Right-sided heart failure is also associated with transverse tubule remodeling in the left ventricle.

Right-sided heart failure is a common consequence of pulmonary arterial hypertension. Overloading the right ventricle results in right ventricular hypertrophy, which progresses to failure in a process characterized by impaired Ca2+ dynamics and force production that is linked with transverse (t)-tubule remodeling. This also unloads the left ventricle, which consequently atrophies. Experimental left-ventricular unloading can result in t-tubule remodeling, but it is currently unclear if this occurs in right-sided heart failure. In this work, we used a model of monocrotaline (MCT)-induced right heart failure in male rats, using confocal microscopy to investigate cellular remodeling of t-tubules, junctophilin-2 (JPH2), and ryanodine receptor-2 (RyR2). We examined remodeling across tissue anatomical regions of both ventricles: in trabeculae, papillary muscles, and free walls. Our analyses revealed that MCT hearts demonstrated a significant loss of t-tubule periodicity, disruption of the normal sarcomere striated pattern with JPH2 labeling, and also a disorganized striated pattern of RyR2, a feature not previously reported in right heart failure. Remodeling of JPH2 and RyR2 in the MCT heart was more pronounced in papillary muscles and trabeculae compared with free walls, particularly in the left ventricle. We find that these structures, commonly used as ex vivo muscle preparations, are more sensitive to the disease process.NEW & NOTEWORTHY In this work, we demonstrate that t-tubule remodeling occurs in the atrophied left ventricle as well as the overloaded right ventricle after right-side heart failure. Moreover, we identify that t-tubule remodeling in both ventricles is linked to sarcoplasmic reticulum remodeling as indicated by decreased labeling periodicity of both the Ca2+ release channel, RyR2, and the cardiac junction-forming protein, JPH2, that forms a link between the sarcoplasmic reticulum and sarcolemma. Studies developing treatments for right-sided heart failure should consider effects on both the right and left ventricle.

Loss of troponin-T labelling in endomyocardial biopsies of cardiac transplant patients is associated with increased rejection grading.

Acute cellular rejection after cardiac transplantation surgery is routinely monitored by pathological assessment of haematoxylin and eosin (H&E) histology of endomyocardial biopsies (EMB). Unfortunately, there is considerable variation in the diagnosis of rejection that has been attributed to the subjectivity involved in assessing the degree of (a) inflammatory infiltrate and (b) myocyte damage. In this work, we sought to investigate the potential of high contrast confocal microscopy for numerically assessing inflammatory infiltrate and myocyte damage in EMB histology. Confocal microscopy was used to capture images from EMB fluorescently labelled for nuclei (DAPI), f-actin (phalloidin), troponin-T (anti-body), and extracellular matrix and cell border (wheat germ agglutinin). Images from 28 biopsy procedures were captured. Standard pathological grading of H&E histology identified the following rejection gradings: 6 0R, 16 1R, 6 2R and no 3R. Confocal imaging was able to identify equivalent features of rejection provided by H&E histology but at higher contrast facilitating quantification. Lymphocytic infiltrate was calculated as the ratio of non-myocyte nuclei to total nuclei. This metric was found to be significantly higher in the biopsies from 2R patients compared to both 1R and 0R patients (P < .05). Myocyte damage was quantified as the loss of troponin-T labelling normalised to f-actin labelling. This metric of myocyte damage found significantly lower amounts of troponin-T in the biopsies from 2R patients compared to those with a 0R rejection grading (P < .05). Confocal imaging and simple image processing routines show potential for numerically assessing both inflammatory infiltrate and myocyte damage in endomyocardial biopsy.

Calcium mishandling impairs contraction in right ventricular hypertrophy prior to overt heart failure.

