Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy.

Well-coordinated activation of all cardiomyocytes must occur on every heartbeat. At the cell level, a complex network of sarcolemmal invaginations, called the transverse-axial tubular system (TATS), propagates membrane potential changes to the cell core, ensuring synchronous and uniform excitation-contraction coupling. Although myocardial conduction of excitation has been widely described, the electrical properties of the TATS remain mostly unknown. Here, we exploit the formal analogy between diffusion and electrical conductivity to link the latter with the diffusional properties of TATS. Fluorescence recovery after photobleaching (FRAP) microscopy is used to probe the diffusion properties of TATS in isolated rat cardiomyocytes: A fluorescent dextran inside TATS lumen is photobleached, and signal recovery by diffusion of unbleached dextran from the extracellular space is monitored. We designed a mathematical model to correlate the time constant of fluorescence recovery with the apparent diffusion coefficient of the fluorescent molecules. Then, apparent diffusion is linked to electrical conductivity and used to evaluate the efficiency of the passive spread of membrane depolarization along TATS. The method is first validated in cells where most TATS elements are acutely detached by osmotic shock and then applied to probe TATS electrical conductivity in failing heart cells. We find that acute and pathological tubular remodeling significantly affect TATS electrical conductivity. This may explain the occurrence of defects in action potential propagation at the level of single T-tubules, recently observed in diseased cardiomyocytes.

Assessment of intramuscular activation patterns using ultrasound M-mode strain.

The intramuscular activation pattern can be connected to the motor unit recruitment strategy of force generation and fatigue resistance. Electromyography has earlier been used in several studies to quantify the spatial inhomogeneity of the muscle activation. We applied ultrasound M-mode strain to study the activation pattern through the tissue deformation. Correlation values of the strain at different force levels were used to quantify the spatial changes in the activation. The assessment was done including the biceps brachii muscle of 8 healthy subjects performing isometric elbow flexion contractions ranging from 0% to 80% of maximum voluntary contraction. The obtained results were repeatable and demonstrated consistent changes of the correlation values during force regulation, in agreement with previously presented EMG-results. Both intra-subject and inter-subject activation patterns of strain were considered along and transverse the fiber direction. The results suggest that ultrasound M-mode strain can be used as a complementary method to study intramuscular activation patterns with high spatial resolution.

Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor.

Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca(2+) release in skeletal muscle and a potentially fatal rise in perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation-contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and Ca(V)1.1, the principal subunit of the L-type Ca(2+) channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca(2+) entry. The Ca(V)1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all Ca(V) channels [Carpenter D, et al. (2009) BMC Med Genet 10:104-115.]. To define the functional consequences of this mutation, we expressed it in dysgenic (Ca(V)1.1 null) myotubes. Unlike previously described MH-linked mutations in Ca(V)1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca(2+) release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in Ca(V)1.1 R174W-expressing myotubes, resting myoplasmic Ca(2+) levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type Ca(V)1.1. Our results indicate that Ca(V)1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca(2+) leak from the SR, and that perturbation of Ca(V)1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers.

Analysis of calcium transients in cardiac myocytes and assessment of the sarcoplasmic reticulum Ca2+-ATPase contribution.

Ca(2+) signaling plays an essential role in several functions of cardiac myocytes. Transient rises and reductions of cytosolic Ca(2+), permitted by the sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) and other proteins, control each cycle of contraction and relaxation. Here we provide a practical method for isolation of neonatal rat cardiac myocytes and measurement of Ca(2+) transients in cultured cardiac myocytes, yielding information on kinetic resolution of the transients, variations of cytosolic Ca(2+) concentrations, and adequacy of intracellular Ca(2+) stores. We also provide examples of experimental perturbations that can be used to assess the contribution of SERCA2 to Ca(2+) signaling.

Assessment of atrial electromechanical coupling characteristics and P-wave dispersion in patients with atrial septal aneurysm.

OBJECTIVE: The aim of this study was to evaluate atrial conduction abnormalities obtained by Doppler tissue imaging (DTI) and electrocardiogram analysis in Atrial septal aneurysm (ASA) patients. METHODS: A total of 30 patients with ASA (11 males/19 females, mean age 29.6 ± 11.3 years) and 25 controls (9 males/16 females, mean age 27.6 ± 9.98 years) were included. Interatrial and intraatrial electromechanical coupling (PA) intervals were measured with DTI. P-wave dispersion (Pd) was calculated from the 12-lead electrocardiogram. Systolic and diastolic left ventricular (LV) functions were measured by using conventional echocardiography and DTI. RESULTS: Atrial electromechanical coupling at the left lateral mitral annulus (PA lateral) was significantly delayed in ASA patients (59.3 ± 4.2 vs. 48.5 ± 1.1 ms, P < 0.0001). Interatrial (PA lateral--PA tricuspid) and intraatrial (PA septum--PA tricuspid) electromechanical coupling interval were significantly longer in ASA patients (26.1 ± 6.2 vs. 14.4 ± 6.75 ms, P < 0.0001 and 9.04 ± 1.1 vs. 5.4 ± 2.5 ms, P < 0.0001). maximum P-wave (Pmax) duration and Pd were significantly longer in ASA patients (98.3 ± 8.1 vs. 86.4 ± 7.8 ms, P < 0.001 and 20.7 ± 0.9 vs. 12.3 ± 1.5, P < 0.0001). Systolic and diastolic left ventricular functions of both groups were comparable. CONCLUSION: This study shows that atrial electromechanical coupling intervals and Pd are delayed in ASA patients.

