Adult Cardiocytes Research Articles

Cardiocyte Functional Data Analysis: A Novel Approach

Current laboratory methods used to assess neonatal and adult cardiocyte function include measurement of gene and protein expression levels, calcium transients, and contractility. Our goal was to develop simple tools to analyze such data readily. We created two MATLAB®-based toolboxes; the Contraction Video Processing (CVP) and the Cardiocyte Functional Response Analysis (CFRA) Toolbox. Videos of contracting cultured cardiocytes are acquired using a digital camera attached to an inverted phase microscope. Video frames are analyzed using digital imaging processing techniques along with several contraction assessment methods available through the CVP toolbox. The CVP offers direct correlation, pixel intensity tracking and Polar Fourier transform methods for the analysis of neonatal cardiocyte contraction. Analysis of adult cardiocytes includes those implemented on neonatal cardiocytes in addition to area boundary tracking, Fourier descriptor analysis, and cell length tracking methods. The resulting contraction records are processed using the CFRA toolbox to provide quantitative analyses of cardiocyte contractility and calcium transient responses. Transient data are obtained by measuring the calcium fluxes using the fluorescent dye Fluo-3, and a Photon Technology fluorometer system running Felix software. Data analysis routines have been created and tailored exclusively to the characteristics and needs of cellular cardiovascular research investigators. The analytical methods created are used to find the onset of contraction, perform signal averaging, and acquire statistical information of functional data. CFRA toolbox contractility processing yields onset time, time-to-peak, duration, and fast and slow recovery times. CFRA toolbox calcium transient signal processing yields onset time, signal intensity, and fast and slow exponential recovery rates associated with SERCA and NCX channels respectively. The toolboxes allow examination of beat-to-beat contractility and calcium transient variations within the same cardiocyte as well as from cell population to population. Supported by NIH/NIGMS SDSU MARC Program 5T34GM008303-22

Introduction of Non-Linear Elasticity Models for the Characterization of Isolated Adult Cardiocyte Contractility

We are exploring the viability of a novel approach to cardiocyte contractility assessment based on biomechanical properties of the cardiac cells, energy conservation principles, and information content measures. We define our measure of cell contraction as being the distance between the shapes of the contracting cell, assessed by the minimum total energy of the domain deformation (warping) of one cell shape into another. To guarantee a meaningful vis-à-vis correspondence between the two shapes, we employ both a data fidelity term and a regularization term. The data fidelity term is based on nonlinear features of the shapes while the regularization term enforces the compatibility between the shape deformations and that of a hyper-elastic material. We tested this approach by assessing the contractile responses in isolated adult rat cardiocytes and contrasted these measurements against two different methods for contractility assessment in the literature. Results show good qualitative and quantitative agreements with these methods as far as frequency, pacing, and overall behavior of the contractions are concerned. We hypothesize that the proposed methodology, once appropriately developed and customized, can provide a framework for computational cardiac cell biomechanics that can be used to integrate both theory and experiment. For example, besides giving a good assessment of contractile response of the cardiocyte, since the excitation process of the cell is a closed system, this methodology can be employed in an attempt to infer statistically significant model parameters for the constitutive equations of the cardiocytes.

Introduction of non-linear elasticity models for characterization of shape and deformation statistics: application to contractility assessment of isolated adult cardiocytes.

BackgroundWe are exploring the viability of a novel approach to cardiocyte contractility assessment based on biomechanical properties of the cardiac cells, energy conservation principles, and information content measures. We define our measure of cell contraction as being the distance between the shapes of the contracting cell, assessed by the minimum total energy of the domain deformation (warping) of one cell shape into another. To guarantee a meaningful vis-à-vis correspondence between the two shapes, we employ both a data fidelity term and a regularization term. The data fidelity term is based on nonlinear features of the shapes while the regularization term enforces the compatibility between the shape deformations and that of a hyper-elastic material.ResultsWe tested the proposed approach by assessing the contractile responses in isolated adult rat cardiocytes and contrasted these measurements against two different methods for contractility assessment in the literature. Our results show good qualitative and quantitative agreements with these methods as far as frequency, pacing, and overall behavior of the contractions are concerned.ConclusionsWe hypothesize that the proposed methodology, once appropriately developed and customized, can provide a framework for computational cardiac cell biomechanics that can be used to integrate both theory and experiment. For example, besides giving a good assessment of contractile response of the cardiocyte, since the excitation process of the cell is a closed system, this methodology can be employed in an attempt to infer statistically significant model parameters for the constitutive equations of the cardiocytes.

