Intrinsic nanomechanical changes in live diabetic cardiomyocytes

Laboratorio de Señalización Celular y Nanobiología, Instituto de Investigaciones Biológicas Clemente Estable, Av. Italia 3318, CP 11600, Montevideo, Uruguay Instituto de Física, Facultad de Ciencias, Universidad de la República, Iguá 4225, CP 11400, Montevideo, Uruguay Departamento de Biología Molecular y Celular, Facultad de Veterinaria, Universidad de la República, Alberto Lasplaces 1550, CP 11600, Montevideo, Uruguay


Introduction
The occurrence of diabetes mellitus (DM) is rapidly increasing.It is estimated an increase in the number of adults with diabetes from 171 million in 2000 to 300 million by 2030 [1] .DM is a well-recognized risk factor for development of heart failure (HF).HF occurs twice as frequently in diabetic men and five times as frequently in diabetic women relative to age-matched controls [2] .Thus, cardiovascular complications such as increased large-artery and coronary atherosclerosis are the leading cause of diabetes-related morbidity and mortality [3,4] .

RESEARCH HIGHLIGHT
To study diabetes, several animal models of diabetes type 1 (TD1) and type 2 (TD2) were developed.In both TD1 and TD2 animal models, functional and structural cardiac alterations or cardiac muscle disorders have been documented [3] .In humans, DM induces diastolic LV dysfunction.This dysfunction is recognized as an important contributor to HF.In patients with diabetes mellitus, an increase in diastolic LV stiffness reduces LV remodeling after myocardial infarction [5,6] .It also raises LV filling pressures at similar filling volumes in HF with both reduced and normal LV ejection fractions [7] .As a consequence, DM patients have a significantly higher incidence of HF after myocardial infarction [5,6,8,9] .Three main mechanisms have been proposed to be responsible for raising myocardial stiffness in DM.These mechanisms are excessive fibrosis [10]   , deposition of advanced glycation end products (AGEs) [11]   , and cardiomyocyte stiffness (resting tension).Resting tension (RT) was measured as the passive force at the same sarcomere length.In previous reports, RT was measured in single cardiomyocytes isolated from frozen human biopsy samples that had been thawed, mechanically disrupted, and permeabilized with Triton X-100 [7,12,13] .Newer data from several disease models obtained from cardiovascular tissue live cells, live cardiomyocytes (including isolated cardiomyocytes from diabetic mice) showed alterations in the cells' intrinsic nanomechanical properties.These alterations must be taken into account to have a more complete understanding of the disease to develop new therapies.In the present report, we will highlight this point.

Diabetes mellitus
Diabetes mellitus is a multivariate metabolic condition that is characterized by cellular dysfunction in the transport and utilization of glucose.Type 1 diabetes, formerly called "insulin-dependent diabetes", is caused by T lymphocyte-mediated autoimmune destruction of the pancreatic β-cells, which results in insufficient insulin production and reduced glucose utilization [14] .Insulin-independent diabetes, commonly known as type 2 diabetes, develops due to decompensatory mechanisms induced by insulin resistance [15] .This maladaptive response leads to hyperinsulinemia, resulting in elevated blood glucose levels (hyperglycemia), impaired cellular glycolysis, and pyruvate oxidation [16] .Diabetes induces several myocardial alterations.These detrimental myocardial alterations could be explained correlatively by the fact that diabetes is associated with known cardiovascular (CV) risk factors such as obesity, hyperlipidemia, thrombosis, myocardial infarction, hypertension, activation of multiple hormone and cytokine systems, autonomic neuropathy, endothelial dysfunction and coronary artery disease [17] .

Diabetic cardiomyopathy
The mechanisms that lead to the development of the diabetic cardiomyopathy are still poorly understood.Cardiomyopathy develops in patients with diabetes independently of coronary disease and hypertension [18] and contributes to the increased mortality and morbidity of the disease [3,19] .The early stages of diabetic cardiomyopathy are associated with reduced diastolic function in 27%-70% of asymptomatic diabetic patients [20][21][22] .At later stages systolic dysfunction and heart failure become evident [23,24] .Diabetic cardiomyopathy develops in both TD1 and TD2 forms of the disease [25,26] .

Pathogenesis of Diabetic Cardiomyopathy
There is a general agreement that the pathogenesis of diabetic cardiomyopathy is multifactorial.Several hypotheses have been proposed.Proposed hypotheses include autonomic dysfunction, metabolic derangements, abnormalities in ion homeostasis, alteration in structural proteins, and interstitial fibrosis [27,28] .Also, it has been reported that sustained hyperglycemia might increase glycation of interstitial proteins such as collagen, resulting in myocardial stiffness and impaired contractility [11,29,30] .

