Natalia S. Torres

Subcellular Structures and Function of Myocytes Impaired During Heart Failure Are Restored by Cardiac Resynchronization Therapy

Rationale: Cardiac resynchronization therapy (CRT) is an established treatment for patients with chronic heart failure. However, CRT-associated structural and functional remodeling at cellular and subcellular levels is only partly understood. Objective: To investigate the effects of CRT on subcellular structures and protein distributions associated with excitation-contraction coupling of ventricular cardiomyocytes. Methods and Results: Our studies revealed remodeling of the transverse tubular system (t-system) and the spatial association of ryanodine receptor (RyR) clusters in a canine model of dyssynchronous heart failure (DHF). We did not find this remodeling in a synchronous heart failure model based on atrial tachypacing. Remodeling in DHF ranged from minor alterations in anterior left ventricular myocytes to nearly complete loss of the t-system and dissociation of RyRs from sarcolemmal structures in lateral cells. After CRT, we found a remarkable and almost complete reverse remodeling of these structures despite persistent left ventricular dysfunction. Studies of whole-cell Ca2+ transients showed that the structural remodeling and restoration were accompanied with remodeling and restoration of Ca2+ signaling. Conclusions: DHF is associated with regional remodeling of the t-system. Myocytes undergo substantial structural and functional restoration after only 3 weeks of CRT. The finding suggests that t-system status can provide an early marker of the success of this therapy. The results could also guide us to an understanding of the loss and remodeling of proteins associated with the t-system. The steep relationship between free Ca2+ and contraction suggests that some restoration of Ca2+ release units will have a disproportionately large effect on contractility.

Activation of reverse Na+–Ca2+ exchange by the Na+ current augments the cardiac Ca2+ transient: evidence from NCX knockout mice

The hypothesis that Na+ influx during the action potential (AP) activates reverse Na+–Ca2+ exchange (NCX) and subsequent entry of trigger Ca2+ is controversial. We tested this hypothesis by monitoring intracellular Ca2+ before and after selective inactivation of INa prior to a simulated action potential in patch‐clamped ventricular myocytes isolated from adult wild‐type (WT) and NCX knockout (KO) mice. First, we inactivated INa using a ramp prepulse to −45 mV. In WT cells, inactivation of INa decreased the Ca2+ transient amplitude by 51.1 ± 4.6% (P < 0.001, n= 14) and reduced its maximum release flux by 53.0 ± 4.6% (P < 0.001, n= 14). There was no effect on diastolic Ca2+. In striking contrast, Ca2+ transients in NCX KO cardiomyocytes were unaffected by the presence or absence of INa (n= 8). We obtained similar results when measuring trigger Ca2+ influx in myocytes with depleted sarcoplasmic reticulum. In WT cells, inactivation of INa decreased trigger Ca2+ influx by 37.8 ± 6% and maximum rate of flux by 30.6 ± 7.7% at 2.5 mm external Ca2+ (P < 0.001 and P < 0.05, n= 9). This effect was again absent in the KO cells (n= 8). Second, exposure to 10 μm tetrodotoxin to block INa also reduced the Ca2+ transients in WT myocytes but not in NCX KO myocytes. We conclude that INa and reverse NCX modulate Ca2+ release in murine WT cardiomyocytes by augmenting the pool of Ca2+ that triggers ryanodine receptors. This is an important mechanism for regulation of Ca2+ release and contractility in murine heart.

Na+ currents are required for efficient excitation-contraction coupling in rabbit ventricular myocytes: A possible contribution of neuronal Na+ channels

Ca2+ transients were activated in rabbit ventricular cells by a sequence of action potential shaped voltage clamps. After activating a series of control transients, Na+ currents (INa) were inactivated with a ramp from −80 to −40 mV (1.5 s) prior to the action potential clamp. The transients were detected with the calcium indicator Fluo‐4 and an epifluorescence system. With zero Na+ in the pipette INa inactivation produced a decline in the SR Ca2+ release flux (measured as the maximum rate of rise of the transient) of 27 ± 4% (n= 9, P < 0.001) and a peak amplitude reduction of 10 ± 3% (n= 9, P < 0.05). With 5 mm Na+ in the pipette the reduction in release flux was greater (34 ± 4%, n= 4, P < 0.05). The ramp effectively inactivates INa without changing ICa, and there was no significant change in the transmembrane Ca2+ flux after the inactivation of INa. We next evoked action potentials under current clamp. TTX at 100 nm, which selectively blocks neuronal isoforms of Na+ channels, produced a decline in SR Ca2+ release flux of 35 ± 3% (n= 6, P < 0.001) and transient amplitude of 12 ± 2% (n= 6, P < 0.05). This effect was similar to the effect of INa inactivation on release flux. We conclude that a TTX‐sensitive INa is essential for efficient triggering of SR Ca2+ release. We propose that neuronal Na+ channels residing within couplons activate sufficient reverse Na+–Ca2+ exchanger (NCX) to prime the junctional cleft with Ca2+. The results can be explained if non‐linearities in excitation–contraction coupling mechanisms modify the coupling fidelity of ICa, which is known to be low at positive potentials.

