Titin hyper-phosphorylation in the peripheral skeletal muscle but not the diaphragm of HFpEF: association with muscle atrophy and dysfunction
Authors: Vahle B , Heilmann L , Schauer A , Augstein A , Winzer E , Mangner N , Linke A , Adams V 
Published: October 2024

Abstract

Background
Titin hypo-phosphorylation is an important regulatory mechanism for myocardial stiffness in heart failure with preserved ejection fraction (HFpEF). In contrast, a hyper-phosphorylation of titin in the peripheral skeletal muscle (SKM) was reported in mouse models exhibiting muscle atrophy. In former studies it, was shown that SKM atrophy occurs in HFpEF and is associated with muscle dysfunction and a fiber type shift from type I, towards type II fibers. However, these alterations were not seen in the diaphragm muscle (Dia) and a fiber type shift towards type I was documented. So far, no information about the phosphorylation status of titin is available for SKM and the Dia and its association with muscle dysfunction in HFpEF.

Purpose
To assess titin phosphorylation in SKM and Dia of a HFpEF animal model and to relate it to muscle function and atrophy.

Methods
To confirm the development of HFpEF echocardiography was performed in female ZSF1-lean (con, n=18) and ZSF1-obese rats (HFpEF, n=18) at the age of 32 weeks. Skeletal muscle function (soleus) was measured in an organ bath setting after the animals were sacrificed. Tissue of the tibialis anterior muscle (TA) and the diaphragm (Dia) was collected and fixed in formalin or snap frozen in liquid nitrogen. To detect titin expression homogenized TA and Dia tissue samples were resolved on a vertical agarose gel. Phosphorylated titin was detected by staining the gel with Pro-Q Diamond, whereas total titin was visualized using Sypro Ruby. Cross sectional area (CSA) of TA and Dia was quantified after Hematoxylin/eosin staining of tissue section. Myosin heavy chain (MHC) ubiquitination was assessed by western blot.

Results
Assessment of SKM function revealed significantly reduced maximal absolute (-12 %) and maximal specific force (-22 %) in HFpEF animals when compared to controls. In the TA a significantly reduced CSA was evident in HFpEF (1911±103 vs. 1394±103 µm2, p=0.002), whereas in the diaphragm an increased CSA was noted (438±11 vs. 560±18 µm2, p&lt;0.001). In TA of HFpEF animals, the proportion of phosphorylated titin was significantly elevated whereas the total amount of titin was reduced (Fig. 1A, C). In addition, a significantly higher proportion of ubiquitinated MHC was detected (Figure 1E). However in the Dia these alterations were not observed, even a higher amount of total titin was seen (Fig. 1B, D, F). Furthermore, a significant negative correlation between phosphorylated titin and absolute force (r=-0.461, p=0.035, Fig. 2A) as well as specific force (r=-0.693, p=0.0005, Fig. 2B) was evident.

Conclusion
In contrast to the heart, the TA showed a titin hyper-phosphorylation in HFpEF, going along with a degradation of total titin. This hyper-phosphorylation could be correlated with a loss of force in the SKM. Additionally a higher ubiquitination of MHC was prevalent in the TA, which may be connected to molecular remodeling and fiber type shift towards a more glycolytic type.