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1.
J Mol Cell Cardiol ; 131: 53-65, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31005484

RESUMO

AIMS: Atrial contractile dysfunction is associated with increased mortality in heart failure (HF). We have shown previously that a metabolic syndrome-based model of HFpEF and a model of hypertensive heart disease (HHD) have impaired left atrial (LA) function in vivo (rat). In this study we postulate, that left atrial cardiomyocyte (CM) and cardiac fibroblast (CF) paracrine interaction related to the inositol 1,4,5-trisphosphate signalling cascade is pivotal for the manifestation of atrial mechanical dysfunction in HF and that quantitative atrial remodeling is highly disease-dependent. METHODS AND RESULTS: Differential remodeling was observed in HHD and HFpEF as indicated by an increase of atrial size in vivo (HFpEF), unchanged fibrosis (HHD and HFpEF) and a decrease of CM size (HHD). Baseline contractile performance of rat CM in vitro was enhanced in HFpEF. Upon treatment with conditioned medium from their respective stretched CF (CM-SF), CM (at 21 weeks) of WT showed increased Ca2+ transient (CaT) amplitudes related to the paracrine activity of the inotrope endothelin (ET-1) and inositol 1,4,5-trisphosphate induced Ca2+ release. Concentration of ET-1 was increased in CM-SF and atrial tissue from WT as compared to HHD and HFpEF. In HHD, CM-SF had no relevant effect on CaT kinetics. However, in HFpEF, CM-SF increased diastolic Ca2+ and slowed Ca2+ removal, potentially contributing to an in-vivo decompensation. During disease progression (i.e. at 27 weeks), HFpEF displayed dysfunctional excitation-contraction-coupling (ECC) due to lower sarcoplasmic-reticulum Ca2+ content unrelated to CF-CM interaction or ET-1, but associated with enhanced nuclear [Ca2+]. In human patients, tissue ET-1 was not related to the presence of arterial hypertension or obesity. CONCLUSIONS: Atrial remodeling is a complex entity that is highly disease and stage dependent. The activity of fibrosis related to paracrine interaction (e.g. ET-1) might contribute to in vitro and in vivo atrial dysfunction. However, during later stages of disease, ECC is impaired unrelated to CF.

2.
J Vis Exp ; (137)2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-30102264

RESUMO

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease. We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling). We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.


Assuntos
Insuficiência Cardíaca/fisiopatologia , Síndrome Metabólica/complicações , Miócitos Cardíacos/metabolismo , Volume Sistólico/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Ratos
4.
J Mol Cell Cardiol ; 115: 10-19, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29289652

RESUMO

Heart failure (HF) with preserved ejection fraction (HFpEF) is present in about 50% of HF patients. Atrial remodeling is common in HFpEF and associated with increased mortality. We postulate that atrial remodeling is associated with atrial dysfunction in vivo related to alterations in cardiomyocyte Calcium (Ca) signaling and remodeling. We examined atrial function in vivo and Ca transients (CaT) (Fluo4-AM, field stim) in atrial cardiomyocytes of ZSF-1 rats without (Ln; lean hypertensive) and with metabolic syndrome (Ob; obese, hypertensive, diabetic) and HFpEF. RESULTS: At 21weeks Ln showed an increased left ventricular (LV) mass and left ventricular end-diastolic pressure (LVEDP), but unchanged left atrial (LA) size and preserved atrial ejection fraction vs. wild-type (WT). CaT amplitude in atrial cardiomyocytes was increased in Ln (2.9±0.2 vs. 2.3±0.2F/F0 in WT; n=22 cells/group; p<0.05). Studying subcellular Ca release in more detail, we found that local central cytosolic CaT amplitude was increased, while subsarcolemmal CaT amplitudes remained unchanged. Moreover, Sarcoplasmic reticulum (SR) Ca content (caffeine) was preserved while Ca spark frequency and tetracaine-dependent SR Ca leak were significantly increased in Ln. Ob mice developed a HFpEF phenotype in vivo, LA area was significantly increased and atrial in vivo function was impaired, despite increased atrial CaT amplitudes in vitro (2.8±0.2; p<0.05 vs. WT). Ob cells showed alterations of the tubular network possibly contributing to the observed phenotype. CaT kinetics as well as SR Ca in Ob were not significantly different from WT, but SR Ca leak remained increased. Angiotensin II (Ang II) reduced in vitro cytosolic CaT amplitudes and let to active nuclear Ca release in Ob but not in Ln or WT. SUMMARY: In hypertensive ZSF-1 rats, a possibly compensatory increase of cytosolic CaT amplitude and increased SR Ca leak precede atrial remodeling and HFpEF. Atrial remodeling in ZSF-1 HFpEF is associated with an altered tubular network in-vitro and atrial contractile dysfunction in vivo, indicating insufficient compensation. Atrial cardiomyocyte dysfunction in vitro is induced by the addition of angiotensin II.

