Cardiac mesenchymal stromal cells are a source of adipocytes in arrhythmogenic cardiomyopathy.

AIM: Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder mainly due to mutations in desmosomal genes, characterized by progressive fibro-adipose replacement of the myocardium, arrhythmias, and sudden death. It is still unclear which cell type is responsible for fibro-adipose substitution and which molecular mechanisms lead to this structural change. Cardiac mesenchymal stromal cells (C-MSC) are the most abundant cells in the heart, with propensity to differentiate into several cell types, including adipocytes, and their role in ACM is unknown. The aim of the present study was to investigate whether C-MSC contributed to excess adipocytes in patients with ACM. METHODS AND RESULTS: We found that, in ACM patients' explanted heart sections, cells actively differentiating into adipocytes are of mesenchymal origin. Therefore, we isolated C-MSC from endomyocardial biopsies of ACM and from not affected by arrhythmogenic cardiomyopathy (NON-ACM) (control) patients. We found that both ACM and control C-MSC express desmosomal genes, with ACM C-MSC showing lower expression of plakophilin (PKP2) protein vs. CONTROLS: Arrhythmogenic cardiomyopathy C-MSC cultured in adipogenic medium accumulated more lipid droplets than controls. Accordingly, the expression of adipogenic genes was higher in ACM vs. NON-ACM C-MSC, while expression of cell cycle and anti-adipogenic genes was lower. Both lipid accumulation and transcription reprogramming were dependent on PKP2 deficiency. CONCLUSIONS: Cardiac mesenchymal stromal cells contribute to the adipogenic substitution observed in ACM patients' hearts. Moreover, C-MSC from ACM patients recapitulate the features of ACM adipogenesis, representing a novel, scalable, patient-specific in vitro tool for future mechanistic studies.

Functional and morphological recovery of dystrophic muscles in mice treated with deacetylase inhibitors.

Pharmacological interventions that increase myofiber size counter the functional decline of dystrophic muscles. We show that deacetylase inhibitors increase the size of myofibers in dystrophin-deficient (MDX) and alpha-sarcoglycan (alpha-SG)-deficient mice by inducing the expression of the myostatin antagonist follistatin in satellite cells. Deacetylase inhibitor treatment conferred on dystrophic muscles resistance to contraction-coupled degeneration and alleviated both morphological and functional consequences of the primary genetic defect. These results provide a rationale for using deacetylase inhibitors in the pharmacological therapy of muscular dystrophies.

High mobility group box 1 is a novel substrate of dipeptidyl peptidase-IV.

AIMS/HYPOTHESIS: High mobility group box 1 (HMGB1) is a cytokine with a key role in tissue regeneration and angiogenesis. Previous studies have shown that topical application of HMGB1 to skin wounds of mouse models of diabetes enhanced vessel density and accelerated wound healing, suggesting that diabetes may affect endogenous HMGB1 functions. Dipeptidyl peptidase IV (DPP-IV/CD26) is a protease whose activity is increased in diabetes and whose inhibition improves glucose tolerance. Since HMGB1 contains potential DPP-IV cleavage sites, we determined whether HMGB1 may be a substrate for DPP-IV and whether DPP-IV-mediated cleavage may alter the biological activity of HMGB1. METHODS: Reversed phase HPLC, mass spectrometry and western blot analyses were performed to analyse and identify HMGB1 peptides generated following DPP-IV digestion. HMGB1 angiogenic functions in the presence of DPP-IV were evaluated in vitro and in vivo. HMGB1 protein was detected in the serum of type 2 diabetic patients before and after treatment with DPP-IV inhibitors. RESULTS: DPP-IV cleaved HMGB1 at its N-terminal region and affected its angiogenic functions. Specifically, DPP-IV inhibited HMGB1-induced endothelial cell migration and capillary-like structure formation, as well as HMGB1-mediated vascular network formation in Matrigel implants in mice. We had previously found that HMGB1 promoted endothelial cell migration through activation of extracellular regulated kinase signalling pathway. Here we showed that such an effect was abolished in the presence of DPP-IV. Finally, the N-terminal truncated form of HMGB1 was detected in the serum of type 2 diabetic patients, in whom DPP-IV inhibitors enhanced the levels of full-length HMGB1. CONCLUSIONS/INTERPRETATION: DPP-IV cleaves HMGB1 and, via this mechanism, inhibits HMGB1 angiogenic activity. Treatment with DPP-IV inhibitors may enhance HMGB1 activity in diabetic patients, thereby improving angiogenesis in this condition.

