Excitation-contraction Coupling Research Articles

Fine tuning contractility: atrial sarcomere function in health and disease.

The molecular mechanisms of sarcomere proteins underlie the contractile function of the heart. Although our understanding of the sarcomere has grown tremendously, the focus has been on ventricular sarcomere isoforms due to the critical role of the ventricle in health and disease. However, atrial-specific or -enriched myofilament protein isoforms, as well as isoforms that become expressed in disease, provide insight into ways this complex molecular machine is fine-tuned. Here, we explore how atrial-enriched sarcomere protein composition modulates contractile function to fulfill the physiological requirements of atrial function. We review how atrial dysfunction negatively affects the ventricle and the many cardiovascular diseases that have atrial dysfunction as a comorbidity. We also cover the pathophysiology of mutations in atrial-enriched contractile proteins and how they can cause primary atrial myopathies. Finally, we explore what is known about contractile function in various forms of atrial fibrillation. The differences in atrial function in health and disease underscore the importance of better studying atrial contractility, especially as therapeutics currently in development to modulate cardiac contractility may have different effects on atrial sarcomere function.

Differential Post-Translational Modifications of Proteins in Bladder Ischemia.

Clinical and basic research suggests that bladder ischemia may be an independent variable in the development of lower urinary tract symptoms (LUTS). We have reported that ischemic changes in the bladder involve differential expression and post-translational modifications (PTMs) of the protein's functional domains. In the present study, we performed in-depth analysis of a previously reported proteomic dataset to further characterize proteins PTMs in bladder ischemia. Our proteomic analysis of proteins in bladder ischemia detected differential formation of non-coded amino acids (ncAAs) that might have resulted from PTMs. In-depth analysis revealed that three groups of proteins in the bladder proteome, including contractile proteins and their associated proteins, stress response proteins, and cell signaling-related proteins, are conspicuously impacted by ischemia. Differential PTMs of proteins by ischemia seemed to affect important signaling pathways in the bladder and provoke critical changes in the post-translational structural integrity of the stress response, contractile, and cell signaling-related proteins. Our data suggest that differential PTMs of proteins may play a role in the development of cellular stress, sensitization of smooth muscle cells to contractile stimuli, and deferential cell signaling in bladder ischemia. These observations may provide the foundation for future research to validate and define clinical translation of the modified biomarkers for precise diagnosis of bladder dysfunction and the development of new therapeutic targets against LUTS.

A Silicon-Based Field-Effect Biosensor for Drug-Induced Cardiac Extracellular Calcium Ion Change Detection.

Calcium ions participate in the regulation of almost all biological functions of the body, especially in cardiac excitation-contraction coupling, acting as vital signaling through ion channels. Various cardiovascular drugs exert their effects via affecting the ion channels on the cell membrane. The current strategies for calcium ion monitoring are mainly based on fluorescent probes, which are commonly used for intracellular calcium ion detection (calcium imaging) and cannot achieve long-term monitoring. In this work, an all-solid-state silicone-rubber ion-sensitive membrane was fabricated on light-addressable potentiometric sensors to establish a program-controlled field-effect-based ion-sensitive light-addressable potentiometric sensor (LAPS) platform for extracellular calcium ion detection. L-type calcium channels blocker verapamil and calcium channel agonist BayK8644 were chosen to explore the effect of ion channel drugs on extracellular calcium ion concentration in HL-1 cell lines. Simultaneously, microelectrode array (MEA) chips were employed to probe the HL-1 extracellular field potential (EFP) signals. The Ca2+ concentration and EFP parameters were studied to comprehensively evaluate the efficacy of cardiovascular drugs. This platform provides more dimensional information on cardiovascular drug efficacy that can be utilized for accurate drug screening.

Cadence Modulation during Eccentric Cycling Affects Perception of Effort But Not Neuromuscular Alterations.