5.
Heart Fail Rev ; 23(1): 27-36, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29038991

RESUMO

Heart failure and atrial fibrillation are common and responsible for significant mortality of patients. Both share the same risk factors like hypertension, ischemic heart disease, diabetes, obesity, arteriosclerosis, and age. A variety of microscopic and macroscopic changes favor the genesis of atrial fibrillation in patients with preexisting heart failure, altered subcellular Ca2+ homeostasis leading to increased cellular automaticity as well as concomitant fibrosis that are induced by pressure/volume overload and altered neurohumoral states. Atrial fibrillation itself promotes clinical deterioration of patients with preexisting heart failure as atrial contraction significantly contributes to ventricular filling. In addition, atrial fibrillation induced tachycardia can even further compromise ventricular function by inducing tachycardiomyopathy. Even though evidence has been provided that atrial functions significantly and independently of confounding ventricular pathologies, correlate with mortality of heart failure patients, rate and rhythm controls have been shown to be of equal effectiveness in improving mortality. Yet, it also has been shown that cohorts of patients with heart failure benefit from a rhythm control concept regarding symptom control and hospitalization. To date, amiodarone is the most feasible approach to restore sinus rhythm, yet its use is limited by its extensive side-effect profile. In addition, other therapies like catheter-based pulmonary vein isolation are of increasing importance. A wide range of heart failure-specific therapies are available with mixed impact on new onset or perpetuation of atrial fibrillation. This review highlights pathophysiological concepts and possible therapeutic approaches to treat patients with heart failure at risk for or with atrial fibrillation.

7.
J Appl Physiol (1985) ; 119(10): 1233-42, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26183480

RESUMO

Left ventricular hypertrophy (LVH) is the most common myocardial structural abnormality associated with heart failure with preserved ejection fraction (HFpEF). LVH is driven by neurohumoral activation, increased mechanical load, and cytokines associated with arterial hypertension, chronic kidney disease, diabetes, and other comorbidities. Here we discuss the experimental and clinical evidence that links LVH to diastolic dysfunction and qualifies LVH as one diagnostic marker for HFpEF. Mechanisms leading to diastolic dysfunction in LVH are incompletely understood, but may include extracellular matrix changes, vascular dysfunction, as well as altered cardiomyocyte mechano-elastical properties. Beating cardiomyocytes from HFpEF patients have not yet been studied, but we and others have shown increased Ca(2+) turnover and impaired relaxation in cardiomyocytes from hypertrophied hearts. Structural myocardial remodeling can lead to heterogeneity in regional myocardial contractile function, which contributes to diastolic dysfunction in HFpEF. In the clinical setting of patients with compound comorbidities, diastolic dysfunction may occur independently of LVH. This may be one explanation why current approaches to reduce LVH have not been effective to improve symptoms and prognosis in HFpEF. Exercise training, on the other hand, in clinical trials improved exercise tolerance and diastolic function, but did not reduce LVH. Thus current clinical evidence does not support regression of LVH as a surrogate marker for (short-term) improvement of HFpEF.


Assuntos
Insuficiência Cardíaca/patologia , Hipertrofia Ventricular Esquerda/patologia , Miocárdio/patologia , Volume Sistólico/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertrofia Ventricular Esquerda/fisiopatologia
8.
Channels (Austin) ; 9(3): 129-38, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25891132

RESUMO

In rabbit atrial myocytes Ca signaling has unique features due to the lack of transverse (t) tubules, the spatial arrangement of mitochondria and the contribution of inositol-1,4,5-trisphosphate (IP3) receptor-induced Ca release (IICR). During excitation-contraction coupling action potential-induced elevation of cytosolic [Ca] originates in the cell periphery from Ca released from the junctional sarcoplasmic reticulum (j-SR) and then propagates by Ca-induced Ca release from non-junctional (nj-) SR toward the cell center. The subsarcolemmal region between j-SR and the first array of nj-SR Ca release sites is devoid of mitochondria which results in a rapid propagation of activation through this domain, whereas the subsequent propagation through the nj-SR network occurs at a velocity typical for a propagating Ca wave. Inhibition of mitochondrial Ca uptake with the Ca uniporter blocker Ru360 accelerates propagation and increases the amplitude of Ca transients (CaTs) originating from nj-SR. Elevation of cytosolic IP3 levels by rapid photolysis of caged IP3 has profound effects on the magnitude of subcellular CaTs with increased Ca release from nj-SR and enhanced CaTs in the nuclear compartment. IP3 uncaging restricted to the nucleus elicites 'mini'-Ca waves that remain confined to this compartment. Elementary IICR events (Ca puffs) preferentially originate in the nucleus in close physical association with membrane structures of the nuclear envelope and the nucleoplasmic reticulum. The data suggest that in atrial myocytes the nucleus is an autonomous Ca signaling domain where Ca dynamics are primarily governed by IICR.


