Human Cardiomyocyte Progenitor Cells in Co-culture with Rat Cardiomyocytes Form a Pro-arrhythmic Substrate: Evidence for Two Different Arrhythmogenic Mechanisms

Abstract

Background: Cardiomyocyte progenitor cells (CMPCs) are a promising cell source for regenerative cell therapy to improve cardiac function after myocardial infarction. However, it is unknown whether undifferentiated CMPCs have arrhythmogenic risks. We investigate whether undifferentiated, regionally applied, human fetal CMPCs form a pro-arrhythmic substrate in co-culture with neonatal rat ventricular myocytes (NRVMs).Method: Unipolar extracellular electrograms, derived from micro-electrode arrays (8 × 8 electrodes) containing monolayers of NRVMs (control), or co-cultures of NRVMs and locally seeded CMPCs were used to determine conduction velocity and the incidence of tachy-arrhythmias. Micro-electrodes were used to record action potentials. Conditioned medium (Cme) of CMPCs was used to distinguish between coupling or paracrine effects.Results: Co-cultures demonstrated conduction slowing (5.6 ± 0.3 cm/s, n = 50) compared to control monolayers (13.4 ± 0.4 cm/s, n = 26) and monolayers subjected to Cme (13.7 ± 0.6 cm/s, n = 11, all p < 0.001). Furthermore, co-cultures had a more depolarized resting membrane than control monolayers (−47.3 ± 17.4 vs. −64.8 ± 7.7 mV, p < 0.001) and monolayers subjected to Cme (−64.4 ± 8.1 mV, p < 0.001). Upstroke velocity was significantly decreased in co-cultures and action potential duration was prolonged. The CMPC region was characterized by local ST-elevation in the recorded electrograms. The spontaneous rhythm was faster and tachy-arrhythmias occurred more often in co-cultured monolayers than in control monolayers (42.0 vs. 5.4%, p < 0.001).Conclusion: CMPCs form a pro-arrhythmic substrate when co-cultured with neonatal cardiomyocytes. Electrical coupling between both cell types leads to current flow between a, slowly conducting, depolarized and the normal region leading to local ST-elevations and the occurrence of tachy-arrhythmias originating from the non-depolarized zone.

In silico Cell Therapy Model Restores Failing Human Myocyte Electrophysiology and Calcium Cycling in Fibrotic Myocardium

“…Nonetheless, across all species, treating myocytes with hCIC HC decreased upstroke velocity (UV), decreased peak action potential voltage (V peak ), depolarized RMP, and increased [Ca 2+ ] i ,max relative to untreated controls; the effects increased with gap junction conductance from 1 to 10 nS, with minimal further changes due to cell fusion. Consistent with in vitro experimental work ( Smit et al, 2017 ), simulated treatment of rat cardiomyocytes with hCIC HC led to prolonged APD, depolarized RMP, and decreased UV when compared to control untreated rat cardiomyocytes ( Figure 3B ). As anecdotally noted in the same study ( Smit et al, 2017 ), we also predicted V peak to be decreased when treating with hCICs.…”

“…Paracrine effects of hCICs on cardiomyocyte electrophysiology and calcium cycling were assumed to be negligible based on findings from Smit et al (2017) and others ( Gray et al, 2015 ; Agarwal et al, 2017 ).…”

“…For example, in addition to promoting angiogenesis and reducing fibrosis (Ranganath et al, 2012), hMSC PS alters excitation-contraction coupling of cardiomyocytes (Mayourian et al, 2018a), causing increased expression of key calcium cycling genes-including sarcoendoplasmic reticulum calcium-ATPase (SERCA) and L-type calcium channel (LTCC)-and thereby increasing cardiomyocyte calcium transient amplitude and contractility (Desantiago et al, 2012;Hwang et al, 2012;Mayourian et al, 2017). By contrast, when studying the paracrine effects of hCICs, Smit et al (2017) observed no significant changes in rat cardiomyocyte action potential morphology, and others reported the exosomal component of the hCIC secretome to have limited uptake by cardiomyocytes with no significant effect on cardiomyocyte calcium cycling (Gray et al, 2015;Agarwal et al, 2017).…”

“…On the other hand, hMSC HC is known to alter cardiomyocyte action potential duration (APD) and tissue-level conduction velocity (Chang et al, 2006;Mayourian et al, 2016). Similarly, hCIC HC was recently found to decrease rat cardiomyocyte upstroke velocity, depolarize resting membrane potential (RMP), and prolong APD (Smit et al, 2017), and human engineered cardiac tissues supplemented with 10% hCICs displayed significant increases in contractility and markers of cardiomyocyte maturation (Murphy et al, 2019). Clearly, there is a complex and cell type-dependent interactome between cardiomyocytes and these non-excitable cells that is difficult to elucidate experimentally.…”

Cardiomyocytes Derived from Human CardiopoieticAmniotic Fluids

“…As reviewed by Petsche Connel 22 , other groups have also reported that AF cells could be guided toward cardiac differentiation: rat or human c-kit + -selected cells may acquire a CM phenotype when co-cultured with rat neonatal CMs, and functional communications develop among the cells of the co-culture 14 , 17 . However, this differentiation process occurs with a very low yield and presents limits deriving from a xenogenic co-culture sysytem 36 . Other evidence suggests that the modulation of WNT 15 signaling or exposure to 5-Aza 14 , 15 may induce a CM phenotype in unselected AF cells, but these protocols were also characterized by low differentiation efficiency, and organized sarcomeric structures and spontaneous contraction were not detected.…”