Browsing by Author "Rubart, Michael"

Now showing 1 - 10 of 21
  • Thumbnail Image
    Item
    Adult Bone Marrow–derived Cells Do Not Acquire Functional Attributes of Cardiomyocytes When Transplanted into Peri-infarct Myocardium
    (Elsevier, 2008-06-01) Scherschel, John A.; Soonpaa, Mark H.; Srour, Edward F.; Field, Loren J.; Rubart, Michael; Microbiology and Immunology, School of Medicine
    (BM) cells after being directly transplanted into the ischemically injured heart remains a controversial issue. In this study, we investigated the ability of transplanted BM cells to develop intracellular calcium ([Ca2+] i ) transients in response to membrane depolarization in situ. Low-density mononuclear (LDM) BM cells, c-kit-enriched (c-kitenr) BM cells, and highly enriched lin– c-kit+ BM cells were obtained from adult transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP), and injected into peri-infarct myocardiums of nontransgenic mice. After 9–10 days the mice were killed, and the hearts were removed, perfused in Langendorff mode, loaded with the calcium-sensitive fluorophore rhod-2, and subjected to two-photon laser scanning fluorescence microscopy (TPLSM) to monitor action potential–induced [Ca2+] i transients in EGFP-expressing donor-derived cells and non-expressing host cardiomyocytes. Whereas spontaneous and electrically evoked [Ca2+] i transients were found to occur synchronously in host cardiomyocytes along the graft–host border and in areas remote from the infarct, they were absent in all of the >3,000 imaged BM-derived cells that were located in clusters throughout the infarct scar or peri-infarct zone. We conclude that engrafted BM-derived cells lack attributes of functioning cardiomyocytes, calling into question the concept that adult BM cells can give rise to substantive cardiomyocyte regeneration within the infarcted heart.
  • Thumbnail Image
    Item
    Arrhythmogenic Calmodulin Mutations Impede Activation of Small-conductance Calcium-Activated Potassium Current
    (Elsevier, 2016-08) Yu, Chih-Chieh; Ko, Jum-Suk; Ai, Tomohiko; Tsai, Wen-Chin; Chen, Zhenhui; Rubart, Michael; Vatta, Matteo; Everett, Thomas H.; George, Alfred L.; Chen, Peng-Sheng; Medicine, School of Medicine
    Background Apamin sensitive small-conductance Ca2+-activated K+ (SK) channels are gated by intracellular Ca2+ through a constitutive interaction with calmodulin. Objective We hypothesize that arrhythmogenic human calmodulin mutations impede activation of SK channels. Methods We studied 5 previously published calmodulin mutations (N54I, N98S, D96V, D130G and F90L). Plasmids encoding either wild type (WT) or mutant calmodulin were transiently transfected into human embryonic kidney (HEK) 293 cells that stably express SK2 channels (SK2 Cells). Whole-cell voltage-clamp recording was used to determine apamin-sensitive current (IKAS) densities. We also performed optical mapping studies in normal murine hearts to determine the effects of apamin in hearts with (N=7) or without (N=3) pretreatment with sea anemone toxin (ATX II). Results SK2 cells transfected with WT calmodulin exhibited IKAS density (in pA/pF) of 33.6 [31.4;36.5] (median and confidence interval 25%-75%), significantly higher than that observed for cells transfected with N54I (17.0 [14.0;27.7], p=0.016), F90L (22.6 [20.3;24.3], p=0.011), D96V (13.0 [10.9;15.8], p=0.003), N98S (13.7 [8.8;20.4], p=0.005) and D130G (17.6 [13.8;24.6], p=0.003). The reduction of SK2 current was not associated with a decrease in membrane protein expression or intracellular distribution of the channel protein. Apamin increased the ventricular APD80 (from 79.6 ms [63.4-93.3] to 121.8 ms [97.9-127.2], p=0.010) in hearts pre-treated with ATX-II but not in control hearts. Conclusion Human arrhythmogenic calmodulin mutations impede the activation of SK2 channels in HEK 293 cells.
