Tissue-specific small heat shock protein 20 activation is not associated with traditional autophagy markers in Ossabaw swine with cardiometabolic heart failure

Abstract

The small heat shock protein 20 (HSPB6) emerges as a potential upstream mediator of autophagy. Although autophagy is linked to several clinical disorders, how HSPB6 and autophagy are regulated in the setting of heart failure (HF) remains unknown. The goal of this study was to assess the activation of the HSPB6 and its association with other well-established autophagy markers in central and peripheral tissues from a preclinical Ossabaw swine model of cardiometabolic HF induced by Western diet and chronic cardiac pressure overload. We hypothesized HSPB6 would be activated in central and peripheral tissues, stimulating autophagy. We found that autophagy in the heart is interrupted at various stages of the process in a chamber-specific manner. Protein levels of HSPB6, Beclin 1, and p62 are increased in the right ventricle, whereas only HSPB6 was increased in the left ventricle. Unlike the heart, samples from the triceps brachii long head showed only an increase in the protein level of p62, highlighting interesting central versus peripheral differences in autophagy regulation. In the right coronary artery, total HSPB6 protein expression was decreased and associated with an increase in LC3B-II/LC3B-I ratio, demonstrating a different mechanism of autophagy dysregulation in the coronary vasculature. Thus, contrary to our hypothesis, activation of HSPB6 was differentially regulated in a tissue-specific manner and observed in parallel with variable states of autophagy markers assessed by protein levels of LC3B, p62, and Beclin 1. Our data provide insight into how the HSPB6/autophagy axis is regulated in a preclinical swine model with potential relevance to heart failure with preserved ejection fraction.NEW & NOTEWORTHY Our study shows that the activation of HSPB6 is tissue specific and associated with variable states of downstream markers of autophagy in a unique preclinical swine model of cardiometabolic HF with potential relevance to HFpEF. These findings suggest that targeted approaches could be an important consideration regarding the development of drugs aimed at this intracellular recycling process.

Keywords: HFpEF; autophagy; coronary vasculature; heart; skeletal muscle.

Fig. 1.

Protein level of HSPB6, phosphorylation of HSPB6 at Ser16, and p-HSPB6/HSPB6 ratio are increased in the LV and RV of Western diet-fed, aortic-banded Ossabaw swine. Representative Western blots of phosphorylated HSPB6 at Serine 16 (p-HSPB6-Ser16) and total HSPB6 from the LV (A), RV (C), TLH (E), and RCA (G). Quantification of p-HSPB6-Ser16, total HSPB6, and p-HSPB6/HSPB6 ratio in the LV (B), RV (D), TLH (F), and RCA (H). Representative CSQ Western blot and its quantification are shown in (I) and (J), respectively. All data are means ± SD, n = 4 or 5, d = effect size. CSQ, calsequestrin; LV, left ventricle; RCA, right coronary artery; RV, right ventricle; TLH, triceps brachii long head.

Fig. 2.

Beclin 1 protein level is increased in RV of Western diet-fed, aortic-banded Ossabaw swine. Representative Western blots of Beclin 1 from the LV (A), RV (C), TLH (E), and RCA (G). Quantification of Beclin 1 in the LV (B), RV (D), TLH (F), and RCA (H). Representative membrane stained with Coomassie blue and its quantification are shown in (I) and (J), respectively. All data are means ± SD, n = 4 or 5, d = effect size. LV, left ventricle; RCA, right coronary artery; RV, right ventricle; TLH, triceps brachii long head.

Fig. 3.

Microtubule-associated proteins 1A/1B light chain 3B (LC3B) conversion is increased in the RCA of Western diet-fed, aortic-banded Ossabaw swine. Representative Western blots of LC3B-I and LC3B-II from the LV (A), RV (C), TLH (E), and RCA (G). Quantification of LC3B-I, LC3B-II, and LC3B-II/LC3B-I ratio in the LV (B), RV (D), TLH (F), and RCA (H). Representative CSQ Western blot and its quantification are shown in (I) and (J), respectively. All data are means ± SD, n = 4 or 5, d = effect size. CSQ, calsequestrin; LV, left ventricle; RCA, right coronary artery; RV, right ventricle; TLH, triceps brachii long head.

Fig. 4.

Sequestosome1/p62 (p62) protein level is elevated in the RV and TLH of Western diet-fed, aortic-banded Ossabaw swine. Representative Western blots of p62 from the LV (A), RV (C), TLH (E), and RCA (G). Quantification of p62 in the LV (B), RV (D), TLH (F), and RCA (H). Representative membrane stained with Coomassie blue and its quantification are shown in (I) and (J), respectively. All data are means ± SD, n = 4 or 5, d = effect size. LV, left ventricle; RCA, right coronary artery; RV, right ventricle; TLH, triceps brachii long head.