Multiscale Finite Element Modeling of Left Ventricular Growth in Simulations of Valve Disease.

Multiscale models of the cardiovascular system are emerging as effective tools for investigating the mechanisms that drive ventricular growth and remodeling. These models can predict how molecular-level mechanisms impact organ-level structure and function and could provide new insights that help improve patient care. MyoFE is a multiscale computer framework that bridges molecular and organ-level mechanisms in a finite element model of the left ventricle that is coupled with the systemic circulation. In this study, we extend MyoFE to include a growth algorithm, based on volumetric growth theory, to simulate concentric growth (wall thickening/thinning) and eccentric growth (chamber dilation/constriction) in response to valvular diseases. Specifically in our model, concentric growth is controlled by time-averaged total stress along the fiber direction over a cardiac cycle while eccentric growth responds to time-averaged intracellular myofiber passive stress over a cardiac cycle. The new framework correctly predicted different forms of growth in response to two types of valvular diseases, namely aortic stenosis and mitral regurgitation. Furthermore, the model predicted that LV size and function are nearly restored (reversal of growth) when the disease-mimicking perturbation was removed in the simulations for each valvular disorder. In conclusion, the simulations suggest that time-averaged total stress along the fiber direction and time-averaged intracellular myofiber passive stress can be used to drive concentric and eccentric growth in simulations of valve disease.

GELBOX: OPEN-SOURCE SOFTWARE TO IMPROVE RIGOR AND REPRODUCIBILITY WHEN ANALYZING GELS AND IMMUNOBLOTS.

GelBox is open-source software that was developed with the goal of enhancing rigor, reproducibility, and transparency when analyzing gels and immunoblots. It combines image adjustments (cropping, rotation, brightness, and contrast), background correction, and band-fitting in a single application. Users can also associate each lane in an image with metadata (for example, sample type). GelBox data files integrate the raw data, supplied metadata, image adjustments, and band-level analyses in a single file to improve traceability. GelBox has a user-friendly interface and was developed using MATLAB. The software, installation instructions, and tutorials, are available at https://campbell-muscle-lab.github.io/GelBox/.

Unfolded Von Willebrand Factor Binds Protein S and Reduces Anticoagulant Activity.

Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.

A multiscale finite element model of left ventricular mechanics incorporating baroreflex regulation.

Cardiovascular function is regulated by a short-term hemodynamic baroreflex loop, which tries to maintain arterial pressure at a normal level. In this study, we present a new multiscale model of the cardiovascular system named MyoFE. This framework integrates a mechanistic model of contraction at the myosin level into a finite-element-based model of the left ventricle pumping blood through the systemic circulation. The model is coupled with a closed-loop feedback control of arterial pressure inspired by a baroreflex algorithm previously published by our team. The reflex loop mimics the afferent neuron pathway via a normalized signal derived from arterial pressure. The efferent pathway is represented by a kinetic model that simulates the net result of neural processing in the medulla and cell-level responses to autonomic drive. The baroreflex control algorithm modulates parameters such as heart rate and vascular tone of vessels in the lumped-parameter model of systemic circulation. In addition, it spatially modulates intracellular Ca2+ dynamics and molecular-level function of both the thick and the thin myofilaments in the left ventricle. Our study demonstrates that the baroreflex algorithm can maintain arterial pressure in the presence of perturbations such as acute cases of altered aortic resistance, mitral regurgitation, and myocardial infarction. The capabilities of this new multiscale model will be utilized in future research related to computational investigations of growth and remodeling.

Cryo-EM structure of the human cardiac myosin filament.

Pumping of the heart is powered by filaments of the motor protein myosin that pull on actin filaments to generate cardiac contraction. In addition to myosin, the filaments contain cardiac myosin-binding protein C (cMyBP-C), which modulates contractility in response to physiological stimuli, and titin, which functions as a scaffold for filament assembly1. Myosin, cMyBP-C and titin are all subject to mutation, which can lead to heart failure. Despite the central importance of cardiac myosin filaments to life, their molecular structure has remained a mystery for 60 years2. Here we solve the structure of the main (cMyBP-C-containing) region of the human cardiac filament using cryo-electron microscopy. The reconstruction reveals the architecture of titin and cMyBP-C and shows how myosin's motor domains (heads) form three different types of motif (providing functional flexibility), which interact with each other and with titin and cMyBP-C to dictate filament architecture and function. The packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps to generate the cardiac super-relaxed state3; how titin and cMyBP-C may contribute to length-dependent activation4; and how mutations in myosin and cMyBP-C might disturb interactions, causing disease5,6. The reconstruction resolves past uncertainties and integrates previous data on cardiac muscle structure and function. It provides a new paradigm for interpreting structural, physiological and clinical observations, and for the design of potential therapeutic drugs.