A recent study showed that cadence modulation during short eccentric cycling exercise affects oxygen consumption (V̇O 2 ), muscular activity (EMG), and perception of effort (PE). This study examined the effect of cadence on V̇O 2 , EMG, and PE during prolonged eccentric cycling and exercise-induced neuromuscular alterations. Twenty-two participants completed three sessions 2-3 wk apart: 1) determination of the maximal concentric peak power output, familiarization with eccentric cycling at two cadences (30 and 60 rpm at 60% peak power output), and neuromuscular testing procedure; 2) and 3) 30 min of eccentric cycling exercise at a cadence of 30 or 60 rpm. PE, cardiorespiratory parameters, and vastus lateralis and rectus femoris EMG were collected during exercise. The knee extensors' maximal voluntary contraction torque, the torque evoked by double stimulations at 100 Hz (Dt100) and 10 Hz (Dt10), and the voluntary activation level were evaluated before and after exercise. V̇O 2 , EMG, and PE were greater at 30 than 60 rpm (all P < 0.05). Maximal voluntary contraction torque, evoked torque, and Dt10/Dt100 ratio decreased (all P < 0.01) without cadence effect (all P > 0.28). Voluntary activation level remained constant after both eccentric cycling exercises ( P = 0.87). When performed at the same power output, eccentric cycling exercise at 30 rpm elicited a greater PE, EMG, and cardiorespiratory demands than pedaling at 60 rpm. Exercise-induced fatigability was similar in both eccentric cycling conditions without neural impairments, suggesting that eccentric cycling seemed to alter more specifically muscular function, such as the excitation-contraction coupling process. In a rehabilitation context, eccentric cycling at 60 rpm seems more appropriate because it will induce lower PE for similar strength loss compared with 30 rpm.

MicroRNA-1 Deficiency Is a Primary Etiological Factor Disrupting Cardiac Contractility and Electrophysiological Homeostasis.

MicroRNA-1 (miR1), encoded by the genes miR1-1 and miR1-2, is the most abundant microRNA in the heart and plays a critical role in heart development and physiology. Dysregulation of miR1 has been associated with various heart diseases, where a significant reduction (>75%) in miR1 expression has been observed in patient hearts with atrial fibrillation or acute myocardial infarction. However, it remains uncertain whether miR1-deficiency acts as a primary etiological factor of cardiac remodeling. miR1-1 or miR1-2 knockout mice were crossbred to produce 75%-miR1-knockdown (75%KD; miR1-1+/-:miR1-2-/- or miR1-1-/-:miR1-2+/-) mice. Cardiac pathology of 75%KD cardiomyocytes/hearts was investigated by ECG, patch clamping, optical mapping, transcriptomic, and proteomic assays. In adult 75%KD hearts, the overall miR1 expression was reduced to ≈25% of the normal wild-type level. These adult 75%KD hearts displayed decreased ejection fraction and fractional shortening, prolonged QRS and QT intervals, and high susceptibility to arrhythmias. Adult 75%KD cardiomyocytes exhibited prolonged action potentials with impaired repolarization and excitation-contraction coupling. Comparatively, 75%KD cardiomyocytes showcased reduced Na+ current and transient outward potassium current, coupled with elevated L-type Ca2+ current, as opposed to wild-type cells. RNA sequencing and proteomics assays indicated negative regulation of cardiac muscle contraction and ion channel activities, along with a positive enrichment of smooth muscle contraction genes in 75%KD cardiomyocytes/hearts. miR1 deficiency led to dysregulation of a wide gene network, with miR1's RNA interference-direct targets influencing many indirectly regulated genes. Furthermore, after 6 weeks of bi-weekly intravenous tail-vein injection of miR1 mimics, the ejection fraction and fractional shortening of 75%KD hearts showed significant improvement but remained susceptible to arrhythmias. miR1 deficiency acts as a primary etiological factor in inducing cardiac remodeling via disrupting heart regulatory homeostasis. Achieving stable and appropriate microRNA expression levels in the heart is critical for effective microRNA-based therapy in cardiovascular diseases.