Characteristics of premature ventricular complexes as correlates of reduced left ventricular systolic function: study of the burden, duration, coupling interval, morphology and site of origin of PVCs.

BACKGROUND: Frequent premature ventricular complexes (PVCs) can cause a decline in left ventricular ejection fraction (LVEF). We investigated whether the site of origin and other PVC characteristics are associated with LVEF. METHODS: We retrospectively studied 70 consecutive patients (mean age 42 ± 17 years, 40 [57%] female) with no other cause of cardiomyopathy undergoing ablation of PVCs. We analyzed the association of a reduced LVEF, defined by LVEF <50% on echocardiography, with features of PVCs obtained from electrocardiography, 24- or 48-hour Holter monitor and electrophysiology study. RESULTS: Patients with reduced LVEF (n = 17) as compared to normal LVEF (n = 53) had an increased burden of PVCs (29.3 ± 14.6% vs 16.7 ± 13.7%, P = 0.004), higher prevalence of nonsustained ventricular tachycardia (VT) [13 (76%) vs 21 (40%), P = 0.01], longer PVC duration (154.3 ± 22.9 vs 145.6 ± 20.8 ms, P = 0.03) and higher prevalence of multiform PVCs [15 (88%) vs 31 (58%), P = 0.04]. There was no significant difference in prevalence of sustained VT, QRS duration of normally conducted complexes, PVC coupling interval, or delay in PVC intrinsicoid deflection. Patients with fascicular PVCs (n = 5) had higher mean LVEF compared to others (66.2 ± 4.0% vs 53.0 ± 10.0%, P = 0.002). There was no association of LVEF with other PVC foci or with left-bundle versus right-bundle branch block morphologies. The threshold burden of PVCs associated with reduced LVEF was lower for right as compared to left ventricular PVCs. CONCLUSION: In addition to the PVC burden, other characteristics like a longer PVC duration, presence of nonsustained VT, multiform PVCs and right ventricular PVCs might be associated with cardiomyopathy.

Chronic and acute exposure of mouse hearts to fatty acids increases oxygen cost of excitation-contraction coupling.

The aim of the present study was to evaluate the underlying processes involved in the oxygen wasting induced by inotropic drugs and acute and chronic elevation of fatty acid (FA) supply, using unloaded perfused mouse hearts from normal and type 2 diabetic (db/db) mice. We found that an acute elevation of the FA supply in normal hearts, as well as a chronic (in vivo) exposure to elevated FA as in db/db hearts, increased myocardial oxygen consumption (MVo2(unloaded)) due to increased oxygen cost for basal metabolism and for excitation-contraction (EC) coupling. Isoproterenol stimulation, on top of a high FA supply, led to an additive increase in MVo2(unloaded), because of a further increase in oxygen cost for EC coupling. In db/db hearts, the acute elevation of FA did not further increase MVo2. Since the elevation in the FA supply is accompanied by increased rates of myocardial FA oxidation, the present study compared MVo2 following increased FA load versus FA oxidation rate by exposing normal hearts to normal and high FA concentration (NF and HF, respectively) and to compounds that either stimulate (GW-610742) or inhibit [dichloroacetate (DCA)] FA oxidation. While HF and NF + GW-610742 increased FA oxidation to the same extent, only HF increased MVo2(unloaded). Although DCA counteracted the HF-induced increase in FA oxidation, DCA did not reduce MVo2(unloaded). Thus, in normal hearts, acute FA-induced oxygen waste is 1) due to an increase in the oxygen cost for both basal metabolism and EC coupling and 2) not dependent on the myocardial FA oxidation rate per se, but on processes initiated by the presence of FAs. In diabetic hearts, chronic exposure to elevated circulating FAs leads to adaptations that afford protection against the detrimental effect of an acute FA load, suggesting different underlying mechanisms behind the increased MVo2 following acute and chronic FA load.

Excitation-contraction coupling in human heart failure examined by action potential clamp in rat cardiac myocytes.

The effect of the loss of the notch in the human action potential (AP) during heart failure was examined by voltage clamping rat ventricular myocytes with human APs and recording intracellular Ca(2+) with fluorescent dyes. Loss of the notch resulted in about a 50% reduction in the initial phase of the Ca(2+) transient due to reduced ability of the L-type Ca(2+) channel to trigger release. The failing human AP increased non-uniformity of cytosolic Ca(2+), with some cellular regions failing to elicit Ca(2+)-induced Ca(2+) release from the sarcoplasmic reticulum. In addition, there was an increase in the occurrence of late Ca(2+) sparks. Monte-Carlo simulations of spark activation by L-type Ca(2+) current supported the idea that the decreased synchrony of Ca(2+) spark production associated with the loss of the notch could be explained by reduced Ca(2+) influx from open Ca(2+) channels. We conclude that the notch of the AP is critical for efficient and synchronous EC coupling and that the loss of the notch will reduce the SR Ca(2+) release in heart failure, without changes in (for example) SR Ca(2+)-ATPase uptake.

Models of cardiac excitation-contraction coupling in ventricular myocytes.

Mathematical and computational modeling of cardiac excitation-contraction coupling has produced considerable insights into how the heart muscle contracts. With the increase in biophysical and physiological data available, the modeling has become more sophisticated with investigations spanning in scale from molecular components to whole cells. These modeling efforts have provided insight into cardiac excitation-contraction coupling that advanced and complemented experimental studies. One goal is to extend these detailed cellular models to model the whole heart. While this has been done with mechanical and electrophysiological models, the complexity and fast time course of calcium dynamics have made inclusion of detailed calcium dynamics in whole heart models impractical. Novel methods such as the probability density approach and moment closure technique which increase computational efficiency might make this tractable.