The road to physiological maturation of stem cell-derived cardiac muscle runs through the sarcomere

Abstract

Recent advances the cardiac biomedical sciences have been propelled forward by the development and implementation of human iPSC-derived cardiac muscle. These notable successes notwithstanding, it is well recognized in the field that a major roadblock persists in the lack of full “adult cardiac muscle-like” maturation of hiPSC-CMs. This Perspective centers focus on maturation roadblocks in the essential physiological unit of muscle, the sarcomere. Stalled sarcomere maturation must be addressed and overcome before this elegant experimental platform can reach the mountaintop of making impactful contributions in disease pathogenesis, drug discovery, and in clinical regenerative medicine.

Human induced pluripotent stem cell derived cardiac myocytes (hiPSC-CMs) have ushered in an exciting platform for advancing basic and clinical sciences [1–5]. From drug discovery to cell-based regenerative medicine therapies, hiPSC-CMs have well noted tremendous therapeutic potential. Before the fullness of basic and clinical impact can be realized, a major barrier in the field centers on advancing hiPSC-CM maturation.

This perspective addresses significant ongoing efforts toward physiological optimization of hiPSC-CM function for bench (basic) and bedside (clinical) applications. It is well recognized that there is a wide range of physiologically relevant cellular processes that must work together, in elegant partnership, to accomplish adult cardiac muscle performance. On this point, the reader is directed to several excellent reviews that have focused hiPSC-CM maturation as it relates to calcium handling, ion channels, cell metabolism, substrate utilization and energetics and cell morphology [1,3,4]. This commentary focuses on maturation-based deficits of the cellular machinery responsible for actually developing and regulating force and motion in cardiac muscle – the sarcomere.

For nearly all cardiac stem cell applications, whether as a platform for drug discovery, in fundamental basic science structure-function studies, or in potential regenerative medicine applications in the clinic, a long sought unmet goal has been to advance the rather primitive immature stem cell-derived cardiac myocytes into bonafide adult cardiac myocytes (Fig. 1) [4]. This is a tall task indeed. Physiological optimization of stem cell-derived cells and tissues represents a major hurdle for advancing progress in regenerative medicine [2]. For example, in Type I diabetes, stem cell-derived β cells are severely limited in terms of physiologically regulated insulin production. In the cardiac drug discovery/ regenerative medicine fields, physiological optimization of stem cell-derived cardiac muscle is a rate-limiting step hindering progress in basic science studies and in clinical applications.

Fig. 1.

Schematic representation of the long and challenging road to hiPSC-CM maturation.

Congenital heart disease (CHD) exacts a tremendous toll on patient morbidity and mortality world-wide [6]. Clinical management of these patients is challenging and limited by the scarcity of new therapies in the pipeline. Clinical trials.gov lists 100s of registered trials currently ongoing for cell-based cardiac repair in adults, including use of organ-derived cells and stem cell-derived cells. A recent review on cardiac repair, focusing on preclinical and clinical trials using cell therapy strategies, concluded that cardiac cell therapy has been extensively studied, however, mechanisms of action and clinical benefits have not yet been established [7]. Complexities in surveying the outcome of these clinical trials are numerous and include distinguishing findings from studies using organ-derived versus stem cell-derived cells. Nonetheless, as surmised in this review [7], continued efforts in preclinical work should facilitate the translation of basic science discoveries in regenerative myocardial therapies to the clinic. Of note, recent preclinical studies using hiPSC-CMs do demonstrate some evidence of engraftment [8]. In this light, there is growing sentiment that the cell intrinsic function of hiPSC-CMs must be physiologically optimized in order to maximize the therapeutic potential of de novo cardiogenesis as required to address severe heart defects. It follows that physiologic optimization of stem cell-derived cardiac tissue presents new opportunities to address intractable diseases of the heart.

Stalled maturation underscores the sub-optimal physiological performance of hiPSC-CMs [1–4]. In the cardiac drug discovery field, the potential of stem cell-derived cardiac muscle is severely hindered by the cell’s sarcomere-intrinsic “failure signature” which mirrors the profile of failing human myocardium [4,9]. This significantly limits cardiac stem cell-based drug discovery and cardiomyopathy etiology studies, and markedly limits therapeutic potential in the clinic. In the diseased human heart, the sarcomere failure signature includes key deficits in essential contractile and regulatory protein isoform expression [10,11]. Critically, the cardiac sarcomere failure profile characteristic of diseased human hearts is well documented in so-called “normal” pluripotent stem cell-derived cardiac muscle. This sarcomere failure profile must be corrected to enable the hiPSC-CM drug discovery/cardiac regenerative medicine field to move forward in both the laboratory and the clinic.

One of the great roadblocks to achieving maturation is that there are no well accepted and implemented standardization profiles or “biomarkers” to track and quantify hiPSC-CM maturation [3]. Difficulties arise from an appreciation of the elegant and highly complex subcellular machinery required to drive the functional performance output of the adult cardiac myocyte. Thus, efforts to advance maturation have logically parsed these complexities into distinct biological bins [3]. These include studies focusing on the genetic fingerprint, ion channels and transporters, calcium handling, metabolic profile, subcellular cell ultrastructure and overall cell morphology (Fig. 1).

