Wayne R. Giles

Nkx2-5 Pathways and Congenital Heart Disease: Loss of Ventricular Myocyte Lineage Specification Leads to Progressive Cardiomyopathy and Complete Heart Block

Human mutations in Nkx2-5 lead to progressive cardiomyopathy and conduction defects via unknown mechanisms. To define these pathways, we generated mice with a ventricular-restricted knockout of Nkx2-5, which display no structural defects but have progressive complete heart block, and massive trabecular muscle overgrowth found in some patients with Nkx2-5 mutations. At birth, mutant mice display a hypoplastic atrioventricular (AV) node and then develop selective dropout of these conduction cells. Transcriptional profiling uncovered the aberrant expression of a unique panel of atrial and conduction system-restricted target genes, as well as the ectopic, high level BMP-10 expression in the adult ventricular myocardium. Further, BMP-10 is shown to be necessary and sufficient for a major component of the ventricular muscle defects. Accordingly, loss of ventricular muscle cell lineage specification into trabecular and conduction system myocytes is a new mechanistic pathway for progressive cardiomyopathy and conduction defects in congenital heart disease.

Functional properties of K + currents in adult mouse ventricular myocytes

Although the K+ currents expressed in hearts of adult mice have been studied extensively, detailed information concerning their relative sizes and biophysical properties in ventricle and atrium is lacking. Here we describe and validate pharmacological and biophysical methods that can be used to isolate the three main time‐ and voltage‐dependent outward K+ currents which modulate action potential repolarization. A Ca2+‐independent transient outward K+ current, Ito, can be separated from total outward current using an ‘inactivating prepulse’. The rapidly activating, slowly inactivating delayed rectifier K+ current, IKur, can be isolated using submillimolar concentrations of 4‐aminopyridine (4‐AP). The remaining K+ current, Iss, can be obtained by combining these two procedures: (i) inactivating Ito and (ii) eliminating IKur by application of low concentration of 4‐AP. Iss activates relatively slowly and shows very little inactivation, even during depolarizations lasting several seconds. Our findings also show that the rate of reactivation of Ito is more than 20‐fold faster than that of IKur. These results demonstrate that the outward K+ currents in mouse ventricles can be separated based on their distinct time and voltage dependence, and different sensitivities to 4‐AP. Data obtained at both 22 and 32°C demonstrate that although the duration of the inactivating prepulse has to be adapted for the recording temperature, this approach for separation of K+ current components is also valid at more physiological temperatures. To demonstrate that these methods also allow separation of these K+ currents in other cell types, we have applied this same approach to myocytes from mouse atria. Molecular approaches have been used to compare the expression levels of different K+ channels in mouse atrium and ventricle. These findings provide new insights into the functional roles of IKur, Ito and Iss during action potential repolarization.

Comparison of contraction and calcium handling between right and left ventricular myocytes from adult mouse heart : a role for repolarization waveform

In the mammalian heart, the right ventricle (RV) has a distinct structural and electrophysiological profile compared to the left ventricle (LV). However, the possibility that myocytes from the RV and LV have different contractile properties has not been established. In this study, sarcomere shortening, [Ca2+]i transients and Ca2+ and K+ currents in unloaded myocytes isolated from the RV, LV epicardium (LVepi) and LV endocardium (LVendo) of adult mice were evaluated. Maximum sarcomere shortening elicited by field stimulation was graded in the order: LVendo > LVepi > RV. Systolic [Ca2+]i was higher in LVendo myocytes than in RV myocytes. Voltage‐clamp experiments in which action potential (AP) waveforms from RV and LVendo were used as the command signal, demonstrated that total Ca2+ influx and myocyte shortening were larger in response to the LVendo AP, independent of myocyte subtypes. Evaluation of possible regional differences in myocyte Ca2+ handling was based on: (i) the current–voltage relation of the Ca2+ current; (ii) sarcoplasmic reticulum Ca2+ uptake; and (iii) mRNA expression of important components of the Ca2+ handling system. None of these were significantly different between RV and LVendo. In contrast, the Ca2+‐independent K+ current, which modulates AP repolarization, was significantly different between RV, LVepi and LVendo. These results suggest that these differences in K+ currents can alter AP duration and modulate the [Ca2+]i transient and corresponding contraction. In summary, these findings provide an initial description of regional differences in excitation–contraction coupling in the adult mouse heart. Evidence that the AP waveform is an important causative factor for these differences is presented.