Activation of the cannabinoid type 2 (CB2) receptor improves cardiac contractile performance in fish, Brycon amazonicus

In addition to their well-known classical effects, cannabinoid CB1 and CB2 receptors have also been involvement in both deleterious and protective actions on the heart under various pathological conditions. While the potential therapeutic applications of the endocannabinoid system in the context of cardiovascular function are indeed a viable prospect, significant debate exists within the literature regarding whether CB1, CB2, or a combination of both receptors exert a favorable influence on cardiac function. Hence, the aim of this study was to investigate the effects of CB1 + CB2 or CB2 agonists on cardiac excitation-contraction (E-C) coupling, utilizing fish (Brycon amazonicus) as an experimental model. The CB2 agonist elicited marked positive inotropic and lusitropic responses in isolated ventricular myocardium, induced cyclic adenosine 3′,5′-monophosphate (cAMP) production, and upregulated critical Ca2+ handling proteins, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). Our current study demonstrated, for the first time, that CB2 receptor activation-induced effects improved the efficiency of Ca2+ cycling, excitation-contraction coupling (E-C coupling), and cardiac performance in under physiological conditions. Hence, CB2 receptors could be considered a potential therapeutic target for modulating cardiac contractile dysfunctions.

Two-pore channels (TPCs) acts as a hub for excitation-contraction coupling, metabolism and cardiac hypertrophy signalling

Ca2+ signaling is essential for cardiac contractility and excitability in heart function and remodeling. Intriguingly, little is known about the role of a new family of ion channels, the endo-lysosomal non-selective cation “two-pore channel” (TPCs) in heart function. Here we have used double TPC knock-out mice for the 1 and 2 isoforms of TPCs (Tpcn1/2−/−) and evaluated their cardiac function. Doppler-echocardiography unveils altered left ventricular (LV) systolic function associated with a LV relaxation impairment. In cardiomyocytes isolated from Tpcn1/2−/- mice, we observed a reduction in the contractile function with a decrease in the sarcoplasmic reticulum Ca2+ content and a reduced expression of various key proteins regulating Ca2+ stores, such as calsequestrin. We also found that two main regulators of the energy metabolism, AMP-activated protein kinase and mTOR, were down regulated. We found an increase in the expression of TPC1 and TPC2 in a model of transverse aortic constriction (TAC) mice and in chronically isoproterenol infused WT mice. In this last model, adaptive cardiac hypertrophy was reduced by Tpcn1/2 deletion. Here, we propose a central role for TPCs and lysosomes that could act as a hub integrating information from the excitation-contraction coupling mechanisms, cellular energy metabolism and hypertrophy signaling.

Macrophage-mediated downregulation of lncRNA Carmn in mouse abdominal aortic aneurysm

The long noncoding RNA (lncRNA) CARMN (cardiac mesoderm enhancer associated noncoding RNA) is a highly conserved lncRNA that expresses primarily by smooth muscle cells (SMCs). Recent literature demonstrates that CARMN plays a critical role in the differentiation and maintaining of the contractile state of vascular SMCs. Because aortic SMCs show diminished contractile proteins in abdominal aortic aneurysms (AAAs), we hypothesize that the expression of CARMN is downregulated in the aortic wall affected by aneurysm. In this study, we analyzed publicly available single-cell or bulk RNA sequencing data comparing healthy and aneurysmal mouse aortic tissues. In both healthy and diseased aortas, Carmn expression was enriched in SMCs characterized by the high expression of SMC-specific contractile proteins including Myh11 and Acta2. Carmn expression levels varied among the sub-clusters of SMCs and consequently along the aortic tree. Comparing to the corresponding sham aorta, aortas from 3 distinct AAA models contained less Carmn. To validate the Carmn downregulation, we induced AAA using the Angiotensin II and CaCl2 models. In situ hybridization showed that Carmn mRNA located in the nuclei of SMCs and became downregulated within a few days following the aneurysm induction. Mechanistically, we tested whether Carmn expression is regulated by infiltrating macrophages --- the predominant inflammatory cells found in aneurysmal tissues --- by treating healthy mouse aortic SMCs with media conditioned by macrophages primed with pro-inflammatory or anti-inflammatory cytokines. PCR analysis showed that inflammatory macrophages reduced the expression of Carmn and contractile genes including Myh11 and Acta2. Taken together, our results from bioinformatic and experimental analyses demonstrate that Carmn is downregulated in different AAA models, likely by inflammatory macrophages. The negative regulation of Carmn in AAA tissues may explain at least in part the loss of SMC contractile state during the pathogenesis of this progressive degenerative disease.

