Differential and conditional activation of PKC-isoforms dictates cardiac adaptation during physiological to pathological hypertrophy

Abstract

A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy) or detrimental (pathological hypertrophy). This study was undertaken to establish the role of different protein kinase-C (PKC) isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week male Balb/c mice (Mus musculus) models, by reverse transcriptase-PCR, western blot analysis and M-mode echocardiography for cardiac function analysis. PKC-δ was significantly induced during pathological hypertrophy while PKC-α was exclusively activated during physiological hypertrophy in our study. PKC-δ activation during pathological hypertrophy resulted in cardiomyocyte apoptosis leading to compromised cardiac function and on the other hand, activation of PKC-α during physiological hypertrophy promoted cardiomyocyte growth but down regulated cellular apoptotic load resulting in improved cardiac function. Reversal in PKC-isoform with induced activation of PKC-δ and simultaneous inhibition of phospho-PKC-α resulted in an efficient myocardium to deteriorate considerably resulting in compromised cardiac function during physiological hypertrophy via augmentation of apoptotic and fibrotic load. This is the first report where PKC-α and -δ have been shown to play crucial role in cardiac adaptation during physiological and pathological hypertrophy respectively thereby rendering compromised cardiac function to an otherwise efficient heart by conditional reversal of their activation.

Figure 1. Assessment of hypertrophy and estimation of collagen in all the experimental groups.

(A) Graph showing HW/BW ratio in all five experimental models: pathological hypertrophy (H), physiological hypertrophy (E), exercise-trained pathological hypertrophy (H^X), mice kept at rest after 4 weeks of exercise training (E^R) and representative control (C) (*p<0.05 for H versus E or C; #p<0.05 for H versus H^X; ¶p<0.05 for E versus E^R). (B) Graph showing cardiomyocyte cross-sectional area (in µm²) in groups C, H, E, H^X, and E^R (***p<0.001 for H versus E or C; ##p<0.01 for H versus H^X; ¶p<0.05 for E versus E^R). (C) Expression profile of pathological hypertrophy markers (ANF and β-MHC) and physiological hypertrophy marker (IGF-1) estimated by RT-PCR. GAPDH was used as loading control. (D) Graph showing ventricular collagen concentration in groups C, H, E, H^X, and E^R estimated by hydroxyproline assay.

Figure 2. Differential expression profile of Protein Kinase-C isoforms.

(A) Western blot analyses showing change in expression of PKC α and PKC-δ (phospho and total) in groups C, H, E, H^X and E^R. PKC-δ cleavage product (41 kD) is exclusive in group H, H^X and E^R. RPL32 was used as loading control. (B) Western blot analyses showing phosphorylation of PKC-δ and PKC-α in adult cardiomyocytes isolated from different experimental groups. RPL32 was used as loading control. (C) Western blot analyses showing changes in expression of phosphorylated and total PKC-δ and PKC-α in exercise withdrawn animals (E^R) rested for different time periods. RPL32 was used as loading control. (D) Immunofluorescence study showing expression of phospho-PKC-δ and -α in different experimental group. Tissue sections showing phospho-PKC-δ expression in panels b, f, j, n and r and phospho-PKC-α in panels b’, f’, j’, n’ and r’ (green fluorescence). Sections were counter stained with alpha sarcomeric actinin antibody (panels c, g, k, o and s for PKC-δ and panels c’, g’, k’, o’ and s’ for PKC -α; red fluorescence). Nuclei were stained with DAPI (panels a, e, i, m and q for PKC-δ and panels a’, e’, i’, m’ and q’ for PKC-α; blue fluorescence) and merged images are shown in panels d, h, l, p and t for PKC-δ and panels d’, h’, l’, p’ and t’ for PKC-α. Increased expression of phospho-PKC-δ was observed in groups H and E^R whereas, phospho-PKC-α expression was induced in group E and H^X (Scale bar = 50 µm, Magnification = 40X).

Figure 3. PKC-δ associated downstream target proteins.

