Effect of floating a gel matrix on mucin release in cultured airway epithelial cells

Confluent cultures of primary hamster tracheal surface epithelial (HTSE) cells grown on a thick collagen gel are highly enriched with secretory cells and constitutively release mucins. In the present experiment, we examined the possible effect of mechanical strain of cultured HTSE cells on the release of mucin. The mechanical strain of cells was accomplished by several methods: 1) by floating the gel from the culture dish by rimming; 2) by treatment with EGTA which interrupts intercellular tight junctions; 3) by treatment with collagenase which disrupts the cell-matrix adhesion; and 4) by mechanically flexing the collagen gel matrix. All these conditions caused increases of mucin release without damage on the plasma membrane. We conclude that a number of mechanical strains which might alter cell shape can stimulate mucin release from cultured HTSE cells. Such a mechanism might be operative in the physiological regulation of airway goblet cell mucin secretion where mechanical strains may be induced on epithelial cells by underlying smooth muscles. © 1993 Wiley-Liss, Inc.     

Magnesium regulates ADP dissociation from myosin V

Processivity in myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. However, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the several potential pathways myosin V takes in the process of ADP release and how actin influences the kinetics of these pathways. We have addressed this issue by examining how magnesium regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a model in which actin accelerates the release of ADP from myosin V by reducing the magnesium affinity of a myosin V-MgADP intermediate. This is likely a consequence of the structural changes that actin induces in myosin to release phosphate. This effect on magnesium affinity provides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase activity and the velocity of movement of this motor.     

Systemic pregabalin attenuates facial hypersensitivity and noxious stimulus-evoked release of glutamate in medullary dorsal horn in a rodent model of trigeminal neuropathic pain

Pregabalin is effective in treating many neuropathic pain conditions. However, the mechanisms of its analgesic effects remain poorly understood. The aim of the present study was to determine whether pregabalin suppresses facial mechanical hypersensitivity and evoked glutamate release in the medullary dorsal horn (MDH) in a rodent model of trigeminal neuropathic pain. Nociceptive mechanical sensitivity was assessed pre-operatively, and then post-operatively 1 h following pregabalin or vehicle (saline) treatment on post-operative days 2 and 5 following infraorbital nerve transection (IONX). In addition, an in vivo microdialysis probe was inserted into the exposed medulla post-operatively and dialysate samples were collected. Glutamate release was then evoked by mustard oil (MO) application to the tooth pulp, and the effects of pregabalin or vehicle were examined on the MDH glutamate release. Glutamate concentrations in the dialysated samples were determined by HPLC, and data analyzed by ANOVA. IONX animals (but not control animals) showed facial mechanical hypersensitivity for several days post-operatively. In addition, tooth pulp stimulation with MO evoked a transient release of glutamate in the MDH of IONX animals. Compared to vehicle, administration of pregabalin significantly attenuated the facial mechanical hypersensitivity as well as the MO-evoked glutamate release in MDH. This study provides evidence in support of recent findings pointing to the usefulness of pregabalin in the treatment of orofacial neuropathic pain.     

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is subject to chemical and mechanical signals, and the mechanisms by which it senses and responds to mechanical signals are poorly understood. Elucidating the role of forces in cell maturation will enable the design of scaffolds that can promote cell adhesion and cytoskeletal coupling to the substrate, and therefore improve the capacity of different neuronal types to regenerate after injury.Here, we describe a method to apply simultaneous force spectroscopy measurements during laser induced cell lesion. We measure tension release in the partially lesioned axon by simultaneous interferometric tracking of an optically trapped probe adhered to the membrane of the axon. Our experimental protocol detects the tension release with piconewton sensitivity, and the dynamic of the tension release at millisecond time resolution. Therefore, it offers a high-resolution method to study how the mechanical coupling between cells and substrates can be modulated by pharmacological treatment and/or by distinct mechanical properties of the substrate.     

