Sex-related differences in cardiac and myofilament function in rats with pressure-overload induced left ventricular hypertrophy

Introduction Recent findings indicate that sex is a major determinant of left ventricular (LV) structure in pressure overload (PO)-induced LV myocardial hypertrophy (LVH). However, data are scare regarding sex-related differences in LV function in case of PO-evoked LVH. Aim Hence, in the present study we aimed at comprehensively investigating sex-related functional differences on the global cardiac level and also on the myofilament level in PO-induced LVH. Method Abdominal aortic banding (AB) was carried out to induce chronic PO for 6 or 12 weeks in male and female rats. Age- and sex-matched sham-operated animals served as controls. The development of LVH was followed by serial echocardiography. The extent of cardiomyocyte hypertrophy and myocardial fibrosis were evaluated by histology. Cardiac function was assessed by pressure-volume analysis. Force measurement was carried out in permeabilized cardiomyocytes to compute myofilament function. Results At week 6, robust myocardial hypertrophy, concentric LV geometry and moderate interstitial fibrosis were detected in both male and female AB rats. This early stage of PO-induced LVH was associated with increased LV contractility (slope of end-systolic pressure-volume relationship [ESPVR, mmHg/μl]: 3.09±0.18 Male-AB-wk6 vs. 1.79±0.22 Male-Sham-wk6 P<0.05; 3.68±0.77 Female-AB-wk6 vs. 1.87±0.21 Female-Sham-wk6 P<0.05) and enhanced myofilament Ca2+ sensitivity in both sexes (pCa50: 5.86±0.01 Male-AB-wk6 vs. 5.73±0.02 Male-Sham-wk6 P<0.05; 5.94±0.03 Female-AB-wk6 vs. 5.73±0.01 Female-Sham-wk6 P<0.05). At week 6, the augmented LV contractility effectively counterbalanced the increased afterload in both male and female AB groups. Hence, ventricular-arterial coupling (VAC) was maintained and LV systolic function was preserved in the AB groups in both sexes. In contrast, at week 12, marked sex differences could be observed. At this later stage, LVH was characterized by eccentric remodeling and intensified collagen accumulation in male AB rats. The initial LV contractility augmentation (slope of ESPVR, mmHg/μl: 1.74±0.13 Male-AB-wk12 vs. 1.31±0.17 Male-Sham-wk12 n.s.) as well as the enhanced myofilament Ca2+ sensitivity (pCa50: 5.78±0.02 Male-AB-wk12 vs. 5.75±0.01 Male-Sham-wk12 n.s.) diminished, leading to impaired VAC and reduced LV systolic function. On the contrary, in female AB rats, cardiac contractility (ESPVR, mmHg/ μl: 3.97±0.50 Female-AB-wk12 vs. 2.08±0.17 Female-Sham-wk12 P<0.05) and myofilament Ca2+ sensitivity (pCa50:5.85±0.02 Female-AB-wk12 vs. 5.78±0.01 Female-Sham-wk12 P<0.05) remained increased, resulting in adequate VAC and preserved LV systolic function at late-stage of PO-induced LVH as well. Conclusion The initially augmented LV contractility and enhanced myofilament Ca2+ sensitivity declines in male but not in female AB rats at later time points. Hence, characteristically different alterations occur in LV systolic function between the two sexes in late-stage of PO-evoked LVH. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): NVKP_16-1-2016-0017.

Abstract MP101: Functional Maintenance of the Sarcomere Requires Bag3-dependent Autophagy

Bcl2-associated athanogene-3 (BAG3) is a pro-autophagy co-chaperone highly expressed in the heart. Clinical studies show BAG3 haploinsufficiency and mutations are associated with heart failure (HF). However, these studies are largely observational and fail to go beyond the observed phenotype and study mechanism. One study in neonatal myocytes suggests a role for BAG3 in structural maintenance of the sarcomere. However, the structural and functional significance of BAG3 in adult myocytes is not known. We found that myofilament BAG3 expression decreases in human heart failure and is associated with impaired myofilament force-generating capacity (F max ). To assess whether rescuing BAG3 levels could restore function, we used a mouse model of HF and treated with BAG3 gene therapy via AAV9. Myofilament function was assessed in skinned cardiomyocytes by force-calcium relationship. HF mice experienced a reduction in F max , but this was fully restored to sham levels by BAG3 gene therapy.To explore mechanism, we used mass spectrometry to identify the BAG3-interactome at the myofilament and found heat shock proteins (HSP) 70 and B8 among the top hits. Immunofluorescence further showed that HSP70, B8, and BAG3 each localized to the sarcomere z-disk. This BAG3-Hsp complex had previously been described to promote ubiquitin-dependent autophagy in skeletal muscle. Notably, in both human HF samples and in the mouse HF model, myofilament ubiquitin levels increased significantly. However, BAG3 gene therapy in HF reduces ubiquitin levels and restores autophagy flux. This suggests that BAG3 serves a role in proteostasis for the sarcomere, which may explain the functional effect of BAG3 gene therapy.To further explore the impact of the BAG3/HSP complex on myofilament function, we used a mouse model with the P209L BAG3 mutation, which had previously been described to disrupt client processing by the complex. We found cardiomyocytes from P209L mice had significantly reduced F max and elevated myofilament ubiquitin levels, suggesting BAG3-dependent autophagy is required to maintain function. Together, our data identify a functional role for BAG3 at the sarcomere and indicate BAG3-mediated autophagy is an important mechanism for maintaining myofilament proteostasis.

