Diastolic calcium is elevated in metabolic recovery of cardiomyocytes expressing elevated levels of the Na+/H+ exchanger

2008 ◽  
Vol 86 (12) ◽  
pp. 850-859 ◽  
Author(s):  
István Baczkó ◽  
Fatima Mraiche ◽  
Peter E. Light ◽  
Larry Fliegel
Keyword(s):  
Intracellular Calcium ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Ischemia Reperfusion ◽  
Metabolic Inhibition ◽  
Subsequent Increase ◽  
Cell Contractility ◽  
Metabolic Recovery ◽  
Single Ventricular Myocytes

In the myocardium, the Na+/H+ exchanger isoform 1 (NHE1) plays a pivotal role in mediating ischemia–reperfusion (I/R) injury by causing intracellular Na+ accumulation that results in a subsequent increase in intracellular calcium (Ca2+ overload). One of the major clinical correlates of I/R injury is contractile dysfunction, in which Ca2+ overload via increased Na+/Ca2+ exchange is a major contributor. To better understand the cellular role of NHE1 during I/R injury, contractile function and calcium transients were measured during metabolic inhibition and recovery in single ventricular myocytes from transgenic mice with elevated NHE1 expression. During normoxic conditions, no differences were seen between NHE1-overexpressing cardiomyocytes and wild-type (WT) cardiomyocytes with respect to fractional cell shortening (FCS), rate of shortening (+dL/dt), and rate of relaxation (–dL/dt). When metabolic recovery followed metabolic inhibition, NHE1-overexpressing ventricular myocytes exhibited a significant increase in FCS (130.2% ± 11.77% baseline) and ±dL/dt (146.93% ± 12.27% baseline). This correlated with a significant increase in the concentration of diastolic intracellular calcium, which was attenuated by the NHE1 inhibitor HOE694. These results indicate that in normoxic conditions, elevated NHE1 expression does not alter contractile function. During metabolic recovery, however, elevated NHE1 expression increased diastolic Ca2+ loading that led to augmented cell contractility.

scholarly journals STAT subtype specificity and ischemic preconditioning in mice: is STAT-3 enough?

2011 ◽  
Vol 300 (2) ◽  
pp. H522-H526 ◽  
Author(s):  
Michael D. Goodman ◽  
Sheryl E. Koch ◽  
Muhammad R. Afzal ◽  
Karyn L. Butler
Keyword(s):  
Ischemic Preconditioning ◽  
Ischemia Reperfusion Injury ◽  
Contractile Function ◽  
Ex Vivo ◽  
Ischemia Reperfusion ◽  
Cardiac Performance ◽  
Subtype Specificity ◽  
Developed Pressure ◽  
Perfused Hearts

The role of other STAT subtypes in conferring ischemic tolerance is unclear. We hypothesized that in STAT-3 deletion alternative STAT subtypes would protect myocardial function against ischemia-reperfusion injury. Wild-type (WT) male C57BL/6 mice or mice with cardiomyocyte STAT-3 knockout (KO) underwent baseline echocardiography. Langendorff-perfused hearts underwent ischemic preconditioning (IPC) or no IPC before ischemia-reperfusion. Following ex vivo perfusion, hearts were analyzed for STAT-5 and -6 phosphorylation by Western blot analysis of nuclear fractions. Echocardiography and postequilibration cardiac performance revealed no differences in cardiac function between WT and KO hearts. Phosphorylated STAT-5 and -6 expression was similar in WT and KO hearts before perfusion. Contractile function in WT and KO hearts was significantly impaired following ischemia-reperfusion in the absence of IPC. In WT hearts, IPC significantly improved the recovery of the maximum first derivative of developed pressure (+dP/d tmax) compared with that in hearts without IPC. IPC more effectively improved end-reperfusion dP/d tmax in WT hearts compared with KO hearts. Preconditioned and nonpreconditioned KO hearts exhibited increased phosphorylated STAT-5 and -6 expression compared with WT hearts. The increased subtype activation did not improve the efficacy of IPC in KO hearts. In conclusion, baseline cardiac performance is preserved in hearts with cardiac-restricted STAT-3 deletion. STAT-3 deletion attenuates preconditioning and is not associated with a compensatory upregulation of STAT-5 and -6 subtypes. The activation of STAT-5 and -6 in KO hearts following ischemic challenge does not provide functional compensation for the loss of STAT-3. JAK-STAT signaling via STAT-3 is essential for effective IPC.