Assuntos
Sinalização do Cálcio , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Citosol/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Mitocôndrias/metabolismo , Miócitos Cardíacos/fisiologia , Coelhos
9.
J Physiol ; 593(6): 1459-77, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25416623

RESUMO

KEY POINTS: Impaired calcium (Ca(2+)) signalling is the main contributor to depressed ventricular contractile function and occurrence of arrhythmia in heart failure (HF). Here we report that in atrial cells of a rabbit HF model, Ca(2+) signalling is enhanced and we identified the underlying cellular mechanisms. Enhanced Ca(2+) transients (CaTs) are due to upregulation of inositol-1,4,5-trisphosphate receptor induced Ca(2+) release (IICR) and decreased mitochondrial Ca(2+) sequestration. Enhanced IICR, however, together with an increased activity of the sodium-calcium exchange mechanism, also facilitates spontaneous Ca(2+) release in form of arrhythmogenic Ca(2+) waves and spontaneous action potentials, thus enhancing the arrhythmogenic potential of atrial cells. Our data show that enhanced Ca(2+) signalling in HF provides atrial cells with a mechanism to improve ventricular filling and to maintain cardiac output, but also increases the susceptibility to develop atrial arrhythmias facilitated by spontaneous Ca(2+) release. ABSTRACT: We studied excitation-contraction coupling (ECC) and inositol-1,4,5-triphosphate (IP3)-dependent Ca(2+) release in normal and heart failure (HF) rabbit atrial cells. Left ventricular HF was induced by combined volume and pressure overload. In HF atrial myocytes diastolic [Ca(2+)]i was increased, action potential (AP)-induced Ca(2+) transients (CaTs) were larger in amplitude, primarily due to enhanced Ca(2+) release from central non-junctional sarcoplasmic reticulum (SR) and centripetal propagation of activation was accelerated, whereas HF ventricular CaTs were depressed. The larger CaTs were due to enhanced IP3 receptor-induced Ca(2+) release (IICR) and reduced mitochondrial Ca(2+) buffering, consistent with a reduced mitochondrial density and Ca(2+) uptake capacity in HF. Elementary IP3 receptor-mediated Ca(2+) release events (Ca(2+) puffs) were more frequent in HF atrial myoctes and were detected more often in central regions of the non-junctional SR compared to normal cells. HF cells had an overall higher frequency of spontaneous Ca(2+) waves and a larger fraction of waves (termed arrhythmogenic Ca(2+) waves) triggered APs and global CaTs. The higher propensity of arrhythmogenic Ca(2+) waves resulted from the combined action of enhanced IICR and increased activity of sarcolemmal Na(+)-Ca(2+) exchange depolarizing the cell membrane. In conclusion, the data support the hypothesis that in atrial myocytes from hearts with left ventricular failure, enhanced CaTs during ECC exert positive inotropic effects on atrial contractility which facilitates ventricular filling and contributes to maintaining cardiac output. However, HF atrial cells were also more susceptible to developing arrhythmogenic Ca(2+) waves which might form the substrate for atrial rhythm disorders frequently encountered in HF.


Assuntos
Sinalização do Cálcio , Acoplamento Excitação-Contração , Átrios do Coração/metabolismo , Insuficiência Cardíaca/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Átrios do Coração/citologia , Masculino , Miócitos Cardíacos/fisiologia , Coelhos
10.
Front Pharmacol ; 5: 35, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24639654

