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Brain is well known for its disproportionate oxygen consumption and high energy-budget for optimal functioning. The decrease in oxygen supply to brain, thus, necessitates rapid activation of adaptive pathways - the absence of which manifest into vivid pathological conditions. Amongst these, oxygen sensing in glio-vascular milieu and H

Hypobaric Hypoxia (HH) is an established risk factor for various neuro-physiological perturbations including cognitive impairment. The origin and mechanistic basis of such responses however remain elusive. We here combined systems level analysis with classical neuro-physiological approaches, in a rat model system, to understand pathological responses of brain to HH. Unbiased ‘statistical co-expression networks’ generated utilizing temporal, differential transcriptome signatures of hippocampus—centrally involved in regulating cognition—implicated perturbation of Glio-Vascular homeostasis during early responses to HH, with concurrent modulation of vasomodulatory, hemostatic and proteolytic processes. Further, multiple lines of experimental evidence from ultra-structural, immuno-histological, substrate-zymography and barrier function studies unambiguously supported this proposition. Interestingly, we show a significant lowering of H2S levels in the brain, under chronic HH conditions. This phenomenon functionally impacted hypoxia-induced modulation of cerebral blood flow (hypoxic autoregulation) besides perturbing the strength of functional hyperemia responses. The augmentation of H2S levels, during HH conditions, remarkably preserved Glio-Vascular homeostasis and key neuro-physiological functions (cerebral blood flow, functional hyperemia and spatial memory) besides curtailing HH-induced neuronal apoptosis in hippocampus. Our data thus revealed causal role of H2S during HH-induced early Glio-Vascular dysfunction and consequent cognitive impairment., Highlights • Glio-Vascular dysfunction temporally precedes Hypobaric Hypoxia (HH) induced neuro-pathological effects. • Exposure to HH significantly lowers the levels of H2S in brain. • Augmentation of H2S, utilizing its donor, preserves Glio-Vascular homeostasis and curtails HH-induced memory impairment. The exposure to Hypobaric Hypoxia (HH) environment (such as that encountered by humans at high altitude) culminates in cognitive impairment in an altitude- and duration-dependent manner. The mechanistic basis for such effects, however, remains elusive. Our present study showed that HH-induced neuro-pathological perturbations are temporally preceded by Glio-Vascular dysfunction and are concomitant with lowered levels of gaseous messenger, H2S, in brain. The maintenance of H2S levels (utilizing a specific donor, NaHS) during hypoxia curtailed HH-induced brain-vascular dysfunction and ensuing neuro-pathological effects (on spatial memory). Interestingly, identification of origin of disease in the present study effectively revealed a possible interventional strategy.

Brain is well known for its disproportionate oxygen consumption and high energy-budget for optimal functioning. The decrease in oxygen supply to brain, thus, necessitates rapid activation of adaptive pathways - the absence of which manifest into vivid pathological conditions. Amongst these, oxygen sensing in glio-vascular milieu and H2S-dependent compensatory increase in cerebral blood flow (CBF) is a major adaptive response. We had recently demonstrated that the levels of H2S were significantly decreased during chronic hypobaric hypoxia (HH)-induced neuro-pathological effects. The mechanistic basis of this phenomenon, however, remained to be deciphered. We, here, describe experimental evidence for marked limitation of cysteine during HH - both in animal model as well as human volunteers ascending to high altitude. We show that the preservation of brain cysteine level, employing cysteine pro-drug (N-acetyl-L-cysteine, NAC), markedly curtailed effects of HH - not only on endogenous H2S levels but also, impairment of spatial reference memory in our animal model. We, further, present multiple lines of experimental evidence that the limitation of cysteine was causally governed by physiological propensity of brain to utilize cysteine, in cystathionine beta synthase (CBS)-dependent manner, past its endogenous replenishment potential. Notably, decrease in the levels of brain cysteine manifested despite positive effect (up-regulation) of HH on endogenous cysteine maintenance pathways and thus, qualifying cysteine as a conditionally essential nutrient (CEN) during HH. In brief, our data supports an adaptive, physiological role of CBS-mediated cysteine-utilization pathway - activated to increase endogenous levels of H2S - for optimal responses of brain to hypobaric hypoxia.