Basic research and clinical applications of non-hematopoietic stem cells, 4-5 April 2008, Tubingen, Germany.

From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to present and discuss the latest proceedings in research on non-hematopoietic stem cells (NHSC). This report presents issues of basic research including characterization, isolation, good manufacturing practice (GMP)-like production and imaging as well as clinical applications focusing on the regenerative and immunomodulatory capacities of NHSC.

Flow-dependent cytosolic acidification of vascular endothelial cells.

Hemodynamic shear stress affects endothelial cell structure and function, but little is known about the signal transduction mechanisms involved in these processes. The effect of laminar shear stress on cytosolic pH (pHi) was examined in rat aortic endothelial cells cultured in glass capillary tubes. Shear stress forces led to a rapid decrease in pHi (maximal effect 0.09 pH unit at 13.4 dynes per square centimeter). Removal of specific ions or addition of exchange inhibitors suggests that in vascular endothelial cells shear stress forces activate both an alkali extruder, sodium ion-independent chloride-bicarbonate ion exchange, and an acid extruder, sodium-hydrogen ion exchange; the net effect in physiologic buffer with the bicarbonate ion is a decrease in pHi.

The Oxidative Stress-Induced miR-200c Is Upregulated in Psoriasis and Correlates with Disease Severity and Determinants of Cardiovascular Risk.

Psoriasis is a chronic inflammatory skin disease associated with reactive oxygen species (ROS) increase and a higher risk of cardiovascular (CV) events. We previously showed that the miR-200 family (miR-200s) is induced by ROS, miR-200c being the most upregulated member responsible for apoptosis, senescence, ROS increase, and nitric oxide decrease, finally causing endothelial dysfunction. Moreover, circulating miR-200c increases in familial hypercholesterolemic children and in plaques and plasma of atherosclerotic patients, two pathologies associated with increased ROS. Given miR-200s' role in endothelial dysfunction, ROS, and inflammation, we hypothesized that miR-200s were modulated in lesional skin (LS) and plasma of psoriatic patients (Pso) and that their levels correlated with some CV risk determinants at a subclinical level. All Pso had severe psoriasis, i.e., Psoriasis Area and Severity Index (PASI) > 10, and one of the following: at least two systemic psoriasis treatments, age at onset < 40 years, and disease duration > 10 years. RNA was extracted from plasma (Pso, N = 29; Ctrl, N = 29) and from nonlesional skin (NLS) and LS of 6 Pso and 6 healthy subject skin (HS) biopsies. miR-200 levels were assayed by quantitative RT-PCR. We found that all miR-200s were increased in LS vs. NLS and miR-200c was the most expressed and upregulated in LS vs. HS. In addition, circulating miR-200c and miR-200a were upregulated in Pso vs. Ctrl. Further, miR-200c positively correlated with PASI, disease duration, left ventricular (LV) mass, LV relative wall thickness (RWT), and E/e', a marker of diastolic dysfunction. Multiple regression analysis indicates a direct association between miR-200c and both RWT and LV mass. Circulating miR-200a correlated positively only with LV mass and arterial pressure augmentation index, a measure of stiffness, although the correlations were nearly significant (P = 0.06). In conclusion, miR-200c is upregulated in LS and plasma of Pso, suggesting its role in ROS increase and inflammation associated with CV risk in psoriasis.

Endothelial cell Ca2+ increases upon tumor cell contact and modulates cell-cell adhesion.

The signal transduction mechanisms involved in tumor cell adhesion to endothelial cells are still largely undefined. The effect of metastatic murine melanoma cell and human prostate carcinoma cell contact on cytosolic [Ca2+] of bovine artery endothelial cells was examined in indo-1-loaded endothelial cell monolayers. A rapid increase in endothelial cell [Ca2+] occurred on contact with tumor cells, but not on contact with 8-microns inert beads. A similar increase in endothelial cell [Ca2+] was observed with human neutrophils or monocyte-like lymphoma cells, but not with endothelial cells, red blood cells, and melanoma cell-conditioned medium. The increase in endothelial cell [Ca2+] was not inhibited by extracellular Ca2+ removal. In contrast, endothelial cell pretreatment with thapsigargin, which releases endoplasmic reticulum Ca2+ into the cytosol and depletes this Ca2+ store site, abolished the cytosolic [Ca2+] rise upon melanoma cell contact. Endothelial cell pretreatment with the membrane-permeant form of the Ca2+ chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid blocked the increase in cytosolic [Ca2+]. Under static and dynamic flow conditions (0.46 dyn/cm2) bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid pretreatment of bovine pulmonary artery endothelial cell monolayers inhibited melanoma cell adhesion to the endothelial cells. Thus, tumor cell contact with endothelial cells induces a rapid Ca2+ release from endothelial intracellular stores, which has a functional role in enhancing cell-cell adhesion.