S-adenosylmethionine attenuates angiotensin II-induced aortic dissection formation by inhibiting vascular smooth muscle cell phenotypic switch and autophagy

It is well known that aortic dissection (AD) is a very aggressive class of vascular diseases. S-adenosylmethionine (SAM) is an autophagy inhibitor with anti-inflammatory and anti-oxidative stress effects; however, the role of SAM in AD is unknown. In this study, we constructed an animal model of AD using subcutaneous minipump continuous infusion of AngII-induced ApoE-/-mice and a cytopathic model using AngII-induced primary vascular smooth muscle cells (VSMCs) to investigate the possible role of SAM in AD. The results showed that mice in the AngII + SAM group had significantly lower AD incidence, significantly prolonged survival, and reduced vascular elastic fiber disruption compared with mice in the AngII group. In addition, SAM significantly inhibited autophagy in vivo and in vitro. Meanwhile, SAM also inhibited the cellular phenotypic switch, mainly by up regulating the expression levels of contractile marker proteins [α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α)] and down regulating the expression levels of synthetic marker proteins [osteoblast protein (OPN), matrix metalloproteinase-2 (MMP2), and matrix metalloproteinase-9 (MMP9)]. Molecularly, SAM inhibited AD formation mainly by activating the PI3K/AKT/mTOR signaling pathway. Using a PI3K inhibitor (LY294002) significantly reversed the protective effect of SAM in AngII-induced mice and VSMCs.Our study demonstrates the protective effect of SAM on mice under AngII-induced AD for the first time. SAM prevented AD formation mainly by inhibiting cellular phenotypic switch and autophagy, and activation of the PI3K/AKT/mTOR signaling pathway is a possible molecular mechanism. Thus, SAM may be a novel strategy for the treatment of AD.

A comparative study of fully implicit staggered and monolithic solution methods. Part II: Coupled excitation–contraction equations of cardiac electromechanics

A comparative study of fully implicit staggered and monolithic solution algorithms for the coupled bidomain equations of cardiac electrophysiology was discussed in Part I (JCAM 407:114021, 2022). In this follow-up study, we further employ the similar approach and present an extensive comparative analysis between the staggered and monolithic solution techniques for the coupled partial differential equations (PDEs) of cardiac electromechanics both in the monodomain and bidomain setting. For the monolithic solution technique, we adopt the work of Göktepe et al. (CM 45: 227-243, 2010) and Dal et al. (CMAME 253: 353-336, 2013), where the governing PDEs of the excitation–contraction problem are solved simultaneously and all rate equations are treated with an implicit time integration scheme. For the staggered scheme, however, we simply suggest a decoupled solution of the governing PDEs while keeping other aspects of the monolithic algorithm the same, such as utilization of the implicit time integration scheme and coupling of ordinary differential equations, which describe the state variables of cardiac electrophysiology, to the parabolic PDE of the electrophysiology. Both solution algorithms are applied to several problems, where we simulate regular planar wave propagations and scroll waves for different spatial and temporal resolution and material parameters. The comparison between the solution schemes is performed in terms of accuracy, efficiency and stability. We reveal that for regular wave propagation the results of different solution algorithms are identical. The only deviation is observed during scroll wave propagation, if the parameters controlling the mutual interaction between the mechanical and electrical field are relatively large and at the same time if the time increment is big, e.g., Δt=2.0ms. Nevertheless, the suggested staggered solution scheme yields almost identical results even for high coupling if smaller time increments are used. Besides, the staggered scheme provides an enormous gain in computational efficiency, particularly, as the number of degrees of freedom is increased and we do not encounter any stability issue arising specifically from the decoupled solution of the governing PDEs. Similar to the conclusion that was made for cardiac electrophysiology in Part I, we report that the suggested fully implicit staggered solution algorithm has a tremendous application potential for computer simulations of excitation–contraction coupling of the heart both in the monodomain and bidomain setting.

Computational Modeling of Cardiac Electrophysiology.

