Isolated Rat Myocytes Research Articles

Cardioprotective effects of alantolactone on isoproterenol-induced cardiac injury and cobalt chloride-induced cardiomyocyte injury.

ObjectivesAlantolactone (AL) is a compound extracted from the roots of Inula Racemosa that has shown beneficial effects in cardiovascular disease. However, the cardioprotective mechanism of AL against hypoxic/ischemic (H/I) injury is still unclear. This research aimed to determine AL’s ability to protect the heart against isoproterenol (ISO)-induced MI injury in vivo and cobalt chloride (CoCl2) induced H/I injury in vitro.MethodsElectrocardiography (ECG), lactate dehydrogenase (LDH), creatine kinase (CK), and cardiac troponin I (cTnI) assays in addition to histological analysis of the myocardium were used to investigate the effects of AL in vivo. Influences of AL on L-type Ca2+ current (ICa-L) in isolated rat myocytes were observed by the patch-clamp technique. Furthermore, cell viability, apoptosis, oxidative stress injury, mitochondrial membrane potential, and intracellular Ca2+ concentration were examined in vitro.ResultsThe results indicated that AL treatment ameliorated the morphological and ECG changes associated with MI, and decreased levels of LDH, CK, and cTnI. Furthermore, pretreatment with AL elevated antioxidant enzyme activity and suppressed ROS production. AL prevented H/I-induced apoptosis, mitochondria damage, and calcium overload while reducing ICa-L in a concentration and time dependent fashion. The 50% inhibiting concentration (IC50) and maximal inhibitory effect (Emax) of AL were 17.29 μmol/L and 57.73 ± 1.05%, respectively.ConclusionAL attenuated MI-related injury by reducing oxidative stress, apoptosis, calcium overload, and mitochondria damage. These cardioprotective effects may be related to the direct inhibition of ICa-L.

8]-Gingerol exerts anti-myocardial ischemic effects in rats via modulation of the MAPK signaling pathway and L-type Ca2+ channels.

Myocardial ischemia (MI) remains the leading cause of mortality worldwide. Therefore, it is urgent to seek the treatment to protect the heart. [8]‐Gingerol (8‐Gin), one of the most active ingredients in ginger, has antioxidant, cardiotonic, and cardiovascular protective properties. The present study elucidated the cardioprotection effects and underlying mechanisms of 8‐Gin in isoproterenol (ISO)‐induced MI. ISO (85 mg/kg/d) was subcutaneously injected for 2 consecutive days to induce acute MI model in rats. Electrocardiography, oxidative stress levels, calcium concentrations, and apoptosis degree were observed. The effects of 8‐Gin on L‐type Ca2+ current (ICa‐L), contraction, and Ca2+ transients were monitored in rat myocytes via patch‐clamp and IonOptix detection systems. 8‐Gin decreased J‐point elevation and heart rate and improved pathological heart damage. Moreover, 8‐Gin reduced the levels of CK, LDH, and MDA, ROS production, and calcium concentrations in myocardial tissue, while increased the activities of SOD, CAT, and GSH. In addition, 8‐Gin down‐regulated Caspase‐3 and Bax expressions, while up‐regulated Bcl‐2 expression. 8‐Gin produced a marked decrease in the expression of p38, JNK, and ERK1/2 proteins. 8‐Gin inhibited ICa‐L, cell contraction, and Ca2+ transients in isolated rat myocytes. The results indicate that 8‐Gin could exert anti‐myocardial ischemic effects, which may be associated with oxidative stress reduction, cardiomyocytes apoptosis inhibition through MAPK signaling pathway, and Ca2+ homeostasis regulation via ICa‐L modulation.

Hypokalemia Promotes Arrhythmia by Distinct Mechanisms in Atrial and Ventricular Myocytes.

