A modified method for isolation of human cardiomyocytes to model cardiac diseases

A good cellular model is crucial to understanding the physiology and pathology of the heart. Due to the difficulty in obtaining heart tissue and in isolating primary cardiomyocytes, most cardiovascular studies to date adopt animal models or iPSCs-derived cardiomyocytes. However, functional and molecular characteristics of the heart vary significantly in human and animals [1]. Indeed, several studies demonstrated evidence for the marked species differences in cardiac ion currents [2, 3], and energy metabolism [4], hampering the clinical translation of findings based on such models. Although iPSCs-derived cardiomyocytes have been successfully generated from patients harboring various diseases, such cardiomyocytes are morphologically and functionally similar to fetal cells, which has become a major and common impediment to their application in modeling late-onset disorders [5]. By contrast, primary human cardiomyocytes are suitable for modeling and studying a broad spectrum of human heart diseases. Isolated myocytes with high purity avoids contamination of other cell types in heart, thereby reducing experimental noise [6].

Of note, cardiomyocytes are susceptible to hypoxia, the slightest exposure to hypoxia can cause changes in ultrastructure [7, 8], posing a great challenge to obtaining viable myocytes. Furthermore, cellular remodeling usually accompanies physiological deterioration [9], rendering myocytes particularly vulnerable to enzymatic digestion.

Enzymatic bulk digestion and the Langendorff method are two major methods for isolation of cardiac myocytes. Bulk digestion consists of enzyme digestion and mechanical agitation of the heart tissue, without requirement for specimen containing vessel structure. But owing to insufficient tissue exposure to the enzyme solution, the yield of rod-shaped myocytes was only 19.0 ± 1.6% [10]. The Langendorff method, invented in 1895, improves cell yield by using retrograde perfusion through the aorta with enzyme-containing solutions [11]. This method was initially applied to isolate cardiomyocytes from adult animal hearts and later modified to isolate human myocytes by placing a small catheter into the artery or a vein of the tissue to perfuse enzyme buffer, with reported yields of 10% to 50% rod-shaped myocytes [12]. As such, this method depends on tissue structure, and is therefore not suitable for tissue fragments from cardiac surgeries.

Considering the limitations of the two methods, we want to explore an efficient isolation method without tissue limitation. We hypothesized that the same hydrodynamic effect of perfusion could be achieved by cutting myocardium into several hundred tissue sections. Using this modified protocol, we isolated human atrial myocytes with 64.8% ± 5.0% rod-shaped cell yield.

Enzymatic bulk digestion and the Langendorff method are two common isolation methods for heart tissue. In spite of some improvements in digestion device of bulk digestion [15], the isolation efficiency is still lower than Langendorff method. Recently, some researchers present a novel, simplified method to isolate the cardiomyocytes from adult mouse heart [13], reducing the technical difficulty. However, the requirement of tissue integrity still limits its application in human sample. Our protocol provides a optimized method to isolate cardiomyocytes from surgery waste with yields comparable to those in published Langendorff-based methods, reflecting practical advantage in processing human heart tissue.

The procedure of isolation is inevitably hypoxic; therefore, cardioprotection is the key to cell yield. We find that the more living myocytes yield from the cardioplegic perfused tissue, a step reported decreasing myocardium oxygen demand [16] as well as fatty acid oxidation [17]. For transport of myocardial specimen, most protocols opt for the Thomas solution [18] or cardioplegic buffer [19, 20] as the transport medium. It has been reported that the UW solution showed beneficial effects on the recovery of myocyte viability compared to the Thomas’ Hospital and glucose-based potassium solutions [21]. Additionally, the superior myocardial protective effects of HTK solution, as compared with conventional St. Thomas crystalloid cardioplegia has been proved [22]. We compared the effect among transport buffers HTK solution, UW solution and cardioplegic buffer, and found that the UW solution results in highest cell yield.

One step of most isolation protocols shared is a period of perfusion with a nominally Ca²⁺-free solution, in order to take advantage of the protective effect of reduced temperature, the initial washing steps, which are usually performed in Ca²⁺-free solution, was performed at room temperature, and not at 37 °C that most other protocols use [10, 23, 24]. Optimization of the entire procedure reduced isolation to around 70 min, effectively shortening ischemia time.

Human atrial tissue is readily available as atrial appendages are often discarded during surgical procedures. Therefore, initial isolation methods of adult human cardiomyocytes were established for atrial cells, and later for ventricular cells [15]. The method described here allows isolation of both atrial and ventricular myocytes with high yields as well as clear striations (Additional file 1: Figure S2). More importantly, the RNA derived from those cells is of high quality (with RNA integrity number higher than 7.5) and qualified for downstream RNA-sequencing (Additional file 1: Figure S3). A surprising finding is that some green-appearing myocytes were round, a morphology usually regarded as cells dying or being stressed or damaged [25]. We speculated these subsets of cells were still alive when exposed to the dye, but soon died and lost the rod-shape morphology. This protocol can also be applied to isolate cardiomyocytes from human heart diseases, such as heart failure, hypertrophic cardiomyopathy etc. Cell excitability (i.e. the capacity of the heart to beat spontaneously) is central to cardiac physiology [26]. We were able to record ion currents, Ca²⁺ transients and action potentials in the cells obtained via this protocol, providing a platform for patch clamp experiments [27]. In addition to excitability, cardiac energy metabolism, which mainly generates ATP from fatty acid oxidation, is also distinctly different from other organs. Alterations in myocardial energy substrate metabolism is known to occur in heart failure, switching from fatty acid oxidation toward predominantly glycolysis [28], providing opportunity for potential intervention [29]. There is compelling evidence indicating that aerobic metabolism in the mitochondria becomes increasingly important as the heart energy transitions, which can be measured via oxygen consumption rate (OCR) [30]. We successfully determined the OCR in the cardiomyocytes isolated using our method. We show that they retain the morbid metabolic characteristics, thus providing a useful tool to study heart metabolism. Finally, cells exhibited sensitivity to drug stimulation, allowing pharmacological experiments and future drug screening.

The described method offers a novel approach to the isolation of viable human cardiomyocytes from surgery waste with expected cell yields. The characteristics of isolated cardiomyocytes are closer to their status in vivo and a series of functional study for cardiovascular research could be performed based on this cell model. We anticipate that this human cell model will expand and accelerate innovative research in related field.