Currently, there are no tailored therapies available for the treatment of right ventricular (RV) hypertrophy, and the cellular mechanisms that underlie the disease are poorly understood. We investigated the cellular changes that occur early in the progression of the disease, when RV hypertrophy is evident, but prior to the onset of heart failure. Intracellular Ca2+ ([Ca2+]i) handling was examined in a rat model of monocrotaline (MCT)-induced pulmonary hypertension and subsequent RV hypertrophy. [Ca2+]i and stress production were measured in isolated RV trabeculae under baseline conditions (1-Hz stimulation, 1.5 mM [Ca2+]o, 37 °C), and in response to inotropic interventions (5-Hz stimulation or 1-µM isoproterenol). Under baseline conditions, MCT trabeculae had impaired Ca2+ release in response to stimulation with a 45% delay in the time-to-peak Ca2+, but there was no difference in the amplitude and decay of the Ca2+ transient, or active stress relative to RV trabeculae from normotensive hearts (CON). Increasing stimulation frequency from 1 to 5 Hz increased stress in CON, but not MCT trabeculae. Similarly, ß-adrenergic stimulation with isoproterenol increased Ca2+ transient amplitude and active stress in CON, but not in MCT trabeculae, despite accelerating Ca2+ transient decay in trabeculae from both groups. During isoproterenol treatment, MCT trabeculae showed increased diastolic Ca2+ leak, which may explain the blunted inotropic response to ß-adrenergic stimulation. Confocal imaging of trabeculae fixed following functional measurements showed that myocytes were on average wider, and transverse-tubule organisation was disrupted in MCT which provides a mechanism to explain the observed slower release of Ca2+.

High serum thrombospondin-1 concentration is associated with slower abdominal aortic aneurysm growth and deficiency of thrombospondin-1 promotes angiotensin II induced aortic aneurysm in mice.

Abdominal aortic aneurysm (AAA) is a common age-related vascular disease characterized by progressive weakening and dilatation of the aortic wall. Thrombospondin-1 (TSP-1; gene Thbs1) is a member of the matricellular protein family important in the control of extracellular matrix (ECM) remodelling. In the present study, the association of serum TSP-1 concentration with AAA progression was assessed in 276 men that underwent repeated ultrasound for a median 5.5 years. AAA growth was negatively correlated with serum TSP-1 concentration (Spearman's rho -0.129, P=0.033). Men with TSP-1 in the highest quartile had a reduced likelihood of AAA growth greater than median during follow-up (OR: 0.40; 95% confidence interval (CI): 0.19-0.84, P=0.016, adjusted for other risk factors). Immunohistochemical staining for TSP-1 was reduced in AAA body tissues compared with the relatively normal AAA neck. To further assess the role of TSP-1 in AAA initiation and progression, combined TSP-1 and apolipoprotein deficient (Thbs1-/-ApoE-/-, n=20) and control mice (ApoE-/-, n=20) were infused subcutaneously with angiotensin II (AngII) for 28 days. Following AngII infusion, Thbs1-/- ApoE-/- mice had larger AAAs by ultrasound (P=0.024) and ex vivo morphometry measurement (P=0.006). The Thbs1-/-ApoE-/- mice also showed increased elastin filament degradation along with elevated systemic levels and aortic expression of matrix metalloproteinase (MMP)-9. Suprarenal aortic segments and vascular smooth muscle cells (VSMCs) isolated from Thbs1-/-ApoE-/- mice showed reduced collagen 3A1 gene expression. Furthermore, Thbs1-/-ApoE-/- mice had reduced aortic expression of low-density lipoprotein (LDL) receptor-related protein 1. Collectively, findings from the present study suggest that TSP-1 deficiency promotes maladaptive remodelling of the ECM leading to accelerated AAA progression.

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 structure-induced 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.

Combining confocal and single molecule localisation microscopy: A correlative approach to multi-scale tissue imaging.

Many biological questions require information at different spatial scales that include molecular, organelle, cell and tissue scales. Here we detail a method of multi-scale imaging of human cardiac tissue by correlatively combining nano-scale data of direct stochastic optical reconstruction microscopy (dSTORM) with cellular and tissue level data provided by confocal microscopy. By utilising conventional fluorescence dyes the same cellular structures can be imaged with both modalities. Human cardiac tissue was first imaged at the nanoscale to identify macro-molecular membrane complexes containing the cardiac muscle proteins junctophilin (JPH) and the ryanodine receptor (RyR). The distribution of these proteins and an additional cell membrane marker (wheat germ agglutinin, WGA) were subsequently imaged by confocal microscopy. By segmenting dSTORM data into membrane and non-membrane components we demonstrate increased colocalization of RyR with JPH at the plasma-membrane as compared to intracellular compartments. Strategies for antibody labelling, quality control, locating and aligning structures between modalities, and analysis of combined multi-scaled data sets are described.

Nanoscale analysis of ryanodine receptor clusters in dyadic couplings of rat cardiac myocytes.