Annexin V staining during reperfusion detects cardiomyocytes with unique properties

2001 ◽  
Vol 281 (5) ◽  
pp. H1931-H1937 ◽  
Author(s):  
Prakash Narayan ◽  
Robert M. Mentzer ◽  
Robert D. Lasley
Keyword(s):  
Propidium Iodide ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Ischemia Reperfusion ◽  
Annexin V ◽  
Cell Width ◽  
Simulated Ischemia ◽  
Propidium Iodide Staining ◽  
Membrane Blebs ◽  
Apoptosis And Necrosis

With the use of markers of sarcolemmal membrane permeability, cardiomyocyte models of ischemic injury have primarily addressed necrotic death during ischemia. In the present study, we used annexin V-propidium iodide staining to examine apoptosis and necrosis after simulated ischemia and simulated reperfusion in rat ventricular myocytes. Annexin V binds phosphatidylserine, a phosphoaminolipid thought to be externalized during apoptosis or programmed cell death. Propidium iodide is a marker of cell necrosis. Under baseline conditions, <1% of cardiomyocytes stained positive for annexin V. After 20 or 60 min of simulated ischemia, there was no increase in annexin V staining, although 60-min simulated ischemia resulted in significant propidium iodide staining. Twenty minutes of simulated ischemia, followed by 20 or 60 min of simulated reperfusion, resulted in 8–10% of myocytes staining positive for annexin V. Annexin V-positive cells retained both rod-shaped morphology and contractile function but exhibited the decreased cell width indicative of cell shrinkage. Baseline mitochondrial free Ca2+(111 ± 14 nM) was elevated in reperfused annexin V-negative cells (214 ± 22 nM), and further elevated in annexin V-positive myocytes (382 ± 9 nM). After 60 min of simulated reperfusion, caspase-3-like activity was observed in ∼3% of myocytes, which had a rounded appearance and membrane blebs. These results suggest that the use of annexin V after simulated ischemia-reperfusion uncovers a population of cardiomyocytes whose characteristics appear to be consistent with cells undergoing apoptosis.

Alterations in nitric oxide-cGMP pathway in ventricular myocytes from obese leptin-deficient mice

2003 ◽  
Vol 285 (5) ◽  
pp. H2111-H2117 ◽  
Author(s):  
Jun Su ◽  
Shengjun Zhang ◽  
James Tse ◽  
Peter M. Scholz ◽  
Harvey R. Weiss
Keyword(s):  
Nitric Oxide ◽  
Guanylyl Cyclase ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Cyclase Activity ◽  
No Production ◽  
Obese Mice ◽  
Cell Contractility ◽  
Dependent Protein ◽  
Guanylyl Cyclase Activity

Leptin is a regulator of body weight and affects nitric oxide (NO) production. This study was designed to determine whether the myocardial NO-cGMP signal transduction system was altered in leptin-deficient obese mice. Contractile function, guanylyl cyclase activity, and cGMP-dependent protein phosphorylation were assessed in ventricular myocytes isolated from genetically obese (B6.V-Lepob) and age-matched lean (C57BL/6J) mice. There were no differences in baseline contraction between the lean and obese groups. After stimulation with the NO donor S-nitroso- N-acetyl-penicillamine (SNAP, 10–6and 10–5M) or a membrane-permeable cGMP analog 8-bromo-cGMP (8-Br-cGMP, 10–6and 10–5M), cell contractility was depressed. However, 8-Br-cGMP had significantly greater effects in obese mice than in lean controls with percent shortening reduced by 47 vs. 39% and maximal rate of shortening decreased by 46 vs. 36%. The negative effects of SNAP were similar between the two groups. Soluble guanylyl cyclase activity was not attenuated. This suggests that the activity of the cGMP-independent NO pathway may be enhanced in obesity. The phosphorylated protein profile of cGMP-dependent protein kinase showed that four proteins were more intensively phosphorylated in obese mice, which suggests an explanation for the enhanced effect of cGMP. These results indicate that the NO-cGMP signaling pathway was significantly altered in ventricular myocytes from the leptin-deficient obese mouse model.

scholarly journals Overexpression of A3 adenosine receptors decreases heart rate, preserves energetics, and protects ischemic hearts

2002 ◽  
Vol 283 (4) ◽  
pp. H1562-H1568 ◽  
Author(s):  
Heather R. Cross ◽  
Elizabeth Murphy ◽  
Richard G. Black ◽  
John Auchampach ◽  
Charles Steenbergen
Keyword(s):  
Heart Rate ◽  
Contractile Function ◽  
Ischemia Reperfusion ◽  
Specific Inhibitor ◽  
Left Ventricular ◽  
Atp Depletion ◽  
Wild Type ◽  
Basal Heart Rate ◽  
Developed Pressure

