Acute Adenoviral Infection Elicits an Arrhythmogenic Substrate Prior to Myocarditis.

BACKGROUND: Viral cardiac infection represents a significant clinical challenge encompassing several etiological agents, disease stages, complex presentation, and a resulting lack of mechanistic understanding. Myocarditis is a major cause of sudden cardiac death in young adults, where current knowledge in the field is dominated by later disease phases and pathological immune responses. However, little is known regarding how infection can acutely induce an arrhythmogenic substrate before significant immune responses. Adenovirus is a leading cause of myocarditis, but due to species specificity, models of infection are lacking, and it is not understood how adenoviral infection may underlie sudden cardiac arrest. Mouse adenovirus type-3 was previously reported as cardiotropic, yet it has not been utilized to understand the mechanisms of cardiac infection and pathology. METHODS: We have developed mouse adenovirus type-3 infection as a model to investigate acute cardiac infection and molecular alterations to the infected heart before an appreciable immune response or gross cardiomyopathy. RESULTS: Optical mapping of infected hearts exposes decreases in conduction velocity concomitant with increased Cx43Ser368 phosphorylation, a residue known to regulate gap junction function. Hearts from animals harboring a phospho-null mutation at Cx43Ser368 are protected against mouse adenovirus type-3-induced conduction velocity slowing. Additional to gap junction alterations, patch clamping of mouse adenovirus type-3-infected adult mouse ventricular cardiomyocytes reveals prolonged action potential duration as a result of decreased IK1 and IKs current density. Turning to human systems, we find human adenovirus type-5 increases phosphorylation of Cx43Ser368 and disrupts synchrony in human induced pluripotent stem cell-derived cardiomyocytes, indicating common mechanisms with our mouse whole heart and adult cardiomyocyte data. CONCLUSIONS: Together, these findings demonstrate that adenoviral infection creates an arrhythmogenic substrate through direct targeting of gap junction and ion channel function in the heart. Such alterations are known to precipitate arrhythmias and likely contribute to sudden cardiac death in acutely infected patients.

Experimentally-guided in silico design of engineered heart tissues to improve cardiac electrical function after myocardial infarction.

Engineered heart tissues (EHTs) built from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) showed promising results for cardiac function restoration following myocardial infarction. Nevertheless, human iPSC-CMs have longer action potential and lower cell-to-cell coupling than adult-like CMs. These immature electrophysiological properties favor arrhythmias due to the generation of electrophysiological gradients when hiPSC-CMs are injected in the cardiac tissue. Culturing hiPSC-CMs on three-dimensional (3D) scaffolds can promote their maturation and influence their alignment. However, it is still uncertain how on-scaffold culturing influences the overall electrophysiology of the in vitro and implanted EHTs, as it requires expensive and time consuming experimentation. Here, we computationally investigated the impact of the scaffold design on the EHT electrical depolarization and repolarization before and after engraftment on infarcted tissue. We first acquired and processed electrical recordings from in vitro EHTs, which we used to calibrate the modeling and simulation of in silico EHTs to replicate experimental outcomes. Next, we built in silico EHT models for a range of scaffold pore sizes, shapes (square, rectangular, auxetic, hexagonal) and thicknesses. In this setup, we found that scaffolds made of small (0.2 mm2), elongated (30° half-angle) hexagons led to faster EHT activation and better mimicked the cardiac anisotropy. The scaffold thickness had a marginal role on the not engrafted EHT electrophysiology. Moreover, EHT engraftment on infarcted tissue showed that the EHT conductivity should be at least 5% of that in healthy tissue for bidirectional EHT-myocardium electrical propagation. For conductivities above such threshold, the scaffold made of small elongated hexagons led to the lowest activation time (AT) in the coupled EHT-myocardium. If the EHT conductivity was further increased and the hiPSC-CMs were uniformly oriented parallel to the epicardial cells, the total AT and the repolarization time gradient decreased substantially, thus minimizing the likelihood for arrhythmias after EHT transplantation.