Myocardial fibrosis, advanced glycation end products deposition and resting tension
Heart failure mortality among diabetic patients is high (between 20 and 45%) [31,32] .Metabolic disturbances associated with diabetes contribute importantly to patients' myocardial dysfunction.It has been observed that an early manifestation of myocardial disfunction is an increase in diastolic left ventricular (LV) stiffness.Excessive diastolic LV stiffness of the diabetic heart was previously primarily attributed to myocardial fibrosis or to myocardial deposition of advanced glycation end products.In 2005, Borbey et al. [12] reported that hypertrophic cardiomyocytes isolated from LV biopsy samples of HF patients with normal LV ejection fraction showed a high resting tension, which correlated with greater in vivo diastolic LV stiffness.With the use of LV endomyocardial biopsies, Loek van Heerebeek et al., 2008 [7] isolated cardiomyocytes from diabetic patients with heart failure and either normal or reduced LV ejection fraction.They assessed myocardial fibrosis, myocardial advanced glycation end product deposition and resting tension.All patients had no coronary artery disease and all had elevated diastolic LV stiffness.Cardiomyocyte stiffness was determined by measuring RT in single isolated cardiomyocytes, but the employed procedure provoked disruption of sarcolemmal and sarcoplasmic membranes.In this preparation, isolated cardiomyocytes are dependent on externally-supplied Ca 2+ for active force development.Alterations of myofilament or cytoskeletal proteins were attributed to be responsible for the elevation of passive force.The hypophosphorylation of titin stiff isoform has been suggested to be responsible for the elevation of passive force in diabetic cardiomyocytes [13] .The mechanisms responsible for the elevated diastolic LV stiffness differed between heart failure patients with normal versus reduced LV ejection fraction.The authors concluded that myocardial deposition of collagen and advanced glycation end products were more important in patients with reduced ejection fraction, while high cardiomyocyte RT was more important in patients with normal ejection fraction [7] .

Mechanotransduction
The majority of cells in organs and tissues are subject of multiple external mechanical stimuli that modulate several aspects of cellular functions and preserve tissue structure and function [33] .Mechanotransduction (MT) describes the cellular processes that translate mechanical stimuli into biochemical signals.This process is fundamental in enabling cells to adapt to their physical surroundings.Several molecular players that are involved in cellular mechanotransduction were identified, including extracellular matrix, membrane receptors, cytoskeletal structures, and multiple signaling pathways [34,35] .The heart has adaptive answers for a great variety of genetic and extrinsic factors to maintain contractile function.When the compensating answers are not possible, cardiac dysfunction take place, leading to cardiomyopathy.The compensatory responses of the heart are mediated by different signaling pathways.Initially, signaling pathways maintain normal contractility but persistent activation of these pathways leads to cardiac dysfunction [36] .
Several apparently unrelated diseases like heart failure, arterial hypertension, cancer, asthma, congenital deafness, sexual dysfunction, urinary incontinence, diabetes, etc., have in common that its etiology or clinical manifestation results from an abnormal MT process [37,38] .Mechanotransduction in cardiomyocytes is complex.Individual muscle cells respond to externally applied mechanical forces, as well as generate internal loads [39] .All these forces are transmitted to adjacent cells and their surrounding extracellular matrix (ECM).MT affects the beat-to-beat regulation of cardiac performance.Proliferation, differentiation, growth, and survival of the cellular components of the human myocardium also are deeply affected by MT [40] .
Transmembrane integrins that link the ECM and intracellular cytoskeleton are important for mechanosensation and mechanotransduction.In cardiomyocytes, several data indicate that the costamere and its related structure, the focal adhesion complex, are critical cytoskeletal elements involved in bidirectional mechanochemical signal transduction.Integrins mechanically transfer forces and initiate biochemical signals between the inside and the outside of the cell in both directions [41][42][43][44] .In cardiomyocytes, costameres are generally considered as the subsarcolemmal protein assemblies that circumferentially align in correspondence with the Z disk of peripheral myofibrils.It was reported that in cardiac muscle, costameres physically couple force-generating by sarcomeres with the sarcolemma [45][46][47] .The cytoskeletal proteins talin, desmin, vinculin, focal adhesion kinase and α-actinin are linked by integrins to the Z disk of the sarcomeres in the costamere structure, which plays an important role in MT [40].
Recently, using atomic force microscopy (AFM) on freshly isolated mice cardiomyocytes, the unbinding force (adhesion force) and adhesion probability between integrins and fibronectin (FN) was quantified and these measurements were correlated with the contractile state as indexed by cell stiffness [48] .In the same report, it was demonstrated that integrin binding to FN is modulated by the contractile state of cardiac myocytes [48] .
As we shall see, pathologies like diabetes, deeply affects the nanomechanical properties of isolated cardiomyocytes, including adhesive force of myocyte sarcolemma [49] .These data strongly 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" [49] .However, at present if structures like costameres (including integrin linkage to the Z disk of the sarcomeres via the cytoskeleton) are affected by diabetes is still unknown.