Deep learning, 3D ultrastructural analysis reveals quantitative differences in platelet and organelle packing in COVID-19/SARSCoV2 patient-derived platelets.

Platelets contribute to COVID-19 clinical manifestations, of which microclotting in the pulmonary vasculature has been a prominent symptom. To investigate the potential diagnostic contributions of overall platelet morphology and their α-granules and mitochondria to the understanding of platelet hyperactivation and micro-clotting, we undertook a 3D ultrastructural approach. Because differences might be small, we used the high-contrast, high-resolution technique of focused ion beam scanning EM (FIB-SEM) and employed deep learning computational methods to evaluate nearly 600 individual platelets and 30 000 included organelles within three healthy controls and three severely ill COVID-19 patients. Statistical analysis reveals that the α-granule/mitochondrion-to-plateletvolume ratio is significantly greater in COVID-19 patient platelets indicating a denser packing of organelles, and a more compact platelet. The COVID-19 patient platelets were significantly smaller -by 35% in volume - with most of the difference in organelle packing density being due to decreased platelet size. There was little to no 3D ultrastructural evidence for differential activation of the platelets from COVID-19 patients. Though limited by sample size, our studies suggest that factors outside of the platelets themselves are likely responsible for COVID-19 complications. Our studies show how deep learning 3D methodology can become the gold standard for 3D ultrastructural studies of platelets.

COVID-19 patients exhibit a range of symptoms including microclotting. Clotting is a complex process involving both circulating proteins and platelets, a cell within the blood. Increased clotting is suggestive of an increased level of platelet activation. If this were true, we reasoned that parts of the platelet involved in the release of platelet contents during clotting would have lost their content and appear as expanded, empty "ghosts." To test this, we drew blood from severely ill COVID-19 patients and compared the platelets within the blood draws to those from healthy volunteers. All procedures were done under careful attention to biosafety and approved by health authorities. We looked within the platelets for empty ghosts by the high magnification technique of electron microscopy. To count the ghosts, we developed new computer software. In the end, we found little difference between the COVID patient platelets and the healthy donor platelets. The results suggest that circulating proteins outside of the platelet are more important to the strong clotting response. The software developed will be used to analyze other disease states.

Guide to assembling a successful K99/R00 application.

The National Institutes of Health's (NIH) K99/R00 Pathway to Independence Award offers promising postdoctoral researchers and clinician-scientists an opportunity to receive research support at both the mentored and the independent levels with the goal of facilitating a timely transition to a tenure-track faculty position. This transitional program has been generally successful, with most K99/R00 awardees successfully securing R01-equivalent funding by the end of the R00 period. However, often highly promising proposals fail because of poor grantsmanship. This overview provides guidance from the perspective of long-standing members of the National Heart, Lung, and Blood Institute's Mentored Transition to Independence study section for the purpose of helping mentors and trainees regarding how best to assemble competitive K99/R00 applications.

Human skeletal myopathy myosin mutations disrupt myosin head sequestration.

Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyze the effects of common MYH7 and MYH2 mutations in the light meromyosin (LMM) region of myosin. Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in silico modeling showed that myosin coiled coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients and fluorescent ATP analog chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with x-ray diffraction measurements to estimate myosin head order, we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofiber mechanics experiments to investigate contractile function showed that myofiber contractility was not affected. These findings indicate that the structural remodeling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.

Effect of the Novel Myotrope Danicamtiv on Cross-Bridge Behavior in Human Myocardium.