The cardiomyocyte origins of diastolic dysfunction: cellular components of myocardial "stiffness".

The impaired ability of the heart to relax and stretch to accommodate venous return is generally understood to represent a state of "diastolic dysfunction" and often described using the all-purpose noun "stiffness." Despite the now common qualitative usage of this term in fields of cardiac patho/physiology, the specific quantitative concept of stiffness as a molecular and biophysical entity with real practical interpretation in healthy and diseased hearts is sometimes obscure. The focus of this review is to characterize the concept of cardiomyocyte stiffness and to develop interpretation of "stiffness" attributes at the cellular and molecular levels. Here, we consider "stiffness"-related terminology interpretation and make links between cardiomyocyte stiffness and aspects of functional and structural cardiac performance. We discuss cross bridge-derived stiffness sources, considering the contributions of diastolic myofilament activation and impaired relaxation. This includes commentary relating to the role of cardiomyocyte Ca2+ flux and Ca2+ levels in diastole, the troponin-tropomyosin complex role as a Ca2+ effector in diastole, the myosin ADP dissociation rate as a modulator of cross bridge attachment and regulation of cross-bridge attachment by myosin binding protein C. We also discuss non-cross bridge-derived stiffness sources, including the titin sarcomeric spring protein, microtubule and intermediate filaments, and cytoskeletal extracellular matrix interactions. As the prevalence of conditions involving diastolic heart failure has escalated, a more sophisticated understanding of the molecular, cellular, and tissue determinants of cardiomyocyte stiffness offers potential to develop imaging and molecular intervention tools.

The benign nature and rare occurrence of cardiac myxoma as a possible consequence of the limited cardiac proliferative/ regenerative potential: a systematic review.

BACKGROUND: Cardiac Myxoma is a primary tumor of heart. Its origins, rarity of the occurrence of primary cardiac tumors and how it may be related to limited cardiac regenerative potential, are not yet entirely known. This study investigates the key cardiac genes/ transcription factors (TFs) and signaling pathways to understand these important questions. METHODS: Databases including PubMed, MEDLINE, and Google Scholar were searched for published articles without any date restrictions, involving cardiac myxoma, cardiac genes/TFs/signaling pathways and their roles in cardiogenesis, proliferation, differentiation, key interactions and tumorigenesis, with focus on cardiomyocytes. RESULTS: The cardiac genetic landscape is governed by a very tight control between proliferation and differentiation-related genes/TFs/pathways. Cardiac myxoma originates possibly as a consequence of dysregulations in the gene expression of differentiation regulators including Tbx5, GATA4, HAND1/2, MYOCD, HOPX, BMPs. Such dysregulations switch the expression of cardiomyocytes into progenitor-like state in cardiac myxoma development by dysregulating Isl1, Baf60 complex, Wnt, FGF, Notch, Mef2c and others. The Nkx2-5 and MSX2 contribute predominantly to both proliferation and differentiation of Cardiac Progenitor Cells (CPCs), may possibly serve roles based on the microenvironment and the direction of cell circuitry in cardiac tumorigenesis. The Nkx2-5 in cardiac myxoma may serve to limit progression of tumorigenesis as it has massive control over the proliferation of CPCs. The cardiac cell type-specific genetic programming plays governing role in controlling the tumorigenesis and regenerative potential. CONCLUSION: The cardiomyocytes have very limited proliferative and regenerative potential. They survive for long periods of time and tightly maintain the gene expression of differentiation genes such as Tbx5, GATA4 that interact with tumor suppressors (TS) and exert TS like effect. The total effect such gene expression exerts is responsible for the rare occurrence and benign nature of primary cardiac tumors. This prevents the progression of tumorigenesis. But this also limits the regenerative and proliferative potential of cardiomyocytes. Cardiac Myxoma develops as a consequence of dysregulations in these key genes which revert the cells towards progenitor-like state, hallmark of CM. The CM development in carney complex also signifies the role of TS in cardiac cells.

