Cyclic strain has antifibrotic effects on the human cardiac fibroblast transcriptome in a human cardiac fibrosis-on-a-chip platform.

In cardiac fibrosis, in response to stress or injury, cardiac fibroblasts deposit excessive amounts of collagens which contribute to the development of heart failure. The biochemical stimuli in this process have been extensively studied, but the influence of cyclic deformation on the fibrogenic behavior of cardiac fibroblasts in the ever-beating heart is not fully understood. In fact, most investigated mechanotransduction pathways in cardiac fibroblasts seem to ultimately have profibrotic effects, which leaves an important question in cardiac fibrosis research unanswered: how do cardiac fibroblasts stay quiescent in the ever-beating human heart? In this study, we developed a human cardiac fibrosis-on-a-chip platform and utilized it to investigate if and how cyclic strain affects fibrogenic signaling. The pneumatically actuated platform can expose engineered tissues to controlled strain magnitudes of 0-25% - which covers the entire physiological and pathological strain range in the human heart - and to biochemical stimuli and enables high-throughput screening of multiple samples. Microtissues of human fetal cardiac fibroblasts (hfCF) embedded in gelatin methacryloyl (GelMA) were 3D-cultured on this platform and exposed to strain conditions which mimic the healthy human heart. The results provide evidence of an antifibrotic effect of the applied strain conditions on cardiac fibroblast behavior, emphasizing the influence of biomechanical stimuli on the fibrogenic process and giving a detailed overview of the mechanosensitive pathways and genes involved, which can be used in the development of novel therapies against cardiac fibrosis.

The circadian clock remains intact, but with dampened hormonal output in heart failure.

BACKGROUND: Circadian (24-h) rhythms are important regulators in physiology and disease, but systemic disease may disrupt circadian rhythmicity. Heart failure (HF) is a systemic disease affecting hormonal regulation. We investigate whether HF affects the rhythmic expression of melatonin and cortisol, main endocrine products of the central clock, and cardiac-specific troponin in patients. We corroborate the functionality of the peripheral clock directly in the organs of translational models, inaccessible in human participants. METHODS: We included 46 HF patients (71.7% male, median age of 60 years, NYHA class II (32.6%) or III (67.4%), ischemic cardiomyopathy (43.5%), comorbidities: diabetes 21.7%, atrial fibrillation 30.4%), and 24 matched controls. Blood was collected at seven time-points during a 24-h period (totalling 320 HF and 167 control samples) for melatonin, cortisol, and cardiac troponin T (cTnT) measurements after which circadian rhythms were assessed through cosinor analyses, both on the individual and the group level. Next, we analysed peripheral circadian clock functionality using cosinor analysis in male animal HF models: nocturnal mice and diurnal zebrafish, based on expression of core clock genes in heart, kidneys, and liver, every 4 h during a 24-h period in a light/darkness synchronised environment. FINDINGS: Melatonin and cortisol concentrations followed a physiological 24-h pattern in both patients and controls. For melatonin, acrophase occurred during the night for both groups, with significantly decreased amplitude (median 5.2 vs 8.8, P = 0.0001) and circadian variation ([maximum]/[minimum]) in heart failure patients. For cortisol, mesor showed a significant increase for HF patients (mean 331.9 vs 275.1, P = 0.017) with a difference of 56.8 (95% CI 10.3-103.3) again resulting in a relatively lower variation: median 3.9 vs 6.3 (P = 0.0058). A nocturnal blood pressure dip was absent in 77.8% of HF patients. Clock gene expression profiles (Bmal, Clock, Per, Cry) were similar and with expected phase relations in animal HF models and controls, demonstrating preserved peripheral clock functionality in HF. Furthermore, oscillations in diurnal zebrafish were expectedly in opposite phases to those of nocturnal mice. Concordantly, cTnT concentrations in HF patients revealed significant circadian oscillations. INTERPRETATION: Central clock output is dampened in HF patients while the molecular peripheral clock, as confirmed in animal models, remains intact. This emphasises the importance of taking timing into account in research and therapy for HF, setting the stage for another dimension of diagnostic, prognostic and therapeutic approaches. FUNDING: Hartstichting.

Pirfenidone Has Anti-fibrotic Effects in a Tissue-Engineered Model of Human Cardiac Fibrosis.

A fundamental process in the development and progression of heart failure is fibrotic remodeling, characterized by excessive deposition of extracellular matrix proteins in response to injury. Currently, therapies that effectively target and reverse cardiac fibrosis are lacking, warranting novel therapeutic strategies and reliable methods to study their effect. Using a gelatin methacryloyl hydrogel, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and human fetal cardiac fibroblasts (hfCF), we developed a multi-cellular mechanically tunable 3D in vitro model of human cardiac fibrosis. This model was used to evaluate the effects of a promising anti-fibrotic drug-pirfenidone-and yields proof-of-concept of the drug testing potential of this platform. Our study demonstrates that pirfenidone has anti-fibrotic effects but does not reverse all TGF-ß1 induced pro-fibrotic changes, which provides new insights into its mechanism of action.

Prognostic biomarker soluble ST2 exhibits diurnal variation in chronic heart failure patients.

AIM: Soluble suppression of tumorigenicity-2 (sST2) is a strong prognostic biomarker in heart failure. The emerging understanding of circadian biology in cardiovascular disease may lead to novel applications in prognosis and diagnosis and may provide insight into mechanistic aspects of the disease-biomarker interaction. So far, it is unknown whether sST2 exhibits a diurnal rhythm. Repeated measurements of sST2 may aid in clinical decision making. The goal of this study was to investigate whether sST2 exhibits diurnal variation in patients with heart failure with reduced ejection fraction (HFrEF) and in control subjects, thereby enhancing its diagnostic and prognostic values. METHODS AND RESULTS: The study comprised 32 subjects: 16 HFrEF patients and 16 controls. Blood was collected at seven subsequent time points during a 24 h time period. sST2, N-terminal pro-B-type natriuretic peptide (NT-proBNP), melatonin, and cortisol were measured from serum. Peak values of sST2 clustered at daytime (modal value: 5 p.m.) in 87.6% of all subjects (81.3% of patients, P = 0.021; 93.8% of controls, P = 0.001), and minimum concentrations at night-time (modal value: 5 a.m.) in 84.4% (87.5% of patients, P = 0.004 81.3% of controls, P = 0.021). A cosinor analysis of mean normalized sST2 values revealed significant cosine shaped 24 h oscillations of patients (P = 0.026) and controls (P = 0.037). NT-proBNP in contrast did not show a diurnal rhythm, while melatonin and cortisol patterns were intact in all subjects. CONCLUSIONS: sST2 exhibits a diurnal rhythm with lower values in the morning than in the late afternoon. This new insight could lead to refinement of its diagnostic and prognostic values through specified and consistent sampling times with repeated measurements. For example, by measuring sST2 during the afternoon, when levels are at their highest, false negatives on prognosis prediction could be avoided.