Background Omecamtiv mecarbil (OM) and danicamtiv both increase myocardial force output by selectively activating myosin within the cardiac sarcomere. Enhanced force generation is presumably due to an increase in the total number of myosin heads bound to the actin filament; however, detailed comparisons of the molecular mechanisms of OM and danicamtiv are lacking. Methods and Results The effect of OM and danicamtiv on Ca2+ sensitivity of force generation was analyzed by exposing chemically skinned myocardial samples to a series of increasing Ca2+ solutions. The results showed that OM significantly increased Ca2+ sensitivity of force generation, whereas danicamtiv showed similar Ca2+ sensitivity of force generation to untreated preparations. A direct comparison of OM and danicamtiv on dynamic cross-bridge behavior was performed at a concentration that produced a similar force increase when normalized to predrug levels at submaximal force (pCa 6.1). Both OM and danicamtiv-treated groups slowed the rates of cross-bridge detachment from the strongly bound state and cross-bridge recruitment into the force-producing state. Notably, the significant OM-induced prolongation in the time to reach force relaxation and subsequent commencement of force generation following rapid stretch was dramatically reduced in danicamtiv-treated myocardium. Conclusions This is the first study to directly compare the effects of OM and danicamtiv on cross-bridge kinetics. At a similar level of force enhancement, danicamtiv had a less pronounced effect on the slowing of cross-bridge kinetics and, therefore, may provide a similar improvement in systolic function as OM without excessively prolonging systolic ejection time and slowing cardiac relaxation facilitating diastolic filling at the whole-organ level.

An in silico study of the effects of left ventricular assist device on right ventricular function and inter-ventricular interaction.

BACKGROUND: Left ventricular assist device (LVAD) is associated with a high incidence of right ventricular (RV) failure, which is hypothesized to be caused by the occurring inter-ventricular interactions when the LV is unloaded. Factors contributing to these interactions are unknown. METHODS: We used computer modeling to investigate the impact of the HeartMate 3 LVAD on RV functions. The model was first calibrated against pressure-volume (PV) loops associated with a heart failure (HF) patient and validated against measurements of inter-ventricular interactions in animal experiments. The model was then applied to investigate the effects of LVAD on (1) RV chamber contractility indexed by V 60 derived from its end-systolic PV relationship, and (2) RV diastolic function indexed by V 20 derived from its end-diastolic PV relationship. We also investigated how septal wall thickness and regional contractility affect the impact of LVAD on RV function. RESULTS: The impact of LVAD on RV chamber contractility is small at a pump speed lower than 4k rpm. At a higher pump speed between 4k and 9k rpm, however, RV chamber contractility is reduced (by ~3% at 6k rpm and ~10% at 9k rpm). The reduction of RV chamber contractility is greater with a thinner septal wall or with a lower myocardial contractility at the LV free wall, septum, or RV free wall. CONCLUSION: RV chamber contractility is reduced at a pump speed higher than 4k rpm, and this reduction is greater with a thinner septal wall or lower regional myocardial contractility. Findings here may have clinical implications in identifying LVAD patients who may suffer from RV failure.

History-dependent muscle resistance to stretch remains high after small, posturally relevant pre-movements.

The contributions of intrinsic muscle fiber resistance during mechanical perturbations to standing and other postural behaviors are unclear. Muscle short-range stiffness is known to vary depending on the current level and history of the muscle's activation, as well as the muscle's recent movement history; this property has been referred to as history dependence or muscle thixotropy. However, we currently lack sufficient data about the degree to which muscle stiffness is modulated across posturally relevant characteristics of muscle stretch and activation. We characterized the history dependence of muscle's resistance to stretch in single, permeabilized, activated, muscle fibers in posturally relevant stretch conditions and activation levels. We used a classic paired muscle stretch paradigm, varying the amplitude of a 'conditioning' triangular stretch-shorten cycle followed by a 'test' ramp-and-hold imposed after a variable inter-stretch interval. We tested low (<15%), intermediate (15-50%) and high (>50%) muscle fiber activation levels, evaluating short-range stiffness and total impulse in the test stretch. Muscle fiber resistance to stretch remained high at conditioning amplitudes of <1% optimal fiber length, L0, and inter-stretch intervals of >1 s, characteristic of healthy standing postural sway. An ∼70% attenuation of muscle resistance to stretch was reached at conditioning amplitudes of >3% L0 and inter-stretch intervals of <0.1 s, characteristic of larger, faster postural sway in balance-impaired individuals. The thixotropic changes cannot be predicted solely on muscle force at the time of stretch. Consistent with the disruption of muscle cross-bridges, muscle resistance to stretch during behavior can be substantially attenuated if the prior motion is large enough and/or frequent enough.