ERRγ agonist under mechanical stretching manifests hypertrophic cardiomyopathy phenotypes of engineered cardiac tissue through maturation.

Engineered cardiac tissue (ECT) using human induced pluripotent stem cell-derived cardiomyocytes is a promising tool for modeling heart disease. However, tissue immaturity makes robust disease modeling difficult. Here, we established a method for modeling hypertrophic cardiomyopathy (HCM) malignant (MYH7 R719Q) and nonmalignant (MYBPC3 G115∗) pathogenic sarcomere gene mutations by accelerating ECT maturation using an ERRγ agonist, T112, and mechanical stretching. ECTs treated with T112 under 10% elongation stimulation exhibited more organized and mature characteristics. Whereas matured ECTs with the MYH7 R719Q mutation showed broad HCM phenotypes, including hypertrophy, hypercontraction, diastolic dysfunction, myofibril misalignment, fibrotic change, and glycolytic activation, matured MYBPC3 G115∗ ECTs displayed limited phenotypes, which were primarily observed only under our new maturation protocol (i.e., hypertrophy). Altogether, ERRγ activation combined with mechanical stimulation enhanced ECT maturation, leading to a more accurate manifestation of HCM phenotypes, including non-cardiomyocyte activation, consistent with clinical observations.

Preclinical Large Animal Porcine Models for Cardiac Regeneration and Its Clinical Translation: Role of hiPSC-Derived Cardiomyocytes.

Myocardial Infarction (MI) occurs due to a blockage in the coronary artery resulting in ischemia and necrosis of cardiomyocytes in the left ventricular heart muscle. The dying cardiac tissue is replaced with fibrous scar tissue, causing a decrease in myocardial contractility and thus affecting the functional capacity of the myocardium. Treatments, such as stent placements, cardiac bypasses, or transplants are beneficial but with many limitations, and may decrease the overall life expectancy due to related complications. In recent years, with the advent of human induced pluripotent stem cells (hiPSCs), newer avenues using cell-based approaches for the treatment of MI have emerged as a potential for cardiac regeneration. While hiPSCs and their derived differentiated cells are promising candidates, their translatability for clinical applications has been hindered due to poor preclinical reproducibility. Various preclinical animal models for MI, ranging from mice to non-human primates, have been adopted in cardiovascular research to mimic MI in humans. Therefore, a comprehensive literature review was essential to elucidate the factors affecting the reproducibility and translatability of large animal models. In this review article, we have discussed different animal models available for studying stem-cell transplantation in cardiovascular applications, mainly focusing on the highly translatable porcine MI model.

Malat1 deficiency prevents neonatal heart regeneration by inducing cardiomyocyte binucleation.

The adult mammalian heart has limited regenerative capacity, while the neonatal heart fully regenerates during the first week of life. Postnatal regeneration is mainly driven by proliferation of preexisting cardiomyocytes and supported by proregenerative macrophages and angiogenesis. Although the process of regeneration has been well studied in the neonatal mouse, the molecular mechanisms that define the switch between regenerative and nonregenerative cardiomyocytes are not well understood. Here, using in vivo and in vitro approaches, we identified the lncRNA Malat1 as a key player in postnatal cardiac regeneration. Malat1 deletion prevented heart regeneration in mice after myocardial infarction on postnatal day 3 associated with a decline in cardiomyocyte proliferation and reparative angiogenesis. Interestingly, Malat1 deficiency increased cardiomyocyte binucleation even in the absence of cardiac injury. Cardiomyocyte-specific deletion of Malat1 was sufficient to block regeneration, supporting a critical role of Malat1 in regulating cardiomyocyte proliferation and binucleation, a landmark of mature nonregenerative cardiomyocytes. In vitro, Malat1 deficiency induced binucleation and the expression of a maturation gene program. Finally, the loss of hnRNP U, an interaction partner of Malat1, induced similar features in vitro, suggesting that Malat1 regulates cardiomyocyte proliferation and binucleation by hnRNP U to control the regenerative window in the heart.

