Direct biologically based biosensing of dynamic physiological function

Dynamic regulation of biological systems requires real-time assessment of relevant physiological needs. Biosensors, which transduce biological actions or reactions into signals amenable to processing, are well suited for such monitoring. Typically, in vivo biosensors approximate physiological function via the measurement of surrogate signals. The alternative approach presented here would be to use biologically based biosensors for the direct measurement of physiological activity via functional integration of relevant governing inputs. We show that an implanted excitable-tissue biosensor (excitable cardiac tissue) can be used as a real-time, integrated bioprocessor to analyze the complex inputs regulating a dynamic physiological variable (heart rate). This approach offers the potential for long-term biologically tuned quantification of endogenous physiological function.     

Cardiovascular and metabolic alterations in mice lacking both beta1- and beta2-adrenergic receptors.

The activation state of beta-adrenergic receptors (beta-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by beta-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of beta1- and/or beta2-AR expression on these homeostatic control mechanisms. Despite total absence of beta1- and beta2-ARs, the predominant cardiovascular beta-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of beta-AR function by beta-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in beta1/beta2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single beta1- and beta2-AR knockouts as well as the beta1-/beta2-AR double knock-out suggest that in the mouse, beta-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the beta1-AR, whereas vascular relaxation and metabolic rate are controlled by all three beta-ARs (beta1-, beta2-, and beta3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular beta3-AR responsiveness are also observed in beta1-/beta2-AR double knockouts. In addition to its ability to define beta-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed.     

Cyclooxygenase-2 induction by adiponectin is regulated by a sphingosine kinase-1 dependent mechanism in cardiac myocytes

The adipose-derived plasma protein, adiponectin (APN), has various protective effects on cardiovascular diseases. In this study, we show that endogenous APN is required for full cyclooxygenase-2 (COX-2) induction by ischemia-reperfusion injury in the heart in vivo. In rat neonatal cardiac myocytes, APN-induced COX-2 expression was reduced by treatment with a sphingosine kinase-1 (SphK-1) inhibitor or siRNA targeting SphK-1. Treatment with a sphingosine-1-phosphate (S1P) receptor antagonist also diminished COX-2 expression in response to APN stimulation. These findings suggest that APN is a physiological regulator of COX-2 signaling in the heart and that this regulation occurs in part via a SphK-1-S1P receptor dependent mechanism in cardiac myocytes.     

The enzymatic inactivation of the fatty acid amide class of signaling lipids

The fatty acid amide (FAA) class of signaling lipids modulates a number of neurobehavioral processes in mammals, including pain, sleep, feeding, and locomotor activity. Representative FAAs include the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. Despite activating several neuroreceptor systems in vitro, most FAAs produce only weak and transient behavioral effects in vivo, presumably due to their expeditious catabolism. This review focuses on one enzyme, fatty acid amide hydrolase (FAAH) that appears to play a major role in regulating the amplitude and duration of FAA signals in vivo. In particular, we will highlight a series of recent papers that have investigated the physiological functions of the mouse and human FAAH enzymes. Collectively, these studies promote FAAH as a central component of FAA signaling pathways, especially those mediated by the endocannabinoid anandamide, and suggest that this enzyme may represent an attractive pharmaceutical target for the treatment of pain and related neurophysiological disorders.     

Exercise-induced functional desensitization of canine cardiac beta-adrenergic receptors

To test the hypothesis that the high levels of endogenous catecholamines associated with strenuous exercise produce functional desensitization of cardiac beta-adrenergic receptors, we measured the bolus chronotropic dose of isoproterenol necessary to produce a 25-beats/min increase in heart rate (CD25) in the resting state and after the return of heart rate to resting levels after 60 min of treadmill running in 13 normal dogs. Immediately after exercise, 12 of 13 dogs were less sensitive to the chronotropic effects of beta-adrenergic receptor stimulation: mean CD25 increased from 1.16 +/- 0.17 to 3.50 +/- 0.98 micrograms (P less than 0.02). A similar reduction in isoproterenol sensitivity was evident regardless of whether testing was performed in the presence or absence of vagal blockade with atropine. By 3 h after exercise, CD25 had returned to the preexercise level, with no further change noted 24 h after exercise. There was no change in the CD25 when measured serially in three unexercised dogs. We conclude that a single bout of dynamic exercise is sufficient to produce a significantly decreased chronotropic responsiveness to isoproterenol. This phenomenon may represent an acute but transient desensitization of cardiac beta-adrenergic receptors.     

