Adult Rabbit Cardiac Myocytes Research Articles

Urocortin II Regulates NFAT Transcription Factor in Adult Rabbit Cardiac Myocytes

Nuclear factor of activated T cells (NFAT) transcription factors play a key role during cellular remodelling associated with hypertrophy and heart failure (HF). We tested the hypothesis that the cardioprotective peptide Urocortin II (UcnII) regulates translocation and activation of NFAT.Rabbit ventricular myocytes were infected with recombinant adenoviruses encoding for NFAT-GFP fusion proteins (isoforms NFATc1 and NFATc3). The subcellular distribution of NFAT was quantified as the ratio of NFATnuc to NFATcyt fluorescence (RNFAT) and nuclear-cytosolic NFAT translocation was expressed as changes of RNFAT. Under basal unstimulated conditions NFATc3 was predominantly localized in the cytoplasm, whereas NFATc1 displayed a nuclear localization. UcnII (100 nM) caused NFATc1 export to the cytoplasm (RNFAT decreased from 2.25 to 1.03 after 24 hrs). UcnII did not affect nuclear-cytosolic NFATc3 distribution.UcnII-induced NFATc1 export to the cytoplasm was significantly (P<0.05) reduced (by 58-79%) by inhibition of guanylate cyclase (ODQ, 10 μM), calcineurin (Cyclosporin A, 1 μM), and GSK3s (Alsterpaullone, 1 μM). Inhibition of PI3K (Wortmannin, 300 nM) and nuclear transport protein crm1 (Leptomycin B, 40 nM) prevented the redistribution of NFATc1 to the cytoplasm completely.In ventricular myocytes from failing rabbit hearts, nuclear localization of NFATc3 was significantly enhanced compared to normal myocytes (RNFAT 0.78 vs. 0.58). Stimulation of HF myocytes with UcnII resulted in a translocation of NFATc3 back to the cytoplasm (RNFAT=0.56). In HF myocytes the nuclear localization of NFATc1 was less pronounced compared to normal myocytes (RNFAT: 1.97 in HF vs. 2.86 in normal ventricular cells). UcnII induced a further redistribution of NFATc1 out of the nucleus (RNFAT=0.97).We conclude that UcnII enhances the export of NFAT from the nucleus to the cytoplasm in ventricular myocytes involving the PI3K/cGMP/PKG/calcineurin- and PI3K/Akt/GSK3s-pathways and could thus positively affect remodeling in hypertrophy and HF.

Beta-Adrenergic Activation Enhances Histone Deacetylase 4 nuclear Localization via Pka and Counters Camkii-Dependent Effects in Adult Rabbit Cardiac Myocytes

Nuclear localization of histone deacetylase 4 (HDAC4), that represses transcription and possibly heart failure by suppressing the transcription factor MEF2, is dependent upon its phosphorylation status. Phosphorylation at three key serines can trigger the nuclear export of HDAC4, while dephosphorylation results in nuclear accumulation. HDAC4 is known to bind CaMKII and phosphorylation by this kinase facilitates nuclear export. A probable PKA phosphorylation site has been identified, suggesting the hypothesis that HDAC4 localization is modulated by both CaMKII and PKA. We tested whether B-adrenergic-induced PKA activity alters HDAC4 localization in adult rabbit ventricular myocytes using confocal imaging and GFP-tagged HDAC4. At baseline, the nuclear to cytosolic ratio of HDAC4 (Nuc/Cyto) was 2.19±.04 (n=638). Myocytes treated with 1µM isoproterenol (Iso) had a 58±8% increase in HDAC4 Nuc/Cyto compared to baseline (n=123). Activation of adenylyl cyclase via forskolin (10µM) produced a similar 50±5% increase in Nuc/Cyto ratio (n=107). CaMKII inhibition at rest with 1µM KN-93 also raised the Nuc/Cyto by 40±6% (n=99), whereas PKA inhibition (2 µM H-89) did not alter baseline HDAC4 localization (n=91). Conversely, phosphatase inhibition (1 µM okadaic acid) decreased basal Nuc/Cyto by 57±5% (n=97). Thus, baseline CaMKII activity limits the degree of nuclear HDAC4 localization, and preventing HDAC4 dephosphorylation exacerbates this. Furthermore, B-adrenergic activation causes HDAC4 nuclear import via PKA (vs other cAMP or B-adrenergic signaling). In conclusion, while CaMKII activates HDAC4 nuclear export, B-adrenergic signaling via PKA favors nuclear retention/import and transcriptional repression in cardiac hypertrophic signaling.