Cardiomyocyte sarcomere length variability: Membrane fluorescence versus second harmonic generation myosin imaging.

Sarcomere length (SL) and its variation along the myofibril strongly regulate integrated coordinated myocyte contraction. It is therefore important to obtain individual SL properties. Optical imaging by confocal fluorescence (for example, using ANEPPS) or transmitted light microscopy is often used for this purpose. However, this allows for the visualization of structures related to Z-disks only. In contrast, second-harmonic generation (SHG) microscopy visualizes A-band sarcomeric structures directly. Here, we compared averaged SL and its variability in isolated relaxed rat cardiomyocytes by imaging with ANEPPS and SHG. We found that SL variability, evaluated by several absolute and relative measures, is two times smaller using SHG vs. ANEPPS, while both optical methods give the same average (median) SL. We conclude that optical methods with similar optical spatial resolution provide valid estimations of average SL, but the use of SHG microscopy for visualization of sarcomeric A-bands may be the "gold standard" for evaluation of SL variability due to the absence of optical interference between the sarcomere center and non-sarcomeric structures. This contrasts with sarcomere edges where t-tubules may not consistently colocalize to Z-disks. The use of SHG microscopy instead of fluorescent imaging can be a prospective tool to map sarcomere variability both in vitro and in vivo conditions and to reveal its role in the functional behavior of living myocardium.

How can the adult zebrafish and neonatal mice teach us about stimulating cardiac regeneration in the human heart?

The proliferative capacity of mammalian cardiomyocytes diminishes shortly after birth. In contrast, adult zebrafish and neonatal mice can regenerate cardiac tissues, highlighting new potential therapeutic avenues. Different factors have been found to promote cardiomyocyte proliferation in zebrafish and neonatal mice; these include maintenance of mononuclear and diploid cardiomyocytes and upregulation of the proto-oncogene c-Myc. The growth factor NRG-1 controls cell proliferation and interacts with the Hippo-Yap pathway to modulate regeneration. Key components of the extracellular matrix such as Agrin are also crucial for cardiac regeneration. Novel therapies explored in this review, include intramyocardial injection of Agrin or zebrafish-ECM and NRG-1 administration. These therapies may induce regeneration in patients and should be further explored.

The heart pumps blood across the body carrying nutrients and oxygen where they are needed. If the heart is damaged (e.g., after a heart attack), it may lose its ability to pump blood, and this can lead to heart failure, where the heart cannot meet the body's needs, leaving the affected person tired and breathless. This occurs because the human heart unfortunately has a limited ability to heal and regain function. Current therapies for heart injuries focus on minimizing the problems resulting from the injury but cannot recover damaged heart tissue. Scientists have found that in contrast to adult human hearts, the hearts of baby mice and zebrafish can repair themselves after injuries and recover normal function. This review highlights some important mechanisms that occur in the hearts of baby mice and zebrafish, which may help contribute to their regenerative abilities. These mechanisms involve small messenger chemicals that stimulate heart cells to replicate and reform normal heart tissues. Further research into these pathways may help develop new therapies for damaged human hearts and help them regain function.

Inhibition of late sodium current via PI3K/Akt signaling prevents cellular remodeling in tachypacing-induced HL-1 atrial myocytes.