Partial reprogramming as a therapeutic approach for heart disease: A state-of-the-art review

Heart disease such as myocardial infarction is the first cause of mortality in all countries. Today, cardiac cell-based therapy using de novo produced cardiac cells is considered as a novel approach for cardiac regenerative medicine. Recently, an alchemy-like approach, known as direct reprogramming or direct conversion, has been developed to directly convert somatic cells to cardiac cells in vitro and in vivo. This cellular alchemy is a short-cut and safe strategy for generating autologous cardiac cells, and it can be accomplished through activating cardiogenesis- or pluripotency-related factors in noncardiac cells. Importantly, pluripotency factors-based direct cardiac conversion, known as partial reprogramming, is shorter and more efficient for cardiomyocyte generation in vitro. Today, this strategy is achievable for direct conversion of mouse and human somatic cells to cardiac lineage cells (cardiomyocytes and cardiac progenitor cells), using transgene free, chemical-based approaches. Although, heart-specific partial reprogramming seems to be challenging for in vivo conversion of cardiac fibroblasts to cardiac cells, but whole organism-based in vivo partial reprogramming ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in mouse. Notably, cardiac cells produced using partial reprogramming strategy can be a useful platform for disease modeling, drug screening and cardiac cell-based therapy, once the safety issues are overcome. Herein, we discuss about all progresses in de novo production of cardiac cells using partial reprogramming-based direct conversion, as well as give an overview about the potential applications of this strategy in vivo and in vitro.     

Live-cell transforms between Ca2+ transients and FRET responses for a troponin-C-based Ca2+ sensor

Genetically encoded Ca²⁺ sensors promise sustained in vivo detection of Ca²⁺ signals. However, these sensors are sometimes challenged by inconsistent performance and slow/uncertain kinetic responsiveness. The former challenge may arise because most sensors employ calmodulin (CaM) as the Ca²⁺-sensing module, such that interference via endogenous CaM may result. One class of sensors that could minimize this concern utilizes troponin C as the Ca²⁺ sensor. Here, we therefore probed the reliability and kinetics of one representative of this class (cyan fluorescence protein/yellow fluorescent protein-fluorescence resonance energy transfer (FRET) sensor TN-L15) within cardiac ventricular myocytes. These cells furnished a pertinent live-cell test environment, given substantial endogenous CaM levels and fast reproducible Ca²⁺ transients for testing sensor kinetics. TN-L15 was virally expressed within myocytes, and Indo-1 acutely loaded to monitor “true” Ca²⁺ transients. This configuration permitted independent and simultaneous detection of TN-L15 and Indo-1 signals within individual cells. The relation between TN-L15 FRET responses and Indo-1 Ca²⁺ transients appeared reproducible, though FRET signals were delayed compared to Ca²⁺ transients. Nonetheless, a three-state mechanism sufficed to map between measured Ca²⁺ transients and actual TN-L15 outputs. Overall, reproducibility of TN-L15 dynamics, coupled with algorithmic transforms between FRET and Ca²⁺ signals, renders these sensors promising for quantitative estimation of Ca²⁺ dynamics in vivo.     

Kyotorphin has a novel action on rat cardiac muscle

Several endogenous peptides for G-protein-coupled receptors have been found to play physiological roles in muscle contraction in addition to their well-demonstrated actions in other tissues. To further identify such peptides, we screened over 400 peptides using an isometric tension assay of rat papillary muscle. Here, we report that kyotorphin, which is known as an analgesic dipeptide, has a cardiac effect. Although kyotorphin had no effect on the twitch tension itself, it inhibited beta-adrenergic agonist isoprenaline-induced increases in twitch tension in a dose-dependent manner. Leu-Arg, a selective antagonist of kyotorphin, reversed this inhibitory effect. The inhibitory effect was also reversed by naloxone, an opioid receptor antagonist. These results suggest that kyotorphin may release opioid peptides from rat cardiac muscle and have an indirect regulatory role in beta-adrenergic action through cross-talk with opioid receptors.     

Efficient in vivo manipulation of alternative pre-mRNA splicing events using antisense morpholinos in mice

Mammalian pre-mRNA alternative splicing mechanisms are typically studied using artificial minigenes in cultured cells, conditions that may not accurately reflect the physiological context of either the pre-mRNA or the splicing machinery. Here, we describe a strategy to investigate splicing of normal endogenous full-length pre-mRNAs under physiological conditions in live mice. This approach employs antisense vivo-morpholinos (vMOs) to mask cis-regulatory sequences or to disrupt splicing factor expression, allowing functional evaluation of splicing regulation in vivo. We applied this strategy to gain mechanistic insight into alternative splicing events involving exons 2 and 16 (E2 and E16) that control the structure and function of cytoskeletal protein 4.1R. In several mouse tissues, inclusion of E16 was substantially inhibited by interfering with a splicing enhancer mechanism using a target protector morpholino that blocked Fox2-dependent splicing enhancers in intron 16 or a splice-blocking morpholino that disrupted Fox2 expression directly. For E2, alternative 3'-splice site choice is coordinated with upstream promoter use across a long 5'-intron such that E1A splices almost exclusively to the distal acceptor (E2dis). vMOs were used to test the in vivo relevance of a deep intron element previously proposed to determine use of E2dis via a two-step intrasplicing model. Two independent vMOs designed against this intronic regulatory element inhibited intrasplicing, robustly switching E1A splicing to the proximal acceptor (E2prox). This finding strongly supports the in vivo physiological relevance of intrasplicing. vMOs represent a powerful tool for alternative splicing studies in vivo and may facilitate exploration of alternative splicing networks in vivo.     

