Myocyte Nuclei Research Articles

Elf-pulsed magnetic fields modulate opioid peptide gene expression in myocardial cells.

Magnetic fields have been shown to affect cell proliferation and growth factor expression in cultured cells. Although the activation of endorphin systems is a recurring motif among the biological events elicited by magnetic fields, compelling evidence indicating that magnetic fields may modulate opioid gene expression is still lacking. We therefore investigated whether extremely low frequency (ELF) pulsed magnetic fields (PMF) may affect opioid peptide gene expression and the signaling pathways controlling opioid peptide gene transcription in the adult ventricular myocyte, a cell type behaving both as a target and as a source for opioid peptides. Prodynorphin gene expression was investigated in adult rat myocytes exposed to PMF by the aid of RNase protection and nuclear run-off transcription assays. In PMF-exposed nuclei, nuclear protein kinase C (PKC) activity was followed by measuring the phosphorylation rate of the acrylodan-labeled MARCKS peptide. The effect of PMF on the subcellular distribution of different PKC isozymes was assessed by immunoblotting. A radioimmunoassay procedure coupled to reversed-phase high performance liquid chromatography was used to monitor the expression of dynorphin B. Here, we show that PMF enhanced myocardial opioid gene expression and that a direct exposure of isolated myocyte nuclei to PMF markedly enhanced prodynorphin gene transcription, as in the intact cell. The PMF action was mediated by nuclear PKC activation but occurred independently from changes in PKC isozyme expression and enzyme translocation. PMF also led to a marked increase in the synthesis and secretion of dynorphin B. The present findings demonstrate that an opioid gene is activated by myocyte exposure to PMF and that the cell nucleus and nuclear embedded PKC are a crucial target for the PMF action. Due to the wide ranging importance of opioid peptides in myocardial cell homeostasis, the current data may suggest consideration for potential biological effects of PMF in the cardiovascular system.

Effect of caspase inhibitors on myocardial infarct size and myocyte DNA fragmentation in the ischemia–reperfused rat heart

Caspase family proteases are recognized as key mediators of apoptosis. However, the role of caspases in the ischemia-reperfused heart remains uncertain. We evaluated the effect of caspase inhibitors on myocardial infarct size and the myocyte DNA fragmentation in the ischemia-reperfused rat hearts. Three groups of Sprague-Dawley rats (n = 7, each) were subjected to 30 min of ischemia followed by 6 h of reperfusion. One of the following drugs: (1) YVAD-aldehyde, a caspase-1-like protease inhibitor (3.5 mg/kg; YVAD), (2) DEVD-aldehyde, a caspase-3-like protease inhibitor (3.5 mg/kg, DEVD), (3) vehicle (140 microliters/kg) was administered intravenously 5 min prior to the ischemia in each group. Myocardial infarct size was defined by triphenyltetrazolium chloride (TTC) staining. Immunohistochemical staining by in situ nick end labeling (TUNEL) of cardiomyocytes and DNA electrophoresis were used for detecting DNA fragmentation. Ultrastructural analysis was done by electron microscopy. The caspase activity was measured in the myocardium of both groups. The percentage of TUNEL-positive myocyte nuclei (%AP) was quantified by microscopy. A ladder pattern was detected by electrophoresis of DNA from the risk area and TUNEL-positive myocytes were seen in the risk area. The %AP was significantly reduced from 20 +/- 1% to 12 +/- 3% by YVAD and to 10 +/- 3% by DEVD (both P < 0.01). However, caspase inhibitors did not significantly change the infarct size. Electronmicrograph showed similar salcolemmal and mitochondrial damage in both group. The caspase activity was blocked by DEVD at 4 h after reperfusion. Myocyte DNA fragmentation and caspase activation was inhibited by caspase inhibitors without reduction of the infarct size in ischemia-reperfused rat hearts.

Catheter-delivered in vivo gene transfer into rat myocardium using the fusigenic liposomal mediated method.

