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Excitation-contraction coupling in ventricular myocytes is enhanced by paracrine signaling from mesenchymal stem cells.

ABSTRACT: In clinical trials mesenchymal stem cells (MSCs) are transplanted into cardiac ischemic regions to decrease infarct size and improve contractility. However, the mechanism and time course of MSC-mediated cardioprotection are incompletely understood. We tested the hypothesis that paracrine signaling by MSCs promotes changes in cardiac excitation-contraction (EC) coupling that protects myocytes from cell death and enhances contractility. Isolated mouse ventricular myocytes (VMs) were treated with control tyrode, MSC conditioned-tyrode (ConT) or co-cultured with MSCs. The Ca handling properties of VMs were monitored by laser scanning confocal microscopy and whole cell voltage clamp. ConT superfusion of VMs resulted in a time dependent increase of the Ca transient amplitude (ConT(15min): ?F/F(0)=3.52±0.38, n=14; Ctrl(15min):? ?F/F(0)=2.41±0.35, n=14) and acceleration of the Ca transient decay (?: ConT: 269±18ms n=14; vs. Ctrl: 315±57ms, n=14). Voltage clamp recordings confirmed a ConT induced increase in I(Ca,L) (ConT: -5.9±0.5 pA/pF n=11; vs. Ctrl: -4.04±0.3 pA/pF, n=12). The change of ? resulted from increased SERCA activity. Changes in the Ca transient amplitude and ? were prevented by the PI3K inhibitors Wortmannin (100nmol/L) and LY294002 (10?mol/L) and the Akt inhibitor V (20?mol/L) indicating regulation through PI3K signal transduction and Akt activation which was confirmed by western blotting. A change in ? was also prevented in eNOS(-/-) myocytes or by inhibition of eNOS suggesting an NO mediated regulation of SERCA activity. Since paracrine signaling further resulted in increased survival of VMs we propose that the Akt induced change in Ca signaling is also a mechanism by which MSCs mediate an anti-apoptotic effect.

SUBMITTER: DeSantiago J

PROVIDER: S-EPMC3570146 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Excitation-contraction coupling in ventricular myocytes is enhanced by paracrine signaling from mesenchymal stem cells.

DeSantiago J J Bare D J DJ Semenov I I Minshall R D RD Geenen D L DL Wolska B M BM Banach K K

Journal of molecular and cellular cardiology 20120323 6

In clinical trials mesenchymal stem cells (MSCs) are transplanted into cardiac ischemic regions to decrease infarct size and improve contractility. However, the mechanism and time course of MSC-mediated cardioprotection are incompletely understood. We tested the hypothesis that paracrine signaling by MSCs promotes changes in cardiac excitation-contraction (EC) coupling that protects myocytes from cell death and enhances contractility. Isolated mouse ventricular myocytes (VMs) were treated with c ...[more]

PMID: 22465692

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Project description:Cardiac excitation-contraction coupling (ECC) is the orchestrated process of initial myocyte electrical excitation, which leads to calcium entry, intracellular trafficking, and subsequent sarcomere shortening and myofibrillar contraction. Neurohumoral ?-adrenergic signaling is a well-established mediator of ECC; other signaling mechanisms, such as paracrine signaling, have also demonstrated significant impact on ECC but are less well understood. For example, resident heart endothelial cells are well-known physiological paracrine modulators of cardiac myocyte ECC mainly via NO and endothelin-1. Moreover, recent studies have demonstrated other resident noncardiomyocyte heart cells (eg, physiological fibroblasts and pathological myofibroblasts), and even experimental cardiotherapeutic cells (eg, mesenchymal stem cells) are also capable of altering cardiomyocyte ECC through paracrine mechanisms. In this review, we first focus on the paracrine-mediated effects of resident and therapeutic noncardiomyocytes on cardiomyocyte hypertrophy, electrophysiology, and calcium handling, each of which can modulate ECC, and then discuss the current knowledge about key paracrine factors and their underlying mechanisms of action. Next, we provide a case example demonstrating the promise of tissue-engineering approaches to study paracrine effects on tissue-level contractility. More specifically, we present new functional and molecular data on the effects of human adult cardiac fibroblast conditioned media on human engineered cardiac tissue contractility and ion channel gene expression that generally agrees with previous murine studies but also suggests possible species-specific differences. By contrast, paracrine secretions by human dermal fibroblasts had no discernible effect on human engineered cardiac tissue contractile function and gene expression. Finally, we discuss systems biology approaches to help identify key stem cell paracrine mediators of ECC and their associated mechanistic pathways. Such integration of tissue-engineering and systems biology methods shows promise to reveal novel insights into paracrine mediators of ECC and their underlying mechanisms of action, ultimately leading to improved cell-based therapies for patients with heart disease.

