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1.
Int J Mol Sci ; 22(21)2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34769252

RESUMO

Involvement of the Toll-like receptor 4 (TLR4) in maladaptive cardiac remodeling and heart failure (HF) upon pressure overload has been studied extensively, but less is known about the role of TLR2. Interplay and redundancy of TLR4 with TLR2 have been reported in other organs but were not investigated during cardiac dysfunction. We explored whether TLR2 deficiency leads to less adverse cardiac remodeling upon chronic pressure overload and whether TLR2 and TLR4 additively contribute to this. We subjected 35 male C57BL/6J mice (wildtype (WT) or TLR2 knockout (KO)) to sham or transverse aortic constriction (TAC) surgery. After 12 weeks, echocardiography and electrocardiography were performed, and hearts were extracted for molecular and histological analysis. TLR2 deficiency (n = 14) was confirmed in all KO mice by PCR and resulted in less hypertrophy (heart weight to tibia length ratio (HW/TL), smaller cross-sectional cardiomyocyte area and decreased brain natriuretic peptide (BNP) mRNA expression, p < 0.05), increased contractility (QRS and QTc, p < 0.05), and less inflammation (e.g., interleukins 6 and 1ß, p < 0.05) after TAC compared to WT animals (n = 11). Even though TLR2 KO TAC animals presented with lower levels of ventricular TLR4 mRNA than WT TAC animals (13.2 ± 0.8 vs. 16.6 ± 0.7 mg/mm, p < 0.01), TLR4 mRNA expression was increased in animals with the largest ventricular mass, highest hypertrophy, and lowest ejection fraction, leading to two distinct groups of TLR2 KO TAC animals with variations in cardiac remodeling. This variation, however, was not seen in WT TAC animals even though heart weight/tibia length correlated with expression of TLR4 in these animals (r = 0.078, p = 0.005). Our data suggest that TLR2 deficiency ameliorates adverse cardiac remodeling and that ventricular TLR2 and TLR4 additively contribute to adverse cardiac remodeling during chronic pressure overload. Therefore, both TLRs may be therapeutic targets to prevent or interfere in the underlying molecular processes.

2.
Physiol Genomics ; 2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34766515

RESUMO

BACKGROUND: Broad cellular functions and diseases including muscular dystrophy, arrhythmogenic right ventricular cardiomyopathy (ARVC5) and cancer are associated with transmembrane protein43 (TMEM43/LUMA). OBJECTIVE: The study aimed to investigate biological roles of TMEM43 through genetic regulation, gene pathways and gene networks, candidate interacting genes and up- or down-stream regulators. METHODS: Cardiac transcriptomes from 40 strains of recombinant inbred BXD mice and two parental strains representing murine genetic reference population (GRP) was applied for genetic correlation, functional enrichment and co-expression network analysis using systems genetics approach. The results were validated in a newly created knock-in Tmem43-S358L mutation mouse model (Tmem43S358L) that displayed signs of cardiac dysfunction, resembling ARVC5 phenotype seen in humans. RESULTS: We found high Tmem43 levels among BXDs with broad variability in expression. Expression of Tmem43 highly negatively correlated with heart mass and heart rate among BXDs, while levels of Tmem43 highly positively correlated with plasma high density lipoproteins (HDL). Through finding differentially expressed genes (DEGs) between Tmem43S358L mutant and wild type (Tmem43WT) lines, 18 pathways (out of 42 found in BXDs GRP) that are involved in ARVC, Hypertrophic cardiomyopathy, Dilated cardiomyopathy, Non-alcoholic fatty liver disease, Alzheimer disease, Parkinson disease and Huntington disease were verified. We further constructed Tmem43-mediated gene network, in which Ctnna1, Adcy6, Gnas, Ndufs6 and Uqcrc2 were significantly altered in Tmem43S358L mice vs Tmem43WT controls. CONCLUSIONS: Our study defined the importance of Tmem43 for cardiac and metabolism related pathways, suggesting that cardiovascular disease-relevant risk factors may also increase risk of metabolic and neurodegenerative diseases via TMEM43-mediated pathways.