An aberrant late sodium current (INa,Late) caused by a mutation in the cardiac sodium channel (Nav1.5) has emerged as a contributor to electrical remodeling that causes susceptibility to atrial fibrillation (AF). Although downregulation of phosphoinositide 3-kinase (PI3K)/Akt signaling is associated with AF, the molecular mechanisms underlying the negative regulation of INa,Late in AF remain unclear, and potential therapeutic approaches are needed. In this work, we constructed a tachypacing-induced cellular model of AF by exposing HL-1 myocytes to rapid electrical stimulation (1.5 V/cm, 4 ms, 10 Hz) for 6 h. Then, we gathered data using confocal Ca2+ imaging, immunofluorescence, patch-clamp recordings, and immunoblots. The tachypacing cells displayed irregular Ca2+ release, delayed afterdepolarization, prolonged action potential duration, and reduced PI3K/Akt signaling compared with controls. Those detrimental effects were related to increased INa,Late and were significantly mediated by treatment with the INa,Late blocker ranolazine. Furthermore, decreased PI3K/Akt signaling via PI3K inhibition increased INa,Late and subsequent aberrant myocyte excitability, which were abolished by INa,Late inhibition, suggesting that PI3K/Akt signaling is responsible for regulating pathogenic INa,Late. These results indicate that PI3K/Akt signaling is critical for regulating INa,Late and electrical remodeling, supporting the use of PI3K/Akt-mediated INa,Late as a therapeutic target for AF.

Adrenoceptor sub-type involvement in Ca2+ current stimulation by noradrenaline in human and rabbit atrial myocytes.

Atrial fibrillation (AF) from elevated adrenergic activity may involve increased atrial L-type Ca2+ current (ICaL) by noradrenaline (NA). However, the contribution of the adrenoceptor (AR) sub-types to such ICaL-increase is poorly understood, particularly in human. We therefore investigated effects of various broad-action and sub-type-specific α- and ß-AR antagonists on NA-stimulated atrial ICaL. ICaL was recorded by whole-cell-patch clamp at 37 °C in myocytes isolated enzymatically from atrial tissues from consenting patients undergoing elective cardiac surgery and from rabbits. NA markedly increased human atrial ICaL, maximally by ~ 2.5-fold, with EC75 310 nM. Propranolol (ß1 + ß2-AR antagonist, 0.2 microM) substantially decreased NA (310 nM)-stimulated ICaL, in human and rabbit. Phentolamine (α1 + α2-AR antagonist, 1 microM) also decreased NA-stimulated ICaL. CGP20712A (ß1-AR antagonist, 0.3 microM) and prazosin (α1-AR antagonist, 0.5 microM) each decreased NA-stimulated ICaL in both species. ICI118551 (ß2-AR antagonist, 0.1 microM), in the presence of NA + CGP20712A, had no significant effect on ICaL in human atrial myocytes, but increased it in rabbit. Yohimbine (α2-AR antagonist, 10 microM), with NA + prazosin, had no significant effect on human or rabbit ICaL. Stimulation of atrial ICaL by NA is mediated, based on AR sub-type antagonist responses, mainly by activating ß1- and α1-ARs in both human and rabbit, with a ß2-inhibitory contribution evident in rabbit, and negligible α2 involvement in either species. This improved understanding of AR sub-type contributions to noradrenergic activation of atrial ICaL could help inform future potential optimisation of pharmacological AR-antagonism strategies for inhibiting adrenergic AF.

Transmural myocardial repair with engineered heart muscle in a rat model of heterotopic heart transplantation - A proof-of-concept study.

Engineered heart muscle (EHM) can be implanted epicardially to remuscularize the failing heart. In case of a severely scarred ventricle, excision of scar followed by transmural heart wall replacement may be a more desirable application. Accordingly, we tested the hypothesis that allograft (rat) and xenograft (human) EHM can also be administered as transmural heart wall replacement in a heterotopic, volume-loaded heart transplantation model. We first established a novel rat model model to test surgical transmural left heart wall repair. Subsequently and in continuation of our previous allograft studies, we tested outcome after implantation of contractile engineered heart muscle (EHM) and non-contractile engineered connective tissue (ECT) as well as engineered mesenchymal tissue (EMT) allografts as transmural heart wall replacement. Finally, proof-of-concept for the application of human EHM was obtained in an athymic nude rat model. Only in case of EHM implantation, remuscularization of the surgically created transmural defect was observed with palpable graft vascularization. Taken together, feasibility of transmural heart repair using bioengineered myocardial grafts could be demonstrated in a novel rat model of heterotopic heart transplantation.

