Dietary protein restriction throughout intrauterine and postnatal life results in potentially beneficial myocardial tissue remodeling in the adult mouse heart

Diet composition impacts metabolic and cardiovascular health with high caloric diets contributing to obesity related disorders. Dietary interventions such as caloric restriction exert beneficial effects in the cardiovascular system, but alteration of which specific nutrient is responsible is less clear. This study investigates the effects of a low protein diet (LPD) on morphology, tissue composition and function of the neonatal and adult mouse heart. Mice were subjected to LPD (8.8% protein) or standard protein diet (SPD, 22% protein) throughout intrauterine and postnatal life. At birth LPD female but not male offspring exhibit reduced body weight whereas heart weight was unchanged in both sexes. Cardiomyocyte cross sectional area was increased in newborn LPD females compared to SPD, whereas proliferation, cellular tissue composition and vascularization were unaffected. Adult female mice on LPD exhibit reduced body weight but normal heart weight compared to SPD controls. Echocardiography revealed normal left ventricular contractility in LPD animals. Histology showed reduced interstitial fibrosis, lower cardiomyocyte volume and elevated numbers of cardiomyocyte and non-myocyte nuclei per tissue area in adult LPD versus SPD myocardium. Furthermore, capillary density was increased in LPD hearts. In conclusion, pre- and postnatal dietary protein restriction in mice causes a potentially beneficial myocardial remodeling

Embryonic cardiomyocytes can orchestrate various cell protective mechanisms to survive mitochondrial stress

Whereas adult cardiomyocytes are highly susceptible to stress, cardiomyocytes in the prenatal heart appear to be rather resistant. To investigate how embryonic cardiomyocytes respond to metabolic stress in vivo, we utilized tissue mosaicism for mitochondrial dysfunction in 13.5dpc mouse hearts. The latter is based on inactivation of the X-linked gene encoding Holocytochrome c synthase (Hccs), which is essential for mitochondrial respiration. In heterozygous heart conditional Hccs knockout females (cHccs(+/-)) random X chromosomal inactivation results in a mosaic of healthy and HCCS deficient cells in the myocardium. Microarray RNA expression analyses identified genes involved in unfolded protein response (UPR) and programmed cell death as differentially expressed in cHccs(+/-) versus control embryonic hearts. Activation of the UPR is localized to HCCS deficient cardiomyocytes but does not involve ER stress pathways, suggesting that it is caused by defective mitochondria. Consistently, mitochondrial chaperones, such as HSP10 and HSP60, but not ER chaperones are induced in defective cells. Mitochondrial dysfunction can result in oxidative stress, but no evidence for excessive ROS (reactive oxygen species) production was observed in cHccs(+/-) hearts. Instead, the antioxidative proteins SOD2 and PRDX3 are induced, suggesting that ROS detoxification prevents oxidative damage in HCCS deficient cardiomyocytes. Mitochondrial dysfunction and unrestricted UPR can induce cell death, and we detected the initiation of upstream events of both intrinsic as well as extrinsic apoptosis in cHccs(+/-) hearts. Cell death is not executed, however, suggesting the activation of antiapoptotic mechanisms. Whereas most apoptosis inhibitors are either unchanged or downregulated in HCCS deficient cardiomyocytes, Bcl-2 and ARC (apoptosis repressor with caspase recruitment domain) are induced. Given that ARC can inhibit both apoptotic pathways as well as necrosis and attenuates UPR, we generated cHccs(+/-) embryos on an Arc knockout background (cHccs(+/-),Arc(-/-)). Surprisingly, the absence of ARC does not induce cell death in embryonic or postnatal HCCS deficient cardiomyocytes and adult cHccs(+/-),Arc(-/-) mice exhibit normal cardiac morphology and function. Taken together, our data demonstrate an impressive plasticity of embryonic cardiomyocytes to respond to metabolic stress, the loss of which might be involved in the high susceptibility of postnatal cardiomyocytes to cell death

Stress-induced dilated cardiomyopathy in a knock-in mouse model mimicking human titin-based disease

Mutations in a variety of myofibrillar genes cause dilated cardiomyopathy (DCM) in humans, usually with dominant inheritance and incomplete penetrance. Here, we sought to clarify the functional effects of the previously identified DCM-causing TTN 2-bp insertion mutation (c.43628insAT) and generated a titin knock-in mouse model mimicking the c.43628insAT allele. Mutant embryos homozygous for the Ttn knock-in mutation developed defects in sarcomere formation and consequently died before E9.5. Heterozygous mice were viable and demonstrated normal cardiac morphology, function and muscle mechanics. mRNA and protein expression studies on heterozygous hearts demonstrated elevated wild-type titin mRNA under resting conditions, suggesting that up-regulation of the wild-type titin allele compensates for the unstable mutated titin under these conditions. When chronically exposed to angiotensin II or isoproterenol, heterozygous mice developed marked left ventricular dilatation (p<0.05) with impaired fractional shortening (p<0.001) and diffuse myocardial fibrosis (11.95 +/- 2.8% versus 3.7 +/-1.1%). Thus, this model mimics typical features of human dilated cardiomyopathy and may further our understanding of how titin mutations perturb cardiac function and remodel the heart

The complex genetics of hypoplastic left heart syndrome.

Congenital heart disease (CHD) affects up to 1% of live births. Although a genetic etiology is indicated by an increased recurrence risk, sporadic occurrence suggests that CHD genetics is complex. Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR-Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD. Nat Genet 2017 Jul; 49(7):1152-59