Modulation of cardiac myocyte beating rate and hypertrophy by cardiac fibroblasts isolated from neonatal rat ventricle.

We compared the determinants of spontaneous activity in explanted neonatal (2-day-old) rat ventricle cells and in reaggregates derived from 15-day-old chick embryos. We studied the beating rate with an optical recording method and the underlying electrical activity with glass microelectrodes using the K current blockers cesium (Cs) and tetraethylammonium, varied Ca concentrations, and the Ca antagonist verapamil. In the rat (i) Cs increased the beating rate that was mediated by an increase in the slope of the diastolic potential, (ii) Ca increased the beating rate dramatically at low and medium concentrations to decrease it again at 8 mM Cao.2This increase in the beating rate was mediated by an increase of the slope of the diastolic depolarization. (iii) The beating rate decreased with verapamil at concentrations between 0.5 and 2.0 μM. The effects of Cs and Ca suggest that an increase in net inward current (block of IK1) underlies the positive chronotropic effect of Cs and that the pacemaker mechanism is determined by a Ca inward current or an IT1 type current modulated by variations of Cai. In the chick reaggregates (i) Cs and tetraethylammonium decreased the beating rate that was mainly brought about by a decrease in the slope of diastolic depolarization. (ii) Ca increased the beating rate but to a lesser degree than in the rat and there was no decrease of the beating rate at higher concentrations. (iii) The increase in the beating rate was not mediated by an increase in the slope of the diastolic potential but mainly by a depolarization of the maximum diastolic potential. (iv) Verapamil inhibited electrogenesis before any change in the diastolic potential was evident. The negative chronotropic effect of Cs and tetraethylammonium is compatible with the notion that a voltage- and time-dependent K current was inhibited and that this current determines the pacemaker. Moreover, the Ca component of the pacemaker mechanism in explanted rat ventricle cells resembles either that of the sinoatrial node or represents triggered activity.Key words: pacemaker mechanism, cultured cardiac cells, K-channel blocker, calcium, verapamil.

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Modulation of the viability of immature cardiac myocytes by cardiac fibroblasts after hypothermic preservation—its values as a technique for evaluation of storage solutions

Cardiology in the Young ◽

10.1017/s1047951100011665 ◽

1995 ◽

Vol 5 (2) ◽

pp. 110-117

Author(s):

Hiroyuki Orita ◽

Manabu Fukasawa ◽

Hideaki Uchino ◽

Kana Fukui ◽

Minoru Kohi ◽

...

Keyword(s):

Cardiac Myocytes ◽

Cardiac Fibroblasts ◽

Complete Recovery ◽

The Other ◽

Neonatal Rat ◽

University Of Wisconsin ◽

Hypothermic Preservation ◽

Storage Solutions ◽

Before And After ◽

Beating Rate

AbstractWe evaluated the modulation of the viability of immature cardiac myocytes by cardiac fibroblasts after hypothermic preservation using three types of storage solutions—saline, University of Wisconsin solution, and MCDB 107 medium. Cardiac myocytes and fibroblasts were isolated from neonatal rat ventricles, and cultures of myocytes only or co-cultures with fibroblasts (myocyte: fibroblast 2:1) were established. On the fourth day of culture, the cultures were incubated at 4 °C for 6, 12, 18 and 24 hours in the different storage solutions. Enzymes were measured in the storage solutions immediately before and after hypothermic incubation. The cultures were then incubated for an additional 24 hours at 37 °C to evaluate the recovery of the myocyte beating rate. The myocyte beating rate in the co-culture groups showed significantly higher recovery ratios than the corresponding groups in which only myocytes were cultured. Complete recovery was observed in the group co-cultured in MCDB medium 24 hours after hypothermic incubation (83.4% of control—beating rate prior to hypothermic incubation) compared to the other co-cultured groups (15.4, 0%, respectively). Release of enzymes in the co-cultures was significantly suppressed compared to the cultured myocytes, and the greatest suppression was found after 24 hours of incubation in MCDB medium (CPK: 36.6 mIU/flask, LDH: 281.2 mIU/flask) compared to the other two co-cultured groups (CPK: 181.1, 281.1; LDH: 501.7, 773.2). Cardiac fibroblasts diminished myocytic injury after hypothermic preservation using various storage solutions, in which MCDB 107 medium showed the best overall protective effect. Thus, cardiac fibroblasts may play an important role in controlling myocytic viability under various hypothermic conditions.