Project description:Pirfenidone (PFD) is used to treat human pulmonary fibrosis. Its administration to animals with distinct forms of cardiovascular disease results in striking improvement in cardiac performance. Here, its functional impact on cardiac myocytes was investigated. Cells were kept 1-2 days under either control culture conditions or the presence of PFD (1 mM). Subsequently, they were subjected to electrical stimulation to assess the levels of contractility and intracellular Ca2+. The PFD treatment promoted an increase in both peak contraction and kinetics of shortening and relaxation. Moreover, the amplitude and kinetics of Ca2+ transients were enhanced as well. Excitation-contraction coupling (ECC) was also investigated, under whole-cell patch-clamp conditions. In keeping with a previous report, PFD increased twofold the density of Ca2+ current (ICa). Notably, a similar increase in the magnitude of Ca2+ transients was also observed. Thus, the gain of ECC was unaltered. Likewise, PFD did not alter the peak amplitude of caffeine-induced Ca2+ release, indicating stimulation of Ca2+-induced-Ca2+-release (CICR) at constant sarcoplasmic reticulum Ca2+ load. A phase-plane analysis indicated that PFD promotes myofilament Ca2+ desensitization, which is being compensated by higher levels of Ca2+ to promote contraction. Interestingly, although the expression of the Na+/Ca2+ exchanger (NCX) was unaffected, the decay of Ca2+ signal in the presence of caffeine was 50% slower in PFD-treated cells (compared with controls), suggesting that PFD downregulates the activity of the exchanger. PFD also inhibited the production of reactive oxygen species, under both, basal conditions and the presence of oxidative insults (acetaldehyde and peroxide hydrogen). Conversely, the production of nitric oxide was either increased (in atrial myocytes) or remained unchanged (in ventricular myocytes). Protein levels of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) were also investigated. eNOS values did not exhibit significant changes. By contrast, a dual regulation was observed for nNOS, which consisted of inhibition and stimulation, in ventricular and atrial myocytes, respectively. In the latter cells, therefore, an up-regulation of nNOS was sufficient to stimulate the synthesis of NO. These findings improve our knowledge of molecular mechanisms of PFD action and may also help in explaining the corresponding cardioprotective effects.

