Study design and main objectives

Understanding the causal biological disease mechanisms enables the development of novel treatment strategies. By harnessing the power of patient-derived material (blood, tissue and iPSCs; see Supplementary Methods in Electronic Supplementary Material) collected in the DECIPHER-PLN cohort, we aim to gain a better understanding of R14∆/+ pathophysiology (Tab. 1). Using phosphoproteomics on heart tissue, relevant data will be collected directly from R14∆/+ carriers. This will be done in combination with in vitro disease modelling utilising iPSC-derived cardiomyocytes (iPSC-CMs), whereby R14∆/+ disease pathophysiology will be studied using a multi-omics approach (phosphoproteomics and proteomics) in combination with extensive functional characterisation (e.g. assays on contractility, calcium handling, metabolism and immunofluorescence) of R14∆/+ iPSC lines. Importantly, these iPSC lines will allow us to better understand why one person in a family develops the R14∆/+ phenotype and the other does not. iPSC-CMs are also an excellent platform for monitoring the response to promising drugs that may eventually enter clinical trials.

Table 1 Overview of DECIPHER-PLN tentative analysis and baseline characteristics of patients with patient-derived R14∆/+ iPSC-lines across disease spectrum

We will test the potential of PLN antisense oligonucleotides, which have been shown to be a promising therapeutic agent to combat R14∆/∆ cardiomyopathy in a murine model [13]. In addition, using R14∆/+ carrier-derived blood samples, we aim to identify biomarkers using metabolomics and the Olink Proteomics Panel (and validate them using other methods, such as enzyme-linked immunosorbent assay) that can distinguish between R14∆/+ carriers across the disease spectrum, which will allow early detection of R14∆/+ disease progression.

Study population

Participants were eligible if they were > 18 years of age, had genetic confirmation of their R14∆/+ carrier status, were capable of adequate communication and were able to provide informed consent. Participants were categorised into either unaffected, early affected or end-stage disease groups based on LV ejection fraction (LVEF), signs and symptoms of HF, ECG and N‑terminal pro-brain natriuretic peptide (NT-proBNP) levels. The unaffected R14∆/+ group had LVEF > 50% and no HF symptoms. The early affected R14∆/+ group showed echocardiographic or ECG abnormalities or NT-proBNP > 200 ng/l without symptomatic HF. The end-stage R14∆/+ group presented with LVEF < 40%, history of sustained ventricular tachycardias (VTs) or symptomatic HF with NT-proBNP > 200 ng/l. For the collection of skin biopsies, additional inclusion criteria were used. Skin biopsies were collected in pairs of two: a pair had to be an age-matched family member (an older unaffected R14∆/+ carrier was acceptable). Exclusion criteria were that R14∆/+ carriers could not participate if they had an extensive skin disorder precluding a biopsy from an unaffected skin area, were known to be allergic to local anaesthetics or had any significant cardiovascular risk factor, non-R14∆/+ cardiometabolic disease or other known gene mutation. For the collection of heart tissues, participants had to undergo LVAD implantation or receive a heart transplant. Patients with other HF aetiologies served as the control group.

Ethical statement

The current study is conducted according to the principles of the Declaration of Helsinki (7th revision, October 2013, Fortaleza, Brazil) and in accordance with the Dutch Medical Research Involving Human Subjects Act (Wet medisch-wetenschappelijk onderzoek met mensen). The scientific advisory board of the University Medical Centre Groningen provided ethical approval for the collection of the blood samples, skin biopsies and human heart tissues (protocol numbers 2020.326 and 2020.327, UMCG Research Register number 202000351; ABR number NL73976.042.20). DECIPHER-PLN has been registered at ClinicalTrials.gov (identifier NCT04978987).

Baseline characteristics

A total of 101 subjects were included: 91 R14∆/+ carriers and 10 patients with end-stage HF of an aetiology other than R14∆/+. Of the 91 R14∆/+ carriers, 21 were classified as unaffected, 42 as early affected and 28 as end-stage R14∆/+. Baseline characteristics of the DECIPHER-PLN cohort are described in Tab. 2. R14∆/+ carriers that advanced across the disease spectrum were older, had higher circulating NT-proBNP and cardiac troponin T levels, had a lower heart rate, systolic and diastolic blood pressure and a higher New York Heart Association (NYHA) class and were more often previously hospitalised for HF. Significantly more devices were used in affected carriers, with 23 of 28 (85%) end-stage R14∆/+ carriers having an implantable cardioverter-defibrillator (ICD), with or without a dual-chamber pacemaker and/or cardiac resynchronisation therapy. End-stage and early affected R14∆/+ carriers had worse RV and LV functions, more LV dilation and higher estimated RV peak pressures. At 24-hour Holter examination, more premature ventricular contractions (PVCs) and non-VTs were observed in affected subjects, with a median number of PVCs of 3434 (interquartile range: 353–7232) in end-stage R14∆/+ carriers and 57% of these subjects showing non-sustained VTs.

Table 2 Baseline characteristics of the DECIPHER-PLN cohortGeneration of patient-derived R14∆/+ iPSC lines across R14∆/+ disease spectrum

To study R14∆/+ disease modifiers, patient-derived skin fibroblasts from end-stage R14∆/+ and unaffected age-matched R14∆/+ family members were successfully reprogrammed into iPSCs (Tab. 1). To confirm successful reprogramming of fibroblasts into the iPSC lineage, iPSCs were stained for the stem cell marker SRY-box transcription factor 2 (SOX2) (Fig. 1). iPSC generated from fibroblasts expressed SOX2 protein, confirming iPSC line generation. iPSC lines were successfully differentiated into the cardiomyocyte lineage expressing α‑actinin and cardiac troponins (Fig. 1).

Fig. 1

Characterisation and differentiation of induced pluripotent stem cells (iPSC) lines from patients with end-stage R14∆/+ (rows 1–3) and unaffected family members with R14∆/+ (rows 4–6). a Immunostaining of 4’,6-diamidino-2-phenylindole (DAPI) and pluripotency marker SRY-box transcription factor 2 (SOX2) to detect iPSCs. b Immunostaining of cardiomyocyte markers α‑actinin and cardiac troponin T (cTnT) to detect iPSC-derived cardiomyocytes

R14∆/+ heart tissue harbours fibrosis, fibrofatty infiltrates and abnormal PLN distribution

Using patient-derived tissue allows us to gain a better understanding of disease pathophysiology and identify therapeutic targets. To this end, heart tissues were collected from end-stage R14∆/+ patients, with patients with other HF aetiologies as the control group. R14∆/+ and control tissues revealed large inter-tissue differences, including regions with seemingly unaffected myocardium, severely fibrotic regions and composite regions consisting of cardiomyocytes, fibrosis and fibrofatty infiltrates (Fig. 2). Immunofluorescent staining of PLN in these tissues revealed abnormal distribution of PLN protein that is specific to R14∆/+ and not detected in control HF tissues.

Fig. 2

Heart tissue characterisation of patients with R14∆/+ cardiomyopathy (rows 1–3) and control dilated cardiomyopathy (DCM) tissue (row 4). Masson staining is shown in first 3 columns revealing inter-tissue differences, including myocardial area, composite area and fibrotic area. Last column shows immunostaining of phospholamban (PLN) in combination with 4’,6-diamidino-2-phenylindole and wheat germ agglutinin stainings