Glycation in the cardiomyocyte.

Glycation is a protein post-translational modification that can occur on lysine and arginine residues as a result of a non-enzymatic process known as the Maillard reaction. This modification is irreversible, so the only way it can be removed is by protein degradation and replacement. Small reactive carbonyl species, glyoxal and methylglyoxal, are the primary glycating agents and are elevated in several conditions associated with an increased risk of cardiovascular disease, including diabetes, rheumatoid arthritis, smoking, and aging. Thus, how protein glycation impacts the cardiomyocyte is of particular interest, to both understand how these conditions increase the risk of cardiovascular disease and how glycation might be targeted therapeutically. Glycation can affect the cardiomyocyte through extracellular mechanisms, including RAGE-based signaling, glycation of the extracellular matrix that modifies the mechanical environment, and signaling from the vasculature. Intracellular glycation of the cardiomyocyte can impact calcium handling, protein quality control and cell death pathways, as well as the cytoskeleton, resulting in a blunted contractility. While reducing protein glycation and its impact on the heart has been an active area of drug development, multiple clinical trials have had mixed results and these compounds have not been translated to the clinic-highlighting the challenges of modulating myocyte glycation. Here we will review protein glycation and its effects on the cardiomyocyte, therapeutic attempts to reverse these, and offer insight as to the future of glycation studies and patient treatment.

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance, often treated via electrical cardioversion. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs, suggesting that defects in contractility occur in AFib and are revealed upon restoration of rhythm. This project aims to define the contractile remodeling that occurs in AFib. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. Atrial cardiomyocytes showed reduced force of contraction, decreased resting tension, and increased calcium sensitivity in skinned single cardiomyocyte studies. These alterations correlated with degradation of myofilament proteins including myosin heavy chain altering force of contraction, titin altering resting tension, and TnI altering calcium sensitivity. We measured degradation of other myofilament proteins including cMyBP-C and actininshowing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. These results provide an understanding of the contractile remodeling that occurs in AFib and provide insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Aims: Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance. Treatment of AFib involves restoration of the atrial electrical rhythm. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs that involves decreased blood flow velocity and reduced atrial contractility. This suggests that defects in contractility occur in AFib and are revealed upon restoration of rhythm. The aim of this project is to define the contractile remodeling that occurs in AFib. Methods and Results: To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. Atrial cardiomyocytes showed reduced force of contraction in skinned single cardiomyocyte calcium-force studies. There were no significant differences in myosin heavy chain isoform expression. Resting tension is decreased in the AFib samples correlating with reduced full-length titin in the sarcomere. We measured degradation of other myofilament proteins including cMyBP-C, actinin, and cTnI, showing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. Skinned cardiomyocytes from AFib atria showed decreased troponin I phosphorylation, consistent with the increased calcium sensitivity that was found within these cardiomyocytes. Conclusions: With these results it can be concluded that AFib causes alterations in contraction that can be explained by both molecular changes occurring in myofilament proteins and overall myofilament protein degradation. These results provide an understanding of the contractile remodeling that occurs in AFib and provides insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.

The structural and functional integrities of porcine myocardium are mostly preserved by cryopreservation.

Structural and functional studies of heart muscle are important to gain insights into the physiological bases of cardiac muscle contraction and the pathological bases of heart disease. While fresh muscle tissue works best for these kinds of studies, this is not always practical to obtain, especially for heart tissue from large animal models and humans. Conversely, tissue banks of frozen human hearts are available and could be a tremendous resource for translational research. It is not well understood, however, how liquid nitrogen freezing and cryostorage may impact the structural integrity of myocardium from large mammals. In this study, we directly compared the structural and functional integrity of never-frozen to previously frozen porcine myocardium to investigate the consequences of freezing and cryostorage. X-ray diffraction measurements from hydrated tissue under near-physiological conditions and electron microscope images from chemically fixed porcine myocardium showed that prior freezing has only minor effects on structural integrity of the muscle. Furthermore, mechanical studies similarly showed no significant differences in contractile capabilities of porcine myocardium with and without freezing and cryostorage. These results demonstrate that liquid nitrogen preservation is a practical approach for structural and functional studies of myocardium.

Pharmacological inhibition of BAG3-HSP70 with the proposed cancer therapeutic JG-98 is toxic for cardiomyocytes.

The co-chaperone Bcl2-associated athanogene-3 (BAG3) maintains cellular protein quality control through the regulation of heat shock protein 70 (HSP70). Cancer cells manipulate BAG3-HSP70-regulated pathways for tumor initiation and proliferation, which has led to the development of promising small molecule therapies, such as JG-98, which inhibit the BAG3-HSP70 interaction and mitigate tumor growth. However, it is not known how these broad therapies impact cardiomyocytes, where the BAG3-HSP70 complex is a key regulator of protein turnover and contractility. Here, we show that JG-98 exposure is toxic in neonatal rat ventricular myocytes (NRVMs). Using immunofluorescence microscopy to assess cell death, we found that apoptosis increased in NRVMs treated with JG-98 doses as low as 10 nM. JG-98 treatment also reduced autophagy flux and altered expression of BAG3 and several binding partners involved in BAG3-dependent autophagy, including SYNPO2 and HSPB8. We next assessed protein half-life with disruption of the BAG3-HSP70 complex by treating with JG-98 in the presence of cycloheximide and found BAG3, HSPB5, and HSPB8 half-lives were reduced, indicating that complex formation with HSP70 is important for their stability. Next, we assessed sarcomere structure using super-resolution microscopy and found that disrupting the interaction with HSP70 leads to sarcomere structural disintegration. To determine whether the effects of JG-98 could be mitigated by pharmacological autophagy induction, we cotreated NRVMs with rapamycin, which partially reduced the extent of apoptosis and sarcomere disarray. Finally, we investigated whether the effects of JG-98 extended to skeletal myocytes using C2C12 myotubes and found again increased apoptosis and reduced autophagic flux. Together, our data suggest that nonspecific targeting of the BAG3-HSP70 complex to treat cancer may be detrimental for cardiac and skeletal myocytes.