Guy Salama

Calmodulin kinase II inhibition protects against structural heart disease

β-Adrenergic receptor (βAR) stimulation increases cytosolic Ca2+ to physiologically augment cardiac contraction, whereas excessive βAR activation causes adverse cardiac remodeling, including myocardial hypertrophy, dilation and dysfunction, in individuals with myocardial infarction. The Ca2+-calmodulin–dependent protein kinase II (CaMKII) is a recently identified downstream element of the βAR-initiated signaling cascade that is linked to pathological myocardial remodeling and to regulation of key proteins involved in cardiac excitation-contraction coupling. We developed a genetic mouse model of cardiac CaMKII inhibition to test the role of CaMKII in βAR signaling in vivo. Here we show CaMKII inhibition substantially prevented maladaptive remodeling from excessive βAR stimulation and myocardial infarction, and induced balanced changes in excitation-contraction coupling that preserved baseline and βAR-stimulated physiological increases in cardiac function. These findings mark CaMKII as a determinant of clinically important heart disease phenotypes, and suggest CaMKII inhibition can be a highly selective approach for targeting adverse myocardial remodeling linked to βAR signaling.

Engraftment of connexin 43-expressing cells prevents post-infarct arrhythmia

Ventricular tachyarrhythmias are the main cause of sudden death in patients after myocardial infarction. Here we show that transplantation of embryonic cardiomyocytes (eCMs) in myocardial infarcts protects against the induction of ventricular tachycardia (VT) in mice. Engraftment of eCMs, but not skeletal myoblasts (SMs), bone marrow cells or cardiac myofibroblasts, markedly decreased the incidence of VT induced by in vivo pacing. eCM engraftment results in improved electrical coupling between the surrounding myocardium and the infarct region, and Ca2+ signals from engrafted eCMs expressing a genetically encoded Ca2+ indicator could be entrained during sinoatrial cardiac activation in vivo. eCM grafts also increased conduction velocity and decreased the incidence of conduction block within the infarct. VT protection is critically dependent on expression of the gap-junction protein connexin 43 (Cx43; also known as Gja1): SMs genetically engineered to express Cx43 conferred a similar protection to that of eCMs against induced VT. Thus, engraftment of Cx43-expressing myocytes has the potential to reduce life-threatening post-infarct arrhythmias through the augmentation of intercellular coupling, suggesting autologous strategies for cardiac cell-based therapy.

Optical Imaging of the Heart

Optical techniques have revolutionized the investigation of cardiac cellular physiology and advanced our understanding of basic mechanisms of electrical activity, calcium homeostasis, and metabolism. Although optical methods are widely accepted and have been at the forefront of scientific discoveries, they have been primarily applied at cellular and subcellular levels and considerably less to whole heart organ physiology. Numerous technical difficulties had to be overcome to dynamically map physiological processes in intact hearts by optical methods. Problems of contraction artifacts, cellular heterogeneities, spatial and temporal resolution, limitations of surface images, depth-of-field, and need for large fields of view (ranging from 2×2 mm2 to 3×3 cm2) have all led to the development of new devices and optical probes to monitor physiological parameters in intact hearts. This review aims to provide a critical overview of current approaches, their contributions to the field of cardiac electrophysiology, and future directions of various optical imaging modalities as applied to cardiac physiology at organ and tissue levels.

NITRIC OXIDE ACTIVATES SKELETAL AND CARDIAC RYANODINE RECEPTORS

The endothelial-derived relaxing factor, nitric oxide (NO.) has been shown to depress force in smooth and cardiac muscles through the activation of guanylyl cyclase and an increase in cGMP. In fast skeletal muscle, NO (i.e. NO-related compounds) elicits a modest decrease in developed force, but in contracting muscles NO increases force by a mechanism independent of cGMP. We now demonstrate an alternative mechanism whereby NO triggers Ca2+ release from skeletal and cardiac sarcoplasmic reticulum (SR). NO delivered in the form of NO gas, NONOates (a class of sulfur-free compounds capable of releasing NO), or S-nitrosothiols (R-SNO) oxidized or transnitrosylated regulatory thiols on the release channel (or ryanodine receptor, RyR), resulting in channel opening and Ca2+ release from skeletal and cardiac SR. The process was reversed by sulfhydryl reducing agents which promoted channel closure and Ca2+ reuptake by ATP-driven Ca2+ pumps. NO did not directly alter Ca(2+)-ATPase activity but increased the open probability of RyRs reconstituted in planar bilayers and inhibited [3H]-ryanodine binding to RyRs. The formation of peroxynitrite or thiyl radicals did not account for the reversible R-SNO-dependent activation of RyRs. Ca2+ release induced by nitric oxide free radicals (NO.) was potentiated by cysteine providing compelling evidence that NO. in the presence of O2 formed nitrosylated cysteine followed by the transnitrosation of regulatory thiols on the RyR to activate the channel. These findings demonstrate direct interactions of NO derivatives with RyRs and a new fundamental mechanism to regulate force in striated muscle.