Hypokalemia occurs in up to 20% of hospitalized patients and is associated with increased incidence of ventricular and atrial fibrillation. It is unclear whether these differing types of arrhythmia result from direct and perhaps distinct effects of hypokalemia on cardiomyocytes. To investigate proarrhythmic mechanisms of hypokalemia in ventricular and atrial myocytes. Experiments were performed in isolated rat myocytes exposed to simulated hypokalemia conditions (reduction of extracellular [K+] from 5.0 to 2.7 mmol/L) and supported by mathematical modeling studies. Ventricular cells subjected to hypokalemia exhibited Ca2+ overload and increased generation of both spontaneous Ca2+ waves and delayed afterdepolarizations. However, similar Ca2+-dependent spontaneous activity during hypokalemia was only observed in a minority of atrial cells that were observed to contain t-tubules. This effect was attributed to close functional pairing of the Na+-K+ ATPase and Na+-Ca2+ exchanger proteins within these structures, as reduction in Na+ pump activity locally inhibited Ca2+ extrusion. Ventricular myocytes and tubulated atrial myocytes additionally exhibited early afterdepolarizations during hypokalemia, associated with Ca2+ overload. However, early afterdepolarizations also occurred in untubulated atrial cells, despite Ca2+ quiescence. These phase-3 early afterdepolarizations were rather linked to reactivation of nonequilibrium Na+ current, as they were rapidly blocked by tetrodotoxin. Na+ current-driven early afterdepolarizations in untubulated atrial cells were enabled by membrane hyperpolarization during hypokalemia and short action potential configurations. Brief action potentials were in turn maintained by ultra-rapid K+ current (IKur); a current which was found to be absent in tubulated atrial myocytes and ventricular myocytes. Distinct mechanisms underlie hypokalemia-induced arrhythmia in the ventricle and atrium but also vary between atrial myocytes depending on subcellular structure and electrophysiology.

<p>Cardioprotective Effect of Monoammonium Glycyrrhizinate Injection Against Myocardial Ischemic Injury in vivo and in vitro: Involvement of Inhibiting Oxidative Stress and Regulating Ca<sup>2+</sup> Homeostasis by L-Type Calcium Channels</p>

PurposeMonoammonium glycyrrhizinate (MAG) is an aglycone of glycyrrhizin that is found in licorice and is often used clinically as an injection to treat liver diseases. However, the effect of MAG injection on cardiac function and its possible cellular mechanisms remain unclear. We explored the protective effects of MAG against myocardial ischemic injury (MII) induced by isoproterenol (ISO), as well as the cellular mechanisms via molecular biology techniques and patch-clamp recording.MethodsA rat model of myocardial ischemia injury was induced by administering ISO (85 mg/kg) subcutaneously for 2 consecutive days. ECG, cardiac functional parameters, CK and LDH levels, SOD and GSH activities, MDA concentration, histological myocardium inspection, mitochondria ultrastructure changes, intracellular calcium concentrations were observed. Influences of MAG on ICa-L and contraction in isolated rat myocytes were observed by the patch-clamp technique.ResultsMAG reduced damage, improved cardiac morphology, inhibited oxidative stress, decreased the generation of reactive oxygen species, and decreased intracellular Ca2+ concentration. Exposure of the rats’ ventricular myocytes to MAG resulted in a concentration-dependent reduction in L-type calcium currents (ICa-L). MAG reduced ICa-L in a consistent and time-dependent fashion with a semi-maximal prohibitive concentration of MAG of 14 μM. MAG also shifted the I-V curve of ICa-L upwards and moved the activation and inactivation curves of ICa-L to the left.ConclusionThe findings indicate that MAG injection exerts a protective influence on ISO-induced MII by inhibiting oxidative stress and regulating Ca2+ homeostasis by ICa-L.

Mechanisms underlying the cardio-protection of total ginsenosides against myocardial ischemia in rats in vivo and in vitro: Possible involvement of L-type Ca2+ channels, contractility and Ca2+ homeostasis

Here we aimed to observe the effects of total ginsenosides (TG) against isoproterenol (ISO) induced myocardial ischemia (MI) and to explore its underlying mechanisms based on L-type Ca2+ current (ICa-L), intracellular Ca2+ ([Ca2+]i) and contraction in isolated rat myocytes. Rat model of MI was induced by subcutaneously injection of ISO (85 mg/kg) for 2 consecutive days. J-point elevation, heart rate, serum levels of creatine kinase (CK) and lactated dehydrogenase (LDH), and heart morphology changes were observed. Influences of TG on ICa-L, [Ca2+]i and contraction in isolated rat myocytes were observed by the patch-clamp technique and IonOptix detection system. TG significantly reduced J-point elevation, heart rate, serum levels of CK and LDH, and improved heart pathologic morphology. TG decreased ICa-L in concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 31.65 μg/mL. TG (300 μg/mL) decreased ICa-L of normal and ischemic ventricular myocytes by 64.33 ± 1.28% and 61.29 ± 1.38% respectively. At 30 μg/mL, TG reduced Ca2+ transient by 21.67 ± 0.94% and cell shortening by 38.43 ± 6.49%. This study showed that TG displayed cardioprotective effects on ISO-induced MI rats and the underlying mechanisms may be related to inhibition of ICa-L, damping of [Ca2+]i and decrease of contractility.