Evidence against prostaglandin modulation of cardioaccelerator nerve activity in the anesthetized dog

The hypothesis that prostaglandins have a modulatory role in adrenergic neurotransmitter release was tested in the anesthetized dog. Inhibition of prostaglandin synthesis with indomethacin (10 mg/kg, i.v.) did not alter positive chronotropic responses to cardioaccelerator nerve stimulation or blood pressure responses to exogenous norepinephrine. In the presence of indomethacin, infusions of PGE₂ (0.01 and 0.1 μg kg⁻¹ min⁻¹) also failed to influence the responses to cardioaccelerator nerve stimulation although the blood pressure responses to exogenous norepinephrine were reduced in a dose-related manner. It was concluded that endogenous prostaglandins and exogenous PGE₂, the purported physiological inhibitor of neurotransmitter release in cardiac tissue, do not play a role in modulating chronotropic responses during cardioaccelerator nerve stimulation in the anesthetized dog.     

Troponin C-based biosensors: a new family of genetically encoded indicators for in vivo calcium imaging in the nervous system

Fluorescence imaging represents a powerful approach for the detection of intracellular Ca(2+) signals in vivo. With appropriate techniques, Ca(2+) signals can be recorded at many levels of complexity, ranging from large scale neuronal networks down to individual presynaptic boutons or postsynaptic spines. Here we review the applicability of genetically encoded Ca(2+) indicators for in vivo Ca(2+) imaging of neural function. We describe some of the recent progress in sensor design and evaluate the performance of the new family of Troponin C-based Ca(2+) indicators. Further, we analyze properties of Ca(2+) biosensors transgenically expressed in various experimental animal models and illustrate their use for measuring somatic and dendritic Ca(2+) signals in neurons of the mammalian brain.     

Hormone multifunctionalities: a theory of endocrine signaling, command and control

A theory is presented outlining how organisms can function and benefit from multifunctionality of hormones in order to enhance greatly the information-carrying potential of endocrine signaling. Hormones are produced continuously as micropulses, and intermittently as larger pulses. It is generally believed that micropulses generate fluctuating basal hormone concentrations, which may consistently elicit particular responses among diverse variables. Evidence is discussed suggesting that in contrast to the hormone micropulses, the larger endogenous hormone pulses may elicit responses which may differ from one pulse to another and may therefore serve different physiological functions. In this paper we postulate that an endogenous hormone pulse is a specific form of a multisignal message that serves a certain physiological function. Different pulses of a hormone may be signals of diverse multisignal messages that serve different functions. A multisignal message may elicit congruous responses by selectively enhancing some actions and suppressing other actions of the component signals. Various roles of signals of multisignal messages are discussed, as well as processes that may be involved in the diversity and selectivity of actions of different pulses of a hormone. Hormones also are converted into other hormones; we analyze how precursor and derived hormones may function independently of each other, and how precursor hormones may give rise to permissive effects. Mechanisms involved in therapeutic and adverse effects of hormone administrations are analyzed, and a strategy is suggested for developing more selective hormonal therapies.     

Genetically engineered microbial biosensors for in situ monitoring of environmental pollution

Microbial biosensors are compact, portable, cost effective, and simple to use, making them seem eminently suitable for the in situ monitoring of environmental pollution. One promising approach for such applications is the fusion of reporter genes with regulatory genes that are dose-dependently responsive to the target chemicals or physiological signals. Their biosensor capabilities, such as target range and sensitivity, could be improved by modification of regulatory genes. Recent uses of such genetically engineered microbial biosensors include the development of portable biosensor kits and high-throughput cell arrays on chips, optic fibers, or other platforms for on-site and on-line monitoring of environmental pollution. This mini-review discusses recent advances in microbial biosensors and their future prospects, with a focus on the development and application of genetically modified microbial biosensors for in situ environmental monitoring.     

