Early and transient gene expression changes in pressure overload-induced cardiac hypertrophy in mice

Cardiac hypertrophy is an important risk factor for cardiac morbidity and mortality. To unravel the underlying pathogenic genetic pathways, we hybridized left ventricular RNA from Transverse Aortic Constriction mice at 48 h, 1 week, and 2, 3, and 8 weeks after surgery to microarrays containing a 15K fetal cDNA collection. Key processes involved an early restriction in the expression of metabolic genes, accompanied by increased expression of genes related to growth and reactivation of fetal genes. Most of these genes returned to basal expression levels during the later, compensated hypertrophic phase. Our findings suggest that compensated hypertrophy in these mice is established by rapid adaptation of the heart at the cost of gene expression associated with metabolic activity, with only temporary expression of possible maladaptive processes. Therefore, the transient early changes may reflect a beneficial response to pressure overload, as deterioration of cardiac hemodynamic function or heart failure does not occur.     

Regression of pressure overload-induced left ventricular hypertrophy in mice

As a prelude to investigating the mechanism of regression of pressure overload-induced left ventricular (LV) hypertrophy (LVH), we studied the time course for the development and subsequent regression of LVH as well as accompanying alterations in cardiac function, histology, and gene expression. Mice were subjected to aortic banding for 4 or 8 wk to establish LVH, and regression was initiated by release of aortic banding for 6 wk. Progressive increase in LV mass and gradual chamber dilatation and dysfunction occurred after aortic banding. LVH was also associated with myocyte enlargement, interstitial fibrosis, and enhanced expression of atrial natriuretic peptide, collagen I, collagen III, and matrix metalloproteinase-2 but suppressed expression of alpha-myosin heavy chain and sarcoplasmic reticulum Ca(2+)-ATPase. Aortic debanding completely or partially reversed LVH, chamber dilatation and dysfunction, myocyte size, interstitial fibrosis, and gene expression pattern, each with a distinct time course. The extent of LVH regression was dependent on the duration of pressure overload, evidenced by the fact that restoration of LV structure and function was complete in animals subjected to 4 wk of aortic banding but incomplete in animals subjected to 8 wk of aortic banding. In conclusion, LVH regression comprises a variety of morphological, functional, and genetic components that show distinct time courses. A longer period of pressure overload is associated with a slower rate of LVH regression.     

Growth/differentiation factor 1 alleviates pressure overload-induced cardiac hypertrophy and dysfunction

Pathological cardiac hypertrophy is a major risk factor for developing heart failure, the leading cause of death in the world. Growth/differentiation factor 1 (GDF1), a transforming growth factor-β family member, is a regulator of cell growth and differentiation in both embryonic and adult tissues. Evidence from human and animal studies suggests that GDF1 may play an important role in cardiac physiology and pathology. However, a critical role for GDF1 in cardiac remodelling has not been investigated. Here, we performed gain-of-function and loss-of-function studies using cardiac-specific GDF1 knockout mice and transgenic mice to determine the role of GDF1 in pathological cardiac hypertrophy, which was induced by aortic banding (AB). The extent of cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. Our results demonstrated that cardiac specific GDF1 overexpression in the heart markedly attenuated cardiac hypertrophy, fibrosis, and cardiac dysfunction, whereas loss of GDF1 in cardiomyocytes exaggerated the pathological cardiac hypertrophy and dysfunction in response to pressure overload. Mechanistically, we revealed that the cardioprotective effect of GDF1 on cardiac remodeling was associated with the inhibition of the MEK–ERK1/2 and Smad signaling cascades. Collectively, our data suggest that GDF1 plays a protective role in cardiac remodeling via the negative regulation of the MEK–ERK1/2 and Smad signaling pathways.     