Allele and species dependent contractile defects by restrictive and hypertrophic cardiomyopathy-linked troponin I mutants

Restrictive cardiomyopathy (RCM) is a debilitating disease characterized by impaired ventricular filling, reduced ventricular volumes, and severe diastolic dysfunction. Hypertrophic cardiomyopathy (HCM) is characterized by ventricular hypertrophy and heightened risk of premature sudden cardiac death. These cardiomyopathies can result from mutations in the same gene that encodes for cardiac troponin I (cTnI). Acute genetic engineering of adult rat cardiac myocytes was used to ascertain whether primary physiologic outcomes could distinguish between RCM and HCM alleles at the cellular level. Co-transduction of cardiac myocytes with wild-type (WT) cTnI and RCM/HCM linked mutants in cTnI's inhibitory region (IR) demonstrated that WT cTnI preferentially incorporated into the sarcomere over IR mutants. The cTnI IR mutants exhibited minor effects in single myocyte Ca 2+-activated tension assays yet prolonged relaxation and Ca 2+ decay. In comparison RCM cTnI mutants in the helix-4/C-terminal region demonstrated a) hyper-sensitivity to Ca 2+ under loaded conditions, b) slowed myocyte mechanical relaxation and Ca 2+ transient decay, c) frequency-dependent Ca 2+-independent diastolic tone, d) heightened myofilament incorporation and e) irreversible cellular contractile defects with acute diltiazem administration. For species comparison, a subset of cTnI mutants were tested in isolated adult rabbit cardiac myocytes. Here, RCM and HCM mutant cTnIs exerted similar effects of slowed myocyte relaxation and Ca 2+ transient decay but did not show variable phenotypes by cTnI region. This study highlights cellular contractile defects by cardiomyopathy mutant cTnIs that are allele and species dependent. The species dependent results in particular raise important issues toward elucidating a unifying mechanistic pathway underlying the inherited cardiomyopathies.

Abstract 198: Endothelin-1 and Phenylephrine Activate Global Protein Kinase D Similarly in Adult Cardiac Myocytes, but Differ Greatly in Nuclear versus Sarcolemmal Targeting

Endothelin-1(ET-1) and phenylephrine (PE) both activate hypertrophic signaling in cardiac myocytes via nuclear export of class II histone deacetylase (HDAC), and protein kinase D (PKD) is critical in mediating this for HDAC5. However, we now find that in adult ventricular myocytes PE-induced HDAC5 nuclear export is more critically PKD-dependent than for ET-1. Here, we use novel FRET-based PKD activity reporter (DKAR) and GFP-tagged PKD (both in adenoviral vectors) expressed in adult rabbit cardiac myocytes, along with confocal and TIRF microcsopy to localize ET-1 vs . PE-induced PKD activation and translocation. On-line PKD activity (sensed by DKAR FRET) was similarly activated by 100 nM ET-1, 10 μM PE or 300 nM PDBu (4.6 ± 0.4, 5.9 ± 0.7 or 5.6 ± 0.6 units) in adult myocytes overexpressing PKD. PKD-GFP expressed in myocytes is relatively uniform and cytosolic at rest (slightly higher at Z-lines), and largely non-nuclear (F nuc /F cyto 0.5 ± 0.03). ET-1 caused rapid sustained PKD recruitment to the plasma membrane (46 ± 3% increase) and also at Z-lines, but only slow and modest PKD nuclear import (12 ± 8% rise in F nuc /F cyto ). In contrast, PE more modestly increased membrane and Z-line recruitment (12 ± 0.9%), but caused more dramatic nuclear translocation (47 ± 4% rise in F nuc /F cyto ). PDBu caused both membrane (120 ± 15% increase) and nuclear localization of PKD (44 ± 4% increase in F nuc /F cyto ). Interestingly, SR Ca depletion by thapsigargin caused an ET-1-like effect, whereas strophantidin did not elicit PKD mobilization. We conclude that ET-1 and PE differ in adult myocyte PKD activation, not in extent, but in cellular localization or translocation. These results are consistent with a more prominent nuclear action of PKD in response to PE vs. ET-1, and with the more critical role of PKD in PE-induced HDAC5 nuclear export.