The contractile properties of cardiac myocytes depend on the calcium (Ca(2+)) released by clusters of ryanodine receptors (RyRs) throughout the myoplasm. Accurate quantification of the spatial distribution of RyRs has previously been challenging due to the comparatively low resolution in optical microscopy. We have combined single-molecule localisation microscopy (SMLM) in a super-resolution modality known as dSTORM with immunofluorescence staining of tissue sections of rat ventricles to resolve a wide, near-exponential size distribution of RyR clusters that lined on average ~57% of the perimeter of each myofibril. The average size of internal couplons is ~63 RyRs (nearly 4 times larger than that of peripheral couplons) and the largest clusters contain many hundreds of RyRs. Similar to previous observations in peripheral couplons, we observe many clusters with one or few receptors; however ≥80% of the total RyRs were detected in clusters containing ≥100 receptors. ~56% of all clusters were within an edge-to-edge distance sufficiently close to co-activate via Ca(2+)-induced Ca(2+) release (100nm) and were grouped into 'superclusters'. The co-location of superclusters with the same or adjacent t-tubular connections in dual-colour super-resolution images suggested that member sub-clusters may be exposed to similar local luminal Ca(2+) levels. Dual-colour dSTORM revealed high co-localisation between the cardiac junctional protein junctophilin-2 (JPH2) and RyR clusters that confirmed that the majority of the RyR clusters observed are dyadic. The increased sensitivity of super-resolution images revealed approximately twice as many RyR clusters (2.2clusters/µm(3)) compared to previous confocal measurements. We show that, in general, the differences of previous confocal estimates are largely attributable to the limited spatial resolution of diffraction-limited imaging. The new data can be used to inform the construction of detailed mechanistic models of cardiac Ca(2+) signalling.

T-tubule disease: Relationship between t-tubule organization and regional contractile performance in human dilated cardiomyopathy.

Evidence from animal models suggest that t-tubule changes may play an important role in the contractile deficit associated with heart failure. However samples are usually taken at random with no regard as to regional variability present in failing hearts which leads to uncertainty in the relationship between contractile performance and possible t-tubule derangement. Regional contraction in human hearts was measured by tagged cine MRI and model fitting. At transplant, failing hearts were biopsy sampled in identified regions and immunocytochemistry was used to label t-tubules and sarcomeric z-lines. Computer image analysis was used to assess 5 different unbiased measures of t-tubule structure/organization. In regions of failing hearts that showed good contractile performance, t-tubule organization was similar to that seen in normal hearts, with worsening structure correlating with the loss of regional contractile performance. Statistical analysis showed that t-tubule direction was most highly correlated with local contractile performance, followed by the amplitude of the sarcomeric peak in the Fourier transform of the t-tubule image. Other area based measures were less well correlated. We conclude that regional contractile performance in failing human hearts is strongly correlated with the local t-tubule organization. Cluster tree analysis with a functional definition of failing contraction strength allowed a pathological definition of 't-tubule disease'. The regional variability in contractile performance and cellular structure is a confounding issue for analysis of samples taken from failing human hearts, although this may be overcome with regional analysis by using tagged cMRI and biopsy mapping.

Next-generation endomyocardial biopsy: the potential of confocal and super-resolution microscopy.

Confocal laser scanning microscopy and super-resolution microscopy provide high-contrast and high-resolution fluorescent imaging, which has great potential to increase the diagnostic yield of endomyocardial biopsy (EMB). EMB is currently the gold standard for identification of cardiac allograft rejection, myocarditis, and infiltrative and storage diseases. However, standard analysis is dominated by low-contrast bright-field light and electron microscopy (EM); this lack of contrast makes quantification of pathological features difficult. For example, assessment of cardiac allograft rejection relies on subjective grading of H&E histology, which may lead to diagnostic variability between pathologists. This issue could be solved by utilising the high contrast provided by fluorescence methods such as confocal to quantitatively assess the degree of lymphocytic infiltrate. For infiltrative diseases such as amyloidosis, the nanometre resolution provided by EM can be diagnostic in identifying disease-causing fibrils. The recent advent of super-resolution imaging, particularly direct stochastic optical reconstruction microscopy (dSTORM), provides high-contrast imaging at resolution approaching that of EM. Moreover, dSTORM utilises conventional fluorescence dyes allowing for the same structures to be routinely imaged at the cellular scale and then at the nanoscale. The key benefit of these technologies is that the high contrast facilitates quantitative digital analysis and thereby provides a means to robustly assess critical pathological features. Ultimately, this technology has the ability to provide greater accuracy and precision to EMB assessment, which could result in better outcomes for patients.

