Am J Physiol Cell Physiol. 2014 Nov 15;307(10):C910-9. doi: 10.1152/ajpcell.00192.2013.

Diabetes increases stiffness of live cardiomyocytes measured by atomic force microscopy nanoindentation.

Benech JC1, Benech N2, Zambrana AI3, Rauschert I3, Bervejillo V3, Oddone N3, Damián JP4.
  • 1Laboratorio de Señalización Celular y Nanobiología, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay; jbenech@iibce.edu.uy juanclaudio.benech@gmail.com.
  • 2Instituto de Física, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay; and.
  • 3Laboratorio de Señalización Celular y Nanobiología, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay;
  • 4Laboratorio de Señalización Celular y Nanobiología, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay; Departamento de Biología Molecular y Celular, Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay.

 

Abstract

Stiffness of live cardiomyocytes isolated from control and diabetic mice was measured using the atomic force microscopy nanoindentation method. Type 1 diabetes was induced in mice by streptozotocin administration. Histological images of myocardium from mice that were diabetic for 3 mo showed disorderly lineup of myocardial cells, irregularly sized cell nuclei, and fragmented and disordered myocardial fibers with interstitial collagen accumulation. Phalloidin-stained cardiomyocytes isolated from diabetic mice showed altered (i.e., more irregular and diffuse) actin filament organization compared with cardiomyocytes from control mice. Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a) pump expression was reduced in homogenates obtained from the left ventricle of diabetic animals compared with age-matched controls. The apparent elastic modulus (AEM) for live control or diabetic isolated cardiomyocytes was measured using the atomic force microscopy nanoindentation method in Tyrode buffer solution containing 1.8 mM Ca(2+) and 5.4 mM KCl (physiological condition), 100 nM Ca(2+) and 5.4 mM KCl (low extracellular Ca(2+) condition), or 1.8 mM Ca(2+) and 140 mM KCl (contraction condition). In the physiological condition, the mean AEM was 112% higher for live diabetic than control isolated cardiomyocytes (91 ± 14 vs. 43 ± 7 kPa). The AEM was also significantly higher in diabetic than control cardiomyocytes in the low extracellular Ca(2+) and contraction conditions. These findings suggest that the material properties of live cardiomyocytes were affected by diabetes, resulting in stiffer cells, which very likely contribute to high diastolic LV stiffness, which has been observed in vivo in some diabetes mellitus patients.

KEYWORDS: atomic force microscopy; diabetes; live cardiomyocyte stiffness

PMID: 25163520

 

Supplemental material

Patients with diabetes develop a cardiomyopathy that is independent of coronary artery disease and hypertension and contributes to the increased morbidity of the disease and patients mortality. The mechanisms that lead to the development of the diabetic cardiomyopathy are poorly understood. Increased diastolic left ventricular (LV) stiffness is an early manifestation of myocardial dysfunction. Excessive diastolic LV stiffness of the diabetic heart is usually attributed to myocardial fibrosis or to myocardial deposition of advanced glycation end products (AGEs). Alteration of the stiffness (resting tension), of the diabetic cardiomyocyte was also proposed to be an important factor contributing to the LV stiffness. These data were obtained from single cardiomyocytes which were isolated from human frozen biopsy samples that were thawed, mechanically disrupted, and incubated with Triton X-100, provoking sarcolemma and sarcoplasmic membrane disruption.

Atomic force microscopy (AFM) allows the study of intact cells’ dynamics and mechanical properties. Different cell events, such as locomotion, differentiation and aging, physiological activation, electromotility, and cell pathology, can be analyzed with this research tool.

Recently, we studied the effect of type 1 diabetes on the nanomechanical properties of live cardiomyocytes by using this technique. For this purpose, type 1 diabetes was induced in mice by streptozotocin administration. Using the AFM nanoindentation method, we obtained the stiffness and work of the adhesive force of live isolated cardiomyocytes from control and diabetic mice. The experiment was performed placing the samples in Tyrode buffer with different ionic compositions. Before and after performing force curves measurements with the AFM, cardiomyocyte viability was controlled by absence of propidium iodide fluorescence (using the inverted microscope coupled to the AFM). Thus, viable cardiomyocytes were selected for all AFM measurements.

Our in vivo data show that 3-months type 1 diabetes causes changes in myocardial fibers of mice hearts (fragmented and disordered), interstitial collagen deposition and reduction in the SERCA2a calcium pump expression as well as changes in F-actin organization. Moreover, the data show that live isolated cardiomyocytes from diabetic mice are stiffer than the control cardiomyocytes in all tested conditions. These results suggest that live cardiac myocytes material properties change after 3 month of diabetes. Adhesive force was 10.5 times higher in diabetic than control cardiomyocytes, suggesting that cardiomyocytes’ sarcolemmas were deeply affected by diabetes. Adhesive force represents the interaction force between the tip and the sample. These results suggest a change (i.e. increase) in the number and/or the activation state of adhesion molecules present in the surface of the diabetic cardiomyocytes. Hence, it is very likely that an intrinsic mechanical change of cardiomyocytes is an important factor of raising myocardial stiffness in vivo.

Changes in extracellular matrix (ECM) properties are well recognized to play a role in myocardial stiffening. Experimental evidences from our work with live isolated cardiomyocytes from control and diabetic mice strongly support that there is a direct mechanical contribution of cardiomyocytes in pathogenic stiffening of the myocardium. Thus, it will be necessary to identify which molecules are responsible for the mentioned increment in stiffness of live diabetic cardiomyocyte as well as which molecules change in cardiomyocyte-ECM interactions. Our data strongly suggest that actin is one of the implicated molecules in stiffness increment. These alterations must be taken into account to have a complete scenario of the disease that allows the development of new therapies.

 

Contact:

Dr. Juan C. Benech

Laboratorio de Señalización Celular y Nanobiología

Instituto de Investigaciones Biológicas Clemente Estable

Av. Italia, 3318. CP 11600,

Montevideo, Uruguay.

Phone: (+) 598 24871616, ext. 107

Email: jbenech@iibce.edu.uy or

juanclaudio.benech@gmail.com

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