Ultrasound-guided Induced Pluripotent Stem Cell-derived Cardiomyocyte Implantation in Myocardial Infarcted Mice.

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Nerve growth factor transfer from cardiomyocytes to innervating sympathetic neurons activates TrkA receptors at the neuro-cardiac junction.

Sympathetic neurons densely innervate the myocardium with non-random topology and establish structured contacts (i.e. neuro-cardiac junctions, NCJ) with cardiomyocytes, allowing synaptic intercellular communication. Establishment of heart innervation is regulated by molecular mediators released by myocardial cells. The mechanisms underlying maintenance of cardiac innervation in the fully developed heart, are, however, less clear. Notably, several cardiac diseases, primarily affecting cardiomyocytes, are associated with sympathetic denervation, supporting the hypothesis that retrograde 'cardiomyocyte-to-sympathetic neuron' communication is essential for heart cellular homeostasis. We aimed to determine whether cardiomyocytes provide nerve growth factor (NGF) to sympathetic neurons, and the role of the NCJ in supporting such retrograde neurotrophic signalling. Immunofluorescence on murine and human heart slices shows that NGF and its receptor, tropomyosin-receptor-kinase-A, accumulate, respectively, in the pre- and post-junctional sides of the NCJ. Confocal immunofluorescence, scanning ion conductance microscopy and molecular analyses, in co-cultures, demonstrate that cardiomyocytes feed NGF to sympathetic neurons, and that this mechanism requires a stable intercellular contact at the NCJ. Consistently, cardiac fibroblasts, devoid of NCJ, are unable to sustain SN viability. ELISA assay and competition binding experiments suggest that this depends on the NCJ being an insulated microenvironment, characterized by high [NGF]. In further support, real-time imaging of tropomyosin-receptor-kinase-A vesicle movements demonstrate that efficiency of neurotrophic signalling parallels the maturation of such structured intercellular contacts. Altogether, our results demonstrate the mechanisms which link sympathetic neuron survival to neurotrophin release by directly innervated cardiomyocytes, conceptualizing sympathetic neurons as cardiomyocyte-driven heart drivers. KEY POINTS: CMs are the cell source of nerve growth factor (NGF), required to sustain innervating cardiac SNs; NCJ is the place of the intimate liaison, between SNs and CMs, allowing on the one hand neurons to peremptorily control CM activity, and on the other, CMs to adequately sustain the contacting, ever-changing, neuronal actuators; alterations in NCJ integrity may compromise the efficiency of 'CM-to-SN' signalling, thus representing a potentially novel mechanism of sympathetic denervation in cardiac diseases.

[Progress in the Role of Mechanical Stimulus in Cardiac Development].

Mechanical stimulus is critical to cardiovascular development during embryogenesis period.The mechanoreceptors of endocardial cells and cardiac myocytes may sense mechanical signals and initiate signal transduction that induce gene expression at a cellular level,and then translate molecular-level events into tissue-level deformations,thus guiding embryo development.This review summarizes the regulatory roles of mechanical signals in the early cardiac development including the formation of heart tube,looping,valve and septal morphogenesis,ventricular development and maturation.Further,we discuss the potential mechanical transduction mechanisms of platelet endothelial cell adhesion molecule 1-vascular endothelial-cadherin-vascular endothelial growth factor receptor 2 complex,primary cilia,ion channels,and other mechanical sensors that affect some cardiac malformations.

Engineered bacterial voltage-gated sodium channel platform for cardiac gene therapy.