Peroxynitrite-Induced Intracellular Ca2+ Depression in Cardiac Myocytes: Role of Sarco/Endoplasmic Reticulum Ca2+ Pump

Several studies have shown that peroxynitrite (ONOO–), formed upon the reaction of •NO and O2–, is increased in many cardiovascular diseases and is detrimental to myocardial function. Proteins associated with Ca2+ homeostasis regulation in the heart may be involved in these effects. Thus, the aim of this study was to elucidate the mechanisms associated with ONOO–-induced effects. We evaluated [Ca2+]i regulation, sarco/endoplasmic reticulum Ca2+- binding proteins, and phosphorylation levels of the ryanodine receptor in isolated rat myocytes. Electrical field-induced intracellular Ca2+ transients and contractions were recorded simultaneously. Myocytes superfused with 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), an ONOO– donor, decreased the amplitude of Ca2+ transients and contraction in a dose-response (1–200 μM) manner. Similarly, SIN-1 increased half-time decay in a concentration-dependent manner. Co-infusion of the ONOO– donor with FeTMPyP (1 μM), an ONOO– decomposition catalyst, inhibited the effects induced by ONOO–. Impaired sarcoplasmic reticulum Ca2+ uptake caused by ONOO– (SIN-1 200 μM) was confirmed by a reduction of caffeine-evoked Ca2+ release along with prolongation of the half-time decay. Surprisingly, ONOO– induced a spontaneous Ca2+ transient that started at the beginning of the relaxation phase and was inhibited by tetracaine. Also, reduced phosphorylation at the ryanodine receptor 2 (RyR2)-Ser-2814 site was observed. In conclusion, deficient sarco/endoplasmic reticulum Ca2+-ATPase-mediated Ca2+ uptake concomitant with augmented Ca2+ release by RyR2 in myocytes may be associated with modification of myocyte Ca2+ handling by ONOO–. Thus, development of cardiac failure in diabetes, nephropathy, or hypertension may be related with elevated ONOO– in cardiac tissue.

A dual role for Nucleolin identified by systems analysis of cardiac chromatin remodelers

To elucidate mechanisms of chromatin reprogramming during cardiac development and disease, we used a systems approach to identify, screen, and validate changes to the nuclear proteome. Quantitative proteomic analysis of chromatin fractions from mouse hearts in basal, hypertrophy or failing conditions identified 338 proteins whose abundances change. One candidate was Nucleolin (Ncl), which increases 3‐fold in hypertrophy.Using a functional screen in zebrafish embryos, we characterized a novel role for Ncl to affect cardiac looping and differentiation. 48‐hour embryos express Ncl in the heart, with in situ hybridization demonstrating BMP4 restricted to the atrial/ventricular boundary. After Morpholino knockdown (KD) of Ncl, BMP4 is found throughout the ventricle, suggesting delayed myocyte differentiation. This is accompanied by defects in chamber looping, with 20% of fish failing to loop or reverse looping, a phenotype seen in 29% of fish after over expression; no changes in myocyte size were observed in KD fish. Delayed hearts exhibit a decreased heart rate and increased fractional shortening. Finally, in isolated rat myocytes, loss of Ncl alters transcription of SERCA, ANF, and β‐MHC, whose expression changes are hallmarks of cardiac hypertrophy. This suggests that changes in Ncl levels affect differentiation during development, whereas in disease it may drive the hypertrophic response. NIH funded.

BKCa++ channel inhibition improves myocardial stunning following hyperkalemic cardioplegic arrest (CP) but not reversible ischemia. Potential role of ROS and GSK3B.