Mathematical modeling and simulation are well-established and powerful tools to integrate experimental data of individual components of cardiac electrophysiology, excitation-contraction coupling, and regulatory signaling pathways, to gain quantitativeandmechanistic insight into pathophysiological processes and guide therapeutic strategies. Here, we briefly describe the processes governing cardiac myocyte electrophysiology and Ca2+ handling and their regulation, as well as action potential propagation in tissue. We discuss the models and methods used to describe these phenomena, including procedures for model parameterization and validation, in addition to protocols for model interrogation and analysis and techniques that account for phenotypic variability and parameter uncertainty. Our objective is to provide a summary of basic concepts and approaches as a resource for scientists training in this discipline and for all researchers aiming to gain an understanding of cardiac modeling studies.

Sex Differences in Mouse Cardiac Electrophysiology Revealed by Simultaneous Imaging of Excitation-Contraction Coupling.

Males and females differ in the basic anatomy and physiology of the heart. Sex differences are evident in cardiac repolarization in humans; women have longer corrected QT and JT intervals. However, the molecular mechanisms that lead to these differences are incompletely understood. Here, we present that, like in humans, sex differences in QT and JT intervals exist in mouse models; female mice had longer corrected QT and JT intervals compared with age-matched males. To further understand the molecular underpinning of these sex differences, we developed a novel technology using fluorescent confocal microscopy that allows the simultaneous visualization of action potential, Ca2+ transients, and contractions in isolated cardiomyocytes at a high temporal resolution. From this approach, we uncovered that females at baseline have increased action potential duration, decreased Ca2+ release and reuptake rates, and decreased contraction and relaxation velocities compared with males. Additionally, males had a shorter overall time from action potential onset to peak contraction. In aggregate, our studies uncovered male and female differences in excitation-contraction coupling that account for differences observed in the EKG. Overall, a better understanding of sex differences in electrophysiology is essential for equitably treating cardiac disease.

Chrysanthemum coronarium L. Extract Attenuates Homocysteine-Induced Vascular Inflammation in Vascular Smooth Muscle Cells.

Hyperhomocysteinemia is a main risk factor for phenotypic modulation of vascular smooth muscle cells (VSMCs) and atherosclerosis. Phenotypic switching and proliferation of VSMCs are related to the progression of vascular inflammation. Chrysanthemum coronarium L. is a leafy vegetable with various biological functions, such as antioxidative, anti-inflammatory, and antiproliferative effects. In this study, we aimed to identify the mechanisms underlying the therapeutic and preventive effects of C. coronarium L. extract (CC) in regulating homocysteine (Hcy)-induced vascular inflammation in human aortic VSMCs. CC did not exhibit cytotoxicity and inhibited Hcy-stimulated VSMC proliferation and migration. In addition, CC promoted Hcy-induced expression of VSMC contractile phenotype proteins, including alpha-smooth muscle actin, calponin, and smooth muscle 22α. CC also decreased Hcy-induced accumulation of reactive oxygen species and expression of inflammatory markers nicotinamide adenine dinucleotide phosphate oxidase-4 and soluble epoxide hydrolase. These results showed that CC attenuates Hcy-induced inflammatory responses, highlighting its potential as a therapeutic or preventive target for Hcy-induced vascular inflammation.

Interstitial cells of Cajal - pacemakers of the gastrointestinal tract.

Gastrointestinal (GI) organs display spontaneous, non-neurogenic electrical, and mechanical rhythmicity that underlies fundamental motility patterns, such as peristalsis and segmentation. Electrical rhythmicity (aka slow waves) results from pacemaker activity generated by interstitial cells of Cajal (ICC). ICC express a unique set of ionic conductances and Ca2+ handling mechanisms that generate and actively propagate slow waves. GI smooth muscle cells lack these conductances. Slow waves propagate actively within ICC networks and conduct electrotonically to smooth muscle cells via gap junctions. Slow waves depolarize smooth muscle cells and activate voltage-dependent Ca2+ channels (predominantly CaV1.2), causing Ca2+ influx and excitation-contraction coupling. The main conductances responsible for pacemaker activity in ICC are ANO1, a Ca2+ -activated Cl- conductance, and CaV3.2. The pacemaker cycle, as currently understood, begins with spontaneous, localized Ca2+ release events in ICC that activate spontaneous transient inward currents due to activation of ANO1 channels. Depolarization activates CaV 3.2 channels, causing the upstroke depolarization phase of slow waves. The upstroke is transient and followed by a long-duration plateau phase that can last for several seconds. The plateau phase results from Ca2+ -induced Ca2+ release and a temporal cluster of localized Ca2+ transients in ICC that sustains activation of ANO1 channels and clamps membrane potential near the equilibrium potential for Cl- ions. The plateau phase ends, and repolarization occurs, when Ca2+ stores are depleted, Ca2+ release ceases and ANO1 channels deactivate. This review summarizes key mechanisms responsible for electrical rhythmicity in gastrointestinal organs.

