Recapitulating human cardio-pulmonary co-development using simultaneous multilineage differentiation of pluripotent stem cells

The extensive crosstalk between the developing heart and lung is critical to their proper morphogenesis and maturation. However, there remains a lack of models that investigate the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing the simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human induced pluripotent stem cells (hiPSCs) via temporal specific tuning of WNT and nodal signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (mTs), and expedited alveolar maturation in the presence of cardiac accompaniment. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage mT effectively segregated from each other with concurrent initiation of cardiac contraction. We expect that our multilineage differentiation model will offer an experimentally tractable system for investigating human cardio-pulmonary interaction and tissue boundary formation during embryogenesis.

Nanoenviroments of the β-Subunit of L-Type Voltage-Gated Calcium Channels in Adult Cardiomyocytes

In cardiomyocytes, Ca2+ influx through L-type voltage-gated calcium channels (LTCCs) following membrane depolarization regulates crucial Ca2+-dependent processes including duration and amplitude of the action potentials and excitation-contraction coupling. LTCCs are heteromultimeric proteins composed of the Cavα1, Cavβ, Cavα2δ and Cavγ subunits. Here, using ascorbate peroxidase (APEX2)-mediated proximity labeling and quantitative proteomics, we identified 61 proteins in the nanoenvironments of Cavβ2 in cardiomyocytes. These proteins are involved in diverse cellular functions such as cellular trafficking, cardiac contraction, sarcomere organization and excitation-contraction coupling. Moreover, pull-down assays and co-immunoprecipitation analyses revealed that Cavβ2 interacts with the ryanodine receptor 2 (RyR2) in adult cardiomyocytes, probably coupling LTCCs and the RyR2 into a supramolecular complex at the dyads. This interaction is mediated by the Src-homology 3 domain of Cavβ2 and is necessary for an effective pacing frequency-dependent increase of the Ca2+-induced Ca2+ release mechanism in cardiomyocytes.

A Stochastic Spatiotemporal Model of Rat Ventricular Myocyte Calcium Dynamics Demonstrated Necessary Features for Calcium Wave Propagation

Calcium (Ca2+) plays a central role in the excitation and contraction of cardiac myocytes. Experiments have indicated that calcium release is stochastic and regulated locally suggesting the possibility of spatially heterogeneous calcium levels in the cells. This spatial heterogeneity might be important in mediating different signaling pathways. During more than 50 years of computational cell biology, the computational models have been advanced to incorporate more ionic currents, going from deterministic models to stochastic models. While periodic increases in cytoplasmic Ca2+ concentration drive cardiac contraction, aberrant Ca2+ release can underly cardiac arrhythmia. However, the study of the spatial role of calcium ions has been limited due to the computational expense of using a three-dimensional stochastic computational model. In this paper, we introduce a three-dimensional stochastic computational model for rat ventricular myocytes at the whole-cell level that incorporate detailed calcium dynamics, with (1) non-uniform release site placement, (2) non-uniform membrane ionic currents and membrane buffers, (3) stochastic calcium-leak dynamics and (4) non-junctional or rogue ryanodine receptors. The model simulates spark-induced spark activation and spark-induced Ca2+ wave initiation and propagation that occur under conditions of calcium overload at the closed-cell condition, but not when Ca2+ levels are normal. This is considered important since the presence of Ca2+ waves contribute to the activation of arrhythmogenic currents.

