Upregulation of beta-actin in left ventricular myocardium of canines with chronic heart failure
R A M E S H Ramesh C Gupta, K R I S T I Kristina J Szekely, K E F E I Kefei Zhang, D A V I D David E Lanfear, S A B B A H Hani N SabbahAbstract
Background
In the heart, β-actin plays a critical cytoskeletal role in non-contractile functions, such as regulating cell shape, growth, and migration, particularly in response to stress. β-Actin is present in the sarcolemma, cytosol, and mitochondria of cardiomyocytes. It is one of the most common proteins used as a normalizing protein, or "loading control," in molecular biology techniques like Western blotting and quantitative PCR. This assumption has been increasingly challenged, and the use of β-actin as a universal normalizing protein may have limitations that must be considered.
Objective
This study tested the hypothesis that β-actin protein level increases in the LV myocardium of dogs with HF and that this increase is also evident in sarcolemma, cytosol, and mitochondria of cardiomyocytes.
Methods
Studies were performed using LV tissue from 7 normal (NL) dogs and 7 dogs with chronic heart failure (HF) produced by intracoronary microembolizations (LV ejection fraction 34 ± 1%). The tissue was separated into homogenate, cytosolic, mitochondrial, and sarcolemma fractions. Western blots using canine-specific β-actin antibody were performed on all fractions. β-Actin protein levels in homogenate, cytosol and sarcolemma were normalized to GAPDH and to porin in mitochondria.
Results
Protein levels of GAPDH and porin were unchanged in dogs with HF compared to NL dogs. β-Actin protein level normalized to GAPDH or to porin was significantly elevated in all cellular fractions of HF dogs compared to NL dogs. Homogenate: (0.137 ± 0.029 vs. 0.064 ± 0.003; p<0.05); cytosol: (0.597 ± 0.066 vs. NL 0.066 ± 0.005, p<0.05); sarcolemma (0.652 ± 0.06 vs. 0.08 ± 0.016, p<0.05); mitochondria: (0.708 ± 0.068 vs. 0.399 ± 0.064, p<0.05) also showed similar trends. These findings suggest a global cytoskeletal surge and redistribution of β-actin in response to progressive myocardial pathology.
Conclusion
Increased β-actin in LV myocardium of dogs with HF reflects adverse cytoskeletal remodeling, a process that contributes to the LV deteriorating structure and function and reversion to fetal programming. Increased levels of β-actin in mitochondria can lead to excessive or uncontrolled fission and an accumulation of fragmented, dysfunctional mitochondria and ultimately impaired oxidative phosphorylation.