We compared the efficacy of four different in vivo hemagglutinating virus of Japan (HVJ)-liposome gene transfer methods, i.e., direct myocardial injection (i.m.), injection into the left ventricular cavity (LV), infusion at the level of the coronary cusps (CI), or injection into the left ventricular cavity with a balloon catheter blocking aortic flow (LV+B) to transfer beta-galactosidase, FlTC-labeled oligodeoxynucleotide (ODN), and/or luciferase genes into the rat heart. I.m. caused highly efficient gene transfer in the limited area around the injection site, which suggests that i.m. may be a suitable method for targeted treatment of focal lesion. In the LV+B group, all rats had myocardial beta-galactosidase staining and fluorescence of FITC-labeled ODN in the nuclei of cardiac myocytes around the coronary arteries and the vasa vasorum, and some transfected myocytes were observed in the middle of the myocardium without any evidence of injury. In contrast, in the CI group, only half of the animals had myocardial expression of beta-galactosidase. In contrast, fluorescence or luciferase activity was present throughout the left ventricle in the LV+B group. However, the percentage of myocytes that exhibited fluorescence was less than 1% of the total ventricular myocyte population and luciferase activity in the LV+B group was 1.6% of that in the i.m. group. No evidence of luciferase expression was observed in brain, lung, liver, kidney, or testis in either the i.m. or LV+B group. These results suggest that HVJ-liposome gene transfer into the myocardium through the coronary arteries using a balloon-catheter technique is safe and has the potential for causing widespread transgene expression with organ-specificity, although the efficiency of gene transfer should be improved.

LPS pretreatment ameliorates peritonitis-induced myocardial inflammation and dysfunction: role of myocytes.

Peritonitis induced by cecal ligation and puncture (CLP) produces a systemic inflammatory response that can be largely mitigated by pretreatment of the animals with lipopolysaccharide (LPS tolerance). Although cells of myeloid origin and endothelial cells have been shown to contribute to the development of LPS tolerance, little is known regarding the potential role of parenchymal cells in this phenomenon. The major aim of the present study was to assess whether cardiac parenchymal cells (myocytes) contribute to the development of LPS tolerance. Six hours after induction of CLP rats were neutropenic and acidotic, the myocardium contained a leukocyte infiltrate [myeloperoxidase (MPO) activity was increased], and myocardial contractile function was impaired (left ventricular developed pressure was decreased). In animals that were pretreated with LPS these manifestations of sepsis were largely reversed. Further studies focused on the responses of cardiac myocytes to CLP and whether myocytes contributed to the development of LPS tolerance. Myocytes were isolated from rat hearts 6 h after induction of CLP. These myocytes 1) exhibited an impaired ability to shorten in response to pacing, 2) contained the nuclear transcription factor NF-kappaB in their nuclei, 3) increased their surface levels of intercellular adhesion molecule-1 (ICAM-1), and 4) were hyperadhesive for neutrophils. All of these events did not occur in myocytes obtained from animals that were pretreated with LPS before induction of CLP. These findings indicate that LPS tolerance can be induced in myocytes with respect to polymorphonuclear leukocyte adhesion, presumably by an inability of CLP to mobilize NF-kappaB to the myocyte nuclei and, thereby, preventing an increase in surface levels of ICAM-1.

Caspase Inhibition Reduces Myocyte Cell Death Induced by Myocardial Ischemia and Reperfusion In Vivo

Myocardial ischemia and reperfusion lead to myocyte cell death, at least in part, by an apoptotic mechanism. Caspases are a conserved family of proteases that play an essential role in the execution of apoptosis; however, their potential contribution to ischemic myocardial cell death is largely unknown. To examine their role in this process, we subjected rabbits to 30 min of coronary artery occlusion followed by 3 h of reperfusion. Immunoblot analyses revealed that caspases-2, -3 and -7 were proteolytically activated during myocardial ischemia and reperfusion in vivo. In addition, the well-characterized caspase substrate poly(ADP-ribose) polymerase (PARP) was selectively cleaved into its signature apoptotic fragment in ischemic/reperfused myocardium. Systemic administration of the broad-spectrum caspase inhibitor acetyl-Tyr-Val-Ala-Asp chloromethylketone (YVAD-cmk, 4.8 mg/kg) partially blocked caspase activation and dramatically reduced the percentage of terminal dUTP deoyxynucleotidyl-transferase nick end-labeling (TUNEL)-positive myocyte nuclei in the infarct region (3.9±0.8%v 13.0±2.2% in control animals, P=0.012). Moreover, YVAD-cmk reduced myocardial infarct size by approximately 31% (31.1±3.3%v 45.3±4.9% in control animals,P =0.032). These results indicate that caspases are critical mediators of myocardial injury induced by ischemia and reperfusion in vivo, and they suggest that caspase inhibition may be therapeutically beneficial in myocardial infarction.