Nuclear factor-kappaB and cAMP response element binding protein mediate opposite transcriptional effects on the Flk-1/KDR gene promoter.

-The vascular endothelial growth factor receptor Flk-1/KDR is highly expressed during development and almost disappears in adult tissues. Despite its biological relevance, little is known about the molecular mechanisms controlling its expression. In the present work, it is shown that cAMP response element binding protein (CREB) and nuclear factor-kappaB (NF-kappaB)-related antigens bind specific sequences in the Flk-1/KDR promoter. Functional studies demonstrate that cAMP represses whereas tumor necrosis factor-alpha, an activator of NF-kappaB, stimulates promoter activity. Histone acetyltransferases (HATs) P/CAF and CBP/p300 together with p65/RelA, the catalytic subunit of NF-kappaB, increase Flk-1/KDR promoter activity 10- to 20-fold. Consistently, inhibition by cAMP is reverted by increasing intracellular HATs and is completely abolished by site-specific mutagenesis of the cAMP response element. In contrast, specific mutations in the NF-kappaB response element abolish responsiveness to p65/RelA and HATs without affecting cAMP-dependent repression. These results suggest that opposing signaling pathways, activating NF-kappaB or CREB and requiring HAT molecules, control Flk-1/KDR promoter activity.

Acidosis inhibits endothelial cell apoptosis and function and induces basic fibroblast growth factor and vascular endothelial growth factor expression.

Endothelial cells are exposed to an acidotic environment in a variety of pathological and physiological conditions. However, the effect of acidosis on endothelial cell function is still largely unknown, and it was evaluated in the present study. Bovine aortic endothelial cells (BAECs) were grown in bicarbonate buffer equilibrated either with 20% CO(2) (pH 7.0, acidosis) or 5% CO(2) (pH 7.4, control). Acidosis inhibited BAEC proliferation in 10% FCS, whereas by day 7 in serum-free medium, cell number was 3-fold higher in acidotic cells than in control cells. Serum deprivation enhanced BAEC apoptosis, and apoptotic cell death was markedly inhibited by acidosis. Additionally, acidosis inhibited FCS-stimulated migration in a modified Boyden chamber assay and FCS-stimulated differentiation into capillary-like structures on reconstituted basement membrane proteins. Conditioned media from BAECs cultured for 48 hours either at pH 7.0 or pH 7.4 enhanced BAEC proliferation and migration at pH 7.4, and both effects were more marked with conditioned medium from BAECs grown in acidotic than in control conditions. Acidosis enhanced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression as well as bFGF secretion, and a blocking bFGF antibody inhibited enhanced BAEC migration in response to conditioned medium from acidotic cells. These results show that acidosis protects endothelial cells from apoptosis and inhibits their proangiogenic behavior despite enhanced VEGF and bFGF mRNA expression and bFGF secretion.

Retinoids induce fibroblast growth factor-2 production in endothelial cells via retinoic acid receptor alpha activation and stimulate angiogenesis in vitro and in vivo.

The effect of retinoic acid (RA) on endothelial cells is still controversial and was examined in the present study. In bovine aortic endothelial cells (BAECs), all-trans RA (ATRA) and 9-cis RA (9CRA), but not 13-cis RA (13CRA), induced fibroblast growth factor-2 (FGF-2) production and exhibited a biphasic dose-dependent effect to enhance BAEC proliferation and differentiation into tubular structures on reconstituted basement membrane proteins (Matrigel); both processes were inhibited by FGF-2-neutralizing antibody. The pan RA receptor (RAR)-selective ligand (E)-4-[2-(5,5,8,8,-tetramethyl-5,6,7,8-tetrahydro-2-naphtalenyl)-1-propenyl] benzoic acid and the RARalpha-selective ligand 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphtyl)-ethenyl] benzoic acid stimulated the production of FGF-2, whereas the addition of the RARalpha-antagonist RO 41-5253 inhibited this effect. In BAECs, the forced expression of RARalpha, but not RARbeta or RARgamma, enhanced FGF-2 production, whereas the RARalpha-dominant negative, Delta403, blocked this effect. Furthermore, RARalpha overexpression directly stimulated BAEC differentiation on Matrigel and potentiated the effects of ATRA in this assay. Finally, ATRA-treated BAECs coinjected with Matrigel subcutaneously in mice induced neovascularization within the Matrigel plug, and ATRA also enhanced angiogenesis in the chicken chorioallantoic membrane assay. In conclusion, RA can stimulate endothelial cell proliferation and differentiation in vitro via enhanced RARalpha-dependent FGF-2 production, and it can also induce angiogenesis in vivo. The full text of this article is available at http://www.circresaha.org.

