Date Published: March 22, 2017
Publisher: Public Library of Science
Author(s): Sathyamangla V. Naga Prasad, Manveen K. Gupta, Zhong-Hui Duan, Venkata Suresh K. Surampudi, Chang-Gong Liu, Ashwin Kotwal, Christine S. Moravec, Randall C. Starling, Dianne M. Perez, Subha Sen, Qingyu Wu, Edward F. Plow, Sadashiva Karnik, Sudhiranjan Gupta.
It is well established that the gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, however, less is known about the reasons behind such global differences. MicroRNAs (miRNAs) are short non-coding RNAs that can target multiple molecules to regulate wide array of proteins in diverse pathways. The goal of the study was to profile alterations in miRNA expression using end-stage human heart failure samples with an aim to build signaling network pathways using predicted targets for the altered miRNA and to determine nodal molecules regulating individual networks. Profiling of miRNAs using custom designed microarray and validation with an independent set of samples identified eight miRNAs that are altered in human heart failure including one novel miRNA yet to be implicated in cardiac pathology. To gain an unbiased perspective on global regulation by top eight altered miRNAs, functional relationship of predicted targets for these eight miRNAs were examined by network analysis. Ingenuity Pathways Analysis network algorithm was used to build global signaling networks based on the targets of altered miRNAs which allowed us to identify participating networks and nodal molecules that could contribute to cardiac pathophysiology. Majority of the nodal molecules identified in our analysis are targets of altered miRNAs and known regulators of cardiovascular signaling. Cardio-genomics heart failure gene expression public data base was used to analyze trends in expression pattern for target nodal molecules and indeed changes in expression of nodal molecules inversely correlated to miRNA alterations. We have used NF kappa B network as an example to show that targeting other molecules in the network could alter the nodal NF kappa B despite not being a miRNA target suggesting an integrated network response. Thus, using network analysis we show that altering key functional target proteins may regulate expression of the myriad signaling pathways underlying the cardiac pathology.
Heart failure is a major cause of morbidity in the elderly across the globe. End-stage heart failure is characterized by significantly perturbed neuro-hormonal and mechanical (hemodynamic) stimuli to the heart. The altered pathological signaling leads to remodeling of the heart with adaptive to maladaptive hypertrophy transitioning into dilated cardiomyopathy (DCM). DCM is the most common and well documented outcome of various deleterious stimuli the heart perceives  leading to heart failure. The myocytes undergo remodeling through activation of intracellular signaling pathways involving a cross-talk between deleterious and compensatory signaling pathways. Despite significant advances in identifying genes and signaling pathways, the overall complexity of the hypertrophic remodeling suggests the involvement of global regulatory mechanisms modulating signaling networks. Increasing body of evidence suggests that small non-coding RNAs such as microRNAs (miRNAs) could play a key role in modulating global signaling networks as they are involved in diverse processes including cell proliferation, cell death, metabolism and neuronal patterning .
Mechanisms that alter global signaling networks underlying endstage DCM in humans are largely unknown. In this study, we have utilized the unique miRNA signature observed in the end-stage human heart failure DCM samples to define global molecular signaling networks regulated by miRNAs that identifies the cross-talk between molecules fundamental to the overall DCM phenotype. Using a relatively large set of human samples and custom miRNA microarray platform, based on our cut-off we have identified 8 differentially expressed miRNAs in DCM compared to non-failing human hearts. In addition to identifying known miRNAs that are dysregulated in heart failure , we also identified a novel miRNA, miRNA 7which has not yet been implicated in cardiac pathophysiology. The signaling networks and functional pathways formed by the predicted targets of these altered miRNAs enabled an unbiased prediction of molecular cardiovascular disease bio-function in understanding heart failure biology within the Ingenuity™ knowledge compendium. Critically the use of the miRNA profile allowed us to identify nodal molecules on the global signaling networks that could be altered in response to miRNA targeting of the peripheral molecules laying the foundation for us to propose a global regulatory role of miRNAs in modulating molecular networks. A key idea that our study shows is that the nodal molecules in the signaling network need not be direct targets for the miRNAs but could still be regulated due alterations in the neighbouring molecules due to miRNA targeting within the network. To further support this idea, we have validated the expression pattern of nodal molecules following meta-analysis of heart failure gene expression fingerprint from the publicly available cardio-genomics database. Validation performed by immunoblotting on some of the nodal molecules further confirms the data from the cardio-genomic database. Finally, we have used TAC mouse model and HL-1 cells to show the regulation of the NF kappa B nodal molecule and the inputs into its regulation by neighbouring miRNA targets within the NF kappa B signaling network.