A Fully-Coupled Electro-Mechanical Whole-Heart Computational Model: Influence of Cardiac Contraction on the ECG

The ECG is one of the most commonly used non-invasive tools to gain insights into the electrical functioning of the heart. It has been crucial as a foundation in the creation and validation of in silico models describing the underlying electrophysiological processes. However, so far, the contraction of the heart and its influences on the ECG have mainly been overlooked in in silico models. As the heart contracts and moves, so do the electrical sources within the heart responsible for the signal on the body surface, thus potentially altering the ECG. To illuminate these aspects, we developed a human 4-chamber electro-mechanically coupled whole heart in silico model and embedded it within a torso model. Our model faithfully reproduces measured 12-lead ECG traces, circulatory characteristics, as well as physiological ventricular rotation and atrioventricular valve plane displacement. We compare our dynamic model to three non-deforming ones in terms of standard clinically used ECG leads (Einthoven and Wilson) and body surface potential maps (BSPM). The non-deforming models consider the heart at its ventricular end-diastatic, end-diastolic and end-systolic states. The standard leads show negligible differences during P-Wave and QRS-Complex, yet during T-Wave the leads closest to the heart show prominent differences in amplitude. When looking at the BSPM, there are no notable differences during the P-Wave, but effects of cardiac motion can be observed already during the QRS-Complex, increasing further during the T-Wave. We conclude that for the modeling of activation (P-Wave/QRS-Complex), the associated effort of simulating a complete electro-mechanical approach is not worth the computational cost. But when looking at ventricular repolarization (T-Wave) in standard leads as well as BSPM, there are areas where the signal can be influenced by cardiac motion of the heart to an extent that should not be ignored.

Carfilzomib Treatment Causes Molecular and Functional Alterations of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes

Background Anticancer therapies have significantly improved patient outcomes; however, cardiac side effects from cancer therapies remain a significant challenge. Cardiotoxicity following treatment with proteasome inhibitors such as carfilzomib is known in clinical settings, but the underlying mechanisms have not been fully elucidated. Methods and Results Using human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) as a cell model for drug‐induced cytotoxicity in combination with traction force microscopy, functional assessments, high‐throughput imaging, and comprehensive omic analyses, we examined the molecular mechanisms involved in structural and functional alterations induced by carfilzomib in hiPSC‐CMs. Following the treatment of hiPSC‐CMs with carfilzomib at 0.01 to 10 µmol/L, we observed a concentration‐dependent increase in carfilzomib‐induced toxicity and corresponding morphological, structural, and functional changes. Carfilzomib treatment reduced mitochondrial membrane potential, ATP production, and mitochondrial oxidative respiration and increased mitochondrial oxidative stress. In addition, carfilzomib treatment affected contractility of hiPSC‐CMs in 3‐dimensional microtissues. At a single cell level, carfilzomib treatment impaired Ca 2+ transients and reduced integrin‐mediated traction forces as detected by piconewton tension sensors. Transcriptomic and proteomic analyses revealed that carfilzomib treatment downregulated the expression of genes involved in extracellular matrices, integrin complex, and cardiac contraction, and upregulated stress responsive proteins including heat shock proteins. Conclusions Carfilzomib treatment causes deleterious changes in cellular and functional characteristics of hiPSC‐CMs. Insights into these changes could be gained from the changes in the expression of genes and proteins identified from our omic analyses.

ACTIN Anchors the Highly Oligomeric DRP1 at Mitochondria-Sarcoplasmic Reticulum Contact Sites in Adult Murine Heart: Its Functional Implication

