Adult Mouse Heart Research Articles

Lamin A precursor localizes to the Z-disc of sarcomeres in the heart and is dynamically regulated in muscle cell differentiation.

The lamin A precursor, prelamin A, requires extensive processing to yield mature lamin A and effect its primary function as a structural filament of the nucleoskeleton. When processing is perturbed, nuclear accumulation of prelamin A is toxic and causes laminopathic diseases such as Hutchinson-Gilford progeria syndrome and cardiomyopathy. However, the physiological role of prelamin A is largely unknown and we sought to identify novel insights about this. Using rodent heart tissue, primary cells and the C2C12 model of myofibrillogenesis, we investigated the expression and localization patterns of prelamin A in heart and skeletal muscle cells. We found that endogenous prelamin A was detectable in mouse heart localized to the sarcomere in both adult mouse heart and isolated neonatal rat cardiomyocytes. We investigated the regulation of prelamin A in C2C12 myofibrillogenesis and found it was dynamically regulated and organized into striations upon myofibril formation, colocalizing with the Z-disc protein α-actinin. These data provide evidence that prelamin A is a component of the sarcomere, underpinning a physiological purpose for unprocessed prelamin A. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Consequences of PDGFRα+ fibroblast reduction in adult murine hearts.

Fibroblasts produce the majority of collagen in the heart and are thought to regulate extracellular matrix (ECM) turnover. Although fibrosis accompanies many cardiac pathologies and is generally deleterious, the role of fibroblasts in maintaining the basal ECM network and in fibrosis in vivo is poorly understood. We genetically ablated fibroblasts in mice to evaluate the impact on homeostasis of adult ECM and cardiac function after injury. Fibroblast-ablated mice demonstrated a substantive reduction in cardiac fibroblasts, but fibrillar collagen and the ECM proteome were not overtly altered when evaluated by quantitative mass spectrometry and N-terminomics. However, the distribution and quantity of collagen VI, microfibrillar collagen that forms an open network with the basement membrane, was reduced. In fibroblast-ablated mice, cardiac function was better preserved following angiotensin II/phenylephrine (AngII/PE)-induced fibrosis and myocardial infarction (MI). Analysis of cardiomyocyte function demonstrated altered sarcomere shortening and slowed calcium decline in both uninjured and AngII/PE-infused fibroblast-ablated mice. After MI, the residual resident fibroblasts responded to injury, albeit with reduced proliferation and numbers immediately after injury. These results indicate that the adult mouse heart tolerates a significant degree of fibroblast loss with a potentially beneficial impact on cardiac function after injury. The cardioprotective effect of controlled fibroblast reduction may have therapeutic value in heart disease.

Multi-species meta-analysis identifies transcriptional signatures associated with cardiac endothelial responses in the ischaemic heart.

Myocardial infarction remains the leading cause of heart failure. The adult human heart lacks the capacity to undergo endogenous regeneration. New blood vessel growth is integral to regenerative medicine necessitating a comprehensive understanding of the pathways that regulate vascular regeneration. We sought to define the transcriptomic dynamics of coronary endothelial cells following ischaemic injuries in the developing and adult mouse and human heart and to identify new mechanistic insights and targets for cardiovascular regeneration. We carried out a comprehensive meta-analysis of integrated single cell RNA-sequencing data of coronary vascular endothelial cells from the developing and adult mouse and human heart spanning healthy and acute and chronic ischaemic cardiac disease. We identified species-conserved gene regulatory pathways aligned to endogenous neovascularisation. We annotated injury-associated temporal shifts of the endothelial transcriptome and validated four genes: VEGF-C, KLF4, EGR1 and ZFP36. Moreover, we showed that ZFP36 regulates human coronary endothelial cell proliferation and defined that VEGF-C administration in vivo enhances clonal expansion of the cardiac vasculature post-myocardial infarction. Finally, we constructed a coronary endothelial cell meta-atlas, CrescENDO, to empower future in-depth research to target pathways associated with coronary neovascularisation. We present a high-resolution single cell meta-atlas of healthy and injured coronary endothelial cells in the mouse and human heart, revealing a suite of novel targets with great potential to promote vascular regeneration, and providing a rich resource for therapeutic development. Myocardial infarction is the leading cause of heart failure, a condition with high morbidity and mortality. Vascular regeneration is vital to tissue survival and functional restoration while a comprehensive understanding of the underpinning mechanisms is lacking. We show that the meta-analysis of single cell RNA-sequencing data excels at deciphering multi-faceted vascular responses conserved between mouse and human following ischaemic injury. We envision that these data will inform future strategies and accelerate therapeutic development for promoting vascular regeneration in the ischaemic heart.

