The Healing of a Heart

                Heart disease continues to be the leading cause of death among Americans and has been for more than 85 years (Murphey et al. 2017). Many of us have a loved one or know someone personally that has had to deal with deteriorating heart health, whether it is due to years of consuming a tasty, yet unhealthy Western diet, leading a sedentary life, or simply as the result of aging. Developing treatments that can improve or even reverse the damage caused by heart disease has been a major part of the medical industry, but in the last few decades, many improvements and scientific breakthroughs have been found specifically using stem cell therapies and regenerative medicine practices as a means of treatment.

                The heart is responsible for pumping blood throughout the body, providing oxygen and nutrients necessary to maintain life. The pumping mechanism that propels blood is due to the unique cellular components of cardiac muscle tissue. There is a cell responsible for conducting the electrical signal known as an autorhythmic cell. It is then propagated across multiple contractile cells using specialized gap junctions to create the pumping motion that effectively empties the heart of blood during contraction and refills upon relaxation.

Heart disease is the primary cause of heart attacks, or myocardial infarctions (MI). An MI is the result of a blockage within an artery responsible for supplying the heart muscle oxygenated blood, seen in Figure 1. As soon as the heart no longer receives the oxygen, cardiac tissue begins to die. This results in a build-up of fibrous scar tissue that reduces the compliance of the heart tissue, which results in less blood being pumped throughout the body with each contraction. This causes other health complications, such as high blood pressure or issues maintaining peripheral blood supply.

Figure 1: Causes of Myocardial Infarction (MI)

The main treatments used today are often a combination of drugs, an angioplasty to remove plaque from arteries, or surgical intervention, such as coronary bypass surgery or heart transplant if the damage is severe (Cambria et al. 2017). While these treatments are the main staples of MI treatment, development of cellular therapies that can reverse the damage caused to the heart tissue could drastically reduce the amount of health deterioration due to decreased cardiac performance, highly invasive surgical intervention, and ultimately the long-term financial cost of treatment.

Currently, there are two different regenerative medicine therapies undergoing clinical trials to determine their efficacy in reducing the detrimental effects of a heart attack: cardiosphere-derived cells (CDCs) and exosome therapies. CDCs are derived from ‘‘cardiospheres,’’ which are multicellular structures that can cultivated from heart tissue, the process outlined in Figure 2, and have many stem cell characteristics (Makkar et al. 2014). Once transplanted into the myocardium, they have paracrine effects that have been shown to reduce fibrosis of the scar tissue, promote angiogenesis, and improve the overall function, as measured by ejection fraction.

Figure 2: Cardiosphere-Derived Cell Isolation Steps, a visual summary

Scar tissue formed due to tissue death is made up of different types of collagen, which is not as contractile as the native heart muscle. This is the cause of hardening and reduced cardiac output post MI. The CDCs were transplanted into myocardium of rats that were fed a high salt diet, which induced hypertension, left ventricular (LV) hypertrophy and diastolic dysfunction, which resulted in fibrosis of the ventricles. Four weeks after treatment with either the CDCs or a placebo of saline, the amount of fibrosis was visualized and quantified using picrosirius red staining. The stain binds to the collagen within the tissue sample and the results demonstrate in Figure 3 that fibrosis was decreased in both the left and right ventricles, returning levels comparable to the statistically normative control (Tseliou et al. 2014).

Figure 3: Fibrosis of the Right and Left Ventricles is Reduced After Treatment with CDCs

Besides fibrosis, the ability of the damaged heart tissue to gain new blood supply through angiogenesis, or the growth of new capillary systems, is also an important therapeutic development. In another study, myocardial infarction was induced in another rat model, this time without the high salt diet, and CDCs were administered (Tseliou et al. 2014). After three weeks, the heart tissue was analyzed for new capillary growth to determine whether the CDCs had any angiogenic effect. In Figure 4AB and 4DE, the dapi stain allows for visualization of the nuclei in all cells, while the vWF stains endothelial cells within the capillaries and the sma stains smooth muscle actin within the microvessels. For Figure 4C and F, the BZ represents the border zone between the infarct and healthy heart tissue, whereas the RZ is a remote zone, far away from the infarct. This gives an idea of the paracrine effects of both the infarct and CDC transplantation on the surrounding heart muscle. After three weeks, the treatment group had significant increases in both capillary and microvessel density compared to the MI control (Gallet et al. 2016).

Figure 4: Angiogenesis of Capillaries and Microvessels in Myocardium after Infarct in the Border Zone and a Remote Zone.

