Sarah Kettlewell

Effects of Adenovirus-Mediated Sorcin Overexpression on Excitation-Contraction Coupling in Isolated Rabbit Cardiomyocytes

&NA; To evaluate the effect of sorcin on cardiac excitation‐contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human sorcin (Ad‐sorcin). A &bgr;‐galactosidase adenovirus (Ad‐LacZ) was used as a control. Fractional shortening in response to 1‐Hz field stimulation (at 37°C) was significantly reduced in Ad‐sorcin‐transfected myocytes compared with control myocytes (2.10±0.05% [n=311] versus 2.42±0.06% [n=312], respectively; P<0.001). Action potential duration (at 20°C) was significantly less in the Ad‐sorcin group (458±22 ms, n=11) compared with the control group (520±19 ms, n=10; P<0.05). In voltage‐clamped, fura 2‐loaded myocytes (20°C), a reduced peak‐systolic and end‐diastolic [Ca2+]i was observed after Ad‐sorcin transfection. L‐type Ca2+ current amplitude and time course were unaffected. Caffeine‐induced Ca2+ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na+‐Ca2+ exchanger (NCX) current revealed a significantly lower SR Ca2+ content and faster Ca2+‐extrusion kinetics in Ad‐sorcin‐transfected cells. Higher NCX activity after Ad‐sorcin transfection was confirmed by measuring the NCX current‐voltage relationship. &bgr;‐Escin‐permeabilized rabbit cardiomyocytes were used to study the effects of sorcin overexpression on Ca2+ sparks imaged with fluo 3 at 145 to 160 nmol/L [Ca2+] using a confocal microscope. Under these conditions, caffeine‐mediated SR Ca2+ release was not different between the two groups. Spontaneous spark frequency, duration, width, and amplitude were lower in sorcin‐overexpressing myocytes. In summary, sorcin overexpression in rabbit cardiomyocytes decreased Ca2+‐transient amplitude predominately by lowering SR Ca2+ content via increased NCX activity. The effect of sorcin overexpression on Ca2+ sparks indicates an effect on the ryanodine receptor that may also influence excitation‐contraction coupling. (Circ Res. 2003;93:132‐139.)

The electrophysiological and mechanical effects of 2,3-butane-dione monoxime and cytochalasin-D in the Langendorff perfused rabbit heart.

Procedures that reduce contraction are used to facilitate optical measurements of membrane potential, but it is unclear to what extent they affect the excitability of the heart. This study has examined the electrophysiological consequences of a range of extracellular [Ca2+] (0.7–2.5 mmol l−1), 2,3‐butane‐dione monoxime (BDM; 1–20 mmol l−1) and cytochalasin‐D (Cyto‐D; 1–5 μmol l−1). Methods. Monophasic action potentials (MAPs) were recorded from the basal epicardial surface of the left ventricle of isolated rabbit hearts. Conduction delay (CD) and time to 90% repolarisation of the monophasic action potential (MAPD90) were measured. The effects of BDM and Cyto‐D on restitution were studied at a [Ca2+] of 1.9 mmol l−1. Restitution curves for MAPD90 were generated using a standard S1–S2 protocol. Results. All manoeuvres decreased left ventricular developed pressure (LVDP): 0.7 mmol l−1 Ca2+ to 74.0 ± 6.1%, 20 mmol l−1 BDM to 4.5 ± 1.0%, and 5 μmol l−1 Cyto‐D to 12.8 ± 3.5% of control value. CD decreased from a control value (33.3 ± 1.0 ms, n= 16) to 93.0 ± 2.2% in 0.7 mmol l−1 Ca2+, but increased to 133.7 ± 10.5% in 20 mmol l−1 BDM and 127.4 ± 10.6% in 5 μmol l−1 Cyto‐D. At 350 ms pacing cycle length, MAPD90 (control = 119.6 ± 1.7 ms n= 16) was prolonged by reduced extracellular [Ca2+]. BDM had no effects on MAPD90 at control pacing rates. Cyto‐D caused a significant prolongation (to 115.0 ± 3.0% of control, n= 6) at the highest concentration studied (5 μmol l−1). Both BDM (20 mmol l−1) and Cyto‐D (3 μmol l−1) flattened the restitution curves but neither agent altered maximum MAPD90. Conclusions. Extracellular [Ca2+] of 1.9 mmol l−1 in conjunction with a moderate dose of Cyto‐D (3 μmol l−1) reduced contractility with minimal effects on action potential duration and conduction at a fixed pacing cycle length. However, both BDM and Cyto‐D had pronounced effects on electrical restitution.

Changes of intra-mitochondrial Ca2+ in adult ventricular cardiomyocytes examined using a novel fluorescent Ca2+ indicator targeted to mitochondria

In this study a Ca(2+) sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca(2+)-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca(2+) sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca(2+) transient. Cytoplasmic (Fura-2) and mitochondrial Ca(2+) (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca(2+) loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca(2+) sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K(d) of approximately 220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca(2+) transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca(2+) release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by approximately 65% and increased the time to 50% decay, whilst cytosolic Ca(2+) transients were unchanged. The mitochondrial Ca(2+) uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca(2+). The mitochondrial-targeted Ca(2+) probe indicates sustained and transient phases of mitochondrial Ca(2+) signal, which are dependent on cytoplasmic Ca(2+) levels and require a functional mCU.

