied by persistent contractile dysfunction.

Mechanical and neurohumoral regulation of adult cardiocyte growth

Culture of adult cardiac myocytes provides an opportunity to directly explore potential regulatory pathways that modulate the growth of the mature heart cell.’ Identifying subcellular events and signal transduction pathways that mediate the growth of the adult cardiac myocyte in response to stresses that provoke cardiac hypertrophy is limited when whole animals are employed, because unraveling“cause from effect” in vivo is often complicated by the cellular heterogeneity of the heart and the likely neurohumoral interactions that exist within the intact rnyoca rd i~m.~-~ Changes in neurotransmitter release, fluctuating plasma hormone levels and attendant alterations in hemodynamic load make it difficult, at best, to precisely determine how postulated chemical and physical signals regulate cellular growth in vivo. Cultured cardiac myocytes provide an experimental paradigm to more definitively identify and characterize putative pathways that control cardiocyte growth in a defined and relatively uncomplicated environment. There are several caveats, however, that should be considered before contemplating the use of such a model system. The production of two-dimensional adult cardiocyte cultures minimizes, in most instances, myocyte-nonmyocyte interactions, alters the geometry of this reestablished “in vitro myocardium” and modifies the extracellular environment (i.e., the culture medium and extracellular matrix) that supports cardiocyte growth. The source of the heart cells employed for study (i.e., are they derived from developing or adult heart) also deserves special consideration. Cardiac myocytes isolated from embryonic, neonatal or adult hearts possess unique properties that make each of them useful for the study of specific aspects of cardiac growth and development. Embryonic heart cells are, perhaps, ideal for exploring cardiogenic determination, while neonatal myocytes are well suited for investigating postnatal myocardial growth. In contrast, adult cardiocytes can be usefully employed to explore the regulation of physiologic and, perhaps, pathophysiologic hypertrophy. The principal focus of this report will be to compare and contrast how mechanical loading and neurohumoral activation modulate the growth of adult cardiac myocytes and, where possible, to determine whether the

Lysosomal integrity in ischemic, hypoxic and recovering heart

The early experiments of de Duve and his colleagues first implicated the lysosome as a potential mediator of ischemic cell injury [1, 2]. Ligating a branch of the hepatic artery was accompanied by a marked subcellular redistribution of lysosomal enzyme activity in the ischemic liver. Since de Duve [1] had recently documented that the lysosome represented a latent storehouse of catabolic acid hydrolases, such alterations in lysosomal properties prompted considerable speculation that these organelles might represent a population of subcellular ‘suicide bags’ which, in theory, could provoke intracellular damage if ‘activated’ by appropriate changes in the intracellular environment [3]. Such provocative observations encouraged numerous investigations aimed at identifying whether alterations in ‘lysosomal integrity’ were, in any way, temporally correlated with evolving ischemic damage in the heart and other tissues [3–7].