The Effects of Mechanical Loading Variations on the Hypertrophic, Anti-Apoptotic, and Anti-Inflammatory Responses of Differentiated Cardiomyocyte-like H9C2 Cells

Figure 1. (A) Cell cycle progression of undifferentiated H9C2 cardiomyoblasts. Percentages of cells in each phase represent a normal cell cycle phenotype of H9C2 cardiomyoblasts. (B,C,N) Expression of cell cycle regulator Cyclin D1 and cardiac-specific markers during H9C2cardiomyoblast differentiation. Quantitative transcriptional analysis of Cyclin D1 revealed its downregulation during the differentiation process of cardiomyoblasts (B), along with the upregulation of the cardiac-specific markers troponin (C) and actin (N) (representative Western blot and immunoblotting quantification of cardiac actin). (D–F) Myogenic regulatory factors’ expression during cardiomyoblast differentiation. Quantitative analysis of the mRNA expression of muscle-specific transcription factors MyoD1, Myogenin, and MRF4 revealed a differential expression pattern, e.g., of early (MyoD1) vs. late (MRF4) myogenic factors in cardiomyoblasts during their differentiation. (L,M) Representative Western blots and immunoblotting quantification of MyoD (L) and Myogenin (M) protein expression are given. The mRNA expression values of each gene of interest have been normalized to the corresponding GAPDH mRNA and are expressed…

Figure 2. (A,B) IGF-1 isoforms’ expression during cardiomyoblasts differentiation. Quantitative analysis of IGF-1Ea and IGF-1Eb mRNA expression in cardiomyoblasts during their differentiation. (C–E) Alterations in the activation of the signalling proteins Akt, Erk1/2, and p38. Representative Western blots and immunoblotting quantification of p-Akt, p-Erk1/2, and p-p38phosphorylation in cardiomyoblasts during their differentiation process. The values of the phosphorylated proteins were normalized to each corresponding GAPDH on the same immunoblot. Significantly different compared to day 0, • p < 0.05; •• p < 0.01; ••• p < 0.001. Significantly different compared to day 3, ▪▪▪ p < 0.001. Mean + SE of three independent experiments performed in triplicate.

Figure 3. (A–C) Effects of cyclic mechanical loading on the expression of myogenic regulatory factors (MRFs). Quantitative analysis of transcriptional responses of (A) MyoD1, (B) Myogenin, and (C) MRF4 in differentiated cardiomyocytes subjected to different mechanical stretching protocols compared to the control (Ctr; nonstretched myotubes). (D,E) The mRNA values of MRFs in stretched myotubes have been normalized to the corresponding GAPDH mRNA and are expressed as fold changes compared to the control. Representative Western blots and immunoblotting quantification of MyoD (D) and Myogenin (E) subjected to the various mechanical loading conditions compared to the control; the proteins of interest were normalized to each corresponding GAPDH on the same immunoblot. The same blots have been used for GAPDH in Figure 3D,E, Figure 5C, and Figure 7A,B, except from the blot of GAPDH in panel A for the 2%/1 Hz stretching protocol. Significantly different compared to the control, * p < 0.05; ** p < 0.01; *** p < 0.001. Significantly different compared to the 2%/0.25 Hz stretching protocol, # p < 0.05. Mean + SE of three independent experiments performed in triplicate.

Figure 4. (A,B) Effects of mechanical stretching on the expression of the IGF-1 isoforms. Quantitative analysis of (A) IGF-1Ea and (B) IGF-1Eb mRNA levels in differentiated cardiomyocytes subjected to different mechanical stretching protocols compared to the control (Ctr; nonstretched myotubes). The transcriptional responses of IGF-1 isoforms in stretched cardiomyocytes have been normalized to the corresponding GAPDH values and are expressed as fold changes compared to the control. (C,D) Effects of mechanical stretch on the expression of muscle atrophy factors. Quantitative analysis of (C) Myostatin and (D) Atrogin 1 mRNA expression in differentiated cardiomyocytes subjected to the various mechanical loading conditions compared to the control (nonstretched myotubes); The mRNA values of the genes of interest in stretched myotubes have been normalized to the corresponding GAPDH mRNA and are expressed as fold changes compared to the control. Significantly different compared to the control, * p < 0.05; ** p < 0.01. Significantly different compared to the 2%/0.25 Hz stretching protocol, # p < 0.05; ## p < 0.01; ### p < 0.001. Significantly different compared to the 12%/ 0.25 Hz stretching…

Figure 5. (A,B) Effects of mechanical loading on the expression of pro-apoptotic factors. Quantitative analysis of (A) FoxO1 and (B) p53 mRNA expression in differentiated cardiomyocytes subjected to various mechanical stretching protocols compared to the control (Ctr; nonstretched myotubes); the transcriptional responses of pro-apoptotic factors in stretched myotubes have been normalized to the corresponding GAPDH mRNA values and are expressed as fold changes compared to the control. (C) Representative Western blots and immunoblotting quantification of p53 subjected to the different stretching protocols compared to the control (nonstretched myotubes). The p53 protein levels were normalized to each corresponding GAPDH on the same immunoblot. The same blots have been used for GAPDH in Figure 3D,E, Figure 5C, and Figure 7A,B, except from the blot of GAPDH in panel A for the 2%/1 Hz stretching protocol, Significantly different compared to the control, * p < 0.05; ** p < 0.01. Significantly different compared to the 2%/0.25 Hz stretching protocol, # p < 0.05; ## p < 0.01. Mean + SE of three independent experiments performed in triplicate.

Figure 6. (A–D) Effects of mechanical loading on the expression of pro-inflammatory factors. Quantitative analysis of (A) NF-kB, (B) IL-6, (C) IL-1β, and (D) TNF-α transcriptional responses of differentiated cardiomyocytes subjected to various mechanical stretching protocols compared to the control (Ctr; nonstretched myotubes). The mRNA values of inflammation-related factors in stretched myotubes have been normalized to the corresponding GAPDH mRNA and are expressed as fold changes compared to the control. Significantly different compared to the control, * p < 0.05; ** p < 0.01; *** p < 0.001. Significantly different compared to 2%/0.25 Hz stretching protocol, # p < 0.05; ### p < 0.001. Significantly different compared to the 12%/0.25 Hz stretching protocol, @@@ p < 0.001. Mean + SE of three independent experiments performed in triplicate.

Figure 7. (A,B) Effects of cyclic mechanical stretching on the activation of the signalling proteins Akt and Erk1/2. Representative Western blots and immunoblotting quantification of (A) p-Erk1/2 and (B) p-Akt phosphorylation in cardiomyoblasts subjected to different mechanical loading protocols compared to the control (Ctr; nonstretched myotubes). The values of the phosphorylated proteins were normalized to each corresponding GAPDH on the same immunoblot. The same blots have been used for GAPDH in Figure 3D,E, in Figure 5C, and in Figure 7A,B except for the blot of GAPDH in (A) for the 2%/1 Hz stretching protocol. Significantly different compared to the control * p < 0.05; ** p < 0.01; *** p < 0.001. Significantly different compared to the 2%/0.25 Hz stretching protocol, # p < 0.05. Significantly different compared to the 2%/1 Hz stretching protocol, $$ p < 0.01. Significantly different compared to the 12%/1 Hz stretching protocol, && p < 0.01. Mean + SE of three independent experiments performed in triplicate.