Faculty Bibliography

Heart muscle excitation-contraction (E-C) coupling is governed by Ca(2+) release units (CRUs) whereby Ca(2+) influx via L-type Ca(2+) channels (Cav1.2) triggers Ca(2+) release from juxtaposed Ca(2+) release channels (RyR2) located in junctional sarcoplasmic reticulum (jSR). Although studies suggest that the jSR protein triadin anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to E-C coupling remains unclear. Here, we identify the role of triadin using mice with ablation of the Trdn gene (Trdn(-/-)). The structure and protein composition of the cardiac CRU is significantly altered in Trdn(-/-) hearts. jSR proteins (RyR2, Casq2, junctin, and junctophilin 1 and 2) are significantly reduced in Trdn(-/-) hearts, whereas Cav1.2 and SERCA2a remain unchanged. Electron microscopy shows fragmentation and an overall 50% reduction in the contacts between jSR and T-tubules. Immunolabeling experiments show reduced colocalization of Cav1.2 with RyR2 and substantial Casq2 labeling outside of the jSR in Trdn(-/-) myocytes. CRU function is impaired in Trdn(-/-) myocytes, with reduced SR Ca(2+) release and impaired negative feedback of SR Ca(2+) release on Cav1.2 Ca(2+) currents (I(Ca)). Uninhibited Ca(2+) influx via I(Ca) likely contributes to Ca(2+) overload and results in spontaneous SR Ca(2+) releases upon beta-adrenergic receptor stimulation with isoproterenol in Trdn(-/-) myocytes, and ventricular arrhythmias in Trdn(-/-) mice. We conclude that triadin is critically important for maintaining the structural and functional integrity of the cardiac CRU; triadin loss and the resulting alterations in CRU structure and protein composition impairs E-C coupling and renders hearts susceptible to ventricular arrhythmias.

Cardiac calsequestrin-null mice (Casq2-/-) display catecholaminergic ventricular tachycardia akin to humans with CASQ2 mutations. However, the specific contribution of Casq2 deficiency to the arrhythmia phenotype is difficult to assess because Casq2-/- mice also show significant reductions in the sarcoplasmic reticulum (SR) proteins junctin and triadin-1 and increased SR volume. Furthermore, it remains unknown whether Casq2 regulates SR Ca2+ release directly or indirectly by buffering SR luminal Ca2+. To address both questions, we examined heterozygous (Casq2+/-) mice, which have a 25% reduction in Casq2 but no significant decrease in other SR proteins. Casq2+/- mice (n=35) challenged with isoproterenol displayed 3-fold higher rates of ventricular ectopy than Casq2+/+ mice (n=31; P<0.05). Programmed stimulation induced significantly more ventricular tachycardia in Casq2+/- mice than in Casq2+/+ mice. Field-stimulated Ca2+ transients, cell shortening, L-type Ca2+ current, and SR volume were not significantly different in Casq2+/- and Casq2+/+ myocytes. However, in the presence of isoproterenol, SR Ca2+ leak was significantly increased in Casq2+/- myocytes (Casq2+/- 0.18+/-0.02 F(ratio) versus Casq2+/+ 0.11+/-0.01 F(ratio), n=57, 60; P<0.01), resulting in a significantly higher rate of spontaneous SR Ca2+ releases and triggered beats. SR luminal Ca2+ measured using Mag-Fura-2 was not altered by Casq2 reduction. As a result, the relationship between SR Ca2+ leak and SR luminal Ca2+ was significantly different between Casq2+/- and Casq2+/+ myocytes (P<0.01). Thus, even modest reductions in Casq2 increase SR Ca2+ leak and cause ventricular tachycardia susceptibility under stress. The underlying mechanism is likely the direct regulation of SR Ca2+ release channels by Casq2 rather than altered luminal Ca2+.

Cardiac calsequestrin (Casq2) is thought to be the key sarcoplasmic reticulum (SR) Ca2+ storage protein essential for SR Ca2+ release in mammalian heart. Human CASQ2 mutations are associated with catecholaminergic ventricular tachycardia. However, homozygous mutation carriers presumably lacking functional Casq2 display surprisingly normal cardiac contractility. Here we show that Casq2-null mice are viable and display normal SR Ca2+ release and contractile function under basal conditions. The mice exhibited striking increases in SR volume and near absence of the Casq2-binding proteins triadin-1 and junctin; upregulation of other Ca2+ -binding proteins was not apparent. Exposure to catecholamines in Casq2-null myocytes caused increased diastolic SR Ca2+ leak, resulting in premature spontaneous SR Ca2+ releases and triggered beats. In vivo, Casq2-null mice phenocopied the human arrhythmias. Thus, while the unique molecular and anatomic adaptive response to Casq2 deletion maintains functional SR Ca2+ storage, lack of Casq2 also causes increased diastolic SR Ca2+ leak, rendering Casq2-null mice susceptible to catecholaminergic ventricular arrhythmias.

