2.3. In-vitro disease modeling in diabetic cardiomyopathy

It has become increasingly clear that also on a cellular level, DMCMP presents as two distinct diseases with a HFrEF and HFpEF phenotype. Early evidence suggests that in the DMCMP HFpEF phenotype collagen and advanced glycation end-products (AGEs) deposition are increased. In contrast, cardiomyocytes in patients with the DMCMP HFrEF phenotype show an overall loss of sarcomeres.

Unfortunately, limited information is available on differences between DMCMP with HFrEF and HFpEF. Elucidating these differences will aid in our understanding of the underlying pathophysiological differences of DMCMP with HFrEF and HFpEF and provide for novel treatment targets. Additionally, it has become increasingly clear that particular DM medications might have beneficial effects in patients with HF. Results from the EMPA-REG trial showed that sGLT2 inhibitors might possible aid in reducing the number of HF hospital readmissions. Novel targets on DMCMP patients with HFrEF and HFpEF will aid in identifying HF patients potentially benefitting from existing treatment for DM.

The overall aim of this proposal is to elucidate the differences between patients with DMCMP presenting with a HFrEF or HFpEF phenotype. We will then investigate mechanistic implications of patient specific treatment targets in in-vitro and in-vivo models.

To approach the above stated aim, we propose four sub aims using a translational approach:

1. We will perform a full blood proteomics screening on age- and sex- matched patients with DMCMP and HFrEF (N=20)/HFpEF(N=20) and compared to age- and sex- matched healthy (absence of coronary artery disease, hypertension, DM and HF) controls (N=20). We will validate our findings in a larger cohort of healthy control patients (N=200) vs. DMCMP patients with HFrEF (N=100) and HFpEF (N=100).
2. Using the top targets found in (1), we will examine the effect of patient specific targets on cardiomyocyte phenotype and function using an in-vitro approach. Here, we will employ human embryonic stem cell derived cardiomyocytes (hESC-CM) as a model using recombinant proteins of targets found in (1) and/or antagonists of targets found in (1) for HFrEF and HFpEF).
3. Following, we will phenotype the hESC-CM and assess response to stimulants (e.g. High glucose; cardiac stretch; oxidative stress; hypoxia) on both RNA (RT-qPCR) and Protein (Western Blot) level using a panel of genes and proteins known to be associated in eccentric and concentric hypertrophy, and extracellular matrix and sarcomere structure. Additionally, we will assess the effect on metabolism of hESC-CM using the Seahorse assay. Lastly, we will study contractility and stiffness of the targeted hESC-CM using an in-house assay. Following our mechanistic studies in (2), we will perform an in-vivo study of the most promising individual targets in HFrEF and HFpEF from (1) & (2). Here, we will produce total knock-out and overexpression mouse models and phenotype mice using echocardiography and study of cardiac tissue. In case of a less-severe phenotype, we will investigate the role of said targets in a pathological setting using a trans-aortic constriction (TAC) model to produce LV dysfunction.
4. Lastly, we will investigate whether targets found in (1), (2) and (3), are associated with new-onset HFrEF and HFpEF. We will measure targets found in individuals from the LIFELINES cohort and investigate a potential predictive association with new-onset HFrEF and HFpEF. Furthermore, we will measure said targets in HF patients with HFrEF and HFpEF (with and without DM) from the BIOSTAT-CHF cohort and investigate whether these novel targets will predict outcomes in these patients.

1. Target identification and validation in patients with DM and HFrEF and HFpEF
2. In-vitro approach to most important targets found in Chapter 1 using hESC derived cardiomyocyte models
3. In-vivo mouse study most promising target in HFrEF and HFpEF
4. Measurement of targets found in patients with HF and association with outcome.

Creating a better understanding of DMCMP with HFrEF and HFpEF will lead to potential novel patient specific treatment targets for these patients. Furthermore, by measuring novel targets in patients with HF, we will identify patients benefiting from a personalized medicine approach (sub aim 4) using existing DM treatments.

This proposal is linked to the following ProminenT domains:

• Disease mechanisms (sub aim [1], [2] and [3])
• Drug development (sub aim [1], [2] and [3])
• Drug application (sub aim [1] and [4])