Palmitic Acid Inhibits Myogenic Activity and Expression of Myosin Heavy Chain MHC IIb in Muscle Cells through Phosphorylation-Dependent MyoD Inactivation

**Background:** Sarcopenia is an age-related loss of muscle mass and strength, with selective atrophy of fast-twitch (type II) muscle fibers. Intramuscular lipid accumulation and lipotoxicity, particularly involving saturated fatty acids like palmitic acid (PA), are implicated in sarcopenia pathogenesis. This study investigated whether PA differentially affects the expression of myosin heavy chain (MHC) isoforms in muscle cells and explored the underlying molecular mechanisms.

**Methods:** C2C12 mouse myoblasts were differentiated into myotubes over 6 days. Cells were treated with 400 μM PA (within physiological plasma range) for 24 hours at various differentiation time points. Outcomes included: morphological assessment of myotube differentiation via immunofluorescence (fusion index, MHC content); mRNA expression of MHC isoforms (Myh1/MHC IId/x, Myh2/MHC IIa, Myh4/MHC IIb, Myh7/MHC I) and myogenic regulatory factors (MyoD, myogenin) by real-time PCR; MHC protein expression by Western blotting; and Myh4 promoter activity using luciferase reporter assays with wild-type and mutant MyoD expression plasmids. Pharmacological inhibitors were used to test pathway involvement: TAK-242 (TLR4 inhibitor), Ro 31-8220 (PKC inhibitor), myriocin (SPT inhibitor), and roscovitine (CDK inhibitor).

**Key Results:** PA treatment significantly reduced the fusion index and average MHC content in differentiated C2C12 cells. PA selectively suppressed Myh4 (MHC IIb) mRNA expression on days 4 and 6 of differentiation, and Myh1 (MHC IId/x) on day 6, while Myh2 (MHC IIa) and Myh7 (MHC I) were unaffected. Myh4 suppression was observed from day 1 through day 6. MyoD and myogenin mRNA were also suppressed by PA. Western blotting confirmed reduced MHC IIb protein, though MHC I protein was also reduced (inconsistent with mRNA data). The TLR4 inhibitor TAK-242 did not prevent PA-induced inhibition of Myh4 expression. Luciferase reporter assays showed that PA significantly reduced Myh4 promoter activity during differentiation. Forced MyoD expression increased Myh4 promoter activity, which was attenuated by PA. Mutant MyoD with serine-to-alanine substitutions at Ser5, Ser200, and Ser262 (MyoD-S/A-2) partially but significantly recovered PA-induced inhibition of Myh4 promoter activity. The PKC inhibitor Ro 31-8220 (0.5 μM) partially diminished PA-induced suppression of Myh4 expression, while myriocin (5 μM) and roscovitine (5 μM) had no effect.

**Clinical Implications:** This study provides a mechanistic link between elevated saturated fatty acids and the selective loss of fast-twitch muscle fibers in sarcopenia. PA suppresses MHC IIb expression through PKC-mediated phosphorylation and inactivation of MyoD, independent of TLR4 signaling. These findings suggest that interventions targeting lipid metabolism or PKC signaling in muscle could potentially mitigate sarcopenia, particularly in the context of age-associated obesity and intramuscular lipid accumulation. However, as an in vitro study using mouse cells, direct translation to human sarcopenia requires further investigation.