We previously demonstrated, in contrast to pure ischemic injury, that inhibition of BKCa channels improves cardiac contractile recovery following CP. We now investigate the mechanism via which BKCa blockade may be protective during CP. Isolated rat hearts and myocytes were subjected to either in vitro ischemia (30 mins) or hyperkalemic CP (2hrs) followed by reperfusion (30 min). Mitochondrial membrane potential (mMP), ROS generation (n=12), and BKCa‐mediated signaling (n=3) were measured in H9c2 cells with or without CP and the BKCa blocker paxilline (pax). CP and ischemia reduced cardiac contractile function in hearts (n>;6) and myocytes (n>;=15). BKCa blockade increased whole heart recovery and myocyte shortening in CP, but not in ischemic groups. Pax did not affect Ca++ transients in myocytes, or indicators of Ca++ signaling and survival in hearts and H9c2 cells (phospho‐PKA, p‐cTnI, PKA, pAkt, pPLB, p‐sHSP). In H9c2 cells, CP reduced mMP and increased ROS, which was partially blocked by pax. Pax increased phosphorylation of GSK3B in both hearts and cells, independent of changes in Akt phosphorylation. In contrast to protective effects of BKCa openers in the setting of ischemic injury, BKCa channel blockers may improve cardiac recovery in the context of hyperkalemic CP. Cardioprotective mechanisms associated with BKCa blockade include normalized ROS and mMP, and increased GSK3B phosphorylation.

Mitochondrial K+ channels are involved in ischemic postconditioning in rat hearts

The mitochondrial calcium-activated potassium channel (mitoK(Ca)) and the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) are both involved in cardiac preconditioning. Here, we examined whether these two channels are also involved in ischemic or pharmacological postconditioning. Using Langendorff perfusion, rat hearts were made hypoxic for 45 min and then reoxygenated for 30 min. Ischemic postconditioning (IPT) was achieved through application of 3 cycles of 10 s of reperfusion and 10 s of ischemia before reoxygenation, with and without paxilline (Pax; a mitoK(Ca) blocker) or 5-hydroxydecanoate (5-HD; a mitoK(ATP) blocker). Pharmacological postconditioning was carried out for 5 min at the onset of reoxygenation using NS1619 (a mitoK(Ca) opener) or diazoxide (Dia; a mitoK(ATP) opener). Pax and 5-HD abolished IPT-induced cardioprotection from reoxygenation injury, whereas administration of NS1619 or Dia significantly improved cardiac contractile activity and reduced aspartate aminotransferase (an index of myocyte injury) release following reoxygenation. In addition, isolated rat myocytes were loaded with tetramethylrhodamine methyl ester (TMRE; fluorescent mitochondrial membrane potential indicator) and 2',7'-dichlorofluorescein [DCFH; fluorescent reactive oxygen species (ROS) indicator] or Fluo-4-acetoxymethyl ester (Fluo-4-AM; fluorescent calcium indicator). When TMRE-loaded myocytes were laser illuminated, the DCFH and Fluo-4 fluorescence increased, and TMRE fluorescence decreased. These effects were significantly inhibited by NS1619 and Dia. We therefore conclude that IPT may protect the heart through activation of mitoK(ATP) and mitoK(Ca) channels, and that opening of these channels at the onset of reoxygenation protects the heart from reoxygenation injury, most likely by reducing excess generation of ROS and the resultant Ca(2+) overload.

Mechanistic distinction between activation and inhibition of (Na++K+)-ATPase-mediated Ca2+ influx in cardiomyocytes

(Na++K+)-ATPase (NKA) mediates positive inotropy in the heart. Extensive studies have demonstrated that the reverse-mode Na+/Ca2+-exchanger (NCX) plays a critical role in increasing intracellular Ca2+ concentration through the inhibition of NKA-induced positive inotropy by cardiac glycosides. Little is known about the nature of the NCX functional mode in the activation of NKA-induced positive inotropy. Here, we examined the effect of an NKA activator SSA412 antibody on 45Ca influx in isolated rat myocytes and found that KB-R7943, a NCX reverse-mode inhibitor, fails to inhibit the activation of NKA-induced 45Ca influx, suggesting that the Ca2+ influx via the reverse-mode NCX does not mediate this process. Nifedipine, an L-type Ca2+ channel (LTCC) inhibitor, completely blocks the activation of NKA-induced 45Ca influx, suggesting that the LTCC is responsible for the moderate increase in intracellular Ca2+. In contrast, the inhibition of NKA by ouabain induces 4.7-fold 45Ca influx compared with the condition of activation of NKA. Moreover, approximately 70% of ouabain-induced 45Ca influx was obstructed by KB-R7943 and only 30% was impeded by nifedipine, indicating that both the LTCC and the NCX contribute to the rise in intracellular Ca2+ and that the NCX reverse-mode is the major source for the 45Ca influx induced by the inhibition of NKA. This study provides direct evidence to demonstrate that the activation of NKA-induced Ca2+ increase is independent of the reverse-mode NCX and pinpoints a mechanistic distinction between the activation and inhibition of the NKA-mediated Ca2+ influx path ways in cardiomyocytes.

