An organ-on-a-chip model for pre-clinical drug evaluation in progressive non-genetic cardiomyopathy

Highlights

• A platform that captures both acute and chronic cardiac responses to Ang II stimulation.

• Recapitulates pathological hallmarks of hypertrophy and fibrosis in non-genetic cardiomyopathy.

• Provides a modular approach that predicts drug toxicity and efficacy for pre-clinical testing.

• Captures distinct drug effects on reversing cardiomyopathy through multiplexed measurements.

• Highlights potential therapeutic application of relaxin for the treatment of cardiac fibrosis.

Abstract

Angiotensin II (Ang II) presents a critical mediator in various pathological conditions such as non-genetic cardiomyopathy. Osmotic pump infusion in rodents is a commonly used approach to model cardiomyopathy associated with Ang II. However, profound differences in electrophysiology and pharmacokinetics between rodent and human cardiomyocytes may limit predictability of animal-based experiments. This study investigates the application of an Organ-on-a-chip (OOC) system in modeling Ang II-induced progressive cardiomyopathy. The disease model is constructed to recapitulate myocardial response to Ang II in a temporal manner. The long-term tissue cultivation and non-invasive functional readouts enable monitoring of both acute and chronic cardiac responses to Ang II stimulation. Along with mapping of cytokine secretion and proteomic profiles, this model presents an opportunity to quantitatively measure the dynamic pathological changes that could not be otherwise identified in animals. Further, we present this model as a testbed to evaluate compounds that target Ang II-induced cardiac remodeling. Through assessing the effects of losartan, relaxin, and saracatinib, the drug screening data implicated multifaceted cardioprotective effects of relaxin in restoring contractile function and reducing fibrotic remodeling. Overall, this study provides a controllable platform where cardiac activities can be explicitly observed and tested over the pathological process. The facile and high-content screening can facilitate the evaluation of potential drug candidates in the pre-clinical stage.

Introduction

The high incidence of non-genetic cardiomyopathy motivates the development of diseased heart-on-a-chip models from otherwise healthy iPSC derived cardiomyocytes. Angiotensin II (Ang II), the main peptide of the renin–angiotensin system (RAS) system, is believed to induce progressive deterioration of organ function. Its upregulation is implicated in various pathological conditions such as cardiac hypertrophy and fibrosis, offering an important mediator for the disease model development [1,2].

A baseline level of Ang II is essential for rapid developmental growth of the left ventricle [3]. However, elevated Ang II provokes signaling perturbations in both cardiomyocytes(CM) and fibroblasts (FB), resulting in a progressive decline in cardiac contractile function over time. Ang II can induce fibrosis indirectly by activating proinflammatory mediators such as cytokines, chemokines, adhesion molecules [4], as well as directly by regulating extracellular matrix synthesis and degradation [5], increasing ventricular wall stiffness and leading to the impaired diastolic function [1].

Infusion in rodent models via an osmotic pump is commonly used to study cardiomyopathy associated with Ang II [6,7]. However, laboratory animals are not always an accurate guide to how a disease develops in the human body due to phylogenetic discrepancy. Of note, responses of the contractility and Ca²⁺ signaling to Ang II stimulation in mice are fundamentally different from human ventricular myocardium [8]. In addition, the procedure is complicated with low-yield; it often cannot outweigh the substantial cost of animal lives [9]. Animal hearts are normally harvested for further study after animal sacrifice at the endpoint of perfusion. The assessment at single time point provides limited insight into the temporal development of cardiac function in response to Ang II.

The in vitro study of cardiac pathogenesis requires a model system which could closely emulate human biology and enable on-line non-invasive measurements of cardiac contractility [10]. However, the conventional monolayer culture usually contains a thin layer of cells growing on 2D structures such as a Petri-dish, which cannot fully recapitulate the important topographic and biomechanical cues in pathological conditions [11] motivating the development of high fidelity organ-on-a-chip models, starting from human iPSC-derived cardiomyocytes.

Our lab previously developed Biowire II platform, an OOC system exhibiting biomechanical and electrophysiological features of healthy and diseased hearts that enables non-invasive tracking of contractile properties [12]. Through precision phosphoproteomics analysis, the Biowire disease model provides a comprehensive profile of molecular pathways and targets involved in hypertrophic and fibrosis remodeling [13]. In direct comparisons of the data sets generated from different model systems, Biowire exhibited over 600 gene set annotations uniquely similar with patient samples, offering clear advantages over animal models for human disease study [13]. A limitation of this disease model is that it solely mimics fibrosis caused by overpopulated resident fibroblasts [14], as it was generated by seeding fibroblasts and cardiomyocytes in a threefold higher ratio compared to the controls. In native hearts, FB:CM ratio is not so profoundly higher at the disease onset. Previous heart-on-a-chip Ang II models relied on neonatal rat cardiomyocytes [15] and demonstrated applicability over a short time scale of acute drug application lasting for several days, additionally they were explored in systems that did not have a capability to enumerate contractile force directly [16].

Herein, we present the Biowire II platform as an OOC model system for studying Ang II-induced cardiomyopathy, capturing the scenario in which exogenous Ang II acts in an independent fashion in stimulating the onset and progression of pathological cardiac remodeling. This disease modeling strategy aims to provoke progressive disease phenotypes, including inotropic responses, diastolic dysfunction, as well as hypertrophic and fibrotic remodeling, akin to disease progression in the native heart. The long-term tissue cultivation lasting for weeks and non-invasive functional readouts enable monitoring of acute and chronic responses to Ang II stimulation. This model presents an opportunity to quantitatively measure the dynamic changes that could not be otherwise identified in animal models. Additionally, the resultant disease model is used to investigate effective compounds for Ang II-induced progressive cardiomyopathy. Using a multipronged approach, various anti-fibrotic compounds, including a standard Angiotensin II receptor blocker (ARB) losartan, a kinase inhibitor saracatinib, and a peptide hormone relaxin, were evaluated based on their efficacy on reversing pathological cardiac remodeling induced by Ang II.