Heart muscle degeneration is a leading cause of debilitation and death in humans. Its primary cause is partial or complete obliteration of the coronary artery, often precipitating acute myocardial infarction (AMI). Long-term ischemia leads to ventricular cell death and myocardial necrosis. Although medication, trans-myocardial revascularization (TMR), and CABG can increase local blood supply and reduce short-term mortality and morbidity, most patients continue suffering decreased ventricular function and chronic heart failure.

Severe myocardial infarction conveys serious complications such as ventricular aneurysm, wall thinning and rupture with fatal consequences. Traditional aneurysm resection improves heart contractility by restoring the original oval structure of the left ventricle. This procedure is reserved only for patients with at least medium-sized ventricles because of volume consideration. Prognosis for these severe heart failure patients is three to six months of life despite CABG. Heart transplantation has been the gold standard for patients with end-stage heart failure, but donor heart non-availability and lifelong immunosuppression dictate its limited usage. It is urgent to explore new therapeutic measures to increase the quality of life and lifespan of these patients.

Chronic ischemia after AMI causes loss of cardiomyocytes, contractile filaments and ventricular contractility. Regeneration attempt by surviving cardiomyocytes consists of undergoing at most four mitotic divisions because the telomeric DNA repeats [1] in these terminally differentiated cells are minimal. Such attempt cannot regenerate enough cardiomyocytes to produce the necessary quantity of contractile filaments such as myosin, actin, troponin and tropomyosin to sustain normal heart contractility. The degenerative heart also transmits biochemical signals to recruit stem cells from the stroma and from the bone marrow to repair the muscle damage. Due to the significant increase in fibroblast growth factor release after infarction, much of the recruited stem cells differentiate to become fibroblasts instead of cardiomyocytes, forming scars and not contractile filaments.

The damaged myocardium needs replenishment of live, genetically normal, myogenic cells to deposit contractile filaments to regain heart function, preferably before fibroblast infiltration. This is when AMT has an advantage over all other cell types [2] [3]. Considering dystrophic and diabetic cardiomyopathies as hereditary, and ischemic cardiomyopathy as genetically predisposed, AMT was designed to genetically repair hereditary degenerative cells and to replenish degenerated cells with live ones. In 1990, AMT was, and still is, the world’s first human gene therapy and the world’s first somatic cell therapy in the treatment of human muscle genetic disease [4].

Myoblasts are differentiated cells destined to become muscles. Unlike cardiomyocytes, myoblasts possess long telomere DNA subunits and can undergo 50 mitotic divisions without loss of myogenic capacity [2] [3] or developing into tumor [5]. The transition from animal experimentation [6] [7] into cardiac clinical trials was largely based on previous demonstration of significant safety and efficacy in the treatment of Duchenne muscular dystrophy in FDA approved Phase II and Phase III clinical trials using allogeneic myoblast allografts [8] [9] [10].

Law et al. reported in the year 2000 the pioneering study of implanting human myoblasts into the porcine hearts with endovascular catheter of the NOGA system [11]. The mechanisms by which allogeneic human myoblasts survived, developed and functioned with the use of cyclosporine immunosuppression were examined through open chest endomyocardial injections of cultured skeletal myoblasts into infarcted porcine myocardium. Three mechanisms were elucidated as proof-of-concept using genetic markers to label the nuclei of the donor myoblasts. Under the influence of hormones and slow contractile activity of the heart, donor myoblasts fused among themselves to form new cardiomyocytes, depositing contractile filaments to improve heart contractility. Others fused with the host cardiomyocytes through natural cell fusion, spontaneously transferring their nuclei into host cardiomyocytes to impart myogenic regeneration. Still others fused to form myotubes that eventually developed into immature skeletal myofibers containing satellite cells. New production of contractile filaments augmented heart contractility [2] [3] [12].

In 2004, Law et al. reported the world’s first two cases of allogeneic myoblast transplantation for myocardial infarction [3] [12]. Results demonstrated that the left ventricular ejection fraction (LVEF), viability, motility, myocardial perfusion, and ventricular wall thickness were significantly improved [3] [12]. The end-diastolic and end-systolic blood volumes were increased without significant arrhythmia. At 12 months after implantation, LVEF was increased by about 40%, and perfusion capacity increased by 38% [3] [12]. More than 300 cases of autologous myoblast transplantation internationally have shown that it was safe and efficacious in treating ischemic cardiomyopathies and heart failure. Approximately 200 cases were injected after thoracotomy and 100 cases were injected with endovascular catheters [10] [12]. Further advance with myoblast clinical trials has been distracted for 17 years by Piero Anversa’s misconduct in cardiac stem cell research in Harvard University until 10/14/2018.

The authors vouch for the accuracy and completeness of the data and analyses and the reporting of adverse events and for the fidelity of the study to the protocol.