Cytosolic Ca2+ triggers early afterdepolarizations and torsade de pointes in rabbit hearts with type 2 long QT syndrome

The role of intracellular Ca2+ (Ca12+) in triggering early afterdepolarizations (EADs), the origins of EADs and the mechanisms underlying Torsade de Pointes (TdP) were investigated in a model of long QT syndrome (Type 2). Perfused rabbit hearts were stained with RH327 and Rhod‐2/AM to simultaneously map membrane potential (Vm) and Ca12+ with two photodiode arrays. The IKr blocker E4031 (0.5 μM) together with 50% reduction of [K+]o and [Mg2+]o elicited long action potentials (APs), Vm oscillations on AP plateaux (EADs) then ventricular tachycardia (VT). Cryoablation of both ventricular chambers eliminated Purkinje fibres as sources of EADs. E4031 prolonged APs (0.28 to 2.3 s), reversed repolarization sequences (base→apex) and enhanced repolarization gradients (30 to 230 ms, n= 12) indicating a heterogeneous distribution of IKr. At low [K+]o and [Mg2+]o, E4031 elicited spontaneous Ca12+and Vm spikes or EADs (3.5 ± 1.9 Hz) during the AP plateau (n= 6). EADs fired ‘out‐of‐phase’ from several sites, propagated, collided then evolved to TdP. Phase maps (Ca12+vs. Vm) had counterclockwise trajectories shaped like a ‘boomerang’ during an AP and like ellipses during EADs, with Vm preceding Ca12+ by 9.2 ± 1.4 (n= 6) and 7.2 ± 0.6 ms (n= 5/6), respectively. After cryoablation, EADs from surviving epicardium (∼1 mm) fired at the same frequency (3.4 ± 0.35 Hz, n= 6) as controls. At the origins of EADs, Ca12+ preceded Vm and phase maps traced clockwise ellipses. Away from EAD origins, Vm coincided with or preceded Ca12+. In conclusion, overload elicits EADs originating from either ventricular or Purkinje fibres and ‘out‐of‐phase’ EAD activity from multiple sites generates TdP, evident in pseudo‐ECGs.

Simultaneous maps of optical action potentials and calcium transients in guinea‐pig hearts: mechanisms underlying concordant alternans

1 The mechanisms underlying electro‐mechanical alternans caused by faster heart rates were investigated in perfused guinea‐pig hearts stained with RH237 and Rhod‐2 AM to simultaneously map optical action potentials (APs) and intracellular free Ca2+ (Ca2+i). 2 Fluorescence images of the heart were focused on two 16 × 16 photodiode arrays to map Ca2+i (emission wavelength (λem) = 585 ± 20 nm) and APs (λem > 715 nm) from 252 sites. Spatial resolution was 0·8 mm × 0·8 mm per diode and temporal resolution 4000 frames s−1. 3 The mean time‐to‐peak for APs and [Ca2+]i was spatially homogeneous (8·8 ± 0·5 and 25·6 ± 5·0 ms, respectively; n= 6). The durations of APs (APDs) and Ca2+i transients were shorter at the apex and progressively longer towards the base, indicating a gradient of ventricular relaxation. 4 Restitution kinetics revealed increasingly longer delays between AP and Ca2+i upstrokes (9·5 ± 0·4 to 11·3 ± 0·4 ms) with increasingly shorter S1‐S2 intervals, particularly at the base, despite nearly normal peak [Ca2+]i. 5 Alternans of APs and Ca2+i transients were induced by a decrease in cycle length (CL), if the shorter CL captured at the pacing site and was shorter than refractory periods (RPs) near the base, creating heterogeneities of conduction velocity. 6 Rate‐induced alternans in normoxic hearts were concordant (long APD with large [Ca2+]i) across the epicardium, with a magnitude (difference between odd‐even signals) that varied with the local RP. Alternans were initiated by gradients of RP, producing alternans of conduction that terminated spontaneously without progressing to fibrillation.

Optical mapping of repolarization and refractoriness from intact hearts

BackgroundHeterogeneities of repolarization (R) across the myocardium have been invoked to explain most reentrant arrhythmias. The measurement of refractory periods (RPs) has been widely used to assess R, but conventional electrode and extrastimulus mapping techniques have not provided reliable maps of RPs. Methods and ResultsGuinea pig hearts were stained with a voltage-sensitive dye to measure fluorescence (F) action potentials (APs) from 124 sites with a photodiode array. AP duration (APD) was defined as the time between depolarization (dF/dt)max and R time points (ie, the time when AP returns to baseline or some percent thereof). However, R time points are difficult to determine because AP downstrokes are often encumbered by drifting baselines and motion artifacts, which make this definition ambiguous. In optical and microelectrode recordings, the second derivative of AP downstrokes is shown to contain an easily detected, unique local maximum. The correlation between the position of this maximum (d2F/dt2)max, and R has been tested during altered AP characteristics induced by changes in cycle length, ischemia, and hypoxia. Under these various modifications of the AP, the time points of (d2F/dt2) max fell at 97.0±2.1% of recovery to baseline. Extrastimulus techniques applied to (1) isolated myocytes, (2) intact hearts, and (3) mathematical simulations indicated that (d2V/dt2)max coincided with the effective RPs of APs. The coincidence of RPs and (d2V/dt2)max was valid within 5 milliseconds, for resting potentials of −75 to −90 mV and extrastimuli three times threshold voltage. ConclusionsThus, optical APs and (d2F/dt2)max can be used to map activation, R, and RPs with AP recordings from a single heartbeat.

