Niall Macquaide

Dyssynchrony of Ca2+ release from the sarcoplasmic reticulum as subcellular mechanism of cardiac contractile dysfunction

Cardiac contractile function depends on coordinated electrical activation throughout the heart. Dyssynchronous electrical activation of the ventricles has been shown to contribute to contractile dysfunction in heart failure, and resynchronization therapy has emerged as a therapeutic concept. At the cellular level, coupling of membrane excitation to myofilament contraction is facilitated by highly organized intracellular structures which coordinate Ca(2+) release. The cytosolic [Ca(2+)] transient triggered by depolarization-induced Ca(2+) influx is the result of a gradable and robust high gain process, Ca(2+)-induced Ca(2+) release (CICR), which integrates subcellular localized Ca(2+) release events. Lack of synchronization of these localized release events can contribute to contractile dysfunction in myocardial hypertrophy and heart failure. Different underlying mechanisms relate to functional and structural changes in sarcolemmal Ca(2+) channels, the sarcoplasmic Ca(2+) release channel or ryanodine receptor, RyR, their intracellular arrangement in close proximity in couplons and the loss of t-tubules. Dyssynchrony at the subcellular level translates in a reduction of the overall gain of CICR at the cellular level and forms an important determinant of myocyte contractility in heart failure.

Unilateral arm strength training improves contralateral peak force and rate of force development.

Neural adaptation following maximal strength training improves the ability to rapidly develop force. Unilateral strength training also leads to contralateral strength improvement, due to cross-over effects. However, adaptations in the rate of force development and peak force in the contralateral untrained arm after one-arm training have not been determined. Therefore, we aimed to detect contralateral effects of unilateral maximal strength training on rate of force development and peak force. Ten adult females enrolled in a 2-month strength training program focusing of maximal mobilization of force against near-maximal load in one arm, by attempting to move the given load as fast as possible. The other arm remained untrained. The training program did not induce any observable hypertrophy of any arms, as measured by anthropometry. Nevertheless, rate of force development improved in the trained arm during contractions against both submaximal and maximal loads by 40–60%. The untrained arm also improved rate of force development by the same magnitude. Peak force only improved during a maximal isometric contraction by 37% in the trained arm and 35% in the untrained arm. One repetition maximum improved by 79% in the trained arm and 9% in the untrained arm. Therefore, one-arm maximal strength training focusing on maximal mobilization of force increased rapid force development and one repetition maximal strength in the contralateral untrained arm. This suggests an increased central drive that also crosses over to the contralateral side.

Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density.

Rationale In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI). Objective To characterize RyR functional properties in relation to TT proximity, at baseline and after MI. Methods Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category. Results In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI. Conclusion TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves.

The effect of exercise training on transverse tubules in normal, remodeled, and reverse remodeled hearts

The response of transverse (T)‐tubules to exercise training in health and disease remains unclear. Therefore, we studied the effect of exercise training on the density and spacing of left ventricle cardiomyocyte T‐tubules in normal and remodeled hearts that associate with detubulation, by confocal laser scanning microscopy. First, exercise training in normal rats increased cardiomyocyte volume by 16% (P < 0.01), with preserved T‐tubule density. Thus, the T‐tubules adapted to the physiologic hypertrophy. Next, we studied T‐tubules in a rat model of metabolic syndrome with pressure overload‐induced concentric left ventricle hypertrophy, evidenced by 15% (P < 0.01) increased cardiomyocyte size. These rats had only 85% (P < 0.01) of the T‐tubule density of control rats. Exercise training further increased cardiomyocyte volume by 8% (P < 0.01); half to that in control rats, but the T‐tubule density remained unchanged. Finally, post‐myocardial infarction heart failure induced severe cardiac pathology, with a 70% (P < 0.01) increased cardiomyocyte volume that included both eccentric and concentric hypertrophy and 55% (P < 0.01) reduced T‐tubule density. Exercise training reversed 50% (P < 0.01) of the pathologic hypertrophy, whereas the T‐tubule density increased by 40% (P < 0.05) compared to sedentary heart failure, but remained at 60% of normal hearts (P < 0.01). Physiologic hypertrophy associated with conserved T‐tubule spacing (∼1.8–1.9 µm), whereas in pathologic hypertrophy, T‐tubules appeared disorganized without regular spacing. In conclusion, cardiomyocytes maintain the relative T‐tubule density during physiologic hypertrophy and after mild concentric pathologic hypertrophy, whereas after severe pathologic remodeling with a substantial loss of T‐tubules; exercise training reverses the remodeling and partly corrects the T‐tubule density. J. Cell. Physiol. 226: 2235–2243, 2011.

