Laser microdissection and capture of pure cardiomyocytes and fibroblasts from infarcted heart regions: perceived hyperoxia induces p21 in peri-infarct myocytes

Myocardial infarction caused by ischemia-reperfusion in the coronary vasculature is a focal event characterized by an infarct-core, bordering peri-infarct zone and remote noninfarct zone. Recently, we have reported the first technique, based on laser microdissection pressure catapulting (LMPC), enabling the dissection of infarction-induced biological responses in multicellular regions of the heart. Molecular mechanisms in play at the peri-infarct zone are central to myocardial healing. At the infarct site, myocytes are more sensitive to insult than robust fibroblasts. Understanding of cell-specific responses in the said zones is therefore critical. In this work, we describe the first technique to collect the myocardial tissue with a single-cell resolution. The infarcted myocardium was identified by using a truncated hematoxylin-eosin stain. Cell elements from the infarct, peri-infarct, and noninfarct zones were collected in a chaotropic RNA lysis solution with micron-level surgical precision. Isolated RNA was analyzed for quality by employing microfluidics technology and reverse transcribed to generate cDNA. Purity of the collected specimen was established by real-time PCR analyses of cell-specific genes. Previously, we have reported that the oxygen-sensitive induction of p21/Cip1/Waf1/Sdi1 in cardiac fibroblasts in the peri-infarct zone plays a vital role in myocardial remodeling. Using the novel LMPC technique developed herein, we confirmed that finding and report for the first time that the induction of p21 in the peri-infarct zone is not limited to fibroblasts but is also evident in myocytes. This work presents the first account of an analytical technique that applies the LMPC technology to study myocardial remodeling with a cell-type specific resolution.     

Characterization of endogenous phosphorylation in isolated cardiac sarcolemma

The cardiac sarcolemma contains kinases which catalyze the incorporation of 32P-phosphate into acid stable and acid precipitable membrane components of low molecular weight. The phosphorylation is not influenced by cyclic AMP or calmodulin. Analysis of phosphorylation products using proteolytic digestion, organic solvent extraction, thin layer chromatography and gel filtration reveals both polypeptides and lipids as kinase substrates. Polypeptides are phosphorylated at their serine and threonine residues, while lipid phosphorylation gives rise to 32P-labelled phosphatidylinositol phosphates and some nonidentified compounds. Phosphorylated polypeptides and phosphorylated lipids do not separate in SDS polyacrylamide gel electrophoresis. On the basis of the fast time course of 32P-phosphate incorporation, it may be supposed that endogenous phosphorylation may play a role in the short term regulation of the cardiac sarcolemmal function.     

Characterization of catecholamine uptake2 in isolated cardiac myocytes

Extraneuronal catecholamine uptake was investigated in isolated quiescent rat myocardial cells. By administration of (³H-)(–)noradrenaline concentration of 22 nmol/l up to 1000 mol/l the following data were obtained: (1) The K_M of the uptake process amounted to 260 mol/l, the V_max to 4.24 nmol/(10 min × mg Protein) corresponding to 179 nmol/(min × g_WWt)(WWT = Wet Weight). (2) The uptake was largely inhibited by the uptake₂-inhibitors corticosterone (100 mol/l), isoprenaline (IC so = 30.6 mol/l), and O-methylisoprenaline (IC₅₀ = 2.1 pmol/l), but not by the uptake₁-inhibitors cocaine (100 mol/l) and desipramine (10 mol/l). (3) The affinity-values K_M and IC₅₀ closely agreed with those already known, but the V_max-value was higher than those obtained in whole rat hearts by a factor of at least 1.79. This is caused presumably by the voltage dependence of the uptake mechanism and the resulting inhibition of uptake 2 during the periods of depolarisation in beating hearts of other studies.     

