We propose that a quantitative

We propose that a quantitative marker for tracking cardiac myocyte maturation resides in the developmentally controlled and irreversible (excepting targeted reprogramming) genetic switch in the sarcomeric TNNI gene. Owing to strict developmental control in all mammalian hearts, including humans, the programmed inactivation of the “fetal” TNNI gene, TNNI1, together in exquisite temporal concert with stoichiometric replacement by the adult gene, TNNI3, represents a unique molecular signature of the mature adult cardiac myocyte in vivo (Bhavsar et al., 1991; Sasse et al., 1993; Siedner et al., 2003). The troponin complex is composed of three subunits—troponin T (TnT), the tropomyosin binding subunit; troponin C (TnC), the calcium binding subunit; and troponin I (TnI), an inhibitory subunit (Dhoot et al., 1978; Ohtsuki et al., 1986; Parmacek and Solaro, 2004)—and controls the interaction of thick and thin filaments in response to alterations in intracellular Ca2+ concentrations (Ebashi, 1983, 1988; Lehman et al., 2001; Metzger and Westfall, 2004). The TnT subunit has been used extensively in attempt to identify the cardiac lineage (Chan et al., 2013b; Hazeltine et al., 2013; Kawamura et al., 2013; Park et al., 2014; Rebuzzini et al., 2013). However, cTnT is also expressed in noncardiac e1 activating enzyme such as smooth muscle cells (Porrello et al., 2011, 2013; Lu et al., 2013; Lundy et al., 2013; Mahmoud et al., 2013; Xin et al., 2013), limiting its use as a cardiac lineage marker. Many other structural, regulatory, morphological, and metabolic markers have been used for cardiac lineage and maturation assignment; however, these are subject to reversion to the fetal program in stress and disease. For example, the MyH6 gene is highly cardiac specific, but a transition in myosin isoform content in disease complicates its use for lineage or maturation assignment (Sasse et al., 1993; Nakao et al., 1997; Reiser et al., 2001) There are two isoforms of TnI in heart muscle, encoded on two separate genes that are expressed under strict developmental control in the mammalian heart (Hunkeler et al., 1991; MacGeoch et al., 1991; Metzger and Westfall, 2004). The TNNI1 gene (slow skeletal TnI/ssTnI/cardiac fetal) is expressed in the sarcomeres of the fetal heart and in late fetal/early neonatal life and then fully extinguished such that there is no ssTnI protein detected in the adult myocardium (Bhavsar et al., 1991; Sasse et al., 1993; Siedner et al., 2003) (Figure 1A). The TNNI3 gene (adult cardiac TnI/cTnI) is activated in fetal/early neonatal life and then is exclusively expressed in the adult myocardium. This strict 1:1 conversion is ideal for a differentiation status marker owing to tight preservation of sarcomere stoichiometry throughout development. This differs, for example, from tracking a marker that increases in maturation because the critical internal control of a reference “fetal” marker decreasing in parallel is lacking. Further, the ssTnI (fetal) to cTnI (adult) isoform stoichiometric conversion does not revert to the “fetal” gene program in cardiac stress or disease (Sasse et al., 1993; Averyhart-Fullard et al., 1994; Huang et al., 2000), in distinction with reversible differentiation status markers currently in use (Razeghi et al., 2001; Lundy et al., 2013). The combined properties of stoichiometric conservation and general irreversibility are unique in combination and make the cTnI: ssTnI protein isoform ratio potentially highly useful as a quantitative marker of maturation status.
Results
Discussion There is tremendous interest and many recent advances in the dynamic field of cardiac stem cell biology. Major breakthroughs in iPSC and reprogramming technologies further enable the potential of stem cell-based therapeutics in heart disease (Takahashi et al., 2007; Ieda et al., 2010). Before potential can be fully realized, several key basic questions remain, including cell lineage assignment and maturation status of the myocytes. A general consensus in the field is that stem cell-derived myocytes are immature; however, there are no widely accepted markers to define and track the precise differentiation status of the emerging myocytes. Accordingly, the field would be advanced by establishing a molecular signature for tracking the differentiation status of stem cell-derived cardiac myocytes. In this light, we propose that the developmentally controlled and irreversible genetic switch in the sarcomeric TNNI genes provides an excellent marker to track the differentiation status of stem cell-derived cardiac myocytes. Owing to strict developmental control in all mammalian hearts, including humans, the programmed inactivation of the “fetal” TNNI gene product, ssTnI, together in exquisite temporal concert with stoichiometric replacement by the adult gene product, cTnI, represents a unique molecular marker of cardiac myocyte maturation status (Bhavsar et al., 1991).