Adenovirus-mediated wild-type p53 expression induces apoptosis and suppresses tumorigenesis of prostatic tumor cells.

The use of replication-deficient adenoviral vectors in gene therapy may become a powerful method to achieve efficient but safe transfer of anti-tumor agents. Introduction of the wild-type p53 gene into tumor cells has, in general, been associated with growth suppression. In this study, infection of androgen-independent human prostate Tsu-pr1 cells lacking functional p53 alleles resulted in high levels of p53 protein within 10-15 h. Cells infected with AdCMV.p53 detached from the substratum, condensed, and exhibited fragmentation of nuclear DNA into nucleosomal units consistent with the process of apoptosis. These effects were evident within 24 h after infection, and the majority of cells had undergone apoptosis by 48 h, whereas cells infected with AdCMV.NLS beta Gal continued to proliferate. Uninfected or AdCMV.NLS beta Gal-infected Tsu-pr1 cells formed tumors in nude mice within 3 weeks after implantation, whereas AdCMV.p53-infected cells failed to form tumors during this period. Therefore, adenoviral-mediated antitumor therapy using the p53 gene is an efficient method to inhibit prostate tumor growth, and agents that target the cellular programmed cell death pathway may be useful in clinical applications.

p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells.

The tumor suppressive effect of p53 is believed to be rooted in its two primary functions: the implementation of cellular growth arrest and the execution of apoptotic cell death. While p53-regulated expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) appears to be central for the implementation of G1 arrest, the participation of p21(Waf1/Cip1) in p53-triggered cell death remains controversial. In the present study, overexpression of p53 in human melanoma SK-MEL-110 cells through use of an adenoviral expression vector (AdCMV.p53) was found to result in apoptosis, while similar infection of primary vascular smooth muscle cells (VSMC) instead resulted in a moderate inhibition of growth. Expression of p21(Waf1/Cip1) was strongly elevated in VSMC, but showed little change in SK-MEL-110 cells, although expression of another p53-regulated gene (GADD45) was comparable in both AdCMV.p53-infected cell types. Evidence that p21(Waf1/Cip1) expression may be required for surviving p53-induced cell death was further supported by the finding that p53 overexpression was highly toxic for p21-deficient mouse embryonal fibroblasts (p21-/- MEFs). In both SK-MEL-110 and p21-/- MEFs, adenovirus-driven ectopic expression of p21(Waf1/Cip1) resulted in a substantial protection against p53-induced apoptosis, indicating that p21(Waf1/Cip1) rescued cells from a path of programmed cell death to one of enhanced survival.

Hydrogen peroxide and superoxide modulate leukocyte adhesion molecule expression and leukocyte endothelial adhesion.

While endothelial oxidant generation and subsequent leukocyte chemotaxis and activation are important mechanisms of tissue damage in ischemic organs, it is not known if oxidant generation may be involved in triggering the subsequent leukocyte-mediated injury which occurs. Questions remain whether particular oxidants and oxygen-free radicals are capable of modulating the expression of leukocyte adhesion molecules and effecting leukocyte endothelial adhesion. Studies were performed to determine the effect of different biologically occurring oxidant molecules and oxygen free radicals including: .O2-, .OH, and H2O2 on the expression of integrin and selectin adhesion molecules on the surface of human PMNs and to determine the effect of these alterations on PMN adhesion to the endothelium. Adhesion molecule expression on the surface of human PMNs was measured by immunofluorescence flow cytometry. Electron paramagnetic resonance spectroscopy was applied to characterize the presence of exogenous free radical generation as well as that from activated PMNs. It was observed that these oxidants can cause up-regulation of CD11b and CD18 expression with shedding of L-selectin. The kinetics and dose-response of these effects were analyzed and their functional significance determined by measuring PMN adhesion to cultured human aortic endothelial monolayers. These studies demonstrate that oxygen free radicals and non-radical oxidants can directly trigger PMN activation and adhesion to vascular endothelium.