Impact of hyperoxia on cardiac pathophysiology

Mechanical ventilation with high oxygen therapy (hyperoxia) is widely implemented in critical care and ICU settings. Although supplemental oxygen is beneficial to treat hypoxia, its use is also associated with poor outcomes and high mortality in patients. Lung injury due to hyperoxia exposure has been well-documented in patients, including in adults and neonates. Thus, lung injury due to hyperoxia has been extensively researched in both preclinical and clinical studies. However, hyperoxia has also been shown to be associated with hemodynamic changes in patients in ICU, including reductions in heart rate, stroke volume, and cardiac output. In addition, certain experimental studies report that hyperoxia exposure in neonates results in cardiac dysfunction in later adult life. Despite this, until recently, the impact of hyperoxia within the heart has not been well studied, or reported, specifically in adult experimental models. To close this significant gap, our lab has sought to clarify hyperoxia-induced cardiac pathophysiology in adult murine models. This review discusses the current findings regarding the cardiovascular impact of hyperoxia exposure.     

Impact of brief oxidant stress on primary adult cardiac fibroblasts

Reperfusion of ischemic myocardium (I/R) is associated with local release of a brief pulse of reactive oxygen species. The purpose of this study was to determine the effects of brief H2O2 stimulation on primary adult cardiac fibroblast phenotype. We demonstrate that brief H2O2 exposure results in transient phosphorylations of p38 and ERK which peaked by 15 min. Proliferation was minimally affected by either H2O2 or MAPK inhibition. Pretreatment with SB203580 or U0126 revealed that p38 enhances or maintains migration rates while ERK retarded migration. Peroxide exposure increased necrosis from 4% at baseline to >12% while reducing apoptosis by 3.5-fold. p38 inhibition resulted in increased necrosis and apoptosis while ERK inhibition had minimal effects. In conclusion, primary adult cardiac fibroblasts exposed to brief H2O2 exhibit an altered phenotype characterized by reduced migration and apoptosis and increased necrosis resulting, in part, from the differential effects of p38 and ERK signaling.     

Characterization of phorbol diester binding to isolated cardiac myocytes

There are specified and saturable binding sites for [20-3H]phorbol-12,13-dibutyrate on enzymatically dissociated rat cardiac myocytes. At 37 degrees C, maximal binding occurs within 20 min, with a KD of 3.9 nM and Bmax of 0.275 pmol/mg. [3H]Phorbol dibutyrate binding is blocked by 12-O-tetradecanoyl phorbol-13-acetate but not by 4 alpha-phorbol or 4 alpha-phorbol-12,13-dibutyrate. Dibucaine, tetracaine, chlorpromazine, and phospholipase C lowered phorbol binding through a competitive mechanism. Similarly, unsaturated (but not saturated) diacylglycerols competed with [3H]phorbol dibutyrate for the binding site. There was a progressive decline in specific binding of phorbol diesters to cardiac myocytes which occurred primarily during the first 3 weeks of postnatal life. Cardiac phorbol diester receptors may mediate protein kinase C-dependent effects on important cellular functions such as Ca2+ transport.     

Heat acclimation: cardiac performance of isolated rat heart

Cardiac performance was studied in the isolated perfused hearts of rats heat acclimated at 34 degrees C (AC) and their age-matched controls (C). The pressure-volume curves during isovolumetric conditions showed a shift to the right in AC compared with C hearts. At similar left ventricular (LV) volumes end-diastolic and peak systolic pressures of AC hearts were lower, but no difference was observed in the maximal pressure developed at the highest LV volumes measured. In both C and AC hearts the developed force decreased as pacing rate increased. AC and C heart responses were the same up to 250 pulses/min. At higher frequencies the amplitude of the developed force of AC hearts was smaller than that of the controls. In accordance the tension produced by very early premature beat reduced in AC compared with C hearts. Since no hypertrophy was observed in AC hearts, it is concluded that heat acclimation results in a change in the intrinsic properties of the AC hearts exhibited by increased compliance, reduced chamber stiffness, and a decrease in the tension developed for each volume load. It is also suggested that at a high beating rate AC hearts fail to restitute its contractility as quickly as C hearts.     

