Inhibitors of the ubiquitin proteasome system block myofibril assembly in cardiomyocytes derived from chick embryos and human pluripotent stem cells

Abstract

Details of sarcomeric protein assembly during de novo myofibril formation closely resemble myofibrillogenesis in skeletal and cardiac myocytes in birds, rodents, and zebrafish. The arrangement of proteins during myofibrillogenesis follows a three-step process: beginning with premyofibrils, followed by nascent myofibrils, and concluding with mature myofibrils (reviewed in Sanger et al., 2017). Assembly and maintenance of myofibrils in living muscle cells. In: Handbook of experimental pharmacology, 2017 [pp. 39-75]. Our aim is to determine if the same pathway is followed in human cardiomyocytes derived from human inducible pluripotent stem cells. We found that the human cardiomyocytes developed patterns of protein organization identical to the three-step series seen in the model organisms cited above. Further experiments showed that myofibril assembly can be blocked at the nascent myofibril by five different inhibitors of the Ubiquitin Proteasome System (UPS) stage in both avian and human cardiomyocytes. With the exception of Carfilzomib, removal of the UPS inhibitors allows nascent myofibrils to proceed to mature myofibrils. Some proteasomal inhibitors, such as Bortezomib and Carfilzomib, used to treat multiple myeloma patients, have off-target effects of damage to hearts in three to 6 % of these patients. These cardiovascular adverse events may result from prevention of mature myofibril formation in the cardiomyocytes. In summary, our results support a common three-step model for the formation of myofibrils ranging from avian to human cardiomyocytes. The Ubiquitin Proteasome System is required for progression from nascent myofibrils to mature myofibrils. Our experiments suggest a possible explanation for the cardiac and skeletal muscle off-target effects reported in multiple myeloma patients treated with proteasome inhibitors.

STED analysis reveals the organization of nonmuscle muscle II, muscle myosin II, and F‐actin in nascent myofibrils

“…The surprising finding was that some muscle myosin II, but not the nonmuscle myosin II, had to be proteolyzed in the nascent myofibril for the mature myofibril to be formed (Wang et al, 2020). Wang et al (2022) have extended their previous studies on the effects of UPS inhibitors on myofibrillogenesis in skeletal muscle cells (Wang et al, 2020) to both embryonic chick cardiomyocytes and human inducible PSC‐derived cardiomyocytes, and obtained similar results, that is, UPS inhibitors block the formation of mature myofibrils at the nascent myofibril stage.…”

“…The Z-bodies of the nascent myofibrils become the initial binding sites for the attachment of the N-termini of titin molecules. The bipolar arrangements of titins in the Z-bands are essential for the binding of the bipolar muscle myosin II filaments in mature myofibrils the first cardiomyocytes isolated from quail precardiac mesoderm (Du et al, 2003); formation of myofibrils inside intact embryonic chick hearts (Du, Sanger, & Sanger, 2008); in cultured neonatal mouse cardiomyocytes (White et al, 2018), and now in cardiomyocytes derived from human induced Pluripotent Stem Cells (hiPSCs) (Wang et al, 2022). This three-step model was also supported by experiments in fixed and living skeletal muscle cells (avian muscle cultures, Sanger et al, 2002;Sanger, Mittal, Pochapin, & Sanger, 1986a, 1986bSanger, Sanger, & Franzini-Armstrong, 2004;Sanger, Wang, Fan, White, & Sanger, 2010;Turnacioglu, Mittal, Dabiri, Sanger, & Sanger, 1997a, 1997bTurnacioglu et al, 1996;Ayoob et al, 2000;Wang et al, 2020Wang et al, , 2018Wang et al, , 2007Stout, Wang, Sanger, & Sanger, 2008; in situ embryonic zebrafish, Sanger, Wang, Holloway, Du, & Sanger, 2009; and mouse muscle cultures, White, Barro, Makarenkova, Sanger, & Sanger, 2014).…”