Pharmacological dissection of the cellular mechanisms associated to the spontaneous and the mechanically stimulated ATP release by mesentery endothelial cells: roles of thrombin and TRPV

Endothelial cells participate in extracellular ATP release elicited by mechanosensors. To characterize the dynamic interactions between mechanical and chemical factors that modulate ATP secretion by the endothelium, we assessed and compared the mechanisms participating in the spontaneous (basal) and mechanically stimulated secretion using primary cultures of rat mesentery endothelial cells. ATP/metabolites were determined in the cell media prior to (basal) and after cell media displacement or a picospritzer buffer puff used as mechanical stimuli. Mechanical stimulation increased extracellular ATP that peaked within 1 min, and decayed to basal values in 10 min. Interruption of the vesicular transport route consistently blocked the spontaneous ATP secretion. Cells maintained in media lacking external Ca²⁺ elicited a spontaneous rise of extracellular ATP and adenosine, but failed to elicit a further extracellular ATP secretion following mechanical stimulation. 2-APB, a TRPV agonist, increased the spontaneous ATP secretion, but reduced the mechanical stimulation-induced nucleotide release. Pannexin1 or connexin blockers and gadolinium, a Piezo1 blocker, reduced the mechanically induced ATP release without altering spontaneous nucleotide levels. Moreover, thrombin or related agonists increased extracellular ATP secretion elicited by mechanical stimulation, without modifying spontaneous release. In sum, present results allow inferring that the spontaneous, extracellular nucleotide secretion is essentially mediated by ATP containing vesicles, while the mechanically induced secretion occurs essentially by connexin or pannexin1 hemichannel ATP transport, a finding fully supported by results from Panx1^?/? rodents. Only the latter component is modulated by thrombin and related receptor agonists, highlighting a novel endothelium-smooth muscle signaling role of this anticoagulant.     

K+ACh channel activation with carbachol increases atrial ANP release

Although it has been known that atrial natriuretic peptide (ANP) release is regulated through muscarinic acetylcholine receptors (mAChR), the mechanism by which this neurotransmitter regulates atrial ANP release is largely unknown. This study tested the hypothesis that K(+)(ACh) channels mediate the action of mAChR on atrial myocyte ANP release. Experiments were performed in perfused beating rabbit atria. Carbachol (CCh), an agonist of cardiac mAChR, increased atrial myocyte ANP release concomitantly with a decrease in stroke volume and intra-atrial pulse pressure in a concentration-dependent manner. Isoproterenol, a beta-adrenoceptor agonist, decreased ANP release concomitantly with an increase in cAMP and mechanical dynamics. In the presence of isoproterenol, the CCh-induced increase in ANP release and decrease in cAMP efflux levels and mechanical dynamics were able to be repeated. The CCh-induced changes were blocked by selective M(2) mAChR antagonists. Tertiapin, a selective G-protein-gated K(+)(ACh) channel blocker, attenuated the CCh-induced increase in ANP release and decrease in mechanical dynamics in a concentration-dependent manner, but without a significant effect on the CCh-induced decrease in cAMP efflux levels. The CCh-induced changes in ANP release and atrial dynamics were inhibited in the atria from pertussis toxin-pretreated rabbits. These findings demonstrate that G-protein-gated K(+)(ACh) channels regulate atrial myocyte ANP release. The present study also shows that mAChR and adrenoceptors have opposing roles in the regulation of ANP release.     

Mechanical stretch induces MMP-2 release and activation in lung endothelium: role of EMMPRIN

High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix metalloproteinases (MMPs). To investigate the mechanism leading to the increased MMP release, we systematically studied the effect of mechanical stretch on human microvascular endothelial cells isolated from the lung. We exposed cells grown on collagen 1 BioFlex plates to sinusoidal cyclic stretch at 0.5 Hz using the Flexercell system with 17-18% elongation of cells. After 4 days of cell stretching, conditioned media and cell lysate were collected and analyzed by gelatin, casein, and reverse zymograms as well as Western blotting. RT-PCR of mRNA extracted from stretched cells was performed. Our results show that 1) cyclic stretch led to increased release and activation of MMP-2 and MMP-1; 2) the activation of MMP-2 was accompanied by an increase in membrane type-1 MMP (MT1-MMP) and inhibited by a hydroxamic acid-derived inhibitor of MMPs (Prinomastat, AG3340); and 3) the MMP-2 release and activation were preceded by an increase in production of extracellular MMP inducer (EMMPRIN). These results suggest that cyclic mechanical stretch leads to MMP-2 activation through an MT1-MMP mechanism. EMMPRIN may play an important role in the release and activation of MMPs during lung injury.     