Ablation of cardiomyocyte-derived BDNF during development causes myocardial degeneration and heart failure in the adult mouse heart

ObjectiveBrain-derived neurotrophic factor (BDNF) and its receptor TrkB-T1 were recently found to be expressed in cardiomyocytes. However, the functional role of cardiomyocyte-derived BDNF in heart pathophysiology is not yet fully known. Recent studies revealed that BDNF-TrkB pathway plays a critical role to maintain integrity of cardiac structure and function, cardiac pathology and regeneration of myocardial infarction (MI). Therefore, the BDNF-TrkB pathway may be a novel target for myocardial pathophysiology in the adult heart.Approach and resultsIn the present study, we established a cardiomyocyte-derived BDNF conditional knockout mouse in which BDNF expression in developing cardiomyocytes is ablated under the control of the Myosin heavy chain 6 (MYH6) promoter. The results of the present study show that ablation of cardiomyocyte-derived BDNF during development does not impair survival, growth or reproduction; however, in the young adult heart, it causes cardiomyocyte death, degeneration of the myocardium, cardiomyocyte hypertrophy, left atrial appendage thrombosis, decreased cardiac function, increased cardiac inflammation and ROS activity, and metabolic disorders, leading to heart failure (HF) in the adult heart and eventually resulting in a decrease in the one-year survival rate. In addition, ablation of cardiomyocyte-derived BDNF during the developmental stage leads to exacerbation of cardiac dysfunction and poor regeneration after MI in adult hearts.ConclusionCardiomyocyte-derived BDNF is irreplaceable for maintaining the integrity of cardiac structure and function in the adult heart and regeneration after MI. Therefore, the BDNF-TrkB pathway will be a novel target for myocardial pathophysiology in the adult heart.

Blood flow modeling reveals improved collateral artery performance during the regenerative period in mammalian hearts.

Collateral arteries bridge opposing artery branches, forming a natural bypass that can deliver blood flow downstream of an occlusion. Inducing coronary collateral arteries could treat cardiac ischemia, but more knowledge on their developmental mechanisms and functional capabilities is required. Here we used whole-organ imaging and three-dimensional computational fluid dynamics modeling to define spatial architecture and predict blood flow through collaterals in neonate and adult mouse hearts. Neonate collaterals were more numerous, larger in diameter and more effective at restoring blood flow. Decreased blood flow restoration in adults arose because during postnatal growth coronary arteries expanded by adding branches rather than increasing diameters, altering pressure distributions. In humans, adult hearts with total coronary occlusions averaged 2 large collaterals, with predicted moderate function, while normal fetal hearts showed over 40 collaterals, likely too small to be functionally relevant. Thus, we quantify the functional impact of collateral arteries during heart regeneration and repair-a critical step toward realizing their therapeutic potential.

Computational insights on coronary artery function.

Collateral arteries may act as natural bypasses that reduce hypoperfusion after a coronary blockage. 3D imaging of neonatal and adult mouse hearts, plus human fetal and diseased adult hearts, is now used to computationally predict flow within the heart, and understand the cardioprotective role of collateral arteries in vivo.

Abstract P2050: Acetyl-coa Synthesis Is Required For Chromatin Remodeling In Myofibroblast Differentiation And Persistence

Heart failure (HF) features fibrotic remodeling by myofibroblasts, a differentiated fibroblast population responsible for wound repair. Discovering mechanisms of myofibroblast formation and function may yield novel targets to delay or reverse HF. Myofibroblasts metabolically reprogram to mediate transcriptional activation of fibrotic genes through chromatin remodeling. We previously reported that α-ketoglutarate production for histone demethylation is essential for cardiac fibrosis, but metabolic regulation of histone acetylation in fibrosis is unclear. ATP-citrate lyase (ACLY) and Acetyl-coenzyme A synthetase 2 (ACSS2) synthesize acetyl-CoA in the cytoplasm and have also been reported to supply substrate for histone acetyltransferases in the nucleus to modify regulatory histone residues. To investigate acetyl-CoA metabolism in myofibroblast formation, we stimulated adult mouse and human cardiac fibroblasts (CFs) with the pro-fibrotic agonist transforming growth factor β (TGFβ) and treated cells with pharmacological inhibitors of ACLY and ACSS2. Either ACLY or ACSS2 inhibition sufficiently reverted differentiated myofibroblasts to a non-fibrotic phenotype, even during continuous TGFβ stimulation. Genetic deletion of ACLY in fibroblasts recapitulated these results. Further, ACLY inactivation was sufficient to decrease global histone acetylation. ACLY deletion, specifically in activated myofibroblasts in the adult mouse heart, slowed LV functional decline and remodeling in a model of pressure overload HF. Our data indicate that ACLY and ACSS2 are necessary for myofibroblast differentiation and persistence. We hypothesize that acetyl-CoA synthesis is necessary for histone acetylation required for activation of myofibroblast genes. Ongoing work will determine the molecular mechanisms of both enzymes in chromatin remodeling and in cardiac injury. In summary, acetyl-CoA producing enzymes are attractive clinical targets to reverse cardiac fibrosis.