A mammalian myocardial cell-free system to study cell cycle reentry in terminally differentiated cardiomyocytes.

Cardiomyocytes withdraw from the cell cycle in the early neonatal period, rendering the adult heart incapable to regenerate after injury. In the present study, we report the establishment of a cell-free system to investigate the control of cell cycle reentry in mammalian ventricular cardiomyocyte nuclei and to specifically address the question of whether nuclei from terminally differentiated cardiomyocytes can be stimulated to reenter S phase when incubated with extracts from S-phase cells. Immobilized cardiomyocyte nuclei were incubated with nuclei and cytoplasmic extract of synchronized H9c2 muscle cells or cardiac nonmyocytes. Ongoing DNA synthesis was monitored by biotin-16-dUTP incorporation as well as proliferating cell nuclear antigen expression and localization. Nuclei and cytoplasmic extract from S-phase H9c2 cells but not from H9c2 myotubes induced DNA synthesis in 92% of neonatal cardiomyocyte nuclei. Coincubation in the presence of cycloheximide indicated that de novo translation is required for the reinduction of S phase. Similar results were obtained with adult cardiomyocyte nuclei. When coincubated with both cytoplasmic extract and nuclei or nuclear extracts of S-phase cells, >70% of adult cardiomyocyte nuclei underwent DNA synthesis. In conclusion, these results demonstrate that postmitotic ventricular myocyte nuclei are responsive to stimuli derived from S-phase cells and can thus bypass the cell cycle block. This cell-free system now makes it feasible to analyze the molecular requirements for the release of the cell cycle block and will help to engineer strategies for regenerative growth in cardiac muscle.

IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1.

Localized synthesis of insulin-like growth factors (IGFs) has been broadly implicated in skeletal muscle growth, hypertrophy and regeneration. Virally delivered IGF-1 genes induce local skeletal muscle hypertrophy and attenuate age-related skeletal muscle atrophy, restoring and improving muscle mass and strength in mice. Here we show that the molecular pathways underlying the hypertrophic action of IGF-1 in skeletal muscle are similar to those responsible for cardiac hypertrophy. Transfected IGF-1 gene expression in postmitotic skeletal myocytes activates calcineurin-mediated calcium signalling by inducing calcineurin transcripts and nuclear localization of calcineurin protein. Expression of activated calcineurin mimics the effects of IGF-1, whereas expression of a dominant-negative calcineurin mutant or addition of cyclosporin, a calcineurin inhibitor, represses myocyte differentiation and hypertrophy. Either IGF-1 or activated calcineurin induces expression of the transcription factor GATA-2, which accumulates in a subset of myocyte nuclei, where it associates with calcineurin and a specific dephosphorylated isoform of the transcription factor NF-ATc1. Thus, IGF-1 induces calcineurin-mediated signalling and activation of GATA-2, a marker of skeletal muscle hypertrophy, which cooperates with selected NF-ATc isoforms to activate gene expression programs.

Ventricular myocardium in control and growth-retarded human fetuses: Growth in different tissue compartments and variation with fetal weight, gestational age, and ventricle size

The aim of this study was to assess the growth of different tissue compartments in ventricular myocardium of control and intrauterine growth-retarded (IUGR) human subjects. Stereological counting and sizing methods were applied to cross-sectional samples of hearts collected post mortem at 16 to 35 (control) and 32 to 42 (IUGR) gestational weeks. Total tissue volumes and total numbers of myocyte, connective tissue, and endothelial cell nuclei were estimated. In control hearts, the volume of each tissue compartment increased linearly over the period of gestation, and this was due to proliferation, because in each case the tissue volume per nucleus remained constant. In IUGR subjects, fetal weight, ventricle volume, myocyte volume, connective tissue volume, and endothelial nuclear number were less than expected for gestational age up to at least 35 weeks. Between this age and 8 postnatal weeks, these variables are predicted to achieve equivalence with values found in control fetuses. Similar deficits were found when variables were related to fetal weight, and it is predicted that equivalence with control values would be reached at weights of between 2.3 and 3.6 kg. No such differences between groups were detected when variables were related to ventricle size. These findings indicate that growth deficits in cardiomyocytes are accompanied, and may be influenced, by developmental delays that involve the intramyocardial interstitium (capillary bed, endothelium, and surrounding connective tissues). The delays and deficits exhibit “catch-up” to control values between 35 weeks and term.