Hydrogen Sulfide (H2S), recently identified as the third endogenously produced gaseous messenger, is a promising therapeutic prospect for multiple cardio-pathological states, including myocardial hypertrophy. The molecular niche of H2S in normal or diseased cardiac cells is, however, sparsely understood. Here, we show that β-adrenergic receptor (β-AR) overstimulation, known to produce hypertrophic effects in cardiomyocytes, rapidly decreased endogenous H2S levels. The preservation of intracellular H2S levels under these conditions strongly suppressed hypertrophic responses to adrenergic overstimulation, thus suggesting its intrinsic role in this process. Interestingly, unbiased global transcriptome sequencing analysis revealed an integrated metabolic circuitry, centrally linked by NADPH homeostasis, as the direct target of intracellular H2S augmentation. Within these gene networks, glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme (producing NADPH) in pentose phosphate pathway, emerged as the critical node regulating cellular effects of H2S. Utilizing both cellular and animal model systems, we show that H2S-induced elevated G6PD activity is critical for the suppression of cardiac hypertrophy in response to adrenergic overstimulation. We also describe experimental evidences suggesting multiple processes/pathways involved in regulation of G6PD activity, sustained over extended duration of time, in response to endogenous H2S augmentation. Our data, thus, revealed H2S as a critical endogenous regulator of cardiac metabolic circuitry, and also mechanistic basis for its anti-hypertrophic effects.

Gelatin zymography, first described by Heussen and Dowdle in the 1980s, is a widely used valuable tool in research and diagnostics. The technique identifies gelatinases by the degradation of their preferential substrate as well as by their molecular weight (kDa). We here describe detailed methodology for the detection of pro- and active- forms of both MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in cells using norepinephrine-stimulated H9c2 cardiomyoblasts as model. An easy to follow step-by-step protocol has been carefully written for reliable results. We also suggest an acceptable method for quantification of gelatin zymograms.

Gelatin zymography, first described by Heussen and Dowdle in the 1980s, is a widely used valuable tool in research and diagnostics. The technique identifies gelatinases by the degradation of their preferential substrate as well as by their molecular weight (kDa). We here describe detailed methodology for the detection of pro- and active- forms of both MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in cells using norepinephrine-stimulated H9c2 cardiomyoblasts as model. An easy to follow step-by-step protocol has been carefully written for reliable results. We also suggest an acceptable method for quantification of gelatin zymograms.

Hydrogen Sulfide (H2S), recently identified as the third endogenously produced gaseous messenger, is a promising therapeutic prospect for multiple cardio-pathological states, including myocardial hypertrophy. The molecular niche of H2S in normal or diseased cardiac cells is, however, sparsely understood. Here, we show that β-adrenergic receptor (β-AR) overstimulation, known to produce hypertrophic effects in cardiomyocytes, rapidly decreased endogenous H2S levels. The preservation of intracellular H2S levels under these conditions strongly suppressed hypertrophic responses to adrenergic overstimulation, thus suggesting its intrinsic role in this process. Interestingly, unbiased global transcriptome sequencing analysis revealed an integrated metabolic circuitry, centrally linked by NADPH homeostasis, as the direct target of intracellular H2S augmentation. Within these gene networks, glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme (producing NADPH) in pentose phosphate pathway, emerged as the critical node regulating cellular effects of H2S. Utilizing both cellular and animal model systems, we show that H2S-induced elevated G6PD activity is critical for the suppression of cardiac hypertrophy in response to adrenergic overstimulation. We also describe experimental evidences suggesting multiple processes/pathways involved in regulation of G6PD activity, sustained over extended duration of time, in response to endogenous H2S augmentation. Our data, thus, revealed H2S as a critical endogenous regulator of cardiac metabolic circuitry, and also mechanistic basis for its anti-hypertrophic effects.