Image Processing Techniques for Assessing Contractility in Isolated Adult and Neonatal Cardiac Myocytes

We propose two computational frameworks for the assessment of contractile responses of enzymatically dissociated adult and neonatal cardiac myocytes. The proposed methodologies are variants of mathematically sound and computationally robust algorithms very well established in the image processing community. The computational pipeline for assessing contractility in adult cardiocytes comprises the following stages: digital video recording of the contracting cell, edge preserving total variation-based image smoothing, segmentation of the smoothed images, contour extraction from the segmented images, shape representation by Fourier descriptors, and contractility assessment.

Proliferating cardiac microtubules

identifying the mechanisms underlying the transition from adaptive cardiac hypertrophy to maladaptive cardiac failure has long been a central goal of basic and clinical cardiovascular science. Such studies at the level of the cardiocyte have focused largely on the cardiac motor, i.e., the

Role of the 5′‐untranslated region in regulating translational efficiency of specific mRNAs in adult cardiocytes

It has been hypothesized that translational efficiency is determined by the amount of secondary structure in the 5'-untranslated region (5'-UTR) of mRNA. Here, we examined whether specific 5'-UTRs with excessive secondary structure selectively regulate translational efficiency in adult cardiocytes. Recombinant adenoviruses were generated to express reporter mRNAs consisting of the 5'-UTR derived from c-jun or ornithine decarboxylase (ODC) fused to beta-galactosidase (betaGal) coding sequence. Each adenovirus expressed GFP mRNA as a control for 5'-UTRs with minimal secondary structure. Subsequently, cardiocytes were electrically stimulated to contract at 1 Hz to accelerate protein synthesis as compared to quiescent controls. Translational efficiency was calculated by measuring protein expression as a function of mRNA levels. Translational efficiency of c-jun/betaGal mRNA increased significantly by 3.7-fold in contracting vs. quiescent cardiocytes, but ODC/betaGal mRNA was unchanged. Contraction increased c-jun/betaGal mRNA levels in polyribosomes by 2.3-fold, which indicates that translational efficiency was enhanced by mobilization. A short, unstructured 5'-UTR was sufficient for efficient translation of betaGal mRNA in quiescent and contracting cardiocytes. GFP mRNA produced similar results. These studies demonstrate that the 5'-UTR functions as a determinant of translational efficiency of specific mRNAs, such as c-jun, that regulate growth of the adult cardiocyte.

Image processing techniques for assessing contractility in isolated adult cardiac myocytes.

We describe a computational framework for the comprehensive assessment of contractile responses of enzymatically dissociated adult cardiac myocytes. The proposed methodology comprises the following stages: digital video recording of the contracting cell, edge preserving total variation-based image smoothing, segmentation of the smoothed images, contour extraction from the segmented images, shape representation by Fourier descriptors, and contractility assessment. The different stages are variants of mathematically sound and computationally robust algorithms very well established in the image processing community. The physiologic application of the methodology is evaluated by assessing overall contraction in enzymatically dissociated adult rat cardiocytes. Our results demonstrate the effectiveness of the proposed approach in characterizing the true, two-dimensional, “shortening” in the contraction process of adult cardiocytes. We compare the performance of the proposed method to that of a popular edge detection system in the literature. The proposed method not only provides a more comprehensive assessment of the myocyte contraction process but also can potentially eliminate historical concerns and sources of errors caused by myocyte rotation or translation during contraction. Furthermore, the versatility of the image processing techniques makes the method suitable for determining myocyte shortening in cells that usually bend or move during contraction. The proposed method can be utilized to evaluate changes in contractile behavior resulting from drug intervention, disease modeling, transgeneity, or other common applications to mammalian cardiocytes.

MRNA cargo no longer on time

heart failure is the most important cause of death in the Western world, and numerous attempts have been undertaken to identify the origin of this disease. Cohn et al. ([1][1]) in 2000 defined cardiac remodeling leading to heart failure as “genome expression resulting in molecular, cellular and

Microtubule-dependent distribution of mRNA in adult cardiocytes

Synthesis of myofibrillar proteins in the diffusion-restricted adult cardiocyte requires microtubule-based active transport of mRNAs as part of messenger ribonucleoprotein particles (mRNPs) to translation sites adjacent to nascent myofibrils. This is especially important for compensatory hypertrophy in response to hemodynamic overloading. The hypothesis tested here is that excessive microtubule decoration by microtubule-associated protein 4 (MAP4) after cardiac pressure overloading could disrupt mRNP transport and thus hypertrophic growth. MAP4-overexpressing and pressure-overload hypertrophied adult feline cardiocytes were infected with an adenovirus encoding zipcode-binding protein 1-enhanced yellow fluorescent protein fusion protein, which is incorporated into mRNPs, to allow imaging of these particles. Speed and distance of particle movement were measured via time-lapse microscopy. Microtubule depolymerization was used to study microtubule-based transport and distribution of mRNPs. Protein synthesis was assessed as radioautographic incorporation of [3H]phenylalanine. After microtubule depolymerization, mRNPs persist only perinuclearly and apparent mRNP production and protein synthesis decrease. Reestablishing microtubules restores mRNP production and transport as well as protein synthesis. MAP4 overdecoration of microtubules via adenovirus infection in vitro or following pressure overloading in vivo reduces the speed and average distance of mRNP movement. Thus cardiocyte microtubules are required for mRNP transport and structural protein synthesis, and MAP4 decoration of microtubules, whether directly imposed or accompanying pressure-overload hypertrophy, causes disruption of mRNP transport and protein synthesis. The dense, highly MAP4-decorated microtubule network seen in severe pressure-overload hypertrophy both may cause contractile dysfunction and, perhaps even more importantly, may prevent a fully compensatory growth response to hemodynamic overloading.

Regulation of Ncx1 Expression: IDENTIFICATION OF REGULATORY ELEMENTS MEDIATING CARDIAC-SPECIFIC EXPRESSION AND UP-REGULATION

The Na+-Ca2+ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the -80 CArG and the -50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the -80 CArG or -50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the -80 CArG transgenic lines. The Ncx1 -50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation.

Regulation of c‐jun mRNA expression in adult cardiocytes by MAP kinase interacting kinase‐1 (MNK1)

Hypertrophic growth of adult myocardium is associated with increased expression of the early response gene c-jun. The purpose of this study was to determine whether eukaryotic initiation factor (elF) 4E (eIF4E) regulates translational efficiency of c-jun mRNA as measured by flux into polysomes. Adult feline cardiomyocytes in primary culture were treated with 0.2 microM 12-O-tetradecanoylphorbol 13-acetate (TPA), and c-jun mRNA was quantified in total, monosome, and polysome fractions by real-time polymerase chain reaction. After 1 h, TPA increased total c-jun mRNA by 10.5-fold. The corresponding flux into polysomes was significantly lower (5-fold). Adenoviral-mediated overexpression of either eIF4E or a nonphosphorylatable mutant (S209/A) did not affect total c-jun mRNA or its flux between monosomes and polysomes. Similar results were obtained following overexpression of the eIF4E kinase Mnk1. Thus, translational efficiency of c-jun mRNA was not affected by changes in activity or amount of eIF4E. In contrast, a kinase-deficient Mnk1 mutant significantly reduced total c-jun mRNA from 9.8-fold to 6.0-fold while flux between monosomes and polysomes remained constant. The decrease in total c-jun mRNA resulted from increased decay of c-jun mRNA incorporated into the polysomes. We conclude that Mnk1 activity stabilizes c-jun mRNA in polysomes independent of eIF4E phosphorylation.

Differential effect of Histone Deacetylase inhibition in neonatal and adult cardiocytes

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate gene expression both by controlling the acetylation state of nucleosomal histones and by directly interacting with transcriptional factors. Deacetylation of nucleosomal histones by HDACs results in transcriptional repression. Class II HDACs have been shown to play a role in cardiac hypertrophy, but most of the work currently published on the molecular mechanisms of how specific HDACs mediate changes in cardiac growth and gene expression have been performed in neonatal cardiocytes. Our hypothesis is that HDACs have a very different effect on the regulation of gene expression and cardiac growth in neonatal cardiocytes than they do in the adult cardiocytes. In this report, we inhibited HDAC activity with trichostatin A (TSA) and examined the hypertrophic maker gene, artial natriuretic factor (ANF), expression and cell growth in both neonatal and adult cardiocytes. Our results showed that TSA inhibited the deacetylation of histone in both neonatal and adult cardiocytes. The expression of ANF was inhibited by TSA in neonatal cardiocytes. Surprisingly, ANF promoter activity is dramatically upregulated by TSA in adult cardiocytes. Treatment with TSA reduced S6 phosphorylation and decreased the level of protein synthesis in neonatal cardiocytes. TSA treatment acts synergistically with a-adrenergic stimulation in adult cardiocytes, resulting in an increasing of S6 phosphorylation and protein synthesis. We conclude that HDACs play a very different role in the regulation of gene expression and cardiac growth in neonatal versus adult cardiocytes.

Inhibition of β-adrenergic receptor trafficking in adult cardiocytes by MAP4 decoration of microtubules

Decreased beta-adrenergic receptor (beta-AR) number occurs both in animal models of cardiac hypertrophy and failure and in patients. beta-AR recycling is an important mechanism for the beta-AR resensitization that maintains a normal complement of cell surface beta-ARs. We have shown that 1) in severe pressure overload cardiac hypertrophy, there is extensive microtubule-associated protein 4 (MAP4) decoration of a dense microtubule network; and 2) MAP4 microtubule decoration inhibits muscarinic acetylcholine receptor recycling in neuroblastoma cells. We asked here whether MAP4 microtubule decoration inhibits beta-AR recycling in adult cardiocytes. [(3)H]CGP-12177 was used as a beta-AR ligand, and feline cardiocytes were isolated and infected with adenovirus containing MAP4 (AdMAP4) or beta-galactosidase (Adbeta-gal) cDNA. MAP4 decorated the microtubules extensively only in AdMAP4 cardiocytes. beta-AR agonist exposure reduced cell surface beta-AR number comparably in AdMAP4 and Adbeta-gal cardiocytes; however, after agonist withdrawal, the cell surface beta-AR number recovered to 78.4 +/- 2.9% of the pretreatment value in Adbeta-gal cardiocytes but only to 56.8 +/- 1.4% in AdMAP4 cardiocytes (P < 0.01). This result was confirmed in cardiocytes isolated from transgenic mice having cardiac-restricted MAP4 overexpression. In functional terms of cAMP generation, beta-AR agonist responsiveness of AdMAP4 cells was 47% less than that of Adbeta-gal cells. We conclude that MAP4 microtubule decoration interferes with beta-AR recycling and that this may be one mechanism for beta-AR downregulation in heart failure.

Regulation of protein synthesis by eIF4E phosphorylation in adult cardiocytes: the consequence of secondary structure in the 5'-untranslated region of mRNA.

In adult cardiocytes, eIF4E (eukaryotic initiation factor 4E) activity and protein synthesis are increased concomitantly in response to stimuli that induce hypertrophic growth. We tested the hypothesis that increases in eIF4E activity selectively improve the translational efficiency of mRNAs that have an excessive amount of secondary structure in the 5'-UTR (5'-untranslated region). The activity of eIF4E was modified in primary cultures of adult cardiocytes using adenoviral gene transfer to increase either the amount of eIF4E or the extent of endogenous eIF4E phosphorylation. Subsequently, the effects of eIF4E on translational efficiency were assayed following adenoviral-mediated expression of luciferase reporter mRNAs that were either 'stronger' (less structure in the 5'-UTR) or 'weaker' (more structure in the 5'-UTR) with respect to translational efficiency. The insertion of G+C-rich repeats into the 5'-UTR doubled the predicted amount of secondary structure and was sufficient to reduce translational efficiency of the reporter mRNA by 48+/-13%. Translational efficiency of the weaker reporter mRNA was not significantly improved by overexpression of wild-type eIF4E when compared with the stronger reporter mRNA. In contrast, overexpression of the eIF4E kinase Mnk1 [MAP (mitogen-activated protein) kinase signal-integrating kinase 1] was sufficient to increase the translational efficiency of either reporter mRNA, independent of the amount of secondary structure in their respective 5'-UTRs. The increases in translational efficiency produced by Mnk1 occurred in association with corresponding decreases in mRNA levels. These findings indicate that the positive effect of eIF4E phosphorylation on translational efficiency in adult cardiocytes is coupled with the stability of mRNA.

Phenotypic consequences of beta1-tubulin expression and MAP4 decoration of microtubules in adult cardiocytes.

In pressure-overload cardiac hypertrophy, microtubule network densification is one cause of contractile dysfunction. Cardiac transcriptional upregulation of beta1-tubulin rather than the constitutive beta4-tubulin and of microtubule-associated protein (MAP)4 accompanies hypertrophy, with extensive microtubule decoration by MAP4. Because MAP4 stabilizes microtubules, and because the isoform-variable carboxy terminus of beta-tubulin binds to MAP4, we wished to determine whether one or both of these proteins has etiologic significance for cardiac microtubule network densification. Recombinant adenoviruses encoding beta1-tubulin, beta4-tubulin, and MAP4 were used to infect isolated cardiocytes. Overexpressed MAP4 caused a shift of tubulin dimers to the polymerized fraction and formation of a dense, stable microtubule network. Overexpressed beta1- or beta4-tubulin had neither any independent effect on these variables nor any effect additive to that of simultaneously overexpressed MAP4. Results from transgenic mice with cardiac overexpression of beta1-tubulin or MAP4 were confirmatory, but unlike the effects of brief adenovirus-mediated MAP4 overexpression in isolated cardiocytes, MAP4 transgenic hearts showed a marked increase in total alpha- and beta-tubulin. Thus MAP4 overexpression caused increased tubulin expression, formation of stable microtubules, and altered microtubule network properties, such that MAP4 upregulation may be one cause for the dense, stable microtubule network characteristic of pressure-overloaded, hypertrophied cardiocytes.

Pathways regulating Na+/Ca2+ exchanger expression in the heart.

The Na(+)/Ca(2+) exchanger (NCX1) is regulated at the transcriptional level in cardiac hypertrophy, ischemia, and failure. Following pressure overload, activation of MAPKs coincides with the kinetics of NCX1 gene upregulation in adult cardiocytes. Using adenoviral gene delivery, we begin to identify the molecular pathways responsible for upregulation of the exchanger gene. Inhibition of ERK with the MEK inhibitor UO126, the ERK protein phosphatase MKP-3, inhibited ERK activation, but only inhibited alpha-adrenergic-induced NCX1 upregulation by 30%. Overexpression of DN-JNK lowered basal NCX1 expression. Overexpression of activated MKK-3 was sufficient for alpha-adrenergic-stimulated upregulation of the reporter gene. Together, this data indicates that (1) JNK mediates basal cardiac expression of the NCX1 gene, (2) ERK and p38 play a role in alpha-adrenergic-stimulated NCX1 upregulation, and (3) p38 activation alone is sufficient for NCX1 upregulation.

Role of MAP kinases in the Na+/Ca2+ exchanger gene expression in feline adult cardiocytes.

Role of MAP kinases in the Na+/Ca2+ exchanger gene expression in feline adult cardiocytes.

Modifications of eukaryotic initiation factor 4F (eIF4F) in adult cardiocytes by adenoviral gene transfer: differential effects on eIF4F activity and total protein synthesis rates.