3.
JACC Clin Electrophysiol ; 7(11): 1345-1347, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34794663
4.
Front Physiol ; 12: 720190, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34675815

RESUMO

Optogenetic technology provides researchers with spatiotemporally precise tools for stimulation, sensing, and analysis of function in cells, tissues, and organs. These tools can offer low-energy and localized approaches due to the use of the transgenically expressed light gated cation channel Channelrhodopsin-2 (ChR2). While the field began with many neurobiological accomplishments it has also evolved exceptionally well in animal cardiac research, both in vitro and in vivo. Implantable optical devices are being extensively developed to study particular electrophysiological phenomena with the precise control that optogenetics provides. In this review, we highlight recent advances in novel implantable optogenetic devices and their feasibility in cardiac research. Furthermore, we also emphasize the difficulties in translating this technology toward clinical applications and discuss potential solutions for successful clinical translation.

5.
Physiol Rep ; 9(15): e14987, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34337891

RESUMO

INTRODUCTION: Doxorubicin (DOX), an anticancer drug used in chemotherapy, causes significant cardiotoxicity. This study aimed to investigate the effects of DOX on mouse cardiac electrophysiology, in conscious versus anesthetized state. METHODS: Male and female C57BL/6 mice were injected with saline, 20 or 30 mg/kg DOX. ECGs were recorded 5 days post-injection in conscious and isoflurane anesthetized states. ECGs were analyzed using a custom MATLAB software to determine P, PR, QRS, QTc, and RR intervals as well as heart rate variability (HRV). RESULTS: ECGs from the same mouse demonstrated P wave and QTc shortening as well as PR and RR interval prolongation in anesthetized versus conscious saline-treated mice. ECG response to DOX was also modulated by anesthesia. DOX treatment induced significant ECG modulation in female mice alone. While DOX20 treatment caused decrease in P and QRS durations, DOX30 treatment-induced QTc and RR interval prolongation in anesthetized but not in conscious female mice. These data suggest significant sex differences and anesthesia-induced differences in ECG response to DOX. HRV measured in time and frequency domains, a metric of arrhythmia susceptibility, was increased in DOX20-treated mice compared to saline. CONCLUSIONS: This study for the first time identifies that the ECG response to DOX is modulated by anesthesia. Furthermore, this response demonstrated stark sex differences. These findings could have significant implications in clinical diagnosis of DOX cardiotoxicity.

6.
Nat Mater ; 20(11): 1559-1570, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34326506

RESUMO

Flexible electronic/optoelectronic systems that can intimately integrate onto the surfaces of vital organ systems have the potential to offer revolutionary diagnostic and therapeutic capabilities relevant to a wide spectrum of diseases and disorders. The critical interfaces between such technologies and living tissues must provide soft mechanical coupling and efficient optical/electrical/chemical exchange. Here, we introduce a functional adhesive bioelectronic-tissue interface material, in the forms of mechanically compliant, electrically conductive, and optically transparent encapsulating coatings, interfacial layers or supporting matrices. These materials strongly bond both to the surfaces of the devices and to those of different internal organs, with stable adhesion for several days to months, in chemistries that can be tailored to bioresorb at controlled rates. Experimental demonstrations in live animal models include device applications that range from battery-free optoelectronic systems for deep-brain optogenetics and subdermal phototherapy to wireless millimetre-scale pacemakers and flexible multielectrode epicardial arrays. These advances have immediate applicability across nearly all types of bioelectronic/optoelectronic system currently used in animal model studies, and they also have the potential for future treatment of life-threatening diseases and disorders in humans.

7.
Nat Biotechnol ; 39(10): 1228-1238, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34183859

RESUMO

Temporary cardiac pacemakers used in periods of need during surgical recovery involve percutaneous leads and externalized hardware that carry risks of infection, constrain patient mobility and may damage the heart during lead removal. Here we report a leadless, battery-free, fully implantable cardiac pacemaker for postoperative control of cardiac rate and rhythm that undergoes complete dissolution and clearance by natural biological processes after a defined operating timeframe. We show that these devices provide effective pacing of hearts of various sizes in mouse, rat, rabbit, canine and human cardiac models, with tailored geometries and operation timescales, powered by wireless energy transfer. This approach overcomes key disadvantages of traditional temporary pacing devices and may serve as the basis for the next generation of postoperative temporary pacing technology.