  • Thumbnail Image
    Item
    Atrial fibrillation and electrophysiology in transgenic mice with cardiac-restricted overexpression of FKBP12
    (American Physiological Society, 2019-02-01) Pan, Zhenwei; Ai, Tomohiko; Chang, Po-Cheng; Liu, Ying; Liu, Jijia; Maruyama, Mitsunori; Homsi, Mohamed; Fishbein, Michael C.; Rubart, Michael; Lin, Shien-Fong; Xiao, Deyong; Chen, Hanying; Chen, Peng-Sheng; Shou, Weinian; Li, Bai-Yan; Medicine, School of Medicine
    Cardiomyocyte-restricted overexpression of FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice develop spontaneous atrial fibrillation (AF). The aim of the present study is to explore the mechanisms underlying the occurrence of AF in αMyHC-FKBP12 mice. Spontaneous AF was documented by telemetry in vivo and Langendorff-perfused hearts of αMyHC-FKBP12 and littermate control mice in vitro. Atrial conduction velocity was evaluated by optical mapping. The patch-clamp technique was applied to determine the potentially altered electrophysiology in atrial myocytes. Channel protein expression levels were evaluated by Western blot analyses. Spontaneous AF was recorded in four of seven αMyHC-FKBP12 mice but in none of eight nontransgenic (NTG) controls. Atrial conduction velocity was significantly reduced in αMyHC-FKBP12 hearts compared with NTG hearts. Interestingly, the mean action potential duration at 50% but not 90% was significantly prolonged in αMyHC-FKBP12 atrial myocytes compared with their NTG counterparts. Consistent with decreased conduction velocity, average peak Na+ current ( INa) density was dramatically reduced and the INa inactivation curve was shifted by approximately +7 mV in αMyHC-FKBP12 atrial myocytes, whereas the activation and recovery curves were unaltered. The Nav1.5 expression level was significantly reduced in αMyHC-FKBP12 atria. Furthermore, we found increases in atrial Cav1.2 protein levels and peak L-type Ca2+ current density and increased levels of fibrosis in αMyHC-FKBP12 atria. In summary, cardiomyocyte-restricted overexpression of FKBP12 reduces the atrial Nav1.5 expression level and mean peak INa, which is associated with increased peak L-type Ca2+ current and interstitial fibrosis in atria. The combined electrophysiological and structural changes facilitated the development of local conduction block and altered action potential duration and spontaneous AF. NEW & NOTEWORTHY This study addresses a long-standing riddle regarding the role of FK506-binding protein 12 in cardiac physiology. The work provides further evidence that FK506-binding protein 12 is a critical component for regulating voltage-gated sodium current and in so doing has an important role in arrhythmogenic physiology, such as atrial fibrillation.
  • Thumbnail Image
    Item
    Cardiac engraftment of genetically-selected parthenogenetic stem cell-derived cardiomyocytes
    (Public Library of Science, 2015) Yang, Tao; Rubart, Michael; Soonpaa, Mark H.; Didié, Michael; Christalla, Peter; Zimmermann, Wolfram-Hubertus; Field, Loren J.; Department of Pediatrics, IU School of Medicine
    Parthenogenetic stem cells (PSCs) are a promising candidate donor for cell therapy applications. Similar to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), PSCs exhibit self-renewing capacity and clonogenic proliferation in vitro. PSCs exhibit largely haploidentical genotype, and as such may constitute an attractive population for allogenic applications. In this study, PSCs isolated from transgenic mice carrying a cardiomyocyte-restricted reporter transgene to permit tracking of donor cells were genetically modified to carry a cardiomyocyte-restricted aminoglycoside phosphotransferase expression cassette (MHC-neor/pGK-hygror) to permit the generation of highly enriched cardiomyocyte cultures from spontaneously differentiating PSCs by simple selection with the neomycin analogue G148. Following engraftment into isogenic recipient hearts, the selected cardiomyocytes formed a functional syncytium with the host myocardium as evidenced by the presence of entrained intracellular calcium transients. These cells thus constitute a potential source of therapeutic donor cells.