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Protection of Cardiac Myocytes from Hypothermic Injury by Cardiac Fibroblasts Isolated from Neonatal Rat Ventricle

Journal of Surgical Research ◽

10.1006/jsre.1993.1199 ◽

1993 ◽

Vol 55 (6) ◽

pp. 654-658 ◽

Cited By ~ 13

Author(s):

Hiroyuki Orita ◽

Manabu Fukasawa ◽

Shigeki Hirooka ◽

Kana Fukui ◽

Minoru Kohi ◽

...

Keyword(s):

Cardiac Myocytes ◽

Cardiac Fibroblasts ◽

Neonatal Rat ◽

Hypothermic Injury ◽

Rat Ventricle

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Abstract 301: Targeting Mixed Lineage Kinases in pathological cardiac fibroblast activation and intercellular communication

Circulation Research ◽

10.1161/res.113.suppl_1.a301 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Emily M Schulz ◽

Melissa L Martin ◽

Val S Goodfellow ◽

Stephen Dewhurst ◽

Harris A Gelbard ◽

...

Keyword(s):

Small Molecule ◽

Cell Activation ◽

Cardiac Myocyte ◽

Myocardial Hypertrophy ◽

Cardiac Fibroblasts ◽

Cardiac Injury ◽

Mitogen Activated Protein Kinase ◽

Neonatal Rat ◽

Tissue Type ◽

Heart Weight

Cardiovascular disease (CVD) and the final clinical iteration, heart failure (HF), affect more than 82 million Americans yearly, affects >5.7 million). Although the cardiac myocyte has long been the focus of cardiac cellular research, our lab and others have demonstrated that pathological activation of cardiac fibroblasts (CF) after onset of disease or cardiac injury is also a key player in HF pathogenesis. Pathologic activation of CFs causes a release of various paracrine and autocrine factors that target cardiomyocytes (CM), local inflammatory cells and the CFs themselves. Elucidating the mechanisms involved in pathological ‘support cell (CF)’- ‘functional cell (CM)’ communication may hold therapeutic promise. Mixed lineage kinase 3 (MLK3) is a stress activated mitogen-activated protein kinase kinase kinase (MAPKKK) involved in pro-apoptotic pathways. Inhibition of MLK3 in HAND results in the attenuation of microglial cell activation, preservation of neuronal function and synaptic structures. The parity between microglia-neuron communication and CF-CM communication and the effects of pathological activation on the respective support cells for each tissue type suggested a role for MLK3 in aberrant CF-CM cross-talk associated with the development and progression of HF. Preliminary data in mice subjected to a pharmacological model of HF (chronic isoproterenol) that were treated with a small molecule inhibitor (URMC-099) of MLK3 demonstrated a reduction in the development of myocardial fibrosis compared to vehicle treated animals. Additionally, small molecule MLK3 inhibition attenuated the development of myocardial hypertrophy as measured by heart weight: body weight and heart weight: tibia length ratios. Studies in cultured neonatal rat ventricular fibroblasts (NRVF) demonstrated that small molecule MLK3 inhibition attenuated CF activation and transition to a myofibroblast phenotype, including reductions of pathologic CF markers such as α-SMA, IL-6, IL-1β, and others. Our data demonstrate that MLK3 plays an important role in pathologic CF-CM communication and myocardial hypertrophy, and suggest that small molecule inhibition of MLK3 holds therapeutic promise for HF.

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Disopyramide phosphate effects on slow and depressed fast responses

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y86-079 ◽

1986 ◽

Vol 64 (4) ◽

pp. 487-491 ◽

Cited By ~ 2

Author(s):

Otto F. Schanne ◽

G. Bkaily ◽

B. Dumais ◽

L. Boutin

Keyword(s):

Spontaneous Activity ◽

Action Potentials ◽

Potassium Conductance ◽

Fast Response ◽

Membrane Depolarization ◽

Neonatal Rat ◽

Slow Response ◽

Beating Rate ◽

Rat Ventricle ◽

Fast Responses

We studied the effects of disopyramide phosphate on explanted neonatal rat ventricle cells exhibiting depressed fast responses or naturally occurring slow response action potentials together with automatic activity. Disopyramide suppressed the spontaneous activity at a concentration of 2.5 μg/mL with a half-maximal value of 10 μg/mL. Before spontaneous activity was lost, there was an increase in beating rate possibly related to membrane depolarization. In depressed fast and slow response action potentials there was an increase in action potential duration (APD) which was consistently found both at the level of the plateau and at 90% repolarization. Comparison of the APD increase observed after disopyramide treatment and that after exposure to 20 mM tetraethylammonium suggested a block of a potassium conductance as a possible cause underlying the change in APD. The [Formula: see text] values of the depressed fast response decreased at constant membrane potential and this was attributed to the local anesthetic effect of the drug. In addition, we report two novel findings: (i) a decrease of [Formula: see text] of the slow response action potentials which may be secondary to membrane depolarization, and (ii) an increase in the duration of slow action potentials, possibly caused by inhibition of a potassium conductance.