Assuntos
Implantes Absorvíveis , Marca-Passo Artificial , Animais , Bloqueio Atrioventricular/terapia , Modelos Animais de Doenças , Cães , Desenho de Equipamento , Humanos , Camundongos , Coelhos , Ratos , Tecnologia sem Fio
9.
Artigo em Inglês | MEDLINE | ID: mdl-34126113

RESUMO

Recent developments in clearing and microscopy enable 3D imaging with cellular resolution up to the whole organ level. These methods have been used extensively in neurobiology, but their uptake in other fields has been much more limited. Application of this approach to the human heart and effective use of the data acquired present challenges of scale and complexity. Four interlinked issues need to be addressed: 1) efficient clearing and labelling of heart tissue, 2) fast microscopic imaging of human-scale samples, 3) handling and processing of multi-terabyte 3D images, and 4) extraction of structural information in computationally tractable structure-based models of cardiac function. Preliminary studies show that each of these requirements can be achieved with the appropriate application and development of existing technologies.

10.
JACC Clin Electrophysiol ; 7(8): 988-999, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33812836

RESUMO

OBJECTIVES: This first-in-human feasibility study was undertaken to translate the novel low-voltage MultiPulse Therapy (MPT) (Cardialen, Inc., Minneapolis, Minnesota), which was previously been shown to be effective in preclinical studies in terminating atrial fibrillation (AF), into clinical use. BACKGROUND: Current treatment options for AF, the most common arrhythmia in clinical practice, have limited success. Previous attempts at treating AF by using implantable devices have been limited by the painful nature of high-voltage shocks. METHODS: Forty-two patients undergoing AF ablation were recruited at 6 investigational centers worldwide. Before ablation, electrode catheters were placed in the coronary sinus, right and/or left atrium, for recording and stimulation. After the induction of AF, MPT, which consists of up to a 3-stage sequence of far- and near-field stimulation pulses of varied amplitude, duration, and interpulse timing, was delivered via temporary intracardiac leads. MPT parameters and delivery methods were iteratively optimized. RESULTS: In the 14 patients from the efficacy phase, MPT terminated 37 of 52 (71%) of AF episodes, with the lowest median energy of 0.36 J (interquartile range [IQR]: 0.14 to 1.21 J) and voltage of 42.5 V (IQR: 25 to 75 V). Overall, 38% of AF terminations occurred within 2 seconds of MPT delivery (p < 0.0001). Shorter time between AF induction and MPT predicted success of MPT in terminating AF (p < 0.001). CONCLUSIONS: MPT effectively terminated AF at voltages and energies known to be well tolerated or painless in some patients. Our results support further studies of the concept of implanted devices for early AF conversion to reduce AF burden, symptoms, and progression.


Assuntos
Fibrilação Atrial , Fibrilação Atrial/cirurgia , Cardioversão Elétrica , Eletrodos , Átrios do Coração , Humanos , Minnesota
11.
Circ Res ; 128(5): e84-e101, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33508947

RESUMO

RATIONALE: Mitral valve prolapse (MVP) is a common valvopathy that leads to mitral insufficiency, heart failure, and sudden death. Functional genomic studies in mitral valves are needed to better characterize MVP-associated variants and target genes. OBJECTIVE: To establish the chromatin accessibility profiles and assess functionality of variants and narrow down target genes at MVP loci. METHODS AND RESULTS: We mapped the open chromatin regions in nuclei from 11 human pathogenic and 7 nonpathogenic mitral valves by an assay for transposase-accessible chromatin with high-throughput sequencing. Open chromatin peaks were globally similar between pathogenic and nonpathogenic valves. Compared with the heart tissue and cardiac fibroblasts, we found that MV-specific assay for transposase-accessible chromatin with high-throughput sequencing peaks are enriched near genes involved in extracellular matrix organization, chondrocyte differentiation, and connective tissue development. One of the most enriched motifs in MV-specific open chromatin peaks was for the nuclear factor of activated T cells family of TFs (transcription factors) involved in valve endocardial and interstitial cell formation. We also found that MVP-associated variants were significantly enriched (P<0.05) in mitral valve open chromatin peaks. Integration of the assay for transposase-accessible chromatin with high-throughput sequencing data with risk loci, extensive functional annotation, and gene reporter assay suggest plausible causal variants for rs2641440 at the SMG6/SRR locus and rs6723013 at the IGFBP2/IGFBP5/TNS1 locus. CRISPR-Cas9 deletion of the sequence including rs6723013 in human fibroblasts correlated with increased expression only for TNS1. Circular chromatin conformation capture followed by high-throughput sequencing experiments provided evidence for several target genes, including SRR, HIC1, and DPH1 at the SMG6/SRR locus and further supported TNS1 as the most likely target gene on chromosome 2. CONCLUSIONS: Here, we describe unprecedented genome-wide open chromatin profiles from human pathogenic and nonpathogenic MVs and report specific gene regulation profiles, compared with the heart. We also report in vitro functional evidence for potential causal variants and target genes at MVP risk loci involving established and new biological mechanisms. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Cromatina/genética , Prolapso da Valva Mitral/genética , Valva Mitral/metabolismo , Polimorfismo de Nucleotídeo Único , Células Cultivadas , Cromatina/metabolismo , Fibroblastos/metabolismo , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Prolapso da Valva Mitral/metabolismo , Telomerase/genética , Tensinas/genética , Transcriptoma
12.
JACC Clin Electrophysiol ; 6(14): 1827-1840, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33357580

