To perfuse or not to perfuse: A new method for adult myocyte isolation to be tested with time

To study molecular and cellular mechanisms for normal cardiac function and cardiac pathology, high-quality isolated adult cardiomyocytes are essential. Isolated cardiomyocytes can be used directly for cellular function studies such as measuring myocyte contraction, cellular electrophysiology, calcium imaging, myofilament calcium sensitivity, cytoskeletal function, drug effects and metabolism. Isolated myocytes can also be cultured for signaling studies with less contamination from non-myocytes. In addition, the isolated cells are also essential for studying the morphology and structure of cardiomyocytes. Furthermore, the function of non-myocytes including cardiac fibroblasts, smooth muscle cells, infiltrated inflammatory cells and endothelial cells in the heart is also an important topic in cardiac biology; and there is an increasing appreciation of the structure and function of these cells in cardiac pathology. Therefore, the development of techniques for heart cell isolation and harvest dates back to almost 40 years ago. In the current issue of Circulation Research, Ackers-Johnson et al (pp. XX-XX) ¹ describes a simple approach for the isolation and culture of adult mouse cardiac myocytes and fibroblasts without the traditional Langendorff-perfusion system.

While techniques for isolation of adult ventricular myocytes from many species including rat ², rabbit ³, and canine ⁴ have been available for almost 40 years, there remains no clear consensus regarding the optimal protocol to isolate and culture viable adult cardiac myocytes. However, at their core, these protocols generally all rely on digestion of the heart with an enzymatic solution ⁵. All myocyte isolation techniques can be essentially categorized into either “chunk” (a small piece of tissue) digestion in an enzymatic solution or coronary artery perfusion with enzymatic solution. The “chunk” digestion technique basically allows the enzymes (collagenases supplemented with other proteases) to chew the extracellular matrix directly and dissociate cardiomyocytes and nonmyocytes from small pieces (normally less than 1mm in diameter) of cardiac tissue. This technique was used in the early days and is still an important method for isolating cardiomyocytes from a very small piece of tissue such as the human biopsy obtained during cardiac surgery ⁶ and a small heart that cannot be cannulated such as fetal and neonatal hearts ⁷. This method normally produces low yield of cardiomyocytes with poor quality from adult tissues.

In comparison, the development of coronary artery perfusion-based isolation technique ^2–4 in the 1970s offers a much-improved approach to obtain adult cardiomyocytes, which is commonly used in the cardiac research community. The coronary artery perfusion technique allows perfusion of a whole heart or a large piece of tissue with large-enough coronary arteries with enzymatic solution flowing through the coronary system. For hearts with aorta large enough for cannulation but also small enough for controlling the cost of enzymes (e.g., feline, rabbit, guinea pig, small-sized monkeys, rat, mouse), the enzymatic solution is usually applied through an aortic cannula with either constant flow produced with a pump or constant pressure by positioning the enzymatic solution above the cannulated hearts, which force the solution to go through the ostia to perfuse the whole heart in a retrograde manner ^2–4. This is usually achieved with a Langendorff perfusion system. For large hearts such as human, canine, and porcine hearts, although it is still possible to perfuse the whole heart to isolate myocytes, it is more economical and practical to just cannulate and perfuse a major coronary artery or a subdivision of a coronary artery and then cut out the perfused region for further digestion ⁸. An interesting new technique to study adult human cardiac myocyte function was recently reported ⁹ and involves the isolation and culture of ventricular tissue slices. However, for most investigators, the most widely used protocols still involve isolation of cardiac myocytes using a Langendorff-based technique.

