Impaired isotonic contractility and structural abnormalities in the diaphragm of congestive heart failure rats

doi:10.1016/j.ijcard.2007.06.080

International Journal of Cardiology

Volume 128, Issue 3, 29 August 2008, Pages 326-335

https://doi.org/10.1016/j.ijcard.2007.06.080 Get rights and content

Abstract

Background

Metabolic alterations and decreased isometric force generation have been demonstrated in different animal models for congestive heart failure (CHF). However, as few morphological examinations have been performed on the CHF diaphragm, it is unknown if structural abnormalities comprise a substrate for diaphragm dysfunction in CHF. Therefore, we investigated CHF diaphragm isometric and isotonic contractility together with the presence of structural abnormalities.

Methods

Isometric twitch (P_t) and maximal (P_o) force, shortening velocity and power generation were determined in diaphragm bundles from rats with CHF, induced by myocardial infarction, and sham-operated rats. Immunofluorescence staining of myosin and sarcolemmal components fibronectin, laminin and dystrophin was performed on diaphragm cryosections. Electron microscopy was used to study the ultrastructure of diaphragm fibres.

Results

P_t and P_o were respectively ∼ 30% and ∼ 20% lower in CHF diaphragm bundles than sham. Maximal shortening velocity was reduced by ∼ 20% and maximal power generation by ∼ 35%. Structural abnormalities were frequently observed in CHF diaphragm fibres and were mainly marked by focal degradation of sarcomeric constituents and expansion of intermyofibrillar spaces with swollen and degenerated mitochondria. Immunofluorescence microscopy showed reduced staining intensities of myosin in CHF diaphragm fibres compared to sham. No differences were found regarding the distribution of fibronectin, laminin and dystrophin, indicating an intact sarcolemma in both groups.

Conclusion

This study demonstrates impaired isometric and isotonic contractility together with structural abnormalities in the CHF diaphragm. The sarcolemma of CHF diaphragm fibres appeared to be intact, excluding a role for sarcolemmal injuries in the development of CHF diaphragm dysfunction.

Introduction

Inspiratory muscle dysfunction is known to occur in patients with congestive heart failure (CHF). Maximal inspiratory pressure is reduced in patients with mild to moderate CHF [1] and has been identified as an independent predictor of mortality [2]. As the diaphragm is the most important inspiratory muscle, several studies investigated functional and metabolic alterations in the CHF diaphragm. In diaphragm biopsies from CHF patients a fibre type redistribution towards slow oxidative fibres was observed [3]. Studies of different CHF animal models confirmed this fibre type shift and described decreased maximal isometric force generation of diaphragm bundles in vitro [4], [5]. In vivo however, the diaphragm does not perform maximum isometric contractions, but shortens against a sub-maximal load. Measuring shortening velocity and power generation of the diaphragm is therefore physiologically more relevant.

CHF is associated with an increased workload on the diaphragm [6], probably as a consequence of reduced compliance of the respiratory system, increased airflow resistance and decreased diffusing capacity [7]. Increased workload on a muscle could result in sarcolemmal damage and sarcomeric disruptions, as observed in the diaphragm after inspiratory loading [8], [9]. Sarcolemmal damage after increased muscle loading has been established by intracellular localization of the extracellular matrix element fibronectin [10] and loss of the sarcolemma-associated protein dystrophin [11]. Sarcomeric disruptions, assessed by electron microscopy, are observed in the diaphragm of patients with Chronic Obstructive Pulmonary Disease (COPD) and increase after inspiratory loading [9]. In CHF, few histologic abnormalities of diaphragm myofibres have been described, and these include decreased cross-sectional areas, increased amount of necrotic fibres and centralisation of nuclei [5], [12]. However, it is unknown if sarcolemmal damage and sarcomeric disruptions are present in the CHF diaphragm.

In the present study we investigated the isometric and isotonic contractile properties of the diaphragm in CHF. We hypothesized that isometric force generation, maximal shortening velocity and power generation are decreased in the diaphragm from rats with CHF, induced by myocardial infarction. In addition, we hypothesized that structural injuries of the sarcolemma and sarcomeres are present in the diaphragm fibres of CHF rats. To test the latter hypothesis, we investigated the ultrastructure of CHF and sham diaphragm fibres with electron microscopy and the immunofluorescence distribution of the contractile protein myosin and the sarcolemmal components fibronectin, dystrophin and laminin.

Section snippets

Experimental model

Male outbred Wistar rats (260 to 300 g) were used in this study. Rats were fed with rat chow (RMH-B 1010, Hope Farms B.V., Woerden, The Netherlands) and water ad libitum. Animals were housed (two per cage) under 12:12 hour light–dark cycle. All procedures were approved by the Animal Ethics Committee, Radboud University Nijmegen, The Netherlands and experiments were performed in an ISO 9001 certified animal facility (Central Animal Laboratory, Radboud University Nijmegen, The Netherlands).