CD95-mediated apoptosis in vivo involves acid sphingomyelinase

Acid sphingomyelinase (ASM) is reported to have an essential function in stress-induced apoptosis although the physiological function of ASM in receptor-triggered apoptosis is unknown. Here, we delineate a pivotal role for ASM in CD95-triggered apoptosis of peripheral lymphocytes or hepatocytes in vivo. We employed intravenous injection of anti-CD4 antibodies or phytohemagglutinin that was previously shown to result in apoptosis of peripheral blood lymphocytes or hepatocytes via the endogenous CD95/CD95 ligand system. Our results demonstrate a high susceptibility in normal mice whereas ASM knock-out mice fail to immunodeplete T cells or develop autoimmune-like hepatitis. Likewise, ASM-deficient mice or hepatocytes and splenocytes ex vivo manifest resistance to anti-CD95 treatment. These results provide in vivo evidence for an important physiological function of ASM in CD95-induced apoptosis.     

Evaluation of permselective membranes for optimization of intracerebral amperometric glutamate biosensors

Monitoring of extracellular brain glutamate concentrations by intracerebral biosensors is a promising approach to further investigate the role of this important neurotransmitter. However, amperometric biosensors are typically hampered by Faradaic interference caused by the presence of other electroactive species in the brain, such as ascorbic acid, dopamine, and uric acid. Various permselective membranes are often used on biosensors to prevent this. In this study we evaluated the most commonly used membranes, i.e. nafion, polyphenylenediamine, polypyrrole, polyaniline, and polynaphthol using a novel silica-based platinum electrode. First we selected the membranes with the highest sensitivity for hydrogen peroxide in vitro and an optimal selectivity against electrochemical interferents. Then we evaluated the performances of these membranes in a short lasting (3-4h) in vivo experiment. We found that best in vitro performance was accomplished with biosensors that were protected by a poly(m-phenylenediamine) membrane deposited onto the platinum electrode by cyclic voltammetry. However, post-implantation evaluation of these membranes showed poor selectivity against dopamine. Combination with a previously applied nafion layer did not protect the sensors against acute biofouling; indeed it was even counter effective. Finally, we investigated the ability of our biosensors to monitor the effect of glutamate transport blocker DL-TBOA on modulating glutamate concentrations in the prefrontal cortex of anaesthetized rats. The optimized biosensors recorded a rapid 35-fold increase in extracellular glutamate, and are considered suitable for further exploration in vivo.     

Regulator of G protein signaling 2 is a functionally important negative regulator of angiotensin II-induced cardiac fibroblast responses

Cardiac fibroblasts play a key role in fibrosis development in response to stress and injury. Angiotensin II (ANG II) is a major profibrotic activator whose downstream effects (such as phospholipase Cβ activation, cell proliferation, and extracellular matrix secretion) are mainly mediated via G(q)-coupled AT(1) receptors. Regulators of G protein signaling (RGS), which accelerate termination of G protein signaling, are expressed in the myocardium. Among them, RGS2 has emerged as an important player in modulating G(q)-mediated hypertrophic remodeling in cardiac myocytes. To date, no information is available on RGS in cardiac fibroblasts. We tested the hypothesis that RGS2 is an important regulator of ANG II-induced signaling and function in ventricular fibroblasts. Using an in vitro model of fibroblast activation, we have demonstrated expression of several RGS isoforms, among which only RGS2 was transiently upregulated after short-term ANG II stimulation. Similar results were obtained in fibroblasts isolated from rat hearts after in vivo ANG II infusion via minipumps for 1 day. In contrast, prolonged ANG II stimulation (3-14 days) markedly downregulated RGS2 in vivo. To delineate the functional effects of RGS expression changes, we used gain- and loss-of-function approaches. Adenovirally infected RGS2 had a negative regulatory effect on ANG II-induced phospholipase Cβ activity, cell proliferation, and total collagen production, whereas RNA interference of endogenous RGS2 had opposite effects, despite the presence of several other RGS. Together, these data suggest that RGS2 is a functionally important negative regulator of ANG II-induced cardiac fibroblast responses that may play a role in ANG II-induced fibrosis development.     

The formin-binding protein 17, FBP17, binds via a TNKS binding motif to tankyrase, a protein involved in telomere maintenance

In acute myelogenous and lymphoid leukemias, rearrangements involving the MLL (mixed lineage leukemia) gene at chromosome 11q23 are frequent. The truncated MLL protein is fused in-frame to a series of partner proteins. We previously identified the formin-binding protein 17 (FBP17) as such an MLL fusion partner. In this study, we explored in vivo physiological interaction partners of FBP17 using a two-hybrid assay and found tankyrase (TNKS), an ADP-ribose polymerase protein involved in telomere maintenance and mitogen-activated protein kinase signaling. We demonstrate that FBP17 binds via a special TNKS-binding motif to tankyrase. The physiological relevance is indicated by co-immunoprecipitation of endogenous proteins in 293T cells.