To determine whether A3 adenosine receptor (A3AR) signaling modulates myocardial function, energetics, and cardioprotection, hearts from wild-type and A3AR-overexpressor mice were subjected to 20-min ischemia and 40-min reperfusion while 31P NMR spectra were acquired. Basal heart rate and left ventricular developed pressure (LVDP) were lower in A3AR-overexpressor hearts than wild-type hearts. Ischemic ATP depletion was delayed and postischemic recoveries of contractile function, ATP, and phosphocreatine were greater in A3AR-hearts. To determine the role of depressed heart rate and to confirm A3AR-specific signaling, hearts were paced at 480 beats/min with or without 60 nmol/l MRS-1220 (A3AR-specific inhibitor) and then subjected to ischemia-reperfusion. LVDP was similar in paced A3AR-overexpressor and paced wild-type hearts. Differences in ischemic ATP depletion and postischemic contractile and energetic dysfunction remained in paced A3AR-overexpressor hearts versus paced wild-type hearts but were abolished by MRS-1220. In summary, A3AR overexpression decreased basal heart rate and contractility, preserved ischemic ATP, and decreased postischemic dysfunction. Pacing abolished the decreased contractility but not the ATP preservation or cardioprotection. Therefore, A3AR overexpression results in cardioprotection via a specific A3AR effect, possibly involving preservation of ATP during ischemia.

Mechanisms of Erythropoietin-Mediated Cardioprotection during Ischemia-Reperfusion Injury: Role of Protein Kinase C Signaling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2907-2907
Author(s):  
Murat O. Arcasoy ◽  
Paul Hanlon ◽  
Ping Fu ◽  
Charles Steenbergen ◽  
Elizabeth Murphy
Keyword(s):  
Protein Kinase ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Contractile Function ◽  
Ischemia Reperfusion ◽  
Cardioprotective Effect ◽  
Isolated Perfused Heart ◽  
Perfused Heart ◽  
Biologic Effects

Abstract The biologic effects of erythropoietin (EPO) are mediated by its cellular receptor EPOR, a member of the cytokine receptor superfamily. EPOR expression in non-hematopoietic cells is associated with novel biologic effects for EPO in diverse organ systems. We recently demonstrated functional EPOR expression in adult rat cardiac myocytes and found that recombinant EPO exerts a rapid cardioprotective effect during ischemia-reperfusion injury of the isolated, perfused heart. Here we investigated the mechanisms of the cardioprotective effect of EPO using Langendorff-perfused rat hearts while left-ventricular-developed pressure (LVDP) was measured continuously to assess contractile function. Hearts were treated directly with EPO in the presence or absence of inhibitors of specific signal transduction pathways prior to normothermic global ischemia followed by reperfusion. Post-ischemic recovery of contractile function was determined by measuring LVDP at the end of reperfusion and expressed as a percentage of the baseline pre-treatment measurement. We investigated EPO-mediated activation of signal transduction pathways in the isolated, perfused heart and observed phosphorylation of p44/p42 MAP kinases ERK 1/2 (Thr202/Tyr204) and protein kinase B/Akt (Ser473), a downstream target of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Furthermore, EPO treatment of the isolated, perfused heart was associated with translocation of protein kinase C (PKC) ε and δ isoforms to the membrane fraction. We investigated the role of specific signaling pathways in EPO-mediated cardioprotection by employing inhibitors targeting PI3K, PKC and MAP kinase kinase (MEK1). PI3K inhibitors LY294002 and wortmannin attenuated EPO-induced phosphorylation of Akt but had no effect on EPO-mediated cardioprotection. MEK1 inhibitor U0126 had no effect on EPO-mediated cardioprotection. The PKC catalytic inhibitor chelerythrine (chel) significantly inhibited EPO-mediated improvement in post-ischemic recovery of LVDP (figure 1). Hearts pre-treated with EPO exhibited significantly improved post-ischemic recovery of LVDP compared to control hearts (mean±SE: 72±3 in EPO-treated versus 35±3% in control hearts, P<0.05 by ANOVA and Bonferroni post-hoc test, n=10 experiments each group) and the protective effect of EPO was significantly inhibited in chel-treated hearts (52±4% in EPO+chel versus 72±3% in EPO-treated hearts, P<0.05, n=10). As a control, treatment of the hearts with chelerythrine alone had no significant effect on LVDP (49±4%) compared to control hearts. These data demonstrate that EPO-mediated activation of the PKC signaling pathway is required for the cardioprotective effect of EPO during ischemia-reperfusion injury. Figure Figure

scholarly journals Testosterone enhances estradiol's cardioprotection in ovariectomized rats