RESUMO

OBJECTIVES: This study sought to investigate the shift of leading pacemaker locations in healthy and failing mammalian hearts over the entire range of physiological heart rates (HRs), and to molecularly characterize spatial regions of spontaneous activity. BACKGROUND: A normal heartbeat originates as an action potential in a group of pacemaker cells known as the sinoatrial node (SAN), located near the superior vena cava. HRs and the anatomical site of origin of pacemaker activity in the adult heart are known to dynamically change in response to various physiological inputs, yet the mechanism of this pacemaker shift is not well understood. METHODS: Optical mapping was applied to ex vivo rat and human isolated right atrial tissues, and HRs were modulated with acetylcholine and isoproterenol. RNA sequencing was performed on tissue areas that elicited spontaneous activity, and comparisons were made to neighboring myocardial tissues. RESULTS: Functional and molecular evidence identified and confirmed the presence of 2 competing right atrial pacemakers localized near the superior vena cava and the inferior vena cava-the superior SAN (sSAN) and inferior SAN (iSAN), respectively-which preferentially control the fast and slow HRs. Both of these regions were evident in non-failing rat and human hearts and maintained spontaneous activity in the rat heart when physically separated from one another. Molecular analysis of these 2 pacemaker regions revealed unique but similar transcriptional profiles, suggesting iSAN dominance when the sSAN is silent. CONCLUSIONS: The presence of 2 spatially distinct dominant pacemakers, sSAN and iSAN, in the mammalian heart clarifies previous identification of migrating pacemakers and corresponding changes in P-wave morphology in mammalian species.


Assuntos
Nó Sinoatrial , Veia Cava Superior , Potenciais de Ação , Animais , Átrios do Coração , Frequência Cardíaca , Humanos , Ratos
13.
PLoS One ; 15(12): e0244687, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33351857

RESUMO

[This corrects the article DOI: 10.1371/journal.pone.0231695.].

14.
Nat Commun ; 11(1): 5555, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144559

RESUMO

It is highly debated how cyclic adenosine monophosphate-dependent regulation (CDR) of the major pacemaker channel HCN4 in the sinoatrial node (SAN) is involved in heart rate regulation by the autonomic nervous system. We addressed this question using a knockin mouse line expressing cyclic adenosine monophosphate-insensitive HCN4 channels. This mouse line displayed a complex cardiac phenotype characterized by sinus dysrhythmia, severe sinus bradycardia, sinus pauses and chronotropic incompetence. Furthermore, the absence of CDR leads to inappropriately enhanced heart rate responses of the SAN to vagal nerve activity in vivo. The mechanism underlying these symptoms can be explained by the presence of nonfiring pacemaker cells. We provide evidence that a tonic and mutual interaction process (tonic entrainment) between firing and nonfiring cells slows down the overall rhythm of the SAN. Most importantly, we show that the proportion of firing cells can be increased by CDR of HCN4 to efficiently oppose enhanced responses to vagal activity. In conclusion, we provide evidence for a novel role of CDR of HCN4 for the central pacemaker process in the sinoatrial node.