Project description:In adult cardiomyocytes (CMs), the Na(+)/Ca(2+) exchanger (NCX) is a well-defined determinant of Ca(2+) homeostasis. Developmentally, global NCX knockout in mice leads to abnormal myofibrillar organization, electrical defects, and early embryonic death. Little is known about the expression and function of NCX in human heart development. Self-renewable, pluripotent human embryonic stem cells (hESCs) can serve as an excellent experimental model. However, hESC-derived CMs are highly heterogeneous. A stably lentivirus-transduced hESC line (MLC2v-dsRed) was generated to express dsRed under the transcriptional control of the ventricular-restricted myosin light chain-2v (MLC2v) promoter. Electrophysiologically, dsRed+ cells differentiated from MLC2vdsRed hESCs displayed ventricular action potentials (AP), exclusively. Neither atrial nor pacemaker APs were observed. While I(Ca-L), I(f), and I(Kr) were robustly expressed, I(Ks) and I(K1) were absent in dsRed+ ventricular hESCCMs. Upon differentiation (7+40 to +90 days), the basal [Ca(2+)](i), Ca(2+) transient amplitude, maximum upstroke, and decay velocities significantly increased (P < 0.05). The I(Ca-L) antagonizer nifedipine (1 microM) decreased the Ca(2+) transient amplitude (to approximately 30%) and slowed the kinetics (by approximately 2-fold), but Ca(2+) transients could still be elicited even after complete ICa-L blockade, suggesting the presence of additional Ca(2+) influx(es). Indeed, Ni(2+)-sensitive INCX could be recorded in 7+40- and +90-day dsRed+ hESC-CMs, and its densities increased from -1.2 +/- 0.6 pA/pF at -120 mV and 3.6 +/- 1.0 pA/pF at 60 mV by 6- and 2-folds, respectively. With higher [Ca(2+)](i), 7+90-day ventricular hESC-CMs spontaneously but irregularly fired transients upon a single stimulus under an external Na(+)-free condition; however, without extracellular Na(+), nifedipine could completely inhibit Ca(2+) transients. We conclude that I(NCX) is functionally expressed in developing ventricular hESC-CMs and contributes to their excitation-contraction coupling.

Project description:BackgroundSecretome profiles of mesenchymal stem cells (MSCs) are reflective of their local microenvironments. These biologically active factors exert an impact on the surrounding cells, eliciting regenerative responses that create an opportunity for exploiting MSCs towards a cell-free therapy for cartilage regeneration. The conventional method of culturing MSCs on a tissue culture plate (TCP) does not provide the physiological microenvironment for optimum secretome production. In this study, we explored the potential of electrospun fiber sheets with specific orientation in influencing the MSC secretome production and its therapeutic value in repairing cartilage.MethodsConditioned media (CM) were generated from MSCs cultured either on TCP or electrospun fiber sheets of distinct aligned or random fiber orientation. The paracrine potential of CM in affecting chondrogenic differentiation, migration, proliferation, inflammatory modulation, and survival of MSCs and chondrocytes was assessed. The involvement of FAK and ERK mechanotransduction pathways in modulating MSC secretome were also investigated.ResultsWe showed that conditioned media of MSCs cultured on electrospun fiber sheets compared to that generated from TCP have improved secretome yield and profile, which enhanced the migration and proliferation of MSCs and chondrocytes, promoted MSC chondrogenesis, mitigated inflammation in both MSCs and chondrocytes, as well as protected chondrocytes from apoptosis. Amongst the fiber sheet-generated CM, aligned fiber-generated CM (ACM) was better at promoting cell proliferation and augmenting MSC chondrogenesis, while randomly oriented fiber-generated CM (RCM) was more efficient in mitigating the inflammation assault. FAK and ERK signalings were shown to participate in the modulation of MSC morphology and its secretome production.ConclusionsThis study demonstrates topographical-dependent MSC paracrine activities and the potential of employing electrospun fiber sheets to improve the MSC secretome for cartilage regeneration.

