Human Engineered Heart Muscles Engraft and Survive Long Term in a Rodent Myocardial Infarction Model.

RATIONALE: Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure. OBJECTIVE: Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model. METHODS AND RESULTS: We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring. CONCLUSIONS: EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.

In vivo vitamin E administration attenuates interleukin-6 and interleukin-1beta responses to an acute inflammatory insult in mouse skeletal and cardiac muscle.

Antioxidants are associated with reduced pro-inflammatory cytokine expression in immune cells and isolated tissues; however, no studies have examined whether short-term vitamin E administration is associated with reduced lipopolysaccharide (LPS)-induced cytokine expression in mouse skeletal and cardiac muscle, in vivo. These experiments tested the hypothesis that vitamin E administration attenuates nuclear factor kappaB (NF-kappaB), IL-6, IL-1beta and tumour necrosis factor alpha (TNFalpha) responses in skeletal and cardiac muscle to an inflammatory challenge induced by systemic LPS. We compared IL-6, IL-1beta and TNFalpha mRNA and protein, activated NF-kappaB and total oxidized proteins in skeletal and cardiac muscle 4 or 24 h after saline or LPS injection in mice receiving vitamin E or placebo for 3 days prior to the insult. Skeletal and cardiac IL-6 mRNA and protein were significantly elevated by LPS in both groups, but responses were significantly lower in vitamin E- compared with placebo-treated mice. In skeletal and cardiac muscle, LPS increased IL-1beta mRNA and protein in placebo- but not vitamin E-treated mice. Lipopolysaccharide-induced levels of cardiac IL-1beta mRNA and protein and skeletal IL-1beta mRNA were lower with vitamin E than placebo. Lipopolysaccharide-induced NF-kappaB activation and increases in total oxidized proteins were attenuated with vitamin E compared with placebo in both tissues. Vitamin E decreased LPS-induced increases in plasma IL-1beta but not IL-6 compared with placebo. The major results provide the first in vivo evidence that short-term vitamin E administration reduces IL-6 and IL-1beta responses to LPS in skeletal and cardiac muscle and prevents LPS-induced increases in NF-kappaB activation and total oxidized proteins.

[Effects of antibodies on contractility of papillary muscle in the rat heart and duration of descending phase of action potential plateau under influence of exogenic arachidonic acid and inhibiting its metabolism]. / Diia antytil na skorotlyvu zdatnist' papiliarnoho m'iaza sertsia shchura ta tryvalist' nyzkhidnoï fazy plato potentsialu diï za umov vplyvu ekzohennoï arakhidonovoï kysloty ta blokuvannia ïï metabolizmu.

The effects of both arachidonic acid (AA) and inhibition of its metabolism on the antibody-induced changes in the mechanical contraction and action potentials of the isolated papillary muscles were studied. It has been shown that antibody-dependent induction of AA oxidation led to the initiation of Ca2+ entry through voltage-dependent Ca2+ channels of a myocyte. Inhibition of the lipoxygenase way of AA peroxidation prevented Ca2+ overload of cardiomyocytes more than inhibition of the cyclooxygenase way, but less than blocking Ca2+ channels.

[The effect of antibodies on the Ca2+ release from the sarcoplasmic reticulum of the papillary muscle of the rat heart]. / Vplyv antytil na vykhid Ca2+ iz sarkoplazmatychnoho retikuluma papiliarnoho m'iaza sertsia shchura.

In experiments of skinned with saponin papillary muscles (PM) of the rat's heart we studied the effects of antibodies (AB), which are specific for the sarcoplasmic reticulum's membranes (SPR). AB (1-5 mg protein/ml) have been established to enhance the amplitude of the isometric PM tension. Inhibition of the Ca2+ release from SPR and its previous release prevented the development of the exciting effect of AB. Thus, AB increase the amplitude of PM contraction due to their intracellular mobilizing action.

Depressed unloaded sarcomere shortening velocity in acute murine coxsackievirus myocarditis: myocardial remodelling in the absence of necrosis or hypertrophy.