Pharmacological activation of plasma‐membrane KATP channels reduces reoxygenation‐induced Ca2+ overload in cardiac myocytes via modulation of the diastolic membrane potential

The opening of cardiac plasma‐membrane ATP‐sensitive K+ channels (pmKATP) can protect the heart against ischaemia/reperfusion injury. We recently demonstrated that the resting membrane potential (Em) of ventricular myocytes strongly modulates reoxygenation‐induced Ca2+ overload. This led to the hypothesis that activation of pmKATP can influence the extent of chemically induced hypoxia (CIH)/reoxygenation Ca2+ overload via hyperpolarization of the diastolic membrane potential of ventricular myocytes. The membrane potential (Em) of isolated rat myocytes was determined using the perforated patch‐clamp technique and DiBac4(3) imaging. Intracellular Ca2+ ([Ca2+]i) was monitored using FURA‐2 imaging. CIH/reoxygenation caused a significant depolarization of Em and a substantial increase in [Ca2+]i. The KATP opener pinacidil (100 μM) and the pmKATP opener P‐1075 (100 μM) hyperpolarized the Em of normoxic myocytes. Pinacidil (100 μM) and P‐1075 (10 and 100 μM), applied during reoxygenation, hyperpolarized Em and prevented reoxygenation‐induced increases in [Ca2+]i. Myocyte hypercontracture and death increased in parallel with an Em depolarization of 10–15 mV and increases in [Ca2+]i. Under these conditions, the selective pmKATP channel inhibitor HMR 1098 further depolarized myocyte membrane potential and increased hypercontracture. In conclusion, activation of pmKATP channels can prevent CIH/reoxygenation‐induced Ca2+ overload via a mechanism that is dependent on hyperpolarization of diastolic membrane potential. Hyperpolarization toward normal resting membrane potential favours the Ca2+ extrusion mode of Na+/Ca2+ exchange.

Resting Membrane Potential Regulates Na+–Ca2+ Exchange-Mediated Ca2+ Overload during Hypoxia–Reoxygenation in Rat Ventricular Myocytes

In the heart, reperfusion following an ischaemic episode can result in a marked increase in [Ca2+]i and cause myocyte dysfunction and death. Although the Na+–Ca2+ exchanger has been implicated in this response, the ionic mechanisms that are responsible have not been identified. In this study, the hypothesis that the diastolic membrane potential can influence Na+–Ca2+ exchange and Ca2+ homeostasis during chemically induced hypoxia–reoxygenation has been tested using right ventricular myocytes isolated from adult rat hearts. Superfusion with selected [K+]o of 0.5, 2.5, 5, 7, 10 and 15 mm yielded the following resting membrane potentials: −27.6 ± 1.63 mV, −102.2 ± 1.89, −86.5 ± 1.03, −80.1 ± 1.25, −73.6 ± 1.02 and −66.4 ± 1.03, respectively. In a second set of experiments myocytes were subjected to chemically induced hypoxia–reoxygenation at these different [K+]o, while [Ca2+]i was monitored using fura‐2. These results demonstrated that after chemically induced hypoxia–reoxygenation had caused a marked increase in [Ca2+]i, hyperpolarization of myocytes with 2.5 mm[K+]o significantly reduced [Ca2+]i (7.5 ± 0.32 vs. 16.9 ± 0.55 %); while depolarization (with either 0.5 or 15 mm[K+]o) significantly increased [Ca2+]i (31.8 ± 3.21 and 20.8 ± 0.36 vs. 16.9 ± 0.55 %, respectively). As expected, at depolarized membrane potentials myocyte hypercontracture and death increased in parallel with Ca2+ overload. The involvement of the Na+–Ca2+ exchanger in Ca2+ homeostasis was evaluated using the Na+–Ca2+ exchanger inhibitor KB‐R7943. During reoxygenation KB‐R7943 (5 μm) almost completely prevented the increase in [Ca2+]i both in control conditions (in 5 mm[K+]o: 2.2 ± 0.40 vs. 10.8 ± 0.14 %) and in depolarized myocytes (in 15 mm[K+]o: −2.1 ± 0.51 vs. 11.3 ± 0.05 %). These findings demonstrate that the resting membrane potential of ventricular myocytes is a critical determinant of [Ca2+]i during hypoxia–reoxygenation. This appears to be due mainly to an effect of diastolic membrane potential on the Na+–Ca2+ exchanger, since at depolarized potentials this exchanger mechanism operates in the reverse mode, causing a significant Ca2+ influx.