Intracellular Dyssynchrony of Diastolic Cytosolic [Ca2+] Decay in Ventricular Cardiomyocytes in Cardiac Remodeling and Human Heart Failure

Rationale: Synchronized release of Ca2+ into the cytosol during each cardiac cycle determines cardiomyocyte contraction. Objective: We investigated synchrony of cytosolic [Ca2+] decay during diastole and the impact of cardiac remodeling. Methods and Results: Local cytosolic [Ca2+] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca2+] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca2+ release. Stimulation of sarcoplasmic reticulum Ca2+ ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca2+] decay (TAUglobal). Na+/Ca2+ exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca2+ uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. Conclusions: In cardiomyocytes, cytosolic [Ca2+] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca2+] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

Selective Modulation of Coupled Ryanodine Receptors During Microdomain Activation of Calcium/Calmodulin-Dependent Kinase II in the Dyadic Cleft

Rationale: In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs. Objective: Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation. Methods and Results: We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca2+ transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca2+ indicator). Ca2+ sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca2+/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca2+/calmodulin-dependent kinase II blockers autocamtide-2–related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca2+/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca2+ transients, frequency-dependent increase of the rate of Ca2+ rise was seen in coupled RyR regions only and abolished by autocamtide-2–related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost. Conclusions: Coupled RyRs have a distinct modulation by Ca2+/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca2+/reactive oxygen species microdomain, and subject to modification with disease.

Selective Modulation of Coupled Ryanodine Receptors During Microdomain Activation of CaMKII in the Dyadic Cleft

Rationale: In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs. Objective: Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation. Methods and Results: We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca2+ transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca2+ indicator). Ca2+ sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca2+/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca2+/calmodulin-dependent kinase II blockers autocamtide-2–related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca2+/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca2+ transients, frequency-dependent increase of the rate of Ca2+ rise was seen in coupled RyR regions only and abolished by autocamtide-2–related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost. Conclusions: Coupled RyRs have a distinct modulation by Ca2+/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca2+/reactive oxygen species microdomain, and subject to modification with disease.

Ryanodine receptor cluster fragmentation and redistribution in persistent atrial fibrillation enhance calcium release

Aims In atrial fibrillation (AF), abnormalities in Ca2+ release contribute to arrhythmia generation and contractile dysfunction. We explore whether ryanodine receptor (RyR) cluster ultrastructure is altered and is associated with functional abnormalities in AF. Methods and results Using high-resolution confocal microscopy (STED), we examined RyR cluster morphology in fixed atrial myocytes from sheep with persistent AF (N = 6) and control (Ctrl; N = 6) animals. RyR clusters on average contained 15 contiguous RyRs; this did not differ between AF and Ctrl. However, the distance between clusters was significantly reduced in AF (288 ± 12 vs. 376 ± 17 nm). When RyR clusters were grouped into Ca2+ release units (CRUs), i.e. clusters separated by <150 nm, CRUs in AF had more clusters (3.43 ± 0.10 vs. 2.95 ± 0.02 in Ctrl), which were more dispersed. Furthermore, in AF cells, more RyR clusters were found between Z lines. In parallel experiments, Ca2+ sparks were monitored in live permeabilized myocytes. In AF, myocytes had >50% higher spark frequency with increased spark time to peak (TTP) and duration, and a higher incidence of macrosparks. A computational model of the CRU was used to simulate the morphological alterations observed in AF cells. Increasing cluster fragmentation to the level observed in AF cells caused the observed changes, i.e. higher spark frequency, increased TTP and duration; RyR clusters dispersed between Z-lines increased the occurrence of macrosparks. Conclusion In persistent AF, ultrastructural reorganization of RyR clusters within CRUs is associated with overactive Ca2+ release, increasing the likelihood of propagating Ca2+ release.