  • Thumbnail Image
    Item
    Cell-Cycle-Based Strategies to Drive Myocardial Repair
    (Springer, 2009-04-02) Zhu, Wuqiang; Hassink, Rutger J.; Rubart, Michael; Field, Loren J.; Pediatrics, School of Medicine
    Cardiomyocytes exhibit robust proliferative activity during development. After birth, cardiomyocyte proliferation is markedly reduced. Consequently, regenerative growth in the postnatal heart via cardiomyocyte proliferation (and, by inference, proliferation of stem-cell-derived cardiomyocytes) is limited and often insufficient to affect repair following injury. Here, we review studies wherein cardiomyocyte cell cycle proliferation was induced via targeted expression of cyclin D2 in postnatal hearts. Cyclin D2 expression resulted in a greater than 500-fold increase in cell cycle activity in transgenic mice as compared to their nontransgenic siblings. Induced cell cycle activity resulted in infarct regression and concomitant improvement in cardiac hemodynamics following coronary artery occlusion. These studies support the notion that cell-cycle-based strategies can be exploited to drive myocardial repair following injury.
  • Thumbnail Image
    Item
    Critical Roles of STAT3 in β-Adrenergic Functions in the Heart
    (American Heart Association, 2016-01-05) Zhang, Wenjun; Qu, Xiuxia; Chen, Biyi; Snyder, Marylynn; Wang, Meijing; Li, Baiyan; Tang, Yue; Chen, Hanying; Zhu, Wuqiang; Zhan, Li; Yin, Ni; Li, Deqiang; Li, Xie; Liu, Ying; Zhang, J. Jillian; Fu, Xin-Yuan; Rubart, Michael; Song, Long-Sheng; Huang, Xin-Yun; Shou, Weinian; Department of Pediatrics, IU School of Medicine
    BACKGROUND: β-Adrenergic receptors (βARs) play paradoxical roles in the heart. On one hand, βARs augment cardiac performance to fulfill the physiological demands, but on the other hand, prolonged activations of βARs exert deleterious effects that result in heart failure. The signal transducer and activator of transcription 3 (STAT3) plays a dynamic role in integrating multiple cytokine signaling pathways in a number of tissues. Altered activation of STAT3 has been observed in failing hearts in both human patients and animal models. Our objective is to determine the potential regulatory roles of STAT3 in cardiac βAR-mediated signaling and function. METHODS AND RESULTS: We observed that STAT3 can be directly activated in cardiomyocytes by β-adrenergic agonists. To follow up this finding, we analyzed βAR function in cardiomyocyte-restricted STAT3 knockouts and discovered that the conditional loss of STAT3 in cardiomyocytes markedly reduced the cardiac contractile response to acute βAR stimulation, and caused disengagement of calcium coupling and muscle contraction. Under chronic β-adrenergic stimulation, Stat3cKO hearts exhibited pronounced cardiomyocyte hypertrophy, cell death, and subsequent cardiac fibrosis. Biochemical and genetic data supported that Gαs and Src kinases are required for βAR-mediated activation of STAT3. Finally, we demonstrated that STAT3 transcriptionally regulates several key components of βAR pathway, including β1AR, protein kinase A, and T-type Ca(2+) channels. CONCLUSIONS: Our data demonstrate for the first time that STAT3 has a fundamental role in βAR signaling and functions in the heart. STAT3 serves as a critical transcriptional regulator for βAR-mediated cardiac stress adaption, pathological remodeling, and heart failure.