Rats

CHF indexes

All rats with infarct sizes > 35% of the left ventricle (n = 17) showed clinical signs of severe heart failure, including ascites, pleural effusion, pulmonary congestion and cardiomegaly. The latter two are supported by increased wet to dry lung weight ratio and heart weight, respectively (Table 1). Cardiac dysfunction was demonstrated by elevated left ventricular end diastolic pressure, decreased left ventricular peak systolic pressure and decreased aortic pressure (Table 1). Diaphragm weight wet

Discussion

The main findings of the present study show that 1) isometric force generation and shortening velocity of diaphragm bundles from CHF rats are decreased, 2) structural abnormalities within the CHF diaphragm fibres are indicated by distinctly decreased myosin immunofluorescence staining and ultrastructural observations of focal degeneration in various stages, and 3) the sarcolemma of these fibres appears not to be injured.

Conflict of interest

All authors declare that they have no potential conflicts of interest.

Acknowledgements

The authors wish to thank Paul H.K. Jap, PhD (Dept. of Anatomy, Radboud University Nijmegen Medical Centre, The Netherlands) for his expertise in the field of light and electron microscopy and Huib Croes (Dept. of Cell Biology, Radboud University Nijmegen Medical Centre, The Netherlands) for kindly providing myosin heavy chain antibodies.

This study is supported by an unrestricted educational grant from Novartis, the Netherlands.

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The transmural infarct area was determined by planimetry (Finsen et al., 2005; Ferreira et al., 2006). Based on previous studies in rodents (Finsen et al., 2005; van Hees et al., 2007, 2008b; Laitano et al., 2016) and the more pronounced diaphragm weakness in patients with severe heart failure with reduced ejection fraction (HFrEF) (Filusch et al., 2011), we studied only MI rats with infarct area > 35% of LV + septum. Thus, MI animals in this study constitute a group with cardiac morphological and functional signs of severe heart failure that show diaphragm contractile dysfunction and atrophy (Kelley et al., 2020).
The diaphragm is the main inspiratory muscle, and the chronic phase post-myocardial infarction (MI) is characterized by diaphragm morphological, contractile, and metabolic abnormalities. However, the mechanisms of diaphragm weakness are not fully understood. In the current study, we aimed to identify the transcriptome changes associated with diaphragm abnormalities in the chronic stage MI. We ligated the left coronary artery to cause MI in rats and performed RNA-sequencing (RNA-Seq) in diaphragm samples 16 weeks post-surgery. The sham group underwent thoracotomy and pericardiotomy but no artery ligation. We identified 112 differentially expressed genes (DEGs) out of a total of 9664 genes. Myocardial infarction upregulated and downregulated 42 and 70 genes, respectively. Analysis of DEGs in the framework of skeletal muscle-specific biological networks suggest remodeling in the neuromuscular junction, extracellular matrix, sarcomere, cytoskeleton, and changes in metabolism and iron homeostasis. Overall, the data are consistent with pathological remodeling of the diaphragm and reveal potential biological targets to prevent diaphragm weakness in the chronic stage MI.
Contractility of myofibrils from the heart and diaphragm muscles measured with atomic force cantilevers: Effects of heart-specific deletion of arginyl-tRNA-protein transferase
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The detailed contractile characteristics of α-MHCAte1 cardiac muscles have not been thoroughly investigated to date. CHF is frequently associated with changes in skeletal muscle functioning, including peripheral and inspiratory muscle weakness [3,4,11,12,19–21,23,25] and an impaired passive elasticity of muscle fibers [3,23]. Human CHF is frequently associated with skeletal muscle involvement, including peripheral and inspiratory muscles [19,25].
Contractile properties of myofibrils from the myocardium and diaphragm in chronic heart failure are not well understood. We investigated myofibrils in a knockout (KO) mouse model with cardiac-specific deletion of arginyl-tRNA–protein transferase (α-MHCAte1), which presents dilated cardiomyopathy and heart failure.
The aim of this study was to test the hypothesis that chronic heart failure in α-MHCAte1 mice is associated with abnormal contractile properties of the heart and diaphragm.
We used a newly developed system of atomic force cantilevers (AFC) to compare myofibrils from α-MHCAte1 and age-matched wild type mice (WT). Myofibrils from the myocardium and the diaphragm were attached to the AFC used for force measurements during activation/deactivation cycles at different sarcomere lengths.
In the heart, α-MHCAte1 myofibrils presented a reduced force during full activation (89 ± 9 nN/μm²) when compared to WT (132 ± 11 nN/μm²), and the decrease was not influenced by sarcomere length. These myofibrils presented similar kinetics of force development (K_act), redevelopment (K_tr), and relaxation (K_rel). In the diaphragm, α-MHCAte1 myofibrils presented an increased force during full activation (209 ± 31 nN/μm²) when compared to WT (123 ± 20 nN/μm²). Diaphragm myofibrils of α-MHCAte1 and WT presented similar K_act, but α-MHCAte1 myofibrils presented a faster K_rel (6.11 ± 0.41 s^− 1 vs 4.63 ± 0.41 s^− 1).
Contrary to our working hypothesis, diaphragm myofibrils from α-MHCAte1 mice produced an increased force compared to myofibrils from WT. These results suggest a potential compensatory mechanism by which the diaphragm works under loading conditions in the α-MHCAte1 chronic heart failure model.
Alterations in Diaphragmatic and Skeletal Muscle in Heart Failure
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Alterations in Diaphragmatic and Skeletal Muscle in Heart Failure
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Heart failure decreases passive tension generation of rat diaphragm fibers
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In an animal model of chronic HF we recently demonstrated that diaphragm fibers display reduced force generating capacity independent of fiber type [8], which implies intrinsic fiber abnormalities. Indeed, electron microscopic investigations on the ultra-structure of HF diaphragm fibers revealed sarcomeric disruptions [9]. For structural stability and optimal active force generation, passive-elastic structures are indispensable [10].
Diaphragm dysfunction is well-known to limit quality of life and prognosis of patients with heart failure (HF), but its underlying mechanisms are not well understood. In an animal model for HF we recently showed that impaired diaphragm contractility arises at the single fiber level and is associated with sarcomeric injuries. For optimal muscle function and sarcomeric stability passive elastic structures, like titin, are indispensable. The current study aimed to investigate if impaired passive elasticity contributes to diaphragm dysfunction in rats with heart failure.
Skinned muscle fibers were isolated from the diaphragm and soleus of rats with chronic HF, induced by left coronary artery ligation and of sham-operated rats. Passive tension–length relationships were determined by applying segmental extension tests. Immunofluorescence was performed on muscle cryosections using antibodies (T12) against a titin epitope near the Z-line. Titin content was determined by SDS-agarose-gel electrophoresis. Titin's mobility on gel was studied to detect changes in titin size.
Passive tension generation upon stretch was significantly reduced (> 35%) in HF diaphragm fibers compared to sham. Immunostaining intensities against titin were reduced in diaphragm cryosections of HF rats compared to sham. Soleus fibers from HF and sham rats did not display differences, neither in passive tension nor in immunostaining. No differences in titin's size were detected in HF and sham diaphragm. Titin content, however, was significantly reduced (∼ 25%) in HF diaphragm.
We conclude that in the diaphragm of HF rats, passive elasticity is impaired, mainly resulting from titin loss.
Locomotor and respiratory muscle abnormalities in HFrEF and HFpEF
2023, Frontiers in Cardiovascular Medicine