Role of protein kinase C in mitochondrial KATP channel-mediated protection against Ca2+ overload injury in rat myocardium.

Growing evidence exists that ATP-sensitive mitochondrial potassium channels (MitoKATP channel) are a major contributor to the cardiac protection against ischemia. Given the importance of mitochondria in the cardiac cell, we tested whether the potent and specific opener of the MitoKATP channel diazoxide attenuates the lethal injury associated with Ca2+overload. The specific aims of this study were to test whether protection by diazoxide is mediated by MitoKATP channels; whether diazoxide mimics the effects of Ca2+ preconditioning; and whether diazoxide reduces Ca2+ paradox (PD) injury via protein kinase C (PKC) signaling pathways. Langendorff-perfused rat hearts were subjected to the Ca2+ PD (10 minutes of Ca2+ depletion followed by 10 minutes of Ca2+ repletion). The effects of the MitoKATP channel and other interventions on functional, biochemical, and pathological changes in hearts subjected to Ca2+ PD were assessed. In hearts treated with 80 micromol/L diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly preserved after Ca2+ PD; peak lactate dehydrogenase release was also significantly decreased, although ATP content was less depleted. The cellular structures were well preserved, including mitochondria and intercalated disks in diazoxide-treated hearts compared with nontreated Ca2+ PD hearts. The salutary effects of diazoxide on the Ca2+ PD injury were similar to those in hearts that underwent Ca2+ preconditioning or pretreatment with phorbol 12-myristate 13-acetate before Ca2+ PD. The addition of sodium 5-hydroxydecanoate, a specific MitoKATP channel inhibitor, or chelerythrine chloride, a PKC inhibitor, during diazoxide pretreatment completely abolished the beneficial effects of diazoxide on the Ca2+ PD. Blockade of Ca2+ entry during diazoxide treatment by inhibiting L-type Ca2+ channel with verapamil or nifedipine also completely reversed the beneficial effects of diazoxide on the Ca2+ PD. PKC-delta was translocated to the mitochondria, intercalated disks, and nuclei of myocytes in diazoxide-pretreated hearts, and PKC-alpha and PKC-epsilon were translocated to sarcolemma and intercalated disks, respectively. This study suggests that the effect of the MitoKATP channel is mediated by PKC-mediated signaling pathway.

Calpain inhibitor-1 reduces infarct size and DNA fragmentation of myocardium in ischemic/reperfused rat heart.

Myocardial ischemia/reperfusion activates a calcium-dependent protease, calpain, in the ischemic myocytes. It is not known whether calpain is involved in the mechanism of ischemia/reperfusion injury in hearts. Thus the purpose of this study was to clarify the effect of a selective calpain inhibitor (CAI) on infarct size and the extent of DNA damage in ischemic/reperfused rat hearts. Rats were divided in four groups (n = 7 each). In saline group, 0.3 ml of saline was administered (i.v.) 10 min before 30-min coronary occlusion followed by 6-h reperfusion. In vehicle group, 0.3 ml of 10% dimethyl sulfoxide (DMSO) was administered 10 min before the 30-min ischemia. CAI (0.5 mg/kg) was administered 10 min before the 30-min ischemia (CAI-A group) and 10 min before the 6-h reperfusion period (CAI-B group). Infarct size was detected with triphenyl tetrazolium chloride, and DNA fragmentation was detected by agarose gel electrophoresis and by in situ nick end labeling (ISEL). Infarct size was significantly smaller in the CAI-A group compared with the vehicle group (13+/-9% vs. 48+/-12%; p < 0.01), and the incidence of ISEL-positive myocyte nuclei in the subendocardial region was significantly reduced in the CAI-A group compared with the vehicle group (26+/-3% vs. 59+/-6%; p < 0.01). However, the effects of CAI in CAI-B group were not significant. Activation of calpain is involved in the mechanism of ischemia/reperfusion injury, and the preischemic administration of CAI was effective in reducing myocardial infarct size and the DNA damage of the myocytes in ischemic/reperfused rat heart.

Apoptosis of cardiac myocytes in Gsalpha transgenic mice.