NGF电纺纤维的体外缓释研究

目的:研究担载神经生长因子(NGF)的静电纺丝纤维的表征,考察NGF电纺纤维对于周围神经修复的效果。方法:将NGF水溶液分散于PLLA溶液,通过W/O乳液法制备静电纺丝纤维,对纤维的形态、力学性能等进行表征,Elisa方法测定NGF的体外释放动力学,Alamer Blue法检测试剂来考察纤维释放液对于PC12细胞增殖的影响。结果:NGF电纺纤维具备良好的形态和力学性质,直径为500-900 nm,纤维具备三维多孔结构。纤维的最大拉伸应力为2.50±0.41 MPa。电纺纤维中NGF在体外可有效释放9天,累积释放量接近3000 pg。细胞活性实验结果显示,第1、3、5、7天释放液的荧光强度与对照组相比有显著差异。结论:担载NGF的乳液法静电纺丝纤维有促进缺损周围神经修复的潜质。     

Mechanosensitive release of parathyroid hormone-related peptide from coronary endothelial cells

10.1152/ajpheart.00925. 2001.-Parathyroid hormone-related peptide (PTHrP) is expressed throughout the cardiovascular system and is able to dilate vessels. This study investigated whether mechanical forces generated by changes in regional perfusion influence PTHrP release from the coronary vascular bed. Experiments were performed in vitro on saline-perfused rat hearts or isolated coronary endothelial cells exposed to cyclic strain and in vivo in anesthetized pigs. In vitro, PTHrP release from saline-perfused rat hearts was strongly correlated with coronary flow (r = 0.84). Increasing coronary flow from 5 to 10 ml/min increased PTHrP release from 442 +/- 42 to 1,563 +/- 167 pg/min. Increasing the viscosity of the perfusate did not change basal PTHrP release. Increasing flow without a concomitant increase in pressure did not lead to an increase in release rate, but reduction in pressure under flow-constant conditions reduced PTHrP release rate. Cyclic strain induced a strain-dependent release of PTHrP from endothelial cells that was inhibited by the addition of a calcium-chelating agent. In vivo, there was a net release of PTHrP in the coronary circulation and decreases in coronary flow and pressure decreased the PTHrP release rate. Bradykinin in the presence of constant pressure increased PTHrP release, probably by increasing the intracellular calcium concentration in coronary endothelial cells. In summary, mechanical forces evoked by blood flow can trigger a constant PTHrP release.     

The effects of mechanical stress on the growth, differentiation, and paracrine factor production of cardiac stem cells

Stem cell therapies have been clinically employed to repair the injured heart, and cardiac stem cells are thought to be one of the most potent stem cell candidates. The beating heart is characterized by dynamic mechanical stresses, which may have a significant impact on stem cell therapy. The purpose of this study is to investigate how mechanical stress affects the growth and differentiation of cardiac stem cells and their release of paracrine factors. In this study, human cardiac stem cells were seeded in a silicon chamber and mechanical stress was then induced by cyclic stretch stimulation (60 cycles/min with 120% elongation). Cells grown in non-stretched silicon chambers were used as controls. Our result revealed that mechanical stretching significantly reduced the total number of surviving cells, decreased Ki-67-positive cells, and increased TUNEL-positive cells in the stretched group 24 hrs after stretching, as compared to the control group. Interestingly, mechanical stretching significantly increased the release of the inflammatory cytokines IL-6 and IL-1β as well as the angiogenic growth factors VEGF and bFGF from the cells in 12 hrs. Furthermore, mechanical stretching significantly reduced the percentage of c-kit-positive stem cells, but increased the expressions of cardiac troponin-I and smooth muscle actin in cells 3 days after stretching. Using a traditional stretching model, we demonstrated that mechanical stress suppressed the growth and proliferation of cardiac stem cells, enhanced their release of inflammatory cytokines and angiogenic factors, and improved their myogenic differentiation. The development of this in vitro approach may help elucidate the complex mechanisms of stem cell therapy for heart failure.     

