Chromosomal localization of the loci responsible for dystrophic cardiac calcinosis in DBA/2 mice.

Dystrophic cardiac calcinosis (DCC) occurs in certain inbred strains of mice, including DBA/2 and C3H/He, and is generally found as an incidental lesion in adult animals at necropsy. Preliminary genetic studies into the cause of DCC have been performed in DBA/2 mice and suggest that DCC is inherited as an autosomal recessive trait involving three or four unlinked genes. To investigate the genetics of DCC further, we produced myocardial cell death by freeze-thaw injury to induce DCC. Experiments were conducted with three F1 hybrids made using three inbred strains of mice (DBA/2J and C3H/HeJ, DCC-susceptible strains; C57BL/6J, DCC-resistant strain) to compare the genetic factors in the development of DCC. We found that DBA/2 and C3H/He mice share the same gene pattern(s) that is responsible for DCC. We determined by backcross linkage analysis in DBA/2 and C57BL/6 mice that at least one recessive locus is responsible for DCC. A haplotype analysis of the backcross data demonstrated that the recessive locus, designated dyscalc1, is located on Chromosome 7, 20.5 cM distal to the centromere. The likely candidate genes for dyscalc1 are discussed. Further understanding of the structure and function of these mutant genes will be beneficial in explaining the molecular pathogenesis of DCC.     

Atrial natriuretic peptide and osteopontin are useful markers of cardiac disorders in mice

Biomarkers are not established for cardiovascular phenotyping in mice. We compared the use of echocardiography with the determination of N-terminal propeptide of the atrial natriuretic peptide (Nt-proANP) and osteopontin (Opn). We measured plasma Nt-proANP and Opn levels in (1) the inbred strains C57BL/6, BALB/c, C3H/He, DBA/2, FVB/N, 129S1/Sv; (2) a surgical model of nonischemic myocardial infarction; and (3) delta-sarcoglycan (Sgcd) and calsarcin 1 [also known as myozenin 2 (Myoz2)] knockout models of cardiomyopathy. Left ventricular function was assessed as fractional shortening (FS) by echocardiography in conscious mice. Plasma Nt-proANP exhibited marked variability and ranged from 0.31 +/- 0.19 (C57BL/6 male mice) to 1.34 +/- 0.43 nmol/l (DBA/2 female mice), depending on sex, age, and genetic background. Opn was less variable than Nt-proANP and was decreased significantly in C3H/He and DBA/2 throughout the 16 wk of study. Nt-proANP increased temporarily in mice with myocardial injury. In contrast, Opn increased in both operated and sham-treated mice. Nt-proANP was inversely correlated with FS and distinguished controls from Sgcd and Myoz2 mutants with 100% sensitivity and 71% specificity. Opn was increased in Sgcd mutants, which exhibited only mildly reduced FS but marked myocardial degeneration and fibrosis. Both of these histologic features were absent in Myoz2 mutants. Nt-proANP is an early marker of cardiac disease and is suitable for age- and sex-matched comparisons between groups of transgenic and matched control mice. Opn is useful to detect inflammatory and degenerative myocardial disorders that may be missed by echocardiography.     

Varying susceptibility of pulmonary cytokine production to lipopolysaccharide in mice

Objectives: The lipopolysaccharide (LPS)-induced acute lung injury (ALI) model has been widely applied for pathophysiological and pharmacological research. The aim of present study is to understand the variation of acute pulmonary inflammation between mouse strains. Methods: The present study investigated the susceptibility of acute production of inflammatory mediators, e.g. cytokines, chemokines and others, to LPS in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J, and C3H/HeN mice. Results: The susceptibility to intra-tracheal challenge with LPS varied between measured variables, durations and strains. General lung hyper-reactive susceptibility to LPS-induced pulmonary production of 6–8 inflammatory mediators followed the order NMRI, Balb/cJ, C3H/HeN, A/J, C57BL/6J, DBA/1J, DBA/2J and CD-1 mice at 4 h, and A/J, C3H/HeN, CD-1, NMRI, C57BL/6J, Balb/cJ, DBA/2J and DBA/1J mice at 24 h. Conclusions: Our data provide information for scientists to consider the proper strain of mice for the measurement of specific inflammatory mediators and to select sensitive or resistant mouse strains for understanding genetic variation in the pathogenesis and for the screening of target-oriented drug development.     