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Impaired isotonic contractility and structural abnormalities in the diaphragm of congestive heart failure rats

Abstract

Background

Methods

Results

Conclusion

Introduction

Section snippets

Experimental model

CHF indexes

Discussion

Conflict of interest

Acknowledgements

J Am Coll Cardiol

Chest

J Mol Cell Cardiol

Diaphragm strength in chronic heart failure

Am J Respir Crit Care Med

Respiratory muscle dysfunction in congestive heart failure: clinical correlation and prognostic significance

Circulation

Chronic congestive heart failure elicits adaptations of endurance exercise in diaphragmatic muscle

Circulation

Congestive heart failure: differential adaptation of the diaphragm and latissimus dorsi

J Appl Physiol

Effects of dilated cardiomyopathy on the diaphragm in the Syrian hamster

Eur Respir J

Diaphragm muscle fiber injury after inspiratory resistive breathing

Am J Respir Crit Care Med

Injury of the human diaphragm associated with exertion and chronic obstructive pulmonary disease

Am J Respir Crit Care Med

Contractile and cellular remodeling in rabbit skeletal muscle after cyclic eccentric contractions

J Appl Physiol

Contractile function, sarcolemma integrity, and the loss of dystrophin after skeletal muscle eccentric contraction-induced injury

Am J Physiol Cell Physiol

Increased compliance in diaphragm muscle of the cardiomyopathic Syrian hamster

J Appl Physiol

Angiotensin-(1–7) attenuates the development of heart failure after myocardial infarction in rats

Circulation

Impaired modulation of sympathetic vasoconstriction in contracting skeletal muscle of rats with chronic myocardial infarctions: role of oxidative stress

Circ Res

Myocardial infarct size and ventricular function in rats

Circ Res

Free radicals in hypoxic rat diaphragm contractility: no role for xanthine oxidase

Am J Physiol Lung Cell Mol Physiol

The heat of shortening and the dynamic constants of muscle

Proc R Soc Lond