Free and bound intracellular calmodulin measurements in cardiac myocytes

Calmodulin (CaM) is a ubiquitous Ca2+ binding protein and Ca2+-CaM activates many cellular targets and functions. While much of CaM is thought to be protein bound, quantitative data in cardiac myocytes is lacking regarding CaM location, [CaM]free and CaM redistribution during changes in [Ca2+]i. Here, we demonstrated that in adult rabbit cardiac myocytes, CaM is highly concentrated at Z-lines (confirmed by Di-8-ANEPPS staining of transverse tubules) using three different approaches: immunocytochemistry (endogenous CaM), Alexa Fluor 488 conjugate CaM (F-CaM) in both permeabilized cells (exogenous CaM) and in patch clamped intact cells (via pipette dialysis). Using 100nM [CaM]free we washed F-CaM into permeabilized myocytes and saw a two-phase (fast and slow) CaM binding curve with a plateau after 40min of F-CaM wash-in. We also measured myocyte [CaM]free using two modified null-point titration methods, finding [CaM]free to be 50–75nM (which is only 1% of total [CaM]). Higher [Ca2+]i increased CaM binding especially in the nucleus and at Z-lines and significantly slowed F-CaM dissociation rate when F-CaM was washed out of permeabilized myocytes. Additionally, in both permeabilized and intact myocytes, CaM moved into the nucleus when [Ca2+]i was elevated, and this was reversible. We conclude that [CaM]free is very low in myocytes even at resting [Ca2+]i, indicating intense competition of CaM targets for free CaM. Bound CaM is relatively concentrated at Z-lines at rest but translocates significantly to the nucleus upon elevation of [Ca2+]i, which may influence activation of different targets and cellular functions.

Dynamic changes in free Ca-calmodulin levels in adult cardiac myocytes

Despite its multifunctional role in cardiac myocyte function, little is known about dynamic changes in activation state of calmodulin (CaM). Thus, the purpose of this study was to develop a tool to measure Ca bound CaM (Ca–CaM) levels in intact cardiac myocytes. For dynamic measurements of Ca–CaM, we generated an adenoviral vector which expresses a cyan and a yellow fluorescent protein linked by a modified version of the Ca–CaM binding domain of avian smooth muscle myosin light chain kinase. Adult rabbit cardiac myocytes were infected with the Ca–CaM sensing probe or simultaneously infected with viruses containing CaM and the Ca–CaM sensing probe for 24–48 h. Myocytes were then field stimulated (1 Hz) and excited at 440 nm with emitted fluorescence measured at 485 and 535 nm. Changes in [Ca–CaM] are expressed as the ratio of 485 nm/535 nm. Small beat-to-beat changes of [Ca–CaM] were detected, but only when CaM was co-expressed with the sensor. However, upon β-adrenergic stimulation with isoproterenol, there was an increase in the amplitude of the signals during each beat (parallel to the shortening, which is an indirect measure of [Ca] i) and also a rise in the diastolic [Ca–CaM]. Total [CaM] measured by both competitive ELISA and semi-quantitative Western blots was 5–6 μM in isolated adult ventricular myocytes. Our results indicate that there are dynamic changes in free Ca–CaM levels (a phasic component tracking [Ca] i) as well as system memory that integrates the [Ca] i signals (a tonic component).