Quantitative Analysis of Cardiac Tissue Including Fibroblasts Using Three-Dimensional Confocal Microscopy and Image Reconstruction: Towards a Basis for Electrophysiological Modeling

Electrophysiological modeling of cardiac tissue is commonly based on functional and structural properties measured in experiments. Our knowledge of these properties is incomplete, in particular their remodeling in disease. Here, we introduce a methodology for quantitative tissue characterization based on fluorescent labeling, 3-D scanning confocal microscopy, image processing and reconstruction of tissue micro-structure at sub-micrometer resolution. We applied this methodology to normal rabbit ventricular tissue and tissue from hearts with myocardial infarction. Our analysis revealed that the volume fraction of fibroblasts increased from 4.83±0.42% (mean ± standard deviation) in normal tissue up to 6.51±0.38% in myocardium from infarcted hearts. The myocyte volume fraction decreased from 76.20±9.89% in normal to 73.48±8.02% adjacent to the infarct. Numerical field calculations on 3-D reconstructions of the extracellular space yielded an extracellular longitudinal conductivity of 0.264±0.082 S/m with an anisotropy ratio of 2.095±1.11 in normal tissue. Adjacent to the infarct, the longitudinal conductivity increased up to 0.400±0.051 S/m, but the anisotropy ratio decreased to 1.295±0.09. Our study indicates an increased density of gap junctions proximal to both fibroblasts and myocytes in infarcted versus normal tissue, supporting previous hypotheses of electrical coupling of fibroblasts and myocytes in infarcted hearts. We suggest that the presented methodology provides an important contribution to modeling normal and diseased tissue. Applications of the methodology include the clinical characterization of disease-associated remodeling.

A modified local control model for Ca2+ transients in cardiomyocytes: junctional flux is accompanied by release from adjacent non-junctional RyRs.

Excitation-contraction coupling in cardiomyocytes requires Ca(2+) influx through dihydropyridine receptors in the sarcolemma, which gates Ca(2+) release through sarcoplasmic ryanodine receptors (RyRs). Ca(2+) influx, release and diffusion produce a cytosolic Ca(2+) transient. Here, we investigated the relationship between Ca(2+) transients and the spatial arrangement of the sarcolemma including the transverse tubular system (t-system). To accomplish this, we studied isolated ventricular myocytes of rabbit, which exhibit a heterogeneously distributed t-system. We developed protocols for fluorescent labeling and triggered two-dimensional confocal microscopic imaging with high spatiotemporal resolution. From sequences of microscopic images, we measured maximal upstroke velocities and onset times of local Ca(2+) transients together with their distance from the sarcolemma. Analyses indicate that not only sarcolemmal release sites, but also those that are within 1 μm of the sarcolemma actively release Ca(2+). Our data also suggest that release does not occur at sites further than 2.5 μm from the sarcolemma. The experimental data are in agreement with results from a mathematical model of Ca(2+) release and diffusion. Our findings can be explained by a modified local control model, which constrains the region of regenerative activation of non-junctional RyR clusters. We believe that this model will be useful for describing excitation-contraction coupling in cardiac myocytes with a sparse t-system, which includes those from diseased heart tissue as well as atrial myocytes of some species.

Potassium channel activators differentially modulate the effect of sodium channel blockade on cardiac conduction

Diminished repolarization reserve contributes to the arrhythmogenic substrate in many disease states. Pharmacological activation of K+ channels has been suggested as a potential antiarrhythmic therapy in such conditions. Having previously demonstrated that IK1 and IKr can modulate cardiac conduction, we tested here the effects of pharmacological IKATP and IKs activation on cardiac conduction and its dependence on the sodium current (INa).

Mitochondrial cardiomyopathies feature increased uptake and diminished efflux of mitochondrial calcium

Calcium (Ca2+) influx into the mitochondrial matrix stimulates ATP synthesis. Here, we investigate whether mitochondrial Ca2+ transport pathways are altered in the setting of deficient mitochondrial energy synthesis, as increased matrix Ca2+ may provide a stimulatory boost. We focused on mitochondrial cardiomyopathies, which feature such dysfunction of oxidative phosphorylation. We study a mouse model where the main transcription factor for mitochondrial DNA (transcription factor A, mitochondrial, Tfam) has been disrupted selectively in cardiomyocytes. By the second postnatal week (10-15day old mice), these mice have developed a dilated cardiomyopathy associated with impaired oxidative phosphorylation. We find evidence of increased mitochondrial Ca2+ during this period using imaging, electrophysiology, and biochemistry. The mitochondrial Ca2+ uniporter, the main portal for Ca2+ entry, displays enhanced activity, whereas the mitochondrial sodium-calcium (Na+-Ca2+) exchanger, the main portal for Ca2+ efflux, is inhibited. These changes in activity reflect changes in protein expression of the corresponding transporter subunits. While decreased transcription of Nclx, the gene encoding the Na+-Ca2+ exchanger, explains diminished Na+-Ca2+ exchange, the mechanism for enhanced uniporter expression appears to be post-transcriptional. Notably, such changes allow cardiac mitochondria from Tfam knockout animals to be far more sensitive to Ca2+-induced increases in respiration. In the absence of Ca2+, oxygen consumption declines to less than half of control values in these animals, but rebounds to control levels when incubated with Ca2+. Thus, we demonstrate a phenotype of enhanced mitochondrial Ca2+ in a mitochondrial cardiomyopathy model, and show that such Ca2+ accumulation is capable of rescuing deficits in energy synthesis capacity in vitro.