Mouse Strain Determines Cardiac Growth Potential

Rationale

The extent of heart disease varies from person to person, suggesting that genetic background is important in pathology. Genetic background is also important when selecting appropriate mouse models to study heart disease. This study examines heart growth as a function of strain, specifically C57BL/6 and DBA/2 mouse strains.

Objective

In this study, we test the hypothesis that two strains of mice, C57BL/6 and DBA/2, will produce varying degrees of heart growth in both physiological and pathological settings.

Methods and Results

Differences in heart dimensions are detectable by echocardiography at 8 weeks of age. Percentages of cardiac progenitor cells (c-kit+ cells) and mononucleated cells were found to be in a higher percentage in DBA/2 mice, and more tri- and quad-nucleated cells were in C57BL/6 mice. Cardiomyocyte turnover shows no significant changes in mitotic activity, however, there is more apoptotic activity in DBA/2 mice. Cardiomyocyte cell size increased with age, but increased more in DBA/2 mice, although percentages of nucleated cells remained the same in both strains. Two-week isoproterenol stimulation showed an increase in heart growth in DBA/2 mice, both at cardiomyocyte and whole heart level. In isoproterenol-treated DBA/2 mice, there was also a greater expression level of the hypertrophy marker, ANF, compared to C57BL/6 mice.

Conclusion

We conclude that the DBA/2 mouse strain has a more immature cardiac phenotype, which correlates to a cardiac protective response to hypertrophy in both physiological and pathological stimulations.     

Sex and strain differences in the hepatotoxic response to acute cocaine administration in the mouse

Cocaine-induced hepatotoxicity was examined in vivo in a dose-responsive manner in C57BL/6Ibg, DBA/2Ibg, C3H/2Ibg, and Balb/cJ mice. Serum glutamic-pyruvic transaminase (SGPT) activities were determined 24 hours after intraperitoneal (IP) administration of cocaine (20 to 100 mg/kg). Significant elevations (100- to 150-fold) in SGPT were observed in male mice receiving cocaine. Significant differences in sensitivity to cocaine-induced hepatotoxicity were found among males of the inbred strains, with Balb being most sensitive and C57BL being least sensitive and C3H and DBA strains exhibiting intermediate sensitivity. Female mice of the four inbred strains were more resistant than males to cocainemediated hepatotoxicity, as indicated by only twofold to tenfold elevations in SGPT values. Among the females, sensitivity of the four inbred strains—as indicated by dose response curves—fell into two categories: the sensitive strains (C3H and C57BL) and the resistant strains (Balb and DBA). Pretreatment of males of the four inbred strains with the P-450 inducer phenobarbital resulted in enhancement of cocaine-mediated hepatotoxicity in the C57BL and Balb but not the C3H and DBA mice. Phenobarbital pretreatment of females of the four inbred strains resulted in enhancement of the hepatotoxic response to cocaine in the C3H, DBA, and Balb mice. Phenobarbital-pretreated C57BL females exhibited a 100% mortality rate after the acute cocaine dose, and thus no determination of hepatotoxicity could be established for them. These data demonstrate sex and strain differences in cocaine-induced hepatotoxicity and suggest that phenobarbital pretreatment does not uniformly enhance the hepatotoxicity of cocaine.     

Myocardial injury after ischemia-reperfusion in mice deficient in Akt2 is associated with increased cardiac macrophage density

Akt2 protein kinase has been shown to promote cell migration and actin polymerization in several cell types, including macrophages. Because migrating macrophages constitute an important inflammatory response after myocardial ischemia, we determined cardiac macrophage expression after ischemia-reperfusion (I/R) injury and cryo-injury in mice lacking Akt2 (Akt2-KO). At 7 days post-I/R, Akt2-KO cardiac tissues showed an increase in immunohistochemical staining for macrophage markers (Galectin 3 and F4/80) compared with wild-type (WT) mice, indicating macrophage density was increased in the injured Akt2-KO myocardium. This change was time dependent because macrophage density was similar between WT and Akt2-KO myocardium at 3 days post-I/R, but by 7 and 14 days post-I/R, macrophage density was significantly increased in Akt2-KO myocardium. Concomitantly, infarct size was larger and cardiac function was reduced in Akt2-KO mice subjected to I/R. However, when cryo-infarction produced similar infarct sizes in the anterior wall in both WT and Akt2-KO mice, macrophage density remained higher in Akt2-KO mouse myocardium, suggesting Akt2 regulates myocardial macrophage density independent of infarct size. Consistently, bone marrow from Akt2-KO mice enhanced myocardial macrophage density in both C57/B6 WT and Akt2-KO recipient mice. Finally, reciprocal ex-vivo coculturing of macrophages and cardiac myocytes showed that activated Akt2-KO peritoneal macrophages had reduced mobility and adhesion when compared with WT littermate controls. Thus, although Akt-2 KO mice did not affect the initial inflammation response after injury and Akt2 deficiency has been shown to impair cell migration or motility in macrophages, our data suggested a novel mechanism in which increasing retention of Akt2-KO macrophages resulted in increasing cardiac Akt2-KO macrophage density in the myocardial space.     

Gene expression changes in the host response between resistant and susceptible inbred mouse strains after influenza A infection

Inbred mouse strains exhibit differences in susceptibility to influenza A infections. However, the molecular mechanisms underlying these differences are unknown. Therefore, we infected a highly susceptible mouse strain (DBA/2J) and a resistant strain (C57BL/6J) with influenza A H1N1 (PR8) and performed genome-wide expression analysis. We found genes expressed in lung epithelium that were specifically down-regulated in DBA/2J mice, whereas a cluster of genes on chromosome 3 was only down-regulated in C57BL/6J. In both mouse strains, chemokines, cytokines and interferon-response genes were up-regulated, indicating that the main innate immune defense pathways were activated. However, many immune response genes were up-regulated in DBA/2J much stronger than in C57BL/6J, and several immune response genes were exclusively regulated in DBA/2J. Thus, susceptible DBA/2J mice showed a hyper-inflammatory response. This response is similar to infections with highly pathogenic influenza virus and may serve as a paradigm for a hyper-inflammatory host response to influenza A virus.     

Bacillary growth, interleukin 2 production defect, and specific antibody secretion governed by different genetical factors in mice infected subcutaneously with Mycobacterium lepraemurium

Different mouse strains were infected subcutaneously in the footpad with 10(7) Mycobacterium lepraemurium (MLM). At various stages of the infection, the number of acid-fast bacilli (AFB) in different organs, spleen cell interleukin 2 production, and specific IgM and IgG serum antibodies to MLM sonicate were assessed. Strains were separable into two distinct groups depending on the number of AFB recovered from the different organs, without any obvious influence of the Bcg gene. Thus C57BL/6, DBA/2, (C57BL/6 X DBA/2)F1 and C3H/Pas mice belonged to the high resistance group and DBA/1, BALB/c, and CBA strains to the low resistance group. Interleukin 2 production was depressed only in C57BL/6 and C3H/Pas mice. Anti-MLM antibody response also markedly varied according to strains, in terms of antibody titers, Ig class distribution, and species specificity, but with a different genetic pattern from that observed for MLM growth control.     

Strain differences in histamine release from mouse peritoneal mast cells induced by compound 48/80 or A23187

Strain differences among BALB/c, BDF1, CDF1, C3 H/He, C57 BL/6, DBA/2, ddy and ICR mice were investigated with respect to the ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80, a Ca2+ dependent histamine releaser, and the Ca2+ ionophore A23187. The ratios of histamine release from mouse peritoneal mast cells induced by compound 48/80 were found to be high in BALB/c, ddY and ICR mice, but low in BDF1, CDF1, C3 H/He, C57 BL/6 and DBA/2 mice. Those induced by Ca2+ ionophore A23187 were high in BALB/c, BDF1, CDF1, C3 H/He, DBA2, ddy and ICR mice but low in C57 BL/6 mice. These results indicate that differences in histamine release from mouse peritoneal mast cells are strain dependent.     

NSD2 promotes ventricular remodelling mediated by the regulation of H3K36me2

Histone lysine methylation plays an important role in the regulation of ventricular remodelling. NSD2 is involved in many types of tumours through enhancing H3K36me2 expression. However, the role of NSD2 in the regulation of histone lysine methylation during ventricular remodelling remains unclear. In this study, we established cardiac hypertrophy model in C57BL/6 mice by transverse aortic constriction and found that histone lysine methylation participated in ventricular remodelling regulation via the up‐regulation of H3K27me2 and H3K36me2 expression. In addition, we constructed transgenic C57BL/6 mice with conditional knockout of NSD2 (NSD2^?/?) in the myocardium. NSD2^?/? C57BL/6 mice had milder ventricular remodelling and significantly improved cardiac function compared with wild‐type mice, and the expression of H3K36me2 but not H3K27me2 was down‐regulated. In conclusion, NSD2 promotes ventricular remodelling mediated by the regulation of H3K36me2.     

Effect of a purified diet on dystrophic cardiac calcinosis in mice

Male and female C3H/HeNCrl mice were divided into test groups and fed either a purified diet (AIN-76A) or a natural ingredient diet (NIH-07). Lesions of dystrophic cardiac calcinosis (DCC) were found to be more prevalent and more severe in mice fed the purified diet. The cardiac changes, which were similar in nature in both groups of mice, consisted of randomly distributed foci of myocardial mineralization and fibrosis. These lesions were not associated with clinical disease or significant alterations in serum calcium, lactic dehydrogenase, aspartate aminotransferase, alkaline phosphatase or creatine kinase levels. We conclude that AIN-76A purified diet should be utilized with caution in toxicology studies which use mice as experimental subjects, especially if the heart is a potential target organ.     

Genetic differences in nicotine-induced conditioned taste aversion

Genetic differences in nicotine-induced conditioned taste aversion were examined using inbred mice. Adult male C57BL/6J, DBA/2J, BALB/cJ and C3H/heJ mice were adapted to a 2-h per day water access regimen. Subsequently, mice received nicotine injections (0.5, 1.0 or 2.0 mg/kg) immediately after 1-h access to a NaCl flavored solution. DBA and C3H mice developed dosedependent aversions to the nicotine-paired flavor. BALB mice showed only minor reductions in intake with no difference between the nicotine dose groups. C57BL mice did not show development of nicotine-induced conditioned taste aversion. These results demonstrate that nicotine's aversive motivational effect is strongly influenced by genotype. Further, genetic sensitivity (DBA mice) or insensitivity (C57BL mice) to nicotine-induced conditioned taste aversion was similar to reports of genetic sensitivity to ethanol's aversive effect measured in this design.     

Strain differences of ether susceptibility in mice

The susceptibility to ether in the following six strains of mice was tested: C57BL/6, DBA/2, BALB/c, C3H/He, ICR and ddY. Mice of 4 weeks old were exposed to a flow of air containing various concentrations of ether for 90 min and the mortalities were assessed. The C57BL/6 strain was the most resistant and the C3H/He strain was the most sensitive to the lethal effect of ether. The susceptibilities of the closed colony mice, ICR and ddY, were intermediate between those of C57BL/6 and C3H/He mice. The DBA/2 and BALB/c strains were more sensitive than these closed colony mice and made up a sensitive group with the C3H/He strain. The LD50 values for ether in male mice of C57BL/6 and C3H/He were 6.0 and 3.1% atm, and in female mice of these strains were 6.6 and 3.2% atm, respectively. The ED50 value of ether which was accompanied by loss of righting reflex after exposure for 10 min was also higher in male C57BL/6 mice than in male C3H/He mice.     

Type 2 MI induced by a single high dose of isoproterenol in C57BL/6J mice triggers a persistent adaptive immune response against the heart

Heart failure is the common final pathway of several cardiovascular conditions and a major cause of morbidity and mortality worldwide. Aberrant activation of the adaptive immune system in response to myocardial necrosis has recently been implicated in the development of heart failure. The ß-adrenergic agonist isoproterenol hydrochloride is used for its cardiac effects in a variety of different dosing regimens with high doses causing acute cardiomyocyte necrosis. To assess whether isoproterenol-induced cardiomyocyte necrosis triggers an adaptive immune response against the heart, we treated C57BL/6J mice with a single intraperitoneal injection of isoproterenol. We confirmed tissue damage reminiscent of human type 2 myocardial infarction. This is followed by an adaptive immune response targeting the heart as demonstrated by the activation of T cells, the presence of anti-heart auto-antibodies in the serum as late as 12 weeks after initial challenge and IgG deposition in the myocardium. All of these are hallmark signs of an established autoimmune response. Adoptive transfer of splenocytes from isoproterenol-treated mice induces left ventricular dilation and impairs cardiac function in healthy recipients. In summary, a single administration of a high dose of isoproterenol is a suitable high-throughput model for future studies of the pathological mechanisms of anti-heart autoimmunity and to test potential immunomodulatory therapeutic approaches.     

Mouse genome-wide association study identifies polymorphisms on chromosomes 4, 11, and 15 for age-related cardiac fibrosis

Dystrophic cardiac calcinosis (DCC), also called epicardial and myocardial fibrosis and mineralization, has been detected in mice of a number of laboratory inbred strains, most commonly C3H/HeJ and DBA/2J. In previous mouse breeding studies between these DCC susceptible and the DCC-resistant strain C57BL/6J, 4 genetic loci harboring genes involved in DCC inheritance were identified and subsequently termed Dyscalc loci 1 through 4. Here, we report susceptibility to cardiac fibrosis, a sub-phenotype of DCC, at 12 and 20 months of age and close to natural death in a survey of 28 inbred mouse strains. Eight strains showed cardiac fibrosis with highest frequency and severity in the moribund mice. Using genotype and phenotype information of the 28 investigated strains, we performed genome-wide association studies (GWAS) and identified the most significant associations on chromosome (Chr) 15 at 72 million base pairs (Mb) (P < 10^?13) and Chr 4 at 122 Mb (P < 10^?11) and 134 Mb (P < 10^?7). At the Chr 15 locus, Col22a1 and Kcnk9 were identified. Both have been reported to be morphologically and functionally important in the heart muscle. The strongest Chr 4 associations were located approximately 6 Mb away from the Dyscalc 2 quantitative trait locus peak within the boundaries of the Extl1 gene and in close proximity to the Trim63 and Cap1 genes. In addition, a single-nucleotide polymorphism association was found on chromosome 11. This study provides evidence for more than the previously reported 4 genetic loci determining cardiac fibrosis and DCC. The study also highlights the power of GWAS in the mouse for dissecting complex genetic traits.     

Natural regulatory T cells mediate the development of cerebral malaria by modifying the pro-inflammatory response

Cerebral malaria (CM) is a severe neurologic complication that arises predominantly in children and non-immune adults infected with Plasmodium falciparum. In the current study, the dynamics of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) and pro- and anti-inflammatory cytokines were analyzed in P. berghei ANKA (P.bANKA)-infected C57BL/6, BALB/c, and DBA/2 mice. We showed that C57BL/6 mice were susceptible to CM, while BALB/c and DBA/2 mice were resistant to CM and succumbed to hyperparasitemia and severe anemia. The proportion and absolute numbers of Tregs in BALB/c and DBA/2 mice were significantly higher than in C57BL/6 mice. The levels of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-6, IL-17 and NO in CM-susceptible C57BL/6 mice were obviously higher than in CM-resistant BALB/c and DBA/2 mice, while the level of the anti-inflammatory cytokine IL-10 was the opposite to that of pro-inflammatory cytokines, confirming that an appropriate balance between pro- and anti-inflammatory immune responses is essential to control the pathogenesis of severe malaria, and Tregs are important regulators if this balance is to be maintained. In vivo depletion of Tregs significantly protected C57BL/6 mice from experimental CM and the production of pro- and anti-inflammatory cytokines was reversed, indicating that this cell population contributes to pathogenesis by modulating the balance of pro- and anti-inflammatory responses. Our data demonstrate that Tregs mediate the incidence and outcome of CM in P.bANKA-infected mice by modifying the pro-inflammatory response.     

Distinctive and critical roles for cellular immunity and immune-inflammatory response in the immunopathology of Sendai virus infection in mice

Respiratory viral infections result in severe pulmonary injury, to which host immune response may be a significant contributor. At present, it is not entirely clear the extent to which lung injury is a necessary consequence of host defense. In this report, we use functional genomics approach to characterize the key roles of cellular immunity and immune-inflammatory response in the immunopathology of Sendai virus infection in resistant C57BL/6J and susceptible DBA/2J mice. Infected mice manifested an immune-inflammatory response characterized by the pulmonary influx of neutrophils and mononuclear cells. DBA/2J mice mounted a vigorous immune response, with significant up-regulation of cytokine/chemokine genes in two successive waves through the course of infection. Whereas, C57BL/6J mice displayed an efficient immune response with less severe pathology and clusters of immune-inflammatory responsive genes were exclusively up-regulated on day 4 in this strain. Overall, DBA/2J mice exhibited a dysregulated hyper-inflammatory cytokine/chemokine cascades that does not limit viral spread resulting in a predisposition to severe lung pathology. This response is similar to severe human respiratory paramyxovirus infections, which will serve as a model for the elucidation of hyper-immune inflammatory response that result to severe immunopathology in respiratory viral infections.     

Anthrax Lethal Toxin Kills Macrophages in a Strain-Specific Manner by Apoptosis or Caspase-1-Mediated Necrosis

Murine macrophages have been classified as either susceptible or nonsusceptible to killing by anthrax lethal toxin (LT) depending upon genetic background. While considered resistant to LT killing, we found that bone marrow-derived macrophages (BMMs) from DBA/2, AKR, and C57BL/6 mice were slowly killed by apoptosis following LT exposure. LT killing was not restricted to in vitro assays, as splenic macrophages were also depleted in LT-injected C57BL/6 mice. Human macrophages, also considered LT resistant, similarly underwent slow apoptosis in response to LT challenge. In contrast, LT triggered rapid necrosis and a broad protein release in BMMs derived from BALB/c and C3H/HeJ, but not C57BL/6 mice. Released proteins included processed interleukin-18, confirming reports of inflammasome and caspase-1 activation in LT-mediated necrosis in macrophages. Complete inhibition of caspase-1 activity was required to block LT-mediated necrosis. Strikingly, minimal residual caspase-1 activity was sufficient to trigger significant necrosis in LT-treated macrophages, indicating the toxicity of caspase-1 in this process. IL-18 release does not trigger cytolysis, as IL-18 is released late and only from LT-treated macrophages undergoing membrane perturbation. We propose that caspase-1-mediated macrophage necrosis is the source of the cytokine storm and rapid disease progression reported in LT-treated BALB/c mice.     

Strain differences in the immune response of mice to homologous sperm-specific lactate dehydrogenase (LDH-C4).

The sperm-specific isozyme of murine lactate dehydrogenase (LDH-C4) was injected into female mice of various strains. Two regulatory phenotypes characterize the resultant immunity to LDH-C4: one is manifested by high, intermediate or low levels of response, the other by the immediate or delayed maturation of peak titer. The response of several strains can be classified as high (SWR, SJL, BABL/c, C3H/He) and intermediate to low (A, CBA, DBA/2, DBA/1, C57BL/6) according to the level of antibody production and cell mediated immunity. BALB/c, SJL and SWR strains are immediate responders while DBA/2 and C3H/He mice are clearly delayed responders. Maturation and magnitude of response do not appear to be related. Both the antibody and cell mediated responses are T-dependent, but are not obviously associated with Ig allotype or H-2 regulation.