BACKGROUND: Action potential duration in the right ventricle is normally shorter than that in the left. We tested the hypothesis that there may be intrinsic differences in the QT and Tp-e (an interval from the peak to the end of the T wave) intervals between the left and right chest leads that can be exaggerated by systemic hypertension but attenuated by pulmonary hypertension in humans. METHODS: Electrocardiograms in the left (V4L-V6L) and right (V4R-V6R) chest leads were obtained in 40 healthy individuals, 29 patients with systemic hypertension and left ventricular hypertrophy, and 15 patients with pulmonary hypertension. RESULTS: In healthy individuals, the corrected QT (QTc) and corrected Tp-e [T(p-e)c] intervals were 421+/-5 and 86+/-3 milliseconds in V4L through V6L, respectively, significantly longer than those recorded from V4R through V6R (383+/-5 and 62+/-4 milliseconds, respectively; P<.01). Left ventricular hypertrophy prolonged the QTc interval in V4L through V6L (456+/-5 milliseconds), exaggerating the difference in the QTc interval between the left and right chest leads (61+/-4 vs 40+/-3 milliseconds in healthy control subjects; P<.01). Left ventricular hypertrophy also resulted in a small but significant increase in the T(p-e)c interval in V4L through V6L (97+/-3 vs 86+/-3 milliseconds in control subjects; P<.05) but exerted no significant effect on the T(p-e)c interval in the right. In contrast, pulmonary hypertension lengthened the QTc interval in the right chest leads, reducing the difference in the QTc interval between the left and right chest leads (3+/-8 vs 40+/-3 milliseconds in control subjects; P<.01). CONCLUSIONS: There are intrinsic differences in the QT and Tp-e intervals between V4L-V6L and V4R-V6R that are significantly amplified by systemic hypertension but markedly attenuated by pulmonary hypertension.

The electrocardiographic (ECG) manifestation of ventricular repolarization includes J (Osborn), T, and U waves. On the basis of biophysical principles of ECG recording, any wave on the body surface ECG represents a coincident voltage gradient generated by cellular electrical activity within the heart. The J wave is a deflection with a dome that appears on the ECG after the QRS complex. A transmural voltage gradient during initial ventricular repolarization, which results from the presence of a prominent action potential notch mediated by the transient outward potassium current (I(to)) in epicardium but not endocardium, is responsible for the registration of the J wave on the ECG. Clinical entities that are associated with J waves (the J-wave syndrome) include the early repolarization syndrome, the Brugada syndrome and idiopathic ventricular fibrillation related to a prominent J wave in the inferior leads. The T wave marks the final phase of ventricular repolarization and is a symbol of transmural dispersion of repolarization (TDR) in the ventricles. An excessively prolonged QT interval with enhanced TDR predisposes people to develop torsade de pointes. The malignant "R-on-T" phenomenon, i.e., an extrasystole that originates on the preceding T wave, is due to transmural propagation of phase 2 reentry or phase 2 early afterdepolarization. A pathological "U" wave as seen with hypokalemia is the consequence of electrical interaction among ventricular myocardial layers at action potential phase 3 of which repolarization slows. A physiological U wave is thought to be due to delayed repolarization of the Purkinje system.

BACKGROUND: Phase 2 reentry caused by heterogeneous loss of the transient outward potassium current (I(to))-mediated epicardial action potential (AP) dome can produce a closely coupled R-on-T extrasystole leading to ventricular fibrillation (VF) under conditions of ST-segment elevation unrelated to ischemia. The present study examined the role of phase 2 reentry in the initiation of VF during early myocardial ischemia. METHODS AND RESULTS: Regional myocardial ischemia was produced in an isolated, arterially perfused canine right ventricular wedge preparation. Transmembrane APs from 2 epicardial sites at each side of the ischemic border were simultaneously recorded together with measurements of extracellular potassium concentration ([K+]o) and a transmural ECG. Loss of the I(to)-mediated epicardial AP dome in the ischemic zone but not in the perfused tissue resulted in phase 2 reentry and associated R-on-T extrasystoles capable of initiating VF in 7 of 15 preparations during the first 3 to 9 minutes of myocardial ischemia, with marked ST-segment elevation and [K+]o accumulation. The I(to) and phase 1 magnitude of epicardium contributed importantly to the onset of VF. Phase 1 magnitude and I(to) density at +30 mV in the group with phase 2 reentry-related R-on-T extrasystoles were 32.2+/-1.3 mV and 30.3+/-0.5 pA/pF (n=7), respectively, significantly greater than those (24.0+/-1.8 mV and 23.2+/-1.0 pA/pF) in the group without the extrasystoles (n=8, P<0.01). CONCLUSIONS: Acute regional myocardial ischemia results in markedly heterogeneous loss of I(to)-mediated epicardial AP domes across the ischemic border, leading to phase 2 reentry. Phase 2 reentry can in turn produce an R-on-T extrasystole capable of initiating VF.