Shear stress downregulation of platelet-derived growth factor receptor-beta and matrix metalloprotease-2 is associated with inhibition of smooth muscle cell invasion and migration.

BACKGROUND: After endovascular injury, smooth muscle cells (SMCs) may be exposed to hemodynamic shear stress (SS), and these forces modulate neointima accumulation. The effect of SS on SMC migration and invasion is unknown, and it was examined in the present study. METHODS AND RESULTS: Bovine aortic SMCs were exposed to laminar SS of 12 dyne/cm(2) for 3 (SS3) or 15 (SS15) hours; control (C3 and C15) SMCs were kept under static conditions. Platelet-derived growth factor (PDGF)-BB-directed SMC migration and invasion were evaluated by a modified Boyden chamber assay with filters coated with either gelatin or reconstituted basement membrane proteins (Matrigel), respectively. SS15 inhibited both SMC migration and invasion (P<0.0001). There was no significant difference between SS3 and C3 cells. Media conditioned with SS15 cells exhibited a reduction in matrix metalloprotease-2 (MMP-2) by zymography and Western analysis. Northern blot analysis revealed no effect of SS15 on MMP-2 mRNA. In contrast, SS15 decreased MMP-2 activator and membrane-type MMP (MT-MMP or MMP-14) mRNA and protein. Furthermore, SS15 decreased PDGF receptor-beta (PDGF-Rbeta) mRNA and protein (P<0.05), and the SS-dependent decrease in PDGF-BB-directed cell migration was rescued by overexpressing PDGF-Rbeta. CONCLUSIONS: SS inhibits SMC migration and invasion via diminished PDGF-Rbeta expression. This effect of SS is associated with decreased MMP-2 secretion and MT-MMP downregulation.

Local delivery of human tissue kallikrein gene accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia.

BACKGROUND: Human tissue kallikrein (HK) releases kinins from kininogen. We investigated whether adenovirus-mediated HK gene delivery is angiogenic in the context of ischemia. METHODS AND RESULTS: Hindlimb ischemia, caused by femoral artery excision, increased muscular capillary density (P:<0.001) and induced the expression of kinin B(1) receptor gene (P:<0.05). Pharmacological blockade of B(1) receptors blunted ischemia-induced angiogenesis (P:<0.01), whereas kinin B(2) receptor antagonism was ineffective. Intramuscular delivery of adenovirus containing the HK gene (Ad. CMV-cHK) enhanced the increase in capillary density caused by ischemia (969+/-32 versus 541+/-18 capillaries/mm(2) for control, P:<0.001), accelerated blood flow recovery (P:<0.01), and preserved energetic charge of ischemic muscle (P:<0.01). Chronic blockade of kinin B(1) or B(2) receptors prevented HK-induced angiogenesis. CONCLUSIONS: HK gene delivery enhances the native angiogenic response to ischemia. Angiogenesis gene therapy with HK might be applicable to peripheral occlusive vascular disease.

Adenovirus-mediated VEGF(121) gene transfer stimulates angiogenesis in normoperfused skeletal muscle and preserves tissue perfusion after induction of ischemia.

BACKGROUND: Administration of angiogenic factors stimulates neovascularization in ischemic tissues. However, there is no evidence that angiogenesis can be induced in normoperfused skeletal muscles. We tested the hypothesis that adenovirus-mediated intramuscular (IM) gene transfer of the 121-amino-acid form of vascular endothelial growth factor (AdCMV.VEGF(121)) could stimulate neovascularization in nonischemic skeletal muscle and consequently attenuate the hemodynamic deficit secondary to surgically induced ischemia. METHODS AND RESULTS: Rabbits and rats received IM injections of AdCMV.VEGF(121), AdCMV.Null, or saline in the thigh, 4 weeks (rabbits) or 2 weeks (rats) before femoral artery removal in the injected limb. In unoperated rats, at the site of injection of AdCMV.VEGF(121), we found 96% and 29% increases in length density of arterioles and capillaries, respectively. Increased tissue perfusion (TP) to the ischemic limb in the AdCMV.VEGF(121) group was documented, as early as day 1 after surgery, by improved blood flow to the ischemic gastrocnemius muscle measured by radioactive microspheres (AdCMV.VEGF(121)=5.69+/-0.40, AdCMV.Null=2.97+/-0.50, and saline=2.78+/-0.43 mL x min(-1) x 100 g(-1), P<0.001), more angiographically recognizable collateral vessels (angioscore) (AdCMV. VEGF(121)=50.58+/-1.48, AdCMV.Null=29.08+/-4.22, saline=11.83+/-1.90, P<0.0001), and improvement of the bioenergetic reserve of the gastrocnemius muscle as assessed by (31)P NMR spectroscopy. Follow-up studies showed that superior TP to the ischemic limb in the AdCMV.VEGF(121) group persisted until it was equalized by spontaneous collateral vessel development in untreated animals. CONCLUSIONS: IM administration of AdCMV.VEGF(121) stimulates angiogenesis in normoperfused skeletal muscles, and the newly formed vessels preserve TP after induction of ischemia.