Nitric oxide induces activation of pkcϵ via a cGMP-dependent mechanism in adult rabbit cardiac myocytes

Nitric oxide induces activation of pkcϵ via a cGMP-dependent mechanism in adult rabbit cardiac myocytes

SERCA2a Overexpression Decreases the Incidence of Aftercontractions in Adult Rabbit Ventricular Myocytes

K. Davia, E. Bernobich, H. K. Ranu, F. del Monte, C. M. N. Terracciano, K. T. MacLeod, D. L. Adamson, B. Chaudhri, R. J. Hajjar and S. E. Harding. SERCA2a Overexpression Decreases the Incidence of Aftercontractions in Adult Rabbit Ventricular Myocytes. Journal of Molecular and Cellular Cardiology (2001) 33, 1005–1015. Slow relaxation and poor contractile response to increasing stimulation frequency in failing human heart have been strongly linked to a decrease in the activity of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a). Restoration of SERCA2a levels using gene transfer has beneficial effects on contractile function but, like β -adrenoceptor stimulation, could potentially produce excess SR Ca2+, arrhythmias and cell death. We have examined the effects of SERCA2a overexpression in adult rabbit cardiac myocytes, and compared changes in relaxation with those following β -adrenoceptor stimulation. Myocytes were infected with an adenovirus carrying both SERCA2a and green fluorescent protein (GFP) for positive identification of infected cells. Myocyte survival was significantly enhanced in the infected cultures. There was a reduction in both time-to-peak contraction and time-to-50% relaxation (R50) 48 h after infection. Time-to-90% relaxation (R90) was particularly improved (non-infected 516±41 ms, AD.SERCA2a-GFP 230±23 ms, n=7 preparations, P<0.001). There was also a decreased incidence of aftercontractions in Ad.SERCA2a-GFP infected myocytes (21±5%v 41±4% in controls, P<0.01). This contrasts with β -adrenoceptor stimulation, which reduced R50 but prolonged R90 by 158±76 ms (P<0.02, n=16). At higher stimulation frequencies (2–3 Hz) contraction amplitude and SR calcium content were increased and diastolic contracture was reduced following SERCA2a overexpression. Overall, increasing levels of SERCA2a resulted in an improvement in systolic and diastolic function and a reduction in cell death and arrhythmic aftercontractions. SERCA2a overexpression therefore lacks the detrimental effects associated with some other inotropic interventions.

Mitochondrial Calcium Transients in Adult Rabbit Cardiac Myocytes: Inhibition by Ruthenium Red and Artifacts Caused by Lysosomal Loading of Ca 2+-Indicating Fluorophores

A cold/warm loading protocol was used to ester-load Rhod 2 into mitochondria and other organelles and Fluo 3 into the cytosol of adult rabbit cardiac myocytes for confocal fluorescence imaging. Transient increases in both cytosolic Fluo 3 and mitochondrial Rhod 2 fluorescence occurred after electrical stimulation. Ruthenium red, a blocker of the mitochondrial Ca 2+ uniporter, inhibited mitochondrial Rhod 2 fluorescence transients but not cytosolic Fluo 3 transients. Thus the ruthenium red-sensitive mitochondrial Ca 2+ uniporter catalyzes Ca 2+ uptake during beat-to-beat transients of mitochondrial free Ca 2+, which in turn may help match mitochondrial ATP production to myocardial ATP demand. After ester loading, substantial amounts of Ca 2+-indicating fluorophores localized into an acidic lysosomal/endosomal compartment. This lysosomal fluorescence did not respond to electrical stimulation. Because fluorescence arose predominantly from lysosomes after the cold loading/warm incubation procedure, total cellular fluorescence failed to track beat-to-beat changes of mitochondrial fluorescence. Only three-dimensionally resolved confocal imaging distinguished the relatively weak mitochondrial signal from the bright lysosomal fluorescence.