Blockade of MyD88 attenuates cardiac hypertrophy and decreases cardiac myocyte apoptosis in pressure overload-induced cardiac hypertrophy in vivo

In this study, we evaluated whether blocking myeloid differentiation factor-88 (MyD88) could decrease cardiac myocyte apoptosis following pressure overload. Adenovirus expressing dominant negative MyD88 (Ad5-dnMyD88) or Ad5-green fluorescent protein (GFP) (Ad5-GFP) was transfected into rat hearts (n = 8/group) immediately followed by aortic banding for 3 wk. One group of rats (n = 8) was subjected to aortic banding for 3 wk without transfection. Sham surgical operation (n = 8) served as control. The ratios of heart weight to body weight (HW/BW) and heart weight to tibia length (HW/TL) were calculated. Cardiomyocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. Cardiac myocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and myocardial interstitial fibrosis was examined by Masson's Trichrome staining. Aortic banding significantly increased the HW/BW by 41.0% (0.44 +/- 0.013 vs. 0.31 +/- 0.008), HW/TL by 47.2% (42.7 +/- 1.30 vs. 29.0 +/- 0.69), cardiac myocyte size by 49.6%, and cardiac myocyte apoptosis by 11.5%, and myocardial fibrosis and decreased cardiac function compared with sham controls. Transfection of Ad5-dnMyD88 significantly reduced the HW/BW by 18.2% (0.36 +/- 0.006 vs. 0.44 +/- 0.013) and HW/TL by 22.3% (33.2 +/- 0.95 vs. 42.7 +/- 1.30) and decreased cardiomyocyte size by 56.8%, cardiac myocyte apoptosis by 76.2%, as well as fibrosis, and improved cardiac function compared with aortic-banded group. Our results suggest that MyD88 is an important component in the Toll-like receptor-4-mediated nuclear factor-kappaB activation pathway that contributes to the development of cardiac hypertrophy. Blockade of MyD88 significantly reduced cardiac hypertrophy, cardiac myocyte apoptosis, and improved cardiac function in vivo.     

gp130 plays a critical role in pressure overload-induced cardiac hypertrophy

gp130, a common receptor for the interleukin 6 family, plays pivotal roles in growth and survival of cardiac myocytes. In the present study, we examined the role of gp130 in pressure overload-induced cardiac hypertrophy using transgenic (TG) mice, which express a dominant negative mutant of gp130 in the heart under the control of alpha myosin heavy chain promoter. TG mice were apparently healthy and fertile. There were no differences in body weight and heart weight between TG mice and littermate wild type (WT) mice. Pressure overload-induced increases in the heart weight/body weight ratio, ventricular wall thickness, and cross-sectional areas of cardiac myocytes were significantly smaller in TG mice than in WT mice. Northern blot analysis revealed that pressure overload-induced up-regulation of brain natriuretic factor gene and down-regulation of sarcoplasmic reticulum Ca(2+) ATPase 2 gene were attenuated in TG mice. Pressure overload activated ERKs and STAT3 in the heart of WT mice, whereas pressure overload-induced activation of STAT3, but not of ERKs, was suppressed in TG mice. These results suggest that gp130 plays a critical role in pressure overload-induced cardiac hypertrophy possibly through the STAT3 pathway.     

Doxycycline inducible expression of SERCA2a improves calcium handling and reverts cardiac dysfunction in pressure overload-induced cardiac hypertrophy

Delayed cardiac relaxation in failing hearts has been attributed to reduced activity and/or expression of sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a). Although constitutive overexpression of SERCA2a has proven effective in preventing cardiac dysfunction, it is unclear whether increasing SERCA2a expression in hearts with preexisting hypertrophy will be therapeutic. To test this hypothesis, we generated a binary transgenic (BTG) system that allows tetracycline-inducible, cardiac-specific SERCA2a expression. In this system (tet-on SERCA2a), a FLAG-tagged SERCA2a transgene is expressed in the presence of doxycycline (Dox) but not in the absence of Dox (2.3-fold more mRNA, 45% more SERCA2a protein). Calcium transients measured in isolated cardiac myocytes from nonbanded Dox-treated BTG mice showed an accelerated calcium decline and an increased systolic Ca2+ peak. Sarcoplasmic reticulum (SR) calcium loading was increased by 45% in BTG mice. In the presence of pressure overload (aortic banding), echocardiographic analysis revealed that expression of SERCA2a-FLAG caused an improvement in fractional shortening. SERCA2a-FLAG expression alleviated the resultant cardiac dysfunction. This was illustrated by an increase in the rate of decline of the calcium transient. Cell shortening and SR calcium loading were also improved in cardiac myocytes isolated from banded BTG mice after SERCA2a overexpression. In conclusion, we generated a novel transgenic mouse that conditionally overexpresses SERCA2a. This model is suitable for both long- and short-term studies of the effects of controlled SERCA2a expression on cardiac function. In addition, inducible overexpression of SERCA2a improved cardiac function and calcium handling in mice with established contractile dysfunction.     