Mechanical stretch-induced serotonin release from pulmonary neuroendocrine cells: implications for lung development

Pulmonary neuroendocrine cells (PNEC) produce amine (serotonin, 5-HT) and peptides (e.g., bombesin, calcitonin) with growth factor-like properties and are thought to play an important role in lung development. Because physical forces are essential for lung growth and development, we investigated the effects of mechanical strain on 5-HT release in PNEC freshly isolated from rabbit fetal lung and in the PNEC-related tumor H727 cell line. Cultures exposed to sinusoidal cyclic stretch showed a significant 5-HT release inhibitable with gadolinium chloride (10 nM), a blocker of mechanosensitive channels. In contrast to hypoxia (Po2 approximately 20 mmHg), stretch-induced 5-HT release was not affected by Ca2+-free medium or nifedipine (50 microM), excluding the exocytic pathway. In H727 cells, stretch failed to release calcitonin, a peptide stored within dense core vesicles (DCV), whereas hypoxia caused massive calcitonin release. 5-HT released by mechanical stretch is derived predominantly from the cytoplasmic pool, because it is rapid ( approximately 5 min) and is releasable from early (20 days of gestation) fetal PNEC containing few DCV. Both mechanical stretch and hypoxia upregulated expression of tryptophan hydroxylase, the rate-limiting enzyme of 5-HT synthesis. We conclude that mechanical strain is an important physiological stimulus for the release of 5-HT from PNEC via mechanosensitive channels with potential effects on lung development and resorption of lung fluid at the time of birth.     

Location of Acid Phosphatase and β-Fructofuranosidase Within Yeast Cell Envelopes

After 16 hr of incubation in a low-phosphate, aerated medium, bakers' yeast was obtained with a high titer of acid phosphatase (EC 3.1.3.2) and beta-fructofuranosidase (EC 3.2.1.26). All of the beta-fructofuranosidase and 75% of the acid phosphatase were easily released by mechanical disruption in a French pressure cell. The cell wall suffered a limited number of cracks, but this was sufficient for the co-release of these enzymes. Both enzymes were subject to autolytic release, although correlation was inconclusive because of the relative instability of acid phosphatase. The data are consistent with the bulk of the two enzymes being located in the periplasmic space. Ethylacetate treatments yielded ghosts with high beta-fructofuranosidase but low acid phosphatase activities. The surviving acid phosphatase was not representative of that in live cells. It was resistant to release by mechanical disruption and showed a high susceptibility to heat inactivation. The beta-fructofuranosidase in live cells and in ethylacetatetreated cells exhibited polydispersity in heat inactivation susceptibility; but the kinetics were indistinguishable, and facile release by mechanical disruption was shown in both cases.     

Effects of doxycycline on articular cartilage GAG release and mechanical properties following impact

The effects of doxycycline were examined on articular cartilage glycosaminoglycan (GAG) release and biphasic mechanical properties following two levels of impact loading at 1 and 2 weeks post-injury. Further, treatment for two continuous weeks was compared to treatment for only the 1st week of a 2-week culture period. Following impact at two levels, articular cartilage explants were cultured for 1 or 2 weeks with 0, 50, or 100 microM doxycycline. Histology, GAG release to the media, and creep indentation biomechanical properties were examined. The "High" (2.8 J) impact level had gross surface damage, whereas "Low" (1.1 J) impact was indiscernible from non-impacted controls. GAG staining decreased after High impact, but doxycycline did not visibly affect staining. High impact resulted in decreased aggregate moduli at both 1 and 2 weeks and increased permeability at 2 weeks, but tissue mechanical properties were not affected by doxycycline treatment. At 1 week, High impact resulted in more GAG release compared to non-impacted controls. However, following High impact, 100 microM doxycycline reduced cumulative GAG release at 1 and 2 weeks by 30% and 38%, respectively, compared to no treatment. Interestingly, there was no difference in GAG release comparing 2 weeks continuous treatment with 1 week on, 1 week off. These results support the hypothesis that doxycycline can mitigate GAG release from articular cartilage following impact loads. However, doxycycline was unable to prevent the loss of tissue stiffness observed post-impact, presumably due to initial matrix damage resulting solely from mechanical trauma.     

Pannexin membrane channels are mechanosensitive conduits for ATP

Intercellular calcium wave propagation initiated by mechanical stress is a phenomenon found in nearly all cell types. The waves utilize two pathways: transfer of InsP3 directly from cell to cell through gap junction channels and release of ATP onto extracellular purinergic receptors. The conduit for ATP has remained elusive and both a vesicular and a channel mediated release have been considered. Here, we describe the properties of single pannexin 1 channels. They have a wide expression spectrum, they are of large conductance and permeant for ATP, and they are mechanosensitive. Hence, pannexins are candidates for the release of ATP to the extracellular space upon mechanical stress.     