Zymography has long been used for the detection of substrate-specific enzyme activity. In situ zymography (ISZ), an adaptation from the conventional substrate zymography, is a widely employed technique useful for the detection, localization, and estimation of enzyme-substrate interactions in tissues. Here, we describe a protocol to detect 'in position' matrix metalloproteinase (MMP) activity in cells utilizing H9c2 cardiomyoblasts as a model. This technique is primarily adopted from the method used for histological sections and is termed as 'Cell in situ Zymography'. It is a simple, sensitive, and quantifiable methodology to assess the functional activity of an enzyme 'on site/in position' in cell culture.

Zymography has long been used for the detection of substrate-specific enzyme activity. In situ zymography (ISZ), an adaptation from the conventional substrate zymography, is a widely employed technique useful for the detection, localization, and estimation of enzyme-substrate interactions in tissues. Here, we describe a protocol to detect 'in position' matrix metalloproteinase (MMP) activity in cells utilizing H9c2 cardiomyoblasts as a model. This technique is primarily adopted from the method used for histological sections and is termed as 'Cell in situ Zymography'. It is a simple, sensitive, and quantifiable methodology to assess the functional activity of an enzyme 'on site/in position' in cell culture.

Developmental processes require a precise spatio-temporal regulation of gene expression wherein a diverse set of transcription factors control the signalling pathways. MicroRNAs (miRNAs), a class of small non-coding RNA molecules have recently drawn attention for their prominent role in development and disease. These tiny sequences are essential for regulation of processes, including cell signalling, cell development, cell death, cell proliferation, patterning and differentiation. The consequence of gene regulation by miRNAs is similar to that by transcription factors (TFs). A regulatory cascade essential for appropriate execution of several biological events is triggered through a combinatorial action of miRNAs and TFs. These two important regulators share similar regulatory logics and bring about a cooperative action in the gene regulatory network, dependent on the binding sites present on the target gene. The review addresses the biogenesis and nomenclature of miRNAs, outlines the mechanism of action and regulation of their expression, and focuses on the combinatorial action of miRNAs and TFs for the expression of genes in various regulatory cascades.

The extracellular matrix is fast emerging as important component mediating cell-cell interactions, along with its established role as a scaffold for cell support. Collagen, being the principal component of extracellular matrix, has been implicated in a number of pathological conditions. However, collagens are complex protein structures belonging to a large family consisting of 28 members in humans; hence, there exists a lack of in depth information about their structural features. Annotating and appreciating the functions of these proteins is possible with the help of the numerous biocomputational tools that are currently available. This study reports a comparative analysis and characterization of the alpha-1 chain of human collagen sequences. Physico-chemical, secondary structural, functional and phylogenetic classification was carried out, based on which, collagens 12, 14 and 20, which belong to the FACIT collagen family, have been identified as potential players in diseased conditions, owing to certain atypical properties such as very high aliphatic index, low percentage of glycine and proline residues and their proximity in evolutionary history. These collagen molecules might be important candidates to be investigated further for their role in skeletal disorders.

The identification of specific target proteins for any diseased condition involves extensive characterization of the potentially involved proteins. Members of a protein family demonstrating comparable features may show certain unusual features when implicated in a pathological condition. Advancements in the field of computational biology and the use of various bioinformatics tools for analysis can aid researchers to comprehend their system of work in primary stages of research. This initial screening can help to reduce time and cost of testing and experimentation in laboratory. Human matrix metalloproteinase (MMP) family of endopeptidases is one such family of 23 members responsible for the remodeling of extracellular matrix (ECM) by degradation of the ECM proteins. Though their role has been implicated in various pathological conditions such as arthritis, atherosclerosis, cancer, liver fibrosis, cardio-vascular and neurodegenerative disorders, little is known about the specific involvement of members of the large MMP family in diseases. A comparative in silico characterization of the MMP protein family has been carried out to analyze their physico-chemical, secondary structural and functional properties. Based on the observed patterns of occurrence of atypical features, we hypothesize that cysteine rich and highly thermostable MMPs might be key players in diseased conditions. Thus, a plausible grouping of disease responsive MMPs that might be considered as promising clinical targets may be done. This study can be used as a fundamental approach to characterize, analyze and screen large protein families for the identification of signature patterns.