RESUMO

Calcium plays a crucial role in excitation-contraction coupling (ECC), but it is also a pivotal second messenger activating Ca(2+)-dependent transcription factors in a process termed excitation-transcription coupling (ETC). Evidence accumulated over the past decade indicates a pivotal role of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca(2+) release in the regulation of cytosolic and nuclear Ca(2+) signals. IP3 is generated by stimulation of plasma membrane receptors that couple to phospholipase C (PLC), liberating IP3 from phosphatidylinositol 4,5-bisphosphate (PIP2). An intriguing aspect of IP3 signaling is the presence of the entire PIP2-PLC-IP3 signaling cascade as well as the presence of IP3Rs at the inner and outer membranes of the nuclear envelope (NE) which functions as a Ca(2+) store. The observation that the nucleus is surrounded by its own putative Ca(2+) store raises the possibility that nuclear IP3-dependent Ca(2+) release plays a critical role in ETC. This provides a potential mechanism of regulation that acts locally and autonomously from the global cytosolic Ca(2+) signal underlying ECC. Moreover, there is evidence that: (i) the sarcoplasmic reticulum (SR) and NE are a single contiguous Ca(2+) store; (ii) the nuclear pore complex is the major gateway for Ca(2+) and macromolecules to pass between the cytosol and the nucleoplasm; (iii) the inner membrane of the NE hosts key Ca(2+) handling proteins including the Na(+)/Ca(2+) exchanger (NCX)/GM1 complex, ryanodine receptors (RyRs), nicotinic acid adenine dinucleotide phosphate receptors (NAADPRs), Na(+)/K(+) ATPase, and Na(+)/H(+) exchanger. Thus, it appears that the nucleus represents a Ca(2+) signaling domain equipped with its own ion channels and transporters that allow for complex local Ca(2+) signals. Many experimental and modeling approaches have been used for the study of intracellular Ca(2+) signaling but the key to the understanding of the dual role of Ca(2+) mediating ECC and ECT lays in quantitative differences of local [Ca(2+)] in the nuclear and cytosolic compartment. In this review, we discuss the state of knowledge regarding the origin and the physiological implications of nuclear Ca(2+) transients in different cardiac cell types (adult atrial and ventricular myocytes) as well as experimental and mathematical approaches to study Ca(2+) and IP3 signaling in the cytosol and nucleus. In particular, we focus on the concept that highly localized Ca(2+) signals are required to translocate and activate Ca(2+)-dependent transcription factors (e.g., nuclear factor of activated T-cells, NFAT; histone deacetylase, HDAC) through phosphorylation/dephosphorylation processes.

11.
Front Physiol ; 5: 517, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25628569

RESUMO

Dyssynchronous local Ca release within individual cardiac myocytes has been linked to cellular contractile dysfunction. Differences in Ca kinetics in adjacent cells may also provide a substrate for inefficient contraction and arrhythmias. In a new approach we quantify variation in local Ca transients between adjacent myocytes in the whole heart. Langendorff-perfused mouse hearts were loaded with Fluo-8 AM to detect Ca and Di-4-ANEPPS to visualize cell membranes. A spinning disc confocal microscope with a fast camera allowed us to record Ca signals within an area of 465 µm by 315 µm with an acquisition speed of 55 fps. Images from multiple transients recorded at steady state were registered to their time point in the cardiac cycle to restore averaged local Ca transients with a higher temporal resolution. Local Ca transients within and between adjacent myocytes were compared with regard to amplitude, time to peak and decay at steady state stimulation (250 ms cycle length). Image registration from multiple sequential Ca transients allowed reconstruction of high temporal resolution (2.4 ± 1.3 ms) local CaT in 2D image sets (N = 4 hearts, n = 8 regions). During steady state stimulation, spatial Ca gradients were homogeneous within cells in both directions and independent of distance between measured points. Variation in CaT amplitudes was similar across the short and the long side of neighboring cells. Variations in TAU and TTP were similar in both directions. Isoproterenol enhanced the CaT but not the overall pattern of spatial heterogeneities. Here we detected and analyzed local Ca signals in intact mouse hearts with high temporal and spatial resolution, taking into account 2D arrangement of the cells. We observed significant differences in the variation of CaT amplitude along the long and short axis of cardiac myocytes. Variations of Ca signals between neighboring cells may contribute to the substrate of cardiac remodeling.

12.
Circ Res ; 113(5): 527-38, 2013 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-23825358

RESUMO

RATIONALE: Synchronized release of Ca²âº into the cytosol during each cardiac cycle determines cardiomyocyte contraction. OBJECTIVE: We investigated synchrony of cytosolic [Ca²âº] decay during diastole and the impact of cardiac remodeling. METHODS AND RESULTS: Local cytosolic [Ca²âº] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca²âº] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca²âº release. Stimulation of sarcoplasmic reticulum Ca²âº ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca²âº] decay (TAUglobal). Na⁺/Ca²âº exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca²âº uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. CONCLUSIONS: In cardiomyocytes, cytosolic [Ca²âº] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca²âº] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.


Assuntos
Sinalização do Cálcio/fisiologia , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/citologia , Miócitos Cardíacos/fisiologia , Remodelação Ventricular/fisiologia , Compostos de Anilina/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Colforsina/farmacologia , Citosol/metabolismo , Diástole , Estimulação Elétrica , Etiocolanolona/análogos & derivados , Etiocolanolona/farmacologia , Humanos , Hipertrofia , Hipertrofia Ventricular Esquerda/fisiopatologia , Indóis/farmacologia , Camundongos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Isquemia Miocárdica/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Éteres Fenílicos/farmacologia , Compostos de Rutênio/farmacologia , Sarcômeros/ultraestrutura , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/genética , Sus scrofa , Suínos
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