Mitochondrial Calcium Transients in Adult Rabbit Cardiac Myocytes: Inhibition by Ruthenium Red and Artifacts Caused by Lysosomal Loading of Ca2+-Indicating Fluorophores

Mitochondrial Calcium Transients in Adult Rabbit Cardiac Myocytes: Inhibition by Ruthenium Red and Artifacts Caused by Lysosomal Loading of Ca2+-Indicating Fluorophores

PKC-dependent activation of p46/p54 JNKs during ischemic preconditioning in conscious rabbits.

A conscious rabbit model was used to study the effect of ischemic preconditioning (PC) on stress-activated kinases [c-Jun NH(2)-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (MAPK)] in an environment free of surgical trauma and attending external stress. Ischemic PC (6 cycles of 4-min ischemia/4-min reperfusion) induced significant activation of protein kinase C (PKC)-epsilon in the particulate fraction, which was associated with activation of p46 JNK in the nuclear fraction and p54 JNK in the cytosolic fraction; all of these changes were completely abolised by the PKC inhibitor chelerythrine. Selective enhancement of PKC-epsilon activity in adult rabbit cardiac myocytes resulted in enhanced activity of p46/p54 JNKs, providing direct in vitro evidence that PKC-epsilon is coupled to both kinases. Studies in rabbits showed that the activation of p46 JNK occurred during ischemia, whereas that of p54 JNK occurred after reperfusion. A single 4-min period of ischemia induced a robust activation of the p38 MAPK cascade, which, however, was attenuated after 5 min of reperfusion and disappeared after six cycles of 4-min ischemia/reperfusion. Overexpression of PKC-epsilon in cardiac myocytes failed to increase the p38 MAPK activity. These results demonstrate that ischemic PC activates p46 and p54 JNKs via a PKC-epsilon-dependent signaling pathway and that there are important differences between p46 and p54 JNKs with respect to the subcellular compartment (cytosolic vs. nuclear) and the mechanism (ischemia vs. reperfusion) of their activation after ischemic PC.

Angiotensin II exacerbates lipopolysaccharide-induced contractile depression in rabbit cardiac myocytes.

In sepsis, lipopolysaccharide (LPS) depresses cardiac function by inducing production of nitric oxide (NO) and its second messenger cGMP. LPS also stimulates ANG II production. We hypothesized that ANG II modulates the cardiac response to LPS. Adult rabbit cardiac myocytes incubated with LPS (10 ng/ml) had increased cardiac cGMP after 6 h (but not within 1 h) [527 +/- 43 vs. 316 +/- 27 (SE) fmol/mg protein in controls, n = 16 each group, P < 0.05]. This was associated with depressed cell shortening with no alterations in Ca2+ transients (indo 1 fluorescence), indicating a decreased myofilament responsiveness to Ca2+. ANG II (100 nM) alone had no effect. However, ANG II with LPS produced higher cGMP levels (1,025 +/- 113 fmol/mg protein, n = 16, P < 0.05 vs. LPS alone), more severe contractile depression, impaired Ca2+ handling, and decreased mitochondrial activity (MTS assay). We conclude that ANG II and LPS have synergistic effects on the activation of NO-cGMP pathways to induce dose-dependent impairments in excitation-contraction coupling in cardiac myocytes.

Phospholamban-to-SERCA2 ratio controls the force-frequency relationship.