Hydrogen Sulfide (H2S), the third gasomessenger to be discovered after nitric oxide (NO) and carbon monoxide (CO), is known for its distinctive health promoting effects on various organ systems, including cardiovascular system. This molecule has now qualified as an authentic mediator of specific cellular signal transduction pathways [1, 2]. The mechanistic insight into its cyto-protective role however remains incompletely understood. To understand the molecular circuitry regulated by H2S augmentation, we utilized an exogenous donor of H2S, Sodium hydrogen sulfide (NaHS), and performed unbiased global transcriptome sequencing (Illumina) in cardiac cells (H9c2 cardiomyoblasts). These experiments yielded differential transcriptome of the cells with varying levels of H2S (with or without NaHS treatment for 6 hrs). We subjected this dataset to multiple pathway mining tools, including gene ontology analysis, functional annotation clustering and co-expression network analysis, to infer biological themes hidden as concerted differential gene expression signatures [3]. We, interestingly, observed common biological processes in different analysis strategies, suggesting authentic, conserved nature of cellular response to H2S. Biological networks, largely associated with metabolic/ redox processes were recognized; within three gross themes - steroid/ isoprenoid biosynthesis, oxidoreductase coenzyme metabolism (representing pentose phosphate pathway, PPP) and glutathione metabolism. Glucose-6- phosphate dehydrogenase (G6PD) - rate-limiting enzyme within PPP stood as the highest degree node in majority of these networks. Also, genes related to oxidative stress and redox signaling were enriched. Interestingly, these pathways appear to be centrally linked by nicotinamide nucleotide cofactor (NADPH) homeostasis. We further supported this proposition at functional level by performing various enzyme activities besides recording cellular NADP/NADPH and GSH levels in established cellular as well as rat model system. In summary, our data suggested profound influence of H2S on integrated cellular metabolic circuitry modulating redox homeostasis in cardiac cells.

Matrix metalloproteinases (MMPs), a family of proteolytic enzymes important in the degradation and turnover of extracellular matrix (ECM) components, result in geometric and structural changes of the myocardium which has become a major mechanism for the progression of various cardiovascular pathologies including atherosclerosis, hypertension and end-organ damage, myocardial infarction, ischemia-reperfusion injury, and hypertrophic cardiomyopathies which ultimately result in heart failure. The natural MMP inhibitors known as Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) also play a major role in regulating the myocardial remodeling by working in a balance with MMPs. An imbalance between MMPs and TIMPs results in disease condition. Restoration of this balance is therefore a promising therapeutic strategy for the treatment of cardiac diseases. Use of synthetic inhibitors to regulate the MMP activity serves as emerging candidates of clinical utility in order to design effective therapeutic strategies. In this chapter we summarize about the MMP family, their involvement in various cardiovascular disease conditions along with the role of MMP inhibitors. We have also mentioned different techniques which can be useful in identification of MMPs and TIMPs and help unveil the mechanisms related to them in disease conditions.

In situ zymography is a unique technique for detection and localization of enzyme–substrate interactions majorly in histological sections. Substrate with quenched fluorogenic molecule is incorporated in gel over which tissue sections are mounted and then incubated in buffer. The enzymatic activity is observed in the form of fluorescent signal. With the advancements in the field of biological research, use of in vitro cell culture has become very popular and holds great significance in multiple fields including inflammation, cancer, stem cell biology and the still emerging 3-D cell cultures. The information on analysis of enzymatic activity in cell lines is inadequate presently. We propose a single-step methodology that is simple, sensitive, cost-effective, and functional to perform and study the ‘in position’ activity of enzyme on substrate for in vitro cell cultures. Quantification of enzymatic activity to carry out comparative studies on cells has also been illustrated. This technique can be applied to a variety of enzyme classes including proteases, amylases, xylanases, and cellulases in cell cultures.