Myocyte Compartment Research Articles

Passive mechanical properties of the left ventricular myocardium and extracellular matrix in hearts with chronic volume overload from mitral regurgitation.

Cardiac volume overload from mitral regurgitation (MR) is a trigger for left ventricular dilatation, remodeling, and ultimate failure. While the functional and structural adaptations to this overload are known, the adaptation of myocardial mechanical properties remains unknown. Using a rodent model of MR, in this study, we discern changes in the passive material properties of the intact and decellularized myocardium. Eighty Sprague‐Dawley rats (350–400 g) were assigned to two groups: (1) MR (n = 40) and (2) control (n = 40). MR was induced in the beating heart by perforating the mitral leaflet with a 23G needle, and rats were terminated at 2, 10, 20, or 40 weeks (n = 10/time‐point). Echocardiography was performed at baseline and termination, and explanted hearts were used for equibiaxial mechanical testing of the intact myocardium and after decellularization. Two weeks after inducing severe MR, the myocardium was more extensible compared to control, however, stiffness and extensibility of the extracellular matrix did not differ from control at this timepoint. By 20 weeks, the myocardium was stiffer with a higher elastic modulus of 1920 ± 246 kPa, and a parallel rise in extracellular matrix stiffness. Despite some matrix stiffening, it only contributed to 31% and 36% of the elastic modulus of the intact tissue in the circumferential and longitudinal directions. At 40 weeks, similar trends of increasing stiffness were observed, but the contribution of extracellular matrix remained relatively low. Chronic MR induces ventricular myocardial stiffening, which seems to be driven by the myocyte compartment of the muscle, and not the extracellular matrix.

Cardiomyocyte‐generated Monocyte Chemoattractant Protein 1 Contributes to Cardiac Inflammation During Adverse Remodeling in Pressure Overload

Attempts to normalize cardiac function in response to stress often lead to cardiovascular diseases associated with adverse remodeling. Cardiac inflammation is a hallmark feature of adverse remodeling. Although it is established that ischemic injury induces cardiac inflammation through release of substances from dying cells, mechanisms for inflammation in the context of nonischemic injury have been elusive. Our lab has previously shown that the cardiomyocyte is the initial site of transcription of pro‐inflammatory cytokines and chemokines such as MCP‐1, IL‐6, Il‐1β and Il‐18, in response to pressure overload and that this occurs in the absence of cell death. In addition, the NLRP3 inflammasome is activated in cardiomyocytes resulting in activation of caspase‐1 and generation of active forms of Il‐1β and IL‐18. Pharmacological blockade of NFκ‐β, MCP‐1 signaling, or NLRP3 inflammasome activation resulted in decreases in cardiac macrophage accumulation, fibrosis, and dysfunction. These results suggest that cardiomyocyte‐generated inflammatory mediators play a key role in adverse cardiac remodeling in response to pressure overload. However, the extent to which cardiomyocytes contribute to immune cell recruitment and remodeling through MCP‐1 and inflammasome generated Il‐1β and IL‐18 remains elusive.To address this question, we made cardiomyocyte‐specific MCP‐1 knockout (MCP1) mice and are currently breeding cardiac specific NLRP‐3 knockout mice. MCP1 and wildtype control (Ctl) mice were subjected to transaortic constriction (TAC) or sham surgery. Blood was collected from the mice at various timepoints and analyzed for serum amylyloid A (SAA) as a marker of systemic inflammation. At the conclusion of 3 or14 days post‐surgery, whole hearts were collected for analysis of gene expression and NLRP3 priming using qPCR. Caspase‐1 activity was used to measure NLRP3 inflammasome activation.Pro‐inflammatory cytokines were upregulated in TAC mice versus sham controls. Inflammasome priming and activation, as measured by IL‐1β and IL‐18 gene expression and caspase‐1 activity respectively, were unaffected by cardiomyocyte‐specific MCP‐1 deletion at 3 days post‐surgery. However, both IL‐1β and IL‐18 gene expression were reduced at 14 days post‐surgery in MCP1 TAC mice compared to Ctl TAC. This coincided with a reduction of CD68+ macrophage accumulation in the myocardium suggesting that infiltrating macrophages, recruited through cardiomyocyte‐generated MCP‐1, contribute to inflammatory gene expression at this time. We are currently assessing inflammasome activation in cardiomyocytes and macrophages following TAC in Ctl and MCP1 mice. Parallel experiments using cardiomyocyte NLRP‐3 KO mice will reveal the importance of inflammasome products made in the myocyte compartment. In other studies, we are assessing the contribution of cardiomyocyte‐generated cytokines and chemokines to systemic inflammation by measuring serum amyloid A. MCP1 TAC animals displayed a reduction in SAA compared to Ctl TAC animals at 14 days post‐surgery suggesting that cardiomyocyte‐derived MCP‐1 can contribute to persistent inflammation in pressure overloaded murine hearts.Support or Funding InformationR01HL145459‐01 (JHB, SM)T32HL007444‐37 (RSR, JC)

Myocyte repolarization modulates myocardial function in aging dogs.

Studies of myocardial aging are complex and the mechanisms involved in the deterioration of ventricular performance and decreased functional reserve of the old heart remain to be properly defined. We have studied a colony of beagle dogs from 3 to 14 yr of age kept under a highly regulated environment to define the effects of aging on the myocardium. Ventricular, myocardial, and myocyte function, together with anatomical and structural properties of the organ and cardiomyocytes, were evaluated. Ventricular hypertrophy was not observed with aging and the structural composition of the myocardium was modestly affected. Alterations in the myocyte compartment were identified in aged dogs, and these factors negatively interfere with the contractile reserve typical of the young heart. The duration of the action potential is prolonged in old cardiomyocytes contributing to the slower electrical recovery of the myocardium. Also, the remodeled repolarization of cardiomyocytes with aging provides inotropic support to the senescent muscle but compromises its contractile reserve, rendering the old heart ineffective under conditions of high hemodynamic demand. The defects in the electrical and mechanical properties of cardiomyocytes with aging suggest that this cell population is an important determinant of the cardiac senescent phenotype. Collectively, the delayed electrical repolarization of aging cardiomyocytes may be viewed as a critical variable of the aging myopathy and its propensity to evolve into ventricular decompensation under stressful conditions.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Cardiomyogenesis in the Aging and Failing Human Heart

Two opposite views of cardiac growth are currently held; one views the heart as a static organ characterized by a large number of cardiomyocytes that are present at birth and live as long as the organism, and the other views the heart a highly plastic organ in which the myocyte compartment is restored several times during the course of life. The average age of cardiomyocytes, vascular endothelial cells (ECs), and fibroblasts and their turnover rates were measured by retrospective (14)C birth dating of cells in 19 normal hearts 2 to 78 years of age and in 17 explanted failing hearts 22 to 70 years of age. We report that the human heart is characterized by a significant turnover of ventricular myocytes, ECs, and fibroblasts, physiologically and pathologically. Myocyte, EC, and fibroblast renewal is very high shortly after birth, decreases during postnatal maturation, remains relatively constant in the adult organ, and increases dramatically with age. From 20 to 78 years of age, the adult human heart entirely replaces its myocyte, EC, and fibroblast compartment ≈8, ≈6, and ≈8 times, respectively. Myocyte, EC, and fibroblast regeneration is further enhanced with chronic heart failure. The human heart is a highly dynamic organ that retains a remarkable degree of plasticity throughout life and in the presence of chronic heart failure. However, the ability to regenerate cardiomyocytes, vascular ECs, and fibroblasts cannot prevent the manifestations of myocardial aging or oppose the negative effects of ischemic and idiopathic dilated cardiomyopathy.

Inhibition by diltiazem of left ventricle collagen proliferation during renovascular hypertension development in rats.

Diltiazem administered in drinking water (0.7 mg mL-1) to Goldblatt two kidney-one clip rats over 16 weeks did not prevent the development of hypertension and left ventricular hypertrophy (LVH). When the collagen content of the left ventricles was assayed (as hydroxyproline), it was found that the fibrosis, characteristic of LVH, was inhibited by diltiazem treatment, despite the fact that hypertension and LVH had developed. This study provides some indirect evidence for the notion that the collagen and myocyte compartments of the myocardium are under separate influences during LVH development in renovascular hypertension.

Abstract 3818: Notch1 Receptor Blockade Results in Dilated Cardiomyopathy in the Neonatal Mouse Heart

The objective of this study was to determine whether Notch1 receptor plays a crucial role in the commitment of c-kit-positive cardiac progenitor cells (CPCs) to the myocyte lineage. This possibility was tested in the neonatal mouse heart because of the dramatic increase in the myocyte compartment early postnatally. CPCs are located within the primitive heart and their number increases progressively with gestational age and after birth. c-kit colocalizes with transcription factors and contractile proteins specific of myocytes, pointing to a lineage relationship between CPCs and myocytes in the prenatal and postnatal heart. Notch1 intracellular domain (N1ICD) was consistently found together with Nkx2.5 suggesting that Notch1 receptor represents an early determinant of myocyte differentiation. The critical function of Notch1 in the generation of myocytes was established by interfering with this pathway through the administration of a γ-secretase inhibitor to newborn mice for 3–7 days; γ-secretase inhibition opposes the cleavage of the active fragment of Notch and initiation of transcription. Treated mice showed a dilated myopathy characterized by decreased muscle mass, wall thinning and impaired fractional shortening and ejection fraction. There was also a marked reduction in the fraction of CPCs and myocytes expressing the Notch1 active fragment N1ICD. Myocyte number decreased 28% while myocyte volume remained constant, suggesting that Notch1 blockade affects myocyte formation and conditions the development of a dilated myopathy. This hypothesis is supported further by a 57% decrease in replicating myocytes positive for Ki67. These anatomical and functional changes were accompanied by downregulation of Nkx2.5 transcripts in the pathologic heart. A perfect consensus site for the target gene of Notch, RBP-Jk, was recognized in the promoter of Nkx2.5. The formation of a functional complex between Nkx2.5 promoter and RBP-Jk protein was demonstrated by electrophoretic mobility assay, chromatin immunoprecipitation and luciferase reporter assay. In conclusion, Notch1 promotes the commitment of CPCs to the myocyte lineage and inhibition of Notch1 interferes with cardiomyogenesis leading to a dilated cardiomyopathy in the postnatal heart.

Region-specific patterns of vascular remodelling occur early in atherosclerosis and without loss of smooth muscle cell markers

Background Vascular remodelling is characterized by increased smooth muscle cell (SMC) proliferation and migration. Coincident with these events, SMC markers of differentiation are known to down-regulate in advanced stages of atherosclerosis, a process known as phenotypic modulation. However, it is not known when this first begins. Here we sought to determine if regions of the mouse aorta with varying susceptibilities for atherosclerosis display differential vascular remodelling and SMC gene expression at the earliest stages of disease. Methods and results LDLrKO mice were fed normal or high cholesterol diet for 0–98 days. In the latter, ORO and H&E staining of arch, thoracic and abdominal aortic sections revealed infrequent occurrences of lipid deposition at d28, but significant region-specific vascular remodelling. Immunostaining for PCNA revealed increased cellular proliferation in the intima and inner media at d28 in all three regions. qRT-PCR of SMC revealed increased expression of SM22α and SM-MHC in the arch by d28, which subsequently decreased by d98. By contrast, eNOS gene expression was consistently decreased in the arch over these times. A temporal increase in macrophage-specific CD68 expression was observed in the arch but not thoracic or abdominal regions. Conclusion Remodelling of the vascular myocyte compartment due to cellular proliferation is an early event in atherosclerosis and is associated with increases in SMC-specific gene expression. These events precede subsequent lesion formation and SMC phenotypic modulation.

The Young Mouse Heart Is Composed of Myocytes Heterogeneous in Age and Function

The recognition that the adult heart continuously renews its myocyte compartment raises the possibility that the age and lifespan of myocytes does not coincide with the age and lifespan of the organ and organism. If this were the case, myocyte turnover would result at any age in a myocardium composed by a heterogeneous population of parenchymal cells which are structurally integrated but may contribute differently to myocardial performance. To test this hypothesis, left ventricular myocytes were isolated from mice at 3 months of age and the contractile, electrical, and calcium cycling characteristics of these cells were determined together with the expression of the senescence-associated protein p16(INK4a) and telomere length. The heart was characterized by the coexistence of young, aged, and senescent myocytes. Old nonreplicating, p16(INK4a)-positive, hypertrophied myocytes with severe telomeric shortening were present together with young, dividing, p16(INK4a)-negative, small myocytes with long telomeres. A class of myocytes with intermediate properties was also found. Physiologically, evidence was obtained in favor of the critical role that action potential (AP) duration and I(CaL) play in potentiating Ca(2+) cycling and the mechanical behavior of young myocytes or in decreasing Ca(2+) transients and the performance of senescent hypertrophied cells. The characteristics of the AP appeared to be modulated by the transient outward K(+) current I(to) which was influenced by the different expression of the K(+) channels subunits. Collectively, these observations at the physiological and structural cellular level document that by necessity the heart has to constantly repopulate its myocyte compartment to replace senescent poorly contracting myocytes with younger more efficient cells. Thus, cardiac homeostasis and myocyte turnover regulate cardiac function.

Predominant fusion of bone marrow-derived cardiomyocytes

Here we address the capacity of bone marrow-derived cells (BMDCs) to trans-differentiate into mature myocytes under the physiological stimulus of exercise training. For this purpose, we have transplanted bone marrow from mice ubiquitously expressing enhanced green fluorescence protein (eGFP) into host mice that have been subjected to a prolonged program of exercise. In all successful bone marrow reconstitutions (greater than 80%), we observed rare but consistent events of bone marrow-derived cardiomyocytes, the frequency of which was unchanged upon exercise training. We have further determined whether these recruited myocytes are a product of trans-differentiation or fusion by the use of a genetic system that distinguishes cell fusion from trans-differentiation in a single-cell assay. We concluded that both in the unchallenged mouse and in the trained specimens, fusion is the most prominent mechanism by which bone marrow-derived cells are observed in the myocyte compartment.

Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes.

To examine effects of cytosolic Na+, K+, and Cs+ on the voltage dependence of the Na+-K+ pump, we measured Na+-K+ pump current (Ip) of ventricular myocytes voltage-clamped at potentials (Vm) from 100 to +60 mV. Superfusates were designed to eliminate voltage dependence at extracellular pump sites. The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na+ concentration ([Na]pip) of 80 mM and a K+ concentration from 0 to 80 mM or with solutions containing Na+ in concentrations from 0.1 to 100 mM and K+ in a concentration of either 0 or 80 mM. When [Na]pip was 80 mM, K+ in pipette solutions had a voltage-dependent inhibitory effect on Ip and induced a negative slope of the Ip-Vm relationship. Cs+ in pipette solutions had an effect on Ip qualitatively similar to that of K+. Increases in Ip with increases in [Na]pip were voltage dependent. The dielectric coefficient derived from [Na]pip-Ip relationships at the different test potentials was 0.15 when pipette solutions included 80 mM K+ and 0.06 when pipette solutions were K+ free.

Broad-spectrum cardioprotection with adenosine

Ischemia-reperfusion results in contractile dysfunction, necrosis, and vascular injury. This postischemic injury is mediated in part by superoxide radical production, neutrophils, dysfunction to ionic pumps, and edema formation. Adenosine is an autacoid released tonically by myocytes, endothelium, and neutrophils; the release of adenosine from the myocyte compartment into the interstitium is increased during ischemia. The major effects of adenosine are mediated by specific receptors identified as A1, A2a, A2b, and A3. Each receptor subtype contributes to physiological responses that influence ischemia-reperfusion injury. Adenosine has potent cardioprotective properties exerted during three major windows of opportunity: pretreatment, ischemia, and reperfusion. The cardioprotective effects exerted during pretreatment and ischemia may involve metabolic changes and hyperpolarization via KATP-channel activation, mediated through A1 receptor mechanisms. The cardioprotective mechanisms exerted during reperfusion involve inhibition of neutrophils directly (superoxide anion generation, expression of adhesion molecules), and by inhibiting activation of the endothelium through A2 receptor-mediated mechanisms, thereby preventing neutrophil-endothelial cell interactions, which initiate the inflammatory-like component of reperfusion injury. Activation of the newly identified A3 receptor has been shown to be cardioprotective partially by inhibition of neutrophil adherence to endothelium and by neutrophil-independent mechanisms. These mechanisms of cardioprotection have been suggested to play major roles in the reduction of infarction and apoptosis after myocardial ischemia, cardioplegic arrest, and subsequent reperfusion. Adenosine has been used as an adjunct to both crystalloid and blood cardioplegia, but its potential as a cardioprotective agent has not been fully explored.

Distinct effects of Rac1 on differentiation of primary avian myoblasts.

Rho family GTPases have been implicated in the regulation of the actin cytoskeleton in response to extracellular cues and in the transduction of signals from the membrane to the nucleus. Their role in development and cell differentiation, however, is little understood. Here we show that the transient expression of constitutively active Rac1 and Cdc42 in unestablished avian myoblasts is sufficient to cause inhibition of myogenin expression and block of the transition to the myocyte compartment, whereas activated RhoA affects myogenic differentiation only marginally. Activation of c-Jun N-terminal kinase (JNK) appears not to be essential for block of differentiation because, although Rac1 and Cdc42 GTPases modestly activate JNK in quail myoblasts, a Rac1 mutant defective for JNK activation can still inhibit myogenic differentiation. Stable expression of active Rac1, attained by infection with a recombinant retrovirus, is permissive for terminal differentiation, but the resulting myotubes accumulate severely reduced levels of muscle-specific proteins. This inhibition is the consequence of posttranscriptional events and suggests the presence of a novel level of regulation of myogenesis. We also show that myotubes expressing constitutively active Rac1 fail to assemble ordered sarcomeres. Conversely, a dominant-negative Rac1 variant accelerates sarcomere maturation and inhibits v-Src-induced selective disassembly of I-Z-I complexes. Collectively, our findings provide a role for Rac1 during skeletal muscle differentiation and strongly suggest that Rac1 is required downstream of v-Src in the signaling pathways responsible for the dismantling of tissue-specific supramolecular structures.