Mapping of epicardial activation in a rabbit model of chronic myocardial infarction

Introduction: This study examines the consequences of a large transmural apical infarct on the epicardial electrical activity in isolated rabbit hearts.

Measuring local gradients of intramitochondrial [Ca(2+)] in cardiac myocytes during sarcoplasmic reticulum Ca(2+) release.

Rationale: Mitochondrial [Ca2+] ([Ca2+]mito) regulates mitochondrial energy production, provides transient Ca2+ buffering under stress, and can be involved in cell death. Mitochondria are near the sarcoplasmic reticulum (SR) in cardiac myocytes, and evidence for crosstalk exists. However, quantitative measurements of [Ca2+]mito are limited, and spatial [Ca2+]mito gradients have not been directly measured. Objective: To directly measure local [Ca2+]mito during normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial [Ca2+]mito gradients. Methods and Results: Using the mitochondrially targeted inverse pericam indicator Mitycam, calibrated in situ, we directly measured [Ca2+]mito during SR Ca2+ release in intact rabbit ventricular myocytes by confocal microscopy. During steady state pacing, &Dgr;[Ca2+]mito amplitude was 29±3 nmol/L, rising rapidly (similar to cytosolic free [Ca2+]) but declining much more slowly. Taking advantage of the structural periodicity of cardiac sarcomeres, we found that [Ca2+]mito near SR Ca2+ release sites (Z-line) versus mid-sarcomere (M-line) reached a high peak amplitude (37±4 versus 26±4 nmol/L, respectively P<0.05) which occurred earlier in time. This difference was attributed to ends of mitochondria being physically closer to SR Ca2+ release sites, because the mitochondrial Ca2+ uniporter was homogeneously distributed, and elevated [Ca2+] applied laterally did not produce longitudinal [Ca2+]mito gradients. Conclusions: We developed methods to measure spatiotemporal [Ca2+]mito gradients quantitatively during excitation–contraction coupling. The amplitude and kinetics of [Ca2+]mito transients differ significantly from those in the cytosol and are respectively higher and faster near the Z-line versus M-line. This approach will help clarify SR-mitochondrial Ca2+ signaling.

Inhibiting Mitochondrial Na+/Ca2+ Exchange Prevents Sudden Death in a Guinea Pig Model of Heart Failure

Rationale: In cardiomyocytes from failing hearts, insufficient mitochondrial Ca2+ accumulation secondary to cytoplasmic Na+ overload decreases NAD(P)H/NAD(P)+ redox potential and increases oxidative stress when workload increases. These effects are abolished by enhancing mitochondrial Ca2+ with acute treatment with CGP-37157 (CGP), an inhibitor of the mitochondrial Na+/Ca2+ exchanger. Objective: Our aim was to determine whether chronic CGP treatment mitigates contractile dysfunction and arrhythmias in an animal model of heart failure (HF) and sudden cardiac death (SCD). Methods and Results: Here, we describe a novel guinea pig HF/SCD model using aortic constriction combined with daily &bgr;-adrenergic receptor stimulation (ACi) and show that chronic CGP treatment (ACi plus CGP) attenuates cardiac hypertrophic remodeling, pulmonary edema, and interstitial fibrosis and prevents cardiac dysfunction and SCD. In the ACi group 4 weeks after pressure overload, fractional shortening and the rate of left ventricular pressure development decreased by 36% and 32%, respectively, compared with sham-operated controls; in contrast, cardiac function was completely preserved in the ACi plus CGP group. CGP treatment also significantly reduced the incidence of premature ventricular beats and prevented fatal episodes of ventricular fibrillation, but did not prevent QT prolongation. Without CGP treatment, mortality was 61% in the ACi group <4 weeks of aortic constriction, whereas the death rate in the ACi plus CGP group was not different from sham-operated animals. Conclusions: The findings demonstrate the critical role played by altered mitochondrial Ca2+ dynamics in the development of HF and HF-associated SCD; moreover, they reveal a novel strategy for treating SCD and cardiac decompensation in HF.

Distinct mPTP activation mechanisms in ischaemia-reperfusion: contributions of Ca2+, ROS, pH, and inorganic polyphosphate.