We used right ventricular papillary muscles to study cellular dysfunction in acute murine coxsackievirus myocarditis. We measured unloaded sarcomere shortening velocity (V0) with laser diffraction (HeNe, lambda = 623.8 nm) 7 days after coxsackievirus infection (M) (n = 7) and after infection + monoclonal antibodies to eliminate T cells (T) (n = 4) and in normals (N) (n = 8). A servomotor rapidly shortened a muscle until slack early in contraction and V0 was measured at the onset of zero force. V0 in N was 4.14 +/- 0.84 microns.s-1 at Sl = 2.08 +/- 0.09 microns, 1.70 +/- 0.33 microns.s-1 at 2.06 +/- 0.08 microns in M and, in preliminary experiments, 4.75 +/- 0.96 microns.s-1 at 2.06 +/- 0.07 microns in T. Resting force and stiffness were normal in M. Ventriculor weights in M and T were the same as N. There was an increase in mononuclear cells in M papillary muscles, but no fibrosis or necrosis. Thus, V0 was markedly reduced in acute viral myocarditis in the absence of tissue disruption or hypertrophy, but not if T cells were absent. Pyrophosphate gel electrophoresis showed a shift from predominantly fast in N to a slow myosin isoform in M. Myosin remodelling and reduced unloaded sarcomere shortening velocity occur early in acute coxsackievirus myocarditis and are dependent on immune responses to the virus, but are not a result of histopathological changes.

[The action of specific antimembrane antibodies on the tonic tension and contractile phases of the heart muscle in rats]. / Diia spetsyfichnykh antymembrannykh antytil na tonichnu napruhu ta fazni skorochennia sertsevoho m'iaza shchura.

Antibodies specific to rat myocardial sarcolemma inhibit phasic contractions and promote an increase in tonic tension of isolated strips of the cardiac muscle in rat. These results indicate that antibodies induce accumulation of intracellular free Ca2+. Analysis of the authors' data and those from literature suggests that specific antibodies inhibit Na(+)-K(+)-pump activity of the cardiac muscle in rat. This conclusion is confirmed by analogy of effects of ouabaine, the well-known inhibitor of Na(+)-K(+)-pump, on the rat cardiac muscle.

Myasthenia gravis. Identification of skeletal and heart muscle antigens not related to the acetylcholine receptor.

Myasthenia gravis (MG) is a neuromuscular disease thought to have an autoimmune etiology. The acetylcholine receptor has been considered the primary site of antibody binding; however, other muscle components may be involved in the pathogenesis of myasthenia gravis. This study describes a hypertonic sucrose extract of skeletal muscle (Muscle-HSE) that reacts with antibodies in myasthenic sera. The active component in Muscle-HSE is not the acetylcholine receptor as demonstrated by the inability of this extract to bind [125I]alpha-bungarotoxin. Muscle-HSE does, however, contain two distinct antigenic components reactive with MG sera. One antigen reacted with 70% (14/20) of myasthenic sera in the passive hemagglutination assay. This antigen was detected in the HSE of both skeletal muscle and heart, and was unaffected by treatment with Triton X-100. The second antigen reacted with 10% (2/20) of MG sera in the complement fixation assay, was unique to skeletal muscle, and was inactivated by Triton X-100.

Reversal of ouabain and acetyl strophanthidin effects in normal and failing cardiac muscle by specific antibody.

Isolated cat right ventricular papillary muscles were used to study the effects of antibodies with high affinity for ouabain and acetyl strophanthidin on myocardium exposed to these cardioactive steroids. Antibodies with average intrinsic affinity constants for ouabain and acetyl strophanthidin of the order of 10(8) M(-1) were raised in rabbits challenged by repeated injection of a conjugate of ouabain covalently linked to a poly D,L-alanyl derivative of human serum albumin. Effects were assessed in terms of time-course and extent of inotropy reversal, influence of experimentally induced ventricular failure, digitalis-antibody concentration relations, influence of digitalis-antibody complex on response to additionally added digitalis, and relation of antibody effects on digitalis-induced automaticity and contracture to reversal of inotropy. Specific antibody (but not control antibody) in 1.1-1.5-fold molar excess over cardioactive steroid concentrations blocked positive inotropic effects of ouabain and acetyl strophanthidin, and gradually reversed established contractile effects of these agents with a mean time for half-reversal of ouabain-induced inotropy of 124+/-6 (SEM) min and 37+/-3 min for half-reversal of acetyl strophanthidin-induced inotropy. Papillary muscles from cats with right ventricular failure induced by chronic pulmonary artery constriction responded similarly. Both normal and failing muscles returned to but not below levels of contractility existing before cardioactive steroid exposure, and time for half-reversal of inotropy by antibody was significantly shorter than time for half-reversal after removal of ouabain or acetyl strophanthidin by muscle bath washout alone. Presence of ouabain- or acetyl strophanthidin-antibody complex did not alter the myocardial contractile response to subsequently added cardioactive steroids. Spontaneous automaticity occurring as a toxic response to ouabain or acetyl strophanthidin in eight muscles was rapidly reversed by specific antibody at a time when positive inotropic effects were still fully manifest. Early contracture was also reversed by specific antibody. These studies provide further support for the concept that cardiac glycoside-specific antibodies are capable of reversing established cellular effects of cardioactive steroids.