Dual mode of action of IP3-dependent SR-Ca2+ release on local and global SR-Ca2+ release in ventricular cardiomyocytes

In heart muscle, the physiological function of IP3-induced Ca2+ release (IP3ICR) from the sarcoplasmic reticulum (SR) is still the subject of intense study. A role of IP3ICR may reside in modulating Ca2+-dependent cardiac arrhythmogenicity. Here we observe the propensity of spontaneous intracellular Ca2+ waves (SCaW) driven by Ca2+-induced Ca2+ release (CICR) in ventricular myocytes as a correlate of arrhythmogenicity on the organ level. We observe a dual mode of action of IP3ICR on SCaW generation in an IP3R overexpression model. This model shows a mild cardiac phenotype and mimics pathophysiological conditions of increased IP3R activity. In this model, IP3ICR was able to increase or decrease the occurrence of SCaW depending on global Ca2+ activity. This IP3ICR-based regulatory mechanism can operate in two “modes” depending on the intracellular CICR activity and efficiency (e.g. SCaW and/or local Ryanodine Receptor (RyR) Ca2+ release events, respectively): a) in a mode that augments the CICR mechanism at the cellular level, resulting in improved excitation-contraction coupling (ECC) and ultimately better contraction of the myocardium, and b) in a protective mode in which the CICR activity is curtailed to prevent the occurrence of Ca2+ waves at the cellular level and thus reduce the probability of arrhythmogenicity at the organ level.

Biophysical reviews top five: voltage-dependent charge movement in nerve and muscle.

The discovery of gating currents and asymmetric charge movement in the early 1970s represented a remarkable leap forward in our understanding of the biophysical basis of voltage-dependent events that underlie electrical signalling that is vital for nerve and muscle function. Gating currents and charge movement reflect a fundamental process in which charged amino acid residues in an ion channel protein move in response to a change in the membrane electrical field and therefore activate the specific voltage-dependent response of that protein. The detection of gating currents and asymmetric charge movement over the past 50 years has been pivotal in unraveling the multiple molecular and intra-molecular processes which lead to action potentials in excitable tissues and excitation-contraction (EC) coupling in skeletal muscle. The recording of gating currents and asymmetric charge movement remains an essential component of investigations into the basic molecular mechanisms of neuronal conduction and muscle contraction.

Exosomal preconditioning of human iPSC-derived cardiomyocytes beneficially alter cardiac electrophysiology during hypoxia

Abstract Introduction and purpose Cardiac-Exosomes (EXO) are nano-sized extracellular vesicles, continuously secreted by the myocardium. Despite previous misconceptions, cardiac-EXO are not just a way for the cell to dispatch waste, but functions as carriers of signaling molecules. Furthermore, the effects of cardiac-EXO signaling during hypoxic stress is not well understood. The present study aimed to determine the biological effects of EXO released from hypoxic cardiomyocytes, focusing on whether the EXO-cargo can benefit recipient cells during hypoxic stress. We hypothesize that cardiomyocytes exposed to hypoxic stress would display advantageous electrophysiological properties after preconditional treatment with isolated cardiac-EXO secreted from cardiomyocytes exposed to hypoxic stress. Methods For preconditional treatment we isolated EXO from human iPSC-derived cardiomyocytes (CM) exposed to hypoxia (1% O2) for 16 h (H-EXO). H-EXO was thereafter administrated as preconditional treatment to a secondary population of CM 24 h before subjection to 16 h of hypoxia. Electrophysiological effects of H-EXO precondition treatment were assessed with a multiwell microelectrode array (MEA) system. Using the MEA-system; field potential, beat period, spike slope, spike amplitude, excitation-contraction (EC) coupling, and cardiac action potentials were recorded and analyzed. Results We found that CM exposed to hypoxic stress conditions (16 hours) displayed significant increase in beat period when preconditioned with H-EXO compared to non-treated CM (+15.7%, p&amp;lt;0.05). Furthermore, precondition with H-EXO resulted in faster EC coupling compared with non-treated CM after 16 h of hypoxia (-25.3%, p&amp;lt;0.05). More efficient EC coupling indicates that the H-EXO recipient cells are less affected by the hypoxic stress. There were no significant changes in field potential, spike slope, spike amplitude and cardiac action potential between the two groups after 16 h of hypoxia. Conclusion The present study report that EXO secreted by CM under hypoxia alter electrophysiological properties in recipient cells exposed to 16 h hypoxic stress. The alterations indicate more efficient cardiomyocyte EC coupling and longer beat period.