The force-frequency relationship (FFR) describes the frequency-dependent potentiation of cardiac contractility. The interaction of the sarcoplasmic reticulum Ca2+-adenosinetriphosphatase (SERCA2) with its inhibitory protein phospholamban (PLB) might be involved in the control of the FFR. The FFR was analyzed in two systems in which the PLB-to-SERCA2 ratio was modulated. Adult rabbit cardiac myocytes were transduced with adenovirus encoding for SERCA2, PLB, and beta-galactosidase (control). After 3 days, the relative PLB/SERCA2 values were significantly different between groups (SERCA2, 0.5; control, 1.0; PLB, 4.5). SERCA2 overexpression shortened relaxation by 23% relative to control, whereas PLB prolonged relaxation by 39% and reduced contractility by 47% (0.1 Hz). When the stimulation frequency was increased to 1.5 Hz, myocyte contractility was increased by 30% in control myocytes. PLB-overexpressing myocytes showed an augmented positive FFR (+78%), whereas SERCA2-transduced myocytes displayed a negative FFR (-15%). A more negative FFR was also found in papillary muscles from SERCA2 transgenic mice. These findings demonstrate that the ratio of phospholamban to SERCA2 is an important component in the control of the FFR.

Confocal Imaging of Both Mitochondrial and Cytosolic Free Ca2+ in Cardiac Myocytes Co-Loaded with Rhod 2 and Fluo 3: Inhibition by Ruthenium Red of Mitochondrial but not Cytosolic Ca2+ Transients

Abstract Previously, we showed rapid mitochondrial Ca2+ transients in adult rabbit cardiac myocytes during the contractile cycle. Ruthenium red, an inhibitor of mitochondrial Ca2+ uptake by the Ca2+ uniporter, inhibits mitochondrial Ca2+ transients in adult rabbit cardiac myocytes during electrical stimulation. Here, we extend this finding to show that ruthenium red inhibition is specific for mitochondrial Ca2+ transients and not cytosolic Ca2+ transients. Ca 2+-tolerant adult rabbit cardiac myocytes were isolated by collagenase and hyaluronidase digestion and loaded in the cold with 2.5 μM Rhod 2-AM for 30 minutes. Subsequently, the cells were incubated in nutrient medium at 37°C for 4-6 hours during which time cytosolic but not mitochondrial Rhod 2 was lost. Subsequently, the myocytes were loaded with 10 μM Fluo 3-AM for 15 minutes at 37°C, conditions that lead to predominantly cytosolic localization of Fluo 3. In sequential scans, the red fluorescence of Rhod 2 and the green fluorescence of Fluo 3 were imaged using a Zeiss LSM 410 laser scanning confocal microscope.

Na-Ca Exchange and the Trigger for Sarcoplasmic Reticulum Ca Release: Studies in Adult Rabbit Ventricular Myocytes

The importance of Na-Ca exchange as a trigger for sarcoplasmic reticulum (SR) Ca release remains controversial. Therefore, we measured whole-cell Ca currents ( I Ca), Na-Ca exchange currents (I NaCa), cellular contractions, and intracellular Ca transients in adult rabbit cardiac myocytes. We found that changing pipette Na concentration markedly affected the relationship between cell shortening (or Ca transients) and voltage, but did not affect the Ca current-voltage relationship. We then inhibited Na-Ca exchange and varied SR content (by changing the number of conditioning pulses before each test pulse). Regardless of SR Ca content, the relationship between contraction and voltage was bell-shaped in the absence of Na-Ca exchange. Next, we rapidly and completely blocked I Ca by applying nifedipine to cells. Cellular shortening was variably reduced in the presence of nifedipine. The component of shortening blocked by nifedipine had a bell-shaped relationship with voltage, whereas the “nifedipine-insensitive” component of contraction increased with voltage. With the SR disabled (ryanodine and thapsigargin pretreatment), I Ca could initiate late-peaking contractions that were ∼70% of control amplitude. In contrast, nifedipine-insensitive contractions could not be elicited in the presence of ryanodine and thapsigargin. Finally, we recorded reverse Na-Ca exchange currents that were activated by membrane depolarization. The estimated sarcolemmal Ca flux occurring by Na-Ca exchange (during voltage clamp steps to +30 mV) was ∼10-fold less than that occurring by I Ca. Therefore, Na-Ca exchange alone is unlikely to raise cytosolic Ca concentration enough to directly activate the myofilaments. We conclude that reverse Na-Ca exchange can trigger SR Ca release. Because of the sigmoidal relationship between the open probability of the SR Ca release channel and pCa, the effects of I Ca and I NaCa may not sum in a linear fashion. Rather, the two triggers may act synergistically in the modulation of SR release.