Mast Cell-Nerve Cell Interaction at Acupoint: Modeling Mechanotransduction Pathway Induced by Acupuncture

Mast cells are found abundant at sites of acupoints. Nerve cells share perivascular localization with mast cells. Acupuncture (mechanical stimuli) can activate mast cells to release adenosine triphosphate (ATP) which can activate nerve cells and modulates pain-processing pathways in response to acupuncture. In this paper, a mathematical model was constructed for describing intracellular Ca²⁺ signal and ATP release in a coupled mast cell and nerve cell system induced by mechanical stimuli. The results showed mechanical stimuli lead to a intracellular Ca²⁺ rise in the mast cell and ATP release, ATP diffuses in the extracellular space (ECS) and activates the nearby nerve cells, then induces electrical current in the nerve cell which spreads in the neural network. This study may facilitate our understanding of the mechanotransduction process induced by acupuncture and provide a methodology for quantitatively analyzing acupuncture treatment.     

Brefeldin A block of integrin-dependent mechanosensitive ATP release from Xenopus oocytes reveals a novel mechanism of mechanotransduction

Many animal cells release ATP into the extracellular medium, and often this release is mechanosensitive. However, the mechanisms underlying this release are not well understood. Using the luciferin-luciferase bioluminescent assay we demonstrate that a Xenopus oocyte releases ATP at a basal rate approximately 0.01 fmol/s, and gentle mechanical stimulation can increase this to 50 fmol/s. Brefeldin A, nocodazole, and progesterone-induced- maturation block basal and mechanosensitive ATP release. These treatments share the common feature of disrupting the Golgi complex and vesicle trafficking to the cell surface and thereby block protein secretion and membrane protein insertion. We propose that ATP release occurs when protein transport vesicles enriched in ATP fuse with the plasma membrane. Collagenase, integrin-binding peptides, and cytochalasin D also block ATP release, indicating that extracellular, membrane and cytoskeletal elements are involved in the release process. Elevation of intracellular Ca(2+) does not evoke ATP release but potentiates mechanosensitive ATP release. Our study indicates a novel mechanism of mechanotransduction that would allow cells to regulate membrane trafficking and protein transport/secretion in response to mechanical loading.     

血管内皮生长因子(VEGF)乳液法电纺纤维膜的体外研究

目的:研究担载血管内皮生长因子(VEGF)的乳液法电纺纤维膜的亲水性能、外观形态和机械性能,纤维膜中VEGF的包封率和体外释放动力学,为评价其能否应用于血管再生领域的研究奠定基础。方法:将VEGF水溶液通过W/O乳液法制备成缓释VEGF的生物可降解的丙交酯-乙交酯共聚物(PLGA)静电纺丝纤维膜,对该纤维膜的接触角、外观形态、机械性能进行表征,Elisa法测定该纤维膜的体外14天的释放行为,分别观察纤维膜释放0天、7天、14天后的电镜图。结果:加入VEGF后,纤维膜的接触角由140.0°减小到136.1°,亲水性增强,具有类似细胞外基质(ECMs)网状结构和良好的力学性能,纤维膜第1天的突释不超过载药量的50%,电镜图下显示纤维膜释放1周时纤维发生断裂。结论:通过乳液法制备的担载VEGF的电纺纤维膜具有良好的物理性能,能够持续缓释VEGF,可作为血管再生的组织工程支架进行深入研究。     

Expression of muscle anabolic and metabolic factors in mechanically loaded MLO-Y4 osteocytes