Therapies for cardiac arrhythmias could greatly benefit from approaches to enhance electrical excitability and action potential conduction in the heart by stably overexpressing mammalian voltage-gated sodium channels. However, the large size of these channels precludes their incorporation into therapeutic viral vectors. Here, we report a platform utilizing small-size, codon-optimized engineered prokaryotic sodium channels (BacNav) driven by muscle-specific promoters that significantly enhance excitability and conduction in rat and human cardiomyocytes in vitro and adult cardiac tissues from multiple species in silico. We also show that the expression of BacNav significantly reduces occurrence of conduction block and reentrant arrhythmias in fibrotic cardiac cultures. Moreover, functional BacNav channels are stably expressed in healthy mouse hearts six weeks following intravenous injection of self-complementary adeno-associated virus (scAAV) without causing any adverse effects on cardiac electrophysiology. The large diversity of prokaryotic sodium channels and experimental-computational platform reported in this study should facilitate the development and evaluation of BacNav-based gene therapies for cardiac conduction disorders.

Analysis of Mitochondrial Function, Structure, and Intracellular Organization In Situ in Cardiomyocytes and Skeletal Muscles.

Analysis of the function, structure, and intracellular organization of mitochondria is important for elucidating energy metabolism and intracellular energy transfer. In addition, basic and clinically oriented studies that investigate organ/tissue/cell dysfunction in various human diseases, including myopathies, cardiac/brain ischemia-reperfusion injuries, neurodegenerative diseases, cancer, and aging, require precise estimation of mitochondrial function. It should be noted that the main metabolic and functional characteristics of mitochondria obtained in situ (in permeabilized cells and tissue samples) and in vitro (in isolated organelles) are quite different, thereby compromising interpretations of experimental and clinical data. These differences are explained by the existence of the mitochondrial network, which possesses multiple interactions between the cytoplasm and other subcellular organelles. Metabolic and functional crosstalk between mitochondria and extra-mitochondrial cellular environments plays a crucial role in the regulation of mitochondrial metabolism and physiology. Therefore, it is important to analyze mitochondria in vivo or in situ without their isolation from the natural cellular environment. This review summarizes previous studies and discusses existing approaches and methods for the analysis of mitochondrial function, structure, and intracellular organization in situ.

Cardioprotective effects of alantolactone on isoproterenol-induced cardiac injury and cobalt chloride-induced cardiomyocyte injury.

OBJECTIVES: Alantolactone (AL) is a compound extracted from the roots of Inula Racemosa that has shown beneficial effects in cardiovascular disease. However, the cardioprotective mechanism of AL against hypoxic/ischemic (H/I) injury is still unclear. This research aimed to determine AL's ability to protect the heart against isoproterenol (ISO)-induced MI injury in vivo and cobalt chloride (CoCl2) induced H/I injury in vitro. METHODS: Electrocardiography (ECG), lactate dehydrogenase (LDH), creatine kinase (CK), and cardiac troponin I (cTnI) assays in addition to histological analysis of the myocardium were used to investigate the effects of AL in vivo. Influences of AL on L-type Ca2+ current (ICa-L) in isolated rat myocytes were observed by the patch-clamp technique. Furthermore, cell viability, apoptosis, oxidative stress injury, mitochondrial membrane potential, and intracellular Ca2+ concentration were examined in vitro. RESULTS: The results indicated that AL treatment ameliorated the morphological and ECG changes associated with MI, and decreased levels of LDH, CK, and cTnI. Furthermore, pretreatment with AL elevated antioxidant enzyme activity and suppressed ROS production. AL prevented H/I-induced apoptosis, mitochondria damage, and calcium overload while reducing ICa-L in a concentration and time dependent fashion. The 50% inhibiting concentration (IC50) and maximal inhibitory effect (Emax) of AL were 17.29 µmol/L and 57.73 ± 1.05%, respectively. CONCLUSION: AL attenuated MI-related injury by reducing oxidative stress, apoptosis, calcium overload, and mitochondria damage. These cardioprotective effects may be related to the direct inhibition of ICa-L.