Abstract P3122: Latent Mouse Model Of HIV-1

Background: Antiretroviral therapy (ART) significantly improves the life span of people living with HIV/AIDS (PLWHA). However, HIV- associated cardiovascular diseases (CVDs) remain one of the leading causes of heart failure and comorbidity in PLWHA. Modern ART regimens reduce viral loads to undetectable levels in HIV+ patients while being less toxic. Nevertheless, HIV proteins are present in the plasma of PLWHA. The HIV protein, Nef, has been found circulating in the plasma of ART-treated patients within extracellular vesicles and in association with immune cells. We hypothesized that circulating HIV Nef proteins may cause an adverse effect on various organ functions. To further understand the effect of Nef on differing organ functions, we have generated a transgenic mouse model which expresses the Nef protein in the presence of Cre recombinase. Methods and Results: Nef transgenic mouse model was generated by cloning HIV-1 Nef ORF in CAG-Lox-CAT vector. Transgenic mice lines were crossed with the alpha myosin heavy chain promoter Cre mice to express Nef protein in the heart. Western blot data validated Nef protein expression in the adult mouse heart. Histological evaluation of the effect of Nef protein expression on heart morphology revealed cardiac hypertrophy. Furthermore, heart function analysis shows that Nef expressing mouse lines have depressed heart function. Conclusion: Our study suggests that Nef causes cardiac dysfunction that mimics cardiomyopathy secondary to latent HIV in PLWHA. Further, this mouse model could be useful in studying the effect of Nef protein in different organs or cells using cell-specific Cre recombinase. Moreover, Nef transgenic mice could be useful in generating new therapeutic tools to control comorbidity in PLWHA.

Proteogenomics reveals sex-biased aging genes and coordinated splicing in cardiac aging.

The risks of heart diseases are significantly modulated by age and sex, but how these factors influence baseline cardiac gene expression remains incompletely understood. Here, we used RNA sequencing and mass spectrometry to compare gene expression in female and male young adult (4 mo) and early aging (20 mo) mouse hearts, identifying thousands of age- and sex-dependent gene expression signatures. Sexually dimorphic cardiac genes are broadly distributed, functioning in mitochondrial metabolism, translation, and other processes. In parallel, we found over 800 genes with differential aging response between male and female, including genes in cAMP and PKA signaling. Analysis of the sex-adjusted aging cardiac transcriptome revealed a widespread remodeling of exon usage patterns that is largely independent from differential gene expression, concomitant with upstream changes in RNA-binding protein and splice factor transcripts. To evaluate the impact of the splicing events on cardiac proteoform composition, we applied an RNA-guided proteomics computational pipeline to analyze the mass spectrometry data and detected hundreds of putative splice variant proteins that have the potential to rewire the cardiac proteome. Taken together, the results here suggest that cardiac aging is associated with 1) widespread sex-biased aging genes and 2) a rewiring of RNA splicing programs, including sex- and age-dependent changes in exon usages and splice patterns that have the potential to influence cardiac protein structure and function. These changes contribute to the emerging evidence for considerable sexual dimorphism in the cardiac aging process that should be considered in the search for disease mechanisms.NEW & NOTEWORTHY Han et al. used proteogenomics to compare male and female mouse hearts at 4 and 20 mo. Sex-biased cardiac genes function in mitochondrial metabolism, translation, autophagy, and other processes. Hundreds of cardiac genes show sex-by-age interactions, that is, sex-biased aging genes. Cardiac aging is accompanied with a remodeling of exon usage in functionally coordinated genes, concomitant with differential expression of RNA-binding proteins and splice factors. These features represent an underinvestigated aspect of cardiac aging that may be relevant to the search for disease mechanisms.