-The stimulatory GTP-binding protein Gsalpha transmits signals from catecholamine receptors to activate adenylyl cyclase and thereby initiate a cascade leading to cardiac chronotropy and inotropy. Transgenic mice overexpressing the Gs alpha subunit (Gsalpha) selectively in their hearts exhibit increased cardiac contractility in response to beta-adrenergic receptor stimulation. However, with aging, these mice develop a cardiomyopathy. This study sought morphological and biochemical evidence that overexpression of Gsalpha is associated with increased myocyte apoptosis in the older animals and to determine whether such overexpression can promote apoptosis of isolated neonatal cardiac myocytes exposed to beta-adrenergic receptor agonists. In the hearts of 15- to 18-month-old Gsalpha transgenic mice, histochemistry and electron microscopy illustrated the existence of numerous myocytes with abnormal nuclei embedded in collagen-rich connective tissue. Terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling (TUNEL, for in situ labeling of DNA breaks) demonstrated that approximately 0.6% of myocyte nuclei contained fragmented DNA. Agarose gel electrophoresis provided further biochemical evidence of apoptosis by showing internucleosomal DNA fragmentation. Cultured cardiac myocytes from newborn Gsalpha transgenic mice showed increased TUNEL staining and internucleosomal DNA fragmentation compared with wild-type controls when treated with the beta-agonist isoproterenol. Thus, enhanced activation of beta-adrenergic signaling by overexpression of Gsalpha in the hearts of transgenic mice induces apoptosis of cardiac myocytes. This represents a potential mechanism that may contribute to the development of cardiomyopathy in this model.

Apoptosis and myocardial infarction.

Myocardial infarction was produced in rats and the contribution of apoptotic and necrotic myocyte cell death was measured quantitatively. Myocyte cell death by apoptosis involved 2.8 million cells at 2 hours after coronary artery occlusion and necrosis only 90,000 cells. Myocyte apoptosis continued to represent the major form of cell death, affecting 6.6 million cells at 4.5 hours, whereas myocyte necrosis peaked at 1 day, including 1.1 million cells. Apoptotic myocyte cell death was also present in the surviving portion of the wall adjacent to and remote from the infarcted myocardium where it peaked at 1-2 days. At this interval, 700/10(6) and 110/10(6) myocyte nuclei were undergoing apoptosis in the non-infarcted tissue bordering on and away from the ischemic area, respectively. Myocyte necrosis was absent in the viable myocardium after infarction. Since mechanical forces produced by pathologic loads may activate apoptosis, papillary muscles were exposed to high levels of resting tension in vitro and the magnitude of cell death in these samples was determined. Overstretching resulted in a 21-fold increase in apoptotic myocyte cell death which was coupled with the formation of reactive oxygen species, side-to-side slippage of myocytes, and depressed tension generation of the myocardium. In conclusion, apoptotic myocyte cell death plays a major role in ventricular remodeling after infarction, but whether physical forces, oxidant stress, architectural rearrangement of myocytes, and impaired force development of the myocardium in vivo are causaly related requires further investigation.

Role of skeletal muscle in thiazolidinedione insulin sensitizer (PPARgamma agonist) action.