The effect of ventricular microvascular dysfunction on cardiomyocyte excitation-contraction coupling and mitochondria

Abstract Introduction Microvascular dysfunction is a hallmark feature of cardiac allograft vasculopathy in HTx patients and associated with heart failure and coronary artery disease. On a cellular level, impaired mitochondrial respiration has been associated with the disease, however the exact mechanisms remain elusive. Objective The aim of this study was to determine the effect of microvascular dysfunction on functional and structural properties of cardiomyocytes isolated from ventricular biopsies of HTx patients. Methods We functionally assessed coronary artery microvasculature using guidewire based coronary flow reserve(CFR)/microvasculatory resistance (IMR) measurements (Abbott) in 13 post-HTx patients with angiographically normal coronary arteries or normal FFR. Ventricular (RV septum) myocardial biopsies were obtained for in-vitro measurements and single ventricular cardiomyocytes were isolated using enzymatic digestion. Cells were electrically stimulated (1 Hz) and subcellular Ca2+ signalling during excitation-contraction coupling was measured using confocal imaging (Fluo-4 AM). In a subset of cardiomyocytes mitochondrial density was quantified upon staining with Mitotracker Red FM (otsu thresholding). Results Microvascular dysfunction (MVD) measured by IMR was present in 6 of 13 patients with a mean IMR of 56±11 and 13±3 in MVD and CTRL, respectively. CFR did not differ between MVD and CTRL. Ca2+ transients during excitation-contraction coupling in isolated ventricular cardiomyocytes from a subset of patients showed unaltered amplitudes (1.2±0.01 vs. 1.3±0.05; a.u. F/F0). In addition, Ca2+ release (i.e. time to peak Ca2+) and Ca2+ removal (i.e. tau) were not significantly different between MVD and CTRL. However, mitochondrial density was significantly increased in MVD vs. CTRL, indicating subcellular changes associated with MVD. Conclusion In-vivo ventricular microvascular dysfunction is associated with preserved excitation-contraction coupling in-vitro, potentially owing to compensatory changes on the mitochondrial level.

Mitochondrial and contractile dysfunction in a swine model of early chronic kidney disease