Assuntos
Relógios Biológicos , AMP Cíclico/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Nó Sinoatrial/patologia , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/complicações , Arritmias Cardíacas/patologia , Relógios Biológicos/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Bradicardia/complicações , Bradicardia/patologia , Carbacol/farmacologia , Eletrocardiografia , Feminino , Células HEK293 , Coração/efeitos dos fármacos , Coração/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes , Nó Sinoatrial/fisiopatologia , Nervo Vago/efeitos dos fármacos , Nervo Vago/fisiopatologia
15.
Am J Physiol Heart Circ Physiol ; 319(5): H1059-H1068, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-33036546

RESUMO

The prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) quickly reached pandemic proportions, and knowledge about this virus and coronavirus disease 2019 (COVID-19) has expanded rapidly. This review focuses primarily on mechanisms that contribute to acute cardiac injury and dysfunction, which are common in patients with severe disease. The etiology of cardiac injury is multifactorial, and the extent is likely enhanced by preexisting cardiovascular disease. Disruption of homeostatic mechanisms secondary to pulmonary pathology ranks high on the list, and there is growing evidence that direct infection of cardiac cells can occur. Angiotensin-converting enzyme 2 (ACE2) plays a central role in COVID-19 and is a necessary receptor for viral entry into human cells. ACE2 normally not only eliminates angiotensin II (Ang II) by converting it to Ang-(1-7) but also elicits a beneficial response profile counteracting that of Ang II. Molecular analyses of single nuclei from human hearts have shown that ACE2 is most highly expressed by pericytes. Given the important roles that pericytes have in the microvasculature, infection of these cells could compromise myocardial supply to meet metabolic demand. Furthermore, ACE2 activity is crucial for opposing adverse effects of locally generated Ang II, so virus-mediated internalization of ACE2 could exacerbate pathology by this mechanism. While the role of cardiac pericytes in acute heart injury by SARS-CoV-2 requires investigation, expression of ACE2 by these cells has broader implications for cardiac pathophysiology.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/enzimologia , Cardiopatias/enzimologia , Peptidil Dipeptidase A/metabolismo , Pericitos/enzimologia , Pneumonia Viral/enzimologia , Internalização do Vírus , Enzima de Conversão de Angiotensina 2 , Animais , COVID-19 , Infecções por Coronavirus/virologia , Cardiopatias/fisiopatologia , Cardiopatias/virologia , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Pericitos/virologia , Pneumonia Viral/virologia , SARS-CoV-2
16.
Toxicol Appl Pharmacol ; 406: 115213, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32877659

RESUMO

The limited availability of human heart tissue and its complex cell composition are major limiting factors for the reliable testing of drug efficacy and toxicity. Recently, we developed functional human and pig heart slice biomimetic culture systems that preserve the viability and functionality of 300 µm heart slices for up to 6 days. Here, we tested the reliability of this culture system for testing the cardiotoxicity of anti-cancer drugs. We tested three anti-cancer drugs (doxorubicin, trastuzumab, and sunitinib) with known different mechanisms of cardiotoxicity at three concentrations and assessed the effect of these drugs on heart slice viability, structure, function and gene expression. Slices incubated with any of these drugs for 48 h showed diminished in viability as well as loss of cardiomyocyte structure and function. Mechanistically, RNA sequencing of doxorubicin-treated tissues demonstrated a significant downregulation of cardiac genes and upregulation of oxidative stress responses. Trastuzumab treatment downregulated cardiac muscle contraction-related genes consistent with its clinically known effect on cardiomyocytes. Interestingly, sunitinib treatment resulted in significant downregulation of angiogenesis-related genes, in line with its mechanism of action. Similar to hiPS-derived-cardiomyocytes, heart slices recapitulated the expected toxicity of doxorubicin and trastuzumab, however, slices were superior in detecting sunitinib cardiotoxicity and mechanism in the clinically relevant concentration range of 0.1-1 µM. These results indicate that heart slice culture models have the potential to become a reliable platform for testing and elucidating mechanisms of drug cardiotoxicity.