Thiazolidinedione (TZD) insulin sensitizers are specific agonists of peroxisome proliferator activated receptor (PPAR)gamma. However, their mechanism of action and the in vivo target tissue(s) that mediate insulin sensitization remain poorly defined. Although PPARgamma messenger RNA expression has been reported in skeletal muscle, the expression of PPARgamma within myocytes in intact muscle tissue has not been examined. An antipeptide PPARgamma antibody was generated; immunohistochemistry was then used to demonstrate that PPARgamma is present within nuclei of myocytes [in both skeletal (white and red fibers) and cardiac tissue (rodent and human)]. The effect of insulin sensitizer treatment on muscle insulin action was studied using ob/ob mice after 4 days dosing with a potent (6 nM PPARgamma Kd) TZD (10 mg/kg x day). 2-deoxyglucose (2-DOG) uptake was then assessed in freshly isolated soleus muscles from lean vs. ob/ob vs. TZD-treated ob/ob mice. In lean mouse muscles, 2-DOG uptake was stimulated by 82%, 95%, 165% (with 25, 100, 2000 microU/ml insulin); muscles from ob/ob were severely insulin resistant (<80% stimulation with 2000 microU/ml insulin). Muscles from TZD-treated ob/ob displayed a normal insulin response with 100 (71%) or 2000 (158%) microU/ml insulin. Additional studies were performed using ZDF rats treated with/without TZD for 7 days. In vivo 2-DOG glucose uptake into soleus, gastrocnemius, and diaphragm muscles was measured during euglycemic-hyperinsulinemic clamp. Compared with lean rats, muscle 2-DOG uptake in ZDF was reduced by 52% (soleus) or 71% (diaphragm). Partial (40-60%) normalization of the reduced 2-DOG uptake was evident in TZD-treated ZDF rats. In contrast to the effect of in vivo treatment on muscle insulin action, preincubation of isolated soleus muscles from naive lean or ob/ob mice for 5 h with 100 nM TZD did not affect insulin-stimulated 2-DOG uptake. We conclude: 1) PPARgamma is expressed in myocytes within skeletal and cardiac muscle. 2) In vivo activation of PPARgamma by treatment of insulin-resistant mice/rats with a potent TZD corrects impaired muscle insulin action. 3) The lack of a direct effect on muscle after 5 h in vitro TZD incubation suggests that changes in insulin action may require a longer duration of PPARgamma activation or that improved muscle insulin sensitivity may result from an indirect in vivo effect of PPARgamma activation (e.g. changes in systemic lipid metabolism).

Role of G1 phase cyclins and cyclin-dependent kinases during cardiomyocyte hypertrophic growth in rats.

Cell cycle regulatory molecules are implicated in cardiomyocyte hypertrophy. We have investigated protein expression of cyclins A, D1-3, and E and cyclin-dependent kinases (CDKs) 2, 4, 5, and 6 in left ventricular (LV) tissues during the development of LV hypertrophy in rats following aortic constriction (AC). Compared with their expression in sham-operated controls (SH), expression of cyclins D2 and D3 and of CDK4 and CDK6 increased significantly from day 3 to day 21 after AC concomitant with increased LV mass. However, no significant difference was observed for CDK2 or CDK5. Cyclins A, D1, and E were undetectable. In vitro kinase activities of CDK4 and CDK6 increased approximately 70% from day 7 to day 14 in AC myocytes compared with SH myocytes (P < 0.03). Fluorescence-activated cell sorter analysis revealed a G0/G1 to G2/M phase progression in AC myocyte nuclei (22.0 +/- 1.1% in G2/M) by day 7 postoperation compared with progression in SH myocyte nuclei (14.0 +/- 0.8% in G2/M; P < 0.01). Thus an upregulation of certain cell cycle regulators is associated with cardiomyocyte hypertrophy.

Numbers of nuclei in different tissue compartments of fetal ventricular myocardium from 16 to 35 weeks of gestation.

The aim of this study was to examine mechanisms of growth in different tissue compartments of the ventricular myocardium of prenatal human hearts. To this end, stereological methods were applied in order to estimate tissue volumes and total numbers of myocyte, connective tissue and endothelial nuclei in hearts collected after death at between 16 and 35 weeks of gestation. Volumes of tissue compartments were obtained after multiplying volume densities (estimated by test-point counting) by ventricular volumes (estimated from ventricular mass and tissue density). Absolute numbers of nuclei were calculated in similar fashion from corresponding nuclear packing densities (estimated using physical disectors). The volumes of all three tissue compartments increased linearly over the period of gestation examined, and in each case, the increase in tissue volume appeared to be due entirely to proliferation. Numbers of all three types of nuclei increased linearly whilst tissue volumes per nucleus remained constant. The net rate of production of myocyte nuclei was 35 x 10(7) per week (2.1 million nuclei per hour). The net rate of production of connective tissue nuclei was 12 x 10(7) per week (0.7 million nuclei per hour) and that for endothelial cell nuclei was 5.1 x 10(7) per week (0.3 million nuclei per hour). Predictions are made about the postnatal ages at which adult ratios of different nuclear types might be attained.