The CDC therapies have demonstrated have beneficial therapeutic indications and are continuing to be thoroughly researched as a means of treatment for future MI patients. However, the main difficulty cellular therapies face is whether the cells can be transplanted without any extraneous effects. If the cells are from a source other than the patient, this opens the possibility of rejection, similarly to tissue and organ transplants. To attempt to circumnavigate this issue, it has been demonstrated that exosomes secreted from CDCs have the same effects in vivo without the need for direct cellular transplantation (Gallet et al. 2017). Exosomes are nanovesicles that are excreted from the CDCs and do not appear to contain active DNA, which is one of the main stimulus of the cellular rejection process. In a study evaluating the efficacy of the CDC-derived exosomes on a larger animal model (porcine), the results, demonstrated in Figure 5, indicate that the exosome treatment significantly reduced the size of the scar left by the infarct. With this result being in a large animal model rather than the previous rat models of MI, this gives more indication that the exosome therapy could be a sustainable therapy option for humans (Gallet et al. 2017). 

Further studies in this area will provide a treatment option that reduces the pathological impact of heart attacks, rather than just treating the resulting symptoms. With the human population over 50 continuing to grow, heart disease is continuing to rise across the United States. Developing these types of cellular therapies are crucial for the health of our country and as the results have demonstrated, the best way to heal a broken heart lies within the future of regenerative medicine.

References:

Cambria E,  Pasqualini FS, Wolint P, Günter J, Steiger J, Bopp A, Hoerstrup SP, and Maximilian Y. Emmert (2017) Translational cardiac stem cell therapy: advancing from first-generation to next-generation cell types. Regenerative Medicine 2(17).

Gallet R, Dawkins J, Valle J, Simsolo E, de Couto G, Middleton R, Tseliou E, Luthringer D, Kreke M, and Rachel R. Smith et all (2017) Exosomes secreted by cardiosphere-derived cells reduce scarring, attenuate adverse remodeling, and improve function in acute and chronic porcine myocardial infarction. Eur Heart J. 38(3):201-211.

Gallet R, de Couto G, Simsolo E, Valle J, Sun B, Liu W, Tseliou E, Zile M, and Eduardo Marban (2016) Cardiosphere-Derived Cells Reverse Heart Failure with Preserved Ejection Fraction in Rats by Decreasing Fibrosis and Inflammation. JACC: Basic to Translational Science 1(1-2).

Makkar RR, Smith RR, Cheng K, Malliaras K, Thomson LE, Berman D, Czer LS, Marbán L, Mendizabal A, Johnston PV, and Stuart D Russell et al. (2104) Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomized phase 1 trial. The Lancet, 379(9819): 895-904.

Murphy SL, Kochanek KD, Xu JQ, Curtin SC (2017) Deaths: Final data for 2015. Hyattsville, MD: NCHS.

Tseliou E, de Couto G, Terrovitis J, Sun B, Weixin L, Marban L, and Eduardo Marban (2014) Angiogenesis, Cardiomyocyte Proliferation and AntiFibrotic Effects Underlie Structural Preservation Post Infarction by Intramyocardially-Injected Cardiospheres. PLOS One 9(2).

Photos:

http://www.onlinejacc.org/content/60/16/1581

Making Muscles

Skeletal muscle accounts for approximately 40% of the total body mass in the human body. This type of muscle is anchored to bone by connective tissues and is involved in the movement of the body. Skeletal muscle has a tremendous ability to heal and regenerate after minor damage such as straining a muscle. When this happens, inflammation in the area activates various signals to recruit cells to the area to aid in repairing the tissue. This includes immune cells which help degrade damaged muscle cells. Next is the repair phase. In muscle tissue there is a population of adult muscle stem cells, called satellite cells, that remain inactive until they receive signals that there is an injury to the muscle. As specialized stem cells, these satellite cells have the ability to become mature muscle cells. When recruited to the damaged area, they fuse with existing heathy muscle cells and help rebuild the tissue. While the satellite cells help rebuild the muscle, cells called fibroblasts also come to the site of injury and produce collagen and connective tissue to replace the extracellular matrix (ECM). The ECM is a meshwork of proteins and molecules secreted by support cells that provides structural stability for surrounding cells as well as regulating intercellular signaling (Matthias et al., 2018). The cells working together can regenerate the muscle tissue. Though this repair mechanism works very well for small injuries, there is a different story when faced with extensive damage.

Volumetric muscle loss (VML) is defined as a large volume of muscle loss which results in permanent functional impairment. These massive injuries can result from combat-related extremity wounds or incidents such as car accidents. As seen in figure 1, extremity wounds compose the greatest percentage of injuries sustained by soldiers and have the largest projected costs of disability benefits (Masini et al., 2009). According to researchers Grogan and Hsu, VML is a significant component of combat related injuries and the cost relating to VML treatments can be in the billions of dollars (Grogan and Hsu, 2011). The prevalence of VML as well as a lack of suitable therapies make it a significant area of study.

Figure 1: Distribution of disability ratings by body region (Masini et al., 2009).