Abstract P2033: Neddylation Regulates Cardiac Mitochondria Homeostasis

Neddylation is a reversible post-translational modification that covalently attaches ubiquitin-like protein NEDD8 to target proteins via NEDD8-specific E1-E2-E3 enzymes. Through regulating the function of various cellular proteins, neddylation impacts diverse cellular processes and disease states. We have previously identified an indispensable role for neddylation in cardiac chamber maturation through sustaining YAP signaling in the developing heart. However, the importance of neddylation in post-mitotic hearts remains unclear. Here we report neddylation as a novel post-translation mechanism regulating mitochondrial homeostasis in adult heart. Inducible deletion of NAE1, a regulatory subunit of NEDD8 E1 enzyme, in adult mouse heart resulted in dilated cardiomyopathy, heart failure, and eventually premature lethality. The severe cardiac phenotype was preceded by accumulation of mitophagic vesicles and mitochondrial elongation, accompanied by pronounced upregulation of mitophagic markers and alterations in mitochondrial dynamics proteins. Moreover, we found that neddylation is required for mitochondrial ubiquitination and turnover. Consequently, pharmacological and genetic inhibition of neddylation accumulated damaged mitochondria, impaired mitochondrial membrane potential, inhibited mitochondrial respiration, and increased cardiomyocyte cell death in both neonatal and adult cardiomyocytes. Mechanistically, neddylation of Cullins is essential for the assembly of a functional multi-subunit Cullin-RING ubiquitin ligase, which appears to mediate mitochondrial ubiquitination following mitochondrial damage. Taken together, our findings suggest that neddylation serves as a novel mechanism surveilling mitochondrial quality and is essential for the function of normal adult heart.

Abstract P2073: Rbx2-CRL5 Mediates Mitochondrial Ubiquitination And Mitophagy To Maintain Myocardial Homeostasis Independent Of Parkin

The ubiquitin (Ub) ligase Parkin has been recognized as a central player in mitophagy. In contrast to its presumed importance in the heart, ablation of Parkin fails to impact homeostatic mitophagy and cardiac function in both young and aging mice, suggesting compensation from other mitochondrial Ub ligases. However, the identities of such additional mitochondrial Ub ligases, as well as their physiological significance in the heart, remain unclear. Here, we demonstrate that Cullin-RING Ub ligase 5 (CRL5) plays a key role in the control of mitochondrial homeostasis in cardiomyocytes. CRL5 belongs to the family of Cullin-RING Ub ligases (CRLs) and consists of the RING box protein RBX2, scaffold protein Cullin 5 (Cul5), and one of 38 substrate receptor proteins. Analysis of the mitochondrial outer membrane-interacting proteome revealed a robust association of CRLs with mitochondria. Pharmacological and genetic perturbations of CRLs inhibited mitochondrial ubiquitination and mitophagy. Subcellular fractionation, live-cell imaging and immunogold electron microscopy further revealed that RBX2 and Cul5, two core components of CRL5, are quickly recruited to mitochondria upon mitochondrial depolarization. Depletion of RBX2 or Cul5 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial function, and increased cell death in cardiomyocytes. Importantly, RBX2 deficiency did not alter Parkin expression, had a more robust inhibitory effect on mitochondrial ubiquitination compared to Parkin deficiency, and remained effective in the absence of Parkin. Moreover, loss of RBX2 attenuated Parkin-mediated ubiquitination, suggesting its dominant, Parkin-independent actions in mitochondria. In vivo , deletion of RBX2 in adult mouse hearts suppressed mitophagic activity and provoked accumulation of damaged mitochondria in myocardium, leading to rapid development of dilated cardiomyopathy and heart failure. Finally, RBX2 was upregulated in pressure overloaded mouse hearts but downregulated in human failing hearts, further supporting its protective role in the heart. Taken together, our data allow us to propose RBX2-CRL5 as a mitochondrial Ub ligase and a physiological regulator of mitochondrial and cardiac integrity.

Restoration of Cardiomyogenesis in Aged Mouse Hearts by Voluntary Exercise.

