**6.2 Cell intrinsic defects in the pathogenesis of HLHS**

The complex genetics of HLHS is further supported by analysis of HLHS mutant mice through the usage of a large-forward genetic screen [5]. Lo et al. recovered 8 HLHS mutant lines with exome sequencing demonstrating no shared mutations among the 8 HLHS lines [5, 84] (**Figure 8**). The results indicate that HLHS is genetically heterogeneous with a multi-genetic etiology. Through extensive analysis of one HLHS mutant line, *Ohia* identified defects in mitochondrial bioenergetics, nitric oxide (NO) metabolism, and cell cycle regulation [5]. The *Ohia* HLHS mouse model exhibits mid-to-late gestation lethality with heart failure characterized by severe pericardial effusion with poor cardiac contractility. This is associated with decreased cardiomyocyte proliferation and increased apoptosis [5]. Ultrastructural analysis showed the myocardium with poorly organized thin myofilaments and altered mitochondrial morphology [5]. Dynamic changes in mitochondria morphology play an important role in the developmentally regulated metabolic switch from glycolysis to oxidative phosphorylation, a process that also plays a critical role in regulating cardiomyocyte differentiation [85]. This entails closure of the mitochondrial permeability transition pore (mPTP) and formation of a mitochondrial transmembrane potential (ΔΨm) mediating oxidative phosphorylation. Using primary cardiomyocyte explants from the E14.5 *Ohia* HLHS mouse heart, Lo et al. measured the ΔΨm, in cardiomyocytes from the right (RV) and left ventricle (LV). A reduction was observed in both the RV and LV cardiomyocytes. To determine whether the abnormal open state of the mPTP is a cell-autonomous defect, mouse induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were used to verify the cardiomyocyte and mitochondrial defects. Those studies indicate that the mitochondrial dysfunction, and proliferation and differentiation defects observed in the *Ohia* HLHS heart tissue are cell autonomous. Hence, the feasibility to model HLHS-HF in iPSC-CM is suggested by studies of the mouse model of HLHS [5]. Furthermore, modeling using human iPSC-CM showed early heart failure (HF) patient iPSC-CM have increased apoptosis, redox stress, and failed antioxidant response. This was associated with mitochondrial permeability transition pore (mPTP) opening, mitochondrial hyper-fusion, and respiration defects. In contrast, iPSC-CM from patients without early-HF had a hyper-elevated antioxidant response with increased mitochondrial fission and mitophagy. Single-cell transcriptomics also showed dichotomization by HF outcome with mitochondrial

#### **Figure 8.**

Ohia *HLHS phenotypes. (A, F) newborn (P0) or E16.5 hearts from wild-type (A) and HLHS mutants (F). Hypoplastic aorta and LV are visible in the HLHS mutant. (B, G)) Histopathology showing the cardiac anatomy of HLHS mutant (G) and littermate control (B) at birth (P0) and E14.5. Compared with controls, the HLHS mutant exhibited hypoplastic aorta and aortic valve atresia, hypertrophied LV with no lumen, and MV stenosis, arrowhead. (C–E, H–J) Ultrasound color-flow imaging of normal fetus (C–E), showing robust flow from the aorta (Ao) and pulmonary artery (PA). In the HLHS mutant (H–J), the aorta showed only a narrow flow stream, whereas the pulmonary artery showed robust flow. 2D imaging revealed hypoplastic LV (H), as compared with the normal-sized LV in the control (C) (modified with permission from reference 5).*

dysfunction and endoplasmic reticulum (ER) stress associated with early-HF. Importantly, oxidative stress and apoptosis associated with early-HF were rescued by sildenafil inhibition of mPTP opening or TUDCA suppression of ER stress. Together, these findings support a new paradigm for modeling clinical outcomes in iPSC-CM, demonstrating that uncompensated mitochondrial oxidative stress underlies early-HF in HLHS [86].