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Angiotensin II-induced protein tyrosine phosphorylation in neonatal rat cardiac fibroblasts.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)32215-9 ◽

1994 ◽

Vol 269 (30) ◽

pp. 19626-19632

Author(s):

W. Schorb ◽

T.C. Peeler ◽

N.N. Madigan ◽

K.M. Conrad ◽

K.M. Baker

Keyword(s):

Angiotensin Ii ◽

Tyrosine Phosphorylation ◽

Cardiac Fibroblasts ◽

Neonatal Rat ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine

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Regenerating Damaged Myocardium: A Review of Stem-Cell Therapies for Heart Failure

10.20944/preprints202109.0087.v1 ◽

2021 ◽

Author(s):

Dihan Fan ◽

Hanrong Wu ◽

Huashan Peng ◽

Kaichao Pan ◽

Rongxue Wu

Keyword(s):

Drug Testing ◽

Cardiac Myocyte ◽

Cardiac Fibroblasts ◽

Heart Diseases ◽

Contributing Factors ◽

Myocardial Repair ◽

Cardiovascular Research ◽

Cell Therapies ◽

Induced Pluripotent ◽

Promising Source

Cardiovascular disease (CVD) is one of the contributing factors to more than one-third of human mortality and the leading cause of death worldwide. Cardiac myocyte death is a fundamental process in cardiac pathologies caused by various heart diseases, including myocardial infarction. Thus, strategies for replacing fibrotic tissue in the infarcted region with functional myocardium have long been a goal of cardiovascular research. This review focuses primarily on induced-pluripotent stem cells (iPSCs), which have emerged as perhaps the most promising source of cardiomyocytes for both therapeutic applications and drug testing. We also briefly summarize other stems- and progenitor-cell populations that have been used for regenerative myocardial therapy and attempt to generate cardiomyocytes directly from cardiac fibroblasts (i.e., transdifferentiation), which, if successful, may enable the pool of endogenous cardiac fibroblasts to be used as an in-situ source of cardiomyocytes for myocardial repair.

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Aucubin Protects against TGFβ1-Induced Cardiac Fibroblasts Activation by Mediating the AMPKα/mTOR Signaling Pathway

Planta Medica ◽

10.1055/s-0043-118663 ◽

2017 ◽

Vol 84 (02) ◽

pp. 91-99 ◽

Cited By ~ 6

Author(s):

Yang Xiao ◽

Wei Chang ◽

Qing-Qing Wu ◽

Xiao-Han Jiang ◽

Ming-Xia Duan ◽

...

Keyword(s):

Cardiac Fibrosis ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Smooth Muscle Actin ◽

Mammalian Target Of Rapamycin ◽

Transforming Growth Factor Β1 ◽

Neonatal Rat ◽

Cell Counting ◽

Compound C ◽

Underlying Mechanisms

AbstractFibrosis is a key feature of various cardiovascular diseases and compromises cardiac systolic and diastolic performance. The lack of effective anti-fibrosis drugs is a major contributor to the increasing prevalence of heart failure. The present study was performed to investigate whether the iridoid aucubin alleviates cardiac fibroblast activation and its underlying mechanisms. Neonatal rat cardiac fibroblasts were incubated with aucubin (1, 10, 20, 50 µM) followed by transforming growth factor β1 (TGFβ1, 10 ng/mL) stimulation for 24 h. Fibrosis proliferation was measured by cell counting kit-8 assay. The differentiation of fibroblasts into myofibroblasts was determined by measuring the expression of α-smooth muscle actin. Then, the expressions levels of cardiac fibrosis-related proteins in myofibroblasts were analyzed by western blot and real-time PCR to confirm the anti-fibrosis effect of aucubin. As a result, aucubin suppressed TGFβ1-induced proliferation in fibroblasts and inhibited the TGFβ1-induced activation of fibroblasts to myofibroblasts. In addition, aucubin further attenuated fibrosis-related protein expression in myofibroblasts. Furthermore, this protective effect was related to increased adenosine 5′-monophosphate-activated protein kinase (AMPK) phosphorylation and decreased mammalian target of rapamycin (mTOR) phosphorylation, which was confirmed by an mTOR inhibitor (rapamycin), an AMPK agonist (AICAR) and an AMPKα inhibitor compound C. Collectively, our findings suggest that aucubin protects against TGFβ1-induced fibroblast proliferation, activation and function by regulating the AMPKα/mTOR signal axis.