The human heart has limited capacity to generate new cardiomyocytes and this capacity declines with age. Because loss of cardiomyocytes may contribute to heart failure, it is crucial to explore stimuli of endogenous cardiac regeneration to favorably shift the balance between loss of cardiomyocytes and the birth of new cardiomyocytes in the aged heart. We have previously shown that cardiomyogenesis can be activated by exercise in the young adult mouse heart. Whether exercise also induces cardiomyogenesis in aged hearts, however, is still unknown. Here, we aim to investigate the effect of exercise on the generation of new cardiomyocytes in the aged heart. Aged (20-month-old) mice were subjected to an 8-week voluntary running protocol, and age-matched sedentary animals served as controls. Cardiomyogenesis in aged hearts was assessed on the basis of 15N-thymidine incorporation and multi-isotope imaging mass spectrometry. We analyzed 1793 cardiomyocytes from 5 aged sedentary mice and compared these with 2002 cardiomyocytes from 5 aged exercised mice, followed by advanced histology and imaging to account for ploidy and nucleation status of the cell. RNA sequencing and subsequent bioinformatic analyses were performed to investigate transcriptional changes induced by exercise specifically in aged hearts in comparison with young hearts. Cardiomyogenesis was observed at a significantly higher frequency in exercised compared with sedentary aged hearts on the basis of the detection of mononucleated/diploid 15N-thymidine-labeled cardiomyocytes. No mononucleated/diploid 15N-thymidine-labeled cardiomyocyte was detected in sedentary aged mice. The annual rate of mononucleated/diploid 15N-thymidine-labeled cardiomyocytes in aged exercised mice was 2.3% per year. This compares with our previously reported annual rate of 7.5% in young exercised mice and 1.63% in young sedentary mice. Transcriptional profiling of young and aged exercised murine hearts and their sedentary controls revealed that exercise induces pathways related to circadian rhythm, irrespective of age. One known oscillating transcript, however, that was exclusively upregulated in aged exercised hearts, was isoform 1.4 of regulator of calcineurin, whose regulation and functional role were explored further. Our data demonstrate that voluntary running in part restores cardiomyogenesis in aged mice and suggest that pathways associated with circadian rhythm may play a role in physiologically stimulated cardiomyogenesis.

Transmembrane protein 121 as a novel inhibitor of cervical cancer metastasis.

Transmembrane protein 121 (TMEM121) is isolated from the chicken heart using subtraction hybridisation. A previous study by the authors indicated that TMEM121 is highly expressed in adult mouse hearts and acts as an inhibitor of pathological cardiac hypertrophy. In the present study, the association between TMEM121 and cancer was investigated using bioinformatics tools, including Tumour Immune Estimation Resource (TIMER) 2.0, cBioPortal, LinkedOmics analysis, Kaplan-Meier plotter and UALCAN analysis. The expression, genetic variation, gene interaction network and co-expression pattern of TMEM121 in tumours were analysed. The results revealed that TMEM121 was expressed in various tumours and significantly downregulated in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) when compared with its expression in paracancerous tissues, whereas the methylation level of its promoter was increased in tumour tissues. Additionally, associations between TMEM121 and the PI3K/AKT signalling pathway, as well as the expression of cancer-related molecules, were detected. The aforementioned bioinformatics analysis suggests that TMEM121 may be involved in the development of cervical cancer. Therefore, gain-of-function and loss-of-function experiments in HeLa cells were conducted to verify the role of TMEM121 in cervical cancer. The assay using Cell Counting Kit-8 (CCK-8) revealed that the cell viability of HeLa cells with TMEM121 overexpression was significantly reduced. High TMEM121 expression inhibited HeLa cell migration, as indicated by the decrease in the cell scratch healing rate. The western blot assay revealed that TMEM121 overexpression downregulated the expression of B-cell lymphoma 2 (BCL-2), cyclin D1, cyclin E2 and phosphorylated (p)-AKT, while upregulating that of p27, E-cadherin and p-p38. When TMEM121 was knocked down, retinoblastoma protein (RB), p53, p27, E-cadherin, p-JNK and p-p38 were inhibited, but cyclin E1 was promoted. By combining bioinformatics and experimental biology in the present study, the results demonstrated for the first time, to the best of our knowledge, that TMEM121 may be a novel inhibitor of cervical cancer that is linked to multiple signalling pathways, paving the way for the development of novel diagnostic and therapeutic strategies.

Age-Independent Cardiac Protection by Pharmacological Activation of Beclin-1 During Endotoxemia and Its Association With Energy Metabolic Reprograming in Myocardium-A Targeted Metabolomics Study.

