Comments for:

---

Response to Bertero, Maack and O’Rourke

Submitter: James D. McCully | james_mccully@hms.harvard.edu

Authors: James D. McCully, Sitaram M. Emani, and Pedro J. del Nido

Harvard Medical School

Published November 26, 2018

In response to the Viewpoint by Bertero, Maack and O’Rourke, there is no magic or tricks in our studies. We based our human trial on the scientific evidence.

The authors ask that we show mitochondria can survive in a high (~1.8 mM) calcium environment. The authors should note that we have demonstrated that donor mitochondria are viable in both the isolated perfused heart, where calcium concentration was 1.7 mM and in  in vitro cell studies where calcium concentration was 1.8 mM as well as in vivo (1-4).  Transplanted mitochondria are rapidly integrated into cardiac cells by endocytosis where they increase total cellular and total tissue ATP content for at least 21 days post-injection (1-5). The authors note that we “convincingly demonstrated” these effects in our experiments showing the restoration of mitochondrial respiration in rho cells (3).  These experiments were performed in media containing 1.8 mM calcium and the transplanted mitochondria increased oxygen consumption and ATP content for 23 days or 53 cell divisions (3). These effects have been replicated in vivo where we have shown that mitochondrial transplantation increases total tissue ATP content in the region in which the mitochondria were delivered and that this increase in total tissue ATP is evident for at least 21 days (2).

The authors also ask us to demonstrate that enough mitochondria must pass through the cell to enter the cardiomyocytes to support contraction. Our data show that mitochondrial transplantation increases regional muscle contraction as evidenced by increased systolic shortening in the region of ischemia. These effects were not present in hearts receiving vehicle alone. Increased contractile function was demonstrated by sonomicrometry, the ‘gold standard’ for evaluation of muscle function and verified by echocardiography (2). The number of mitochondria apparent in hearts is much higher than the authors suggest as we show thin tissue sections and the absolute number of mitochondria taken up by cardiomyocytes was underestimated due to the limitations of the imaging employed (1,2). Our recent studies show that 43.52% of mitochondria are found within cardiomyocytes in culture (5).

The authors claim that in the clinical experience ischemic injury is “complete”.  To state that the preclinical experience has no relevance to the clinical experience is an oversimplification.  Ischemia does not equal infarct, and myocardium can still be viable and salvageable, as was demonstrated by increase in ejection fraction by nearly 50% in some patients.  Clearly randomized prospective trials are necessary to validate our observations (6,7).

References:

1. McCully JD, Cowan DB, Pacak CA, Toumpoulis IK, Dayalan H, Levitsky S. Injection of Isolated Mitochondria During Early Reperfusion for Cardioprotection.  Am. J. Phys. Heart Circ. Phys 2009, 296: 94-105. 

2. Masuzawa A, Black KM, Pacak  CA, Ericsson M, Barnett RJ,  Drumm C, Seth P, Bloch DB, Levitsky S, Cowan DB, McCully JD. Transplantation of autologously-derived mitochondria protects the heart from ischemia-reperfusion injury. Am. J. Phys. Heart Circ. Physiol 2013;304 H966-H982. 

3. Pacak AP, Preble JM, Kondo H, Seibel P, Levitsky S, del Nido PJ, Cowan DB, McCully JD. Actin-Dependent Mitochondrial Internalization in Cardiomyocytes: Evidence for Rescue of Mitochondrial Function. Biol. Open  2015; 4:622-626.

4. Cowan DB, Yao R, Akurathi V,  Snay ER, Thedsanamoorthy JK, Zurakowski D, Ericsson M, Friehs I, Wu Y, Levitsky S,  del Nido  PJ, Packard AB, McCully JD. Intracoronary Delivery of Mitochondria to the Ischemic Heart for Cardioprotection. PLoS One. 2016 Aug 8;11(8):e0160889. 

5. Cowan DB, Yao R, Thedsanamoorthy JK, Zurakowski D, del Nido PJ, McCully JD. Transit and fusion of exogenous mitochondria in human heart cells. Sci Rep. 2017 Dec 12;7(1):17450. 

6. Emani SM, Piekarski BL, Harrild D, del Nido  PJ, McCully JD. Autologous Mitochondria Transplantation for Ventricular Dysfunction following Myocardial Ischemia-Reperfusion Injury. J Thorac Cardiovasc Surg 2017; 154: 286-289. 

7. Emani SM, McCully JD. Mitochondrial transplantation: applications for pediatric patients with congenital heart disease. Transl Pediatr. 2018; 7:169-175.