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Regenerating Damaged Myocardium: A Review of Stem-Cell Therapies for Heart Failure

Cells ◽

10.3390/cells10113125 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3125

Author(s):

Dihan Fan ◽

Hanrong Wu ◽

Kaichao Pan ◽

Huashan Peng ◽

Rongxue Wu

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Cardiac Myocyte ◽

Cardiac Fibroblasts ◽

Heart Diseases ◽

Contributing Factors ◽

Myocardial Repair ◽

Cardiovascular Research ◽

Cell Therapies ◽

Promising Source

Cardiovascular disease (CVD) is one of the contributing factors to more than one-third of human mortality and the leading cause of death worldwide. The death of cardiac myocyte is a fundamental pathological process in cardiac pathologies caused by various heart diseases, including myocardial infarction. Thus, strategies for replacing fibrotic tissue in the infarcted region with functional myocardium have long been a goal of cardiovascular research. This review begins by briefly discussing a variety of somatic stem- and progenitor-cell populations that were frequently studied in early investigations of regenerative myocardial therapy and then focuses primarily on pluripotent stem cells (PSCs), especially induced-pluripotent stem cells (iPSCs), which have emerged as perhaps the most promising source of cardiomyocytes for both therapeutic applications and drug testing. We also describe attempts to generate cardiomyocytes directly from cardiac fibroblasts (i.e., transdifferentiation), which, if successful, may enable the pool of endogenous cardiac fibroblasts to be used as an in-situ source of cardiomyocytes for myocardial repair.

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Abstract 13837: Adrenergic Receptors β2 and β3 Transduce Differential Signals in Cardiac Fibroblasts

Circulation ◽

10.1161/circ.132.suppl_3.13837 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Kota Tonegawa ◽

Hiroyuki Nakayama ◽

Hiromi Igarashi ◽

Sachi Matsunami ◽

Nao Hayamizu ◽

...

Keyword(s):

Heart Failure ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Cell Types ◽

Neonatal Rat ◽

Rt Pcr ◽

Β Blocker ◽

Selective Agonists ◽

Selective Blocker ◽

Camkii Activation

Background: Cardiac fibroblasts (CFs) are the most prevalent cell types in heart and play important roles in cardiac remodeling. While the roles of β-adrenergic receptor (βAR) signaling in cardiomyocytes (CMs) are well characterized, those in CFs remain to be elusive due to lack of convenient method to assess those signaling. There are three subtypes of, βAR β1, β2, β3 and β2AR is reported to be expressed in CFs by which enhances cell proliferation and production of inflammatory cytokines. Clinical efficacy of non-selective β blocker carvedilol for heart failure (HF) surpasses that of β1 selective blocker metoprolol, suggesting critical roles of β2 and β3AR in the pathogenesis of HF. Objective: To elucidate the signaling downstream βARs in CFs in heart. Methods and Results: Caveolae is an important microdomain for signal transduction, such as βAR, present in CMs or CFs. To elucidate βAR signaling of caveolae in CFs, we generated a fusion protein composed of phospholamban (PLN) and caveolin3 (Cav3) representing PKA activation as phosphorylation at S16 of PLN and CaMKII as that at T17 in caveolae. Thus, activation of PKA or CaMKII is detectable by anti-phospho-S16 or T17 antibody, respectively. In neonatal rat CFs (NRCFs) infected PLN-Cav3 adenovirus, stimulation by isoproterenol (ISO) led to enhanced phosphorylation of both S16 and T17, suggesting PKA and CaMKII activation in caveolae of CFs. RT-PCR analyses showed β2AR and β3AR were present in NRCFs. Stimulation with β2AR selective agonists activated both PKA and CaMKII, while β3AR elicited solely PKA activation, analyzed by using β3AR selective agonist/antagonist. In addition, in order to examine the significance of βAR stimulation for heart failure, we administered ISO continuously for two weeks in β2ARKO mice. As a result, fibrosis was suppressed in β2ARKO mice compared with wild-type mice (0.35% vs 2.37%, p<0.05) suggesting critical roles of β2AR in development of cardiac fibrosis caused by βAR stimulation in mice. Conclusions: Both β2 and β3AR are expressed in NRCFs and transduce distinct signaling and β2AR selective stimulation elicit development of cardiac fibrosis via activation of CaMKII signaling. Thus, selective βAR regulation could be potential novel anti-fibrotic therapeutics in HF.

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