Nanomechanical properties in living cardiovascular tissue cells
With AFM is possible to study the dynamics and mechanical properties of intact cells.Cell events like locomotion, differentiation and aging, physiological activation, electromotility, and cell pathology, can be analyzed by AFM [48,[50][51][52][53][54][55] .Using AFM, the effects of aging and obesity on vascular smooth muscle cell stiffness [50,54,55] and aging on cardiomyocyte stiffness [52] have been analyzed.Qiu, H et al., 2010 [54] measured vascular smooth muscle cell stiffness (VSMCs) and a reconstituted tissue model, using VSMCs from aorta of young and old male monkeys, finding that aortic stiffness increases by 200% in vivo.The Apparent Elastic Modulus (AEM) increased significantly in old (41.7±0.5 kPa) versus young (12.8±0.3 kPa) VSMCs but this increase disappeared after disassembly of the actin cytoskeleton with cytochalasin D. Stiffness of VSMCs in the reconstituted tissue model was also higher in old (23.3±3.0 kPa) than in young (13.7±2.4 kPa) monkeys.To investigate the pathophysiology that links obesity to aortic stiffening, Chen, J.Y, et al., 2013 [50] analyzed 2-and 3-month-old obese mice (ob/ob) and obese humans.They demonstrated that obesity resulted in aortic stiffening in both species, and established a causal relationship between lysyl oxidase down-regulation and aortic stiffening.In another interesting study [55] , AFM was used to measure elasticity and adhesion´s Young's modulus as assessed by fibronectin or anti-beta 1 integrin interaction with the VSMC surface.VSMC from old monkey cells had a 612% increase in elastic modulus, and a 200% increase in adhesion (unbinding force) relative to control young monkey cells.

Nanomechanical changes in live isolated cardiomyocytes
The first demonstration of a significant increase in the AEM of single aging cardiac myocytes was published by Lieber et al. in 2004 [52] .They proposed the novel concept that the mechanism mediating LV diastolic dysfunction in aging hearts is at least in part myocyte-intrinsic.With AFM-nanoindentation, they studied old male rats to see the effect of aging in cardiac myocytes [52] .A significant increase of the cardiac myocytes AEM in older animals was found, registering 35.1± 0.7 kPa for control young rat cells and 42.5±1.0kPa for old rat cells.

Nanomechanical change in living cardiomyocytes in heart failure (post-ischemic condition)
Recently, sarcolemmal surface topography and physical properties were directly examined using Atomic Force Microscopy in living cardiomyocytes, from healthy and failing mouse hearts [56] .The presence of highly organized crests and hollows along myofilaments in isolated healthy cardiomyocytes was observed.In the study, sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows.This was correlated with the presence of few packed subsarcolemmal mitochondria (SSM) as observed by electron microscopy.At the end-stage post-ischemic condition (15 days post-myocardial infarction), sarcolemma disorganization was reported with a general loss of crest/hollow periodicity and a significant increase in cell surface stiffness.Electron microscopy observation revealed the total depletion of SSM while some interfibrillar mitochondria (IFM) heaps could be visualized beneath the membrane.The authors concluded that increased stiffness of the cardiomyocyte surface was related to atypical IFM heaps while SSM died during HF progression.