With the advent of transgenic mouse and increasingly transgenic rat technology, the need to develop simple and highly reproducible techniques to isolate and culture adult mouse cardiac myocytes and other cardiac cells would greatly advance our repertoire of tools to study cardiovascular disease mechanisms. However, unlike myocyte isolation from large mammal hearts, isolation of myocytes from rodent hearts is hampered by distinct rodent cardiomyocyte physiology. For example, rodent myocytes have higher intracellular sodium favoring calcium influx through the sodium/calcium exchange when the heart is not beating, rendering rodent myocytes prone to calcium overload when the heart stops beating and under hypoxic condition ¹⁰. Therefore, the search for an optimized protocol for myocyte isolation from murine hearts has never stopped. Several labs have published techniques to optimize isolation and culture of adult mouse cardiac myocytes ^11–14, but once again the core of these protocol relies on retrograde perfusion of the heart with an enzymatic solution using a Langendorff perfusion system. Overall, adoption of these protocols posts an intimidating challenge to a new researcher given the small size of mouse hearts and the fragility of adult mouse cardiac myocytes in culture. To overcome these challenges, Ackers-Johnson et al have detailed a new technique that does not rely on a Langendorff based system, and propose several modifications of existing protocols they claim will increase yield and survival in culture. This novel approach for isolation and culture of adult mouse cardiac myocytes and fibroblasts eschews the traditional Langendorff-perfusion based methods in favor of a method whereby enzymatic digestion is achieved through intra-ventricular injection to achieve coronary perfusion and cell dissociation. The advantages attributed to this new procedure are the relative ease and quick pace of the procedure without the technical skill, cost of specialized equipment, and logistical requirements inherent when using a Langendorff system.

While the principle of cardiomyocyte isolation from hearts sounds simple, to achieve high quality and high yield of myocytes from adult hearts requires careful procedure to avoid myocyte death/malfunction before and during the isolation. As mentioned above, the distinct physiological properties of rodent (including murine) myocytes make them more susceptible for hypoxia and Ca2+ overload. This can be exacerbated during the cannulation in the Langendorff-based method, which can only be improved through skill and experience, thus posting a steep learning curve to a new researcher. The new method reported by Ackers-Johnson et al ¹ applies several steps that could greatly reduce hypoxia and calcium overload. The new method rapidly applies perfusion buffer containing EDTA through a syringe to wash out the blood in the body and reduce extracellular calcium. This is followed by a rapid switch to a washing buffer and digestion solution to perfuse the heart from the LV. These maneuvers will ensure that there is little blot clot in the coronary system and the rapid pace of these steps will reduce the likelihood of hypoxia and calcium overload and thus increase the survival of cardiac myocytes before the isolation. Although the study did not directly compare the myocytes and other cells isolated with the new method and those cells isolated with the traditional Langendorff-based method, the data show that the quality of cells may be comparable. More importantly, the new method lowers the threshold of skill and experience that are demanded to produce high quality cardiomyocytes with a Langendorff system, making it readily adopted by different groups in cardiac research field.

The new method still has several limitations and disadvantages. An inherent disadvantage of this new method is that the perfusion is manually controlled and flow rate control is difficult to achieve while a Langendorff system can either maintain constant pressure or constant flow rate. As the authors point out, a syringe pump may help. In Langendorff perfusion-based methods, the acceleration of perfusion rate under constant pressure is often considered as an indication of good digestion and can be used reliably as a benchmark for complete digestion, which is often appreciated by learners. Authors mention a series of signs of complete digestion include a noticeable reduction in resistance to injection pressure and loss of shape and rigidity of the heart, etc., which can still post a challenge to a researcher with limited experience. Meanwhile, perfusing a whole heart through inserting a needle into the LV repeatedly still requires care and training to avoid the rupture and leak of the ventricular walls because of the small size of the heart and the thin ventricular walls. This could be improved by inserting a needle connected to a flexible silicon tube that can be easily disassembled from a syringe; and thus repetitive piercing of the ventricular wall can be avoided. It is also critical to maintain the integrity of aorta to prevent the leak from the damaged aorta. Another limit of the new method is that it may not be effectively used for isolating myocytes from some diseased hearts such as an infarcted heart with large and thin scar that can be easily ruptured, and a heart with mitral valve defect or insufficiency. At last, this new method may not be suitable for isolating heart cells from animals larger than mice. Taken together, this new method for isolating adult myocytes from mice might be useful for cardiac research field with clear understanding of its limitations. Additional studies are warranted to definitively demonstrate its utility to the field.