Whereas, these are all appreciated as essential elements that together define the cellular and molecular underpinnings of the adult cardiac myocyte, these are ancillary, in terms of the cardiac myocyte raison d’être, that is, to produce force and motion. In this light, the road to cardiac stem cell-derived maturation runs through the sarcomere – the functional unit of muscle (Fig. 1).

Fundamental to the sarcomere are its regulatory, structural and effector functions [11–13]. The sarcomere consists of a highly organized assembly of literally millions (if not more) of individual contractile and regulatory proteins highly organized into inter-digitating thin and thick myofilaments, together with a host of Z-line and M-line proteins and elastic elements (titin). The precision of the protein packaging required in the sarcomere is readily appreciated by the near-crystalline alignment of these myofilaments. The thin myofilaments are critical for regulatory control of sarcomere activation and inactivation [12]. The thin myofilament structure of cardiac muscle is dominated by actin, a globular protein that under physiological conditions polymerizes into elongated filaments of double helical strands. Residing in the major groove formed between the coiled actin strands is α tropomyosin (α Tm), an elongated 284 amino acid, α-helical protein that dimerizes to form an intertwining polypeptide chain. Thus, in the myofilament, α Tm exists as a coiled-coil. Each Tm spans seven actin monomers and is polymerized head to tail along the length of the actin filament. Associated with each Tm is whole troponin, a globular protein consisting of three subunits: 1) troponin C (TnC), the Ca²⁺ binding subunit, 2) troponin I (TnI), the inhibitory subunit, and 3) troponin T (TnT), the subunit that binds to tropomyosin. In the absence of Ca²⁺, tropomyosin is thought to sterically hinder cross-bridge formation, and under this condition the thin filament is considered to be in a blocked state. Addition of Ca²⁺ and its binding to TnC allows strong cross-bridge formation.

At a molecular level, the Ca²⁺-TnC complex leads to strengthening of the interaction between TnC and TnI and weakening of the interaction between TnI and actin, which causes tropomyosin to move deeper into the groove between the actin strands, thus revealing actin binding sites for myosin attachment [12]. In the presence of Ca²⁺, the thin filament has been further characterized as residing in either a closed state where myosin can bind, but not produce force, or an open state where myosin can bind and then isomerize to a strong binding, force generating state. The occupancy of cross-bridges in the closed or open states is thus highly dependent upon [Ca²⁺]. The precise role of Tm in regulating transitions between thin filament states (blocked, closed, open) has not been fully resolved. Further, it is unknown how mutant α Tm molecules may specifically alter occupancy of these states or specific transitions among these states. It is further appreciated that Ca²⁺, TnI and myosin all serve as activating ligands of the cardiac sarcomere [14,15].

In normal human heart development, sarcomere maturation is essential and involves several critical protein isoform transitions. Most notable and unique is the irreversible gene switch from TNNI1, encoding the fetal slow skeletal troponin I (ssTnI) isoform, to TNNI3, encoding the adult cardiac TnI (cTnI) isoform [4,9]. This genetic switch occurs in all mammals and typically completes shortly after birth, including in the human heart. This isoform switch is critical to refine and optimize the physiological contractile function of the human heart [12]. This is particularly evident in muscle relaxation, especially in the fight-or-flight physiological response wherein the sarcomere is producing greater force in a shorter amount of time, thus necessitating significantly faster relaxation performance. Dramatically, cardiac sarcomeres deficient in this essential isoform switch are severely defective, causing severe heart failure and early death in humans [10]. Other key sarcomere proteins also shift their isoform profile during development; however, these, including myosin and titin isoforms, are also subject to isoform shifts in heart disease [16–18], making it difficult to implement their use as quantitative markers of cardiac maturation.

HiPSC-CMs studied to date are severely deficient in sarcomere maturation, most notably quantified in the stalled TNNI1 to TNNI3 protein isoform transition [9,19,20]. Thus, in terms of sarcomere maturation, hiPSC-CMs mirrors the sarcomere profile of the failing human heart muscle [9,21]. The sarcomere failure profile of stem cell-derived cardiac muscle must be corrected to enable the cardiac drug discovery/regenerative medicine fields to move forward in the laboratory and clinic.

Myosin is the molecular motor that dominates the composition of the thick myofilaments [12]. The molecular motor myosin heavy chain (MyHC) converts chemical energy into work in the heart. The human heart contains two MyHC isoforms: α-MyHC and β-MyHC. In the healthy human heart, β-MyHC is the dominant and energetically economical isoform. In the healthy human heart, α-MyHC, which is the more powerful isoform, is critical and accounts for ~15% of the total sarcomeric MyHC [18]. In failing human hearts, there is complete loss of the powerful α-MyHC isoform. Gain of function studies using gene transfer techniques demonstrate the key role of MyHC isoforms on cardiac muscle performance [22]. The hypothesis that follows is that the α-MyHC isoform is the essential physiological “pilot light” in the sarcomere, necessary for robust heart function. It follows that loss of α-MyHC contributes to the sarcomere failure signature by directly diminishing performance of the failing human heart. The working hypothesis in the cardiac stem cell field is that ongoing cell-based therapies for the diseased heart are unsuccessful in part because the delivered cells/new myocytes have sarcomeres with a “failure signature” of poor energetics and sub-optimal contractile isoforms [4]. Before full clinical success can be achieved, optimization of sarcomere-centered hiPSC-CM maturation is essential.