The antioxidant tempol attenuates pressure overload-induced cardiac hypertrophy and contractile dysfunction in mice fed a high-fructose diet

We have previously shown that high-sugar diets increase mortality and left ventricular (LV) dysfunction during pressure overload. The mechanisms behind these diet-induced alterations are unclear but may involve increased oxidative stress in the myocardium. The present study examined whether high-fructose feeding increased myocardial oxidative damage and exacerbated systolic dysfunction after transverse aortic constriction (TAC) and if this effect could be attenuated by treatment with the antioxidant tempol. Immediately after surgery, TAC and sham mice were assigned to a high-starch diet (58% of total energy intake as cornstarch and 10% fat) or high-fructose diet (61% fructose and 10% fat) with or without the addition of tempol [0.1% (wt/wt) in the chow] and maintained on the treatment for 8 wk. In response to TAC, fructose-fed mice had greater cardiac hypertrophy (55.1% increase in the heart weight-to-tibia length ratio) than starch-fed mice (22.3% increase in the heart weight-to-tibia length ratio). Treatment with tempol significantly attenuated cardiac hypertrophy in fructose-fed TAC mice (18.3% increase in the heart weight-to-tibia ratio). Similarly, fructose-fed TAC mice had a decreased LV area of fractional shortening (from 38+/-2% in sham to 22+/-4% in TAC), which was prevented by tempol treatment (33+/-3%). Markers of lipid peroxidation in fructose-fed TAC hearts were also blunted by tempol. In conclusion, tempol significantly blunted markers of cardiac hypertrophy, LV remodeling, contractile dysfunction, and oxidative stress in fructose-fed TAC mice.     

PYK2 expression and phosphorylation increases in pressure overload-induced left ventricular hypertrophy

Proline-rich tyrosine kinase 2 (PYK2) is a member of the focal adhesion kinase (FAK) family of nonreceptor protein tyrosine kinases. PYK2 has been implicated in linking G protein-coupled receptors to activation of mitogen-activated protein kinase cascades and cellular growth in a variety of cell types. To determine whether PYK2 expression and phosphorylation is altered in left ventricular (LV) myocardium undergoing LV hypertrophy (LVH) and heart failure in vivo, suprarenal abdominal aortic coarctation was performed in 160-g male Sprague-Dawley rats. Immunohistochemistry and Western blotting were performed on LV tissue 1, 8, and 24 wk after aortic banding. Aortic banding produced sustained hypertension and gradually developing LVH. PYK2 levels were increased 1.8 +/- 0.2-, 2.7 +/- 0.6-, and 2.0 +/- 0.2-fold in 1-, 8-, and 24-wk banded animals compared with their respective sham-operated controls. The increase in PYK2 expression was paralleled by an increase in PYK2 phosphorylation, both of which preceded the development of LVH. Immunohistochemistry revealed that enhanced PYK2 expression occurred predominantly in the cardiomyocyte population. Furthermore, there was a high degree of correlation (R = 0.75; P < 0.001) between the level of PYK2 and the degree of LVH in 24-wk sham and banded animals. In contrast, FAK levels and FAK phosphorylation were not increased before the development of LVH. However, there was a high degree of correlation (R = 0.68; P < 0.001) between the level of FAK and the degree of LVH in 24-wk sham and banded rats. There was also a significant increase in the ratio of phosphospecific anti-FAK to FAK at this time point. These data are consistent with a role for PYK2 in the induction of pressure overload-induced cardiomyocyte hypertrophy, and suggest that PYK2 and FAK have distinctly different roles in LVH progression.     

