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Background Mutations to the co‐chaperone protein BAG3 (B‐cell lymphoma‐2–associated athanogene‐3) are a leading cause of dilated cardiomyopathy (DCM). These mutations often impact the C‐terminal BAG domain (residues 420–499), which regulates heat shock protein 70‐dependent protein turnover via autophagy. While mutations in other regions are less common, previous studies in patients with DCM found that co‐occurrence of 2 BAG3 variants (P63A, P380S) led to worse prognosis. However, the underlying mechanism for dysfunction is not fully understood. Methods and Results In this study, we used proteomics, Western blots, and myofilament functional assays on left ventricular tissue from patients with nonfailing, DCM, and DCM with BAG363/380 to determine how these mutations impact protein quality control and cardiomyocyte contractile function. We found dysregulated autophagy and increased protein ubiquitination in patients with BAG363/380 compared with nonfailing and DCM, suggesting impaired protein turnover. Expression and myofilament localization of BAG3‐binding proteins were also uniquely altered in the BAG3,63/380 including abolished localization of the small heat shock protein CRYAB (alpha‐crystallin B chain) to the sarcomere. To determine whether these variants impacted sarcomere function, we used cardiomyocyte force‐calcium assays and found reduced maximal calcium‐activated force in DCM and BAG363/380. Interestingly, myofilament calcium sensitivity was increased in DCM but not with BAG363/380, which was not explained by differences in troponin I phosphorylation. Conclusions Together, our data support that the disease‐enhancing mechanism for BAG3 variants outside of the BAG domain is through disrupted protein turnover leading to compromised sarcomere function. These findings suggest a shared mechanism of disease among pathogenic BAG3 variants, regardless of location.

Summary: General control of amino acid synthesis 5-like 1 (GCN5L1) was previously identified as a key regulator of protein lysine acetylation in mitochondria. Subsequent studies demonstrated that GCN5L1 regulates the acetylation status and activity of mitochondrial fuel substrate metabolism enzymes. However, the role of GCN5L1 in response to chronic hemodynamic stress is largely unknown. Here, we show that cardiomyocyte-specific GCN5L1 knockout mice (cGCN5L1 KO) display exacerbated heart failure progression following transaortic constriction (TAC). Mitochondrial DNA and protein levels were decreased in cGCN5L1 KO hearts after TAC, and isolated neonatal cardiomyocytes with reduced GCN5L1 expression had lower bioenergetic output in response to hypertrophic stress. Loss of GCN5L1 expression led to a decrease in the acetylation status of mitochondrial transcription factor A (TFAM) after TAC in vivo, which was linked to a reduction in mtDNA levels in vitro. Together, these data suggest that GCN5L1 may protect from hemodynamic stress by maintaining mitochondrial bioenergetic output.

I.v. administration of a high-affinity carbon monoxide–binding (CO-binding) molecule, recombinant neuroglobin, can improve survival in CO poisoning mouse models. The current study aims to discover how biochemical variables of the scavenger determine the CO removal from the RBCs by evaluating 3 readily available hemoproteins, 2,3-diphosphoglycerate stripped human hemoglobin (StHb); N-ethylmaleimide modified hemoglobin (NEMHb); and equine myoglobin (Mb). These molecules efficiently sequester CO from hemoglobin in erythrocytes in vitro. A kinetic model was developed to predict the CO binding efficacy for hemoproteins, based on their measured in vitro oxygen and CO binding affinities, suggesting that the therapeutic efficacy of hemoproteins for CO poisoning relates to a high M value, which is the binding affinity for CO relative to oxygen (KA,CO/KA,O2). In a lethal CO poisoning mouse model, StHb, NEMHb, and Mb improved survival by 100%, 100%, and 60%, respectively, compared with saline controls and were well tolerated in 48-hour toxicology assessments. In conclusion, both StHb and NEMHb have high CO binding affinities and M values, and they scavenge CO efficiently in vitro and in vivo, highlighting their therapeutic potential for point-of-care antidotal therapy of CO poisoning.

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF.

Abstract The global burden of heart failure following myocardial ischemia‐reperfusion (IR) injury is a growing problem. One pathway that is key to understanding the progression of myocardial infarction and IR injury is the endoplasmic reticulum (ER) stress pathway, which contributes to apoptosis signaling and tissue death. The role of calreticulin in the progression of ER stress remains controversial. We hypothesized that calreticulin induction drives proapoptotic signaling in response to ER stress. We find here that calreticulin is upregulated in human ischemic heart failure cardiac tissue, as well as simulated hypoxia and reoxygenation (H/R) and thapsigargin‐mediated ER stress. To test the impact of direct modulation of calreticulin expression on ER stress‐induced apoptosis, human cardiac‐derived AC16 cells with stable overexpression or silencing of calreticulin were subjected to thapsigargin treatment, and markers of apoptosis were evaluated. It was found that overexpression of calreticulin promotes apoptosis, while a partial knockdown protects against the expression of caspase 12, CHOP, and reduces thapsigargin‐driven TUNEL staining. These data shed light on the role that calreticulin plays in apoptosis signaling during ER stress in cardiac cells.

: Aims We have previously shown that activation of leptin signalling in the heart reduces cardiac morbidity and mortality after myocardial infarction (MI). In the present study, we tested the hypothesis that leptin signalling limits cardiac apoptosis after MI through activation of signal transducer and activator of transcription (STAT)-3 responsive anti-apoptotic genes, including B-cell lymphoma (bcl)-2 and survivin, that serve to downregulate the activity of caspase-3. : Methods and results Hearts from C57BL/6J and three groups of leptin-deficient Ob/Ob mice (food-restricted, ad libitum, and leptin-repleted) were examined 4 weeks after permanent left coronary artery ligation or sham operation. Inflammatory and apoptotic cell number was determined in cardiac sections by immunostaining. Expression of cardiac bcl-2, survivin, and pro and active caspase-3 was determined and correlated with in vitro caspase-3 activity. In the absence of MI, both lean and obese leptin-deficient mice exhibited increased cardiac apoptosis compared with wild-type mice. After MI, the highest rates of apoptosis were seen in the infarcted tissue of lean and obese Ob/Ob mice. Further, leptin-deficient hearts, as well as hearts from wild-type mice treated with the STAT-3 inhibitor WP1066, exhibited blunted anti-apoptotic bcl-2 and survivin gene expression, and increased caspase-3 protein expression and activity. The increased caspase-3 activity and apoptosis in hearts of leptin-deficient mice after MI was significantly attenuated in Ob/Ob mice replete with leptin, reducing apoptosis to levels comparable to that observed in wild-type mice after MI. : Conclusion These results demonstrate that intact leptin signalling post-MI acts through STAT-3 to increase anti-apoptotic bcl-2 and survivin gene expression and reduces caspase-3 activity, consistent with a cardioprotective role of leptin in the setting of chronic ischaemic injury. [ABSTRACT FROM AUTHOR]

RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p