Pannexin 1 Constitutes the Large Conductance Cation Channel of Cardiac Myocytes

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca(2+) release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca(2+) release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.

Heptanol triggers cardioprotection via mitochondrial mechanisms and mitochondrial potassium channel opening in rat hearts

To investigate mechanisms behind heptanol (Hp)-induced infarct size reduction and in particular if protection by pre-treatment with Hp is triggered through mitochondrial mechanisms. Langendorff perfused rat hearts, isolated mitochondria and isolated myocytes were used. Infarct size, mitochondrial respiration, time to mitochondrial permeability transition pore (MPTP) opening and AKT and glycogen synthase kinase 3 beta (GSK-3β) phosphorylation were examined. Pre-treatment with Hp reduced infarct size from 29.7 ± 3.4% to 12.6 ± 2.1%. Mitochondrial potassium channel blockers 5-hydroxy decanoic acid (5HD) blocking mitoK(ATP) and paxilline (PAX) blocking mitoK(Ca) abolished cardioprotective effect of Hp (Hp + 5HD 36.7 ± 2.9% and Hp + PAX 40.2 ± 2.8%). Hp significantly reduced respiratory control ratio in both subsarcolemmal and interfibrillar mitochondria in a dose-dependent manner (0.5-5.0 mm). The ADP oxygen ratio was also significantly reduced by Hp (2 mm). Laser scanning confocal microscopy of tetramethylrhodamine-loaded isolated rat myocytes using line scan mode showed that Hp increased time to MPTP opening. Western blot analysis showed that pre-treatment with Hp increased phosphorylation of AKT and GSK-3β before ischaemia and after 30 min of global ischaemia. Pre-treatment with Hp protects the heart against ischaemia-reperfusion injury. This protection is most likely mediated via mitochondrial mechanisms which initiate a signalling cascade that converges on inhibition of opening of MPTP.

Hydrogen Peroxide Generated From Cardiac Myocytes Impacts Metabolic Dilation in Coronary Arterioles

During oxidative cardiac metabolism, the myocardium produces reactive oxygen species, such as superoxide and hydrogen peroxide (H(2)O(2)). We hypothesized H(2)O(2) is a coronary metabolic dilator linking regulation of coronary tone with myocardium metabolism. Dilation of isolated, pressurized coronary arterioles (76 +/- 10 microm, diameter) in reaction to supernatant collected from enzymatically isolated cardiac myocytes was measured. Isolated rat myocytes were stimulated electrically [unpaced or stimulated at 200, 400 beats/min (bpm)]. H(2)O(2) was significantly generated by pacing (400 bpm n = 11, 9.3 +/- 0.4 microM P < 0.01, versus unpaced) and the addition of this supernatant caused vasodilation (500 microL to 2 mL bath, 14.6 +/- 0.7%, P < 0.01 versus unpaced). Supernatant from unpaced myocytes was not vasoactive. To clarify the source of H(2)O(2), myocytes were also stimulated at 400 bpm following treatment with Mn-TBAP (25 microM), which mimics the action of Mn-SOD, and apocynin (3 mM), an NADPH oxidase inhibitor (n = 11, each). Mn-TBAP increased H(2)O(2) generation in myocyte supernatant stimulated at 400 bpm (12.2 +/- 0.8 microM, P < 0.01 versus 400 bpm stimulation only). Treatment of the myocytes with Mn-TBAP augmented vasodilation by the stimulated myocyte supernatant (19.6 +/- 1.1%, P < 0.01 versus untreated myocyte supernatant). Apocynin did not alter vasodilation to myocyte supernatant. These results suggest that the main source of superoxide by metabolic stimuli is cardiac myocytes and Mn-SOD is a scavenger from superoxide to H(2)O(2). We conclude that H(2)O(2) is a key metabolic vasodilator produced by myocardium.