Project description:BackgroundHuman induced pluripotent stem cells-derived myogenic progenitors develop functional and ultrastructural features typical of skeletal muscle when differentiated in culture. Besides disease-modeling, such a system can be used to clarify basic aspects of human skeletal muscle development. In the present study, we focus on the development of the excitation-contraction (E-C) coupling, a process that is essential both in muscle physiology and as a tool to differentiate between the skeletal and cardiac muscle. The occurrence and maturation of E-C coupling structures (Sarcoplasmic Reticulum-Transverse Tubule (SR-TT) junctions), key molecular components, and Ca2+ signaling were examined, along with myofibrillogenesis.MethodsPax7+-myogenic progenitors were differentiated in culture, and developmental changes were examined from a few days up to several weeks. Ion channels directly involved in the skeletal muscle E-C coupling (RyR1 and Cav1.1 voltage-gated Ca2+ channels) were labeled using indirect immunofluorescence. Ultrastructural changes of differentiating cells were visualized by transmission electron microscopy. On the functional side, depolarization-induced intracellular Ca2+ transients mediating E-C coupling were recorded using Fura-2 ratiometric Ca2+ imaging, and myocyte contraction was captured by digital photomicrography.ResultsWe show that the E-C coupling machinery occurs and operates within a few days post-differentiation, as soon as the myofilaments align. However, Ca2+ transients become effective in triggering myocyte contraction after 1 week of differentiation, when nascent myofibrils show alternate A-I bands. At later stages, myofibrils become fully organized into adult-like sarcomeres but SR-TT junctions do not reach their triadic structure and typical A-I location. This is mirrored by the absence of cross-striated distribution pattern of both RyR1 and Cav1.1 channels.ConclusionsThe E-C coupling machinery occurs and operates within the first week of muscle cells differentiation. However, while early development of SR-TT junctions is coordinated with that of nascent myofibrils, their respective maturation is not. Formation of typical triads requires other factors/conditions, and this should be taken into account when using in-vitro models to explore skeletal muscle diseases, especially those affecting E-C coupling.

Project description:Mitochondrial Ca2+ ([Ca2+]m) regulates oxidative phosphorylation and thus contributes to energy supply and demand matching in cardiac myocytes. Mitochondria take up Ca2+ via the Ca2+ uniporter (MCU) and extrude it through the mitochondrial Na+/Ca2+ exchanger (mNCE). It is controversial whether mitochondria take up Ca2+ rapidly, on a beat-to-beat basis, or slowly, by temporally integrating cytosolic Ca2+ ([Ca2+]c) transients. Furthermore, although mitochondrial Ca2+ efflux is governed by mNCE, it is unknown whether elevated intracellular Na+ ([Na+]i) affects mitochondrial Ca2+ uptake and bioenergetics. To monitor [Ca2+]m, mitochondria of guinea pig cardiac myocytes were loaded with rhod-2-acetoxymethyl ester (rhod-2 AM), and [Ca2+]c was monitored with indo-1 after dialyzing rhod-2 out of the cytoplasm. [Ca2+]c transients, elicited by voltage-clamp depolarizations, were accompanied by fast [Ca2+]m transients, whose amplitude (delta) correlated linearly with delta[Ca2+]c. Under beta-adrenergic stimulation, [Ca2+]m decay was approximately 2.5-fold slower than that of [Ca2+]c, leading to diastolic accumulation of [Ca2+]m when amplitude or frequency of delta[Ca2+]c increased. The MCU blocker Ru360 reduced delta[Ca2+]m and increased delta[Ca2+]c, whereas the mNCE inhibitor CGP-37157 potentiated diastolic [Ca2+]m accumulation. Elevating [Na+]i from 5 to 15 mmol/L accelerated mitochondrial Ca2+ decay, thus decreasing systolic and diastolic [Ca2+]m. In response to gradual or abrupt changes of workload, reduced nicotinamide-adenine dinucleotide (NADH) levels were maintained at 5 mmol/L [Na+]i, but at 15 mmol/L, the NADH pool was partially oxidized. The results indicate that (1) mitochondria take up Ca2+ rapidly and contribute to fast buffering during a [Ca2+]c transient; and (2) elevated [Na+]i impairs mitochondrial Ca2+ uptake, with consequent effects on energy supply and demand matching. The latter effect may have implications for cardiac diseases with elevated [Na+]i.

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Excitation-contraction coupling in ventricular myocytes is enhanced by paracrine signaling from mesenchymal stem cells.

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Excitation-contraction coupling in ventricular myocytes is enhanced by paracrine signaling from mesenchymal stem cells.

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