Detection of Parvovirus B19 genome in human heart tissue samples.

OBJECTIVE: Identifying viral genomes in human heart tissues is critical for disease diagnosis and assessment of cardiovascular damage. Human heart tissue samples obtained during a biopsy procedure are routinely used to test for the presence of viruses, as guided by clinical manifestations and prognosis. Furthermore, heart tissue samples obtained post-mortem or during a cardiac transplant procedure serve as a valuable research tool, as they allow for an in-depth assessment of cardiac pathology that can aid in our understanding of molecular pathways associated with disease. Because viral nucleic acid constitutes only a small portion of each sample's genetic material, appropriate methods are necessary for positive viral genome identification. RESULTS: Snap-frozen heart tissue samples obtained either post-mortem or during a cardiac transplant procedure were used to develop conditions for detection of Parvovirus B19. Briefly, total DNA was isolated from the heart tissue under varying conditions. A PCR-based assay with Parvovirus B19 specific primers was implemented to detect the presence of the viral genome, followed by Sanger Sequencing. The mechanical disruption of the heart tissue, as well as the cardiac tissue processing methods, had a significant effect on the DNA quality and the ability to detect the Parvovirus B19 genome.

Basic science methods for the characterization of variants of uncertain significance in hypertrophic cardiomyopathy.

With the advent of next-generation whole genome sequencing, many variants of uncertain significance (VUS) have been identified in individuals suffering from inheritable hypertrophic cardiomyopathy (HCM). Unfortunately, this classification of a genetic variant results in ambiguity in interpretation, risk stratification, and clinical practice. Here, we aim to review some basic science methods to gain a more accurate characterization of VUS in HCM. Currently, many genomic data-based computational methods have been developed and validated against each other to provide a robust set of resources for researchers. With the continual improvement in computing speed and accuracy, in silico molecular dynamic simulations can also be applied in mutational studies and provide valuable mechanistic insights. In addition, high throughput in vitro screening can provide more biologically meaningful insights into the structural and functional effects of VUS. Lastly, multi-level mathematical modeling can predict how the mutations could cause clinically significant organ-level dysfunction. We discuss emerging technologies that will aid in better VUS characterization and offer a possible basic science workflow for exploring the pathogenicity of VUS in HCM. Although the focus of this mini review was on HCM, these basic science methods can be applied to research in dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM), arrhythmogenic cardiomyopathy (ACM), or other genetic cardiomyopathies.

Predominant myosin superrelaxed state in canine myocardium with naturally occurring dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a naturally occurring heart failure condition in humans and dogs, notably characterized by a reduced contractility and ejection fraction. As the identification of its underlying cellular and molecular mechanisms remain incomplete, the aim of the present study was to assess whether the molecular motor myosin and its known relaxed conformational states are altered in DCM. For that, we dissected and skinned thin cardiac strips from left ventricle obtained from six DCM Doberman Pinschers and six nonfailing (NF) controls. We then used a combination of Mant-ATP chase experiments and X-ray diffraction to assess both energetic and structural changes of myosin. Using the Mant-ATP chase protocol, we observed that in DCM dogs, the amount of myosin molecules in the ATP-conserving conformational state, also known as superrelaxed (SRX), is significantly increased when compared with NF dogs. This alteration can be rescued by applying EMD-57033, a small molecule activating myosin. Conversely, with X-ray diffraction, we found that in DCM dogs, there is a higher proportion of myosin heads in the vicinity of actin when compared with NF dogs (1,0 to 1,1 intensity ratio). Hence, we observed an uncoupling between energetic (Mant-ATP chase) and structural (X-ray diffraction) data. Taken together, these results may indicate that in the heart of Doberman Pinschers with DCM, myosin molecules are potentially stuck in a nonsequestered but ATP-conserving SRX state, that can be counterbalanced by EMD-57033 demonstrating the potential for a myosin-centered pharmacological treatment of DCM.NEW & NOTEWORTHY The key finding of the present study is that, in left ventricles of dogs with a naturally occurring dilated cardiomyopathy, relaxed myosin molecules favor a nonsequestered superrelaxed state potentially impairing sarcomeric contractility. This alteration is rescuable by applying a small molecule activating myosin known as EMD-57033.