Background We showed that Beclin-1-dependent autophagy protects the heart in young and adult mice that underwent endotoxemia. Herein, we compared the potential therapeutic effects of Beclin-1 activating peptide, TB-peptide, on endotoxemia-induced cardiac outcomes in young adult and aged mice. We further evaluated lipopolysaccharide (lipopolysaccharide)-induced and TB-peptide treatment-mediated alterations in myocardial metabolism. Methods and Results C57BL/6J mice that were 10 weeks and 24 months old were challenged by lipopolysaccharide using doses at which cardiac dysfunction occurred. Following the treatment of TB-peptide or control vehicle, heart contractility, circulating cytokines, and myocardial autophagy were evaluated. We detected that TB-peptide boosted autophagy, attenuated cytokines, and improved cardiac performance in both young and aged mice during endotoxemia. A targeted metabolomics assay was designed to detect a pool of 361 known metabolites, of which 156 were detected in at least 1 of the heart tissue samples. Lipopolysaccharide-induced impairments were found in glucose and amino acid metabolisms in mice of all ages, and TB-peptide ameliorated these alterations. However, lipid metabolites were upregulated in the young group but moderately downregulated in the aged by lipopolysaccharide, suggesting an age-dependent response. TB-peptide mitigated lipopolysaccharide-mediated trend of lipids in the young mice but had little effect on the aged. (Study registration: Project DOI: https://doi.org/10.21228/M8K11W). Conclusions Pharmacological activation of Beclin-1 by TB-peptide is cardiac protective in both young and aged population during endotoxemia, suggest a therapeutic potential for sepsis-induced cardiomyopathy. Metabolomics analysis suggests that an age-independent protection by TB-peptide is associated with reprograming of energy production via glucose and amino acid metabolisms.

Interrelation between α-Cardiac Actin Treadmilling and Myocardin-Related Transcription Factor-A Nuclear Shuttling in Cardiomyocytes.

Myocardin-related transcription factors (MRTFs) play a central role in the regulation of actin expression and cytoskeletal dynamics that are controlled by Rho GTPases. SRF is a ubiquitous transcription factor strongly expressed in muscular tissues. The depletion of SRF in the adult mouse heart leads to severe dilated cardiomyopathy associated with the down-regulation of target genes encoding sarcomeric proteins including α-cardiac actin. The regulatory triad, composed of SRF, its cofactor MRTFA and actin, plays a major role in the coordination of the nuclear transcriptional response to adapt actin filament dynamics associated with changes in cell shape, and contractile and migratory activities. Most of the knowledge on the regulation of the SRF–MRTF–Actin axis has been obtained in non-muscle cells with α-actin and smooth muscle cells with α-smooth actin. Here, we visualized for the first time by a time-lapse video, the nucleocytoplasmic shuttling of MRTFA induced by serum or pro-hypertrophic agonists such as angiotensin II, phenylephrine and endothelin-1, using an MRTFA-GFP adenovirus in cultures of neonatal rat cardiomyocytes. We showed that an inhibitor of the RhoA/ROCK signaling pathway leads to an α-cardiac actin polymerization disruption and inhibition of MRTFA nucleocytoplasmic shuttling. Moreover, inhibition of the PI3K/Akt signaling pathway also prevents the entry of MRTFA into the nuclei. Our findings point out a central role of the SRF–MRTFA–actin axis in cardiac remodeling.

Pathophysiological study of the loss of function of the Tbx5 gene in the ventricular conduction system