AIMS The mitochondrial permeability transition pore (mPTP) plays a central role for tissue damage and cell death during ischaemia-reperfusion (I/R). We investigated the contribution of mitochondrial inorganic polyphosphate (polyP), a potent activator of Ca(2+)-induced mPTP opening, towards mPTP activation and cardiac cell death in I/R. METHODS AND RESULTS A significant increase in mitochondrial free calcium concentration ([Ca(2+)]m), reactive oxygen species (ROS) generation, mitochondrial membrane potential depolarization (ΔΨm), and mPTP activity, but no cell death, was observed after 20 min of ischaemia. The [Ca(2+)]m increase during ischaemia was partially prevented by the mitochondrial Ca(2+) uniporter (MCU) inhibitor Ru360 and completely abolished by the combination of Ru360 and the ryanodine receptor type 1 blocker dantrolene, suggesting two complimentary Ca(2+) uptake mechanisms. In the absence of Ru360 and dantrolene, mPTP closing by polyP depletion or CSA decreased mitochondrial Ca(2+) uptake, suggesting that during ischaemia Ca(2+) can enter mitochondria through mPTP. During reperfusion, a burst of endogenous polyP production coincided with a decrease in [Ca(2+)]m, a decline in superoxide generation, and an acceleration of hydrogen peroxide (H2O2) production. An increase in H2O2 correlated with restoration of mitochondrial pHm and an increase in cell death. mPTP opening and cell death on reperfusion were prevented by antioxidants Trolox and MnTBAP [Mn (III) tetrakis (4-benzoic acid) porphyrin chloride]. Enzymatic polyP depletion did not affect mPTP opening during reperfusion, but increased ROS generation and cell death, suggesting that polyP plays a protective role in cellular stress response. CONCLUSIONS Transient Ca(2+)/polyP-mediated mPTP opening during ischaemia may serve to protect cells against cytosolic Ca(2+) overload, whereas ROS/pH-mediated sustained mPTP opening on reperfusion induces cell death.

Negative Inotropy of the Gastric Proton Pump Inhibitor Pantoprazole in Myocardium From Humans and Rabbits Evaluation of Mechanisms

Background— Proton pump inhibitors are used extensively for acid-related gastrointestinal diseases. Their effect on cardiac contractility has not been assessed directly. Methods and Results— Under physiological conditions (37°C, pH 7.35, 1.25 mmol/L Ca2+), there was a dose-dependent decrease in contractile force in ventricular trabeculae isolated from end-stage failing human hearts superfused with pantoprazole. The concentration leading to 50% maximal response was 17.3±1.3 &mgr;g/mL. Similar observations were made in trabeculae from human atria, normal rabbit ventricles, and isolated rabbit ventricular myocytes. Real-time polymerase chain reaction demonstrated the expression of gastric H+/K+–adenosine triphosphatase in human and rabbit myocardium. However, measurements with BCECF-loaded rabbit trabeculae did not reveal any significant pantoprazole-dependent changes of pHi. Ca2+ transients recorded from field-stimulated fluo 3–loaded myocytes (F/F0) were significantly depressed by 10.4±2.1% at 40 &mgr;g/mL. Intracellular Ca2+ fluxes were assessed in fura 2–loaded, voltage-clamped rabbit ventricular myocytes. Pantoprazole (40 &mgr;g/mL) caused an increase in diastolic [Ca2+]i by 33±12%, but peak systolic [Ca2+]i was unchanged, resulting in a decreased Ca2+ transient amplitude by 25±8%. The amplitude of the L-type Ca2+ current (ICa,L) was reduced by 35±5%, and sarcoplasmic reticulum Ca2+ content was reduced by 18±6%. Measurements of oxalate-supported sarcoplasmic reticulum Ca2+ uptake in permeabilized cardiomyocytes indicated that pantoprazole decreased Ca2+ sensitivity (Kd) of sarcoplasmic reticulum Ca2+ adenosine triphosphatase: control, Kd=358±15 nmol/L; 40 &mgr;g/mL pantoprazole, Kd=395±12 nmol/L (P<0.05). Pantoprazole also acted on cardiac myofilaments to reduced Ca2+-activated force. Conclusions— Pantoprazole depresses cardiac contractility in vitro by depression of Ca2+ signaling and myofilament activity. In view of the extensive use of this agent, the effects should be evaluated in vivo.

Inositol‐1,4,5‐trisphosphate induced Ca2+ release and excitation–contraction coupling in atrial myocytes from normal and failing hearts

Impaired calcium (Ca2+) signalling is the main contributor to depressed ventricular contractile function and occurrence of arrhythmia in heart failure (HF). Here we report that in atrial cells of a rabbit HF model, Ca2+ signalling is enhanced and we identified the underlying cellular mechanisms. Enhanced Ca2+ transients (CaTs) are due to upregulation of inositol‐1,4,5‐trisphosphate receptor induced Ca2+ release (IICR) and decreased mitochondrial Ca2+ sequestration. Enhanced IICR, however, together with an increased activity of the sodium–calcium exchange mechanism, also facilitates spontaneous Ca2+ release in form of arrhythmogenic Ca2+ waves and spontaneous action potentials, thus enhancing the arrhythmogenic potential of atrial cells. Our data show that enhanced Ca2+ signalling in HF provides atrial cells with a mechanism to improve ventricular filling and to maintain cardiac output, but also increases the susceptibility to develop atrial arrhythmias facilitated by spontaneous Ca2+ release.

Adrenergic signaling regulates mitochondrial Ca2+ uptake through Pyk2-dependent tyrosine phosphorylation of the mitochondrial Ca2+ uniporter.

AIMS Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. RESULTS α1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. INNOVATION Our data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. CONCLUSION The α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells.