Abstract-Upregulation of ␣B-crystallin (CryAB), a small heat shock protein, is associated with a variety of diseases, including the desmin-related myopathies. CryAB, which binds to both desmin and cytoplasmic actin, may participate as a chaperone in intermediate filament formation and maintenance, but the physiological consequences of CryAB upregulation are unknown. A mutation in CryAB, R120G, has been linked to a familial desminopathy. However, it is unclear whether the mutation is directly causative. We created multiple transgenic mouse lines that overexpressed either murine wild-type CryAB or the R120G mutation in cardiomyocytes. Overexpression of wild-type CryAB was relatively benign, with no increases in mortality and no induction of desmin-related cardiomyopathy even in a line in which CryAB mRNA expression was increased Ϸ104-fold and the protein level increased by 11-fold. In contrast, lines expressing the R120G mutation were compromised, with a high-expressing line exhibiting 100% mortality by early adulthood. Modest expression levels resulted in a phenotype that was strikingly similar to that observed for the desmin-related cardiomyopathies. The desmin filaments in the cardiomyocytes were overtly affected, myofibril alignment was significantly impaired, and a hypertrophic response occurred at both the molecular and cellular levels. The data show that the R120G mutation causes a desminopathy, is dominant negative, and results in cardiac hypertrophy. Key Words: transgenic Ⅲ heart disease Ⅲ mouse Ⅲ cardiac Ⅲ genetics T he small heat shock-related protein ␣B-crystallin (CryAB) was originally discovered and classified as a lens protein. 1 CryAB is also found in nonlenticular tissues and is abundant in cardiac and skeletal muscle. 2,3 CryAB binds both desmin and cytoplasmic actin and possesses molecular chaperone function in vitro. 4 -6 When a cell is subjected to stress, CryAB transits from the cytosol onto the cytoskeleton. 7 Phosphorylation by mitogen-activated protein kinase, p38, and other kinases may regulate this translocation and presumably its chaperone function. 8,9 The upregulation of the gene and subsequent accumulation of CryAB occurs in a number of cardiac disorders including familial hypertrophic cardiomyopathy and desminopathy, 10 -12 as well as degenerative neural pathologies such as Alexander and Alzheimer diseases. 2,13 However, the pathophysiological significance, if any, of CryAB protein upregulation in muscle remains obscure.A missense mutation (R120G) of CryAB has recently been linked to familial desmin-related myopathy (DRM), a disease that is characterized by intrasarcoplasmic accumulation of desmin. 14 Restrictive, hypertrophic, and dilated cardiomyopathies have all been observed in the desminopathies and often result in death. 12,15 Overexpression of R120G-CryAB in a muscle cell line caused formation of electron-dense aggregates containing CryAB in the center and desmin at the periphery. 14 However, there is no direct in vivo evidence, outside of linkage analysis, proving that th...

Mutations in the gene encoding the thiazide-sensitive Na ؉ -Cl ؊ cotransporter (NCC) of the distal convoluted tubule cause Gitelman's syndrome, an inherited hypokalemic alkalosis with hypomagnesemia and hypocalciuria. These metabolic abnormalities are secondary to the deficit in NaCl reabsorption, but the underlying mechanisms are unclear. To gain a better understanding of the role of NCC in sodium and fluid volume homeostasis and in the pathogenesis of Gitelman's syndrome, we used gene targeting to prepare an NCC-deficient mouse. Null mutant (Ncc ؊/؊ ) mice appear healthy and are normal with respect to acid-base balance, plasma electrolyte concentrations, serum aldosterone levels, and blood pressure. Ncc ؊/؊ mice retain Na ؉ as well as wild-type mice when fed a Na ؉ -depleted diet; however, after 2 weeks of Na ؉ depletion the mean arterial blood pressure of Ncc ؊/؊ mice was significantly lower than that of wild-type mice. In addition, Ncc ؊/؊ mice exhibited increased renin mRNA levels in kidney, hypomagnesemia and hypocalciuria, and morphological changes in the distal convoluted tubule. These data indicate that the loss of NCC activity in the mouse causes only subtle perturbations of sodium and fluid volume homeostasis, but renal handling of Mg 2؉ and Ca 2؉ are altered, as observed in Gitelman's syndrome.

Angiotensin II (Ang II), a potent hypertrophic stimulus, causes significant increases in TGFb1 gene expression. However, it is not known whether there is a causal relationship between increased levels of TGF-beta1 and cardiac hypertrophy. Echocardiographic analysis revealed that TGF-beta1-deficient mice subjected to chronic subpressor doses of Ang II had no significant change in left ventricular (LV) mass and percent fractional shortening during Ang II treatment. In contrast, Ang II-treated wild-type mice showed a >20% increase in LV mass and impaired cardiac function. Cardiomyocyte cross-sectional area was also markedly increased in Ang II-treated wild-type mice but unchanged in Ang II-treated TGF-beta1-deficient mice. No significant levels of fibrosis, mitotic growth, or cytokine infiltration were detected in Ang II-treated mice. Atrial natriuretic factor expression was approximately 6-fold elevated in Ang II-treated wild-type, but not TGF-beta1-deficient mice. However, the alpha- to beta-myosin heavy chain switch did not occur in Ang II-treated mice, indicating that isoform switching is not obligatorily coupled with hypertrophy or TGF-beta1. The Ang II effect on hypertrophy was shown not to result from stimulation of the endogenous renin-angiotensis system. These results indicate that TGF-beta1 is an important mediator of the hypertrophic growth response of the heart to Ang II.

The sarco(endo)plasmic reticulum Ca 2؉ -ATPase isoform 2 (SERCA2) gene encodes both SERCA2a, the cardiac sarcoplasmic reticulum Ca 2؉ pump, and SERCA2b, which is expressed in all tissues. To gain a better understanding of the physiological functions of SERCA2, we used gene targeting to develop a mouse in which the promoter and 5 end of the gene were eliminated. Mating of heterozygous mutant mice yielded wild-type and heterozygous offspring; homozygous mutants were not observed. RNase protection, Western blotting, and biochemical analysis of heart samples showed that SERCA2 mRNA was reduced by ϳ45% in heterozygous mutant hearts and that SERCA2 protein and maximal velocity of Ca 2؉ uptake into the sarcoplasmic reticulum were reduced by ϳ35%. Measurements of cardiovascular performance via transducers in the left ventricle and right femoral artery of the anesthetized mouse revealed reductions in mean arterial pressure, systolic ventricular pressure, and the absolute values of both positive and negative dP/dt in heterozygous mutants. These results demonstrate that two functional copies of the SERCA2 gene are required to maintain normal levels of SERCA2 mRNA, protein, and Ca 2؉ sequestering activity, and that the deficit in Ca 2؉ sequestering activity due to the loss of one copy of the SERCA2 gene impairs cardiac contractility and relaxation. The SERCA21 gene encodes two Ca 2ϩ -transporting ATPases, SERCA2a and SERCA2b, which differ in their C-terminal sequences as a result of alternative splicing (1-3). SERCA2a is expressed at highest levels in heart, where it plays a central role in cardiomyocyte Ca 2ϩ handling required for excitation/ contraction coupling (reviewed in Ref. 4). Contraction is initiated by an increase in Ca 2ϩ concentrations around the myofibrils, which occurs as Ca 2ϩ is released from the SR or enters the cell via channels in the sarcolemma. SERCA2a pumps Ca 2ϩ out of the cytosol and into the SR, thereby contributing to the low diastolic Ca 2ϩ levels required for relaxation and replenishing Ca 2ϩ stores needed for the next contraction. SERCA2a serves a similar function in slow twitch skeletal muscle and is also expressed in some smooth muscles (5). In contrast to the limited tissue distribution and organ-specific function of SERCA2a, SERCA2b is expressed in all tissues and it has been suggested that it plays an essential housekeeping role (1), although it undoubtedly serves some organ-specific functions as well.An important role for SERCA2a in cardiac function is well established. Recent studies have shown that the levels of SERCA2a are decreased in several animal models of cardiac hypertrophy and human heart failure (reviewed in Refs. 6 and 7). However, it is currently unknown whether, and to what extent, the reductions in SERCA2a levels contribute to altered contractile function. In addition, it is unclear whether there are homeostatic mechanisms within the cardiac myocyte that are capable of sensing perturbations in the levels of pump activity and adjusting its expression accordingly. These are i...

Upregulation of desmin protein at moderate levels is not detrimental. However, the D7-des mutation is dominant negative, and expression of the mutant protein leads to the appearance of aggregates that are characteristic of and diagnostic for human desmin-related cardiomyopathy.

Inhibiting FN polymerization or cardiac fibroblast gene expression attenuates pathological properties of MFs in vitro and ameliorates adverse cardiac remodeling and fibrosis in an in vivo model of heart failure. Interfering with FN polymerization may be a new therapeutic strategy for treating cardiac fibrosis and heart failure.

In vitro, fibroblast growth factor-2 (FGF2) has been implicated in cardiomyocyte growth and reexpression of fetal contractile genes, both markers of hypertrophy. However, its in vivo role in cardiac hypertrophy during pressure overload is not well characterized. Mice with or without FGF2 (Fgf2 +/+ and Fgf2 -/-, respectively) were subjected to transverse aortic coarctation (AC). Left ventricular (LV) mass and wall thickness were assessed by echocardiography preoperatively and once a week postoperatively for 10 weeks. In vivo LV function during dobutamine stimulation, cardiomyocyte cross-sectional area, and recapitulation of fetal cardiac genes were also measured. AC Fgf2 -/-mice develop significantly less hypertrophy (4-24% increase) compared with AC Fgf2 +/+ mice (41-52% increase). Cardiomyocyte cross-sectional area is significantly reduced in AC Fgf2 -/-mice. Noncoarcted (NC) and AC Fgf2 -/-mice have similar β-adrenergic responses, but those of AC Fgf2 +/+ mice are blunted. A lack of mitotic growth in both AC Fgf2 +/+ and Fgf2 -/-hearts indicates a hypertrophic response of cardiomyocytes. Consequently, FGF2 plays a major role in cardiac hypertrophy. Comparison of α-and β-cardiac myosin heavy chain mRNA and protein levels in NC and AC Fgf2 +/+ and Fgf2 -/-mice indicates that myosin heavy chain composition depends on hemodynamic stress rather than on FGF2 or hypertrophy, and that isoform switching is transcriptionally, not posttranscriptionally, regulated.

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