Bedside ultrasound of the internal jugular vein to assess fluid status and right ventricular function: The POCUS-JVD study.

BACKGROUND: Accurate estimation of fluid status is important in the management of heart failure patients, however, the current methods for bedside assessment can be unreliable or impractical for daily use. METHODS: Non-ventilated patients were enrolled immediately prior to scheduled right heart catheterization (RHC). Using M-mode, IJV maximum (Dmax) and minimum (Dmin) anteroposterior diameters were measured during normal breathing, while supine. Respiratory variation in diameter (RVD) was calculated as [(Dmax - Dmin)/Dmax] in percentage. Collapsibility with sniff maneuver (COS) was assessed. Lastly, inferior vena cava (IVC) was assessed. Pulmonary artery pulsatility index (PAPi) was calculated. Data was obtained by five investigators. RESULTS: Total 176 patients were enrolled. Mean BMI was 30.5 kg/m2, LVEF 14-69% (range), 38% with LVEF ≤35%. The POCUS of IJV could be performed in all patients in <5 min. Increasing RAP demonstrated progressive increase in IJV and IVC diameters. For high filling pressure (RAP ≥10 mmHg), an IJV Dmax ≥1.2 cm or IJV-RVD < 30% had specificity >70%. Combining the POCUS of IJV to physical examination improved the combined specificity to 97% for RAP ≥10 mmHg. Conversely, a finding of IJV-COS was 88% specific for normal RAP (<10 mmHg). An IJV-RVD <15% is suggested as a cutoff for RAP ≥15 mmHg. The performance of IJV POCUS was comparable to IVC. For RV function assessment, IJV-RVD < 30% had 76% sensitivity and 73% specificity for PAPi <3, while IJV-COS was 80% specific for PAPi ≥3. CONCLUSION: POCUS of IJV is an easy to perform, specific and reliable method for volume status estimation in daily practice. An IJV-RVD < 30% is suggested for estimation of RAP ≥10 mmHg and PAPi <3.

Right Ventricular Sarcomere Contractile Depression and the Role of Thick Filament Activation in Human Heart Failure With Pulmonary Hypertension.

BACKGROUND: Right ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in patients with heart failure with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. We thus sought to characterize RV myocyte contractile depression in HFrEF-PH, identify those components reflected by clinical RV indices, and uncover underlying biophysical mechanisms. METHODS: Resting, calcium-, and load-dependent mechanics were prospectively studied in permeabilized RV cardiomyocytes isolated from explanted hearts from 23 patients with HFrEF-PH undergoing cardiac transplantation and 9 organ donor controls. RESULTS: Unsupervised machine learning using myocyte mechanical data with the highest variance yielded 2 HFrEF-PH subgroups that in turn mapped to patients with decompensated or compensated clinical RV function. This correspondence was driven by reduced calcium-activated isometric tension in decompensated clinical RV function, whereas surprisingly, many other major myocyte contractile measures including peak power and myocyte active stiffness were similarly depressed in both groups. Similar results were obtained when subgroups were first defined by clinical indices, and then myocyte mechanical properties in each group compared. To test the role of thick filament defects, myofibrillar structure was assessed by x-ray diffraction of muscle fibers. This revealed more myosin heads associated with the thick filament backbone in decompensated clinical RV function, but not compensated clinical RV function, as compared with controls. This corresponded to reduced myosin ATP turnover in decompensated clinical RV function myocytes, indicating less myosin in a crossbridge-ready disordered-relaxed (DRX) state. Altering DRX proportion (%DRX) affected peak calcium-activated tension in the patient groups differently, depending on their basal %DRX, highlighting potential roles for precision-guided therapeutics. Last, increasing myocyte preload (sarcomere length) increased %DRX 1.5-fold in controls but only 1.2-fold in both HFrEF-PH groups, revealing a novel mechanism for reduced myocyte active stiffness and by extension Frank-Starling reserve in human heart failure. CONCLUSIONS: Although there are many RV myocyte contractile deficits in HFrEF-PH, commonly used clinical indices only detect reduced isometric calcium-stimulated force, which is related to deficits in basal and recruitable %DRX myosin. Our results support use of therapies to increase %DRX and enhance length-dependent recruitment of DRX myosin heads in such patients.