Coxsackievirus B3-Induced Myocarditis: Perforin Exacerbates Disease, But Plays No Detectable Role in Virus Clearance

Viral myocarditis is remarkably common, being detected in approximately 1% of unselected asymptomatic individuals. Many cases are attributable to enteroviral infection, and in particular to coxsackievirus B3. The underlying pathogenesis is controversial, but most studies admit the important immunopathological role of infiltrating CD8+ (cytotoxic) T lymphocytes (CTLs). We have previously shown that CTLs play conflicting roles in coxsackievirus B (CVB) myocarditis; they assist in controlling virus replication, but also are instrumental in causing the extensive inflammatory disease, which often results in severe myocardial scarring. A role for perforin, the major CTL cytolytic protein, in CVB myocarditis has been suggested, but never proven. In the present study we use perforin knockout (PKO) mice to show that perforin plays a major role in CVB infection; in broad terms, perforin is important in immunopathology, but not in CVB clearance. For example, PKO mice are better able to withstand a normally lethal dose of CVB (100% survival of PKO mice compared with 90% death in +/+ littermates). In addition, PKO mice given a nonlethal dose of CVB develop only a mild myocarditis, whereas their perforin+ littermates have extensive myocardial lesions. The myocarditis in PKO mice resolves more quickly, and these mice show minimal histological sequelae; in contrast, late in disease the perforin+ mice develop severe myocardial fibrosis. PKO mice, despite lacking this major CTL effector function, can control the infection and eradicate the virus; growth kinetics and peak CVB titers are indistinguishable in PKO and perforin+ mice. Therefore, the immunopathological and antiviral effects of CTLs can be uncoupled by ablation of perforin; this offers a promising target for therapy of myocarditis. Furthermore, we evaluate the possible roles of apoptosis, and of chemokine expression, in CVB infection. In perforin+ mice, apoptotic cells are detected within the inflammatory infiltrate, whereas in their PKO counterparts, apoptotic myocyte nuclei are seen. Chemokine expression in both PKO and perforin+ mice precedes and parallels the course of myocarditis. Several chemokines are detectable earlier in PKO mice than in perforin+ mice, but PKO mice show reduced peak levels, and chemokine expression decays sooner. In particular, MIP-1alpha expression is barely detectable at any time point in PKO mice, but it is readily identified in perforin+ animals, peaking just before the time of maximal myocarditis; this is particularly interesting, given that MIP-1alpha knockout mice are resistant to CVB myocarditis, but remain able to control viral infection. Thus, the chemokine pathway offers a second route of intervention to diminish myocarditis and its sequelae, while permitting the host to eradicate the virus.

Cardiac and extracardiac expression of Csx/Nkx2.5 homeodomain protein.

Csx/Nkx2.5 is an evolutionary conserved homeobox gene related to the Drosophila tinman gene, which is essential for the dorsal mesoderm formation. Expression of Csx/Nkx2.5 mRNA is the earliest marker for heart precursor cells in all vertebrates so far examined. Previous studies have demonstrated that Csx/Nkx2.5 mRNA is highly expressed in the heart and at lower levels in the spleen, tongue, stomach, and thyroid in the murine embryo. Since some developmental genes are regulated by posttranscriptional mechanisms, we analyzed the developmental pattern of Csx protein expression at the single-cell level using Csx-specific antibodies. Immunohistochemical analysis of murine embryos at 7.8 days post coitum revealed that Csx protein is strongly expressed in the nucleus of endodermal and mesodermal cells in the cardiogenic plate. Subsequently, in the heart, Csx protein was detected only in the nucleus of myocytes of the atrium and the ventricle through the adult stage. During the fetal period, Csx protein expression in the nucleus was also noted in the spleen, stomach, liver, tongue, and anterior larynx. Unexpectedly, confocal microscopy revealed that Csx immunoreactivity was detected only in the cytoplasm of a subset of cranial skeletal muscles. Csx protein was not detected in the thyroid glands. The expression of Csx protein in all organs was markedly downregulated after birth except in the heart. These results raise the possibility that Csx/Nkx2.5 may play a role in the early developmental process of multiple tissues in addition to its role in early heart development.