In VML, due to the volume of muscle lost, the signals that normally recruit satellite cells to help rebuild the muscle are not present. However, fibroblasts still migrate to the area and produce large amounts of collagen. This results in scar tissue formation which can prevent the muscle from contracting uniformly and cause greatly reduced functionality of the injured muscle (Grasman et al., 2015). Currently, the standard-of-care treatment is free functional muscle transfer. This requires taking muscle tissue from a healthy section of the body and transplanting it to the area of VML to attempt to restore movement. Figure 2 demonstrates a free functioning muscle transfer where muscle from the thigh was transplanted to the leg (Lin et al., 2007). Drawbacks of this method are that it requires healthy donor tissue, which may be limited on a patient suffering from severe injuries. There is also the consideration of creating two wound areas, as well as aesthetic concerns.  

Figure 2: (Left) A 46-year-old female presented with major muscle loss on the left leg. (Center) Diagram on the area of thigh to be used for free functioning muscle transfer. (Right) Leg at 8 months post-surgery (Lin et al., 2007).

Looking toward new solutions for VML, some researchers have postulated that using a scaffold, a structure designed to support another material, that mimics the muscle’s natural environment could provide environmental cues to recruit satellite cells. This would theoretically allow the large injury of the muscle to regenerate in the same manner as a small injury. One group, Sicari et al., has shown positive results with this method using a scaffold composed of a natural ECM. As seen in figure 3, after implanting mice with this ECM scaffold, the VML mice treated with ECM had a significantly higher rate of recruitment of satellite cells than the untreated mice. The treated mice also showed new formation of skeletal muscle cells while the untreated did not show any signs of new muscle formation (Sicari et al., 2014).

Figure 3: Recruitment rate of satellite cells in VML mice treated with the ECM scaffold, untreated VML mice, and an uninjured mouse (Sicari et al. 2014).

Some groups are also including cells in their scaffold to create a bioconstruct with potentially more regenerative effects. Recently published work by Quarta et al. utilizes a biological scaffold suffused with cells. The group used satellite cells, referred to here as muscle stem cells (MuSC), and muscle resident cells (MRC), which are other cell types found in proximity with the satellite cells. As seen in figure 4, they showed that by including the MRCs, the total muscle area significantly increased rather than using MuSCs alone (Quarta et al., 2017). By including a scaffold to mimic the natural environment and the different types of cells, the group has created a bioconstruct that is very physiologically similar to the native muscle. 

Figure 4: Total muscle area between mice treated with satellite cells, referred to here as muscle stem cells (MuSC), and muscle resident cells (MRC) (Quarta et al., 2017).

New research on treatments for VML are encouraging. The concept of utilizing the body’s own ability to regenerate muscle has shown promising results. However, there is still progress to be made. Some future directions for research would be to focus on scaling up the size of these constructs and work on further integrating them with the body.  

References

Grasman, J. M., Zayas, M. J., Page, R. L. and Pins, G. D. (2015). Biomimetic scaffolds for regeneration of volumetric muscle loss in skeletal muscle injuries. Acta Biomater. 25, 2–15.

Grogan, B. F. and Hsu, J. R. (2011). Volumetric Muscle Loss. J. Am. Acad. Orthop. Surg. 19, 35–37.

Lin, C. H., Lin, Y. Te, Yeh, J. T. and Chen, C. T. (2007). Free functioning muscle transfer for lower extremity posttraumatic composite structure and functional defect. Plast. Reconstr. Surg. 119, 2118–2126.

Masini, B. D., Waterman, S. M., Wenke, J. C., Owens, B. D., Hsu, J. R. and Ficke, J. R. (2009). Resource utilization and disability outcome assessment of combat causalities from operation Iraqi freedom and operation enduring freedom. J. Orthop. Trauma 23, 261–266.

Matthias, N., Hunt, S. D., Wu, J., Lo, J., Smith Callahan, L. A., Li, Y., Huard, J. and Darabi, R. (2018). Volumetric muscle loss injury repair using in situ fibrin gel cast seeded with muscle-derived stem cells (MDSCs). Stem Cell Res. 27, 65–73.

Quarta, M., Cromie, M., Chacon, R., Blonigan, J., Garcia, V., Akimenko, I., Hamer, M., Paine, P., Stok, M., Shrager, J. B., et al. (2017). Bioengineered constructs combined with exercise enhance stem cell-mediated treatment of volumetric muscle loss. Nat. Commun. 8, 1–17.

Sicari, B. M., Rubin, J. P., Dearth, C. L., Wolf, M. T., Ambrosio, F., Boninger, M., Turner, N. J., Weber, D. J., Simpson, T. W., Wyse, A., et al. (2014). An Acellular Biologic Scaffold Promotes Skeletal Muscle Formation in Mice and Humans with Volumetric Muscle Loss. Sci. Transl. Med. 6, 234ra58-234ra58.