Adipocyte-mediated electrophysiological remodeling of human stem cell - derived cardiomyocytes.

Adipocytes normally accumulate in the epicardial and pericardial layers around the human heart, but their infiltration into the myocardium can be proarrhythmic. METHODS AND RESULTS: Human adipose derived stem/stromal cells and human induced pluripotent stem cells (hiPSC) were differentiated, respectively into predominantly white fat-like adipocytes (hAdip) and ventricular cardiomyocytes (CMs). Adipocytes cultured in CM maintenance medium (CM medium) maintained their morphology, continued to express adipogenic markers, and retained clusters of intracellular lipid droplets. In contrast, hiPSC-CMs cultivated in adipogenic growth medium displayed abnormal cell morphologies and more clustering across the monolayer. Pre-plated hiPSC-CMs co-cultured in direct contact with hAdips in CM medium displayed prolonged action potential durations, increased triangulation, slowed conduction velocity, increased conduction velocity heterogeneity, and prolonged calcium transients. When hAdip-conditioned medium was added to monolayer cultures of hiPSC-CMs, results similar to those recorded with direct co-cultures were observed. Both co-culture and conditioned medium experiments resulted in increases in transcript abundance of SCN10A, CACNA1C, SLC8A1, and RYR2, with a decrease in KCNJ2. Human adipokine immunoblots revealed the presence of cytokines that were elevated in adipocyte-conditioned medium, including MCP-1, IL-6, IL-8 and CFD that could induce electrophysiological changes in cultured hiPSC-CMs. CONCLUSIONS: Co-culture of hiPSC-CMs with hAdips reveals a potentially pathogenic role of infiltrating human adipocytes on myocardial tissue. In the absence of structural changes, hAdip paracrine release alone is sufficient to cause CM electrophysiological dysfunction mirroring the co-culture conditions. These effects, mediated largely by paracrine mechanisms, could promote arrhythmias in the heart.

Surfaceome mapping of primary human heart cells with CellSurfer uncovers cardiomyocyte surface protein LSMEM2 and proteome dynamics in failing hearts.

Cardiac cell surface proteins are drug targets and useful biomarkers for discriminating among cellular phenotypes and disease states. Here we developed an analytical platform, CellSurfer, that enables quantitative cell surface proteome (surfaceome) profiling of cells present in limited quantities, and we apply it to isolated primary human heart cells. We report experimental evidence of surface localization and extracellular domains for 1,144 N-glycoproteins, including cell-type-restricted and region-restricted glycoproteins. We identified a surface protein specific for healthy cardiomyocytes, LSMEM2, and validated an anti-LSMEM2 monoclonal antibody for flow cytometry and imaging. Surfaceome comparisons among pluripotent stem cell derivatives and their primary counterparts highlighted important differences with direct implications for drug screening and disease modeling. Finally, 20% of cell surface proteins, including LSMEM2, were differentially abundant between failing and non-failing cardiomyocytes. These results represent a rich resource to advance development of cell type and organ-specific targets for drug delivery, disease modeling, immunophenotyping and in vivo imaging.

Altered Electrical, Biomolecular, and Immunologic Phenotypes in a Novel Patient-Derived Stem Cell Model of Desmoglein-2 Mutant ARVC.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a progressive heart condition which causes fibro-fatty myocardial scarring, ventricular arrhythmias, and sudden cardiac death. Most cases of ARVC can be linked to pathogenic mutations in the cardiac desmosome, but the pathophysiology is not well understood, particularly in early phases when arrhythmias can develop prior to structural changes. Here, we created a novel human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of ARVC from a patient with a c.2358delA variant in desmoglein-2 (DSG2). These DSG2-mutant (DSG2Mut) hiPSC-CMs were compared against two wildtype hiPSC-CM lines via immunostaining, RT-qPCR, Western blot, RNA-Seq, cytokine expression and optical mapping. Mutant cells expressed reduced DSG2 mRNA and had altered localization of desmoglein-2 protein alongside thinner, more disorganized myofibrils. No major changes in other desmosomal proteins were noted. There was increased pro-inflammatory cytokine expression that may be linked to canonical and non-canonical NFκB signaling. Action potentials in DSG2Mut CMs were shorter with increased upstroke heterogeneity, while time-to-peak calcium and calcium decay rate were reduced. These were accompanied by changes in ion channel and calcium handling gene expression. Lastly, suppressing DSG2 in control lines via siRNA allowed partial recapitulation of electrical anomalies noted in DSG2Mut cells. In conclusion, the aberrant cytoskeletal organization, cytokine expression, and electrophysiology found DSG2Mut hiPSC-CMs could underlie early mechanisms of disease manifestation in ARVC patients.

The Strongest Risk Factor for Operative Mortality in Acute Type A Aortic Dissection is Acidosis: Validation of Risk Model.

Multiple risk factors for operative mortality in the setting of acute type A aortic dissection (ATAAD) have been described. Recently, the combination of severe acidosis and malperfusion was found to significantly impact operative mortality following surgery for ATAAD and a treatment algorithm was proposed. The purpose of this study is to validate these findings in our institution. A retrospective chart review was performed for patients who underwent ATAAD repair between Feb 1997 and Jan 2018. Preoperative nadir pH, bicarbonate, base deficit, organ malperfusion, and other relevant parameters were collected. Multivariable logistic regression was performed to evaluate operative mortality. A total of 298 patients underwent ATAAD repair. The highest operative mortality (18/49; 37%) was noted in patients with severe acidosis (base deficit ≤ -10). There were 96 patients (32%) with malperfusion. In patients with abdominal malperfusion, this trend is even more pronounced. Multivariable logistic regression showed that severe acidosis is associated with higher operative mortality, odds ratio of 13.9 (P = 0.001). The presence of diabetes and advanced age were also associated with higher operative mortality. These findings validate the previously reported findings that severe acidosis is a strong predictor of operative mortality, and risk increases with associated organ malperfusion. This supports the suggestion that base deficit, which is easily performed at the bedside, should be used clinically to predict operative mortality and should be collected in aortic dissection databases.

Engineered Heart Slice Model of Arrhythmogenic Cardiomyopathy Using Plakophilin-2 Mutant Myocytes.

IMPACT STATEMENT: Genetic heart diseases such as arrhythmogenic cardiomyopathy (AC), a common genetic cause of sudden cardiac death, can be modeled using patient-specific induced pluripotent stem cell-derived cardiac myocytes (CMs). However, it is important to culture these cells in a multicellular syncytium with exposure to surrounding matrix cues to create more accurate and robust models of the disease due to the importance of cell-cell and cell-matrix interactions. The engineered heart slice, constructed by seeding CMs on intact decellularized matrix slices, allows molecular and functional studies on an aligned multilayered syncytium of CMs. This study reveals the potential for an improved disease-in-a-dish model of AC.