Patients with Holt-Oram syndrome suffer from cardiac conduction disorders and are often associated with mutations in the gene encoding the cardiac transcription factor Tbx5. In mice, it has been shown that Tbx5 is essential for maintaining the structure and function of the atrioventricular node and His bundle. Conditional deletion of Tbx5 in the adult mouse heart is associated with conduction defects, slowing of the ventricular conduction system (VCS), QRS prolongation, bundle branch blocks and atrial fibrillation. However, there were no data on the morphological and functional consequences of a cardiac deletion of Tbx5 during embryonic development. The aim is to analyze the morphological and functional consequences in the adult heart of the deletion of the Tbx5 gene in the progenitor cells of the cardiac VCS at the fetal stage. Tbx5 gene was inactivated in VCS progenitors using Cx40-CreERT2:Tbx5 floxed/floxed transgenic mice. Cre-mediated recombination was induced by oral gavage of pregnant females at E13-14 during trabecular compaction. Surface ECGs were achieved monthly until 3 months of age, when immunofluorescence staining was performed on mouse hearts. In 3-week-old Tbx5Fl/Fl mice ( n = 16), ECG analysis showed a decrease in P- ( P < 0.01) and T-wave amplitudes ( P < 0.01) and an increased QRS interval duration ( P < 0.05) compared to controls ( n = 12). At 2 months, the mice did not show any worsening of the phenotype. In addition, severe functional defects in Tbx5Fl/Fl mice, including atrial susceptibility, extrasystoles, and arrhythmias were observed by 3 weeks of age. Moreover, left bundle branches block and slowing of ventricular depolarization were also detected in Tbx5Fl/Fl mice. A β-adrenergic stimulation revealed a large tendency for atrial fibrillation and extrasystoles in 88% of Tbx5Fl/Fl mice against 50% of Tbx5Fl/+ mice and 43% of control mice. In 2-month-old Tbx5Fl/Fl mice, Purkinje Fiber cells composing the fast-VCS are still present, however, they lose the expression of Contactin-2 and Connexin-40. Furthermore, we observed a drastic loss of Connexin-43 in Tbx5-deleted contractile cardiomyocytes that are close to the VCS, which may have resulted in decreased signal propagation. In this study, we demonstrated that Tbx5 deletion in VCS progenitors prevents their maturation and hence the expression of rapid conduction markers causing disturbances in the propagation of ventricular electrical activity.

Proteomic profiling of concurrently isolated primary microvascular endothelial cells, pericytes, and vascular smooth muscle cells from adult mouse heart

The microcirculation serves crucial functions in adult heart, distinct from those carried out by epicardial vessels. Microvessels are governed by unique regulatory mechanisms, impairment of which leads to microvessel-specific pathology. There are few treatment options for patients with microvascular heart disease, primarily due to limited understanding of underlying pathology. High throughput mRNA sequencing and protein expression profiling in specific cells can improve our understanding of microvessel biology and disease at the molecular level. Understanding responses of individual microvascular cells to the same physiological or pathophysiological stimuli requires the ability to isolate the specific cell types that comprise the functional units of the microcirculation in the heart, preferably from the same heart, to ensure that different cells have been exposed to the same in-vivo conditions. We developed an integrated process for simultaneous isolation and culture of the main cell types comprising the microcirculation in adult mouse heart: endothelial cells, pericytes, and vascular smooth muscle cells. These cell types were characterized with isobaric labeling quantitative proteomics and mRNA sequencing. We defined microvascular cell proteomes, identified novel protein markers, and confirmed established cell-specific markers. Our results allow identification of unique markers and regulatory proteins that govern microvascular physiology and pathology.

Endothelial Cell Sprouting in Coronary Collateral Growth

Rationale– Coronary collateral growth is a natural bypass for ischemic heart diseases. It offers tremendous therapeutic benefit, but the process of coronary collateral growth is incompletely understood due to limited preclinical murine models that would enable interrogation of its mechanisms and processes via genetic modification and lineage tracing. Understanding the processes by which coronary collaterals develop can unlock new therapeutic strategies for ischemic heart disease.Objective– To develop a murine model of coronary collateral growth by repetitive ischemia (RI) and investigate the process of coronary collateral growth in adult hearts by lineage tracing.Methods and Results– A murine model of coronary collateral growth was developed using short episodes of repetitive ischemia. Repetitive ischemia resulted in robust collateral growth in adult mouse hearts, validated by high‐resolution micro‐computed tomography. ROSA mT/mG reporter mice with Apln‐CreERT2 transgene were used for lineage tracing of sprouting endothelial cells to determine cellular contributions to coronary collateral growth. Two‐photon and confocal microscopy imaging revealed RI‐induced coronary collateral growth initiated from sprouting Apelin+GFP+endothelial cells. Newly formed capillaries in the collateral‐dependent zone expanded in diameter upon RI stimulation and arterialized with smooth muscle cell recruitment to form mature coronary collateral arteries. Notably, pre‐existing coronary arteries and arterioles were not Apelin+ GFP+, and all Apelin+endothelial cells arose from sprouting capillaries. HIF1α, VEGFR2, CXCR4, MCP‐1, and JAG1 signaling in the collateral‐dependent zone were also upregulated during coronary collateral growth.Conclusions– We developed a murine model of RI‐induced coronary collateral growth, and our data show sprouting EC contribute to coronary collateral growth in adult hearts. Sprouting angiogenesis is shown for the first time to arterialize into mature blood vessels of larger diameter in response to repetitive ischemia in adult hearts.