(A) Western blot analysis with the nuclear protein revealed significantly increased translocation of cleaved PKC-δ (41 kD) to nucleus in group H, H^X and E^R compared to either C or E. Significantly increased phospho-p53 (at Ser 46 and Ser 15) and total p53 was observed in group H and E^R compared to E or C whereas significantly reduced phospho-p53 (at Ser 46 and Ser 15) and total p53 was observed in groups E and H^X compared to H or E. RPL32 and Lamin B were used as loading control for cytosolic proteins and nuclear proteins respectively. (B) Subcellular fractionation followed by western blot analyses with mitochondrial protein showing significantly increased translocation of cleaved PKC-δ (41 kD) to mitochondria in group H, H^X and E^R compared to either C or E along with increased expression of PLS3, t-Bid, Bax, cytochrome-c proteins. RPL32 and COX IV were used as loading control for cytosolic proteins and mitochondrial proteins respectively. (C) Western blot analysis showingcleavage of caspase-3 and PARP in group H and E^R compared to C or E. RPL32 was used as loading control. Caspase-3 activity assay showing similar changes in all the experimental groups. No significant difference in caspase-3 activity was detected between groups E and C (*p<0.05 for H versus C; ###p<0.001 for H versus H^X; ¶¶p<0.01 for E versus E^R). (D) Subcellular fractionation followed by western blot analyses showed significantly increased translocation of cleaved PKC-δ (41 kD) to mitochondria along with caspase-3 cleavage in adult cardiomyocytes isolated from group H and E^R compared to either C or E or H^X. RPL32 was used as loading control. (E) Western blot analysis showing phosphorylation status of STAT3 and P38 MAPK in groups C, E, E^R and H.

Figure 4. Inhibition of PKC-δ reduces expression of downstream targets.

(A) Western blot analysis showing successful down regulation in expression of both phospho and total PKC-δ along with significant decrease in the level of bax, cytosolic cytochrome-c and PARP cleavage in mice treated with PKC-δ siRNA in group H. RPL32 was used as loading control. (B) Graph showing caspase-3 and caspase-9 activities in the two experimental groups (C and H) treated with siRNA and nonspecific siRNA. Significant decrease in caspase-3 and caspase-9 activities occurred in mice treated with PKC-δ siRNA compared to mice treated with nonspecific siRNA [*p<0.05 for H (nonspecific siRNA) versus H (PKC-δ siRNA)].

Figure 5. PKC-α and survival kinases.

(A) Western blot analysis showing phospho-Akt to Akt and phospho-ERK-1/2 to ERK-1/2 ratio to be significantly increased in group E compared to either H or C. phospho-Akt/Akt and phospho-ERK-1/2 to ERK-1/2 ratio was significantly decreased in group E^R compared to E. RPL32 was used as loading control. (B) Graph showing caspase-9 activity in groups C, E, E^R and H. (*p<0.05 for H versus E; ¶p<0.05 for E versus E^R). (C) Western blot analyses reveal successful knockdown of phospho and total PKC-α along with significant decrease in the phosphorylation level of Akt and ERK-1/2 in mice treated with PKC-α siRNA. RPL32 was used as loading control. (D) Phosphorylation of PKC-α and Akt was observed by western blot analysis in adult cardiomyocytes isolated from group E and H^X compared to C, H and E^R. RPL32 was used as loading control.

Figure 6. Reversal of PKC isoforms modulates hypertrophy regulators.

(A) Western blot analysis showing inhibition of PKC-α in physiological hypertrophy mice with PKC-α siRNA resulting in significant phosphorylation of PKC-δ and translocation of active PKC-δ (41 kD), cytosolic cytochrome-c and caspase-3 cleavage compared to nonspecific siRNA treated physiological hypertrophy mice. RPL32 was used as loading control. (B) Graph showing PKC-α siRNA treatment during physiological hypertrophy generation showed significant increase in caspase-3 and caspase-9 activities compared to nonspecific siRNA treatment during physiological hypertrophy [*p<0.05 for E (nonspecific siRNA) versus E (PKC-α siRNA)]. (C) Western blot analyses showing significantly increased phosphorylation of PKC-α, Akt and ERK-1/2 and PKC-α expression in pathological hypertrophy mice treated with PKC-δ siRNA. RPL32 was used as loading control.

Figure 7. Effect of PKC-δ down regulation on collagen synthesis during pathological hypertrophy.

(A) Graph showing ventricular collagen concentration in groups C, H and H + PKC-δ siRNA (in vivo) and in groups C, Ang-II and Ang-II + PKC-δ siRNA (in vitro) estimated by hydroxyproline assay. [***p<0.01 for H (nonspecific siRNA) versus H (PKC-δ siRNA) and **p<0.01 for Ang-II (nonspecific siRNA) versus Ang-II (PKC-δ siRNA)]. (B) Western blot analysis showing status of phospho PKC-δ, phospho P38, phospho STAT3 (Tyr 705 and Ser 727), STAT3 following PKC-δ siRNA treatment along with pathological hypertrophy (H) in comparison to hypertrophy alone in vivo as well as in vitro. RPL32 was used as loading control.