Contractility and cell motility depend on accurately controlled assembly of the actin cytoskeleton. Formins are a large group of actin assembly proteins that nucleate and elongate new actin filaments. Some formins may cap filaments while others sever or bundle filaments. The formin homology domain-containing protein (FHOD) family of formins is critical to the formation of the fundamental contractile unit in muscle, the sarcomere. Specifically, mammalian FHOD3L plays an essential role in cardiomyocytes. Despite our knowledge of FHOD3L’s importance in cardiomyocytes, its biochemical and cellular activities remain poorly understood. It was proposed that FHOD-family formins act by capping and bundling, as opposed to assembling new filaments. Here, we demonstrate that human FHOD3L nucleates actin and rapidly but briefly elongates filaments after temporarily pausing elongation. We designed function-separating mutants that enabled us to distinguish which biochemical roles are required in the cell. We found that FHOD3L’s elongation activity, but not its nucleation, capping, or bundling activity, is necessary for proper sarcomere formation and contractile function in neonatal rat ventricular myocytes. The results of this work provide new insight into the mechanisms by which formins build specific structures and will contribute to knowledge regarding how cardiomyopathies arise from defects in sarcomere formation and maintenance.

Protein post-translational modifications (PTMs) can regulate biological processes by altering an amino acid's bulkiness, charge, and hydrogen bonding interactions. Common modifi- cations include phosphorylation, methylation, acetylation, and ubiquitylation. Although a primary focus of studying PTMs is understanding the effects of a single amino acid modification, the possibility of additional modifications increases the complexity. For example, substrate recognition motifs for arginine methyltransferases and some serine/threonine kinases overlap, leading to potential enzymatic crosstalk. In this study we have shown that the human family of formin homology domain-containing proteins (Fhods) contain a substrate recognition motif specific for human protein arginine methyltransferase 7 (PRMT7). In particular, PRMT7 methylates two arginine residues in the diaphanous autoinhibitory domain (DAD) of the family of Fhod proteins: R1588 and/or R1590 of Fhod3 isoform 4. Additionally, we confirmed that S1589 and S1595 in the DAD domain of Fhod3 can be phosphorylated by Rho/ROCK1 kinase. Significantly, we have determined that if S1589 is phosphorylated then PRMT7 cannot subsequently methylate R1588 or R1590. In contrast, if R1588 or R1590 of Fhod3 is methylated then ROCK1 phosphorylation activity is only slightly affected. Finally, we show that the interaction of the N-terminal DID domain can also inhibit the methylation of the DAD domain. Taken together these results suggest that the family of Fhod proteins, potential in vivo substrates for PRMT7, might be regulated by a combination of methylation and phosphorylation.

Background/Objectives: Carvedilol is an adrenergic blocker FDA-approved to improve outcomes in heart failure with reduced ejection fraction. Clinical trials examining whether carvedilol may be cardioprotective in the setting of cancer therapy-induced heart failure have generated mixed results that may depend on the cancer regimen, tumor, or comorbidities. Methods: To investigate the therapeutic potential of carvedilol to mitigate doxorubicin cardiotoxicity in cardiomyocytes, myocardial tissue, and in vivo, independent of confounding factors in clinical studies, we utilized disease-free cardiac slices and cardiomyocytes from mice, dogs, and human in vitro, and in wildtype mice injected with doxorubicin in vivo. We further evaluated the impact of carvedilol in dogs with cancer receiving doxorubicin. Results: In primary canine and murine cardiac slices, carvedilol treatment restored autophagy and prevented apoptosis from doxorubicin. Carvedilol restored mitochondrial energetics in human, canine, and murine models. In wildtype mice challenged with doxorubicin, carvedilol prevented declines in cardiac function and alterations in cardiac structure. In pet dogs with cancer and undergoing doxorubicin treatment, carvedilol was beneficial in preserving cardiac function and structure. Conclusions: Carvedilol activates cardioprotective autophagy, arrests doxorubicin-induced cell death, and improves energetics and cardiac structure and function across species.