Age and hypertrophy related changes in contractile post-rest behavior and action potential properties in isolated rat myocytes

"Physiological" aging as well as early and progressive cardiac hypertrophy may affect action potential (AP) pattern, contractile function, and Ca(2+) handling. We hypothesize that contractile function is disturbed in hypertrophy from early stages and is differently affected in aged myocardium. In vivo function, cardiomyocyte contractile behavior and APs were compared in Wistar-Kyoto (WIS) rats and spontaneously hypertensive rats (SHR) at different ages and degrees of hypertrophy (3-4, 9-11, 20-24 months). Post-rest (PR) behavior was used to investigate the relative contribution of the sarcoplasmic reticulum (SR) and the Na/Ca exchanger (NCX) to cytosolic Ca(2+) removal. APs were recorded by whole-cell current-clamp and sarcomere shortening by video microscopy. Cyclopiazonic acid was used to suppress Ca(2+) ATPase (SERCA) function. Heart weight/body weight ratio was increased in SHR versus WIS within all age groups. Myocyte steady state (SS) shortening amplitude was reduced in young SHR versus WIS. Aging led to a significant decay of SS contractile amplitude and relengthening velocity in WIS, but the PR potentiation was maintained. In contrast, aging in SHR led to a decrease of PR potentiation, while SS contraction and relengthening velocity increased. APD(50%) was always prolonged in SHR versus WIS. With aging, APD(50%) increased in both WIS and SHR, but was still shorter in WIS. However, in old WIS the late AP portion (APD(90%)) was prolonged. Ca(2+) handling and AP properties are disturbed progressively with aging and with increasing hypertrophy. Decreased amplitude of shortening and velocity of relengthening in aged WIS may be attributed to reduced SERCA function. In SHR, an increase in SR leak and shift towards transmembraneous Ca handling via NCX may be responsible for the changes in contractile function. A prolonged APD(90%) in aged WIS may be an adaptive mechanism to preserve basal contractility. Therefore, the effects on contractile parameters and AP are different in hypertrophy and aging.

Endothelin-1 mobilizes profilin-1-bound PIP2 in cardiac muscle.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key down-stream substrate of the endothelin signaling pathway and plays a role in regulating protein function at the membrane-cytoskeletal interface. However, the dynamic properties of distinct pools of PIP2 are poorly understood, especially for PIP2 that is bound to cytoskeletal proteins. We investigated the effects of endothelin-1 (ET-1) stimulation on protein-bound PIP2 in cardiac muscle. Isolated rat myocytes and homogenized mouse ventricles were exposed to 10 nM ET-1 for varying time periods and protein-bound PIP2 was analyzed using an anti-PIP2 antibody and Western blotting. Several cytoskeletal proteins were found to contain tightly bound PIP2, including profilin-1 (approximately 15 kDa), capZ (approximately 32 kDa), gCap39, (approximately 39 kDa) and alpha-actinin (approximately 106 kDa). Interestingly, ET-1 pretreatment reduced the amount of PIP2 bound to profilin-1 by 46% after 15 mins, followed by a recovery to near basal levels after 60 mins. ET-1 had no effect on capZ-, gCap39-, or alpha-actinin-bound PIP2 levels. To further explore the dynamics of PIP2 binding, brefeldin-A (BFA) was used to disrupt PIP2 binding to ADP-ribosylation factors and to impair receptor internalization. Pretreatment with 1 microM BFA increased the PIP2 signal on profilin-1 x 54% after 15 mins, followed by a decline to subbasal levels after 60 mins. Like ET-1, BFA had no effect on levels of PIP2 bound to capZ or to alpha-actinin. Taken together, the data indicate that profilin-1 binds PIP2 dynamically and may serve as a key regulator of the balance between cytoskeletal integrity and PIP2 availability for Ca2+/PKC signaling in the heart.