Lack of physical activity results in muscle atrophy and bone loss, which can be counteracted by mechanical loading. Similar molecular signaling pathways are involved in the adaptation of muscle and bone mass to mechanical loading. Whether anabolic and metabolic factors regulating muscle mass, i.e., insulin-like growth factor-I isoforms (IGF-I Ea), mechano growth factor (MGF), myostatin, vascular endothelial growth factor (VEGF), or hepatocyte growth factor (HGF), are also produced by osteocytes in bone in response to mechanical loading is largely unknown. Therefore, we investigated whether mechanical loading by pulsating fluid flow (PFF) modulates the mRNA and/or protein levels of muscle anabolic and metabolic factors in MLO-Y4 osteocytes. Unloaded MLO-Y4 osteocytes expressed mRNA of VEGF, HGF, IGF-I Ea, and MGF, but not myostatin. PFF increased mRNA levels of IGF-I Ea (2.1-fold) and MGF (2.0-fold) at a peak shear stress rate of 44Pa/s, but not at 22Pa/s. PFF at 22 Pa/s increased VEGF mRNA levels (1.8- to 2.5-fold) and VEGF protein release (2.0- to 2.9-fold). Inhibition of nitric oxide production decreased (2.0-fold) PFF-induced VEGF protein release. PFF at 22 Pa/s decreased HGF mRNA levels (1.5-fold) but increased HGF protein release (2.3-fold). PFF-induced HGF protein release was nitric oxide dependent. Our data show that mechanically loaded MLO-Y4 osteocytes differentially express anabolic and metabolic factors involved in the adaptive response of muscle to mechanical loading (i.e., IGF-I Ea, MGF, VEGF, and HGF). Similarly to muscle fibers, mechanical loading enhanced expression levels of these growth factors in MLO-Y4 osteocytes. Although in MLO-Y4 osteocytes expression levels of IGF-I Ea and MGF of myostatin were very low or absent, it is known that the activity of osteoblasts and osteoclasts is strongly affected by them. The abundant expression levels of these factors in muscle cells, in combination with low expression in MLO-Y4 osteocytes, provide a possibility that growth factors expressed in muscle could affect signaling in bone cells.     

Application of multiple forms of mechanical loading to human osteoblasts reveals increased ATP release in response to fluid flow in 3D cultures and differential regulation of immediate early genes

ATP is actively released into the extracellular environment from a variety of cell types in response to mechanical stimuli. This is particularly true in bone where mechanically induced ATP release leads to immediate early gene activation to regulate bone remodelling; however there is no consensus as to which mechanical stimuli stimulate osteoblasts the most. To elucidate which specific type(s) of mechanical stimuli induce ATP release and gene activation in human osteoblasts, we performed an array of experiments using different mechanical stimuli applied to both monolayer and 3D cultures of the same osteoblast cell type, SaOS-2. ATP release from osteoblasts cultured in monolayer significantly increased in response to turbulent fluid flow, laminar fluid flow and substrate strain. No significant change in ATP release could be detected in 3D osteoblast cultures in response to cyclic or static compressive loading of osteoblast-seeded scaffolds, whilst turbulent fluid flow increased ATP release from 3D cultures of osteoblasts to a greater degree than observed in monolayer cultures. Cox-2 expression quantified using real time PCR was significantly lower in cells subjected to turbulent fluid flow whereas c-fos expression was significantly higher in cells subjected to strain. Load-induced signalling via c-fos was further investigated using a SaOS-2 c-fos luciferase reporter cell line and increased in response to substrate strain and turbulent fluid flow in both monolayer and 3D, with no significant change in response to laminar fluid flow or 3D compressive loading. The results of this study demonstrate for the first time strain-induced ATP release from osteoblasts and that turbulent fluid flow in 3D up regulates the signals required for bone remodelling.     

Model of mechanical alternans in the mammalian myocardium

A model is proposed to elucidate the cause and mechanism of mechanical alternans in cardiac muscle in terms of discrete calcium movements. Mechanical alternans, the cause of which lies within the borders of excitation-contraction-coupling (ECC), is analyzed. In this case, the "input" of the ECC system (the action potentials and intervals) is constant while the "output" (contractile force) oscillates between two constant values, indicating that the system has a "memory" with two "internal states". It is proposed that these two "states" are associated with a part of the sarcoplasmic reticulum ("releasable terminal") containing the readily releasable calcium. A mechanism of "calcium-concentration-dependent threshold" is suggested to govern the "release function", i.e. the release of calcium from the "releasable terminal" to the myofilaments. The "release function" is analyzed in both the linear and the non-linear cases and its implication on the initiation of sustained and transient mechanical alternans are described. The dependence of mechanical alternans on a disturbance is also explained. The model response resembles the experimental observations of mechanical alternans in mammalian myocardium in the following manners: abrupt transition from low to high heart rates, slow progressive acceleration of rate, variations in persistence at subthreshold rates, effect of premature and delayed beat following the small and large beats, restitution curves, and transient mechanical alternans initiated by a delayed beat.