Characterization of mono-, di- and triacylglycerol lipase activities in the isolated rat heart

The lipolytic activities of heart tissue towards full and partial acylglycerols were characterized. Tissue lysosomal, acid lipase activity (pH 4.8) was inhibited by high salt, protamine sulfate, NaF, MgATP, Triton X-100, serum and the esterase-inhibitor diethylparanitrophenyl phosphate. The tissue neutral triacylglycerol lipase activity (pH 7.4) was recovered predominantly in the microsomal and soluble fractions and exhibited essentially identical properties towards activators (serum, apolipoprotein C-II) and reagents (NaCl, Triton X-100, NaF, MgATP and diethylparanitrophenyl phosphate) relative to vascular lipoprotein lipase, except for protamine sulfate which increased the serum-stimulated neutral triacylglycerol lipase activity. Triacylglycerol hydrolysis at acid pH was incomplete, whereas at neutral pH full hydrolysis occurred. Myocardial mono- and diacylglycerol lipase activities, with pH optima of 8.0 and 7.4, respectively, were recovered in the microsomal fraction. They differed immunologically from neutral lipase and lipoprotein lipase and did not bind to heparin-Sepharose 4B. They were kinetically different, partially inhibited by NaCl and differentially affected by protamine sulfate. NaF, Triton X-100 and diethylparanitrophenyl phosphate. Our data suggest that endogenous hydrolytic activity against full and partial acylglycerols is mediated by separate enzymes.     

Use of micromachined probes for the recording of cardiac electrograms in isolated heart tissues

Micromachined probes, with iridium (Ir) microelectrodes on silicon shanks, were evaluated to assess their suitability for cardiac electrogram recording. The electrochemical activation (anodic oxidation) procedure for the circular Ir microelectrode was investigated using the square wave potential according to the electrode size, number of cycles, and cathodic-anodic potential level of the square wave. Increase in the charge storage capacity was pronounced either in smaller electrodes or with higher potential level of the square wave. The electrode impedance reduced in a similar manner with increasing number of cycle irrespective of the electrode size. With either lower potential level (-0.70/+0.60 V) or smaller number of cycle (200 cycles) than those for the activation of stimulating electrode, the likelihood of overactivation of the recording microelectrode can be minimized. These anodic IrOx film (AIROF) microelectrodes were used for the recording of extracellular electrograms in two different ex vivo cardiac tissue preparations. A single-shank microprobe was applied to the left ventricle of a mouse heart. Both the spontaneous and paced transmural responses propagating between epicardium and endocardium were obtained. Longitudinal cardiac wavefronts propagating along the rabbit papillary muscle were also recorded with a unique multiple-shank design. The measured mean amplitude and the propagation velocity of the extracellular voltage were 12.2 +/- 1.8 mV and 58.9 +/- 2.2 cm/s, respectively (n = 27). These microprobes with precisely defined electrode spacing make a useful tool for the spatial and temporal mapping of electrical properties in isolated heart tissues ex vivo.     

Implication of connexins 40 and 43 in functional coupling between mouse cardiac fibroblasts in primary culture

Cardiac fibroblasts contribute to the structure and function of the myocardium. However their involvement in electrophysiological processes remains unclear; particularly in pathological situations when they proliferate and develop fibrosis. We have identified the connexins involved in gap junction channels between fibroblasts from adult mouse heart and characterized their functional coupling. RT-PCR and Western blotting results show that mRNA and proteins of connexin40 and connexin43 are expressed in cultured cardiac fibroblasts, while Cx45 is not detected. Analysis of gap junctional communications established by these connexins with the gap-FRAP technique demonstrates that fibroblasts are functionally coupled. The time constant of permeability, k, calculated from the fluorescence recovery curves between cell pairs is 0.066 ± 0.005 min^− 1 (n = 65). Diffusion analysis of Lucifer Yellow through gap junction channels with the scrape-loading method demonstrates that when they are completely confluent, a majority of fibroblasts are coupled forming an interconnecting network over a distance of several hundred micrometers. These data show that cardiac fibroblasts express connexin40 and connexin43 which are able to establish functional communications through homo and/or heterotypic junctions to form an extensive coupled cell network. It should then be interesting to study the conditions to improve efficiency of this coupling in pathological conditions.     

