Examination of the Effects of Heterogeneous Organization of RyR Clusters, Myofibrils and Mitochondria on Ca2+ Release Patterns in Cardiomyocytes

Spatio-temporal dynamics of intracellular calcium, [Ca2+]i, regulate the contractile function of cardiac muscle cells. Measuring [Ca2+]i flux is central to the study of mechanisms that underlie both normal cardiac function and calcium-dependent etiologies in heart disease. However, current imaging techniques are limited in the spatial resolution to which changes in [Ca2+]i can be detected. Using spatial point process statistics techniques we developed a novel method to simulate the spatial distribution of RyR clusters, which act as the major mediators of contractile Ca2+ release, upon a physiologically-realistic cellular landscape composed of tightly-packed mitochondria and myofibrils.We applied this method to computationally combine confocal-scale (~ 200 nm) data of RyR clusters with 3D electron microscopy data (~ 30 nm) of myofibrils and mitochondria, both collected from adult rat left ventricular myocytes. Using this hybrid-scale spatial model, we simulated reaction-diffusion of [Ca2+]i during the rising phase of the transient (first 30 ms after initiation). At 30 ms, the average peak of the simulated [Ca2+]i transient and of the simulated fluorescence intensity signal, F/F0, reached values similar to that found in the literature ([Ca2+]i 1 μM; F/F0 5.5). However, our model predicted the variation in [Ca2+]i to be between 0.3 and 12.7 μM (~3 to 100 fold from resting value of 0.1 μM) and the corresponding F/F0 signal ranging from 3 to 9.5. We demonstrate in this study that: (i) heterogeneities in the [Ca2+]i transient are due not only to heterogeneous distribution and clustering of mitochondria; (ii) but also to heterogeneous local densities of RyR clusters. Further, we show that: (iii) these structureinduced heterogeneities in [Ca2+]i can appear in line scan data. Finally, using our unique method for generating RyR cluster distributions, we demonstrate the robustness in the [Ca2+]i transient to differences in RyR cluster distributions measured between rat and human cardiomyocytes

Cardiac mitochondrial function depends on BUD23 mediated ribosome programming.

Efficient mitochondrial function is required in tissues with high energy demand such as the heart, and mitochondrial dysfunction is associated with cardiovascular disease. Expression of mitochondrial proteins is tightly regulated in response to internal and external stimuli. Here we identify a novel mechanism regulating mitochondrial content and function, through BUD23-dependent ribosome generation. BUD23 was required for ribosome maturation, normal 18S/28S stoichiometry and modulated the translation of mitochondrial transcripts in human A549 cells. Deletion of Bud23 in murine cardiomyocytes reduced mitochondrial content and function, leading to severe cardiomyopathy and death. We discovered that BUD23 selectively promotes ribosomal interaction with low GC-content 5'UTRs. Taken together we identify a critical role for BUD23 in bioenergetics gene expression, by promoting efficient translation of mRNA transcripts with low 5'UTR GC content. BUD23 emerges as essential to mouse development, and to postnatal cardiac function

Transverse tubule remodelling: a cellular pathology driven by both sides of the plasmalemma?

Transverse (t)-tubules are invaginations of the plasma membrane that form a complex network of ducts, 200–400 nm in diameter depending on the animal species, that penetrates deep within the cardiac myocyte, where they facilitate a fast and synchronous contraction across the entire cell volume. There is now a large body of evidence in animal models and humans demonstrating that pathological distortion of the t-tubule structure has a causative role in the loss of myocyte contractility that underpins many forms of heart failure. Investigations into the molecular mechanisms of pathological t-tubule remodelling to date have focused on proteins residing in the intracellular aspect of t-tubule membrane that form linkages between the membrane and myocyte cytoskeleton. In this review, we shed light on the mechanisms of t-tubule remodelling which are not limited to the intracellular side. Our recent data have demonstrated that collagen is an integral part of the t-tubule network and that it increases within the tubules in heart failure, suggesting that a fibrotic mechanism could drive cardiac junctional remodelling. We examine the evidence that the linkages between the extracellular matrix, t-tubule membrane and cellular cytoskeleton should be considered as a whole when investigating the mechanisms of t-tubule pathology in the failing heart

Modeling the heart

Quantitative prediction over multiple space and time scales using computer models of the electrical activity in the mammalian heart, based on membrane and intracellular ion transport and binding dynamics, digital histology, and three-dimensional cardiac anatomy and architecture

P939Identifying predictive electrocardiographic features for ventricular fibrillation during acute myocardial infarction in an ovine model

British Heart FoundationMyocardial infarction (MI) is a leading cause of mortality with complications such as left ventricular dysfunction and arrhythmias. Despite the well characterized clinical characteristics of MI, little is known about the cellular remodeling that occurs. Altered calcium (Ca2+) handling in disease can give rise to Ca2+ waves resulting in a transient inward current via the Na+- Ca2+ exchanger. Under certain conditions this causes membrane depolarization and potentially triggered arrhythmias. Since abnormal intracellular Ca2+ handling leads to both dysfunction and arrhythmias, an ovine MI model was established to simultaneously investigate cellular and in vivo alterations that occur post-MI. Methods: Young female sheep (∼18 months) were randomly allocated as control or for MI induction surgery. A minimally invasive technique was used to create the infarct by occluding the left anterior descending coronary artery. Electrocardiograms (ECG) were recorded at intervals. At 8 weeks, ventricular cells were isolated from the infarct border zone (BZ) and loaded with a Ca2+ indicator to study the systolic calcium transient, calcium sparks and occurrence of calcium waves. Statistical significance was taken as p < 0.05, determined using t-test, Friedman’s test and Fisher’s exact test. N= animals, n = cells. Results: The majority of animals had ventricular rhythm abnormalities at around 30 minutes post coronary occlusion (N = 8/11). However, the severity of the ventricular arrhythmias was variable between animals. Preliminary data suggests the animals which had ventricular fibrillation (VF) (N = 3) had ECG evidence of QT interval prolongation at 30 minutes post occlusion (618 ± 42ms), reperfusion (516 ± 46ms) and at the end of surgery (625 ± 109ms) compared to baseline (324 ± 6ms). Animals without VF (N = 3) demonstrated QT prolongation on reperfusion (562 ± 44ms) compared to baseline (348 ± 31ms). In animals with VF, the T peak-T end interval also increased at 30 minutes post occlusion (115 ± 13ms) compared to baseline (35 ± 5ms). Overall, the QT interval remained unchanged at 8 weeks compared to baseline along with the QRS duration, PR and RR interval with the exception of the ST segment at week 1 which was elevated by 0.05 ± 0.01mV (N = 7). On a cellular level, preliminary data indicated increased propensity for Ca2+ waves in MI BZ compared to control cells (37% vs 49%) and larger amplitude Ca2+ sparks (control = 1.65 ± 0.03, MI BZ 2.13 ± 0.11. N= 38 control, 5 MI BZ) in the BZ compared to control. Conclusion: The alteration in the QT and T peak-T end interval at 30 minutes post occlusion is reflective of alterations in ventricular repolarization and may be related to the incidence of more severe arrhythmias during MI. The increased propensity for Ca2+ waves and larger amplitude Ca2+ sparks in BZ suggests Ca2+ handling remodeling. Given the proarrythmic characteristics of aberrant calcium release, this may explain complications seen post-MI

Three-dimensional MRCP with respiratory triggering versus conventional MRCP in the evaluation of IPMT of the collateral branches

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Imaging pesato in diffusione nello studio dell\u2019addome: possibili applicazioni cliniche

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