E1A stimulates FGF-2 release promoting differentiation of primary endothelial cells.

Basic Fibroblast Growth Factor (FGF-2) is a growth and survival factor and represents one of the most potent differentiation agents of vascular system. In the present study we describe that adenoviral oncoprotein E1A regulates FGF-2 production and determines the acquisition of a pro-angiogenic phenotype in primary bovine aortic endothelial cells (BAEC). Following their transfection, wild type E1A proteins 12S and 13S (wtE1A) stimulated BAEC to differentiate on reconstituted basement membrane matrix (Matrigel). This outcome was paralleled by invasion and migration enhancement in wtE1A-transfected cells. This stimulating effect was absent with the E1A mutant dl646N. Accordingly, zymography and RT - PCR analyses showed that matrix metalloproteinase-9 protein- and mRNA-levels increased following wtE1A transfection. Interestingly, wtE1A-transfected BAEC showed FGF-2 mRNA- and protein-levels higher than controls. Further, FGF-2 neutralization reduced the amount of MMP-9 released in the supernatant of E1A-transfected cells and strongly inhibited BAEC differentiation, thus suggesting that wtE1A activates BAEC by a mechanism, at least partially, dependent on a FGF-2 autocrine/paracrine loop.

Spontaneous Ca2+ release from the sarcoplasmic reticulum limits Ca2+-dependent twitch potentiation in individual cardiac myocytes. A mechanism for maximum inotropy in the myocardium.

We hypothesized that the occurrence of spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), in diastole, might be a mechanism for the saturation of twitch potentiation common to a variety of inotropic perturbations that increase the total cell Ca. We used a videomicroscopic technique in single cardiac myocytes to quantify the amplitude of electrically stimulated twitches and to monitor the occurrence of the mechanical manifestation of spontaneous SR Ca2+ release, i.e., the spontaneous contractile wave. In rat myocytes exposed to increasing bathing [Ca2+] (Cao) from 0.25 to 10 mM, the Cao at which the peak twitch amplitude occurred in a given cell was not unique but varied with the rate of stimulation or the presence of drugs: in cells stimulated at 0.2 Hz in the absence of drugs, the maximum twitch amplitude occurred in 2 mM Cao; a brief exposure to 50 nM ryanodine before stimulation at 0.2 Hz shifted the Cao of the maximum twitch amplitude to 7 mM. In cells stimulated at 1 Hz in the absence of drugs, the maximum twitch amplitude occurred in 4 mM Cao; 1 microM isoproterenol shifted the Cao of the maximum twitch amplitude to 3 mM. Regardless of the drug or the stimulation frequency, the Cao at which the twitch amplitude saturated varied linearly with the Cao at which spontaneous Ca2+ release first occurred, and this relationship conformed to a line of identity (r = 0.90, p = less than 0.001, n = 25). The average peak twitch amplitude did not differ among these groups of cells. In other experiments, (a) the extent of rest potentiation of the twitch amplitude in rat myocytes was also limited by the occurrence of spontaneous Ca2+ release, and (b) in both rat and rabbit myocytes continuously stimulated in a given Cao, the twitch amplitude after the addition of ouabain saturated when spontaneous contractile waves first appeared between stimulated twitches. A mathematical model that incorporates this interaction between action potential-mediated SR Ca2+ release and the occurrence of spontaneous Ca2+ release in individual cells predicted the shape of the Cao-twitch relationship observed in other studies in intact muscle. Thus, the occurrence of spontaneous SR Ca2+ release is a plausible mechanism for the saturation of the inotropic response to Ca2+ in the intact myocardium.