Stretch induced accumulation of total Ca and Na in cytosol and nucleus: a comparison between cardiac trabeculae and isolated myocytesThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

By means of electron probe microanalysis (EPMA), we quantified changes in total sodium [Na] and calcium [Ca] concentration owing to the following: (i) local axial stretch (LAS) of isolated rat myocytes and (ii) end-to-end stretch (ETES) of rat ventricular trabeculae. For LAS, the distance between patch pipette and a cell-attached stylus was increased by maximally 20%; this activated a nonselective cationic current I(SAC) of approximately -0.5 nA, which was blocked by streptomycin. Trabeculae were stretched end-to-end from 85% L(max) to L(max). Stretch increased cytosolic [Na](total) by 34% in isolated myocytes (p < 0.001) and by 43% in trabeculae (p < 0.001). The increment in nuclear [Na](total) was 21% in myocytes (p < 0.01) and 20% in trabeculae (p < 0.001). Stretch increased [Ca](total) in isolated myocytes, in both cytosol (from 0.63 +/- 0.09 to 1.09 +/- 0.20 mmol/L, p < 0.05) and nucleus (from 0.33 +/- 0.05 to 0.64 +/- 0.13 mmol/L, p < 0.05). In trabeculae, the stretch-induced increment of 51% in cytosolic [Ca](total) remained nonsignificant (p < 0.15). In the nucleus, [Ca](total) did not change. We interpret the difference of stretch on nuclear calcium in myocytes vs. trabeculae with the assumption that LAS, but not ETES, produces shear-stress components that translate the mechanical stimulus deeply into the cell where it may modulate [Ca](total) by signals independent of I(SAC).

Parvalbumin corrects slowed relaxation in adult cardiac myocytes expressing hypertrophic cardiomyopathy-linked alpha-tropomyosin mutations.

Hypertrophic cardiomyopathy mutations A63V and E180G in alpha-tropomyosin (alpha-Tm) have been shown to cause slow cardiac muscle relaxation. In this study, we used two complementary genetic strategies, gene transfer in isolated rat myocytes and transgenesis in mice, to ascertain whether parvalbumin (Parv), a myoplasmic calcium buffer, could correct the diastolic dysfunction caused by these mutations. Sarcomere shortening measurements in rat cardiac myocytes expressing the alpha-Tm A63V mutant revealed a slower time to 50% relengthening (T50R: 44.2+/-1.4 ms in A63V, 36.8+/-1.0 ms in controls; n=96 to 108; P<0.001) when compared with controls. Dual gene transfer of alpha-Tm A63V and Parv caused a marked decrease in T50R (29.8+/-1.0 ms). However, this increase in relaxation rate was accompanied with a decrease in shortening amplitude (114.6+/-4.4 nm in A63+Parv, 137.8+/-5.3 nm in controls). Using an asynchronous gene transfer strategy, Parv expression was reduced (from approximately 0.12 to approximately 0.016 mmol/L), slow relaxation redressed, and shortening amplitude maintained (T50R=33.9+/-1.6 ms, sarcomere shortening amplitude=132.2+/-7.0 nm in A63V+PVdelayed; n=56). Transgenic mice expressing the E180G alpha-Tm mutation and mice expressing Parv in the heart were crossed. In isolated adult myocytes, the alpha-Tm mutation alone (E180G+/PV-) had slower sarcomere relengthening kinetics than the controls (T90R: 199+/-7 ms in E180G+/PV-, 130+/-4 ms in E180G-/PV-; n=71 to 72), but when coexpressed with Parv, cellular relaxation was faster (T90R: 36+/-4 ms in E180G+/PV+). Collectively, these findings show that slow relaxation caused by alpha-Tm mutants can be corrected by modifying calcium handling with Parv.

Pharmacological activation of plasma-membrane KATP channels reduces reoxygenation-induced Ca(2+) overload in cardiac myocytes via modulation of the diastolic membrane potential.