The relationship between temperature and calcium in acute cell damage after exposure to radiofrequency or thermal energy in isolated neonatal and adult rabbit cardiac myocytes.

Radiofrequency (RF) ablation is a nonsurgical technique using catheter-directed RF energy for treating cardiac arrhythmias in children and adults. Previous reports have suggested that sequestration of calcium (Ca2+) by the sarcoplasmic reticulum may partially protect mature cardiac myocytes from the effects of RF energy. The purposes of this study were to determine whether differences exist between neonatal and adult myocyte responses to RF energy and if myocyte damage is a Ca2+-dependent process. Because immature myocardium is functionally deficient in sarcoplasmic reticulum, we hypothesized that immature myocytes would be more susceptible to damage induced by RF energy. Isolated ventricular myocytes were obtained from neonatal and adult New Zealand White rabbits by enzymatic dissociation, then placed in a perfusion chamber designed to deliver RF energy or a heated perfusate solution. Measurements of bath temperature, cell morphology, and contractile response to electrical stimuli were recorded. RF energy application associated with increased perfusate temperature resulted in cell death, but not when the temperature rise was inhibited. Thus, the acute damage to cells exposed to RF energy appears to be mediated by thermal energy. After exposure to thermal energy, neonatal cells underwent contracture at lower temperatures than did adult cells. Perfusion with solutions containing low Ca2+ concentrations, comparable to intracellular diastolic Ca2+ levels, had a protective effect for both neonatal and adult myocytes. These findings indicate that acute cell damage after exposure to RF energy is mediated by a Ca2+-dependent process. Furthermore, immature myocardium is particularly susceptible to RF-mediated cell damage, possibly secondary to reduced Ca2+ sequestration by the sarcoplasmic reticulum.

Mitochondrial Ca2+ transients in cardiac myocytes during the excitation-contraction cycle: effects of pacing and hormonal stimulation.

Using laser scanning confocal microscopy, our objective was to measure mitochondrial, nuclear, and cytosolic free ionized Ca2+ in adult rabbit cardiac myocytes loaded with Ca2+-indicating fluorophores. When myocytes were loaded with Fluo 3 at 37 degrees C, the fluorophore was loaded extensively into the cytosol and nucleus, but poorly into mitochondria, and Fluo 3 fluorescence transients after field stimulation were confined to the cytosol and nucleus. In contrast, after loading at 4 degrees C, Fluo 3 also entered mitochondria, and large transients of mitochondrial Fluo 3 fluorescence then occurred after stimulation. Isoproterenol (1 microM) increased the magnitude of Ca2+ transients and their subsequent rate of decay, an effect more marked in the cytosol and nucleus than in mitochondria. As pacing frequency was increased from 0.5 to 2 Hz, diastolic mitochondrial Ca2+ rose markedly in the absence but not in the presence of isoproterenol. Resting Ca2+ estimated by Indo 1 ratio imaging using UV/visible laser scanning confocal microscopy was about 200 nM in all compartments. During field stimulation, Ca2+ transiently increased to 671, 522, and 487 nM in cytosol, interfibrillar mitochondria, and perinuclear mitochondria, respectively. Isoproterenol increased these respective peak values to 1280, 750, and 573 nM. These results were consistent with those obtained in Fluo 3 experiments. We conclude that rapid mitochondrial Ca2+ transients occur during excitation-contraction coupling in adult rabbit cardiac myocytes, which may be important in matching mitochondrial metabolism to myocardial ATP demand during changes in cardiac output.

Endothelin-1 ameliorates contractile depression by lipopolysaccharide in cardiac myocytes.