Single adult rabbit and rat cardiac myocytes retain the Ca2+- and species-dependent systolic and diastolic contractile properties of intact muscle.

The systolic and diastolic properties of single myocytes and intact papillary muscles isolated from hearts of adult rats and rabbits were examined at 37 degrees C over a range of stimulation frequencies and bathing [Ca2+]o (Cao). In both rabbit myocytes and intact muscles bathed in 1 mM Cao, increasing the frequency of stimulation from 6 to 120 min-1 resulted in a positive staircase of twitch performance. During stimulation at 2 min-1, twitch performance also increased with increases in Cao up to 20 mM. In the absence of stimulation, both rabbit myocytes and muscles were completely quiescent in less than 15 mM Cao. Further increases in Cao caused the appearance of spontaneous asynchronous contractile waves in myocytes and in intact muscles caused scattered light intensity fluctuations (SLIF), which were previously demonstrated to be caused by Ca2+-dependent spontaneous contractile waves. In contrast to rabbit preparations, intact rat papillary muscles exhibited SLIF in 1.0 mM Cao. Two populations of rat myocytes were observed in 1 mM Cao: approximately 85% of unstimulated cells exhibited low-frequency (3-4 min-1) spontaneous contractile waves, whereas 15%, during a 1-min observation period, were quiescent. In a given Cao, the contractile wave frequency in myocytes and SLIF in intact muscles were constant for long periods of time. In both intact rat muscles and myocytes with spontaneous waves, in 1 mM Cao, increasing the frequency of stimulation from 6 to 120 min-1 resulted, on the average, in a 65% reduction in steady state twitch amplitude. Of the rat myocytes that did not manifest waves, some had a positive, some had a flat, and some had a negative staircase; the average steady state twitch amplitude of these cells during stimulation at 120 min-1 was 30% greater than that at 6 min-1. In contrast to rabbit preparations, twitch performance during stimulation at 2 min-1 saturated at 1.5 mM Cao in both intact rat muscles and in the myocytes with spontaneous waves. We conclude that the widely divergent, Ca2+-dependent systolic and diastolic properties of intact rat and rabbit cardiac muscle are retained with a high degree of fidelity in the majority of viable single myocytes isolated from the myocardium of these species, and that these myocytes are thus a valid model for studies of Ca2+-dependent excitation-contraction mechanisms in the heart.

The interaction of electrically stimulated twitches and spontaneous contractile waves in single cardiac myocytes.

Spontaneous myofilament motion that propagates within cells as a contractile wave is a manifestation of localized Ca2+ release from sarcoplasmic reticulum (SR). At 37 degrees C, when bathing [Ca2+] (Cao) is 1.0 mM, rat myocytes exhibit contractile waves at rest and the interwave interval averages 9.1 +/- 1.5 s (n = 6). We determined whether there was an interaction between this type of SR Ca2+ release and that induced by electrical stimulation to cause a twitch, and whether such an interaction had functional significance. Progressive decreases in SR Ca2+ loading effected by graded concentrations of caffeine produced proportional decreases in the mechanical amplitude of the twitch and of the spontaneous contractile wave. Regular electrical stimulation in physiologic Cao abolished the waves and, after stimulation, waves did not reappear for a period of time (delay interval). Over a range of stimulation frequencies (6-72 min-1), the delay interval ranged from 11.4 +/- 3.6 to 12.4 +/- 1.7 s and was usually greater than the interwave interval at rest. The delay interval for a wave to occur after a twitch was reduced in the presence of increased Cao, glycosides, or catecholamines. When the interstimulus interval exceeded the delay interval, waves could appear between twitches and had a marked effect of shortening the duration of the action potential and decreasing the amplitude of the subsequent twitch. The magnitude of this effect varied inversely with time (up to 2 s) between the onset of the spontaneous diastolic wave and the subsequent stimulated twitch. A reduction of the interstimulus interval to less than the delay interval prevented the occurrence of diastolic waves. These results demonstrate the presence of an interaction between spontaneous and action potential-mediated Ca2+ release, which can be interpreted on the basis of a common Ca2+ pool and perhaps common release mechanisms. This interaction can explain many of the known effects of electrical stimulation on phenomena that are thought to result from spontaneous Ca2+ oscillations in intact tissue.