Mitochondrial reorganization is rate-limiting for direct cardiac reprogramming

Direct cardiac reprogramming of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) has emerged as a promising potential therapeutic for preserving cardiac function after ischemic injury. Remaining barriers to clinical translation include low conversion efficiency and relative immaturity of iCMs. A major phenotypic distinction between CFs and native CMs is the latter’s reliance on mitochondrial energetics facilitated by their high mitochondrial fusion (joining) activity relative to fission (dividing) activity. However, how mitochondria reorganize during reprogramming remains understudied. We combined metabolic flux assays with novel microscopy techniques to characterize mitochondrial energetics and morphology over a time course of cardiac reprogramming with Mef2c, Gata4 and Tbx5 (MGT). We found that MGT reprogramming induces increased mitochondrial respiration and fusion. However, this reorganization occurs only at later reprogramming time points. Hypothesizing that mitochondrial reorganization represents a rate-limiting step in reprogramming, we then performed a loss of function screen for a panel of genes known to regulate mitochondrial morphology. We found that the fusion apparatus is essential for successful iCM conversion and identified the fission regulator Mtfr1l as a powerful barrier to reprogramming. Using transcriptomic analysis and concomitant knockdown of the Mtfr1l target Opa1, the effector of inner membrane fusion, we showed that loss of Mtfr1l dramatically improves reprogramming efficiency and the maturity of nascent iCMs by facilitating a metabolic switch toward more interconnected and energetically active mitochondria. This study represents a major step forward in understanding reprogramming mechanisms and is the first report of Mtfr1l as a significant barrier to iCM conversion and maturation.