Human Cardiac Mesenchymal Stem Cells Remodel in Disease and Can Regulate Arrhythmia Substrates

Abstract

Background: The mesenchymal stem cell (MSC), known to remodel in disease and have an extensive secretome, has recently been isolated from the human heart. However, the effects of normal and diseased cardiac MSCs on myocyte electrophysiology remain unclear. We hypothesize that in disease the inflammatory secretome of cardiac human MSCs (hMSCs) remodels and can regulate arrhythmia substrates.

Methods: hMSCs were isolated from patients with or without heart failure from tissue attached to extracted device leads and from samples taken from explanted/donor hearts. Failing hMSCs or nonfailing hMSCs were cocultured with normal human cardiac myocytes derived from induced pluripotent stem cells. Using fluorescent indicators, action potential duration, Ca2+ alternans, and spontaneous calcium release (SCR) incidence were determined.

Results: Failing and nonfailing hMSCs from both sources exhibited similar trilineage differentiation potential and cell surface marker expression as bone marrow hMSCs. Compared with nonfailing hMSCs, failing hMSCs prolonged action potential duration by 24% (P<0.001, n=15), increased Ca2+ alternans by 300% (P<0.001, n=18), and promoted spontaneous calcium release activity (n=14, P<0.013) in human cardiac myocytes derived from induced pluripotent stem cells. Failing hMSCs exhibited increased secretion of inflammatory cytokines IL (interleukin)-1β (98%, P<0.0001) and IL-6 (460%, P<0.02) compared with nonfailing hMSCs. IL-1β or IL-6 in the absence of hMSCs prolonged action potential duration but only IL-6 increased Ca2+ alternans and promoted spontaneous calcium release activity in human cardiac myocytes derived from induced pluripotent stem cells, replicating the effects of failing hMSCs. In contrast, nonfailing hMSCs prevented Ca2+ alternans in human cardiac myocytes derived from induced pluripotent stem cells during oxidative stress. Finally, nonfailing hMSCs exhibited >25× higher secretion of IGF (insulin-like growth factor)-1 compared with failing hMSCs. Importantly, IGF-1 supplementation or anti-IL-6 treatment rescued the arrhythmia substrates induced by failing hMSCs.

Conclusions: We identified device leads as a novel source of cardiac hMSCs. Our findings show that cardiac hMSCs can regulate arrhythmia substrates by remodeling their secretome in disease. Importantly, therapy inhibiting (anti-IL-6) or mimicking (IGF-1) the cardiac hMSC secretome can rescue arrhythmia substrates.

Keywords: COVID-19; arrhythmia; insulin-like growth factor 1; interleukin-6; mesenchymal stem cell.

Figure 1:

Shown is a myocardial tissue sample obtained from a cardiac device lead extraction procedure as it exists on the tip of the lead after extraction (Panel A) and after removal from the tip (Panel B). In both panels, the arrow points to the tissue sample. Shown at the bottom are cardiac MSCs from device leads from a non-failing heart (Panel C), and cardiac hMSCs from device leads from a failing heart (Panel D) after isolation and plating.

Figure 2:

Examples of osteoblasts (Alizarin Red, top), adipocytes (Oil Red, middle), and chondroblasts (Alcian Blue, bottom) differentiated from plastic adherent cells isolated from the bone marrow (positive control), a device lead extracted from a patient with a normal EF (non-failing hMSC, middle left) and a failing heart (Failing hMSC, middle right), and human dermal fibroblasts (negative control). After 3 weeks, a similar differentiation potential was observed in all groups, except dermal fibroblasts.

Figure 3:

Surface marker expression by flow cytometry for BM (bone marrow, n=5), non-failing (n=3) and failing hMSCs (n=9). All three MSC types had characteristic MSC surface marker expression (positive for CD 90 and CD 105 and negative for CD45). CD 117 and CD 133 surface expression are shown for further characterization.

Figure 4:

Shown are Ca2+ alternans (ALT) and APD measured in hCM under normal conditions when co-cultured with cardiac hMSCs from non-failing hearts (nonFailing hMSCs) and failing hearts (Failing hMSC). Example traces in Panel A show Ca2+ alternans (left) and APD (right). Summary data are shown in Panel B. For the summary data, filled circles represent samples from device leads and empty circles are from explanted/donor hearts. Levels of significance are shown in the figure.

Figure 5:

Shown are examples of Ca2+ transient recordings from hCM with H₂O₂ and co-cultured with either failing (top) or non-failing cardiac hMSCs (bottom) during the termination of rapid pacing. Spontaneous calcium release (SCR) activity (arrow) was observed with failing but not non-failing cardiac hMSCs.

Figure 6:

Shown are Ca2+ alternans (ALT) measured under conditions of oxidative stress (H₂O₂) in hCM alone or when also co-cultured with cardiac hMSCs from a non-failing heart (nonFailing hMSC) and failing heart (Failing hMSC). All conditions include H₂O₂ and hCM. Example traces are shown in Panel A and summary data in Panel B. For the summary data, filled circles represent samples from device leads and empty circles are from explanted/donor hearts. For reference, hCM in the absence of H₂O₂ is shown. There was no significant difference between hCM and non-failing hMSCs. Otherwise, levels of significance are shown in the figure.

Figure 7:

IL-1β and IL-6 effect on arrhythmia substrates. Panel A shows the effect of IL-1β (n=6) and IL-6 (n=5) on average Ca2+ alternans (Ca2+ ALT), APD50 and APD90 when administered to hCM alone. Levels of significance are compared to hCM where * p < 0.001 (for Ca2+ ALT, n=23), * p = 0.002 (for APD50, n=6), * p < 0.02 (for APD90, n=6). Panel B shows ELISA results for IL-1β (left) and IL-6 (right) in separate populations of bone marrow (BM) hMSCs, non-failing cardiac hMSCs, failing cardiac hMSCs, and hCM alone (n=4). Levels of significance are compared to BM hMSC where * p < 0.001 (for IL-1β), * p = 0.008 (for IL-6). Panel C shows the rescue of Ca2+ alternans induced by failing hMSCs (n=18) with anti-IL-6 (n=18) treatment. Levels of significance are compared to hCM (n=12) where * p < 0.001.

Figure 8:

Panel A shows the effect of IGF-1 (n=6) and VEGF (n=5) on average Ca2+ alternans when incubated with hCM alone. Also shown are the effects of IGF-1 when incubated with Wortmannin (WORT), an inhibitor of PI3K (n=5). All conditions include hCM. Levels of significance are * p < 0.006 compared to hCM alone (n=38). Panel B shows ELISA results for IGF-1 in separate populations of bone marrow (BM) hMSCs (n=4), non-failing hMSCs (n=8), failing hMSCs (n=8), and hCM alone (n=4). Levels of significance are * p < 0.0001 compared to BM hMSC and † p < 0.0001 compared to non-failing hMSC. Panel C shows IGF-1 supplementation rescues the failing phenotype induced by failing hMSCs without oxidative stress. Ca2+ ALT (n=15), APD50 (n=6) and APD90 (n=6) are shown for hCM alone, hCM co-cultured with failing cardiac hMSC (n=21), and with the addition of IGF-1 (n=5). Levels of significance are compared to hCM where * p < 0.001 and † p = 0.05 (for Ca2+ ALT) and † p = 0.08 (non-significant for APD50) and * p = 0.004 (for APD90).