New insights into the structure and function of couplons

It is widely accepted that the calcium transient in heart is a summation of many Ca2+ release events or sparks (Cheng et al. 1993). Each local release event is produced by a structure called a couplon (Stern et al. 1997), defined as ‘the functional grouping of RyRs and dihydropyridine receptors, (DHPRs; also known as L-type Ca channels) (and other junctional SR proteins) which may act in concert during EC coupling’ (Franzini-Armstrong et al. 1999). Calcium that enters the couplon through DHPRs triggers Ca2+ release from a cluster of RyRs (ryanodine receptors, sarcoplasmic reticulum Ca2+ release channels), thus producing a local release event. Each couplon obeys local control, which can explain, for example, the gradation of Ca2+ transients with trigger magnitude. The number of recruited couplons determines the transient amplitude. The number of DHPRs found in a couplon is of central importance, but is difficult to investigate experimentally. It seems certain that, at least in rabbits, multiple DHPRs must be present in each couplon because the probability of activation of local Ca2+ release events exceeds the probability that an individual DHPR will open during the action potential (Inoue & Bridge, 2003).

Blockade of CaMKII depresses conduction preferentially in the right ventricular outflow tract and promotes ischemic ventricular fibrillation in the rabbit heart

Calcium/calmodulin-dependent protein kinase II (CaMKII) regulates the principle ion channels mediating cardiac excitability and conduction, but how this regulation translates to the normal and ischemic heart remains unknown. Diverging results on CaMKII regulation of Na+ channels further prevent predicting how CaMKII activity regulates excitability and conduction in the intact heart. To address this deficiency, we tested the effects of the CaMKII blocker KN93 (1 and 2.75 μM) and its inactive analog KN92 (2.75 μM) on conduction and excitability in the left (LV) and right (RV) ventricles of rabbit hearts during normal perfusion and global ischemia. We used optical mapping to determine local conduction delays and the optical action potential (OAP) upstroke velocity (dV/dtmax). At baseline, local conduction delays were similar between RV and LV, whereas the OAP dV/dtmax was lower in RV than in LV. At 2.75 μM, KN93 heterogeneously slowed conduction and reduced dV/dtmax, with the largest effect in the RV outflow tract (RVOT). This effect was further exacerbated by ischemia, leading to recurrent conduction block in the RVOT and early ventricular fibrillation (at 6.7 ± 0.9 vs. 18.2 ± 0.8 min of ischemia in control, P < 0.0001). Neither KN92 nor 1 μM KN93 depressed OAP dV/dtmax or conduction. Rabbit cardiomyocytes isolated from RVOT exhibited a significantly lower dV/dtmax than those isolated from the LV. KN93 (2.75 μM) significantly reduced dV/dtmax in cells from both locations. This led to frequency-dependent intermittent activation failure occurring predominantly in RVOT cells. Thus CaMKII blockade exacerbates intrinsically lower excitability in the RVOT, which is proarrhythmic during ischemia.NEW & NOTEWORTHY We show that calcium/calmodulin-dependent protein kinase II (CaMKII) blockade exacerbates intrinsically lower excitability in the right ventricular outflow tract, which causes highly nonuniform chamber-specific slowing of conduction and facilitates ventricular fibrillation during ischemia. Constitutive CaMKII activity is necessary for uniform and safe ventricular conduction, and CaMKII block is potentially proarrhythmic.

Na + -Ca 2+ Exchange Currents

The chapter on Na-Ca exchange comprises a total of 16 sections. Since this chapter describes exchange currents, most of the chapter is devoted to NCX1 the cardiac isoform of the exchanger in which most of the measurements of exchange current have been performed. The first section is an introduction that outlines the discovery of the exchange process in both heart and squid axons. The authors proceed to discuss the energetics of the process including the classic studies of Reeves and Hale. There is a detailed section on the methods for measuring exchange currents including difficulties associated with these which is followed by sections describing the isolation of the exchange current, its ionic dependencies and its regulation by a variety of processes. An extensive section presents the cloning and subsequent elucidation or the primary structure of the exchange and there is a discussion of the relationship between structure and function. The chapter concludes with a description of the exchange mechanism, the voltage dependents of components of the mechanism and, finally, the involvement of exchange in cellular physiology particularly cardiac excitation contraction coupling.