1. The opening of cardiac plasma-membrane ATP-sensitive K(+) channels (pmK(ATP)) can protect the heart against ischaemia/reperfusion injury. We recently demonstrated that the resting membrane potential (E(m)) of ventricular myocytes strongly modulates reoxygenation-induced Ca(2+) overload. This led to the hypothesis that activation of pmK(ATP) can influence the extent of chemically induced hypoxia (CIH)/reoxygenation Ca(2+) overload via hyperpolarization of the diastolic membrane potential of ventricular myocytes. 2. The membrane potential (E(m)) of isolated rat myocytes was determined using the perforated patch-clamp technique and DiBac(4)(3) imaging. Intracellular Ca(2+) ([Ca(2+)](i)) was monitored using FURA-2 imaging. 3. CIH/reoxygenation caused a significant depolarization of E(m) and a substantial increase in [Ca(2+)](i). The K(ATP) opener pinacidil (100 microm) and the pmK(ATP) opener P-1075 (100 microm) hyperpolarized the E(m) of normoxic myocytes. Pinacidil (100 microm) and P-1075 (10 and 100 microm), applied during reoxygenation, hyperpolarized E(m) and prevented reoxygenation-induced increases in [Ca(2+)](i). 4. Myocyte hypercontracture and death increased in parallel with an E(m) depolarization of 10-15 mV and increases in [Ca(2+)](i). Under these conditions, the selective pmK(ATP) channel inhibitor HMR 1098 further depolarized myocyte membrane potential and increased hypercontracture. 5. In conclusion, activation of pmK(ATP) channels can prevent CIH/reoxygenation-induced Ca(2+) overload via a mechanism that is dependent on hyperpolarization of diastolic membrane potential. Hyperpolarization toward normal resting membrane potential favours the Ca(2+) extrusion mode of Na(+)/Ca(2+) exchange.

β-Adrenergic receptor blockade modulates Bcl-X S expression and reduces apoptosis in failing myocardium

The mechanisms by which β-adrenergic receptor (β-AR) blockade modulates apoptosis in heart failure (HF) are unclear. We examined the impact of β-AR blockade with metoprolol on myocardial remodeling, apoptosis, pro-apoptotic (Fas, Fas ligand, Bax, and Bcl-X S) and anti-apoptotic (Bcl-X Land Bcl-2) gene expression, and Bcl-X L and Bcl-X S protein in post-infarction HF in rats. In untreated rats, there was significant ( P < 0.001) LV dilatation and systolic dysfunction compared to sham. Myocardial apoptosis was significantly increased ( P < 0.005). Fas, Bax, and Bcl-2 mRNA expression was unchanged. However, Fas ligand mRNA and Bcl-X S mRNA and protein, all undetectable in sham, were markedly elevated ( P < 0.001), whereas Bcl-X L mRNA and protein was unchanged. Immunohistochemistry confirmed increased Bcl-X S staining in failing myocardium, with unchanged Bcl-X L. Metoprolol treatment resulted in: (1) improved LV remodeling ( P < 0.025), (2) reduced myocardial apoptosis ( P < 0.005), and (3) selective reduction in myocardial Bcl-X S expression ( P < 0.001) without change in Fas, Fas ligand, Bax, Bcl-2, or Bcl-X L. Studies in isolated rat myocytes revealed that prolonged isoproterenol (ISO) stimulation significantly increased Bcl-X S protein, reducing the Bcl-X L/X S ratio and myocyte survival ( P < 0.005). ISO-induced Bcl-X S expression was significantly attenuated ( P < 0.001) by both metoprolol and CGP20712A, a β 1-AR selective antagonist, but not by ICI118,551, a β 2-AR selective antagonist. We conclude that adrenergic activation, such as occurs in HF, increases pro-apoptotic Bcl-X S expression via the β 1-AR. β-AR blockade in HF reduces myocardial apoptosis; attenuation of Bcl-X S expression may be one mechanism underlying this effect.

Metabolic regulation of coronary vascular tone: role of endothelin-1.

Coronary tone is determined by a balance between endogenously produced endothelin and metabolic dilators. We hypothesized that coronary vasodilation during augmented metabolism is the net result of decreased endothelin production and increased production of vasodilators. Isolated rat myocytes were stimulated at 0, 200, and 400 beats/min to modify metabolism. Supernatant from these preparations was added to isolated coronary arterioles with and without blocking vasoactive pathways (adenosine, bradykinin, and endothelin). Chronically instrumented swine were studied while resting and running on a treadmill before and after endothelin type A (ET(A)) receptor blockade. The vasodilatory properties of the supernatant increased with increased stimulation frequencies. Combined blockade of adenosine and bradykinin receptors abolished vasodilation in response to supernatant of stimulated myocytes. ET(A) blockade increased vasodilation to supernatant of unstimulated myocytes but did not affect dilation to supernatant of myocytes stimulated at 400 beats/min. In vivo, ET(A) blockade resulted in coronary vasodilation at rest, which waned during exercise. Thus endothelin has a tonic constrictor influence through the ET(A) receptor at low myocardial metabolic demand but its influence decreased during increased metabolism.