Comprehensive analyses of the inotropic compound omecamtiv mecarbil in rat and human cardiac preparations.

Omecamtiv mecarbil (OM), a myosin activator, was reported to induce complex concentration- and species-dependent effects on contractile function, and clinical studies indicated a low therapeutic index with diastolic dysfunction at concentrations above 1 µM. To further characterize effects of OM in a human context and under different preload conditions, we constructed a setup that allows isometric contractility analysis of human induced pluripotent stem cell (hiPSC)-derived engineered heart tissues (EHTs). The results were compared with effects of OM on the very same EHTs measured under auxotonic conditions. OM induced a sustained, concentration-dependent increase in time to peak under all conditions (maximally two- to threefold). Peak force, in contrast, was increased by OM only in human, but not rat EHTs and only under isometric conditions, varied between hiPSC lines and showed a biphasic concentration dependency with maximal effects at 1 µM. Relaxation time tended to fall under auxotonic and strongly increased under isometric conditions, again with biphasic concentration dependency. Diastolic tension concentration dependently increased under all conditions. The latter was reduced by an inhibitor of the mitochondrial sodium calcium exchanger (CGP-37157). OM induced increases in mitochondrial oxidation in isolated cardiomyocytes, indicating that OM, an inotrope that does not increase intracellular and mitochondrial Ca2+, can induce mismatch between an increase in ATP and ROS production and unstimulated mitochondrial redox capacity. Taken together, we developed a novel setup well suitable for isometric measurements of EHTs. The effects of OM on contractility and diastolic tension are complex with concentration-, time-, species- and loading-dependent differences. Effects on mitochondrial function require further studies.NEW & NOTEWORTHY We developed a novel setup allowing precise control of preload of EHT and characterized effects of the myosin activator OM. OM not only exerted contraction-slowing and positive inotropic effects but also increased diastolic tension. Effect size and direction varied between species, auxotonic and isometric conditions, concentration, and time. We also observed OM-induced increase of mitochondrial ROS, which has not been observed before and may explain part of the effects on contractility.

Chronic spinal cord injury functionally repaired by direct implantation of encapsulated hair-follicle-associated pluripotent (HAP) stem cells in a mouse model: Potential for clinical regenerative medicine.

Chronic spinal cord injury (SCI) is a highly debilitating and recalcitrant disease with limited treatment options. Although various stem cell types have shown some clinical efficacy for injury repair they have not for SCI. Hair-follicle-associated pluripotent (HAP) stem cells have been shown to differentiate into neurons, Schwan cells, beating cardiomyocytes and many other type of cells, and have effectively regenerated acute spinal cord injury in mouse models. In the present report, HAP stem cells from C57BL/6J mice, encapsulated in polyvinylidene fluoride membranes (PFM), were implanted into the severed thoracic spinal cord of C57BL/6J or athymic nude mice in the early chronic phase. After implantation, HAP stem cells differentiated to neurons, astrocytes and oligodendrocytes in the regenerated thoracic spinal cord of C57BL/6J and nude mice. Quantitative motor function analysis, with the Basso Mouse Scale for Locomotion (BMS) score, demonstrated a significant functional improvement in the HAP-stem-cell-implanted mice, compared to non-implanted mice. HAP stem cells have critical advantages over other stem cells: they do not develop teratomas; do not loose differentiation ability when cryopreserved and thus are bankable; are autologous, readily obtained from anyone; and do not require genetic manipulation. HAP stem cells therefore have greater clinical potential for SCI repair than induced pluripotent stem cells (iPSCs), neuronal stem cells (NSCs)/neural progenitor cells (NPCs) or embryonic stem cells (ESCs). The present report demonstrates future clinical potential of HAP-stem-cell repair of chronic spinal cord injury, currently a recalcitrant disease.