Potential role of the microvasculature in progression of heart failure

After cardiac injury, there are changes in the cardiac myocyte morphology, function, matrix, and molecular gene expression. These all play an important role in remodeling of the injured heart, contributing to the progression toward heart failure. The role of the microvasculature in the progression toward heart failure is less well characterized. However, laboratory studies have established that there are important interactions between the microvascular endothelium and the myocyte. Furthermore, in a multitude of animal models of heart failure and cardiomyopathy, there is always an association with microvascular abnormalities. Reversal of these abnormalities is also associated with improvement in the cardiomyopathy. Major mediators that likely play an important role in the microvasculature include endothelin and nitric oxide. These are elaborated by both endothelium and myocyte compartments of the myocardium. Preliminary clinical studies already demonstrate that microvascular ischemia may have prognostic power in patients with nonischemic dilated cardiomyopathy. Results from these studies showed a reduction in mortality from treatment with amlodipine, suggesting a possible benefit based on changes in the microvasculature.

Post-ischaemic cardiac dilatation and remodelling: reperfusion injury of the interstitium.

Cardiac size and geometry have an important influence on clinical prognosis in heart disease. The cardiac interstitium would appear to play a major role in modulating muscle configuration after ischaemic insults. Ischaemic reperfusion injury of the heart should not be viewed as confined to the myocyte compartment. There are major events occurring in the interstitial compartment which could ultimately determine the long-term configuration and topography of the heart. Thus, permanent plastic changes in cardiac dimensions appear to evolve after initial alterations in the collagen matrix. The physiological, cellular, biochemical, and molecular considerations in the cardiac interstitium are quite different from those of the myocyte compartment. Accordingly, therapeutic interventions to modulate cardiac geometry and remodelling may differ.

Loss of intermediate-sized coronary arteries and capillary proliferation after left ventricular failure in rats

To determine the adaptation of coronary vasculature and microvasculature to cardiac failure, renal hypertension was produced in rats, and the animals were killed 8 mo later when severe impairment in left ventricular function was present. By use of a new morphometric approach, it could be demonstrated that length densities of arteries from 6 to 20 microns in luminal diameter decreased by 23 and 26% in the midmyocardium and endomyocardium, respectively, whereas arteries ranging from 21 to 40 microns were reduced by 59, 55, and 46% in the outer, middle, and inner layers, respectively, of the left ventricular wall. In contrast, capillary density increased by 29 and 38% in the epimyocardium and endomyocardium, respectively. Capillary proliferation resulted in a 15% decrease in average diffusion distance for oxygen to the myocyte compartment of the tissue. Despite these opposite effects that may tend to compensate each other, the volume percent of collagen in the wall augmented by 106%. In conclusion, differences exist in the response of the coronary vascular tree to long-term renal hypertension that may impair coronary resistance and flow without affecting the capillary network and the oxygenation potential of muscle cells.

Morphometric Analysis of the Infarcted Heart

To determine whether left ventricular hypertrophy following myocardial infarction leads to a complete or incomplete reconstitution of myocardial mass, the left coronary artery in rats was ligated and the animals sacrificed 30 days later. Infarcts affecting an average 43% of the ventricle were characterized by a 90% hypertrophic growth of the remaining myocardium that was inadequate for a full restoration of ventricular tissue. Myocyte hypertrophy, evaluated by changes in mean cell volume per nucleus, was again insufficient for a total recovery of the myocyte compartment of the ventricle. These observations suggest that infarcts comprising nearly 50% of the ventricle produce a sufficiently large stress on the spared myocytes to stimulate their maximal hypertrophic reserve capacity. Cardiac muscle cells, however, appear to be unable to offset by cellular hypertrophy alone the loss of mass induced by infarcts of this size. The inadequate compensatory response of the myocytes could be the underlying structural mechanism responsible for impaired ventricular function in large infarcts.