Alteration in myosatellite cell commitment with muscle maturation

Myosatellite cells are myoblasts found between the basal lamina and sarcolemma of myofibers of postnatal mice. The extent to which these cells are programmed, upon differentiation, to express isoforms of contractile protein genes specific to the type of fiber with which they are associated has been evaluated in vitro using myosatellite cells derived from the soleus and the extensor digitorum longus muscles (EDL) of 4-day-old and adult transgenic mice, which express nuclear localizing beta-galactosidase (nlsbeta-gal) under the control of the promoter and 3' enhancer of the gene encoding fast myosin light chain 3F (MLC3F) (Kelly et al. [1995] J. Cell Biol. 129:383-396). Cultures were allowed to differentiate either as myocytes (mononucleated cells), to prevent possible modification of the myosatellite phenotype by other myonuclei in mosaic myotubes, or as myotubes. Transgene expression was age related, with 90% and 70% of the myocytes derived from the neonatal EDL and soleus muscles (muscles that had not yet achieved their mature phenotype), respectively, having nuclei encoding beta-gal; 61% and 32% of the myocyte nuclei derived from myosatellite cells of the adult EDL (a fast muscle) and the adult soleus muscle (a mixed muscle containing many slow myofibers), respectively, expressed this transgene. Because myosatellite cells found in adult muscles are the progeny of those found in the neonate, an alteration of myosatellite cell commitment to express this transgene occurs with muscle maturation. Because expression of the transgene in neonatal and adult muscle in vivo reflects the expression of the endogenous MLC3F gene (Kelly et al. [1995] J. Cell Biol. 129:383-396), it is likely that expression of the transgene by differentiated myosatellite cells reflects the extent of commitment of these cells to produce MLC3F. A hypothesis is presented that MLC3F is widely expressed in developing muscles but eliminated in myofibers that undergo maturation toward a slower phenotype.

Pacing-induced heart failure in dogs enhances the expression of p53 and p53-dependent genes in ventricular myocytes.

Rapid ventricular pacing in dogs is characterized by a dilated myopathy in which myocyte cell death by apoptosis may play a significant role in the impairment of cardiac pump function. However, the molecular mechanisms implicated in the modulation of programmed cell death under this setting remain to be identified. Moreover, questions have been raised on the specificity and sensitivity of the histochemical detection of DNA strand breaks in nuclei by the terminal deoxynucleotidyl transferase (TdT) reaction. Changes in the expression of Bcl-2 and Bax and their transcriptional regulator, p53, were determined by Western blot analysis in myocytes isolated from dogs affected by pacing-induced heart failure. A mobility shift assay for p53 binding activity was also performed. In addition, apoptosis was measured by confocal microscopy, which allowed the simultaneous detection of chromatin alterations and DNA damage. p53 DNA binding activity to the bax promoter was increased in nuclear extracts from myocytes obtained from failing hearts, and this response was associated with enhanced expression of Bax protein, 52%, and attenuation of Bcl-2, -92%. Immunolabeling of p53 in myocyte nuclei, measured by confocal microscopy, was 100% higher in cells from paced hearts. The combination of the TdT assay and confocal microscopy demonstrated that 20 myocyte nuclei per 10(6) were undergoing apoptosis in control myocardium and 4000 per l0(6) after pacing. Moreover, DNA laddering was shown in myocytes by agarose gel electrophoresis of DNA fragments. The activation of p53 and p53-dependent genes may be critical in the modulation of myocyte apoptosis in pacing-induced heart failure.

Histopathologic effects of meconium on human umbilical artery and vein: In vitro study

Objective To determine the histopathologic effects of meconium on human umbilical artery and vein. Methods Umbilical cords from six patients with uncomplicated, singleton, term gestations were obtained immediately after delivery. One centimeter segments from each cord were flushed, isolated, placed in either modified Krebs solution alone or modified Krebs solution with various concentrations (1%, 10%, or 25%) of fresh meconium, and then incubated at pH 7.2, temperature 37°C, PCO2 50–55 mmHg, PO2 40–45 mmHg for 1, 6, 12, or 24 h. The specimens were then fixed, stained, and examined under light microscopy. Results Umbilical arteries exhibited focal vacuolation of the endothelium after exposure of the cord to meconium. Umbilical veins revealed: 1) endothelial loss proportionate to the meconium concentration at all intervals (P < 0.05), 2) increased density or focal absence of the internal elastic lamina, and 3) focal loss of myocyte nuclei. Rare nonpigmented macrophages and increased mast cells were identified in the Wharton's jelly. Conclusions Exposure to meconium in vitro results in histopathologic changes in the umbilical artery and vein. J. Matern.-Fetal Med. 6:356–361, 1997. © 1997 Wiley-Liss, Inc.