In adult feline cardiocytes, increases in eukaryotic initiation factor 4F (eIF4F) activity are correlated with accelerated rates of total protein synthesis produced in response to increased load. Adenoviral gene transfer was employed to increase either eIF4F complex formation or the phosphorylation of eIF4E on Ser-209. To simulate load,cardiocytes were electrically stimulated to contract (2 Hz,5 ms pulses). Non-stimulated cardiocytes were used as controls.Adenovirus-mediated overexpression of wild-type eIF4E increased the total eIF4E pool by 120-140% above endogenous levels after 24 h and produced a corresponding increase in eIF4F content.However, it did not accelerate total protein synthesis rates inquiescent cardiocytes; neither did it potentiate the increase produced by contraction. To modify the affinity of eIF4F, cardiocytes were infected with a mutant (eIF4E/W56F) with a decreased binding affinity for the mRNA cap. Overexpression of eIF4E/W56F increased the quantity of eIF4F but the rate of total protein synthesis was decreased inquiescent and contracting cardiocytes. Overexpression of a mutant that blocked eIF4E phosphorylation (eIF4E/S209A) increased the quantity ofeIF4F without any significant effect on total protein synthesis rates in quiescent or contracting cardiocytes. Overexpression of the eIF4Ekinase Mnk-1 increased eIF4E phosphorylation without a corresponding increase in eIF4F complex formation or in the rate of total protein synthesis. We conclude the following: (1) eIF4F assembly is increased by raising eIF4E levels via adenoviral gene transfer; (2) the capbinding affinity of eIF4F is a rate-limiting determinant for total protein synthesis rates; and (3) increases in the quantity of eIF4Falone or in eIF4E phosphorylation are not sufficient to accelerate total protein synthesis rates.

Modifications of eukaryotic initiation factor 4F (eIF4F) in adult cardiocytes by adenoviral gene transfer: differential effects on eIF4F activity and total protein synthesis rates

In adult feline cardiocytes, increases in eukaryotic initiation factor 4F (eIF4F) activity are correlated with accelerated rates of total protein synthesis produced in response to increased load. Adenoviral gene transfer was employed to increase either eIF4F complex formation or the phosphorylation of eIF4E on Ser-209. To simulate load, cardiocytes were electrically stimulated to contract (2Hz, 5ms pulses). Non-stimulated cardiocytes were used as controls. Adenovirus-mediated overexpression of wild-type eIF4E increased the total eIF4E pool by 120–140% above endogenous levels after 24h and produced a corresponding increase in eIF4F content. However, it did not accelerate total protein synthesis rates in quiescent cardiocytes; neither did it potentiate the increase produced by contraction. To modify the affinity of eIF4F, cardiocytes were infected with a mutant (eIF4E/W56F) with a decreased binding affinity for the mRNA cap. Overexpression of eIF4E/W56F increased the quantity of eIF4F but the rate of total protein synthesis was decreased in quiescent and contracting cardiocytes. Overexpression of a mutant that blocked eIF4E phosphorylation (eIF4E/S209A) increased the quantity of eIF4F without any significant effect on total protein synthesis rates in quiescent or contracting cardiocytes. Overexpression of the eIF4E kinase Mnk-1 increased eIF4E phosphorylation without a corresponding increase in eIF4F complex formation or in the rate of total protein synthesis. We conclude the following: (1) eIF4F assembly is increased by raising eIF4E levels via adenoviral gene transfer; (2) the cap binding affinity of eIF4F is a rate-limiting determinant for total protein synthesis rates; and (3) increases in the quantity of eIF4F alone or in eIF4E phosphorylation are not sufficient to accelerate total protein synthesis rates.

Cardiocyte cytoskeleton in hypertrophied myocardium.

The Frank-Starling mechanism, by which load directly regulates muscle length and thus performance is the means by which the mechanics and energetics of cardiac muscle are regulated on a beat-to-beat basis. When this short-term compensation for increased load is insufficient, the long-term compensation of cardiac hypertrophy ensues. The simplest and most direct mechanism for load regulation of cardiac mass would obtain if an analog of the short-term Frank-Starling mechanism of functional regulation operated in the long-term time domain of mass regulation; that is, if heart muscle were able to directly transduce increased load into growth. It is now clear that load does indeed serve as a direct regulator of cardiac mass in the adult. Cardiac hypertrophy, at the levels of intact animal, isolated tissue, and cultured cells, is a direct response of the adult mammalian cardiocyte to increased load, modified by but without the requisite involvement of factors external to the cell. The extent to which such hypertrophy is compensatory is critically dependent on the type of hemodynamic overload that serves as the hypertrophic stimulus. Thus, cardiac hypertrophy is not intrinsically maladaptive; rather, it is the nature of the inducing load rather than hypertrophy itself that is responsible for the frequent deterioration of initially compensatory hypertrophy into the congestive heart failure state. As one example reviewed here of this load specificity of maladaptation, increased microtubule network density is a persistent feature of severely pressure overloaded, hypertrophied and failing myocardium which imposes a viscous load on active myofilaments during contraction.