“…The third and final step of myofibrillogenesis consists of alignment of muscle myosin II filaments into A‐bands, typical of mature myofibrils (Huxley, 1963), alignments of titin molecules stretching from the Z‐bands to the middle of A‐bands (Sanger & Sanger, 2001), and the loss of nonmuscle myosin II (Wang, Fan, Sanger, & Sanger, 2018). Support for this three‐step model was also obtained from studies of myofibrillogenesis in cultured cardiomyocytes isolated from adult rats and cats (LoRusso, Rhee, Sanger, & Sanger, 1997); in living embryonic chick cardiomyocytes expressing either GFP‐alpha‐actinin (Dabiri, Turnacioglu, Sanger, & Sanger, 1997), or GFP‐truncated titins (Turnacioglu, Mittal, Dabiri, Sanger, & Sanger, 1997a, 1997b; Turnacioglu, Mittal, Sanger, & Sanger, 1996; Ayoob, Turnacioglu, Mittal, Sanger, & Sanger, 2000); in the first cardiomyocytes isolated from quail precardiac mesoderm (Du et al, 2003); formation of myofibrils inside intact embryonic chick hearts (Du, Sanger, & Sanger, 2008); in cultured neonatal mouse cardiomyocytes (White et al, 2018), and now in cardiomyocytes derived from human induced Pluripotent Stem Cells (hiPSCs) (Wang et al, 2022). This three‐step model was also supported by experiments in fixed and living skeletal muscle cells (avian muscle cultures, Sanger et al, 2002; Sanger, Mittal, Pochapin, & Sanger, 1986a, 1986b; Sanger, Sanger, & Franzini‐Armstrong, 2004; Sanger, Wang, Fan, White, & Sanger, 2010; Turnacioglu, Mittal, Dabiri, Sanger, & Sanger, 1997a, 1997b; Turnacioglu et al, 1996; Ayoob et al, 2000; Wang et al, 2020, 2018, 2007; Wang, Sanger, & Sanger, 2005; Wang et al, 2005; Stout, Wang, Sanger, & Sanger, 2008; in situ embryonic zebrafish, Sanger, Wang, Holloway, Du, & Sanger, 2009; and mouse muscle cultures, White, Barro, Makarenkova, Sanger, & Sanger, 2014).…”

Comparison of incorporation of wild type and mutated actins into sarcomeres in skeletal muscle cells: A fluorescence recovery after photobleaching study

“…Our studies of sarcomeric protein localization during myofibril assembly in skeletal and cardiac muscle cells (reviewed in Sanger et al, 2005, 2017; White, Barro, Makarenkova, Sanger, & Sanger, 2014; White et al, 2018) led us to propose that premyofibrils and nascent myofibrils are precursors of mature myofibrils (Rhee et al, 1994; Sanger et al, 2017). With FRAP analysis we found that exchange of sarcomeric proteins between a cytoplasmic pool and sarcomere subunits takes place in each myofibril stage, but is more dynamic in the early stages of assembly (premyofibril and nascent myofibril) than in mature myofibrils (Wang et al, 2005, 2007, 2008, 2018, 2020, 2022; Wang, Dube, Mittal, Sanger, & Sanger, 2011).…”

“…The wt-YFP-α-actin (a) and the H40Y YFP-α-actin (d) exhibit identical recoveries for the plus and minus ends of the actin filaments. The G15R YFP-α-actin (b) and cytochalasin-D treated myotubes expressing wt-YFP-α-actin (d) reveal decreased incorporation of α-actin at the plus ends, and increased recoveries at the minus ends of the thin filaments compared to the wt-YFP-α-actin (a) and H40Y YFP-α-actin (d) in myotubes assembly (premyofibril and nascent myofibril) than in mature myofibrils (Wang et al, 2005(Wang et al, , 2007(Wang et al, , 2008(Wang et al, , 2018(Wang et al, , 2020(Wang et al, , 2022Wang, Dube, Mittal, Sanger, & Sanger, 2011).…”