Expression profiling reveals differences in metabolic gene expression between exercise-induced cardiac effects and maladaptive cardiac hypertrophy

While cardiac hypertrophy elicited by pathological stimuli eventually leads to cardiac dysfunction, exercise-induced hypertrophy does not. This suggests that a beneficial hypertrophic phenotype exists. In search of an underlying molecular substrate we used microarray technology to identify cardiac gene expression in response to exercise. Rats exercised for seven weeks on a treadmill were characterized by invasive blood pressure measurements and echocardiography. RNA was isolated from the left ventricle and analysed on DNA microarrays containing 8740 genes. Selected genes were analysed by quantitative PCR. The exercise program resulted in cardiac hypertrophy without impaired cardiac function. Principal component analysis identified an exercise-induced change in gene expression that was distinct from the program observed in maladaptive hypertrophy. Statistical analysis identified 267 upregulated genes and 62 downregulated genes in response to exercise. Expression changes in genes encoding extracellular matrix proteins, cytoskeletal elements, signalling factors and ribosomal proteins mimicked changes previously described in maladaptive hypertrophy. Our most striking observation was that expression changes of genes involved in beta-oxidation of fatty acids and glucose metabolism differentiate adaptive from maladaptive hypertrophy. Direct comparison to maladaptive hypertrophy was enabled by quantitative PCR of key metabolic enzymes including uncoupling protein 2 (UCP2) and fatty acid translocase (CD36). DNA microarray analysis of gene expression changes in exercise-induced cardiac hypertrophy suggests that a set of genes involved in fatty acid and glucose metabolism could be fundamental to the beneficial phenotype of exercise-induced hypertrophy, as these changes are absent or reversed in maladaptive hypertrophy.     

Zerumbone prevents pressure overload-induced left ventricular systolic dysfunction by inhibiting cardiac hypertrophy and fibrosis

BackgroundCardiac hypertrophy and fibrosis are hallmarks of cardiac remodeling and are involved functionally in the development of heart failure (HF). However, it is unknown whether Zerumbone (Zer) prevents left ventricular (LV) systolic dysfunction by inhibiting cardiac hypertrophy and fibrosis.PurposeThis study investigated the effect of Zer on cardiac hypertrophy and fibrosis in vitro and in vivo.Study Design/methodsIn primary cultured cardiac cells from neonatal rats, the effect of Zer on phenylephrine (PE)-induced hypertrophic responses and transforming growth factor beta (TGF-β)-induced fibrotic responses was observed. To determine whether Zer prevents the development of pressure overload-induced HF in vivo, a transverse aortic constriction (TAC) mouse model was utilized. Cardiac function was evaluated by echocardiography. The changes of cardiomyocyte surface area were observed using immunofluorescence staining and histological analysis (HE and WGA staining). Collagen synthesis and fibrosis formation were measured by scintillation counter and picrosirius staining, respectively. The total mRNA levels of genes associated with hypertrophy (ANF and BNP) and fibrosis (Postn and α-SMA) were measured by qRT-PCR. The protein expressions (Akt and α-SMA) were assessed by western blotting.ResultsZer significantly suppressed PE-induced increase in cell size, mRNA levels of ANF and BNP, and Akt phosphorylation in cardiomyocytes. The TGF-β-induced increase in proline incorporation, mRNA levels of Postn and α-SMA, and protein expression of α-SMA were decreased by Zer in cultured cardiac fibroblasts. In the TAC male C57BL/6 mice, echocardiography results demonstrated that Zer improved cardiac function by increasing LV fractional shortening and reducing LV wall thickness compared with the vehicle group. ZER significantly reduced the level of phosphorylated Akt both in cultured cardiomyocytes treated with PE and in the hearts of TAC. Finally, Zer inhibited the pressure overload-induced cardiac hypertrophy and cardiac fibrosis.ConclusionZer ameliorates pressure overload-induced LV dysfunction, at least in part by suppressing both cardiac hypertrophy and fibrosis.     