Rationale: Mitochondrial fission and fusion are relatively infrequent in adult cardiomyocytes compared to other cell types. This is surprising considering that proteins involved in mitochondrial dynamics are highly expressed in the heart. It has been previously reported that dynamin related protein 1 (DRP1) has a critical role in mitochondrial fitness and cardiac protection. Cardiac DRP1 ablation in the adult heart evokes a progressive dilated cardiac myopathy and lethal heart failure. Nevertheless, the conditional cardiacspecific DRP1 knock out animals present a significantly longer survival rate compared with global DRP1 KO models. We have described before the great importance for cardiac physiology of the strategic positioning of mitochondrial proteins in the cardiac tissue. Therefore, we hypothesize that DRP1 plays a regulatory role in cardiac physiology and mitochondrial fitness by preferentially accumulating at mitochondria and junctional sarcoplasmic reticulum (jSR) contact sites, where the high Ca2+ microdomain is formed during excitation-contraction (EC) coupling. Objective: This study aims to determine whether mitochondria-associated DRP1 is preferentially accumulated in the mitochondria and jSR contact sites and if indeed this is the case, what is the mechanism responsible for such a biased distribution and what is the functional implication. Methods and Results: Using high-resolution imaging approaches, we found that mitochondria-associated DRP1 in cardiomyocytes was localized in the discrete regions where T-tubule, jSR, and mitochondria are adjacent to each other. Western blot results showed that mitochondria-bound DRP1 was restricted to the mitochondria-associated membranes (MAM), with undetectable levels in purified mitochondria. Furthermore, in comparison to the cytosolic DRP1, the membrane-bound DRP1 in SR and MAM fractions formed high molecular weight oligomers. In both electrically paced adult cardiomyocytes and Langendorff-perfused beating hearts, the oscillatory Ca2+ pulses preserved MAM-associated DRP1 accumulation. Interestingly, similar to DRP1, all mitochondria-bound βACTIN only exists in MAM and not in the purified mitochondria. Additionally, co-immunoprecipitation pulls down both DRP1 and βACTIN together. Inhibition of βACTIN polymerization with Cytochalasin D disrupts the tight association between DRP1 and βACTIN. In cardiac specific DRP1 knockout mouse after 6 weeks of tamoxifen induction the cardiomyocytes show disarray of sarcomere, a decrease of cardiac contraction, loss of mitochondrial membrane potential significantly decreased spare respiratory capacity, and frequent occurrence of earl after contraction, suggesting the heart is susceptible for failure and arrhythmias. Despite of this phenotype, DRP1icKo animal have a longer life spam than other DRP1 KO models. We also observed that DRP1icKO. Strikingly, DRP1 levels are is only modestly decreased in the MAM when compared with the rest of the cellular fractions. These preserved levels were accompanied with preservation of the mitochondrial pool in the MAM fraction obtained from the DRP1icKO hearts. Conclusions: The results show that in adult cardiomyocytes, mitochondria bound DRP1 clusters in high molecular weight protein complexes at MAM. This clustering is fortified by EC coupling mediated Ca2+ transients and requires its interaction with βACTIN. Together with the better preserved dRP1 levels in the DRP1icKO model in the MAM, we conclude that DRP1 is anchored in mitochondria-SR interface through βACTIN and position itself to play a fundamental role in regulating mitochondrial quality control in the working heart.

Acute exposure to the chemotherapy cisplatin has a biphasic effect in cardiac contractility

Cancer and cardiovascular diseases are the main causes of death in Uruguay and developed countries. In clinical practice, there is often the need to administrate chemotherapy with cisplatin (CTP) to patients with cardiovascular comorbidities. The aim of this work is to characterize the possible detrimental effects in cardiac function by the acute exposition to CPT using isolated heart and cardiomyocytes from guinea pigs (Cavia porcellus). All the procedures regarding animal experimentation were performed following approved protocols by the university ethics committee. Isolated hearts were placed in a Langendorff system and perfused with Tyrode 1.8 mM Ca2+ as control medium, or with extracellularly added CPT (0–100 µM). Tension was recorded with a gauge force transducer attached to the papillary muscle and electrical responses were measured with Ag-AgCl electrodes placed in surface extremes near the papillary muscle. Cardiomyocytes were isolated by enzymatic methods. Data were obtained by patch clamp and confocal microscopy with Rhodamine and Fluo dyes sensitive to Ca2+ binding. Non-parametric t tests were used for data comparison. The best fit of Hill’s equation to dose–response curves was done using nonlinear regression methods. In isolated hearts, CPT showed a biphasic effect over the development of tension, increasing up to 5–10 µM to decrease at higher concentrations. In isolated cardiomyocytes, Ca2+ currents were stimulated and inhibited by CPT in a similar dose. Confocal microscopy showed an increment and a reduction of relative fluorescence of the calcium-sensitive dyes with CPT as well. Our results suggest that CPT may affect cardiac contraction and automatism upon acute exposure of the heart, presumably by blocking L-type (Cav1.2) calcium channels and interference with molecules involved in maintaining the homeostasis of intracellular Ca2+.