Enzyme release and mitochondrial activity in reoxygenated cardiac muscle: relationship with oxygen-induced lipid peroxidation

The aim of this work was to precisely determine the sites of the peroxidative action on unsatured lipids by oxygen-derived free radicals and the lytic cell damage on reoxygenated perfused hearts. The cellular load of lipid peroxidation products (malondialdehyde) during the reoxygenation was dependent on PO2. This unfavorable biochemical response was linked to creatine kinase leakage, alteration of coronary flow and mitochondrial injury. When an enzymatic (superoxide dismutase, 290 IU/minute) or tripeptide scavenger of oxygen radicals (reduced glutathione, 0.5 mmol/l) was administered at the end of hypoxia and during reoxygenation, the abnormal intolerance of hypoxic heart to molecular oxygen was significantly weakened; the load of lipid peroxides load, enzyme release, and vascular alteration were all reduced. Moreover, mitochondrial activity was enhanced and the oxygen-induced uncoupling of mitochondrial remained limited: both the respiratory control ratio (RCR) and the ADP/O ratio were higher than in control reoxygenated hearts. The inhibition by rotenone (100 mumol/l) of reoxidation of electron chain transfer during oxygen readmission also reduced the unfavorable cardiac accumulation of lipid peroxidation products and the release of creatine kinase. These data demonstrate that in the oxygen paradox, the peroxidative attack on lipids plays an important role in inducing alterations of sarcolemmal permeability and mitochondrial activity. An uncontrolled reactivation of oxidative function of mitochondria during reoxygenation enhances the synthesis of oxygen-derived free radicals and triggers the peroxidation of cardiac lipids resulting in irreversible injury to cellular and intracellular membranes.     

Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts

Erythropoietin has recently been shown to have effects beyond hematopoiesis such as prevention of neuronal and cardiac apoptosis secondary to ischemia. In this study, we evaluated the in vivo protective potential of erythropoietin in the reperfused rabbit heart following ventricular ischemia. We show that "preconditioning" with erythropoietin activates cell survival pathways in myocardial tissue in vivo and adult rabbit cardiac fibroblasts in vitro. These pathways, activated by erythropoietin in both whole hearts and cardiac fibroblasts, are also activated acutely by ischemia/reperfusion injury. Moreover, in vivo studies indicate that erythropoietin treatment either prior to or during ischemia significantly enhances cardiac function and recovery, including left ventricular contractility, following myocardial ischemia/reperfusion. Our data indicate that a contributing in vivo cellular mechanism of this protection is mitigation of myocardial cell apoptosis. This results in decreased infarct size as evidenced by area at risk studies following in vivo ischemia/reperfusion injury, translating into more viable myocardium and less ventricular dysfunction. Therefore, erythropoietin treatment may offer novel protection against ischemic heart disease and may act, at least in part, by direct action on cardiac fibroblasts and myocytes to alter survival and ventricular remodeling.     

Sex differences in murine cardiac pathophysiology with hyperoxia exposure

Hyperoxia (>90% oxygen) is commonly implemented in mechanically ventilated patients. Reports suggest that hyperoxia is directly associated with in-hospital mortality in ventilated patients. Certain studies also show that mortality in women undergoing mechanical ventilation is significantly higher than that in men. Additionally, females are predisposed to certain cardiac electrophysiological risks, including QTc prolongation. In this study, we assessed the impact of hyperoxia in male and female mice (C57BL/6J) at age 8–10 weeks. On completion of either hyperoxia or normoxia exposures, physical, hemodynamic, biochemical, functional, electrophysiological, and molecular assessments were conducted. Hyperoxia-exposed mice lost a significant amount of body mass, compared with normoxia controls, in both sexes. However, while both genders developed brady-arrhythmia after hyperoxia exposure, female mice exhibited significantly reduced heart rates compared with males, with significantly elevated RR intervals. Additionally, 50% mortality was observed in females, whereas no mortality was reported in males. Furthermore, unlike in male mice, we observed no hypertrophy upon hyperoxia exposure in female mice. We reported that both hyperoxia-treated male and female mice exhibit significant hyperdynamic left ventricular ejection fraction, which is marked by % ejection fraction > 70 compared with the normoxia controls. We also noted significant reductions in stroke volume and cardiac output in both mice with hyperoxia. Surface ECG also demonstrated that hyperoxia exposure significantly augments RR, PR, QRS, QTc, and JT intervals in both sexes. Molecular analysis of left ventricular tissue demonstrated dysregulation of potassium ion channels in hyperoxia-treated males and females. In summary, we determined that sex differences are present with 72 hr hyperoxia exposure.     