Pirfenidone increases transverse tubule length in the infarcted rat myocardium.

Transverse (t)-tubule remodelling is a prominent feature of heart failure with reduced ejection fraction (HFrEF). In our previous research, we identified an increased amount of collagen within the t-tubules of HFrEF patients, suggesting fibrosis could contribute to the remodelling of t-tubules. In this research, we tested this hypothesis in a rodent model of myocardial infarction induced heart failure that was treated with the anti-fibrotic pirfenidone. Confocal microscopy demonstrated loss of t-tubules within the border zone region of the infarct. This was documented as a reduction in t-tubule frequency, area, length, and transverse elements. Eight weeks of pirfenidone treatment was able to significantly increase the area and length of the t-tubules within the border zone. Echocardiography showed no improvement with pirfenidone treatment. Surprisingly, pirfenidone significantly increased the thickness of the t-tubules in the remote left ventricle of heart failure animals. Dilation of t-tubules is a common feature in heart failure suggesting this may negatively impact function but there was no functional loss associated with pirfenidone treatment. However, due to the relatively short duration of treatment compared to that used clinically, the impact of long-term treatment on t-tubule structure should be investigated in future studies.

Fibrosis and impaired Ca2+ signalling in heart failure.

Fibrosis and impaired Ca2+ signalling are two prominent features of the failing heart that are generally considered as separate entities. Our discovery of increased amounts of collagen (types I, III, and VI) within the lumen of the transverse (T)-tubules in the failing heart suggests they may be directly linked. T-tubules are plasma membrane invaginations that facilitate a rapid transmission of the action potential deep within the myocyte where they facilitate a synchronous Ca2+ release that triggers contraction. T-tubule remodelling causing impaired Ca2+ release and contraction in heart failure with reduced ejection fraction is well established. However, what drives this mechanism is less clear. In this commentary, I will briefly outline the evidence that supports the role of excessive collagen disposition driving t-tubule remodelling in the failing heart.

Correction to: Imaging tools for assessment of myocardial fibrosis in humans: the need for greater detail.

[This corrects the article DOI: 10.1007/s12551-020-00738-w.].

Mapping system for coregistration of cardiac MRI and ex vivo tissue sampling.

PURPOSE: To design a method suitable for obtaining tissue samples from regions of different function as ascertained by magnetic resonance imaging (MRI). MATERIALS AND METHODS: In vivo MRI was used to create azimuthal projections of the heart from dilated cardiomyopathy transplant patients with the cardiac valves in the center and four concentric rings representing the septum and free wall. Tagged MRI could identify regions of different contractile strength that were then transferred onto the map projection. The resulting tissue sampling map was used to guide dissection of tissue samples from the explanted heart for analysis by electron microscopy (EM) as well as provide samples for subsequent mRNA analysis. Accuracy of the sampling was determined in a sheep heart using 17 fiduciary markers glued to the epicardial surface. RESULTS: Tagged MRI identified areas of "normal" (%S(c) -11), "poor" (%S(c) -4) and "failed" contraction (%S(c) +3). The mapping method we developed enabled straightforward sampling of these regions after surgical excision. EM showed good tissue preservation while the test of accuracy using the fiduciary markers showed a sampling accuracy of 0.3 ± 3.7 mm. This was similar to the resolution of tagged MRI images themselves. CONCLUSION: The methods we have developed can accurately guide tissue sampling for ex vivo tissue analysis.

Mechanisms of reduced contractility in an animal model of hypertensive heart failure.