Nanomechanical changes in live isolated diabetic cardiomyocyte
In our study [49] , the effect of TD1 on the nanomechanical properties of live cardiomyocytes was analyzed.Diabetes was induced in CD1 mice by streptozotocin (STZ) administration.This is a well-known animal model to induce TD1 [3,[57][58][59] .All diabetic animals had high blood glucose levels and developed severe diabetes symptoms, including polyuria, polydipsia, and reduced weight gain.Histological examination of myocardium from mice that were diabetic for 3 months showed "disorder 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 irregular and diffuse actin filaments relative to cardiomyocytes from control mice" [49]   .LV Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA2a) pump expression was reduced in diabetic animals relative to age-matched controls.Multiple functional and structural cardiac muscle alterations were reported in STZ-induced TD1 animals [3,57,58,60,61] .Moreover, in agreement with our own data interstitial collagen accumulation was observed [61] and SERCA2a expression was reduced [61,62] .Atomic Force Microscopy has been previously employed to measure the viscoelastic response of many cell types [51] .Sufficient cell thickness is an important factor in determining the feasibility of AEM.The smallest cardiomyocyte heights present in our preparation were 2.5 ± 1.4 µm for control and 3.2 ± 1.5 µm for diabetic cardiomyocytes while data were analyzed with an indentation of 0.1 µm.Thus, since the AFM tip never indents more than 10% of the cell thickness, substrate contribution can be neglected [51] .On the other hand, previous reports showed that cardiomyocytes are softer in the nuclear region relative to the periphery [63] .To avoid this complication, "all force curves were taken at the middle of the longitudinal axis.Proper force curves with an indentation-and-retraction rate of 6 m/s were obtained in the force calibration mode at selected points.A low frequency (1-Hz) was set as this frequency was found to minimize not only hysteresis but also drag force" [49] .It also allowed us to maximize the number of force curves that were captured as previously described [64][65][66][67] .
The AFM technique has been proven in its ability to detect cytoskeletal changes [68] .When applied in live isolated cardiomyocytes, AFM indentation measurements register changes in the myocyte sarcolemma, sarcomeric skeleton and cytoskeletal proteins including tubulin, desmin and actin.AEM measurements of live control or diabetic isolated cardiomyocytes were performed with the nanoindentation method in Tyrode buffer solution (TyBS) with different ionic compositions.For the normal physiological condition, TyBS with 1.8 mM Ca 2+ was used.For the low extracellular Ca 2+ condition, TyBS with 100 nM free Ca 2+ was used.The Values are means ± SD; 800-1,200 force curves were obtained from 4-6 cells from 3 mice.Cardiomyocytes isolated from control and diabetic mice were plated on polylysine-coated glass microslide chambers as described in Benech et al, 2014 [49] .Attached cardiomyocytes were incubated in Tyrode buffer solution with different ionic compositions: normal physiological condition (1.8 mM Ca 2+ and 5.4 mM KCl), low extracellular Ca 2+ condition (100 nM free Ca 2+ and 5.4 mM KCl), or contraction condition (1.8 mM Ca 2+ and 140 mM KCl).Normalized Young's modulus histograms were obtained from all experimental conditions.Each histogram was fitted with a Gaussian curve.Statistical significance is as follows: P< 0.05 (by Student's t-test) for a vs. b, c vs. d, e vs. f, a or c vs. e, and b or d vs. f; no significant difference for a vs. c and b vs. d.Reprinted with permission of [49] .
free Ca 2+ concentration was calculated using the apparent Ca-EGTA association constants and a computer program as previously described [69,70] .For the contraction condition, cells were incubated in high-K+ TyBS with 1.8 mM Ca 2+ .For more information regarding complete ionic composition of used TyBS see Benech JC et al. 2014 [49] .The absence of Propidium Iodide (PI) fluorescence was used to select viable cardiomyocytes.All AFM data were obtained from viable cardiomyocytes.
To calculate the Young modulus in normal physiological conditions, a normalized histogram was obtained from all diabetic and control samples.Each histogram was fitted with a Gaussian curve in order to retrieve the mean value and the standard Young modulus deviation.The AEM of diabetic cardiomyocytes (   = 91 ± 14 kPa ) was significantly increased relative to that of control cardiomyocytes (  = 43 ± 7 kPa).A normalized histogram for the work of the adhesive force was also obtained from all diabetic and control samples.Each histogram was fitted again with a Gaussian curve to obtain the mean value and the standard deviation.The results were: γ a d = 2.2± 0.8 ×10 -4 J/m 2 , and    = 0.21 ± 0.04 × 10 −4 J/m 2 for the diabetic and control cells respectively.The AEMs of control and diabetic isolated cardiomyocytes were also measured in TyBS containing low extracellular Ca2+ (100 nm), see Table 1.Under the conditions described above, the AEM of diabetic cardiomyocytes (   = 103 ± 12kPa ) was significantly higher than that of control cardiomyocytes (   = 39 ± 6kPa).Cytosolic calcium