Cardioprotection by Cu, Zn-superoxide dismutase is lost at high doses in the reoxygenated heart

Limited dose-response curves for superoxide dismutase (SOD) were assessed in isolated and in vivo hearts. SOD at 2.3, 7, 20, or 50 mg/L suppressed CK release in Langendorff rat hearts by 61%, 63%, 72%, and 30%, respectively. SOD at 0.5, 1, 5, and 50 mg/L suppressed LDH release in Langendorff rabbit hearts by 32%, 48%, 54%, and −12%, respectively. In rabbit hearts subjected to coronary artery ligation and reperfusion in vivo, SOD at 2, 5,or 15 mg/kg reduced infarct size by 10%, 30% or 19%, respectively, while 50 mg/kg increased infarct size by 28%. In conclusion, while SOD was protective at low doses in all models, protection was lost at higher doses in the isolated rat and rabbit hearts, and exacerbation of damage was seen in the in vivo rabbit hearts.     

Characterization of cardiac hypertrophy and heart failure due to volume overload in the rat.

Alterations in general characteristics and morphology of the heart, as well as changes in hemodynamics, myosin heavy chain isoforms, and beta-adrenoceptor responsiveness, were determined in Sprague-Dawley rats at 1, 2, 4, 8, and 16 wk after aortocaval fistula (shunt) was induced by the needle technique. Three stages of cardiac hypertrophy due to volume overload were recognized during the 16-wk period. Developing hypertrophy occurred within the first 2 wk after aortocaval shunt was induced and was characterized by a rapid increase of cardiac mass in both left and right ventricles. Compensated hypertrophy occurred between 2 and 8 wk after aortocaval shunt where normal or mild depression in hemodynamic function was observed. Decompensated hypertrophy or heart failure occurred between 8 and 16 wk after aortocaval shunt and was characterized by circulatory congestion, decreased in vivo and in vitro cardiac function, and a shift in myosin heavy chain isozyme expression. However, the positive inotropic effect of isoproterenol was augmented at all times during the 16-wk period. Characterization of beta-adrenoceptor binding in failing hearts at 16 wk revealed a significant increase in beta(1)-receptor density, whereas beta(2)-receptor density was unchanged. Consistent with this, basal adenylyl cyclase activity was significantly increased, and both isoproterenol- and forskolin-stimulated adenylyl cyclase activities were also increased. These results indicate that upregulation of beta-adrenoceptor signal transduction is a unique feature of cardiac hypertrophy and failure induced by volume overload.     

Characterization of autoantigenic sites on isolated dog heart mitochondria.

1. Anti-heart mitochondria autoantibodies were developed in serum from dogs following experimental myocardial infarction. 2. Heart mitochondria frozen and thawed repeatedly in a sucrose/Tris-chloride buffer retained both their functional integrity as measured by the respiratory control ratio and their ability to serve as an antigen in a complement fixation test. Mitochondria frozen and thawed in a potassium chloride/Tris-chloride buffer lost both their functional integrity and their autoantigenic activity after one freeze-thaw cycle. 3. Extraction of the heart mitochondria with acetone/water mixtures to remove phospholipids from the membrane led to a complete loss of the ability of the mitochondria to react in the complement fixation test but did not affect the ability of the membranes to bind autoantibody in absorption experiments. 4. Treatment of the mitochondrial membranes with increasing concentrations of trypsin caused a loss of up to approximately 50% of the membrane protein with a gradual decrease in the autoantigenic activity of the membrane without impairment of the ability of the membrane to bind autoantibody. 5. Removal of up to 90% of the sialic acid of the mitochondrial membrane with neuraminidase resulted in a considerable increase in the complement-fixing autoantigenic activity of the membrane without changing the apparent ability of the membrane to bind autoantibody in absorption experiments. 6. Exposure of mitochondrial membranes to autoantibody and complement caused an inhibition of both an inner mitochondrial membrane enzyme, i.e. cytochrome oxidase (48%) and an outer mitochondrial membrane enzyme, i.e. NADH cytochrome c reductase (rotenone insensitive) (37%).