Effect of pressure overload-induced hypertrophy on the expression and localization of p38 MAP kinase isoforms in the mouse heart

p38 mitogen-activated protein kinases (MAPKs) are serine/threonine specific protein kinases that respond to cellular stress and regulate a broad range of cellular activities. There are four major isoforms of p38 MAPK: α, β, γ, and δ. To date, the prominent isoform in heart has been thought to be p38α. We examined the expression of each p38 isoform at both the mRNA and protein level in murine heart. mRNA for all four p38 isoforms was detected. p38γ and p38δ were expressed at protein levels comparable to p38α and 38β, respectively. In the early phase of pressure-overload hypertrophy (1–7 days after constriction of the transverse aorta), the abundance of p38β, p38γ and p38δ mRNA increased; however, no corresponding changes were detected at the protein level. Confocal immunofluorescence studies revealed p38α and p38γ in both the cytoplasm and nucleus. In the established phase of hypertrophy induced by chronic pressure overload (7–28 days after constriction of the transverse aorta), p38γ immunoreactivity accumulated in the nucleus whereas the distribution of p38α remained unaffected. Hence, both p38α and p38γ are prominent p38 isoforms in heart and p38γ may play a role in mediating the changes in gene expression associated with cardiac remodeling during pressure-overload hypertrophy.     

Switch from actin alpha1 to alpha2 expression and upregulation of biomarkers for pressure overload and cardiac hypertrophy in taurine-deficient mouse heart

Taurine is the most abundant free amino acid in heart muscle and protects against heart failure. In the present study, the consequences of hereditary taurine deficiency on cardiac gene expression were examined in 2- and 15-16-month-old taurine transporter knockout (taut(-/-)) mice using a mouse-specific DNA microarray. This oligonucleotide-based microarray contains probes for 251 genes with relevance for heart function. Of these, 163 probes exhibited a reproducible hybridization signal and were analyzed. alpha-Actin type 1 mRNA levels were 70% lower in the heart of young and older taut(-/-) mice compared to wild-type controls. Interestingly, the hearts of taut(-/-) mice showed a switch from alpha-actin 1 to alpha-actin 2 expression, as confirmed by real-time PCR and Western blot analysis. In addition, mRNA levels of biomarkers for pressure overload and hypertension were upregulated in taut(-/-) hearts, i.e., atrial natriuretic factor (+848%), brain natriuretic peptide (+90%), cardiac ankyrin repeat protein (+118%), and procollagen 1a1, 1a2 and 3a1 (+40% at least). These results point to a stress situation in the heart of taut(-/-) mice under laboratory conditions, and it can be speculated that taut(-/-) hearts may be even more susceptible to failure in the wild when under exogenous stress.     

Melatonin ameliorates pressure overload-induced cardiac hypertrophy by attenuating Atg5-dependent autophagy and activating the Akt/mTOR pathway

Cardiac hypertrophy, including hypertension and valvular dysfunction, is a pathological feature of many cardiac diseases that ultimately leads to heart failure. Melatonin confers a protective role against pathological cardiac hypertrophy, but the underlying mechanisms remain elusive. In the present study, we hypothesized that melatonin protects against pressure overload-induced cardiac hypertrophy by attenuating Atg5-dependent autophagy and activating the Akt/mTOR pathway. Male C57BL/6 mice that received adenovirus carrying cardiac-specific Atg5 (under the cTNT promoter; Ad-cTNT-Atg5) underwent transverse aortic constriction (TAC) or sham operation and received an intraperitoneal injection of melatonin (10 mg/kg/d), vehicle or LY294002 (10 mg/kg/d) for 8 weeks. Melatonin treatment for 8 weeks markedly attenuated cardiac hypertrophy and restored impaired cardiac function, as indicated by a decreased HW/BW ratio, reduced cell cross-sectional area and fibrosis, downregulated the mRNA levels of ANP, BNP, and β-MHC and ameliorated adverse effects on the LVEF and LVFS. Melatonin treatment also inhibited apoptosis and alleviated autophagy dysfunction. Furthermore, melatonin inhibited Akt/mTOR pathway activation, while these effects were blocked by LY294002. In addition, the effect of melatonin regulation on TAC-induced autophagy dysfunction was inhibited by LY294002 or cardiac-specific Atg5 overexpression. As expected, Akt/mTOR pathway inhibition or cardiac-specific Atg5 overexpression restrained melatonin alleviation of pressure overload-induced cardiac hypertrophy. These results demonstrated that melatonin ameliorated pressure overload-induced cardiac hypertrophy by attenuating Atg5-dependent autophagy and activating the Akt/mTOR pathway.     