Dilatated Cardiomyopathy In Two Month Old Puppy

Background: Canine dilated cardiomyopathy (DCM) is a disease that results in a decreased ability of the cardiac contraction to generate pressure to pump blood through the vascular system. DCM is characterized by dilation of the ventricles with ventricular wall thinning. Purpose: The DCM case in Indonesia is rarely reported; therefore, this paper contains information about dilatated cardiomyopathy in a 2-month-old puppy. Case Analyze: A two-month-old local dog arrived with a complaint about coughing, loss of appetite, fatigue, and swelling on extremities, also having a history of seizures and bloody diarrhea. Physical examination shows that the patient breathes using abdominal type and polypnea, tachycardia pulse, pale mucose, and dehydration. Electrocardiogram result shows tachycardia sinus and abnormality in the depression of ST-segment. Radiography examination shows heart dilation and liquid accumulation in the thoracic cavity and abdomen. Hematology routine examination shows microcytic hyperchromic anemia, leucocytosis, and eosinophilia. Feces examination resulted in negative. Pathology anatomy examination show dilatated cardio, pulmonum hepatization, fluid accumulation in the thoracic cavity and abdomen cavity. Result: According to anamnesis, clinical examination, laboratory examination, and anatomy pathology examination can be concluded that the dog, in this case, is diagnosed with dilatated cardiomyopathy.

PRC1 Stabilizes Cardiac Contraction by Regulating Cardiac Sarcomere Assembly and Cardiac Conduction System Construction

Cardiac development is a complex process that is strictly controlled by various factors, including PcG protein complexes. Several studies have reported the critical role of PRC2 in cardiogenesis. However, little is known about the regulation mechanism of PRC1 in embryonic heart development. To gain more insight into the mechanistic role of PRC1 in cardiogenesis, we generated a PRC1 loss-of-function zebrafish line by using the CRISPR/Cas9 system targeting rnf2, a gene encoding the core subunit shared by all PRC1 subfamilies. Our results revealed that Rnf2 is not involved in cardiomyocyte differentiation and heart tube formation, but that it is crucial to maintaining regular cardiac contraction. Further analysis suggested that Rnf2 loss-of-function disrupted cardiac sarcomere assembly through the ectopic activation of non-cardiac sarcomere genes in the developing heart. Meanwhile, Rnf2 deficiency disrupts the construction of the atrioventricular canal and the sinoatrial node by modulating the expression of bmp4 and other atrioventricular canal marker genes, leading to an impaired cardiac conduction system. The disorganized cardiac sarcomere and defective cardiac conduction system together contribute to defective cardiac contraction. Our results emphasize the critical role of PRC1 in the cardiac development.

Cardiac sarcomere mechanics in health and disease

The sarcomere is the fundamental structural and functional unit of striated muscle and is directly responsible for most of its mechanical properties. The sarcomere generates active or contractile forces and determines the passive or elastic properties of striated muscle. In the heart, mutations in sarcomeric proteins are responsible for the majority of genetically inherited cardiomyopathies. Here, we review the major determinants of cardiac sarcomere mechanics including the key structural components that contribute to active and passive tension. We dissect the molecular and structural basis of active force generation, including sarcomere composition, structure, activation, and relaxation. We then explore the giant sarcomere-resident protein titin, the major contributor to cardiac passive tension. We discuss sarcomere dynamics exemplified by the regulation of titin-based stiffness and the titin life cycle. Finally, we provide an overview of therapeutic strategies that target the sarcomere to improve cardiac contraction and filling.