Assuntos
Cardiotoxicidade , Cardiotoxinas/efeitos adversos , Coração/efeitos dos fármacos , Modelos Biológicos , Técnicas de Cultura de Tecidos , Adulto , Idoso , Animais , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Doxorrubicina/efeitos adversos , Feminino , Coração/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Pessoa de Meia-Idade , Suínos , Trastuzumab/efeitos adversos
17.
Nat Biomed Eng ; 4(10): 997-1009, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32895515

RESUMO

The rigidity and relatively primitive modes of operation of catheters equipped with sensing or actuation elements impede their conformal contact with soft-tissue surfaces, limit the scope of their uses, lengthen surgical times and increase the need for advanced surgical skills. Here, we report materials, device designs and fabrication approaches for integrating advanced electronic functionality with catheters for minimally invasive forms of cardiac surgery. By using multiphysics modelling, plastic heart models and Langendorff animal and human hearts, we show that soft electronic arrays in multilayer configurations on endocardial balloon catheters can establish conformal contact with curved tissue surfaces, support high-density spatiotemporal mapping of temperature, pressure and electrophysiological parameters and allow for programmable electrical stimulation, radiofrequency ablation and irreversible electroporation. Integrating multimodal and multiplexing capabilities into minimally invasive surgical instruments may improve surgical performance and patient outcomes.


Assuntos
Cateteres Cardíacos , Eletrônica/instrumentação , Monitorização Intraoperatória/instrumentação , Monitorização Intraoperatória/métodos , Animais , Ablação por Cateter , Eletroporação , Desenho de Equipamento , Feminino , Análise de Elementos Finitos , Ventrículos do Coração/cirurgia , Humanos , Pressão , Coelhos , Temperatura
19.
Am J Physiol Heart Circ Physiol ; 319(4): H775-H786, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32822209

RESUMO

The efficacy of an anthracycline antibiotic doxorubicin (DOX) as a chemotherapeutic agent is limited by dose-dependent cardiotoxicity. DOX is associated with activation of intracellular stress signaling pathways including p38 MAPKs. While previous studies have implicated p38 MAPK signaling in DOX-induced cardiac injury, the roles of the individual p38 isoforms, specifically, of the alternative isoforms p38γ and p38δ, remain uncharacterized. We aimed to determine the potential cardioprotective effects of p38γ and p38δ genetic deletion in mice subjected to acute DOX treatment. Male and female wild-type (WT), p38γ-/-, p38δ-/-, and p38γ-/-δ-/- mice were injected with 30 mg/kg DOX and their survival was tracked for 10 days. During this period, cardiac function was assessed by echocardiography and electrocardiography and fibrosis by Picro Sirius Red staining. Immunoblotting was performed to assess the expression of signaling proteins and markers linked to autophagy. Significantly improved survival was observed in p38δ-/- female mice post-DOX relative to WT females, but not in p38γ-/- or p38γ-/-δ-/- male or female mice. The improved survival in DOX-treated p38δ-/- females was associated with decreased fibrosis, increased cardiac output and LV diameter relative to DOX-treated WT females, and similar to saline-treated controls. Structural and echocardiographic parameters were either unchanged or worsened in all other groups. Increased autophagy, as suggested by increased LC3-II level, and decreased mammalian target of rapamycin activation was also observed in DOX-treated p38δ-/- females. p38δ plays a crucial role in promoting DOX-induced cardiotoxicity in female mice by inhibiting autophagy. Therefore, p38δ targeting could be a potential cardioprotective strategy in anthracycline chemotherapy.NEW & NOTEWORTHY This study for the first time identifies the sex-specific roles of the alternative p38γ and p38δ MAPK isoforms in promoting doxorubicin (DOX) cardiotoxicity. We show that p38δ and p38γ/δ systemic deletion was cardioprotective in female but not in male mice. Cardiac structure and function were preserved in DOX-treated p38δ-/- females and autophagy marker was increased.


Assuntos
Doxorrubicina , Cardiopatias/prevenção & controle , Proteína Quinase 13 Ativada por Mitógeno/deficiência , Miocárdio/enzimologia , Animais , Autofagia/efeitos dos fármacos , Cardiotoxicidade , Modelos Animais de Doenças , Feminino , Fibrose , Técnicas de Inativação de Genes , Cardiopatias/enzimologia , Cardiopatias/genética , Cardiopatias/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Quinase 12 Ativada por Mitógeno/deficiência , Proteína Quinase 12 Ativada por Mitógeno/genética , Proteína Quinase 13 Ativada por Mitógeno/genética , Miocárdio/patologia , Fatores Sexuais , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos
20.
J Vis Exp ; (160)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32628156

RESUMO

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.


Assuntos
Cálcio/fisiologia , Técnicas Eletrofisiológicas Cardíacas , Função Ventricular , Ventrículos do Coração , Humanos , Técnicas In Vitro , Potenciais da Membrana , Microtomia
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