For the first time, AMT in adjunct use with CABG and cyclosporine, was demonstrated to be safe and efficacious in sustaining the lifespan of end-stage heart failure patients, who suffered myocardial infarction with ventricular aneurysm, for up to 2 years, with significant improvement in LVEF (TableS7, Figure 1, Figure 2(A)), NYHA cardiac function (Central Illustration 1, Figure 5) and quality of life. It was the original design to define a treatment modality that could 100% sustain the survival of the severe heart failure patients having no more than 6-months of life expectance, and such design was demonstrated to be successful. The statistically significant increases in the mean LVEF by 20.1% at 6 months after treatment, and by 19.3% at 2 years after treatment compared

favorably to those reported by all other studies using autologous myoblasts 17 - 33. As the trend of improvement indicated, statistically significant difference in LVDd and CO before versus after treatment would have become apparent if more subjects were enrolled.

The ultimate demonstration of AMT efficacy by itself should be from randomized, double-blinded studies involving more subjects of dystrophic cardiomyopathies, diabetic cardiomyopathy, or dilated cardiomyopathies that would not necessitate CABG.

This study showed that white blood cell count, neutrophil count, total bilirubin, alanine aminotransferase, aspartate aminotransferase, total red blood cells, hemoglobin, platelets, total protein, renal function, fibrinogen, prothrombin time, and partial prothrombin time before versus after treatment of myoblasts were not different (P > 0.05). On repeated follow-ups after the operation, there were no deaths, malignant arrhythmias, or viral infections. The results demonstrated that allogeneic myoblast transplantation had no significant impact on various important indicators of the human body, and there was no serious adverse reaction. Although occasional ventricular premature beats occurred in four subjects after CABG, all symptomatic treatments were traced to the sinus and were related to electrolyte disturbance, myocardial damage and the primary disease. None of the subjects experienced malignant arrhythmia or ventricular tachycardia.

AMT represented the earliest human cell therapy for heart disease [11]. Without significant knowledge of myoblast manufacture, quality control and cell transplant techniques, many clinicians rushed into clinical studies. Menasche, et al. first reported feasibility and safety data on ischemic heart failure patients, implanting 650 million to one billion autologous impure myoblast cells with overly large number of injections into the infarctions during CABG [17] [18]. Follow-up studies showed that patients had significantly improved left ventricular function and NYHA function level, decreased ventricular remodeling, and increased myocardial tolerance confirmed by histology. However, 4 patients had delayed episodes of sustained left ventricular tachycardia [18]. During this period, similar reports continued to appear around the world with mixed results.

In most of the reports, myoblast purity was determined using CD56+, an antibody that reacts with fibroblasts, neurons, and myoblasts indiscriminately. A common pitfall of myoblast culture is fibroblast contamination. Since myoblast doubling time is about 21 hours and fibroblast doubling time is about 15 hours, fibroblast growth often overtakes the myoblast culture [13] [14]. Without published documentation of quality controls, authors of these studies were implanting very impure myoblasts. Fibroblasts produce scars but not contractile filaments. These scars created numerous barriers to electric coupling and rhythmic synchronization of ventricular contraction. This pitfall, together with the physical trauma induced by overly large number of injections, were largely responsible for the malignant arrhythmia and tachycardia reported in some early patients, not to mention the gross GMP non-compliance in quality control of myoblast identity, viability, purity, quantity, potency, no mycoplasma, no endotoxins and no bacteria [19] - [33]. The latter explained cell death of up to 90% that affected the safety and efficacy of myoblast transplantation [34]. Clinical studies confirmed that CABG with transplantation of impure and non-viable myoblasts produced ventricular tachycardia [26]. The intercalated disc, the basic unit for the transmission and synchronization of electrical activity and mechanical function between adjacent cardiac fibers were not found, and its absence constituted a risk of malignant arrhythmia after transplantation with substandard myoblasts [35]. Therefore, the safety and efficacy of myoblast transplantation depend largely on high quality control of myoblast production and the technique of implantation.

Heartbeat is myogenic in origin and is initiated by pacemaker activity in the sinoatrial node. As depolarization sweeps through the atrioventricular node, the depolarization excites the Purkinje fibers of the bundle of His, which, in turn, signals the ventricles to contract rhythmically. Heart function would be impaired if the rhythmic action potentials do not synchronize the fiber contractions. In the regenerative heart with AMT, excitation of the newly formed and heterokaryotic cardiomyocytes [2] [3] [12] remained unchanged because there was little change in gap junctions for current flow. However, where new skeletal myofiber [2] [3] [12] were formed, presumably at the inner border of the infarction, such heterogenicity might create aberrant electric activities such as arrhythmia, especially when earlier studies reported absence of gap junction protein connexin 43, a marker protein responsible for electrical coupling between cardiomyocytes [36].