Electrophysiological evidence for a gradient of G protein-gated K+ current in adult mouse atria

Whole cell current and voltage clamp techniques were used to examine the properties of acetylcholine‐sensitive K+ current (IKACh) in myocytes from adult mouse atrium. Superfusion of a maximal dose of carbachol (CCh; 10 μM) caused a substantial increase in K+ current in all myocytes examined. The current–voltage (I–V) relation of maximally activated IKACh exhibited weak inward rectification. Consequently, CCh increased the amount of depolarising current necessary to evoke action potentials (APs), and APs evoked in CCh had significantly shorter durations than control APs (P<0.05). The effects of CCh on K+ current and on AP properties were blocked by the muscarinic receptor antagonist methoctramine (1 μM). ACh (10 μM) activated a K+ current with identical properties to that activated by CCh, as did the A1 receptor agonist adenosine (100 μM). Right atrial myocytes had significantly more IKACh than left atrial myocytes (P<0.05), regardless of whether IKACh was evoked by superfusion of muscarinic or A1 receptor agonists. IKACh current density was significantly higher in SA node myocytes than either right or left atrial myocytes. These data identify a gradient of IKACh current density across the supraventricular structures of mouse heart. This gradient, combined with the heterogeneous distribution of parasympathetic innervation of the atria, may contribute to the proarrhythmic ability of vagal nerve stimulation to augment dispersion of atrial refractoriness.

Changes in extracellular K+ concentration modulate contractility of rat and rabbit cardiac myocytes via the inward rectifier K+ current IK1

The mechanisms underlying the inotropic effect of reductions in [K+]o were studied using recordings of membrane potential, membrane current, cell shortening and [Ca2+]i in single, isolated cardiac myocytes. Three types of mammalian myocytes were chosen, based on differences in the current density and intrinsic voltage dependence of the inwardly rectifying background K+ current IK1 in each cell type. Rabbit ventricular myocytes had a relatively large IK1 with a prominent negative slope conductance whereas rabbit atrial cells expressed much smaller IK1, with little or no negative slope conductance. IK1 in rat ventricle was intermediate in both current density and slope conductance. Action potential duration is relatively short in both rabbit atrial and rat ventricular myocytes, and consequently both cell types spend much of the duty cycle at or near the resting membrane potential. Rapid increases or decreases of [K+]o elicited significantly different inotropic effects in rat and rabbit atrial and ventricular myocytes. Voltage‐clamp and current‐clamp experiments showed that the effects on cell shortening and [Ca2+]i following changes in [K+]o were primarily the result of the effects of alterations in IK1, which changed resting membrane potential and action potential waveform. This in turn differentially altered the balance of Ca2+ efflux via the sarcolemmal Na+–Ca2+ exchanger, Ca2+ influx via voltage‐dependant Ca2+ channels and sarcoplasmic reticulum (SR) Ca2+ release in each cell type. These results support the hypothesis that the inotropic effect of alterations of [K+]o in the heart is due to significant non‐linear changes in the current–voltage relation for IK1 and the resulting modulation of the resting membrane potential and action potential waveform.