Hand-in-hand with significant technological advances in stem cell-cardiac muscle generation and development are the dramatic discoveries centering on directed genetic engineering of chromosomally organized DNA in live cells and organisms. Advances in protein and RNA-directed endonucleases to specific addresses within the genome opens the door to precision gene editing in hiPSCs. Germane to this review, recent works have investigated implementing multiple genetic engineering tools and principles to bypass developmental roadblocks in the sarcomere hindering hiPSC-CM maturation.

Bedada et al., [19] established the TNNI1/TNNI3 protein isoform transition as a reliable, quantitative and physiologically relevant gene switch for tracking sarcomere maturation in hiPSC-CMs. Here, shown across multiple hiPSC lines, they demonstrated that hiPSC-CMs are stalled in a fetal-like TNNI isoform profile (TNNI1). The fetal/embryonic TNNI protein isoform content in the sarcomere persisted over long time periods in culture, at least 9.5 months of hiPSC-CMs beating in culture. They subsequently showed via direct adenoviral gene transfer that it was possible to gain the full TNNI3 isoform profile present in all mammalian adult heart muscle, including in the adult human heart.

More recently, Wheelwright et al., [9] designed and engineered a drug inducible sarcomere maturation gene switch system using genome editing in hiPSC-CMs. Here, physiologically relevant TNNI3 gene induction was demonstrated under precise temporal control. HiPSC-CMs having TNNI3 protein isoform stoichiometrically integrated into the sarcomeres directly caused enhanced physiologically relaxation performance in the beating cardiac myocytes. This is highly reminiscent of the acquisition of lusitropic function in adult human myocardium essential for robust dynamic range of heart function, including the essential heart performance component to the fight or flight stress response.

These findings substantiate the premise that sarcomere maturation is necessary for acquisition of physiological maturation in hiPSC-CMs. They further demonstrated that the key TNNI isoform switch is not a master regulator of sarcomere/global hiPSC-CM maturation. It was shown that induction of TNNI3 isoform in itself did not cause a maturation “domino” effect by advancing other aspects of adult myocardial maturation (e.g., induction of the physiological MyHC profile) [9].

Because induction of the adult TNNI3 isoform did not overcome other delayed maturation isoform switching processes in the sarcomere, it is clear a multifocal approach will be necessary to advance the adult sarcomere signature in hiPSC-CMs. Of note, the aforementioned genetic profile in MyHC isoforms is critical to cardiac muscle performance in health and disease. In contrast to the TNNI switch, the MyHC isoform is reversible and a hallmark of sarcomere dysfunction in disease [18,23]. In this light, it has been reported that stem cell-derived cardiac muscle is dominated by the β-MyHC [24]. Future efforts to genetically engineer α-MyHC isoform expression in hiPSC-CMs will be essential to mirror the α-MyHC: β-MyHC sarcomeric ratio in healthy human heart muscle.

New tools and approaches to track sarcomere maturation non-invasively would provide a significant boost to the field. This would be particularly important for effective drug discovery and disease modeling structure-function studies. In this light, Miki et al., [20] recently developed an elegant approach to track non-invasively a key sarcomere maturation roadblock, notably the stoichiometrically preserved TNNI1 to TNNI3 gene switch transition required to adult cardiac myocyte sarcomere functionality. Here they used a CRISPR/Cas9 genome editing system via a homologous recombination to accomplish TNNI locus knockin, wherein hiPSCs were engineered at the TNNI1 locus to formTNNI1EmGFP and at the TNNI3 locus to form TNNI3mCherry. In this setting, a hiPSC double reporter was thus developed to detect the critical troponin switch for cardiac maturation. They used this system in a drug discovery platform to identify novel drugs and small molecules that could serve to advance sarcomere maturation in hiPSC-CMs. Extensive drug screening led to two compounds to enhance cardiac maturation, notably an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 (SKP2) inhibitor. They report that the ERRγ agonist also enhanced the metabolic maturation of hiPSC-CMs via reduced reliance on glycolysis. This novel non-invasive screening strategy for tracking sarcomeric maturation is an excellent addition to the growing number of tools seeking to advance hiPSC-CMs for drug discovery and regenerative medicine applications.

The road to physiological hiPSC-CM maturation is a long one. To date, it seems clear that a single Master regulator switch, gene or molecule for accomplishing physiologically relevant maturation is highly unlikely. Rather, a multifaceted, unified focus on the myriad elements culminating in advancing maturation will most likely be required to attain the elusive adult cardiac muscle signature. With the recognition of the sarcomere as the centerpiece of heart muscle, in frame with known sarcomere maturation deficits in hiPSC-CMs, it seems reasonable to exert greater focus on clearing barriers impeding sarcomere maturation as the field climbs farther up the mountainside toward complete physiologically relevant maturation of hiPSC-CMs.