1. Alterations in intracellular Ca(2+) homeostasis have frequently been implicated as underlying the contractile dysfunction of failing hearts. Contraction in cardiac muscle is due to a balance between sarcolemmal (SL) and sarcoplasmic reticulum (SR) Ca(2+) transport, which has been studied in single cells and small tissue samples. However, many studies have not used physiological temperatures and pacing rates, and this could be problematic given different temperature dependencies and kinetics for transport processes. 2. Spontaneously-hypertensive rats (SHR) and their age-matched Wistar Kyoto controls (WKY) provide an animal model of hypertensive failure with many features in common to heart failure in humans. Steady-state measurements of Ca(2+) and force showed that peak stress was reduced in trabeculae from failing SHR hearts in comparison to WKY, although the Ca(2+) transients were bigger and decayed more slowly. 3. Dynamic Ca(2+) cycling was investigated by determining the recirculation fraction (RF) of activator Ca(2+) through the SR between beats during recovery from experimental protocols that potentiated twitch force. No difference in RF between rat strains was found, although the RF was dependent on the potentiation protocol used. 4. Superfusion with 10 mmol/L caffeine and 0 mmol/L [Ca(2+)](o) was used to measure SL Ca(2+) extrusion. The caffeine-induced [Ca(2+)](i) transient decayed more slowly in SHR trabeculae, suggesting that SL Ca(2+) extrusion was slower in SHR. 5. An ultrastructural immunohistochemical analysis of left ventricular free wall sections using confocal microscopy showed that t-tubule organization was disrupted in myocytes from SHR, with reduced labelling of the SR Ca(2+) -ATPase and Na(+) -Ca(2+) exchanger in comparison to WKY, with the latter possibly related to a lower fraction of t-tubules per unit cell volume. 6. We suggest that although Ca(2+) transport is altered in the progression to heart failure, force development is not limited by the amplitude of the Ca(2+) transient. Despite slower SR Ca(2+) transport, the recirculation fraction and dynamic response to a change of inotropic state minimally altered changes in the SHR model because there was a similar slowing in Ca(2+) extrusion across the surface membrane.

Biophysical reviews' "Meet the Councillor Series": a brief profile of David J. Crossman.

I am the New Zealand representative on the IUPAB council. I have research interest in the biophysics of calcium release in the cardiac myocyte and use advance fluorescence microscopy, particularly super-resolution methods to characterise the subcellular remodelling that occurs in the failing heart. However, my career started in the marine biology field, which highlights the circuitous pathways that science can take. In the new council, I am responsible for Social Networks and Scientific Dissemination and look forward to increasing engagement across the diversity that is biophysics.

Women in science symposium.

A women in science symposium was held at the combined 20th International Union for Pure and Applied Biophysics (IUPAB) Congress, 45th Annual Brazilian Biophysical Society (SBBf) Meeting and 50th Annual Brazilian Society for Biochemistry and Molecular Biology (SBBq) Meeting. There were five excellent speakers from prominent scientist from around the globe that included Frances Separovic (University of Melbourne, Australia), Pimchai Chaiyen (Vidyasirimedhi Institute of Science and Technology (VISTEC), Thailand), Lauren Arendse (University of Cape Town, South Africa), Milagros Medina (University of Zaragoza, Spain) and Carla Mattos (Northeastern University, USA). Each speaker was asked to reflect on their career and challenges they overcome to attain professional success. What followed was a fascinating and thought-provoking exposé on the careers of these five incredibly talented and strong women.

Disruption of transverse-tubular network reduces energy efficiency in cardiac muscle contraction.

AIM: Altered organization of the transverse-tubular network is an early pathological event occurring even prior to the onset of heart failure. Such t-tubular remodelling disturbs the synchrony and signalling between membranous and intracellular ion channels, exchangers, receptors and ATPases essential in the dynamics of excitation-contraction coupling, leading to ionic abnormality and mechanical dysfunction in heart disease progression. In this study, we investigated whether a disrupted t-tubular network has a direct effect on cardiac mechano-energetics. Our aim was to understand the fundamental link between t-tubular remodelling and impaired energy metabolism, both of which are characteristics of heart failure. We thus studied healthy tissue preparations in which cellular processes are not altered by any disease event. METHODS: We exploited the "formamide-detubulation" technique to acutely disrupt the t-tubular network in rat left-ventricular trabeculae. We assessed the energy utilization by cellular Ca2+ cycling and by crossbridge cycling, and quantified the change of energy efficiency following detubulation. For these measurements, trabeculae were mounted in a microcalorimeter where force and heat output were simultaneously measured. RESULTS: Following structural disorganization from detubulation, muscle heat output associated with Ca2+ cycling was reduced, indicating impaired intracellular Ca2+ homeostasis. This led to reduced force production and heat output by crossbridge cycling. The reduction in force-length work was not paralleled by proportionate reduction in the heat output and, as such, energy efficiency was reduced. CONCLUSIONS: These results reveal the direct energetic consequences of disrupted t-tubular network, linking the energy disturbance and the t-tubular remodelling typically observed in heart failure.

Nanoscale Organisation of Ryanodine Receptors and Junctophilin-2 in the Failing Human Heart.