Lipopolysaccharide (LPS) induces cardiac depression by activating nitric oxide pathways to increase guanosine 3',5'-cyclic monophosphate (cGMP), a second messenger of nitric oxide. Endothelin-1 (ET-1) may interact with nitric oxide pathways. We hypothesized that ET-1 modulates LPS-induced contractile depression in cardiac myocytes. Adult rabbit cardiac myocytes exposed to LPS (10 ng/ml) developed decreased cell shortening after 6 h, with an increase in cardiac cGMP levels [606 +/- 36 (SE) fmol/mg protein] compared with control myocytes (360 +/- 26 fmol/mg protein, P < 0.05). LPS effects were completely blocked by coincubation with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (1 mM). Coincubation with ET-1 (10 nM) attenuated the contractile depression and increase in cGMP with LPS (482 +/- 28 fmol/mg protein, P < 0.05 vs. LPS alone). ET-1 alone did not alter cGMP levels (350 +/- 30 fmol/mg protein). ET-1 effects on contractile function were blocked by BQ-123 (10 microM), a selective ET-1 type A receptor antagonist. We conclude that ET-1 ameliorates LPS-induced contractile depression in cardiac myocytes by attenuating LPS effects on nitric oxide-cGMP pathways.

Selective Loading of Rhod 2 into Mitochondria Shows Mitochondrial Ca2+Transients during the Contractile Cycle in Adult Rabbit Cardiac Myocytes

A strategy of cold loading of the Ca2+-indicating fluorophore Rhod 2-AM followed by warm incubation was developed to selectively label mitochondria of adult rabbit cardiac myocytes. After electrical stimulation, mitochondrial Rhod 2 fluorescence observed by confocal microscopy increased and then rapidly decayed to baseline. In regions between mitochondria, the fluorescent transients were small or absent. Subsequent addition of calcium ionophore increased mitochondrial but not cytosolic fluorescence, confirming the mitochondrial localization of Rhod 2. These experiments directly demonstrate rapid mitochondrial free Ca2+transients during the contractile cycle in rabbit cardiac myocytes.

Negative inotropic effect of adrenomedullin in isolated adult rabbit cardiac ventricular myocytes.

Adrenomedullin (AM) is a potent vasodilator peptide. AM-induced vasodilatation is mediated by an increase of NO as well as cAMP. Both AM and binding sites for this peptide have been found in cardiac tissue, indicating the possible existence of an autocrine or paracrine system of AM in the heart. Myocytes were isolated by use of retrograde coronary perfusion with physiological solution containing collagenase and hyaluronidase from adult rabbit ventricles. Contraction of cardiac myocytes was traced with a video motion detector, and [Ca2+]i was measured with indo 1 at 37 degrees C. The Ica was measured with a whole-cell patch clamp at 23 degrees C. AM and calcitonin gene-related peptide (CGRP), another member of the same peptide family, showed a concentration-dependent negative inotropic effect (10(-7) mol/L AM: contraction amplitude, 64 +/- 7% of control; [Ca2+]i, 52 +/- 5% of control; n = 10; 10(-6) mol/L CGRP: contraction amplitude, 64 +/- 25%; [Ca2+]i, 70 +/- 3%; n = 5; mean +/- SD). Ica was decreased to 60 +/- 39% by superfusion with AM after the cessation of NG-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor. Pretreatment with L-NMMA (10 mumol/L) abolished the negative inotropic effect of AM, whereas switching from AM+L-NMMA to AM+L-arginine (1 mmol/L) restored it. Superfusion with 8-bromo-cGMP also showed a negative inotropic effect. AM significantly increased the intracellular content of cGMP, a second messenger of NO, but not that of cAMP. AM (10 nmol/L) blunted the effect of 1 mumol/L forskolin. AM has a negative inotropic effect and decreased both [Ca2+]i and Ica, with these effects being at least party mediated via the L-arginine-NO pathway in adult rabbit ventricular myocytes.