2011 ◽  
Vol 212 (1) ◽  
pp. 61-69 ◽  
Author(s):  
Aiying Liu ◽  
Liping Gao ◽  
Shoulei Kang ◽  
Ying Liu ◽  
Chuanying Xu ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Contractile Function ◽  
Ischemia Reperfusion ◽  
Testosterone Replacement ◽  
Ovariectomized Rats ◽  
Sham Operation ◽  
Protective Effects ◽  
Sprague Dawley ◽  
Ovx Rats ◽  
Ldh Release

After menopause, the development of cardiovascular disease (CVD) is due not only to estrogen decline but also to androgen decline. This study examined the effects of either estradiol (E2) or testosterone replacement alone or E2–testosterone combination on isolated myocytes in ovariectomized (Ovx) rats subjected to ischemia/reperfusion (I/R). Furthermore, we determined whether the effects are associated with β2-adrenoceptor (β2-AR). Five groups of adult female Sprague–Dawley rats were used: Sham operation (Sham) rats, bilateral Ovx rats, Ovx rats with E2 40 μg/kg per day (Ovx+E), Ovx rats with testosterone 150 μg/kg per day (Ovx+T), and Ovx rats with E2 40 μg/kg per day+testosterone 150 μg/kg per day (Ovx+E/T). We determined the lactate dehydrogenase (LDH) release, percentage of rod-shaped cells and apoptosis of ventricular myocytes from rats of all groups subjected to I/R. Then, we determined the above indices and contractile function with or without a selective β2-AR antagonist ICI 118 551. We also determined the expression of β2-AR. Our data show that either E2 or testosterone replacement alone or E2 and testosterone in combination decreased the LDH release, increased the percentage of rod-shaped cells, reduced apoptotic cells (%), and combination treatment appeared to be more effective than either E2 or testosterone replacement alone. ICI 118 551 abolished the effects of the three. Combination supplementation also enhanced the expression of β2-AR. We concluded that in Ovx rats, testosterone enhances E2's cardioprotection, while E2 and testosterone in combination was more effective and the protective effects may be associated with β2-AR. The study highlights the potential therapeutic application for CVD in postmenopausal women.

Ischemic preconditioning of the whole heart confers protection on subsequently isolated ventricular myocytes

2008 ◽  
Vol 294 (1) ◽  
pp. H524-H531 ◽  
Author(s):  
Glenn C. Rodrigo ◽  
Nilesh J. Samani
Keyword(s):  
Ischemic Preconditioning ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Ischemia Reperfusion ◽  
Isolated Cells ◽  
Cellular Models ◽  
Whole Heart ◽  
Intact Heart

Current cellular models of ischemic preconditioning (IPC) rely on inducing preconditioning in vitro and may not accurately represent complex pathways triggered by IPC in the intact heart. Here, we show that it is possible to precondition the intact heart and to subsequently isolate individual ventricular myocytes that retain the protection triggered by IPC. Myocytes isolated from Langendorff-perfused hearts preconditioned with three cycles of ischemia-reperfusion were exposed to metabolic inhibition and reenergization. Injury was assessed from induction of hypercontracture and loss of Ca2+ homeostasis and contractile function. IPC induced an immediate window of protection in isolated myocytes, with 64.3 ± 7.6% of IPC myocytes recovering Ca2+ homeostasis compared with 16.9 ± 2.4% of control myocytes ( P < 0.01). Similarly, 64.1 ± 5.9% of IPC myocytes recovered contractile function compared with 15.3 ± 2.2% of control myocytes ( P < 0.01). Protection was prevented by the presence of 0.5 mM 5-hydroxydecanoate during the preconditioning stimulus. This early protection disappeared after 6 h, but a second window of protection developed 24 h after preconditioning, with 54.9 ± 4.7% of preconditioned myocytes recovering Ca2+ homeostasis compared with 12.6 ± 2.9% of control myocytes ( P < 0.01). These data show that “true” IPC of the heart confers both windows of protection in the isolated myocytes, with a similar temporal relationship to in vivo preconditioning of the whole heart. The model should allow future studies in isolated cells of the protective mechanisms induced by true ischemia.

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