Abstract Background Numerous clinical trials have demonstrated that patients with kidney damage have an increased chance of developing heart failure, and the risk is compounded in people with diabetes. Cardiac remodeling occurs at very early stages of kidney injury but studies undertaken to pinpoint the common biomarkers of cardiorenal signaling are still inadequate, which may be due to the fact that research done on rodent models does not fully reflect the complexity of the disease. We and our collaborators established swine models for diabetes (DM- insulin gene INS mutation) and chronic kidney disease (CKD- renal microembolisation) to overcome the translational gap associated with rodent models. Purpose In order to provide mechanistic as well as therapeutic insights, we used proteomic analyses to elucidate the pathophysiological effects of DM and CKD on the heart. Methods We used microspheres to embolise both kidneys of wild type and diabetic swine at 10-12 weeks of age and 6 months later we performed quantitative LC-MS-MS based proteomic analyses of left ventricular tissue from these four groups (wild type (WT), wild type with CKD (WT_CKD), diabetic (DM) and diabetic with CKD (DM_CKD). STRING bioinformatics software was used to identify significantly enriched pathways. Results Masson Trichrome staining confirmed the presence of kidney damage and fibrosis in CKD animals (Fig1 A). Quantitative proteomic analysis detected significant differences in the left ventricle of the WT or DM swine when compared to those with CKD using a volcano plot (Fig1B,C). STRING pre-ranked functional analysis showed pathway enrichments in both the comparisons (Fig2A,B). We found that proteins involved in the remodeling of the extracellular matrix (ECM), such as C3, F9, Col6A2, and FBN1, were downregulated in both groups (WT CKD and DM CKD as compared to WT and DM respectively). Interestingly, in WT_CKD we found that mitochondrial proteins like cytochrome oxidase (MT-CO2) and ATP5 synthase subunit were downregulated suggesting mitochondrial dysfunction. Strong indicators of this dysfunction include pathways with altered regulation of reactive oxygen species like hydrogen peroxide and substantial downregulation of contractile proteins like phospholamban, which maintains the Ca2+ homeostasis. DM_CKD on the other hand when compared to DM suggested presence of an inflammatory milieu with alterations of metabolic, wound healing and immune cell regulation pathways. Conclusions Our proteomic data in swine model provides strong evidence that mild CKD can induce early alterations in contractile proteins of the heart due to mitochondrial dysfunction along with ECM remodeling. In the presence of diabetes, metabolic and immune cells play a more predominant role in early part of the disease. Thus, we aim to further our understanding by applying our proteomic findings in conjunction with functional hemodynamic data in this swine model of cardiorenal disease.Figure 1Figure 2

Temporal proteomic and phosphoproteomic profiling of the left ventricle in a novel rat model of ischemia/reperfusion with or without ischemic preconditioning

Abstract Background The relationship between ischemic preconditioning (IPC) and myocardial stunning is incompletely understood. Preconditioning has been shown to protect against ischemic injury, whereas myocardial stunning, i.e. transient contractile dysfunction in the setting of ischemia, can lead to severe heart failure and has been considered a deleterious process. To understand this relationship, we recently developed a novel rat model of ischemia-reperfusion (I/R) injury in the presence or absence of IPC. In the presence of IPC (2 cycles of 5 min of I/R followed by 13.5 min of ischemia), we observe a profound myocardial stunning at 4h of reperfusion which completely resolves at 48h. In the absence of IPC, we observe necrosis at 4h of reperfusion and sustained injury at 48h. Despite extensive studies on IPC and myocardial stunning in the setting of I/R, the molecular basis of the relationship between these phenomena are still unknown. Purpose To investigate alterations in protein expression after IPC and myocardial stunning in a novel rat model of I/R injury. Methods We utilized tandem mass tags and mass spectrometry to identify temporal changes in the proteomic- and phosphoproteomic-profiles in left ventricular (LV) tissue from rat hearts following 13.5 min of ischemia and 4h of reperfusion in the presence of absence of IPC. Results Proteomics analysis of the LV tissues collected at baseline showed that 115 proteins were differentially expressed between IPC and non-IPC groups. We observed abundant changes in sarcoplasmic proteins, sarcoplasmic reticulum-related proteins, and actin-based cell projection proteins. Most of these proteins are implicated in arylesterase activity, haemoglobin binding, and nitrogen metabolism. At 4h, differentially expressed proteins were predominantly related to wound healing, coagulation, and lipase activity. Phosphoproteomic analysis at baseline showed that 686 phosphosites were differentially regulated between IPC and non-IPC groups. Aberrant phosphorylation patterns mostly occurred in sarcomere proteins, Z-disc, I-band, contractile fiber associated with actin binding, and the thyroid hormone signalling pathway. At 4h, 520 phosphosites differed between IPC and non-IPC rats; these phosphosites were abundant in contractile fiber proteins, myofibril proteins, and sarcomere proteins associated with calcium signalling and cardiomyopathies. Conclusions This study demonstrates marked changes in proteins and phosphosites related to lipase activity, haemoglobin binding, calcium signalling, and the cardiac contractile apparatus in IPC versus non-IPC groups. This extensive catalogue of novel proteins and phosphosites associated with IPC and myocardial stunning in the setting of I/R injury sets the stage for future manipulation of myocardial stunning and preconditioning in both experimental and clinical settings.