Cardiomyocyte‐specific Adhesion G Protein‐Coupled Receptor F5 mediates cardiac homeostasis and contractility

G‐protein coupled receptors (GPCRs) serve as pharmacological targets for a third of all currently prescribed drugs, including gold‐standard heart failure (HF) therapeutics. However, the second largest class of GPCRs, adhesion GPCRs (aGPCRs), have yet to be established as clinical targets, representing an area of highly unexplored druggable potential. RNA‐sequencing analysis of left ventricle (LV) tissue isolated from healthy hearts of adult mice identified ADGRF5 as the most highly expressed aGPCR, which was validated in isolated adult mouse cardiomyocytes (AMCM) and shown to increase in response to transaortic constriction (TAC) surgery. To determine the impact of ADGRF5 on cardiac parameters normally or during HF, we generated constitutive cardiomyocyte‐specific (αMHC‐Cre‐driven) ADGRF5 knockout mice (CM‐ADGRF5‐KO), which displayed normal cardiac structure and function at 12 weeks of age, as assessed via echocardiography, gravimetrics and immunohistochemistry. However, over time CM‐ADGRF5‐KO mice developed worsened cardiac function and remodeling with increased mortality, even in the absence of pathologic insult, suggesting ADGRF5 provides a homeostatic regulatory function in the heart. Further, we observed that CM‐ADGRF5‐KO mice experienced an accelerated decrease in cardiac function following transaortic constriction injury (TAC) surgery with greater pathologic remodeling. To identify the mechanistic underpinnings which underlie these phenotypes, we have identified an increase in NFAT‐luciferase activity following stimulation of CM‐ADGRF5 using a synthetic ligand, GAP10, to activate CM‐ADGRF5, in vitro. This suggests that the receptor couples to Gαq signaling to mediate its effects, an intriguing finding which requires further investigation. In all, these results suggest that ADGRF5 plays an integral role in the maintenance of cardiac health normally and during the development of HF.

Frequency-Dependent Properties of the Hyperpolarization-Activated Cation Current, If, in Adult Mouse Heart Primary Pacemaker Myocytes.

A number of distinct electrophysiological mechanisms that modulate the myogenic spontaneous pacemaker activity in the sinoatrial node (SAN) of the mammalian heart have been investigated extensively. There is agreement that several (3 or 4) different transmembrane ionic current changes (referred to as the voltage clock) are involved; and that the resulting net current interacts with direct and indirect effects of changes in intracellular Ca2+ (the calcium clock). However, significant uncertainties, and important knowledge gaps, remain concerning the functional roles in SAN spontaneous pacing of many of the individual ion channel- or exchanger-mediated transmembrane current changes. We report results from patch clamp studies and mathematical modeling of the hyperpolarization-activated current, If, in the generation/modulation of the diastolic depolarization, or pacemaker potential, produced by individual myocytes that were enzymatically isolated from the adult mouse sinoatrial node (SAN). Amphotericin-mediated patch microelectrode recordings at 35 °C were made under control conditions and in the presence of 5 or 10 nM isoproterenol (ISO). These sets of results were complemented and integrated with mathematical modeling of the current changes that take place in the range of membrane potentials (−70 to −50 mV), which corresponds to the ‘pacemaker depolarization’ in the adult mouse SAN. Our results reveal a very small, but functionally important, approximately steady-state or time-independent current generated by residual activation of If channels that are expressed in these pacemaker myocytes. Recordings of the pacemaker depolarization and action potential, combined with measurements of changes in If, and the well-known increases in the L-type Ca2+ current, ICaL, demonstrated that ICaL activation, is essential for myogenic pacing. Moreover, after being enhanced (approximately 3-fold) by 5 or 10 nM ISO, ICaL contributes significantly to the positive chronotropic effect. Our mathematical model has been developed in an attempt to better understand the underlying mechanisms for the pacemaker depolarization and action potential in adult mouse SAN myocytes. After being updated with our new experimental data describing If, our simulations reveal a novel functional component of If in adult mouse SAN. Computational work carried out with this model also confirms that in the presence of ISO the residual activation of If and opening of ICaL channels combine to generate a net current change during the slow diastolic depolarization phase that is essential for the observed accelerated pacemaking rate of these SAN myocytes.