The disrupted organisation of the ryanodine receptors (RyR) and junctophilin (JPH) is thought to underpin the transverse tubule (t-tubule) remodelling in a failing heart. Here, we assessed the nanoscale organisation of these two key proteins in the failing human heart. Recently, an advanced feature of the t-tubule remodelling identified large flattened t-tubules called t-sheets, that were several microns wide. Previously, we reported that in the failing heart, the dilated t-tubules up to ~1 µm wide had increased collagen, and we hypothesised that the t-sheets would also be associated with collagen deposits. Direct stochastic optical reconstruction microscopy (dSTORM), confocal microscopy, and western blotting were used to evaluate the cellular distribution of excitation-contraction structures in the cardiac myocytes from patients with idiopathic dilated cardiomyopathy (IDCM) compared to myocytes from the non-failing (NF) human heart. The dSTORM imaging of RyR and JPH found no difference in the colocalisation between IDCM and NF myocytes, but there was a higher colocalisation at the t-tubule and sarcolemma compared to the corbular regions. Western blots revealed no change in the JPH expression but did identify a ~50% downregulation of RyR (p = 0.02). The dSTORM imaging revealed a trend for the smaller t-tubular RyR clusters (~24%) and reduced the t-tubular RyR cluster density (~35%) that resulted in a 50% reduction of t-tubular RyR tetramers in the IDCM myocytes (p < 0.01). Confocal microscopy identified the t-sheets in all the IDCM hearts examined and found that they are associated with the reticular collagen fibres within the lumen. However, the size and density of the RyR clusters were similar in the myocyte regions associated with t-sheets and t-tubules. T-tubule remodelling is associated with a reduced RyR expression that may contribute to the reduced excitation-contraction coupling in the failing human heart.

Cardiac mechanical efficiency is preserved in primary cardiac hypertrophy despite impaired mechanical function.

Increased heart size is a major risk factor for heart failure and premature mortality. Although abnormal heart growth subsequent to hypertension often accompanies disturbances in mechano-energetics and cardiac efficiency, it remains uncertain whether hypertrophy is their primary driver. In this study, we aimed to investigate the direct association between cardiac hypertrophy and cardiac mechano-energetics using isolated left-ventricular trabeculae from a rat model of primary cardiac hypertrophy and its control. We evaluated energy expenditure (heat output) and mechanical performance (force length work production) simultaneously at a range of preloads and afterloads in a microcalorimeter, we determined energy expenditure related to cross-bridge cycling and Ca2+ cycling (activation heat), and we quantified energy efficiency. Rats with cardiac hypertrophy exhibited increased cardiomyocyte length and width. Their trabeculae showed mechanical impairment, evidenced by lower force production, extent and kinetics of shortening, and work output. Lower force was associated with lower energy expenditure related to Ca2+ cycling and to cross-bridge cycling. However, despite these changes, both mechanical and cross-bridge energy efficiency were unchanged. Our results show that cardiac hypertrophy is associated with impaired contractile performance and with preservation of energy efficiency. These findings provide direction for future investigations targeting metabolic and Ca2+ disturbances underlying cardiac mechanical and energetic impairment in primary cardiac hypertrophy.

A new twist in cardiac muscle: dislocated and helicoid arrangements of myofibrillar z-disks in mammalian ventricular myocytes.

Using deconvolved confocal microscopy of fluorescently labeled markers for z-disks, t-tubules and ryanodine receptors, we have examined sarcomere organization in cardiac myocytes from rat, rabbit and human. We show that sarcomeres exhibit dislocations in registration and occasionally more complex helicoidal topology. This organization was present at both slack ( approximately 1.8 microm) and long sarcomere lengths ( approximately 2.2 microm). Misregistrations in z-disks persisted over 15-20 sarcomere lengths and appeared to arise primarily from variations in fiber direction; particularly as myofibrils pass around nuclei. In addition, myofibrils twist along the cell length. T-tubules generally follow the sarcomere z-disks although additional elements bridging adjacent myofibrils and along the length of the myofibril are present to varying degrees in all cells. Ryanodine receptors (the sarcoplasmic reticulum Ca(2+) release channel) are generally located within 250 nm of the local plane containing t-tubules and z-disks, but a small fraction ( approximately 2%) is found on longitudinal elements of the t-system between z-disks. The results are discussed with respect to the possible role(s) of such complex z-disk organization and z-disk dislocations in the maintenance of cell structure and sarcomere assembly. In addition, the non-planar organization of z-disks may be important in the propagation of local Ca(2+) waves which may have a useful role in helping maintain the uniformity of sarcomere activation in the presence of t-tubule remodeling.