Enhanced Dispersion of Repolarization and Refractoriness in Transgenic Mouse Hearts Promotes Reentrant Ventricular Tachycardia

The heterogeneous distribution of ion channels in ventricular muscle gives rise to spatial variations in action potential (AP) duration (APD) and contributes to the repolarization sequence in healthy hearts. It has been proposed that enhanced dispersion of repolarization may underlie arrhythmias in diseases with markedly different causes. We engineered dominant negative transgenic mice that have prolonged QT intervals and arrhythmias due to the loss of a slowly inactivating K(+) current. Optical techniques are now applied to map APs and investigate the mechanisms underlying these arrhythmias. Hearts from transgenic and control mice were isolated, perfused, stained with di-4-ANEPPS, and paced at multiple sites to optically map APs, activation, and repolarization sequences at baseline and during arrhythmias. Transgenic hearts exhibited a 2-fold prolongation of APD, less shortening (8% versus 40%) of APDs with decreasing cycle length, altered restitution kinetics, and greater gradients of refractoriness from apex to base compared with control hearts. A premature impulse applied at the apex of transgenic hearts produced sustained reentrant ventricular tachycardia (n=14 of 15 hearts) that did not occur with stimulation at the base (n=8) or at any location in control hearts (n=12). In transgenic hearts, premature impulses initiated reentry by encountering functional lines of conduction block caused by enhanced dispersion of refractoriness. Reentrant VT had stable (>30 minutes) alternating long/short APDs associated with long/short cycle lengths and T wave alternans. Thus, optical mapping of genetically engineered mice may help elucidate some electrophysiological mechanisms that underlie arrhythmias and sudden death in human cardiac disorders.

Merocyanine 540 as an optical probe of transmembrane electrical activity in the heart

Frog hearts stained with merocyanine 540 shows a 1.5 to 2.0 percent increase in fluorescence intensity at 585 nanometers during the cardiac action potential when excited with a 540-nonometer light beam. Fluorometric action potentials similar to those recorded with intracellular microelectrodes in pacemaker, atrial, and ventricular tissues were recorded by focusing a 1-millimeter excitation beam on various regions of the heart. The signal-to-noise ratio for a single action potential ranged between 10/1 and 40/1. In spontaneously pacing hearts the slower rate of rise of the fluorescence action potential is due to the slow propagation of the electrical signal. In solutions containing normal calcium concentrations the fluorometric signal is altered by contractions. Merocyanine 540 is biologically inert as it stains the cardiac cell membrane and acts as a sensitive optical probe of the change in transmembrane potential.

Propagated Endothelial Ca2+ Waves and Arteriolar Dilation In Vivo : Measurements in Cx40BAC-GCaMP2 Transgenic Mice

To study endothelial cell (EC)- specific Ca2+ signaling in vivo we engineered transgenic mice in which the Ca2+ sensor GCaMP2 is placed under control of endogenous connexin40 (Cx40) transcription regulatory elements within a bacterial artificial chromosome (BAC), resulting in high sensor expression in arterial ECs, atrial myocytes, and cardiac Purkinje fibers. High signal/noise Ca2+ signals were obtained in Cx40BAC-GCaMP2 mice within the ventricular Purkinje cell network in vitro and in ECs of cremaster muscle arterioles in vivo. Microiontophoresis of acetylcholine (ACh) onto arterioles triggered a transient increase in EC Ca2+ fluorescence that propagated along the arteriole with an initial velocity of ≈116 &mgr;m/s (n=28) and decayed over distances up to 974 &mgr;m. The local rise in EC Ca2+ was followed (delay, 830±60 ms; n=8) by vasodilation that conducted rapidly (mm/s), bidirectionally, and into branches for distances exceeding 1 mm. At intermediate distances (300 to 600 &mgr;m), rapidly-conducted vasodilation occurred without changing EC Ca2+, and additional dilation occurred after arrival of a Ca2+ wave. In contrast, focal delivery of sodium nitroprusside evoked similar local dilations without Ca2+ signaling or conduction. We conclude that in vivo responses to ACh in arterioles consists of 2 phases: (1) a rapidly-conducted vasodilation initiated by a local rise in EC Ca2+ but independent of EC Ca2+ signaling at remote sites; and (2) a slower complementary dilation associated with a Ca2+ wave that propagates along the endothelium.