Protein Kinase D Plays a Crucial Role in Maintaining Cardiac Homeostasis by Regulating Post-Translational Modifications of Myofilament Proteins.

Protein kinase D (PKD) enzymes play important roles in regulating myocardial contraction, hypertrophy, and remodeling. One of the proteins phosphorylated by PKD is titin, which is involved in myofilament function. In this study, we aimed to investigate the role of PKD in cardiomyocyte function under conditions of oxidative stress. To do this, we used mice with a cardiomyocyte-specific knock-out of Prkd1, which encodes PKD1 (Prkd1loxP/loxP; αMHC-Cre; PKD1 cKO), as well as wild type littermate controls (Prkd1loxP/loxP; WT). We isolated permeabilized cardiomyocytes from PKD1 cKO mice and found that they exhibited increased passive stiffness (Fpassive), which was associated with increased oxidation of titin, but showed no change in titin ubiquitination. Additionally, the PKD1 cKO mice showed increased myofilament calcium (Ca2+) sensitivity (pCa50) and reduced maximum Ca2+-activated tension. These changes were accompanied by increased oxidation and reduced phosphorylation of the small myofilament protein cardiac myosin binding protein C (cMyBPC), as well as altered phosphorylation levels at different phosphosites in troponin I (TnI). The increased Fpassive and pCa50, and the reduced maximum Ca2+-activated tension were reversed when we treated the isolated permeabilized cardiomyocytes with reduced glutathione (GSH). This indicated that myofilament protein oxidation contributes to cardiomyocyte dysfunction. Furthermore, the PKD1 cKO mice exhibited increased oxidative stress and increased expression of pro-inflammatory markers interleukin (IL)-6, IL-18, and tumor necrosis factor alpha (TNF-α). Both oxidative stress and inflammation contributed to an increase in microtubule-associated protein 1 light chain 3 (LC3)-II levels and heat shock response by inhibiting the mammalian target of rapamycin (mTOR) in the PKD1 cKO mouse myocytes. These findings revealed a previously unknown role for PKD1 in regulating diastolic passive properties, myofilament Ca2+ sensitivity, and maximum Ca2+-activated tension under conditions of oxidative stress. Finally, we emphasized the importance of PKD1 in maintaining the balance of oxidative stress and inflammation in the context of autophagy, as well as cardiomyocyte function.

Increased Expression of Proinflammatory Genes in Peripheral Blood Cells Is Associated with Cardiac Cachexia in Patients with Heart Failure with Reduced Ejection Fraction.

Background: Cardiac cachexia (CC) in chronic heart failure with reduced ejection fraction (HFrEF) is characterized by catabolism and inflammation predicting poor prognosis. Levels of responsible transcription factors like signal transducer and activator of transcription (STAT)1, STAT3, suppressor of cytokine signaling (SOCS)1 and SOCS3 in peripheral blood cells (PBC) are underinvestigated in CC. Expression of mediators was related to patients' functional status, body composition (BC) and metabolic gene expression in skeletal muscle (SM). Methods: Gene expression was quantified by qRT-PCR in three cohorts: non-cachectic patients (ncCHF, n = 19, LVEF 31 ± 7%, BMI 30.2 ± 5.0 kg/m2), cachectic patients (cCHF; n = 18, LVEF 27 ± 7%, BMI 24.3 ± 2.5 kg/m2) and controls (n = 17, LVEF 70 ± 7%, BMI 27.6 ± 4.6 kg/m2). BC was assessed by dual-energy X-ray absorptiometry. Blood inflammatory markers were measured. We quantified solute carrier family 2 member 4 (SLC2A4) and protein degradation by expressions of proteasome 20S subunit beta 2 and calpain-1 catalytic subunit in SM biopsies. Results: TNF and IL-10 expression was higher in cCHF than in ncCHF and controls (all p < 0.004). cCHF had a lower fat mass index (FMI) and lower fat-free mass index (FFMI) compared to ncCHF and controls (p < 0.05). STAT1 and STAT3 expression was higher in cCHF vs. ncCHF or controls (1.1 [1.6] vs. 0.8 [0.9] vs. 0.9 [1.1] RU and 4.6 [5.5] vs. 2.5 [4.8] vs. 3.0 [4.2] RU, all ANOVA-p < 0.05). The same applied for SOCS1 and SOCS3 expression (1.1 [1.5] vs. 0.4 [0.4] vs. 0.4 [0.5] and 0.9 [3.3] vs. 0.4 [1.1] vs. 0.8 [0.9] RU, all ANOVA-p < 0.04). In cCHF, higher TNF and STAT1 expression was associated with lower FMI (r = 0.5, p = 0.053 and p < 0.05) but not with lower FFMI (p > 0.4). In ncCHF, neither cytokine nor STAT/SOCS expression was associated with BC (all p > 0.3). SLC2A4 was upregulated in SM of cCHF vs. ncCHF (p < 0.03). Conclusions: Increased STAT1, STAT3, SOCS1 and SOCS3 expression suggests their involvement in CC. In cCHF, higher TNF and STAT-1 expression in PBC were associated with lower FMI. Increased SLC2A4 in cachectic SM biopsies indicates altered glucose metabolism.

Metabolic remodeling in cardiac hypertrophy and heart failure with reduced ejection fraction occurs independent of transcription factor EB in mice.

Background: A metabolic shift from fatty acid (FAO) to glucose oxidation (GO) occurs during cardiac hypertrophy (LVH) and heart failure with reduced ejection fraction (HFrEF), which is mediated by PGC-1α and PPARα. While the transcription factor EB (TFEB) regulates the expression of both PPARGC1A/PGC-1α and PPARA/PPARα, its contribution to metabolic remodeling is uncertain. Methods: Luciferase assays were performed to verify that TFEB regulates PPARGC1A expression. Cardiomyocyte-specific Tfeb knockout (cKO) and wildtype (WT) male mice were subjected to 27G transverse aortic constriction or sham surgery for 21 and 56 days, respectively, to induce LVH and HFrEF. Echocardiographic, morphological, and histological analyses were performed. Changes in markers of cardiac stress and remodeling, metabolic shift and oxidative phosphorylation were investigated by Western blot analyses, mass spectrometry, qRT-PCR, and citrate synthase and complex II activity measurements. Results: Luciferase assays revealed that TFEB increases PPARGC1A/PGC-1α expression, which was inhibited by class IIa histone deacetylases and derepressed by protein kinase D. At baseline, cKO mice exhibited a reduced cardiac function, elevated stress markers and a decrease in FAO and GO gene expression compared to WT mice. LVH resulted in increased cardiac remodeling and a decreased expression of FAO and GO genes, but a comparable decline in cardiac function in cKO compared to WT mice. In HFrEF, cKO mice showed an improved cardiac function, lower heart weights, smaller myocytes and a reduction in cardiac remodeling compared to WT mice. Proteomic analysis revealed a comparable decrease in FAO- and increase in GO-related proteins in both genotypes. A significant reduction in mitochondrial quality control genes and a decreased citrate synthase and complex II activities was observed in hearts of WT but not cKO HFrEF mice. Conclusions: TFEB affects the baseline expression of metabolic and mitochondrial quality control genes in the heart, but has only minor effects on the metabolic shift in LVH and HFrEF in mice. Deletion of TFEB plays a protective role in HFrEF but does not affect the course of LVH. Further studies are needed to elucidate if TFEB affects the metabolic flux in stressed cardiomyocytes.