Optimal cutting temperature medium embedding and cryostat sectioning are valid for cardiac myofilament function assessment

Myocardial tissue in optimal cutting temperature (OCT) fixation and cryostat sectioning was tested as a means of storing and preparing tissue for myofilament function analysis in relation to conventional liquid nitrogen freezing and dissection. Actomyosin interaction, Ca2+ force activation, and passive compliance were tested. The study concluded that OCT storage and cryostat sectioning do not interfere with the actomyosin cross-bridge dynamics or Ca2+ activation but that absolute tension values suffer and may not be investigated by this method.

Estrogen but not testosterone preserves myofilament function from doxorubicin-induced cardiotoxicity by reducing oxidative modifications

Here, we aimed to explore sex differences and the impact of sex hormones on cardiac contractile properties in doxorubicin (DOX)-induced cardiotoxicity. Male and female Sprague-Dawley rats were subjected to sham surgery or gonadectomy and then treated or untreated with DOX (2 mg/kg) every other week for 10 wk. Estrogen preserved maximum active tension (Tmax) with DOX exposure, whereas progesterone and testosterone did not. The effects of sex hormones and DOX correlated with both altered myosin heavy chain isoform expression and myofilament protein oxidation, suggesting both as possible mechanisms. However, acute treatment with oxidative stress (H2O2) or a reducing agent (DTT) indicated that the effects on Tmax were mediated by reversible myofilament oxidative modifications and not only changes in myosin heavy chain isoforms. There were also sex differences in the DOX impact on myofilament Ca2+ sensitivity. DOX increased Ca2+ sensitivity in male rats only in the absence of testosterone and in female rats only in the presence of estrogen. Conversely, DOX decreased Ca2+ sensitivity in female rats in the absence of estrogen. In most instances, this mechanism was through altered phosphorylation of troponin I at Ser23/Ser24. However, there was an additional DOX-induced, estrogen-dependent, irreversible (by DTT) mechanism that altered Ca2+ sensitivity. Our data demonstrate sex differences in cardiac contractile responses to chronic DOX treatment. We conclude that estrogen protects against chronic DOX treatment in the heart, preserving myofilament function. NEW & NOTEWORTHY We identified sex differences in cardiotoxic effects of chronic doxorubicin (DOX) exposure on myofilament function. Estrogen, but not testosterone, decreases DOX-induced oxidative modifications on myofilaments to preserve maximum active tension. In rats, DOX exposure increased Ca2+ sensitivity in the presence of estrogen but decreased Ca2+ sensitivity in the absence of estrogen. In male rats, the DOX-induced shift in Ca2+ sensitivity involved troponin I phosphorylation; in female rats, this was through an estrogen-dependent mechanism.

Pim-1 Kinase Phosphorylates Cardiac Troponin I and Regulates Cardiac Myofilament Function

Background/Aims: Pim-1 is a serine/threonine kinase that is highly expressed in the heart, and exerts potent cardiac protective effects through enhancing survival, proliferation, and regeneration of cardiomyocytes. Its myocardial specific substrates, however, remain unknown. In the present study, we aim to investigate whether Pim-1 modulates myofilament activity through phosphorylation of cardiac troponin I (cTnI), a key component in regulating myofilament function in the heart. Methods: Coimmunoprecipitation and immunofluorescent assays were employed to investigate the interaction of Pim-1 with cTnI in cardiomyocytes. Biochemical, site directed mutagenesis, and mass spectrometric analyses were utilized to identify the phosphorylation sites of Pim1 in cTnI. Myofilament functional assay using skinned cardiac fiber was used to assess the effect of Pim1-mediated phosphorylation on cardiac myofilament activity. Lastly, the functional significance of Pim1-mediated cTnI in heart disease was determined in diabetic mice. Results: We found that Pim-1 specifically interacts with cTnI in cardiomyocytes and this interaction leads to Pim1-mediated cTnI phosphorylation, predominantly at Ser23/24 and Ser150. Furthermore, our functional assay demonstrated that Pim-1 induces a robust phosphorylation of cTnI within the troponin complex, thus leading to a decreased Ca2+ sensitivity. Insulin-like growth factor 1 (IGF-1), a peptide growth factor that has been shown to stimulate myocardial contractility, markedly induces cTnI phosphorylation at Ser23/24 and Ser150 through increasing Pim-1 expression in cardiomyocytes. In a high-fat diabetic mice model, the expression of Pim1 in the heart is significantly decreased, which is accompanied by a decreased phosphorylation of cTnI at Ser23/24 and Ser150, further implicating the pathological significance of the Pim1/cTnI axis in the development of diabetic cardiomyopathy. Conclusion: Our results demonstrate that Pim-1 is a novel kinase that phosphorylates cTnI primarily at Ser23/24 and Ser150 in cardiomyocytes, which in turn may modulate myofilament function under a variety of physiological and pathophysiological conditions.