concentration was not measured.However, at low extracellular Ca 2+ in non-stimulated viable cardiomyocytes it is conceivable that cytosolic Ca 2+ concentration did not change.Therefore, very likely these cardiomyocytes were at rest.On the other hand, previous works reported no difference between resting Ca2+ concentrations of control and diabetic cardiomyocytes [71] .Thus, likely the difference in the AEM between diabetic and control cardiomyocytes is not related to the contractile state in those conditions.The difference seems to be related to modifications promoted by diabetes in the material properties of cardiomyocytes.This conclusion is supported by changes detected in F-actin organization when isolated control and diabetic cardiomyocytes stained with phalloidin were compared [49] ."Control cardiomyocytes showed regular and well-defined actin organization, while the diabetic ones showed more diffuse and irregular actin disposition" [49] .On the other hand, the AEM of control or diabetic cardiomyocytes measured in low extracellular Ca 2+ condition and the normal physiological condition showed not significant differences (see Table 1).The absence of significant differences in the AEM could be explained by the fact that only viable cardiomyocytes were selected to perform all AFM measurements.However, a dramatic change in the AEM was observed (in both control and diabetic cardiomyocytes) when high potassium (140 mM) was included in TyBS containing 1.8 mM calcium (contraction condition).In control cardiomyocytes, the AEM was 106 ± 5 kPa and a significantly higher value of AEM was meassured in diabetic cardiomyocytes, 324± 11 kPa.This dramatic change can be explained by the increase in cytosolic Ca 2+ concentration promoted by the opening of membrane Ca2+ channels and cardiomyocyte contraction (contraction condition).The AEM of diabetic cardiomyocytes under this condition was significantly higher than the control ones (see Table 1).Therefore, diabetic cardiomyocytes were stiffer than the control ones in all tested conditions.These results support the idea that the intrinsic "mechanical properties of live cardiomyocytes were affected by diabetes" [49] .As mentioned before, previous studies reported that excessive fibrosis, advanced glycation end products (AGEs) deposition, and high cardiomyocyte stiffness contribute to increased myocardial stiffness in DM [7,12,13] .Our data showed that, the AEM mean value for diabetic cardiomyocytes was higher than the control one.Thus, our data are in concordance with the previously mentioned reports studying isolated cardiomyocytes with disrupted membranes.In our studies, live cardiomyocyte membranes were kept undisrupted.This suggest, that diabetes caused changes in cardiomyocyte membranes and probably contributing to the increased AEM.Additionally, changes in F-actin in isolated cardiomyocytes of diabetic mice may be at least partially responsible for the higher AEM of the diabetic cardiomyocytes.
Recently, it was reported that primary neonatal rat cardiomyocytes in culture (under diabetic conditions) increased their stiffness but not fibroblasts [72] .This is important, since data obtained using AFM in an in vitro model are in concordance with data obtained with live isolated diabetic [49] .
We also found that adhesive force was higher in diabetic cardiomyocytes than in control ones.Adhesive force represents the interaction force between the tip and the sample.The change in adhesive force was 10.5-fold from γ a c = 0.21 ± 0.04 × 10 −4 J/m 2 in control cardiomyocytes to γ a d = 2.2± 0.8 ×10 -4 J/m 2 in diabetic cardiomyocytes.This data suggest that cardiomyocytes' sarcolemmas were greatly affected by diabetes.Additionally, these results suggest "there is a change (i.e.increase) in the number and/or the activation state of adhesion molecules present in the surface of the diabetic cardiomyocytes" [49] .

Perspectives
Our data showed that 3 months of TD1 diabetes provokes changes in myocardial fibers of mice hearts.In diabetic mice hearts, fibers were fragmented and disordered with interstitial collagen deposition.Reduction in SERCA2a calcium pump expression and changes in F-actin organization relative to control were observed.Moreover, the data showed that live isolated diabetic cardiomyocytes are stiffer than control cardiomyocytes in all tested conditions, suggesting that material properties of live cardiomyocytes change with diabetes.Hence, it is very likely that an intrinsic mechanical change of cardiomyocytes is an important factor of raising myocardial stiffness in the whole heart.
Changes in ECM properties have long been recognized to play a role in myocardial stiffening.Our data, obtained in isolated live cardiomyocytes from control and diabetic mice, strongly support that the cardiomyocyte mechanical properties directly contribute to the pathogenic stiffening of the myocardium.Thus, it will be necessary to identify which molecules are responsible for the increase in stiffness, as well as which molecules change in cardiomyocyte-ECM interactions.Our data strongly suggest that actin is one of the implicated molecules.These alterations must be taken into account in order to have a complete scenario of the disease and to develop new therapies and interventions.