Activation of the cardiac Na+–Ca2+ exchanger by sorcin via the interaction of the respective Ca2+-binding domains

Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca2+ binding. The sarcolemmal Na+/Ca2+ exchanger (NCX1) may be an important sorcin target in cardiac muscle. In this study, RNAi knockdown of sorcin, purified sorcin or sorcin variants was employed in parallel measurements of: (i) NCX activity in isolated rabbit cardiomyocytes using electrophysiological techniques and (ii) sorcin binding to the NCX1 calcium binding domains (CBD1 and (iii) using surface plasmon resonance and gel overlay techniques. Sorcin is activated by Ca2+ binding to the EF3 and EF2 regions, which are connected by the D helix. To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca2+ due to a mutation at EF3. Downregulation of sorcin decreased and supplementation with wt sorcin (3 μM) increased NCX activity in isolated cardiomyocytes. The relative stimulatory effects of the sorcin variants were: W105G > wt sorcin > Sorcin Calcium Binding Domain (SCBD) > W99G > E124A. Sorcin binding to both CBD1 and 2 was observed. In the presence of 50 µM Ca2+, the interaction with CBD1 followed the order W105G > SCBD > wt sorcin > W99G > E124A. In sorcin, the interacting surface can be mapped on the C-terminal Ca2+-binding domain in the D helix region comprising W99. The fast association/dissociation rates that characterize the interaction of sorcin with CBD1 and 2 may permit complex formation/dissociation during an excitation/contraction cycle.

Assessment of Sarcoplasmic Reticulum Ca2+ depletion during spontaneous Ca2+ waves in isolated permeabilized rabbit ventricular Cardiomyocytes

In this study, Ca2+ release due to spontaneous Ca2+ waves was measured both from inside the sarcoplasmic reticulum (SR) and from the cytosol of rabbit cardiomyocytes. These measurements utilized Fluo5N-AM for intra-SR Ca2+ from intact cells and Fluo5F in the cytosol of permeabilized cells. Restricted subcellular volumes were resolved with the use of laser-scanning confocal microscopy. Local Ca2+ signals during spontaneous Ca2+ release were compared with those induced by rapid caffeine application. The free cytoplasmic [Ca2+] increase during a Ca2+ wave was 98.1% +/- 0.3% of that observed during caffeine application. Conversion to total Ca2+ release suggested that Ca2+ release from a Ca2+ wave was not significantly different from that released during caffeine application (104% +/- 6%). In contrast, the maximum decrease in intra-SR Fluo-5N fluorescence during a Ca2+ wave was 82.5% +/- 2.6% of that observed during caffeine application. Assuming a maximum free [Ca2+] of 1.1 mM, this translates to a 96.2% +/- 0.8% change in intra-SR free [Ca2+] and a 91.7% +/- 1.6% depletion of the total Ca2+. This equates to a minimum intra-SR free Ca2+ of 46 +/- 7 microM during a Ca2+ wave. Reduction of RyR2 Ca2+ sensitivity by tetracaine (50 microM) reduced the spontaneous Ca2+ release frequency while increasing the Ca2+ wave amplitude. This did not significantly change the total depletion of the SR (94.5% +/- 1.1%). The calculated minimum [Ca2+] during these Ca2+ waves (87 +/- 19 microM) was significantly higher than control (p < 0.05). A computational model incorporating this level of Ca2+ depletion during a Ca2+ wave mimicked the transient and sustained effects of tetracaine on spontaneous Ca2+ release. In conclusion, spontaneous Ca2+ release results in substantial but not complete local Ca2+ depletion of the SR. Furthermore, measurements suggest that Ca2+ release terminates when luminal [Ca2+] reaches approximately 50 microM.