Phosphorylation of phospholamban at threonine-17 reduces cardiac adrenergic contractile responsiveness in chronic pressure overload-induced hypertrophy

Physiological hemodynamic stress, such as aerobic exercise, is intermittent and requires an increase in Ca2+ -dependent contractility through sympathetic nervous system activation. Pathological hemodynamic stress, such as hypertension, is persistent and requires sustained increases in cardiac function. Over time, this causes left ventricular hypertrophy (LVH)-reduced responsiveness to sympathetic stimulation. In this study, we examined the hypothesis that blunted in vivo adrenergic contractile responsiveness in pressure overload (PO)-induced cardiac hypertrophy is caused by abnormalities in the abundance and/or basal phosphorylation state of Ca2+ regulatory proteins. PO, induced by aortic constriction, caused concentric LVH or dilated LVH. Only animals with dilation exhibited a decrease in baseline left ventricle function [fractional area change (FAC); measured with echocardiography]. All PO animals had a reduced contractile response to adrenergic agonists (increase in FAC with 40 microg.kg(-1).min(-1) dobutamine, control 0.30 +/- 0.04, n = 5 vs. banded 0.10 +/- 0.03, n = 10; P < 0.01). PO animals had reduced phospholamban (PLB) protein abundance (P = 0.07, not significant) and increased PLB phosphorylation at the calmodulin-dependent kinase II (CaMKII)-specific site (PLB-Thr17, P < 0.05) but not at the protein kinase A-specific site (PLB-Ser16). PLB-Thr17 phosphorylation was inversely correlated with dobutamine-induced increases in contractility in PO animals (r2 = 0.81, P < 0.05). Continuous induction of Ca2+ transients in isolated ventricular myocytes for 24 h increased phosphorylation at PLB-Thr17 and diminished inotropic responsiveness and PLB-Ser16 phosphorylation after exposure to isoproterenol (P < 0.05). These data show that reduced adrenergic responsiveness in feline PO hypertrophy and failure involves increases in basal PLB-Thr17 phosphorylation, suggesting that activation of CaMKII in PO hypertrophy contributes to defective adrenergic reserve in compensated LVH and early heart failure.     

Glycogen synthase kinase-3alpha reduces cardiac growth and pressure overload-induced cardiac hypertrophy by inhibition of extracellular signal-regulated kinases

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase having multiple functions and consisting of two isoforms, GSK-3alpha and GSK-3beta. Pressure overload increases expression of GSK-3alpha but not GSK-3beta. Despite our wealth of knowledge about GSK-3beta, the function of GSK-3alpha in the heart is not well understood. To address this issue, we made cardiac-specific GSK-3alpha transgenic mice (Tg). Left ventricular weight and cardiac myocyte size were significantly smaller in Tg than in non-Tg (NTg) mice, indicating that GSK-3alpha inhibits cardiac growth. After 4 weeks of aortic banding (transverse aortic constriction (TAC)), increases in left ventricular weight and myocyte size were significantly smaller in Tg than in NTg, indicating that GSK-3alpha inhibits cardiac hypertrophy. More severe cardiac dysfunction developed in Tg after TAC. Increases in fibrosis and apoptosis were greater in Tg than in NTg after TAC. Among signaling molecules screened, ERK phosphorylation was decreased in Tg. Adenovirus-mediated overexpression of GSK-3alpha, but not GSK-3beta, inhibited ERK in cultured cardiac myocytes. Knockdown of GSK-3alpha increased ERK phosphorylation, an effect that was inhibited by PD98059, rottlerin, and protein kinase Cepsilon (PKCepsilon) inhibitor peptide, suggesting that GSK-3alpha inhibits ERK through PKC-MEK-dependent mechanisms. Knockdown of GSK-3alpha increased protein content and reduced apoptosis, effects that were abolished by PD98059, indicating that inhibition of ERK plays a major role in the modulation of cardiac growth and apoptosis by GSK-3alpha. In conclusion, up-regulation of GSK-3alpha inhibits cardiac growth and pressure overload-induced cardiac hypertrophy but increases fibrosis and apoptosis in the heart. The anti-hypertrophic and pro-apoptotic effect of GSK-3alpha is mediated through inhibition of ERK.     