  • Thumbnail Image
    Item
    Dishevelled-associated activator of morphogenesis 1 (Daam1) is required for heart morphogenesis
    (2011-01) Li, Deqiang; Hallett, Mark A.; Zhu, Wuqiang; Rubart, Michael; Liu, Ying; Yang, Zhenyun; Chen, Hanying; Haneline, Laura S.; Chan, Rebecca J.; Schwartz, Robert J.; Field, Loren J.; Atkinson, Simon J.; Shou, Weinian
    Dishevelled-associated activator of morphogenesis 1 (Daam1), a member of the formin protein family, plays an important role in regulating the actin cytoskeleton via mediation of linear actin assembly. Previous functional studies of Daam1 in lower species suggest its essential role in Drosophila trachea formation and Xenopus gastrulation. However, its in vivo physiological function in mammalian systems is largely unknown. We have generated Daam1-deficient mice via gene-trap technology and found that Daam1 is highly expressed in developing murine organs, including the heart. Daam1-deficient mice exhibit embryonic and neonatal lethality and suffer multiple cardiac defects, including ventricular noncompaction, double outlet right ventricles and ventricular septal defects. In vivo genetic rescue experiments further confirm that the lethality of Daam1-deficient mice results from the inherent cardiac abnormalities. In-depth analyses have revealed that Daam1 is important for regulating filamentous actin assembly and organization, and consequently for cytoskeletal function in cardiomyocytes, which contributes to proper heart morphogenesis. Daam1 is also found to be important for proper cytoskeletal architecture and functionalities in embryonic fibroblasts. Biochemical analyses indicate that Daam1 does not regulate cytoskeletal organization through RhoA, Rac1 or Cdc42. Our study highlights a crucial role for Daam1 in regulating the actin cytoskeleton and tissue morphogenesis.
  • Thumbnail Image
    Item
    The Effects of Refractive Index Mismatch on Multiphoton Fluorescence Excitation Microscopy of Biological Tissue
    (2010-08-31T18:42:10Z) Young, Pamela Anne; Rubart, Michael; Decca, Ricardo S.; Bacallao, Robert L.; Dunn, Kenneth William
    Introduction: Multiphoton fluorescence excitation microscopy (MPM) is an invaluable tool for studying processes in tissue in live animals by enabling biologists to view tissues up to hundreds of microns in depth. Unfortunately, imaging depth in MPM is limited to less than a millimeter in tissue due to spherical aberration, light scattering, and light absorption. Spherical aberration is caused by refractive index mismatch between the objective immersion medium and sample. Refractive index heterogeneities within the sample cause light scattering. We investigate the effects of refractive index mismatch on imaging depth in MPM. Methods: The effects of spherical aberration on signal attenuation and resolution degradation with depth are characterized with minimal light absorption and scattering using sub-resolution microspheres mounted in test sample of agarose with varied refractive index. The effects of light scattering on signal attenuation and resolution degradation with depth are characterized using sub-resolution microspheres in kidney tissue samples mounted in optical clearing media to alter the refractive index heterogeneities within the tissue. Results: The studies demonstrate that signal levels and axial resolution both rapidly decline with depth into refractive index mismatched samples. Interestingly, studies of optical clearing with a water immersion objective show that reducing scattering increases reach even when it increases refractive index mismatch degrading axial resolution. Scattering, in the absence of spherical aberration, does not degrade axial resolution. The largest improvements in imaging depth are obtained when both scattering and refractive index mismatch are reduced. Conclusions: Spherical aberration, caused by refractive index mismatch between the immersion media and sample, and scattering, caused by refractive index heterogeneity within the sample, both cause signal to rapidly attenuate with depth in MPM. Scattering, however, seems to be the predominant cause of signal attenuation with depth in kidney tissue. Kenneth W. Dunn, Ph.D., Chair
  • Thumbnail Image
    Item
    Electrical coupling between ventricular myocytes and myofibroblasts in the infarcted mouse heart
    (European Society of Cardiology, 2018-03-01) Rubart, Michael; Tao, Wen; Lu, Xiao-Long; Conway, Simon J.; Reuter, Sean P.; Lin, Shien-Fong; Soonpaa, Mark H.; Medicine, School of Medicine