Plasma membrane KATP channel-mediated cardioprotection involves posthypoxic reductions in calcium overload and contractile dysfunction: mechanistic insights into cardioplegia

Our recent data demonstrate that activation of pmKATP channels polarizes the membrane of cardiomyocytes and reduces Na+/Ca2+ exchange‐mediated Ca2+ overload. However, it is important that these findings be extended into contractile models of hypoxia/reoxygenation injury to further test the notion that pmKATP channel activation affords protection against contractile dysfunction and calcium overload. Single rat heart right ventricular myocytes were enzymatically isolated, and cell contractility and Ca2+ transients in field‐stimulated myocytes were measured in a cellular model of metabolic inhibition and reoxygenation. Activation of pmKATP with P‐1075 (5µM) or inhibition of the Na+/Ca2+ exchanger with KB‐R7943 (5 µM) reduced reoxygenation‐induced diastolic Ca2+ overload and improved the rate and magnitude of posthypoxic contractile recovery during the first few minutes of reoxygenation. Moreover, diastolic Ca2+ overload and posthypoxic contractile dysfunction were aggravated in ventricular myocytes either subjected to specific blockade of pmKATP with HMR1098 (20 µM) or expressing the dominant‐negative pmKATP construct Kir6.2(AAA) in the presence of P‐1075. Our results suggest that a common mechanism, involving resting membrane potential‐modulated increases in diastolic [Ca2+]i, is responsible for the development of contractile dysfunction during reoxygenation following metabolic inhibition. This novel and highly plausible cellular mechanism for pmKATP‐mediated cardioprotection may have direct clinical relevance as evidenced by the following findings: a hypokalemic polarizing cardioplegia solution supplemented with the pmKATP opener P‐1075 improved Ca2+ homeostasis and recovery of function compared with hyperkalemic depolarizing St. Thomas cardioplegia following contractile arrest in single ventricular myocytes and working rat hearts. We therefore propose that activation of pmKATP channels improves posthypoxic cardiac function via reductions in abnormal diastolic Ca2+ homeostasis mediated by reverse‐mode Na+/Ca2+ exchange.

Measurements of Electrophysiological Effects of Components of Acute Ischemia in Langendorff-Perfused Rat Hearts Using Voltage-Sensitive Dye Mapping

Introduction: This study was carried out to evaluate optical mapping in the presence of cytochalasin‐D as a method for measuring electrophysiological responses in general, and in particular the responses to acute ischemia in the Langendorff‐perfused rat heart. Cytochalasin‐D is commonly used to reduce contraction for the purpose of suppressing motion artifacts in voltage‐sensitive dye recordings of cardiac membrane potential.

ATX-II effects on the apparent location of M cells in a computational model of a human left ventricular wedge.

Introduction: The apparent location of the myocytes (M cells) with the longest action potential duration (APD) in a canine left ventricular (LV) wedge have been reported to shift after application of a sea anemone toxin, ATX‐II. This toxin slows inactivation of INa and thus prolongs APD. Thus, M cells may exhibit dynamic functional states, rather than being a static, anatomically discrete, myocyte population. In this study, we attempted to further define and understand this phenomenon using a mathematical model of the human ventricular myocyte action potential incorporated into an in silico “wedge” preparation. Our simulations demonstrate that even under conditions of a fixed population and ratio of epicardial, M, and endocardial myocytes, the apparent anatomical position (transmural location) of the myocytes with the longest APD can shift following ATX‐II treatment. This arises because the ATX‐II effect, modeled as a small increase in the late or persistent Na+ current, and consequent prolongation of APD significantly changes the electrotonic interactions between ventricular myocytes in this LV wedge preparation.