Cryo-EM structure of the human cardiac myosin filament.

Pumping of the heart is powered by filaments of the motor protein myosin, which pull on actin filaments to generate cardiac contraction. In addition to myosin, the filaments contain cardiac myosin-binding protein C (cMyBP-C), which modulates contractility in response to physiological stimuli, and titin, which functions as a scaffold for filament assembly 1 . Myosin, cMyBP-C and titin are all subject to mutation, which can lead to heart failure. Despite the central importance of cardiac myosin filaments to life, their molecular structure has remained a mystery for 60 years 2 . Here, we have solved the structure of the main (cMyBP-C-containing) region of the human cardiac filament to 6 Å resolution by cryo-EM. The reconstruction reveals the architecture of titin and cMyBP-C for the first time, and shows how myosin's motor domains (heads) form 3 different types of motif (providing functional flexibility), which interact with each other and with specific domains of titin and cMyBP-C to dictate filament architecture and regulate function. A novel packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps generate the cardiac super-relaxed state 3 , how titin and cMyBP-C may contribute to length-dependent activation 4 , and how mutations in myosin and cMyBP-C might disrupt interactions, causing disease 5, 6 . A similar structure is likely in vertebrate skeletal myosin filaments. The reconstruction resolves past uncertainties, and integrates previous data on cardiac muscle structure and function. It provides a new paradigm for interpreting structural, physiological and clinical observations, and for the design of potential therapeutic drugs.

Right Ventricular Sarcomere Contractile Depression and the Role of Thick Filament Activation in Human Heart Failure with Pulmonary Hypertension.

Rationale: Right ventricular (RV) contractile dysfunction commonly occurs and worsens outcomes in heart failure patients with reduced ejection fraction and pulmonary hypertension (HFrEF-PH). However, such dysfunction often goes undetected by standard clinical RV indices, raising concerns that they may not reflect aspects of underlying myocyte dysfunction. Objective: To determine components of myocyte contractile depression in HFrEF-PH, identify those reflected by clinical RV indices, and elucidate their underlying biophysical mechanisms. Methods and Results: Resting, calcium- and load-dependent mechanics were measured in permeabilized RV cardiomyocytes isolated from explanted hearts from 23 HFrEF-PH patients undergoing cardiac transplantation and 9 organ-donor controls. Unsupervised machine learning using myocyte mechanical data with the highest variance yielded two HFrEF-PH subgroups that in turn mapped to patients with depressed (RVd) or compensated (RVc) clinical RV function. This correspondence was driven by reduced calcium-activated isometric tension in RVd, while surprisingly, many other major myocyte contractile measures including peak power, maximum unloaded shortening velocity, and myocyte active stiffness were similarly depressed in both groups. Similar results were obtained when subgroups were first defined by clinical indices, and then myocyte mechanical properties in each group compared. To test the role of thick-filament defects, myofibrillar structure was assessed by X-ray diffraction of muscle fibers. This revealed more myosin heads associated with the thick filament backbone in RVd but not RVc, as compared to controls. This corresponded to reduced myosin ATP turnover in RVd myocytes, indicating less myosin in a cross-bridge ready disordered-relaxed (DRX) state. Altering DRX proportion (%DRX) affected peak calcium-activated tension in the patient groups differently, depending on their basal %DRX, highlighting potential roles for precision-guided therapeutics. Lastly, increasing myocyte preload (sarcomere length) increased %DRX 1.5-fold in controls but only 1.2-fold in both HFrEF-PH groups, revealing a novel mechanism for reduced myocyte active stiffness and by extension Frank-Starling reserve in human HF. Conclusions: While there are multiple RV myocyte contractile deficits In HFrEF-PH, clinical indices primarily detect reduced isometric calcium-stimulated force related to deficits in basal and recruitable %DRX myosin. Our results support use of therapies to increase %DRX and enhance length-dependent recruitment of DRX myosin heads in such patients.