    Aims: Recent studies have demonstrated electrotonic coupling between scar tissue and the surrounding myocardium in cryoinjured hearts. However, the electrical dynamics occurring at the myocyte-nonmyocyte interface in the fibrotic heart remain undefined. Here, we sought to develop an assay to interrogate the nonmyocyte cell type contributing to heterocellular coupling and to characterize, on a cellular scale, its voltage response in the infarct border zone of living hearts. Methods and results: We used two-photon laser scanning microscopy in conjunction with a voltage-sensitive dye to record transmembrane voltage changes simultaneously from cardiomyocytes and adjoined nonmyocytes in Langendorff-perfused mouse hearts with healing myocardial infarction. Transgenic mice with cardiomyocyte-restricted expression of a green fluorescent reporter protein underwent permanent coronary artery ligation and their hearts were subjected to voltage imaging 7-10 days later. Reporter-negative cells, i.e. nonmyocytes, in the infarct border zone exhibited depolarizing transients at a 1:1 coupling ratio with action potentials recorded simultaneously from adjacent, reporter-positive ventricular myocytes. The electrotonic responses in the nonmyocytes exhibited slower rates of de- and repolarization compared to the action potential waveform of juxtaposed myocytes. Voltage imaging in infarcted hearts expressing a fluorescent reporter specifically in myofibroblasts revealed that the latter were electrically coupled to border zone myocytes. Their voltage transient properties were indistinguishable from those of nonmyocytes in hearts with cardiomyocyte-restricted reporter expression. The density of connexin43 expression at myofibroblast-cardiomyocyte junctions was ∼5% of that in the intercalated disc regions of paired ventricular myocytes in the remote, uninjured myocardium, whereas the ratio of connexin45 to connexin43 expression levels at heterocellular contacts was ∼1%. Conclusion: Myofibroblasts contribute to the population of electrically coupled nonmyocytes in the infarct border zone. The slower kinetics of myofibroblast voltage responses may reflect low electrical conductivity across heterocellular junctions, in accordance with the paucity of connexin expression at myofibroblast-cardiomyocyte contacts.
  • Thumbnail Image
    Item
    In situ three-dimensional reconstruction of mouse heart sympathetic innervation by two-photon excitation fluorescence imaging
    (Elsevier, 2014-01-15) Freeman, Kim; Tao, Wen; Sun, Hongli; Soonpaa, Mark H.; Rubart, Michael; Department of Medicine, IU School of Medicine
    Background Sympathetic nerve wiring in the mammalian heart has remained largely unexplored. Resolving the wiring diagram of the cardiac sympathetic network would help establish the structural underpinnings of neurocardiac coupling. New Method We used two-photon excitation fluorescence microscopy, combined with a computer-assisted 3-D tracking algorithm, to map the local sympathetic circuits in living hearts from adult transgenic mice expressing enhanced green fluorescent protein (EGFP) in peripheral adrenergic neurons. Results Quantitative co-localization analyses confirmed that the intramyocardial EGFP distribution recapitulated the anatomy of the sympathetic arbor. In the left ventricular subepicardium of the uninjured heart, the sympathetic network was composed of multiple subarbors, exhibiting variable branching and looping topology. Axonal branches did not overlap with each other within their respective parental subarbor nor with neurites of annexed subarbors. The sympathetic network in the border zone of a 2-week-old myocardial infarction was characterized by substantive rewiring, which included spatially heterogeneous loss and gain of sympathetic fibers and formation of multiple, predominately nested, axon loops of widely variable circumference and geometry. Comparison with Existing Methods In contrast to mechanical tissue sectioning methods that may involve deformation of tissue and uncertainty in registration across sections, our approach preserves continuity of structure, which allows tracing of neurites over distances, and thus enables derivation of the three-dimensional and topological morphology of cardiac sympathetic nerves. Conclusions Our assay should be of general utility to unravel the mechanisms governing sympathetic axon spacing during development and disease.