Reduced cardiac hypertrophy and altered blood pressure control in transgenic rats with the human tissue kallikrein gene.

To evaluate the cardiovascular actions of kinins, we established a transgenic rat line harboring the human tissue kallikrein gene, TGR(hKLK1). Under the control of the zinc-inducible metallothionein promoter, the transgene was expressed in most tissues including the heart, kidney, lung, and brain, and human kallikrein was detected in the urine of transgenic animals. Transgenic rats had a lower 24-h mean arterial pressure in comparison with control rats, which was further decreased when their diet was supplemented with zinc. The day/night rhythm of blood pressure was significantly diminished in TGR(hKLK1) animals, whereas the circadian rhythms of heart rate and locomotor activity were unaffected. Induction of cardiac hypertrophy by isoproterenol treatment revealed a marked protective effect of the kallikrein transgene because the cardiac weight of TGR(hKLK1) increased significantly less, and the expression of atrial natriuretic peptide and collagen III as markers for hypertrophy and fibrosis, respectively, were less enhanced. The specific kinin-B2 receptor antagonist, icatibant, abolished this cardioprotective effect. In conclusion, the kallikrein-kinin system is an important determinant in the regulation of blood pressure and its circadian rhythmicity. It also exerts antihypertrophic and antifibrotic actions in the heart.     

Rho-kinase inhibition reverses impaired Ca2+ handling and associated left ventricular dysfunction in pressure overload-induced cardiac hypertrophy

Recent studies have implicated a relationship between RhoA/ROCK activity and defective Ca²⁺ homeostasis in hypertrophic hearts. This study investigated molecular mechanism underlying ROCK inhibition-mediated cardioprotection against pressure overload-induced cardiac hypertrophy, with a focus on Ca²⁺ homeostasis.Cardiac hypertrophy model was established by performing transverse aortic constriction (TAC) in 8-week-old male rats. Groups were assigned as SHAM, TAC and TAC + Fas (rats undergoing TAC and treated with fasudil). Rats in the TAC + Fas group were administered fasudil (5 mg/kg/day), and rats in the SHAM and TAC groups were treated with vehicle for 10 weeks. Electrophysiological recordings were obtained from isolated left ventricular myocytes and expression levels of proteins were determined using western blotting. Rats in the TAC group showed remarkable cardiac hypertrophy, and fasudil treatment significantly reversed this alteration. TAC + Fas myocytes showed significant improvement in reduced contractility and Ca²⁺ transients. Moreover, these myocytes showed restoration of slow relaxation rate and Ca²⁺ reuptake. Although L-type Ca²⁺ currents did not change in TAC group, there was a significant reduction in the triggered Ca²⁺ transients which was reversed either by long-term fasudil treatment or incubation of TAC myocytes with fasudil. The hearts of rats in the TAC group showed a significant decrease in ROCK1, ROCK2, RyR2 protein levels and p-PLB^S16/T17/SERCA2 ratio and increase in RhoA expression and MLC phosphorylation. However, fasudil treatment largely reversed TAC-induced alterations in protein expression.Thus, our findings indicate that upregulation of the RhoA/ROCK pathway is significantly associated with cardiac hypertrophy-related Ca²⁺ dysregulation and suggest that ROCK inhibition prevents hypertrophic heart failure.