Conceptually, myoblast transplantation should not cause arrhythmia if the well-researched standard operation procedures were followed. The thresholds of excitation for cardiac and skeletal myofibers are similar, i.e. between +40 mV to +50 mV depolarization. Whereas the cardiomyocyte action potential is triggered with an increase in Ca²⁺ conductance into the cell, the skeletal myofiber action potential is triggered with an increase of Na⁺ conductance. As Ca²⁺ has a greater ionic size than Na⁺ and thus lower ionic mobility, the action potential of cardiomyocytes has a longer duration (~250 ms) than that of skeletal myofiber (~1.5 ms). This duration difference is advantageous because the same myocardial depolarization can simultaneously and synchronously stimulate the cardiac and skeletal myofibers through direct excitation contraction coupling. Since the action potentials of skeletal myofibers are of short duration, they will merge into the longer compound action potential of the heart. The skeletal myofibers will cease to fire and stop contracting once Cl⁻ efflux hyperpolarization of the myocardium reaches approximately +40 mV. Since 90% of the transplanted myoblasts developed to become cardiomyocytes or heterokaryotic cardiomyocytes [2] [3] [12], and only a small amount of skeletal myofibers were formed, the electrophysiologic treatise explains why none of the subjects in the current study experienced malignant arrhythmia from the myoblast transplantation.

Abraham et al. [37] demonstrated in vitro that connexin 43 was expressed when exogenous connexin 43 gene was transferred into myoblasts. Electrophysiological studies had also found that there was a synchronized instantaneous calcium current between skeletal muscle myotubes and adjacent cardiomyocytes, further confirming that after transplantation, cardiomyocytes, heterokaryotic cardiomyocytes and skeletal myotubes were simultaneously activated through the same excitation-contraction coupling. Simultaneous contraction could not only promote the formation of gap junctions, but also effectively reduced the incidence of arrhythmia. In addition, a sodium ionic current was detected [38], confirming that some myoblasts developed to become myotubes and immature myofiber [2] [3] [12]. Skeletal myofibers are known to adapt to the frequency of electric excitation to which they are subjected. Under the influence of hormones and slow contractile activity of the heart, these immature skeletal myofibers developed characteristics of cardiomyocytes.

The regenerative heart with myoblasts was endowed with a greater number of myogenic cells capable of mitosis and was prepared to regenerate upon injury. These cells produced more contractile filaments to augment heart contractility. The latter is fundamental to the quality of life and the lifespan of patients suffering various forms of cardiovascular diseases. Being pluripotent, embryonic or adult stem cells exhibit uncontrolled differentiation into various lineages to produce bone, cartilage, fat, connective tissue, skeletal and heart muscles. Until scientists can accurately define the specific transcriptional factors and pathways to guide stem cell differentiation into adequate quantity of cardiomyocytes, the use of stem cell injection into the human heart would have a risk-benefit ratio much higher than the use of myoblasts.

Myoblasts are differentiated cells destined to become muscles. Further studies are necessary to better define the efficacy of myoblast transplantation itself, preferably through transplanting male donor myoblasts into female subjects, and using the Y-chromosome to track the development of implanted allogeneic myoblasts in the host. Position papers in 2017 highlighted numerous developing cell therapies for severe heart failure with neither governmental approval nor endorsement [39] [40]. Heart transplant has remained the generally accepted treatment for end-stage patients. With an estimate of over 50 million heart failure patients worldwide, only a few thousand donor hearts were available for transplants last year. Patients who survive heart transplants need to be immunosuppressed for life with not only compromised quality of life but also constant life-threat of COVID-19.

This study demonstrated survival and engraftment of allogeneic myoblasts in patients with ischemic cardiomyopathy. The improvement of heart function and the quality of life of patients was the result of a combination of CABG and cell therapy. A large randomized clinical trial will be required to demonstrate the efficacy and safety of AMT.

The authors thank the Cell Therapy Institute for free supplies of human myoblasts.

This work was supported by the National Distinguished Expert Award of China and the Cell Therapy Institute Endowment Fund awarded to Prof. Peter K. Law, and by the Discipline Construction Fund of Xinxiang Medical College awarded to Prof. Wenbin Li.

Prof. Li, Prof. Law and Dr. Wang contributed equally and are co-first authors. W.L., H.Q., F.W. performed surgery; H.G., B.Z. provided postoperative intensive care; K.W., L.L., F.L. collected data and conducted statistical analyses; Q.S. provided patient coordination; H.F. conducted extracorporeal circulation, J.L., Y.L., Y.Y. conducted postoperative follow-up; P.L. supervised myoblast manufacture; D.C. provided myoblast quality control; D.M.L provided project coordination; P.K.L. designed project and analyzed data, provided free licenses and myoblasts, and wrote manuscript with K.W.

All authors read and approved the final manuscript and declared no conflicts of interest regarding the publication of this manuscript.

Li, W., Wang, K., Shi, Q., Lu, P., Chen, B.S.D., Zhang, B., Qi, H., Wang, F., Fan, H., Guo, H., Lu, L., Lu, F., Liu, J., Li, Y., Yang, Y., Law, D.M. and Law, P.K. (2021) Two-Year Heart Failure Study with Allogeneic Myoblast Transplantation. Open Journal of Regenerative Medicine, 10, 1-18. https://doi.org/10.4236/ojrm.2021.101001

NYHA grading before versus after treatment.